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MX2007003341A - Inorganic selenium for treatment of cancer. - Google Patents

Inorganic selenium for treatment of cancer.

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Publication number
MX2007003341A
MX2007003341A MX2007003341A MX2007003341A MX2007003341A MX 2007003341 A MX2007003341 A MX 2007003341A MX 2007003341 A MX2007003341 A MX 2007003341A MX 2007003341 A MX2007003341 A MX 2007003341A MX 2007003341 A MX2007003341 A MX 2007003341A
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MX
Mexico
Prior art keywords
cancer
selenate
cells
akt
agent
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MX2007003341A
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Spanish (es)
Inventor
Niall Corcoran
Christopher Hovens
Anthony Costello
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Velacor Therapeutics Pty Ltd
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Priority claimed from AU2004905422A external-priority patent/AU2004905422A0/en
Application filed by Velacor Therapeutics Pty Ltd filed Critical Velacor Therapeutics Pty Ltd
Priority claimed from PCT/AU2005/000111 external-priority patent/WO2006032074A1/en
Publication of MX2007003341A publication Critical patent/MX2007003341A/en

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention discloses the use of selenate or its pharmaceutically acceptable salts, especially in supranutritional amounts, in methods and compositions for inhibiting the growth or proliferation of tumor cells. The present invention also discloses the use of selenate or its pharmaceutically acceptable salts in combination with one or both of a hormone ablation therapy and a cytostatic agent or cytotoxic agent, for inhibiting the growth or proliferation of tumor cells. In certain embodiments, the methods of the invention are useful for treating or preventing cancers, especially cancers in which the Akt signaling pathway is activated, such as prostate cancer. Additionally, the present invention discloses the use of selenate or its pharmaceutically acceptable salts in combination with a hormone-ablation therapy and optionally a cytostatic agent or cytotoxic agent in methods and compositions for treating hormone-dependent cancers.

Description

ORGANIC SELENIO FOR CANCER TREATMENT FIELD OF THE INVENTION This invention relates in general to the use of selenate or its pharmaceutically acceptable salts, especially in supra-nutritional quantities, in methods and compositions for inhibiting the growth or proliferation of tumor cells. The present invention also relates to the use of selenate or a pharmaceutically acceptable salt thereof in combination with at least one of a hormone ablation therapy, a cytostatic agent or cytotoxic agent, to inhibit the growth or proliferation of tumor cells. In certain embodiments, the methods of the invention are useful for treating or preventing cancers, especially cancers wherein the Akt signaling pathway is activated, such as prostate cancer. In addition, the present invention relates to the use of selenate or a pharmaceutically acceptable salt thereof in combination with a hormone ablation therapy and optionally a cytostatic agent or a cytotoxic agent in methods and compositions for treating hormone dependent cancers.
BACKGROUND OF THE INVENTION There has been a lot of interest in the use of selenium compounds as cancer preventive agents. Studies that have expanded over the last 20 years have documented its cancer preventive action in tumors of the mammary gland (Ip, C. 1981, Cancer Res. 41: 4386-4390), colon (Reddy et al., 1981, Res Cancer). 47: 5901-5904), lung and prostate (Clark et al 1996, Jama 276; 1957-1963). Both Phase II and III clinical trials for the prevention of prostate cancer using an organic selenomethionine are currently under investigation (Nelson et al., 1999, supra). The selenium compounds used in chemoprevention studies can be broadly classified into inorganic and organic selenium forms. The typical form of inorganic selenium, sodium selenite, (Na2Se03) is relatively toxic, causing damage to the DNA of single and double chain suture rupture, while the typical organic selenium entity, seleniomethionine (SeMet) is relatively non-toxic and it does not harm DNA (Lu ef ai 1995, supra, Sinha et al 1996, supra, Stewart et al 1999, supra). Organic selenomethionine, the main constituent of a selenium diet, is incorporated into cellular selenoproteins, such as thioredoxin reductase and glutathione peroxidase through a series of complex intermediates (Alian et al., 1999, Annu Rev. Nutr. 19: 1 -16). Since this selenocysteine-containing selenium, such as antioxidant glutathione peroxidase and redox-regulatory thioredoxins, are involved in cellular responses to mutagenic oxidant stress, it has long been proposed that supra-nutritional levels of selenium consumption promote anticarcinogenic cellular activity promoting a cellular reductive environment (Alian er al., 1999, supra). However, it has become very clear that the nature of the anti-tumorigenic action of the selenium compounds depends on the chemical form in which the element is administered (IP, C. 1998, J. Nutr.128: 1845-1854 ). Organic selenium in the form of methylselenic acid induces G1 arrest, as well as DNA fragmentation and caspase-mediated cleavage of PARP, which are two markers of apoptosis, in prostate cancer cells DU145 (Jiang et al., 2001). , Cancer Res. 61: 3062-3070). In contrast, selenite induces S-phase arrest and apoptotic DNA fragmentation, which is independent of caspase function (Jiang et al., 2002, Mol.Cancer Ther. 1: 1059-1066). Differences have also been reported for inorganic selenium compounds, selenite and selenate inhibiting the growth of lymphocytes through different mechanisms (Spyrou et al., 1996, Cancer Res. 56: 4407-4412). These differences can be explained in part through variations in metabolism and bioavailability between an in vitro cell culture and in vivo studies (Dong et al 2003, Cancer Res. 63: 52-59). Hormone-dependent cancers include tumor cells that have hormone receptors and the growth and proliferation of these tumor cells is enhanced by the presence of the hormone. Hormone ablation therapy, either Surgical or chemical, it is used to stop, at least over a period of time, the growth and proliferation of tumor cells in hormone-dependent tumors, such as prostate, testicular, thyroid, breast, ovarian and uterine cancers. Prostate cancer is a cancer prevalent in human men and the treatment of patients with advanced prostate carcinoma growth typically involves medical or surgical castration (Huggins, C. and Hodges, CV 1941, Cancer Res. 1: 293-297) . Up to 80% of patients demonstrate a temporary response lasting an average of 12-18 months, before continuous tumor growth is evident despite castration levels of testosterone (Petrylak, DP 1999, Urology 54:30 -35). Once independent androgen growth is established, the average life expectancy is 9-12 months. Although traditional therapy can improve pain rates and increase quality of life, they offer no significant survival benefit. The molecular mechanisms that underline the transition to androgen independent growth are incompletely understood. Changes in androgen receptor signaling play an important role, including over-regulation of receptor expression, promiscuous ligand binding as well as transactivation of ligand-independent receptor through other cascades of growth signaling (Feldman, BJ and Feldman, D. 2001, Nat. Rev. Cancer 1: 34-45).
Concomitantly, the prostate cancer cells acquire a number of mechanisms that protect them from induced cell death, partly explaining their chemoresistance (Gurumurthy et al., 2001, Cancer Metastasis Rev. 20: 225-243). The role of the P13K / Akt trajectory in this is greatly recognized. Akt is a serine / threonine kinase that is activated in response to phosphorylation of P13K stimulated by membrane receptor. Akt, in turn, regulate the activity of several proteins involved in the control of apoptosis, including the transcription factors of Forkhead (FKHR), Bad, Caspase 9, GSK-3β and Mtor (Vivanco, I. and Sawyers, CL 2002 , Nat. Rev. Cancer 2: 289-501). Akt over activity is common in androgen-independent prostate cancer, usually as a result of the hypofunction of PTEN, a phosphatase that inactivates PI3K, and is sufficient to induce androgen-independent growth (Whang et al., 1998, p. Proc. Nati, Acad. Sci. USA 95: 5246-5250). The resistance to radiotherapy is tumor cells has also been linked with the over-regulation of the trajectory of PI3K / Akt Soderlund et al. 2005, Int. J. Oncol, 26: 25-32, Zhan et al, 2004, Histol. Histopathol., 19: 915-923; Tanno ef al. 2004, Cancer Res., 64: 3486-3490; Li eí al. 2004, Oncogene, 23: 4594-4602; McKenna et al., 2003, Genes Chromosomes Cancer, 28: 330-338; Liang ef ai. 2003, Mol. Cancer Ther. 2: 353-360). Interest in the clinical use of compounds containing selenium as a chemopreventive agent has been disseminated after of Clark's publication ef al. (1996, supra). These results have disseminated a number of human clinical trials using supra-nutritional selenomethionine as a chemopreventive agent for prostate cancer. (Meuillet et al., 2004, J. Cell Biochem. 91: 443-458). However, given the relatively short intervention time compared to the long natural history of prostate cancer, it is possible that instead of preventing the transformation of normal prostate epithelium to neoplasia, selenium probably inhibits the growth of malignant cells (Corcoran et al. al., 2004, J. Ul. 171: 907-910). The evidence is to accumulate that selenium anticancer activity is not related to the element per se but depends on its chemical form (Menter et al., 2000, Epidemiol Cancer, Biomarkers Prev. 9: 1171-1182, Jiang et al., 2002, supra; Kim et al., 2003, Biochem, Pharmacol 66: 2301-2311). The mechanism of this activity is also unclear. Nevertheless, it has been observed that there are marked differences in tumor progression of human prostate tumor cells in vivo, depending on the selenium compound administered. For example, inorganic sodium selenate significantly reduces tumor progression compared to organic selenomethionine, methylselenocysteine or selenized yeast (Corcoran ef al.2004, supra). In addition, certain selenium compounds, such as sodium selenite, have been found to be toxic to cells when used at certain doses (Jiang et al.2002, supra). Therefore, there is a need to identify forms of selenium compounds to treat or prevent the growth of cancer. In particular, there is a need to understand the selective molecular underlying mechanisms of action of various selenium compounds on cancer cells with a view to designing more effective therapeutic regimens.
BRIEF DESCRIPTION OF THE INVENTION The present invention is in part dedicated to the discovery that a specific type of inorganic selenium compound, primarily selenate, significantly inhibits tumor cell proliferation, including the proliferation of hormone-independent tumor cells and hormone-dependent tumor cells, especially when It uses high amounts or supra-nutritional amounts, as compared to other selenium compounds. It has also been found that selenate and its pharmaceutically acceptable salts have an inhibitory effect on tumor cells, especially prostate tumor cells, where the path of Akt signaling is activated, and have a strong synergistic inhibitory effect on the growth of tumor cells. when used in combination with at least one of a cytostatic agent, a cytotoxic agent and radiotherapy that is optionally administered with a radiosensitizing agent. In addition, it has been found that selenate and its pharmaceutically acceptable salts have an inhibitory effect on the hormone-dependent tumor cell growth, when used in combination with a hormone ablation therapy and optionally one or more of a cytotoxic agent, a cytotoxic agent and a radiotherapy that is optionally administered with a radiosensitizing agent. Accordingly, in one aspect, the present invention provides methods for inhibiting the growth or proliferation of tumor cells, wherein the Akt signaling pathway is activated. These methods generally comprise exposing the tumor cells to an amount of inhibition of activation of the Akt signaling pathway of selenate or a pharmaceutically acceptable salt thereof. In some embodiments, activation of the Akt signaling pathway involves the activation of at least one selected member of Akt, mTOR, GSK-3β and FKHR. In illustrative examples of this type, activation of the Akt signaling pathway involves Akt phosphorylation (eg, phosphorylation of the Thr 308 and Ser 473 residues of Akt). In other modalities, the activation of the Akt signaling path involves the inactivation of PTEN. In some embodiments, the signaling path of Akt is over-activated. In some embodiments, the amount of selenate or its pharmaceutically acceptable salt, to which the tumor cells are exposed, is a supra-nutritional amount. Conveniently, the tumor cells were selected from oral squamous cell carcinoma, thyroid cancer, hepatocellular carcinoma, prostate carcinoma, fibrosarcoma, ovarian carcinoma, uterine or endometrial cancer, pancreatic carcinoma, stomach cancer, breast cancer, lung cancer, renal cell carcinoma, colon cancer, melanoma, acute leukemia, and brain cancer (eg, astrocytoma and glioblastoma) ) in specific modalities, the tumor cells are prostate cancer cells, include hormone-independent prostate cancer cells. In another aspect, the present invention provides methods for treating cancer, especially a hormone-independent cancer or a hormone-dependent cancer, in a subject. These methods generally comprise administering to the subject a therapeutically effective amount of selenate or a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutically effective amount is a supra-nutritional amount. In specific modalities, cancer is prostate cancer, especially hormone-independent or hormone-dependent prostate cancer. In this way, in a related aspect, the invention provides methods for treating prostate cancer, especially a hormone-independent or hormone-dependent prostate cancer, which comprises administering to a subject in need of such treatment, a therapeutically effective amount of selenate or a pharmaceutically acceptable salt thereof. In another aspect, the present invention provides methods for inhibiting tumor cell growth dependent on hormone, which comprises exposing the tumor cells to an inhibitory amount of selenate hormone-dependent tumor cell growth or a pharmaceutically acceptable salt thereof and a hormone ablation therapy. In yet another aspect, the present invention provides methods for treating a hormone dependent cancer in a subject, comprising administering a therapeutically effective amount of selenate or a pharmaceutically acceptable salt thereof in combination with a hormone ablation therapy. Hormone-dependent cancers are conveniently selected from androgen-dependent cancers and estrogen-dependent cancer. In certain embodiments, the hormone-dependent cancer is an androgen-dependent cancer such as prostate cancer. In some modalities, the hormone-dependent cancer is selected from prostate cancer, testicular cancer, breast cancer, ovarian cancer, uterine cancer, endometrial cancer, thyroid cancer and pituitary cancer. In a further aspect, the present invention provides the use of selenate or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a cancer wherein the Akt signaling pathway is activated. In still another aspect, the present invention provides the use of selenate or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a cancer, wherein the signaling pathway of Akt is activated, wherein the cancer is different from a cancer selected for PC-3 prostate cancer, 3B6 lymphoma, BL41 lymphoma and HTB123 / DU4473 breast tumor. In another aspect, the present invention provides the use of selenate or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a hormone-dependent cancer, wherein the selenate or its pharmaceutically acceptable salt is formulated to be administered in combination with therapy. of hormone ablation. In still another aspect, the present invention provides pharmaceutical compositions for treating or preventing cancer. The compositions generally comprise a supra-nutritional amount of selenate or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. In some embodiments of the methods and uses widely described herein, the selenate or its pharmaceutically acceptable salt is administered in combination with at least one cytostatic agent, a cytotoxic agent or a radiotherapy that is optionally administered with a radio sensitizing agent. In other embodiments, the selenate is formulated in a composition with at least one cytostatic agent or cytotoxic agent. In still other embodiments, the selenate is formulated in a composition with a radiosensitizing agent to be used in combination with radiotherapy.