CN104338134A - Autophagy inhibitor and arginase medicine compound and application thereof - Google Patents

Abstract

The invention relates to the field of biological pharmacy, and provides a new medicine compound for treating tumors, and particularly relates to an autophagy inhibitor and arginase medicine compound and application thereof. The medicine compound is used for treating the tumors. According to the medicine compound, the antineoplastic activity of arginase can be obviously strengthened in vitro; the immunogenicity of a person for recombing the arginase can be reduced; and the half-life period of the arginase in vivo can be prolonged. The arginase modified by poly-sialic acid is provided; and the arginase modified by the poly-sialic acid is characterized in that the active activated poly-sialic acid is reacted with the arginase under the condition that sodium cyanoborohydride exists to form conjugates. By means of the autophagy inhibitor and arginase medicine compound, the killing effect of the arginase on the tumors can be strengthened, the curative effect of the arginase can be strengthened, and the curative effects of the arginase on treating the melanin tumor and the breast cancer can be strengthened.

Description

A kind of autophagy inhibitor and arginase medicinal composition and uses thereof

Technical field

The invention belongs to medical art, relate to the medicinal composition of new treatment tumor; Be specifically related to a kind of autophagy inhibitor and arginase medicinal composition and uses thereof; This medicinal composition is used for the treatment of tumor, especially treats melanoma and hepatocarcinoma.

Background technology

Arginase is a kind of key enzyme participating in ornithine cycle in mammalian liver, is ornithine and carbamide in ornithine cycle by conversion of Arginine.Prior art discloses arginine is the essential amino acids maintaining Partial tumors Growth of Cells, and in tumor cell, arginine-level is lower than after certain value, will cause cell death.Arginase can the degraded arginine of differential high efficient in vivo and in vitro.Arginine has important function in the normal physiological function of human body, is a kind of half must aminoacid, normal somatic cell can by ornithine cycle by other amino acid converting be arginine, thus offset exogenous arginine shortage; But, for the tumor cell that part arginine relies on, owing to lacking the key enzyme in ornithine cycle process, when exogenous arginine deprivation, self cannot synthesize arginine, finally cause cell death.Arginase can be degraded arginine in vivo and in vitro, causes the death of neoplastic cells that arginine relies on, and on normal cell without impact.In having research to test in vitro, find [the 1. Lam TL such as the remarkable killing hepatoma cell of arginase energy, melanoma cell, pancreatic cancer cell and leukaemia, Wong GK, Chong HC, Cheng PN, Choi SC, Chow TL, et al.Recombinant human arginase inhibits proliferation of human hepatocellular carcinoma by inducing cell cycle arrest.Cancer letters.2009, 277:91-100. is Hernandez CP 2., Morrow K, Lopez-Barcons LA, Zabaleta J, Sierra R, Velasco C, et al.Pegylated arginase I:a potential therapeutic approach in T-ALL.Blood.2010, 115:5214-21. 3. Hsueh EC, Knebel SM, Lo WH, Leung YC, Cheng PN, Hsueh CT.Deprivation of arginine by recombinant human arginase in prostate cancer cells.Journal of hematology & oncology.2012, 5:17.] the first phase clinical effectiveness of arginase Hepatoma therapy shows its good patent medicine performance [Yau T, Cheng PN, Chan P, Chan W, Chen L, Yuen J, et al.A phase l dose-escalating study of pegylated recombinant human arginase l (Peg-rhArgl) in patients with advanced hepatocellular carcinoma.Investigational new drugs.2012.], it is reported, the treatment research that arginase is used for hepatocarcinoma completes phase ii clinical trial, show good application prospect, CMM (cutanous malignant melanoma, CMM) sickness rate is in rising trend, shows according to the online data in National Cancer research center: the white American CMM sickness rate rises to 22.5/100000 of calendar year 2001 from 7.5/100000 in 1973.In China, melanoma sickness rate becomes to increase trend, although most early stage patient can be cured by operation, for progressive stage, the usual poor effect of chemotherapy, thus finds new therapeutic strategy particularly important.