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graphic and photographic representation showing that sodium selenate inhibits the proliferation of prostate tumor cells, in an orthotopic mouse model. Six-week-old male BALB / c hairless mice were injected into the dorsal lateral prostate with 1 x 10 6 PC-3 cells. Ten animals per group then received either 5 ppm of sodium selenate (NaSe) or no treatment (With) in the water to drink for 5 weeks. The animals were then sacrificed selectively. (A) Figure 1 (A) graphically indicates that the weight of the prostate glands containing the tumor was reduced in the mice that received sodium selenate. (B) Figure 1 (B) graphically indicates that tumor volumes, measured with a Vernier caliper, were reduced slightly (but not significantly) in mice that received sodium selenate. (C) The retroperitoneum was then explored under cephalad extension at the level of the renal vein and the lymph nodes (L.N.no) greater than 0.5 mm were counted. Figure 1 (C) indicates the number of lymph nodes greater than 0.5 mm that were reduced in mice that received sodium selenate. The results illustrate the +/- SE average. Figures 1 (A) and 1 (C) have p < 0.05 against control. (D) Figure 1 (D) provides a compilation of BrdU positive cell nuclei and Tunnel of prostate tumor samples. The results represent the +/- SE mean per high energy field for four tumor samples from the groups without treatment (With) and with sodium selenate (NaSe). (E) Figure 1 (E) provides an immunohistochemical sample representative of BrdU positive nuclei from prostate tumor tissue samples without treatment (control) and with sodium selenate (NaSe), at a magnification of 100x. Figure 2 is a graphical and photographic representation showing that sodium selenate inhibits proliferation of prostate carcinoma cell by inducing a G1 cell cycle block. (A) 2 x 1 O5 PC-3 cells were plated in a 6-well plate and incubated for 16 hours. Then sodium selenate was added at the indicated concentrations and incubation was continued for the indicated time points. The nonadherent and adherent cell fractions were then harvested and the total viable cell numbers were combined and counted through the Trypan Blue Exclusion assay. Figure 1 (A) graphically indicates the effect of different concentrations of sodium selenate on numbers of viable cells. (B) 2.5 x 104 PC-3 cells were plated in a 24-well plate and 16 hours later they were exposed to 0.05, 0.1, or 0.25 nM of sodium selenate for 36 hours, then the cell-labeling reagent was added. BrdU / FrdU proliferation (Roche) for 16 more hours. The cells were washed, fixed and stained for nuclear incorporation of BrdU / FrdU, and DAP1 for the identification of cell nuclei. The results are shown in Figure 2 (B). (C) 2 x 105 PC-3 cells were seeded in a T25 flask and incubated for 16 hours. The cells they were then washed and covered in a serum-free medium for an additional 48 hours. The cells were then re-stimulated with fresh serum (FCS, 10%) containing 0.0, 0.1, 0.25, or 0.5 mM of sodium selenate for 24 or 48 hours. Cells were then harvested, fixed and stained with proprioiodide, and FACS analysis was performed. The graphical representation of mean percentages of cells in the G1 and G2 / M phases of the cell cycle for three independent experiments is shown in Figure 2 (D). (E) The cell proliferation was analyzed through the MTT assay. 1 x 104 PC-3 cells were plated in a 96-well plate and incubated for 16 hours. Then indicated concentrations of sodium selenate or selenomethionine were added and 73 hours later the cell proliferative index was measured through the MTT assay. The results are shown in Figure 2 (E). For all results, the values shown represent + S.D. average of at least three independent experiments. Figure 3 is a photographic representation showing that sodium selenate induces up-regulation of the cell cycle inhibitory proteins and dephosphorylation of the retinoblastoma protein. (A) 7.5 × 10 5 PC-3 cells were seeded in 6-centimeter dishes, after 16 hours of asynchronous growth, the cells were treated with 0.5 mM of sodium selenate for the indicated times, then the cells were lysed in buffer. ELB pH, and equal amounts of whole cell lysates (75 μg) were separated on 12% SDS-PAGE gels and the membranes were probed with the indicated primary antibodies. The results are shown in Figure 3 (A). (B) 5 x 10 5 PC-3 cells were plated in a 6-well plate, incubated for 10 hours, washed, and serum-deprived for 16 hours, then treated with sodium selenate (0.5 mM) for 10 hours. time points shown. The cells were lysed in pH buffer of ELB and the whole cell lysates (75 μg) were loaded in 10% SDS-PAGE geies and the membranes were probed with the indicated antibodies. The results are shown in Figure 3 (B). (C) 2 x 105 PC-3 cells were plated in a 6-well plate (incubated for 16 hours and then treated with sodium selenate (Na2SeO4, 0.5 mM) or selenomethionine SeMet, 0.5 mM) for the indicated times and Whole cell lysates (75 μg) were operated on 12.5% SDS-PAGE gels and membranes were probed with the indicated antibodies. The results are shown in Figure 3 (C). Figure 4 is a photographic representation showing that sodium selenate, but not selenomethionine, inhibits the chronic activation of Akt in PTEN-deficient PC-3 cells. (A) PC-3 cells were deprived of serum for 16 hours, then sodium senate (Na2Se0, 0.5 mM) was added during the indicated periods and whole cell lysates (75 μg) were loaded on SDS-PAGE gels 10% and the membranes were probed with phospho-specific Akt antibodies and pan-Akt. The results are shown in Figure 4 (A). (B) Identical lysates (75 μg) were probed from (A) with Phospho-specific PDK1 antibodies and tubulin ßlll antibody as a load control. These results are shown in Figure 4 (B). (C) PC-3 cells were deprived of serum for 16 hours, then sodium selenate (Na2SeO, 0.5 mM, for 10 minutes) of LY294002 (50 μM for 1 hour) alone or in combination was added, then the cells were lysed and the whole cell lysates (75 μg) were resolved on 10% SDS-PAGE gels and the membranes were probed with phospho-specific antibodies and pan Akt. The results are shown in Figure 4 (C). (D) PC-3 cells were deprived of serum for 16 hours, then treated with either sodium selenate (Na2Se04, 0.5 mM) or selenomethionine (SeMet, 0.5 mM) for the different periods of time indicated, then equal amounts of whole cell lysates (75 μg) were stirred on 10% SDS-PAGE gels and the membranes were probed with phospho-specific antibodies and pan Akt. The results are shown in Figure 4 (D). Figure 5 is a graphical and photographic representation showing that sodium selenate, but not selenomethionine, induces nuclear translocation of the Forkhead transcription factor and sub-regulation of effector protein activity of the PI3K cell survival path. (A) PC-3 cells were transfected with the GFP-Forkhead expression construct (GFP-FKHR, 0.8 μg) with Lipofectamine 2000 reagent, after 24 hours they were serum starved for 16 hours, then treated either with sodium selenate (Na2SeO, 0.5 mM), or selenomethionine (SeMet, 0.5 mM) for 3 hours, or with LY294002 (10 μM) for 1 hour. Cells were then fixed and nuclear and cytoplasmic GFP fluorescence status was observed. DAPI staining indicates cell nuclei. The results are shown in Figure 5 (A). (B) Figure 5 (B) provides a graphic illustration of + S.D. average of results in (A). All experiments were performed in triplicate and at least 70 transfected cells were classified for each treatment. (C), (D) PC-3 cells were deprived of serum for 16 hours, then treated with sodium selenate (Na2SeO4, 0.5 mM) for the indicated periods of time and whole cell lysates (75 μg in ( C) and 100 μg in (D)) were resolved on SDS-PAGE gels and the membranes were probed with the indicated antibodies. Tubulin ßlll served as a charge control in (C). PC-3 cells with asynchronous growth were treated with either sodium selenate (Na2Se04) at the indicated concentrations with or without LY294002 (10 μM) for 72 hours. The cells were then processed for the proliferative index, using the MTT assay. The results illustrate the + S.D. average of at least three independent experiments. Figure 6 is a graphical and photographic representation showing in vivo results of treatment of prostate tumor in mice with selenium in the form of selenate alone or in combination with taxol (paclitaxel). Treatments include: Cont (control group), selenium (sodium selenate) and taxol (paclitaxel). The control group was treated with paclitaxel solubilization carrier, cremophor, and ethanol without paclitaxel. The Y axis of the graph indicates the weight of the prostate tumor (mg). The figures show ± SD average. Figure 7 is a photographic representation showing tissue sections of prostate tumors treated with taxol (paclitaxel) alone or taxol in combination with sodium selenate. The sodium selenate and paclitaxel are synergized to reduce the size of the prostate tumor. Figure 7 shows images of sections of a tumor from animals treated only with paclitaxel (taxol) against animals treated with a combination of sodium selenate and paclitaxei (taxol + selenate). Tumor sections were stained with HE. Both images were taken at the same amplification and were directly comparable. Figure 8 is a graphical representation showing in vivo results of the treatment of prostate tumors in mice with taxol (paclitaxel) (T) alone or in combination with sodium selenate (S + T). The Y axis of the graph indicates the volume of the prostate tumor (mm3). The Figures represent the + SD average. Figure 9 is a graphical representation illustrating the effects of cell toxicity of 5 μM or 50 μM of sodium selenate or sodium selenite measured through Trypan Blue Exclusion after 24 hours, 48 hours, 72 hours and 96 hours. The toxicity of selenite and selenate cells was measured through Trypan Blue Exclusion. The results illustrate the ± SD average of three independent experiments. The percentage of viable cells was compared with total cell numbers in each sample and indicated in the y axis, and the treatment in the x axis. Figure 10 is a photographic representation having the effect of taxol (paclitaxel) at 1 μg / mL or 10 μg / mL or 500 μM sodium selenate (equivalent to a dose of 19 mg / kg) or 500 μM sodium selenite ( equivalent to a dose of 18 mg / kg) during the indicated times in PC-3 cells. The treated cell lysates were operated on an SDS-PAGE gel and then stained and probed with the indicated antibodies. It shows that selenite induces DNA damage while selenate and paclitaxel do not. The whole-cell lysates of treated PC-3 cells were transferred to PVDF membranes and probed with a histone H2A.X antibody specific for phosphorylation (P H2A.X) and then the membrane was separated and re-probed with a β-antibody. -tubulin as a load control (tubulin). Figure 11 is a graphical representation showing the effects of different selenium compounds on Akt activation. Treatments: control (with); sodium selenate (ATE); selenoso acid (sel acid); sodium selenite (ITE); selenium dioxide (Se02); Selenium sulfide (SeS2); methyl selenocysteine (MSC); and selenocysteine (SeC). The relative active Akt signal intensity correlated with the total Akt protein levels is illustrated in the y-axis. The graph indicates that only sodium selenate (ATE) inhibits Akt activation, reducing levels of phosphorylated Akt below control levels (con). In contrast, selenoso acid (sel acid), sodium selenite (ITE), dioxide selenium (Se02), selenium sulfide (SeS2), methyl selenocysteine (MSC), selenocysteine (SeC) all induce activation of Akt above the control levels (con). Figure 12 is a photographic representation showing the effects of a high dose of sodium selenite and sodium selenate on Akt activation. Sodium selenite (ITE) 500 μM does not inhibit the activation of Akt compared to a similar dose of sodium selenate (ATE). The lysates of treated PC-3 cells were run on SDS-PAGE gels and stained and probed with the P-Akt antibody of the specific phosphorylation Akt antibody (Ser 408). Figure 13 is a photographic representation showing the results of treatment of PC-3 cells with taxol (paclitaxel) at 1, 10, 100 ng / mL and 1 μg / mL, 10 μg / mL or sodium selenate (ATE) at 100 μM, 250 μM or 500 μM (equivalent to a dose of 4-19 mg / kg) or sodium selenite (ITE) at 100 μM, 250 μM or 500 μM) (equivalent to a dose of 3.6-18 mg / kg) ) for 16 hours. The treated cell lysates were operated on an SDS-PAGE gel and stained and probed with specific antibodies directed to cleaved PARP protein and β-Tubulin (control). Taxol and selenate are shown to induce cleavage of the pro-apoptotic PARP protein while selenite did not. Selenite also induces a marked degradation of cellular β-Tubulin underlining its cellular toxicity. Figure 14 is a graphic and photographic representation showing the percentage of inhibition of growth of parental LNCaP cells developed in the presence or absence of androgen after 3 days of treatment with sodium selenate (50 μM). 5 × 10 4 human-prostate-sensitive androgen LNCaP cells were seeded in a 6-well plate and 8 hours later treated with 50 μM of sodium selenate, or without selenate (control). The cells were harvested at 72 hours after the addition of selenate and viable cell counts were determined as analyzed by Trypan Blue staining. Figure 15 is a graphical representation showing the percentage of growth inhibition of the CCS LNCaP cell line developed in the presence or absence of androgen after 3 days of treatment with sodium selenate (50 μM). 5 x 10 4 CCS LNCaP independent cells of prostate cancer androgen were seeded in a 6-well plate and 8 hours later treated with either 50 μM of sodium selenate, or without selenate (control). The cells were harvested at 72 hours after the addition of sodium selenate and the cell counts of viable cells were determined as analyzed by Trypan Blue staining.
Figure 16 is a graphical representation showing the time course of cell proliferation for parental LNCaP cells (normal serum, NS LNCaP) in the presence of androgen, after treatment with sodium selenate (50 μM) or LY294002 (10 μM) . 1 x 105 human prostate cancer NS-LNCaP sensitive cells were seeded in a 6-well plate and 8 hours later treated with sodium selenate (5 μM), LY294002 (10 μM), or no treatment (control) . The cells are harvested at 3 days, 5 days and 9 days after the addition of selenate or LY294002 and the cell counts of viable cells were determined as analyzed by Trypan Blue staining. Figure 17 is a graphical representation showing the time course of cell proliferation for parental NS LNCaP cells developed in the absence of androgen (in serum separated with carbon, CSS), after treatment with sodium selenate (5 μM) or LY294002 (10 μM). 1 × 10 5 NS LNCaP cells sensitive to human prostate cancer were plated in a 6-well plate and 8 hours later treated with 5 μM sodium selenate, 10 μM LY294002, or no treatment (control). The cells were harvested at 3 days, 5 days and 9 days after the addition of selenate or LY294002 and the cell counts of viable cells were determined as analyzed by Trypan Blue staining. Figure 18 is a graphical representation showing the time course of cell proliferation for androgen-independent LNCaP (CSS LNCap) cells in the presence of androgen (in normal serum, NS), after treatment with sodium selenate (5 μM ) or LY294002 (10 μM). 1 x 1 O5 CSS independent LNCaP androgen human prostate cancer cells were plated in a 6-well plate and 8 hours later treated with 5 μM of sodium selenate, 10 μM of LY294002, or without treatment (control). The cells were harvested at 3 days, 5 days and 9 days after the addition of sodium selenate or LY294002 and viable cell counts were determined according to analyzed by Trypan Blue staining. Figure 19 is a graphical representation showing the time course of cell proliferation for androgen-independent LNCaP (CSS LNCap) cells grown in the absence of androgen (in serum separated with carbon, CSS), after treatment with sodium selenate (5 μM) or LY294002 (10 μM). 1 x 105 human prostate cancer androgen independent LNCaP CSS cells were seeded in a 6-well plate and 8 hours later treated with either 5 μM of sodium selenate or LY294002 (10 μM), or no treatment (control ). The cells were harvested at 3 days, 5 days and 9 days after the addition of sodium selenate or LY294002 and the cell counts of viable cells were determined as analyzed by Trypan Blue staining. Figure 20 is a graphical representation showing the time course of cell proliferation of Q293 cells developed in the presence of androgen (in normal serum, NS), after treatment with sodium selenate (5 μM) or LY294002 (10 μM ) or without treatment (control). 1 × 10 5 Q293 human kidney epithelial cells were seeded in a 6-well plate and 8 hours later treated with either 5 μM of sodium selenate or (10 μM) LY294002, or no treatment (control). Cells were harvested at 3 days, 5 days and 9 days after treatment with sodium selenate or LY294002 and the cell counts of viable cells were determined as analyzed by Trypan Blue staining.
Figure 21 is a graphical representation showing the time course of cell proliferation of Q293 cells developed in the absence of androgen (in serum separated from charcoal, CSS), after treatment with sodium selenate (5 μM) or LY294002 ( 10 μM). 1 × 10 5 Q293 human kidney epithelial cells were seeded in a 6-well plate, and 8 hours later treated with 5 μM of sodium selenate or 10 μM LY294002, or without treatment (control). The cells were harvested at 3 days, 5 days and 9 days after the addition of sodium selenate or LY294002 and the cell counts of viable cells were determined as analyzed by Trypan Blue staining.
DETAILED DESCRIPTION OF THE INVENTION /. Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning commonly understood by those skilled in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below. The articles "a, an" and "some, some" are used here to refer to one or more than one (ie, at least one) of the grammatical objects of the article. By way of example, "an element" means an element or more than one element. As used herein, the term "approximately" refers to an amount, level, value, dimension, size or quantity that varies as much as 30%, 20%, or 10% to an amount, level, value, dimension, size or reference quantity. As used herein, the term "inhibiting amount of Akt signaling pathway activation" in the context of treating or preventing a cancer or inhibiting the growth of tumor cells, represents the administration of a number or series of doses of selenate. , which is effective to antagonize the Akt signaling path, including the prevention or reduction of Akt activation by preventing or reducing the expression of Akt or a member upstream of the path, or by reducing the level or functional activity of a product of expression of the Akt gene or of a gene member upstream of the path, or by preventing the phosphorylation of Akt. The amount will vary depending on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the formulation of the composition, the determination of the medical situation, and other important factors. It is believed that the amount will fall within a relatively broad scale that can be determined through routine testing. In specific embodiments, an inhibitory amount of Akt signaling path activation is a supra-nutritional amount of selenate.
By "androgen" is meant a hormone that promotes the development of male sexual characteristics. Non-limiting examples of androgens include testosterone, androstenedione, dihydroepiandrosterone and dihydrotestosterone. The term "androgen-dependent cancer" or "androgen-dependent tumor cell" refers to a cancer or tumor cell that depends on an androgen for the survival, growth and / or proliferation of the cell. Typically, an "androgen-dependent cancer" results from the excessive accumulation of an androgen (i.e., testosterone or another androgenic hormone), increased sensitivity of androgen receptors to androgen, or an increase in androgen-stimulated transcription, and generally will benefit from a reduction in androgen stimulation. The term "androgen-independent cancer" or "androgen-independent tumor cell" refers to a cancer or tumor cell that is insensitive to the presence or absence of androgens.
The phrases "cancer where the pathway of Akt signaling is activated" and "tumor cells where the pathway of Akt signaling is activated" refer to cancers and tumor cells where the survival path of key cell, the path of PTEN / P13K / Akt, is unregulated. Without wishing to be bound to any theory or mode of operation, it is considered that the activation of phosphoinositide 3-kinase PI3K, induced by diverse trophic signals, leads to the accumulation of sub-membrane of the phosphorylated lipid products, phosphatidylinositol-3,4,5-triphosphate (PIP3) and phosphotidylisonitol-3,4-bisphosphate (PIP2) (Vanhaesebroeck et al 2001, Annu Rev. Biochem.; Cantley ef to 2002, Science, 296: 1655). The increased concentration of these phospholipids in the submembrane micro-environment, in turn, leads to the accumulation of phosphoinositide-dependent kinases (PDK-1 and PDK-2), leading to the activation of serine / threonine kinase, Akt. The PTEN tumor suppressor protein normally acts as an important negative regulator of PI3K through its action as a lipid phosphatase that converts PIP3 back to PIP2. Inactivation of PTEN or loss of PTEN function results in chronic activation of the Akt signaling path. The tumor cells in which Akt is activated or in which PTEN is inactivated include various types of tumor cells, including carcinoma. For example, cancers wherein Akt is activated or where PTEN is inactivated include, but are not limited to, oral squamous cell carcinoma, thyroid cancer, pituitary cancer, hepatocellular cancer including hepatocellular carcinoma, prostate cancer including carcinoma of the prostate, testicular cancer, fibrosarcoma, ovarian cancer including ovarian carcinoma, uterine cancer including endometrial cancer, pancreatic cancer including pancreatic carcinoma, stomach cancer, breast cancer, lung cancer, renal cell carcinoma, colon cancer, melanoma, Acute leukemia and brain cancer (for example, astrocytoma and glioblastoma). In some modalities, the Cancer is a hematopoietic neoplastic disorder, including diseases that involve hyperplastic / neoplastic cells of hematopoietic origin, such as: lymphoma and a lymphocytic leukemia. Non-limiting examples of lymphomas include: T-cell lymphomas (including peripheral T-cell lymphomas, adult T-cell leukemia / lymphomas (ATL), cutaneous T-cell lymphomas (CTCLs), large granular lymphocytic leukemias (LGFs); B cell, Hodgkin's lymphoma and a non-Hodgkin's lymphoma Illustrative examples of lymphocytic leukemias include: poorly differentiated acute leukemias, e.g., acute megakaryoblastic leukemia; myeloid disorders, including, but not limited to, acute promyeloid leukemia (APML) , acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML); lymphoid malignancies, including, but not limited to, acute lymphoblastic leukemia (ALL) which includes ALL of line B and ALL of T line, chronic lymphocytic leukemia ( CLL), prolymphocytic leukemin (PLL), hairy cell leukemia (HLL) and Waldenstrom macroglobulemia (WM) .The phrases "cancer where Akt's trajectory is "over-activated", "cancer where Akt is over-activated" and the like refer to a cancer wherein not only Akt is over-expressed, but Akt kinase activity is positively enhanced by phosphorylation of Thr residues. 308 and Ser 473. This is illustrated by the isoform of Akt Akt2, wherein in breast cancer cell lines and prostate cancer the levels of mRNA expression of Akt3 are 2-4 times higher in normal cells but the Akt3 kinase activity is raised about 20-60 times (Nakatani ef al 1999, J. Biol. Chem.274: 21528). Akt is usually over-active in tumors or cancers resistant to drugs or refractory. Examples of cancers wherein Akt is over-active or constitutively active include, but are not limited to, androgen-independent prostate cancer and breast cancer deficient in estrogen receptor. The term "carcinoma" as used herein refers to a form of cancer that develops into epithelial cells covering or lining organs such as the skin, uterus, lungs, chest or prostate. Carcinomas can, but need not, directly invade nearby organs or metastasize to distant sites such as the liver, lymph nodes or bones. Through this specification and the claims that follow unless the context requires otherwise, the word "comprises" and variations such as "comprising" and "comprising", will be understood to imply the inclusion of an established integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "cytostatic agent" refers to a substance that can inhibit cell proliferation or cell division without necessarily killing the cell. Conveniently, the cytostatic agent inhibits the proliferation of cancer cells. The term "cytotoxic agent" or "cytotoxic therapy" as used herein, refers to a substance therapy that is dangerous to cells and finally cause the death of the cell. In some embodiments, the cytotoxic agent rapidly damages the dividing cells such as cancer cells and causes the death of the cancer cell, especially the death of the cancer cell as long as it does not cause damage to or causes less damage to non-cancerous cells. An example of a cytotoxic therapy is radiotherapy. As used herein, the terms "drug resistant" and "refractory" refer to a cancer or tumor cell that is not sensitive or partially not sensitive to treatments normally used to treat cancer or kill the tumor cell. The terms "hormone ablation" and "hormone ablation therapy" refer to the total abstention of hormones that may be required for the survival and growth of cancer cells. Hormone ablation can be achieved through surgical removal of hormone producing organs such as testes or ovaries or can be achieved chemically with compounds that interfere with hormone biosynthesis or secretion, compounds that antagonize or block hormone receptors and in some way prevent the hormone from exerting its biological effect. For example, the conversion of testosterone to the more active dihydrotestosterone can be blocked by an inhibitor of 5 alpha-reductase, such as finasteride. The term "hormone-dependent cancer" or "hormone-dependent tumor cell" refers to a cancer or tumor cell that depends on the presence of a hormone for survival, growth and / or proliferation. Hormone-dependent cancers include, but are not limited to, prostate cancer, testicular cancer, breast cancer, ovarian cancer, uterine cancer, endometrial cancer, thyroid cancer and pituitary cancer. As used herein, the term "hormone-dependent tumor cell growth inhibitory amount" in the context to treat or prevent a cancer or inhibit the growth of tumor cells refers to the administration of a quantity or series of doses of selenate, which is effective to inhibit the growth and / or proliferation of cancer or tumor cells or to cause tumor cell death. The amount will vary depending on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, the formulation of the composition, the determination of the medical situation, and other important factors. It is expected that the amount will fall within a relatively broad scale that can be determined by routine trials. In specific embodiments, an inhibitory amount of hormone-dependent tumor cell growth is a supra-nutritional amount of selenate. As used herein, the term "in combination with" refers to the treatment of cancer or exposure of a tumor cell to at least two agents, so that its effects on the cancer or tumor cell occurs, at least in part, during the same period of time. The administration of at least two agents can occur simultaneously in a single composition, or each agent can be simultaneously or sequentially administered in separate compositions. The phrase "inhibit growth of tumor cells" refers to the fact that tumor cell growth ceases or is reduced and cell proliferation or cell division ceases or is reduced. This is also known as cytostostasis. The growth of tumor cells can be measured in terms of weight or volume or cell number or cellular metabolic activity, i.e., MTT assay. By "pharmaceutically acceptable carrier" is meant a solid or liquid filler, diluent or encapsulating substance that can be safely used in topical, local or systemic administration. The term "pharmaceutically acceptable salt" as used herein, in relation to selenate, refers to metal ion salts that are toxicologically safe for administration to humans and animals. For example, suitable metal ion salts of selenate include, but are not limited to, sodium, potassium, magnesium, calcium, iron, nickel and zinc salts. A preferred salt of selenate is the sodium salt, Na2Se04. The term "radiotherapy" as used herein, refers to the treatment or exposure of a cancer or cancer cells such as tumor cells to high energy radiation. The effectiveness of radiotherapy can be improved through selenate or its pharmaceutically acceptable salt. In addition, radiotherapy can be further improved through the administration of a radiosensitization agent. Illustrative examples of radiosensitizing agents include, but are not limited to, efaproxiral, etanidazole, fluosol, misonidazole, nimorazol, temoporfin and tirapazamine. The terms "subject" or "individual" or "patient", used interchangeably herein, refer to any subject, in particular a vertebrate subject, and more particularly a mammal, for whom prophylaxis or treatment is desired. Suitable vertebrate animals that fall within the scope of the invention include, but are not limited to, primates, birds, farmyard animals (e.g., pigs, sheep, cows, horses, donkeys), laboratory test animals (e.g. , rabbits, mice, rats, guinea pigs, hamsters), pets (for example, cats and dogs) and captive wild animals (eg, foxes, deer, dingoes (wild dogs)). A preferred subject is a human being in need of cancer prophylaxis treatment, wherein the pathway of Akt signaling is activated or of a hormone-dependent cancer, especially prostate cancer. However, it will be understood that the aforementioned terms do not imply that the symptoms are present. The term "supra-nutritional" as used herein, refers to an amount that is greater than the quantity considered a nutritional requirement. In adults, the recommended dietary ration of selenium is 55 μ / day. Pregnant and lactating women have a recommended daily allowance of 60.70 μ / day.