But the arginase in vivo factor such as low-affinity and shorter half-life have impact on the antitumor curative effect of medicine.Therefore, the anti-tumor activity improved in arginase body will contribute to the application of its antitumor further.

Polysialic acids is that N-acetyl-neuraminate connects unique carbohydrate of glycosidic bond poly formation by N-, research finds that polysialic acids polypeptide or protein are under the prerequisite keeping activity, peptides hydrolysis significantly can be reduced after modification, prolong half-life, reduce immunogenicity (Gregoriadis G, Fernandes A, Mital M, McCormack B:Polysialic acids:potential in improving the stability and pharmacokinetics of proteins and other therapeutics.Cell Mol Life Sci 2000, 57 (13-14): 1964-1969).Fernandes etc. utilize polysialic acids to modify L-ASP, the antigenicity of the rear enzyme of modification and immunogenicity are all lower than asparaginase, but the antitryptic hydrolysis ability of enzyme and vitro half-lives obviously strengthen (Fernandes AI, Gregoriadis G:The effect of polysialylation on the immunogenicity and antigenicity of asparaginase:implication in its pharmacokinetics.Int J Pharm2001 after modifying, 217 (1-2): 215-224), Lin Yi etc. carry out polysialic acids modification to copper-zinc superoxide dismutase, the stability experiment result of modifying afterproduct shows, polysialic acids modifies the ph stability that can improve copper-zinc superoxide dismutase, heat stability and resistance to enzymic degradation ability (Wu JR, Lin Y, Zheng ZY, Lin CC, Zhan XB, Shen YQ:Improvement of the CuZn-superoxide dismutase enzyme activity and stability as a therapeutic agent by modification with polysialic acids.Biotechnol Lett2010, 32 (12): 1939-1945).In addition polysialic acids regulates neural cell development by the adhesion changing nervous system nerves adhesion molecule, the maintenance of neurocyte function is played an important role (Weber M, Modemann S, Schipper P, Trauer H, Franke H, Illes P, Geiger KD, Hengstler JG, Kleemann WJ:Increased polysialic acid neural cell adhesion molecule expression in human hippocampus of heroin addicts.Neuroscience2006; 138 (4): 1215-1223; Angata K, Huckaby V, Ranscht B, Terskikh A, Marth JD, Fukuda M:Polysialic acid-directed migration and differentiation of neural precursors are essential for mouse brain development.Mol Cel l Biol2007; 27 (19): 6659-6668).

Cell autophagy (autophagy) is again II type programmed death (type II programmed cell death), it is the phenomenon of common in most eukaryotes " self-digestion " (cellular degradation), organelle impaired or unnecessary in cell and albumen generation nucleotide can be decomposed, the small-molecule substance such as aminoacid synthesizes new protein for cell, and can maintain the stable of microenvironment in cell.Research finds itself and various diseases, and especially the development relationship of tumor is close.The difference of lysosome intracavitary is transported to according to intracellular substrate, mammalian cell autophagy can be divided into three kinds of modes: the autophagy (chaperone-mediated autophagy, CMA) of large autophagy (macroautophagy), little autophagy (microautophagy) and molecular chaperones mediation.The large autophagy (hereinafter referred to as autophagy) of main general introduction and tumor development and treat relation the closest [ sridhar s, botbol Y, macian F, et al.Autophagy and disease:always two sides to a problem. j Pathol.2012; 226 (2): 255-73.].Autophagy is the biological process of endochylema macromolecular substances and organelle a large amount of degraded in duplicature encapsulation bubble.The large activation of prior art process be wherein divided into 4 stages [ mart í nez-Borra J, l ó pez-Larrea C.Autophagy and self-defense. adv Exa Med Biol.2012; 738:169-84.].Autophagy can produce stress to cell to outside environment change and various stimulation.Can be there is the autophagy of reduced levels in cell, claim basic autophagy under growth conditions.But once be subject to extraneous stimulation, as hunger, anoxia, high temperature, high-cell density or somatomedin are deprived, the level of cell autophagy will raise rapidly.As when nutrient substance lacks, in cell autophagy energy decomposer, non-viable non-apoptotic cell device generation aminase etc. synthesize new protein for cell, maintain the survival of cell [1. piacentini m, d ' Eletto M, falasca L, et al.Transglutaminase2at the crossroads between cell death and survival. adv Enzymol Relat Areas Mol Biol.2011; 78:197-246; 2. cook KL, shajahan aN, clarke R.Autophagy and endocrine resistance in breast cancer. exnertRev Anticancer Ther.2011; 11 (8): 1283-94.; 3. wirawan E, vanden berghe T, lippens S, et al.Autophagy:for better or for worse. cell res.2012; 22 (1): 43-61.].