A supra-nutritional amount of selenium provides selenium to a subject at approximately the recommended daily ration. For example, a supra-nutritional amount of selenium can be from 0.15 mg / kg to 20.0 mg / kg, 0.1 mg / kg to 14 mg / kg, 0.1 mg / kg to 13 mg / kg, 0.1 mg / kg to 12 mg / kg, 0.1 mg / kg to 10 mg / kg, 0.1 mg / kg to 9 mg / kg, 0.1 mg / kg to 8 mg / kg, 0.1 mg / kg to 7 mg / kg, 0.1 mg / kg to 6 mg / kg, 0.15 mg / kg to 5 mg / kg, 0.15 mg / kg to 5 mg / kg, 0.15 mg / kg to 4 mg / kg, 0.15 mg / kg to 3 mg / kg, 0.15 mg / kg to 2 mg / kg, 0.15 mg / kg to 1 mg / kg, especially 0.1 mg / kg to 14 mg / kg, and more especially 0.15 mg / kg to 5 mg / kg, As used herein, the term "therapeutically effective amount" in the context of treating or preventing cancer or inhibiting the growth of tumor cells refers to the administration of a quantity of selenate or a pharmaceutically acceptable salt thereof, either in an individual dose or as part of a series of doses, which is effective to inhibit the growth and / or proliferation of cancer or tumor cells or to cause the death of cancer or tumor cells. The effective amount will vary depending on the health and physical condition of the individual to be treated, the taxonomic group of the individual to be treated, and the formulation of the composition, the determination of medical situations and other important factors. It is expected that the amount will fall within a relatively broad scale that can be determined through routine trials. In specific modalities, a therapeutically effective amount is a supra-nutritional amount. 2. Methods to inhibit the growth and proliferation of tumor cells and to treat cancer. The present invention is predicted in part in the determination that selenate, as opposed to other forms of selenium such as selenite, is effective to inhibit the growth or proliferation of tumor cells, where the Akt signaling path is activated. Accordingly, in one aspect, the present invention provides methods for inhibiting the growth or proliferation of tumor cells, wherein the Akt signaling pathway is activated, wherein the methods generally comprise exposing the tumor cells to an inhibitory amount of the activation. of Akt signaling pathway of selenate or a pharmaceutically acceptable salt thereof. Conveniently, the amount of selenate or its pharmaceutically acceptable salt is a supra-nutritional amount, which is generally from about 0.015 mg / kg to 20.0 mg / kg, usually from about 0.1 mg / kg to 14 mg / kg and more usually from about 0.15 mg / kg to 5 mg / kg. The present invention can be used effectively against various types of tumor cells and cancers, including carcinomas, illustrative examples, cancers wherein Akt is activated or where PTEN is inactivated, include, but are not limited to, oral squamous cell carcinoma, cancer thyroid cancer, pituitary cancer, hepatocellular cancer including hepatocellular carcinoma, prostate cancer including prostate carcinoma, testicular cancer, fibrosarcoma, ovarian cancer including ovarian carcinoma, uterine cancer including endometrial cancer, pancreatic cancer including pancreatic carcinoma, stomach cancer, breast cancer, lung cancer, renal cell carcinoma, colon cancer, melanoma, water leukemia and cancer brain (for example, astrocytoma and glioblastoma). In some embodiments, cancer is a hematopoietic neoplastic disorder, which includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin, such as: lymphoma and lymphocytic leukemia. Nonlimiting examples of lymphomas include: T-cell lymphomas (including peripheral T-cell lymphomas, leukemia / T-cell lymphomas in adults (ATL), cutaneous T-cell lymphomas (CTCLs), large granular lymphocytic leukemias (LGFs); B cell, Hodgkin lymphoma and non-Hodgkin's lymphoma Illustrative examples of lymphocytic leukemia include: poorly differentiated acute leukemias, eg, acute megakaryoblastic leukemia; myeloid disorders, including, but not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CNL); lymphoid malignancies, including, but not limited to, watery lymphoblastic leukemia (ALL) that includes line B ALL and T line ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) and Waldstrom macroglobulinemia (WM). In specific embodiments, the tumor cells are prostate cancer or carcinoma cells. In this way, in one aspect related, the invention provides methods for treating a cancer wherein Akt is activated, wherein the methods generally comprise administering to a subject in need thereof, an inhibiting amount of the activation of Akt signaling pathway of selenate or a pharmaceutically salt acceptable of it. In some embodiments, the amount of selenate or its pharmaceutically acceptable sai is a supra-nutritional amount as broadly defined. In other embodiments, the cancer is prostate cancer, especially a drug-resistant prostate cancer or an androgen-independent prostate cancer. In some embodiments, the tumor cell or cancer is drug resistant. In some embodiments, the tumor cell or cancer has an over-active Akt pathway (eg, androgen-independent prostate cancer and estrogen receptor deficient breast cancer.) In certain embodiments, the selenate or its pharmaceutically acceptable salt is administered in combination with at least one cytostatic agent or cytotoxic agent.Non-limiting examples of cytostatic agents are selected from: (1) microtubule stabilizing agents such as, but not limited to, taxanes, paclitaxel, docetaxel, epothilones and laulimaiides; kinase inhibitors, illustrative examples of which include Iressa®, Gleeve, Tarceva ™, (Erlotinib CHI), BAY-43-9006, tyrosine kinase subgroup inhibitors of division kinase domain receptor (e.g. , PTK787 / ZK222584 and SU11248); (3) antibodies activated in receptor kinase, which include, but are not limited to, Trastuzumab (herceptin®), Cetuximab (Erbitux®), Bevacizumab (Avastin ™), Rituximab (ritusan®), Pertuzumab (Omnitarg ™); (4) mTOR pathway inhibitors, illustrative examples of which include rapamycin and CCI-778; (5) Apo2L / Trail, antiangiogenic agents such as, but not limited to, endostatin, combrestatin, angioestatin, thrombospondin, and vascular endothelial growth inhibitor (VEGI); (6) antineoplastic immunotherapy vaccines, representative examples of which include activated T cells, non-specific immune reinforcing agents (ie, interferons, interleukins); (7) antibiotic cytotoxic agents such as, but not limited to, doxorubicin, bleomycin, dactinomycin, daunorubicin, epirubicin, mitomycin and metozantrone; (8) alkylating agents, illustrative examples of which include Melphalan, Carmustine, Lomustine, Cyclophosphamide, Ifosfamide, Chlorambucil, Photomustine, Busulfan, Temozolomide and Tiotepa; (9) hormonal antineoplastic agents, non-limiting examples of which include Nilutamide, Cyproterone Acetate, Anastrazole, Exemestane, Tamoxifen, Raloxifene, Bicalutamide, Aminoglutethimide, Leuprorelin Acetate, Tiromifen Citrate, Letrozole, Flutamide, Megestrol Acetate and Acetate Goserelin; (10) gonadal hormones such as, but not limited to, Cyproterone acetate and Medoxiprogesterone acetate; (11) Antimetabolites, illustrative examples of which include Cytarabine, Fluoouracil, Gemcitabine, Tpotecan, Hydroxyurea, Thioguanine, Methotrexate, Colaspase, Raltitrexed and Capicitabine; (12) anabolic agents such as, but not limited to, Nandrolone; (13) adrenal steroidal hormones, illustrative examples of which include methylprednisolone acetate, dexamethasone, hydrocortisone, prednisolone and prednisone; (14) neoplastic agents such as, but not limited to, Irinotecan, carboplatin, Cisplatin, Oxaliplatin, Etoposide and Dacarbazine; and (15) topoisomerase inhibitors, illustrative examples of which include topotecan and irinotecan. In some embodiments, the cytostatic agent is a nucleic acid molecule, conveniently a recombinant antisense nucleic acid molecule or siRNA. In other embodiments, the cytostatic agent is a peptide or polypeptide. In other modalities more, the cytostatic agent is a small molecule. The cytostatic agent may be a cytotoxic agent that is suitably modified to improve the consumption or supply of the agent. Non-limiting examples of said modified cytotoxic agents include, but are not limited to, pegylated or albumin labeled cytotoxic drugs. In specific embodiments, the cytostatic agent is a microtubule stabilizing agent, especially a taxane and preferably paclitaxel. In some embodiments, the cytotoxic agent is selected from anthracyclines such as idarubicin, doxorubicin, epirubicin, daunorubicin and mitozantrone, CMF agents such as cyclophosphamide, methotrexate and 5-fluorouracil or other cytotoxic agents such as cisplatin, carboplatin, bleomycin, topotecan, irinotecan, melphalan, chlorambucil, vincristine, vinblastine and mitomycin-C. The present invention also discloses the discovery that selenate and its pharmaceutically acceptable salts have an inhibitory effect on hormone-dependent tumor cell growth when used in combination with a hormone ablation therapy and optionally a cytostatic agent or cytotoxic agent. Accordingly, another aspect of the present invention provides methods for treating a hormone-dependent cancer in a subject, wherein the methods generally comprise administering a therapeutically effective amount of selenate or a pharmaceutically acceptable salt thereof, in combination with a therapy of hormone ablation. Conveniently, the amount of selenate or a pharmaceutically acceptable salt thereof is a supra-nutritional amount of selenate, as broadly defined above. In some embodiments, the hormone-dependent cancer is selected from prostate cancer, testicular cancer, breast cancer, ovarian cancer, endometrial cancer, uterine cancer, thyroid cancer or pituitary cancer, especially prostate cancer or breast cancer. Hormone ablation therapy can be any therapy that deprives the cancer or tumor cells of hormones required for survival, growth and / or proliferation of cancer or tumor cells. Hormone ablation therapy can be achieved surgically through the removal of hormone producing organs such as testes or ovaries. Alternatively, hormone ablation therapy can be achieved chemically with compounds that interfere with the biosynthesis or secretion of hormone, compounds that antagonize or block hormone receptors or in some way prevent the hormone from exerting its biological effect. Illustrative examples of chemical hormone ablation therapy include GnRH agonists or antagonists such as Cetrorelix, agents that interfere with the androgen receptor including non-steroidal agents such as Bicalutamide and steroidal agents such as Cyproterone, and agents that interfere with biosynthesis and steroids such as Ketoconazole. Chemical agents suitable for use in combination with selenate or its pharmaceutically acceptable salts as hormone ablation therapy for prostate cancer include, but are not limited to, non-steroidal anti-androgens such as Nilutamide, Bicalutamide and Flutamide; GnRH agonists such as Goserelin acetate, leuprorelin and triptorelin; 5-alpha-reductase inhibitors such as finasteride; and cyproterone acetate. Chemical agents suitable for use in combination with selenate or its pharmaceutically acceptable salts as hormone ablation therapy in breast cancer include, but are not limited to, aromatase inhibitors such as Anastrazole; Exemestane, Tamoxifen, Aminoglutethimide, Toremifene citrate, Letrozole, Megestrol acetate and Goserelin acetate. Suitable chemical agents to be used in combination with selenate or its pharmaceutically acceptable salts as hormone ablation therapy in ovarian and uterine cancers, including endometrial cancer, include, but are not limited to, progestins such as Megestrol acetate, levonorgestrol and norgestrol. . In some embodiments, the method for treating a hormone-dependent cancer further comprises administering a cytostatic agent such as those defined above, or a cytotoxic agent. A preferred cytostatic agent is a microtubule stabilizing agent, especially a taxane, and more especially paclitaxel. Certain embodiments of the present invention are directed to methods of treating cancer in a subject, said methods generally comprising administering to the subject a therapeutically effective amount of selenate or its pharmaceutically acceptable salt. To practice these methods, the person who manages the subject can determine the effective dose of the form of selenate or its pharmaceutically acceptable salts for the condition and particular circumstances of the subject. A therapeutically effective amount of selenate is one that is effective for the treatment or prevention of cancer, including prevention of incurring a symptom (eg, proliferation of cancer cells), keeping such symptoms in check, and / or treating associated existing symptoms. with cancer (for example, pain, fluid development, urinary retention, nausea, indigestion, gas, appetite, changes in bowel habits and weight loss). In some embodiments, the therapeutically effective amount is a supra-nutritional amount of selenate or its pharmaceutically acceptable salt. In specific modalities, the selenate is conveniently in the form of the salt, sodium selenate (Na2Se04). Next, modes of administration, amounts of selenate administered, and selenate formulations will be discussed for use in the methods of the present invention. If the cancer has been treated, it is determined by measuring one or more diagnostic parameters indicative of the course of the disease, compared with adequate control. In the case of a human subject, an "appropriate control" may be the individual before treatment, or it may be a human being (eg, one of matching age or similar control) treated with a placebo. According to the present invention, the treatment of cancer includes and includes without limitation: (i) preventing cancer in a subject who may be predisposed to cancer but has not yet been diagnosed with cancer and, therefore, the treatment constitutes a treatment prophylactic for cancer; (ii) inhibit tumorigenesis, that is, stop the development of cancer; or (iii) relieve the symptoms that result from cancer. The methods of the present invention are suitable for treating an individual who has been diagnosed with cancer, who is suspected of having cancer, who knows that he is susceptible and who is considered likely to develop a cancer, or who is considered likely to develop a recurrence of a previously treated cancer. In specific embodiments of the above methods, the cancer is prostate cancer, especially a drug-resistant prostate cancer or androgen-independent prostate cancer and the treatment optionally further comprises the administration of a cytostatic agent such as those defined above (eg, an agent of microtubule stabilization such as paclitaxel) or a cytotoxic agent. In other embodiments, prostate cancer is an androgen-sensitive prostate cancer and the treatment is optionally administered in combination with a hormone ablation therapy and / or a cytostatic agent as defined above, or a cytotoxic agent or radiotherapy optionally together with a radiosensitization agent. Conveniently, hormone ablation therapy is selected from surgical castration, finasteride, Nilutamide, Cyproterone acetate, Bicolutamide, Leuprorelin acetate, Flutamide and Goserelin acetate. Preferably, the cytostatic agent is a microtubule stabilizing agent, especially a taxane, and more especially paclitaxel. Illustrative subjects for treatment with the methods of the invention are vertebrates, especially mammals. In certain embodiments, the subject is selected from the group consisting of humans, sheep, cattle, horses, cattle, pigs, dogs and cats.
A preferred subject is a human being. The selenate or its pharmaceutically acceptable salt can be formulated following any number of techniques known in the field of anti-cancer drug delivery. The selenate or its pharmaceutically acceptable salts can, of course, be administered through a number of means keeping in mind that all formulations are not suitable for each route of administration. The selenate or its pharmaceutically acceptable salts can be administered in liquid or solid form. The application can be oral, rectal, nasal, topical (including buccal and sublingual), or through inhalation. The selenate or its pharmaceutically acceptable salts can be administered together with conventional pharmaceutically acceptable auxiliaries, vehicles and / or diluents. Solid forms of application include tablets, capsules, powders, pills, pills, suppositories and granular forms of administration. They may also include carriers or additives such as flavors, colorants, diluents, softeners, binders, preservatives, strength agents and / or closure materials. Liquid forms of administration include solutions, suspensions and emulsions. These can also be offered together with the aforementioned additives. The solutions and suspensions of selenate or its pharmaceutically acceptable salt, assuming a suitable viscosity to facilitate the use, can be injected. The suspensions too viscous for injection can be implanted using devices designed for such purposes, if necessary. Sustained release forms are generally administered through parenteral or enteric means. Parenteral administration is another route of administration of the selenate or a pharmaceutically acceptable salt thereof, used to practice the invention. "Parenteral" includes formulations suitable for injection and for nasal, vaginal, rectal and oral administration. The administration of selenate or its pharmaceutically acceptable salts may involve a prolonged oral dose formulation. Oral dose formulations are preferably administered once a day to three times a day, in the form of a sustained release capsule or tablet, or alternatively as an aqueous-based solution. The selenate or its pharmaceutically acceptable salt can be administered intravenously, either daily, continuously, once a week to other times a week. The administration of selenate or its pharmaceutically acceptable salts can include daily administration, preferably once a day in the form of a sustained release capsule or tablet, or once a day as an aqueous solution. Combinations of selenate or its pharmaceutically acceptable salt and at least one cltostatic agent or cytotoxic agent can be administered in solid or liquid form in a single formulation or composition or in formulations or compositions separated. In some embodiments, the selenate or its pharmaceutically acceptable salt and the cytostatic agent (s) or cytotoxic agent (s) are administered orally as a single tablet or capsule or separate tablets or capsules. In other embodiments, the selenate or its pharmaceutically acceptable salt and the cytostatic agent (s) or cytotoxic agent (s) are administered intravenously in a single composition or separate compositions. The methods of the present invention can be employed in combination with other known treatments for cancer, for example, but not limited to, surgery, chemotherapy and radiotherapy. In some embodiments, selenate or its pharmaceutically acceptable salt is used in combination with radiotherapies, such as, but not limited to, conformational external beam radiation therapy (50-100 Gray given as fractions for 4-8 weeks), either single-shot or fractionated, high-speed dose brachytherapy, permanent interstitial brachytherapy, systemic radioisotope (eg, Strontium 89). In some modalities, radiotherapy can be administered in combination with a radiosensitization agent. Illustrative examples of radiosensitizing agents include, but are not limited to, efaproxiral, etanidazole, fluosol, misonidazole, nimorazol, temoporfin and tirapazamine. In other embodiments, selenate or its pharmaceutically acceptable salt is used in combination with lumpectomy. The present invention also provides compositions pharmaceuticals for treating or preventing cancer, which generally comprise a supra-nutritional amount, conveniently from about 0.5 mg to 1.0 g of selenate or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In some embodiments, the selenate or its pharmaceutically acceptable salt is in an amount of about 5.0 mg to about 700 mg. In illustrative examples, the selenate or its pharmaceutically acceptable salt is in an amount of about 7.5 mg to 250 mg, especially 50 mg to 200 mg, for example, 100 to 150 mg for an individual daily dose. The pharmaceutical composition can be for the treatment of a cancer wherein Akt is activated or over-active or a hormone-dependent cancer. In some embodiments, the pharmaceutical compositions are useful for treating prostate cancer, especially a drug-resistant or an androgen-independent prostate cancer. In some modalities, the pharmaceutical compositions further comprise at least one cytostatic agent or a cytotoxic agent. In other embodiments, the pharmaceutical compositions further comprise a chemical hormone ablating agent. In still other embodiments, the pharmaceutical compositions further comprise at least one cytostatic agent and / or a cytotoxic agent and a chemical hormone ablating agent. In other embodiments, the pharmaceutical compositions further comprise a radiosensitizing agent for use with radiotherapy.
The pharmaceutical composition of the present invention can include any of the additional components that are non-immunogenic and biocompatible with selenate, as well as capable of bioabsorption, biodegradation, elimination as an intact molecule. The formulation can be supplied in a ready-to-use form or it can be supplied as a sterile powder or liquid that requires the addition of a vehicle before administration. If sterility is desired, the formulation can be made under sterile conditions, the individual components of the mixture can be sterile, or the formulation can be sterile filtered before use. Said solution may also contain suitable pharmaceutically acceptable vehicles, such as, but not limited to, pH regulators, salts, excipients, preservatives, etc. In some embodiments, sustained release oral formulations are used to administer selenate or its pharmaceutically acceptable salt in the methods of the invention. These formulations in general comprise selenate or its pharmaceutically acceptable salt having a reduced solubility in order to delay absorption into the bloodstream. In addition, these formulations may include other components, agents, vehicles, etc., which may also serve to delay the absorption of selenate or its pharmaceutically acceptable salt. You can also use microencapsulation, polymeric trapping systems, and osmotic pumps, which may or may not be bioerodible, to allow delayed or controlled diffusion of the selenate or a pharmaceutically acceptable salt thereof from a capsule or matrix. Selenate or its pharmaceutically acceptable salts can be used alone or as part of another agent. Accordingly, the invention also contemplates an agent comprising selenate or a pharmaceutically acceptable salt thereof for the treatment of a cancer wherein Akt is activated, or for the treatment of a hormone-dependent cancer, wherein the agent is formulated to be administered in combination with hormone ablation therapy. In order that the nature of the present invention may be more clearly understood and put into practical use, its preferred particular embodiments will now be described with reference to the following non-limiting examples.
EXAMPLES EXAMPLE 1 PROGRESSION SUPPRESSION OF PROSTATE TUMOR WITH SODIUM SELENATE INHIBITING THE CTIVATION OF ROTEIN KINASE B / AKT Materials and Methods Cell Culture Cell culture experiments involved a PC-3 cell line, which was obtained from the American Type Culture Collection (Manassas, Virginia, USA). The cells were cultured in routine form in RPMI 1641 (Invitrogen) supplemented with 10% fetal bovine serum and a 1% antibiotic / antifungal mixture (Invitrogen). The cells were maintained at 37 ° C in 5% C02. Sodium selenate and selenomethionine (Sigma) were made as 10mM supply solutions in distilled water and sterilized by filter before dilution in the medium for in vitro experiments.
Animal Experimentation Animal experimentation involved the use of hairless mice BALB / c. on day one of the experiment, 6-week-old BALB / c hairless mice were anaesthetized with an intraperitoneal injection of ketamine 100 mg / kg and xylazine 20 mg / kg. Under amplification, 1 × 10 6 PC-3 cells with greater than 95% viability were injected into the dorsolateral prostate, essentially as described by Corcoran, N.M., Najdovska, M., and Costello, A.J. (2004), J. Urol, 171: 907-910. On day 3 of the experiment, all mice were switched to the minimum selenium diet D19101 with an analyzed selenium content of 0.07 ppm (Research Diets Inc., New Brunswick, New Jersey, USA). The mice were then randomly assigned to receive either 5 ppm selenium as sodium selenate in drinking water or unsupplemented water. After 5 weeks of supplementation the mice were sacrificed in a special way and then weighed and the glands were measured of prostate containing the tumor with a Vernier caliper. The retroperitoneum was then explored under cephalad amplification at the level of the renal veins and lymph nodes measuring greater than 0.5 mm identified. The degree of apoptosis in the tissue samples was determined using a TUNNEL cell death detection equipment (in situ) (Roche Applied Science) according to the manufacturer's instructions. The number of cells held in high energy coffee (x400) was counted in 10 randomly selected fields in areas where the tension necrosis of HY &E of adjacent sections was absent. The rate of tumor cell proliferation was determined through incorporation of BrdU in vivo. Two hours before sacrifice, the mice were injected with 50 mg / kg BrdU (Sigma) intraperitoneally. 5 μm sections were cut, fixed in absolute methanol at 4 ° C for 10 minutes. The sections were rehydrated in PBS and incubated in 2N HCl for 1 hour at 37 ° C to denature the DNA. The acid was neutralized by immersing the slides in 0.1 M borate pH buffer (pH 8.5). After 3 washes with PBS the samples were incubated with a monoclonal antibody for bromodeoxyueidine (BrdU) (Roche Applied Science) at a concentration of 5 μg / mL diluted in 0.1% BSA in PBS for 1 hour at room temperature, then overnight at 4 ° C. The samples were washed 3 times in PBS and incubated for 1 hour with a mixture of immunoglobulin biotinylated link (DAKO Corporation). After 3 washes in PBS, the samples were incubated with streptavidin-horseradish peroxidase (BD Biosciences) and immunostaining revealed DAB (Enhanced Liquid Substrate System, Sigma). Ten high-energy fields (x400) were randomly selected and the number of brown stained cells counted.
Growth curve PC-3 PC-3 growth curves were obtained allowing the overnight binding of 2 x 1 O5 PC-3 cells after 16 hours, the medium was changed to include sodium selenate in the presence of serum at indicated concentrations and allowed to believe until the specified time points. The supernatants and cells were harvested, combined and the viable cells were determined by Trypan Blue Blue exclusion assay. The experiments were carried out in triplicate.
BrdU and Immunofluorescence Assays BrdU and immunofluorescence incorporation assays were performed by plating 2.5 x 10 3 PC-3 cells and leaving them to bind overnight. After 16 hours, the medium was changed to include sodium selenate at the indicated concentrations in the presence of serum. After 36 hours the medium containing the sodium selenate was cooled with 1 μL / mL of Cell Proliferation Marking Reagent (BrdU / FrdU, Amersham Biosciences). The cells were incubated for a further 16 hours, then washed 3 times in PBS and fixed in 4% PFA for 10 minutes at room temperature. Anti-BrdU and mouse monoclonal antibodies were used as primary antibodies and Alexa anti-mouse 488, as secondary antibodies. The percentage of cells incorporating BrdU was determined by counting the number of nuclei stained green by the number of DAPI positive cells. One hundred DAPI-positive cells were counted per cover and the experiment was performed in triplicate.
Determination of the cell cycle block level The determination of the cell cycle block level involved plating 5 x 10 5 PC-3 cells that were synchronized through serum deprivation for 48 hours before sodium selenate was added to the cell cycle. the fresh serum containing the medium at the indicated concentrations. The cells were allowed to grow to the indicated time points. They were then harvested, washed in PBS and fixed in 70% ice-cold ethanol for 15 minutes. The cells were washed with PBS and resuspended in PBS containing 40 μg / mL of propidium iodide (Sigma) and 100 μg / L of RNase. DNA histograms were generated for each reading and the proportion of cells present in the G1 and G2 / M peaks was determined. The results were obtained for 3 independent experiments.
MTT Growth Assay MTT growth assays involved plating 1 x 103 PC-3 cells per well in a 96-well plate and allowing the cells to attach overnight. At 16 hours the medium was replaced with fresh medium containing the indicated concentration of sodium selenate selenomethionine.
For the experiment that determined the additional effect of inhibition of PI3K in the inhibition of growth of sodium selenate, LY294002 (Promega, Madison, Wl, USA) was added at a concentration of 50 μM with sodium selenate. The controls for this experiment received an equal volume of diluent LY294002 (DMSO). Then, the MTT growth assays were performed according to the manufacturer's protocol (Sigma).
Antibodies and Immunostaining The following antibodies were obtained from Cell Signaiing Technology (Beverly, MA, USA) unless otherwise indicated: anti-p27? Lp1 (bd Pharmingen), anti-p21CIP1, ANTI-CYCLIN di, ANTI-CYCLIN d3 , ANTI-CDK4, ANTI-CDK6, ANTI-PHOSPHO rb (Ser807 / 811), anti-RB, anti-phospho Akt (Ser473), anti-phospho Akt (Thre308), anti-Akt, anti-phospho-PDKI (Ser241) , anti-phospho-PDKI (Tyr373 / 376), anti-phospho-GSK-3ß, anti-phospho mTOR (Ser2448), anti-mTOR, anti-ßlll tubulin (Promega). To determine the effect of sodium selenate on the cell cycle regulatory promoter, 5x105 cells were treated PC-3 with 0.5 mM of sodium selenate during the indicated times. To determine the effect of selenate on the phosphorylation of the retinoblastoma protein, 2x105 cells were allowed to bind overnight, then they were deprived of serum for 24 hours before being treated with 0.5 mM of sodium selenate for the indicated time. To determine the effect of selenate on the phosphorylation of proteins involved in the PI3K / Akt signaling cascade, 5x105 PC-3 cells were allowed to bind for 10 hours. The cells were deprived of serum for 16 hours and then treated with 0.5 mM of sodium selenate for the indicated times. Cells were lysed in ELB (250 mM NaCl, 50 mM HEPES pH 7.0, 5 mM EDTA, 0.5 mM DTT, 0.2% TX100, 20 mM sodium fluoride, 2 mM sodium pervanadate, Complete Protease Inhibitor cocktail (Roche )). The lists were centrifuged for 15 minutes at 4 ° C. The protein concentration was determined using the BCA system (Sigma) and equal amounts of protein were loaded into each lane of an SDS-polyacrylamide gel. Proteins were transferred onto PVDF membranes (Millipore) and detected with anti-mouse or anti-rabbit secondary antibodies coupled to horseradish peroxidase (HRP) and chemiluminescence using the SuperSignal West Dura (Pierce). The membranes were separated using the Restore Western Blot Stripping Buffer (Pierce) pH Regulator.
Cell Transfection and Immunofluorescence The day before transfection, 5 x 105 PC-3 cells were plated in 24-well plates and transfected with 0.8 μg of the pcDNA3-GFP-FKHRwt construct (a type of gift from Dr Bill Sellers). , Dana-Farber Institute, Boston) using Lipofectamine 2000 (Invitrogen). The cells were permeabilized in 0.2% Triton X-100 and the nuclear contents were stained with DAPI. The coverslips were mounted with a fluorescent mounting medium (DAKO). The cellular distribution of the green fluorescence was determined in double fluorescent cells using a Leica epifluorescent microscope.
Statistical Analysis The data are provided as + SE medium unless otherwise indicated. The differences between groups were analyzed (Figure 1A-D) using the Student's test with assumed importance to p < 0.05.