Research announcement autophagy can be degraded and be folded the protein of mistake, the organelle etc. of damage, delays the generation of body aging.Collective's diseases associated with senescence one neurodegenerative diseases can be classified as protein conformation mistake disease, and the protein generally owing to folding mistake in a large number piles up in cell thus trigger cell toxicity causes.Research shows, a large amount of senile disease, add neurodegenerative diseases and dislike part tumor all with cell autophagy closely related [1. mart í nez-Borra J, l ó pez-Larrea C.Autophagy and self-defense. adv Exp Med Biol.2012; 738:169-84.; 2. caballero B, coto-Montes A.An insight into the role of autophagy in cell responses in the aging and neurodegenerative brain. histol Histopathol.2012; 27 (3): 263-75.; 3. mendelsohn AR, larrick JW.Rapamycin as an antiaging therapeutic?: targeting mammalian target of rapamycin to treat Hutchinson-Gilford progeria and neurodegenerative diseases. rejuvenation Res.2011; 14 (4): 437-41.].

It is reported, nematode growth developmental defect, the aging of autophagygene disappearance or sudden change are accelerated and shorten the life-span; And autophagy also participates in the generation of fruit bat metamorphosis.In addition autophagy mammalian adult individuality organize allelotaxis and differentiation in also play important function [ mizushima N, komatsu M.Autophagy:renovation of cells and tissues.Cell.2011; 147 (4): 728-41.].In addition as the one of programmed cell death, cell autophagy directly or indirectly causes cell death by number of ways.[ Denton D， Nicolson S， Kumar S.Cell death by autophagy：facts and apparent artefacts. Cell Death Differ.2012；19(1)：87-95.]。Research shows, the generation of tumor is very close with the relation of development and autophagy.

In general, because cell autophagy is conducive to the survival of cell, but the generation development of autophagy to tumor cell is not still come to a conclusion at present.The autophagy initial stage can as tumorigenic a kind of restraining factors, some known tumor-inhibiting factor, such as PTEN, TSCl and TSC2 can activate autophagy, and protein degradation can be made to reduce to the suppression of autophagy, anabolism increases, and finally causes former cancerous cell continuous proliferation.Although autophagy ability is had nothing in common with each other before canceration, after canceration, its autophagy ability all weakens most of tumor cell (as liver, pancreas, breast carcinoma etc.).Autophagy lack can cause autophagy substrate p62 gather by NF-κ B signal pathway cause tumor formed [ trocoli A, d javaheri-Mergny M.The complex interplay between autophagy and NF-κ B signaling pathways in cancer cells. am J Cancer Res.2011; 1 (5): 629-49.].But tumor growth to a certain extent time, especially when also not forming enough blood vessels in tumor and providing nutrition for its amplification, tumor cell also can overcome the environment existence of malnutrition and hypoxia by autophagy.Research shows, rises to 37% lacking in serum or amino acid whose situation the autophagy part in about 3h, HeLa cell from 4%.This also illustrates mechanism that autophagy under the conditions such as malnutrition is also a kind of self-protection of tumor cell [ baldwin AS.Regulation of cell death and autophagy by IKK and NF-κ B:critical mechanisms in immune function and cancer. immunol Rev.2012; 246 (1): 327-45.].