All statistical analyzes were performed using SPSS 9.05 for Windows (SPSS, Chicago, Illinois).
Results The results indicated that sodium selenate inhibits cell proliferation of prostate carcinoma in an orthotopic mouse model. A comparison of animals treated with sodium selenate with the control group was made as follows: The prostate weights containing the mean tumor were compared as shown in Figure 1A and the average tumor volumes were compared as shown in Figure 1B. The mean number of lymph node metastases was also determined, sodium selenate showed significant inhibition of primary tumor growth, compared with controls determined by the prostate weight index, while differences in tumor volume between the two groups they just failed to achieve importance. The number of retroperitoneal lymph nodes was significantly reduced in the sodium selenate group compared to the controls (Figure 1 C). Inhibition of tumor growth caused by sodium selenate may have occurred due to an inhibitory effect on tumor cell proliferation or through an increase in tumor cell apoptosis. To distinguish these two mechanisms, the incorporation of the nucleotide analog, BrdU in proliferation cells and for apoptotic markers with the Tunnel assay in the control prostates and treated with sodium selenate, was analyzed. The sodium selenate significantly impaired the incorporation of BrdU in prostate tumors against the controls (Figure 1D, E), but the apoptosis between the two groups was not significantly different (Figure 1D). These results indicated that sodium selenate acts to prevent tumor cell proliferation instead of improving tumor cell death. The results also indicated that sodium selenate stops prostate carcinoma cells in the G1 phase of the cell. To determine whether the inhibitory effect of sodium selenate on tumor progression observed in vivo, could lead to an inhibition of tumor cell growth in vitro, human prostate carcinoma PC-3 cells were seeded and cultured for 16 hours. The cells were then incubated in the presence or absence of concentrations of the increase in sodium selenate. The total cell numbers 24, 48 and 72 hours after incubation were counted. As indicated in Figure 2A, the number of PC-3 cells cultured in the absence of sodium selenate was increased evenly over the 72 hour period. In contrast, the PC-3 cell numbers were significantly reduced in a dose-dependent manner in the presence of sodium selenate. Between the 0.01 and 0.1 mM doses, the rate of increase in cell number was prevented, while the higher doses of 0.25 and 0.5 mM, all increases in cell number were blocked. These results suggested that sodium selenate interferes with cell cycle progression; PC-3 cells were seeded essentially as described above, at concentrations of sodium selenate ranging from 0.05 to 0.25 mM, for 54 hours. The incorporation of the fluorescent nucleotide analogs BrdU / FrdU in cells progressing through the G1 / S phase was then determined through an immunofluorescence microscope. A typical image of cells exposed to 0.25 mM sodium selenate for 54 hours is shown in Figure 2B. Exposure to this dose of sodium selenate severely impeded G1 / S progression of PC-3 cells as determined by positive BrdU / FrdU nuclei. The distribution of the cell cycle in the presence or absence of increasing concentrations of sodium selenate through flow cytometry was also analyzed. A typical histogram of the cell cycle is shown in Figure 2C and the average percentage heats in Figure 2D. The treatment of PC-3 cells with 0.25 and 0.5 mM sodium selenate for 24 hours increased the percentage of the G1 cell population by 55% in the control group to a scale of 69-70% in the samples treated with sodium selenate (Figure 2D). Conversely, incubation in the presence of increasing concentrations of sodium selenate reduced the percentage of cells to 19% and 24% from the control of 28% in the G2 / M phase. Treatment with sodium selenate over a period of 48 hours increased the percentage of the cell population in G1 in a dose-dependent and time-dependent manner, while drug treatment reduced the percentage of PC-3 cells in the G2 / M phase. The chemopreventive effect of selenium cancer can not be fully explained by the presence of the selenium trace element, and it has become enormously clear that the selenium compound by itself is important in this activity. (Corcoran eí al 2004, supra; Kim e to 2003, supra). The effects of inorganic sodium selenate with organic selenomethionine on the proliferation of PC-3 cells were compared using the MTT assay as an index measure proliferative As shown in Figure 2E, sodium selenate at doses of 0.25 and 0.5 mM markedly reduced proliferation of PC-3 cells to the 72-hour period, whereas selenomethionine was less effective in reducing cell proliferation to them. dose .. A light but reproducible reinforcement in the cell number at the lowest dose of sodium selenate, 0.01 mM, was observed (Figure 2E). In summary, these data indicate that sodium selenate inhibits cell proliferation by preventing cell cycle entry from G1 to S phase. The results also indicated that sodium selenate, but not selenomethionine, induced overgrowth. regulation of cell cycle inhibitory proteins. The ordered progression of proliferation cells through G1 was regulated mainly through the sequential activation of the cyclin D / cdk4 / cdk6 kinase complex which regulates the phosphorylation of Rb and the release of the transcription factor E2F. To determine the effect of treatment with sodium selenate on key cell cycle regulatory proteins, PC-3 cells were treated essentially as previously described, with 0.5mM of sodium selenate for different periods of up to 24 hours. Afterwards, total cellular proteins were resolved and analyzed through immunostaining assays for the presence of cyclin D1, D3cdk4 and cdk6. Cyclin D1 expression levels fell significantly from the 14 hour period, while cdk4 levels reached a peak at 6 hours and declined in a time dependent manner at 24 hours (Figure 3A). The CDK inhibitors such as as p27 and p21 CIP 1 / WAF1 are important negative regulators of cell cycle progression. These molecules block CDK kinase activity by binding to the cyclin D / CDK complex in the G1 phase, preventing the phosphorylation of members of the Rb gene family and the transition from the G1 to S phase. To determine the effect of sodium selenate on these cell cycle inhibitors, the expression levels of p27? lp1 and p21CIP1 in the same lysates of the PC-3 cell were analyzed, as described above. Treatment with sodium selenate increased p27?, P1 levels in a time-dependent manner until 24 hours when levels declined, while a marked increase in p21CIP1 levels was observed, reaching a peak at 6 hours and declining afterwards (Figure 3A). A marked decline in phosphorylation of Rb (ser807 / 811) in a time-dependent manner in PC-3 cells treated with sodium selenate under the same conditions was also observed (Figure 3B). The effects of organic versus inorganic selenium compounds on the levels of the cell cycle inhibitor protein p27? Lp1 were compared in Pc-3 cells. As shown in Figure 3C, selenomethionine had little effect on the levels of p27KIP1 during the period of time analyzed, while sodium selenate, markedly reinforced total protein levels p27K! P1 as compared to control levels ( 0 hours) and selenomethonine (Figure 3C). These data indicate the reduction in cyclin D1 levels and Rb phosphorylation as well as the increase concomitant in the cell cycle inhibitory proteins p27KIP1 and p2-] Cip./wAF. They probably play an important role in the regulation of G1 arrest induced by the inorganic selenium compound, and that organic selenium is unable to induce these effects. The results of the present also indicate that sodium selenate, but not selenomethionine, potently inhibits the activation of protein B / Akt kinase. The loss of regulation of the survival path of the PI3K cell through the loss of the PTEN tumor suppressor protein is a common marker of human neoplasia (Vivanco, I. and Sawyers, CL, 2002, supra), particularly Prostate tumors (Visakorpi, 1999, Ann.Chir.Gynaecol, 88: 11-16; Li eí al 1997, Science, 275: 1943-1947). The trajectory of PI3K through its Akt effector kinase plays an important role in regulating cell proliferation, preventing the degradation of cyclin D1, and negatively influencing the expression of cell cycle inhibitor proteins p27 IP1 and p2l¡wAF. cip, (Graff et al. 2002, J. Biol. Chem., 275: 24500-24505). PC-3 cells have lost expression of PTEN and have a constitutively activated PI3K pathway activity (Chakraborty et al 2001, Cancer Res., 61: 7255-7263, Beresford et al 2001, J. Interferon Cytokine Res., 21: 313 -322), particularly the Akt kinase activity. To determine if sodium selenate can interfere with the activity of the PI3K pathway, PC-3 cells were treated with sodium selenate (0.5 mM) for several exposure times. The state of phosphorylation of Akt in whole-cell isatos was determined using specific activation antibodies. As shown in Figure 4A, sodium selenate induced a temporary boost in the phosphorylation of Akt in Ser473 and Thr308 in 10 minutes of exposure to inorganic selenium. This reinforcement was then followed by a marked and prolonged deactivation of Akt, while the total levels of cellular Akt (pan Akt) remained essentially unchanged (Figure 3A). Akt was phosphorylated in Thr308 on the cell membrane through PDK1 (Vanhaesebroeck, B. and Alessi, D.R. 2000, Biochem. J., 346 Pt3: 561-576). To determine if sodium selenate acted to sub-regulate the PDKI activity upstream of Akt, the phosphorylation status of PDK1 was analyzed using phosphol-specific PDK1 ser241 and tyr373 / 376 antibodies. As shown in Figure 4B the exposure of PC-3 cells to 0.5 mM sodium selenate 6 hours had no effect on the phosphorylation state of PDK1, indicating that sodium selenate does not inhibit Akt activation by blocking activity of kinase PDK1. To determine if the transient reinforcement in Akt activation observed with sodium selenate was dependent on components upstream of the PI3K pathway, PC-3 cells were treated with sodium selenate (0.5 mM) for 10 minutes, with or without treatment (1 hour) with PI3K inhibitor LY294002 (50μM). As seen in Figure 4C, treatment with sodium selenate for 10 minutes reinforced the activation of Akt as determined by phospho-specific antibodies. However, this reinforcement was completely blocked by pretreatment with LY294002, indicating that the transient reinforcement in Akt phosphorylation induced by sodium selenate requires upstream components of the PI3K path. The effects of inorganic selenium compounds on Akt activation in PC-3 cells were compared by treating cells with 0.5 mM of sodium selenate or selenomethionine for several time points and determined the phosphorylation of Akt in Ser473. As shown in Figure 4D, selenomethionine was able to affect the phosphorylation of Akt in all measured time periods, whereas sodium selenate profoundly inhibited Akt activation. The results also indicated that sodium selenate inhibits effectors current below cell survival, PI3K path. Potential effectors of the PI3K signaling downstream of the PTEN tumor suppressor include a number of Akt kinase substrates, such as BAD, caspase 9, IKKa and the Forkhead transcription factors FKHR, FKHRLI and AFX (Biggs, et al. 1999, Proc. Nati, Acad. ScU USA, 96: 7421-7426, Brunet et al 1999, Cell, 96: 857-868, Cardone et al 1998, Science, 282: 1318-1321, Datta et al 1997, Cell, 91: 231-241, Kops et al 1999, Nature, 398: 630-634, Ozes et al 1999, Nature, 401: 82-85, Tang et al 1999, J. Biol. Chem., 274: 16741-16746) .
The members of the Forkhead transcription factor family were de-regulated and inactivated in null PTEN cells, being aberrantly localized in the cytoplasm, where they can not activate transcription (Nakamura et al 2000, Mol. Cell. Biol, 20: 8969-8982). The re-introduction of the PTEN function in said cells induces the translocation of FKHR towards the nucleus, finally causing the G1 arrest in cells lacking PTEN (Nakamura ef ai 2000, supra). Therefore, inhibition with sodium selenate of Akt activation in PC-3 cells lacking PTEN should similarly induce the re-location of FKHR from the cytoplasm to the nucleus. PC-3 cells transfected with a GFP-FKHR expression construct were treated with LY294002 (50 μM, 1 hour) or sodium selenate (0.5 mM, 3 hours) or selenomethionine (0.5 mM, 3 hours) and the effects of the treatments in the cellular localization of the Forkhead fluorescent protein. As shown in Figures 5A and B, the GFP-FKHR fusion protein was located in the cytoplasm of untreated control PC-3 cells. Treatment with LY294002 or sodium selenate induced a marked re-localization of the GFP-FKHR fusion protein from the cycloplasm to the nucleus. However, selenomethionine was unable to induce the relocation of GFP-FKHR from the cytoplasm to the nucleus. These results confirm the selectivity between selenium compounds to block Akt signaling by inhibiting the activation of substrate protein Akt key, FKHR. Two additional downstream effectors / substrates of Akt are glycogen-synthase-kinase-3β, GSK-3β, (Mouie et al 1997, J. Biol. Chem., 272: 7713-7719; Van Weeren et al. 1998, J. Biol. Chem., 273: 13150-13156) and the mammalian target of rapamycin, mTOR protein (Nave ef al 1999, Biochem J., 344 Pt2: 427-431, Sekulic et al 2000, Cancer Res., 60: 3504-3513). To determine if sodium selenate can also activate these Akt substrates, PC-3 cells were treated with sodium selenate (0.5 mM) for several periods of time and whole cell lysates were probed with phospho-specific antibodies to GSK- 3ß and mTOR. As shown in Figures 5C and 5D, sodium selenate induced a decline in the phosphorylation status of both mTOR and GAK-3β.
Discussion The above results demonstrate the effects of sodium selenate supplementation at a high dose in hormone refractory prostate cancer. Sodium selenate was shown to inhibit PC-3 cell proliferation by inducing G1 arrest, in a dose-dependent and time-dependent manner, but did not increase the level of apoptosis. The arrest of G1 was accompanied by an increase in the expression of cell cycle inhibitory proteins p27KIP1 and p2? CIP1 / AF1 and p0r a reduction in the expression of cyclin D1. Rb phosphorylation was also suppressed by selenate. The selenate, in contrast to selenomethionine, markedly reduced the active levels of the effector PI3K, Akt. L activation of effectors downstream key of the PI3K / Akt path, mTOR, GSK-3β and the transcription factor FKHR were also inhibited by selenate, but not by selenomethionine. These results demonstrate a specific inhibitory effect of inorganic selenate on the path of PI3K / Akt, suggesting that this compound can be used in therapies for the treatment of cancer, particularly cancer caused by over-activation of the PBK / Akt path, such as tumors lacking PTEN. The molecular mechanisms involved in the anti-tumorigenic effect of sodium selenate against selenomethionine have also been produced in this study. Inorganic sodium selenate inhibited tumor progression in vivo by preventing celar proliferation without having any effect on apoptosis. A dose-dependent and time-dependent reduction in PC-3 cell proliferation was observed with sodium selenate capable of inhibiting phase progression S of these cells inducing the arrest of G1 without increasing the proportion of apoptotic cells observed by flow cytometry.
Progression through the cell cycle was controlled through CDKs, whose activity was inhibited by CDK inhibitors. The progression through the G1-S transition was postulated to be controlled by the activity of G1 cyclins and CDKs. Cyclins such as cdkl stimulate the progression of G1 cell cycle (Baldin ei to 1993, Genes Dev., 7: 812-821), while Rb seems to be a key downstream target by coupling the cell cycle procedure to the regulation of gene (Weintraub ef al 1995, Nature, 375: 812-815). The Rb phosphorylation through cdk4 / cdk6 / cyclin D complexes disrupts the Rb / E2F complex allowing E2F to activate genes required for DNA synthesis and cell cycle progression (Harbor et al 1999, Cell, 98: 859-869 ). The results of the present indicate that the levels of cyclin D1 were reduced, as did the phosphorylation of Rb in Ser807 / 811, while the levels of cdk6 and cdk4 remained relatively unchanged. These results demonstrate that selenate at a high dose induces a G1 arrest in PC-3 cells by interfering specifically with the levels and activation of key cell cycle progression protein. It is shown that p27KIP1 levels rise after exposure to a high dose of selenate, but not with a high dose of selenomethionine. Elevated p27KIP1 levels are a key marker and necessary for the arrest of Gl on a normal cell scale after serum deprivation (Coats et al 1996, Science, 272: 877-880) or contact inhibition (Polyak et al 1994, Cell 78: 59-66). p27KIP1 plays a central role as a negative regulator of cell cycle progression, therefore, the high levels of this protein provide a logical basis for the observed Gl arrest induced by selenate. Expression of P21CIP1 also leads to the arrest of Gl by inhibiting the cyclin / CDK complexes and inhibiting DNA synthesis (Johnson, G.G. and Walker, C.L. 1999, Annu.Rev Pharmacol.Ticotol.Co 39: 295-312). The trajectory of PI3K has a central role in many cellular functions that belong to the proliferation and survival and the de-regulation of this matter, through the loss of PTEN tumor suppressor protein, is a common event in prostate malignancies (Whang ef al 1998, Proc. Nati. Acad. Sci. USA 95: 5246-5250). Activation of the PI3K pathway is required for the induction of cyclin D1 expression (Muise-Helmericks et al 1998, J. Biol. Chem. 273: 29864-29872) and the inhibition of this pathway with the specific inhibitor of PI3K LY294002 has recently been reported as leading to the arrest of G1 in prostate carcinoma cells DU145 and PC-3 (Gao, et al 2003, Biochem, Biophys, Res. Commun., 310: 1124-1132). A principal downstream effector of the PI3K pathway is the protein B / Akt kinase. Akt is constitutively activated in prostate tumors lacking PTEN, such as PC-3 cells. The results of the present indicate that selenate at a high dose but not selenomethionine, can markedly reduce the levels of activated Akt present in these cells, an effect that acts specifically at the activated Akt level since no change in the activation of PDK1 it was detected, a kinase rather directly induces activation of Akt downstream of PI3K (Vivanco, I. and Sawyers, CL 2003, supra). These results suggest that selenate can act to reinforce the dephosphorylation of Akt, through a poorly characterized phosphatase. Interestingly, the effects of selenato on ¡a Akt activation seem to follow two different modes of action. Initially, selenate induces a reinforcement in Akt phosphorylation. This reinforcement is dependent on PI3K since it is inhibited by pre-treatment with LY294002. This transient reinforcement is then followed by a deep, long-lasting inhibition of Akt activity, which does not involve components upstream of Akt, since the proliferative inhibition of sodium selenate at a high dose was not improved by LY294002. Sodium selenate is also able to prevent the activation of the downstream Akt effectors, mTOR and GSK-3β, as well as the transcription factor FKHR, according to the inhibitory effect of selenate that targets the key PI3K effector kinase, Akt. Methylselenic acid also induces G1 arrest of DU-145 cells as well as apoptosis through a trajectory measured by caspase in 24 hours of treatment (Jiang et al 2001, supra). Doses as low as 3μM induced the arrest of G1 but this was not associated with apoptosis or with reduced Akt phosphorylation (Jiang et al 2002, supra). However, higher doses of 5μM lead to a dose-dependent decline in Akt activation as well as in the initiation of apoptosis measured by caspase. Inhibition of the PI3K pathway with LY294003 failed to induce apoptosis in DU-145 cells, indicating that the block in the PI3K pathway induced by methylselenic acid was not involved in the induction of apoptosis induction (Jiang et al 2002, supra). A low dose of methylselenic acid, inducing G1 arrest, Independent of Akt Inhibition and low dose apoptosis but blocking Akt and inducing apoptosis at higher doses, it is in sharp contrast to the results herein. Sodium selenate induced the arrest of G1 through the inhibition of the PI3K pathway, specifically at the Akt level, without induction of apoptosis. Therefore, it may appear that the anti-proliferative effects of selenate are more focused than those observed with other selenium compounds. Based on the findings herein and given that chronic Akt activation is associated with resistance to chemotherapy in cancer models in vivo (Tanaka et al 2000, Oncogene, 19: 5406-5412), an attractive mechanical basis is provided for the use of selenate with chemotherapeutic agents in combination therapy in tumor models.
EXAMPLE 2 PROSTATE TUMOR GROWTH REDUCTION DUE TO EFFECT SINERGÍSTICO DE SELENATO DE SODIO WITH PACLITAXEL Materials and Methods 1 x 10 6 PC-3 prostate tumor cells were injected into the prostate of hairless mice. After three days, they were administered 5 ppm selenium in the form of sodium selenate to water to drink from mice receiving selenate. Paclitaxel was administered at 10 mg / kg intraperitoneally once a week in a solution of 10% Cremophor EL / 25% ethanol / 65% PBS to those animals that received paclitaxel. 10 animals were used per group. After 5 weeks after the injection, the animals were sacrificed, the prostates removed, the appendages dissected and the tumors weighed. The retroperitoneum for lymphadenopathy was explored and lymph nodes with a diameter greater than 0.5mm were removed. The tumors were then sectioned stepwise at 50 micron steps and the tumor volumes were calculated from the digitalized images using the standard volumetric form (a + b2 / 2).
Results Figure 6 demonstrates that the seiane synergizes with paclitaxel to reduce prostate tumor weights. The control group received the solubilization vehicle paclitaxel Cremophor and ethanol without paclitaxel. It is known that Cremophor has some antitumor effect that probably represents the fact that the anti-tumor effect of selenate alone was not reduced compared to the control in this experiment. The results shown in Figure 6 indicate that selenate and paclitaxel synergize to reduce tumor weights greater than the additive effects of selenate and paclitaxel alone. The selenate inhibits a key cell survival trajectory, the PI3K / Akt trajectory. This trajectory is over-regulated in a high proportion of human tumors and the over-activation of this trajectory is highly correlated with the development of chemoresistance to chemotherapeutic agents. The results of the present indicate that the co-treatment of prostate tumors in vivo with a combination therapy of selenate and paclitaxel can induce a greater anti-tumor effect than any treatment alone. Selenate can interfere with the ability of tumor cells to induce chemoresistance to the chemotherapeutic drug, paclltaxel. Therefore, prostate tumors were induced in hairless mice by injecting PC-3 cells into the prostates of the animals and subsequently treating the animals with selenate or paclitaxel alone or in combination for 5 weeks and then the tumors were analyzed. As shown in Figure 6, the combination therapy had a marked synergistic effect to reduce tumor weights greater than the additive effect of any treatment alone. The control group received paclitaxel solubilization carrier Cremophor EL and ethanol without paclitaxel. It is known that Cremophor EL has an anti-tumor effect that probably represents the fact that selenate alone was not reduced compared to the control in this experiment. The synergistic effects that were observed are greater in vivo than in vitro. This supports the concept that the selenate is having anti-angiogenic effect in vascular endothelial cells as well as in the growth of tumor cells per se and that this combined effect can only be observed in vivo and represents the synergistic effect. Figure 7 shows sections of tumor tissue. It can see that selenate in combination with paclitaxel has a synergistic effect that significantly reduces tumor size and volume, compared to paclitaxel alone. This important synergistic effect of selenate and paclitaxel is also evident in the graphs of tumor volumes indicated in Figure 8. Tumor volumes of animals treated only with paclitaxel and animals treated with combination therapy were measured to determine the effects of the agents in the growth of prostate tumor. The combination therapy had a marked effect on tumor size reduction as shown in Figure 7, and also had a marked effect on tumor volumes (Figure 8). This effect was statistically significant (P <; 0.05). Therefore, the in vivo data shown in Figures 6 to 8 demonstrate the synergistic effect of selenate with paclitaxel to reduce weights and volumes of prostate tumor. This effect was greater than the additive effects of the agent alone demonstrating the synergistic effects of the two compounds. This supports the finding that selenate is having an anti-angiogenic effect on vascular endothelial cells as well as on the growth of tumor cells and that this combined effect observed in vivo represents the synergistic effect. Selenate alone had a greater effect in reducing the density of microvessel in tumors than in tumor volumes, indicating a direct anti-angiogenic effect.
EXAMPLE 3 COMPARATIVE TESTS BETWEEN SELENATO AND SELENITA Materials and Methods Cell toxicity Cell toxicity after treatment with 5 μM or 50 μM of sodium selenate or sodium selenite involved measuring cell toxicity through Trypan Blue Exclusion. 5x103 human prostate cancer PC-3 cells were seeded in a 24-well plate and 8 hours later treated with either 5 μM or 50 μM selenate or selenite. The dose scale of 5 μM or 50 μM is equivalent to 0.1 mg / kg to 1.25 mg / kg. The cells were harvested at 24, 48, 72 and 96 hours after the addition of selenate or selenite and the percentage of viable cells determined as determined by Trypan Blue staining was determined. The results shown in the graphs of Figure 9 illustrate the midpoints and SD of three independent experiments. Figure 9 shows that selenate is cytostatic, while selenite at similar concentrations is cytotoxic. Therefore, selenite may be unsuitable for combination therapy in animals. In order to distinguish between the relative cytotoxic effects of selenate and selenite, PC-3 human prostate carcinoma cells were treated with two different concentrations of selenate at 5 μM or 50 μM (equivalent to a dose of 0.2 to 1.9 mg / kg ) or 5 μM or 50 μM selenite (equivalent to a dose of 0.18 to 1.8 mg / kg). All Cells in the tissue culture cavities were then harvested sequentially at periods of between 24 and 96 hours after the addition of the treatments. The percentage of viable cells was then determined using the Trypan Blue exclusion assay. The selenate at all time points and at both lower and higher concentrations was non-cytotoxic to these cells. The percentages of viable cells in the samples treated with selenate were equivalent to the untreated control samples (Figure 9). This effect was maintained across all time points and reached statistical significance for a number of time points (see time points marked with an asterisk, Figure 9). The selenate had a demonstrated cytostatic effect on the cells, whereas selenite had a cytotoxic effect.
Cell Genotoxicity 5 × 10 5 PC-3 cells were plated in a 6-well plate and 24 hours later the cells were treated with paclitaxel at 1 μg / mL or 10 μg / mL or 500 μM selenate (19 mg / kg) or 500 μM selenite (18 mg / kg) during the indicated times. The cells were then washed in PBS, lysed in pH buffer of ELB lysis and the whole cell lysates were operated on an SDS-PAGE gel and stained and probed with the indicated antibodies. Histone H2AX phosphorylation occurred minutes after DNA damage, typically through double or single strand DNA structure breaks and is a very sensitive reading of DNA damage.
To determine the differences between seinate, selenite and the taxane, paclitaxel to induce DNA damage, PC-3 cells were treated with these compounds for increasing periods of time, and then lysed. Protein extracts were probed for histone H2A.X phosphorylation using an antibody-specific phosphorylation. The results are illustrated in Figure 10. Figure 10 indicates that selenite is genotoxic, including breaks in DNA structure whereas selenate and paclitaxel are not. Even at a high concentration of selenate (500 μM, equivalent to a dose of 19 mg / kg), as well as increasing concentrations of paclitaxel did not induce any DNA damage as determined by the phosphorylation of Histone H2A.X. In contrast, selenite at the same concentration (500 μM, equivalent to a dose of 18 mg / kg) induces DNA damage in 30 minutes of treatment, an effect that increases with time despite the general toxic effects of this treatment as illustrated by the concomitant reduction in β-tubuin levels in the same samples (Figure 10).
Activation of Akt 5x105 PC-3 cells were plated in a 6-well plate and 24 hours later the cells were deprived of serum for 16 hours, then treated with the indicated selenium compounds all at 125 μM (equivalent to a scale of dose of 4-9 mg / kg) for 6 hours. The cells were then washed with PBS, lysed in pH regulator ELB lysis and the whole cell lysates were run on an SDS-PAGE gel and stained and probed, first with a specific activation Akt antibody (Ser408), then the stains were separated and returned to probe with a specific antibody Akt total (pan Akt) as a load control. The signal intensities were scanned from the developed films and the intensities of active Akt correlated to total Akt levels were plotted. To compare the effects of different selenium compounds on the Akt activation state, PC-3 cells were treated with seven different selenium compounds as shown in Figure 11. Figure 11 indicates that only sodium selenate (ATE) ) inhibits the activation of Akt, reducing levels of phosphorylated Akt below control levels (con). In contrast, selenosic acid (Sel acid), sodium selenite (ITE), selenium dioxide (Se02), selenium sulfide (SeS2), methyl seienocysteine (MSC), selenocysteine (SeC) all induced Akt activation from above of control levels (with). To determine the effects of a high dose of selenite, 5x105 PC-3 cells were plated in a 6-well plate and 24 hours later the cells were deprived of serum for 16 hours then treated with sodium selenite or sodium selenate. at 500 μM (equivalent to a dose scale of 18-19 mg / kg) for 1 hour. After the cells in PBS, they were lysed in pH buffer of ELB lysis and the whole cell lysates were run in a SDS-PAGE gel and were graphed and probed first with a specific activation Akt antibody (Ser408). Figure 12 indicates that even at a higher dose, 500 μM sodium selenite (ITE) did not inhibit Akt activation compared to a similar high dose of sodium selenate (ATE). To determine if high dose levels of selenite might be able to inhibit Akt activation levels similar to those observed with selenate, PC-3 cells were treated with 500 μM (18-19 mg / kg) of sodium selenate or selenite of sodium for 1 hour and I activation of Akt was determined using an antibody-specific phosphorylation. This period of time was selected since the selenite at 500 μM (18 mg / kg) does not induce degradation of general protein at this point in time. As shown in Figure 12, selenate inhibited Akt activation compared to untreated control cells while selenite was unable to inhibit Akt activation.
Apoptosis 5 × 10 5 PC-3 cells were plated in a 6-well plate and 24 hours later the cells were treated with paclitaxel a 1, 10, 100 ng / mL and 1 μg / mL, 101 μg / mL or sodium selenate at 100 μM, 250 μM or 500 μM (equivalent to 4 to 19 mg / kg) or sodium selenite a 100 μM, 250 μM or 500 μM (equivalent of 3.6 to 18 mg / kg) for 16 hours. The cells were then washed in PBS, lysed in ELB pH lysis buffer and the whole cell lysates were ran on a SDS-PAGE gel and were graphed and probed with anti-split PARP (PARP) and anti-β-tubulin (tubulin) specific antibodies. Agents that induce genotoxic damage produce apoptotic programs from p53-dependent mechanisms as demonstrated for selenite (Jiang, C. et al, 2004 Mol.Can.Ther.3: 877). Microtubule stabilizing agents such as paclitaxel induce apoptosis through the intrinsic apoptotic pathway. To differentiate the pro-apoptotic mechanisms of seienato and selenite, PC-3 cells were treated with increasing concentrations of these compounds for 16 hours and the levels of the apoptotic marker protein were analyzed., PARP unfolded. Figure 13 indicates that selenate and selenite induce apoptosis through different mechanisms. Paclitaxel and selenate induce cleavage of the pro-apoptotic PARP protein, whereas selenite does not. Selenite also induces a marked degradation of cell tubulin underlying its cellular toxicity. Therefore, it seems that selenate and selenite induce apoptosis through different mechanisms, as evidenced by their effects on PARP and -tubulin, all data show a clear differentiation in the effect of several selenium compounds on the trajectory of Akt. .
EXAMPLE 4 5 × 10 4 cells LNCaP sensitive cells were seeded androgen of human prostate cancer in 6-well plates, and 8 hours later treated with 50μM of sodium selenate or not (control). The cells were harvested at 72 hours after the addition of the compounds and viable cell counts were determined as determined by Trypan Blue staining. As shown in Figure 14, selenate synergizes with androgen ablation to reduce paternal LNCaP cell proliferation after 72 hours of treatment.
EXAMPLE 5 5 x 10 4 independent human and prostate cancer LNCaP CSS cells were seeded in a 6-well plate and 8 hours later treated with either 50 μM selenate or not (control). Cells were harvested at 72 hours after the addition of sodium selenate and viable cell counts were determined as determined by Trypan Blue staining. As shown in Figure 15, the selenate synergism with androgen ablation to reduce the proliferation of CSS LNCaP cell after 72 hours of treatment. Androgen-independent cells are still more sensitive to selenate treatment than LNCaP cells are parental cells.
EXAMPLE 6 1 x 10 5 NS-LNCaP cells sensitive to androgen were seeded of human prostate cancer in a 6-well plate and 8 hours later were treated with either 5 μM of sodium selenate or 10 μM of LY294002. The cells were harvested at the indicated periods after the addition of sodium selenate or Ly294002 and viable cell counts were determined as determined by Trypan Blue staining. As shown in Figure 16, selenate at only 5 μM markedly reduces (p <0.95) the proliferation of sensitive androgen NS LNCaP cell after 9 days of treatment.
EXAMPLE 7 1 x 105 human-prostate cancer NS LNCaP cells were seeded in a 6-well plate and 8 hours later treated with either 5 μM of sodium selenate or 10 μM of LY294002. The cells were harvested at the indicated periods after the addition of the compounds and the viable cell counts were determined as determined by Trypan Blue staining. As shown in Figure 17 selenate alone at 5 μM synergizes with androgen ablation to markedly reduce (p <0.95) the proliferation of androgen-sensitive NS LNCaP cell after 9 days of treatment.
EXAMPLE 8 1 x 105 independent CSS LNCaP cells were seeded Human prostate cancer androgen in a 6-well plate and 8 hours later were treated with either 5 μM sodium selenate or 10 μM LY294002. The cells were harvested at the indicated periods after the addition of the sodium seyenate or LY294002 and viable cell counts were determined as determined by Trypan Blue staining. As shown in Figure 18, selenate alone at 5 μM markedly reduces (p <0.95) the proliferation of androgen-independent CSS LNCaP cell after 9 days of treatment, even in the presence of testosterone.
EXAMPLE 9 1 x 105 human-prostate cancer androgen independent LNCaP cells were seeded in a 6-well plate and 8 hours later treated with either 5 μM of sodium selenate or 10 μM of LY294002. The cells were harvested at the indicated periods after the addition of sodium selenate or LY294002 and the viable cell counts were determined as determined by Trypan Blue staining. As shown in Figure 19, selenate alone at 5 μM synergizes with androgen ablation to markedly reduce (p <0.95) the proliferation of androgen-sensitive CSS LNCaP cell after 9 days of treatment.
EXAMPLE 10 1 x 1 O5 human kidney epithelial Q293 cells were seeded in a 6-well plate and 8 hours later treated with either 5 μM of sodium selenate or 10 μM of LY294002. The cells were harvested at the indicated periods after the addition of sodium selenate or LY294002 and viable cell counts were determined as determined by Trypan Blue staining. As shown in Figure 20, selenate at 5 μM does not significantly affect Q293 cell proliferation whereas PI3K inhibitor, LY294002 significantly affects proliferation (p <0.95) after 9 days of treatment. The inhibitory effect of selenates is therefore cell-specific.
EXAMPLE 11 1 x 1 O5 human kidney epithelial cells 0293 were seeded in a 6-well plate and 8 hours later treated with either 5 μM of sodium selenate or 10 μM of LY294002. The cells were harvested at the indicated periods after the addition of sodium selenate or LY294002 and viable cell counts were determined as determined by Trypan Blue staining. As shown in Figure 21, selenate at 5 μM does not significantly affect the proliferation of Q293 cells even when combined with androgen alloy (developed in a CSS medium) while the inhibitor PI3K, LY294002 significantly effected proliferation (p <0.95) after 9 days of treatment. The inhibitory effect of selenates is therefore cell-specific.
Materials and Methods for Examples 4 to 11 Cell Culture The parental androgen sensitive parental LNCaP cell line was obtained from the American Type Culture Collection (Manassas, Virginia, USA) and cultured routinely in RPMI 1641 (Invitrogen) supplemented they are 10% fetal bovine serum and 1% antibiotic / antifungal mixture (Invitrogen). The cells were maintained at 37 ° C in 5% C02. To select androgen-independent LNCaP cells, LNCaPs were cultured in RPMI 1641 (Invitrogen) in 5% carbon separate serum (CSS) (Invitrogen) which removed all detectable testosterone traces for 6-8 weeks and the lines of LNCaP cell obtained that were now able to proliferate freely in the absence of testosterone. These cells are referred to as CSS LNCaPs. Q293 cells are a human kidney epithelial cell line routinely cultured in a DMEM medium (Invitrogen) in 5% fetal bovine serum and an antibiotic, 1% antifungal mixture (Invitrogen). Sodium selenate was made in 10 mM supply solutions in distilled water and a sterilized filter before dilution in the media for in vitro experiments. The inhibitor PI3K, LY294002 was dissolved in DMSO to a supply solution of 50mM and diluted in the cell culture medium for in vitro experiments.
Cell Growth Curve Between 5x104 (Figure 1-2) and 1x105 LNCaP cells, CSS LNCaP or Q293 were allowed to bind overnight. After a few hours, the medium was changed to include sodium selenate or LY294002 in the presence of either normal serum or separated carbon serum (CSS) as indicated and allowed to develop until the specified time points. The supernatants and cells were then harvested, combined and viable cells were determined by Trypan Blue exclusion assay. The experiments were carried out in triplicate.
Statistical Analysis The data are presented as ± SE medium unless otherwise indicated. The differences between the treatments and the control group were analyzed using the t-pair test with an assumed importance to p < 0.05. The asterisks represent values significantly different from the corresponding control value. A statistical analysis was performed using the software SPSS 9.05 for Windows (SPSS, Chicago, Illinois). The description of each patent, patent application, and publication cited herein is incorporated herein by reference in its entirety.
The citation of any reference herein should not be construed as an admission that such reference is available as "prior art" in the present application. Through the specification, the object has been to describe the preferred embodiments of the invention without limiting the invention to any modality or group of specific features. Those skilled in the art, therefore, will appreciate that, in view of the description, various changes and modifications may be made to the particular embodiments illustrated without departing from the scope of the present invention. All these modifications and changes are intended to be included within the scope of the appended claims.

Claims (43)

1. A method for inhibiting the growth of a tumor cell, wherein the Akt signaling pathway is activated, the method comprises exposing the tumor cell to an inhibiting amount of the Akt signaling activation of selenate or a pharmaceutically acceptable salt thereof.
2. A method according to claim 1, wherein the tumor cell is one wherein Akt is over-active.
3. A method according to claim 1, wherein the tumor cell is a prostate tumor cell.
4. A method according to claim 3, wherein the growth of the tumor cell is independent of androgen or chemo-resistant.
5. A method according to claim 1, wherein the inhibitory amount of activation of Akt signaling pathway of selenate or its pharmaceutically acceptable salt is from about 0.015 mg / kg to about 20.0 mg / kg.
6. A method according to claim 1, further comprising exposing the tumor cell to a cytostatic agent or cytotoxic agent.
7. A method according to claim 6, wherein the cltostatic agent is a microtubule stabilizing agent.
8. A method according to claim 7, wherein the microtubule stabilizing agent is paclitaxel.
9. A method according to claim 1, further comprising exposing the tumor cells to radiotherapy, optionally together with a radiosensitizing agent.
10. A method for treating a cancer, wherein the Akt signaling pathway is activated, the method comprising administering to a subject in need of such treatment, an inhibiting amount of activation of Akt signaling pathway of selenate or a pharmaceutically salt acceptable of it.
11. A method according to claim 10, wherein the cancer is one in which Akt is over-active.
12. A method according to claim 10, wherein the cancer is prostate cancer.
13. A method according to claim 12, wherein the cancer is an androgen-independent prostate cancer or a cyst-resistant prostate cancer.
14. A method according to claim 10, wherein the activating amount of Akt signaling pathway of selenate is a supra-nutritional amount.
15. A method according to claim 10, wherein the inhibiting amount of signaling path activation Selenate Akt is from about 0.015 mg / kg to about 20.0 mg / kg.
16. A method according to claim 10, wherein the selenate is in the form of sodium selenate.
17. A method according to claim 10, which further comprises administering a cytostatic agent or a cytotoxic agent.
18. A method according to claim 17, wherein the cytostatic agent is a microtubule stabilizing agent.
19. A method according to claim 18, wherein the microtubule stabilizing agent is paclitaxel.
20. A method according to claim 10, further comprising administering radiotherapy, optionally in combination with a radiosensitizing agent.
21. A method for treating a hormone-dependent cancer in a subject, the method comprising administering a therapeutically effective amount of selenate or a pharmaceutically acceptable salt thereof in combination with hormone ablation therapy.
22. A method according to claim 21, wherein the therapeutically effective amount of selenate or its pharmaceutically acceptable salt is a supra-nutritional amount.
23. A method according to claim 21, wherein the therapeutically effective amount of selenate or its pharmaceutically acceptable salt is from about 0.015 mg / kg to about 20 mg / kg.
24. A method according to claim 21, wherein the selenate is in the form of sodium selenate.
25. A method according to claim 21, wherein the hormone-dependent cancer is selected from a cancer androgen-dependent or an estrogen-dependent cancer.
26. A method according to claim 21, wherein the hormone-dependent cancer is selected from prostate cancer, testicular cancer, breast cancer, ovarian cancer, uterine cancer, endometrial cancer, thyroid cancer or pituitary cancer.
27. A method according to claim 21, wherein the hormone-dependent cancer is an androgen-dependent prostate cancer.
28. A method according to claim 21, further comprising the administration of a cytostatic agent or a cytotoxic agent.
29. A method according to claim 28, wherein the cytostatic agent is a microtubule stabilizing agent.
30. A method according to claim 29, wherein the microtubule stabilizing agent is paciitaxel.
31. A method according to claim 21, further comprising administering radiotherapy, optionally together with a radiosensitizing agent.
32. A method for treating prostate cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of selenate or a pharmaceutically acceptable salt thereof.
33. A method according to claim 32, wherein the prostate cancer is an androgen-independent prostate cancer or a chemo-resistant prostate cancer.
34. A method according to claim 32, wherein the therapeutically effective amount is a supra-nutritional amount.
35. A method according to claim 32, wherein the therapeutically effective amount of selenate or its pharmaceutically acceptable sai is from about 0.015 mg / kg to about 20.0 mg / kg.
36. A method according to claim 32, wherein the selenate is in the form of sodium selenate.
37. A method according to claim 32, further comprising administering a cytostatic agent or a cytotoxic agent.
38. A method according to claim 37, wherein the cytostatic agent is a microtubule stabilizing agent.
39. A method according to claim 38, wherein the microtubule stabilizing agent is paclitaxel.
40. A method according to claim 32, further comprising administering radiotherapy, optionally in combination with a radiosensitizing agent.
41. The use of selenate or its pharmaceutically acceptable salt in the manufacture of a medicament for treating cancer, wherein the path of Akt signaling is activated.
42. The use of selenate or its pharmaceutically acceptable salt in the manufacture of a medicament for treating cancer, wherein the path of Akt signaling is activated, wherein the cancer is different from a cancer selected for prostate cancer PC-3, lymphoma 3B6, lymphoma BL41 and mammary tumor HTB123 / DU 4475.
43. The use of selenate or its pharmaceutically acceptable salt in the manufacture of a medicament for treating a hormone-dependent cancer , where the drug is formulated for administration with hormone ablation therapy.
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