Separately have research to disclose, the effect that the tumor cell autophagy of antitumor drug induction embodies roughly may be summarized to be two kinds: be the protection to tumor cell in most cases, also can kill and wound tumor cell in some cases.Research shows that chemotherapeutics 5-FU and monoclonal antibody drug Herceptin (Trastuzumab) and Cetuximab (Cetuximab) all can Induces Autophagy significantly, and suppress the cell autophagy produced by these 3 kinds of medicines significantly can increase the sensitivity of tumor cell to treatment, [1. Vazquez-Martin A, Oliveras-Ferraros C, Menendez JA.Autophagy Facilitates the Development of Breast Cancer Resistance to the Anti-HER2Monoclonal Antibody Trastuzumab.PLoS One.2009, 4 (7): e6251., 2. li j, hou N, faried A, tsutsumi s, et al.Inhibition of Autophagy by3-MA Enhances the Effect of5-FU-Induced Apoptosis in Colon Cancer Cells.Ann Surg Oncol.2009, 16 (3): 761-771,

Up to now, there is not yet the relevant report of people's recombinant arginase inducing tumor cell autophagy and cell autophagy inhibitor and people's recombinant arginase therapeutic alliance tumor.

Summary of the invention

The object of this invention is to provide a kind of medicinal composition for the treatment of tumor newly, be specifically related to a kind of autophagy inhibitor and arginase medicinal composition and uses thereof; This medicinal composition is used for the treatment of tumor, especially treats melanoma and hepatocarcinoma.This medicinal composition significantly can strengthen the anti-tumor activity of arginase in vitro; The immunogenicity of people's recombinant arginase can be reduced, extend the arginase half-life in vivo.

The present invention is based on the chemotherapeutics being mainly used in oncotherapy at present mostly can produce drug resistance and have stronger side effect; Wherein, prove widely although the antitumor action of arginase obtains, in its body, affinity is lower, and effective dose is comparatively large, there is the toxic and side effects that some are potential, therefore, the invention provides a kind of autophagy inhibitor and arginase medicinal composition; This medicinal composition can be strengthened arginase activities thus reduce its consumption and toxic and side effects; This medicinal composition makes compound medicine or compositions by using one or several autophagy inhibitors and arginase, carries out administering drug combinations or sequential administration, strengthens the antitumor curative effect of arginase.

The present invention is also based on cell autophagy and various diseases, and especially the development relationship of tumor is close, and manifest cell autophagy in research of the present invention has resistant function to people's recombinant arginase in the therapeutic process of arginase to melanoma and breast carcinoma; And use autophagy inhibitor can add strong man's recombinant arginase to melanoma, pulmonary carcinoma, cerebroma, breast carcinoma, lymphoma, leukemia and myeloma, the especially curative effect of melanin and breast carcinoma.

Specifically, autophagy inhibitor of the present invention and arginase medicinal composition, is characterized in that, is made up of one or more autophagy inhibitors and arginase;

In the present invention, cell autophagy inhibitor includes but not limited to: 3-MA (3-Methyladenine), wortmannin (wortmannin), LY294002, cycloheximide, Ba Faluo mycin Al (Bafilomycin Al), NH4Cl, chloroquine (Chloroquine) and hydroxychloroquine (hydroxychloroquine); In embodiments of the invention, preferred cell autophagy inhibitor is: 3-MA (3-Methyladenine), chloroquine (Chloroquine) and hydroxychloroquine (hydroxychloroquine).

Arginase described in the present invention comprises the arginase asked and be not limited to Mus source arginase, people's recombinant arginase and various long-actingization and modify, and includes but not limited to people's recombinant arginase that polyethyleneglycol modified people's recombinant arginase, polysialic acids are modified.

In the present invention, adopt the polysialic acids of activation to carry out chemical modification to arginase, in embodiments of the invention, carry out reaction by single aldehyde radical of the polysialic acids end activated the 7th and the amino of arginase and obtain the long-acting arginase of chemical modification.

In the present invention, described cell autophagy inhibitor and described arginase can be made into medicinal composition, compound medicine or sequential use, are used for the treatment of malignant tumor; Wherein, offset the antagonism to arginase treatment that tumor causes due to cell autophagy by suppressing the cell autophagy of arginase induction, thus significantly strengthen arginase to the therapeutic effect of tumor.

In the present invention, described tumor includes but not limited to melanoma, breast carcinoma, lymphoma, leukemia, hepatocarcinoma, pulmonary carcinoma and myeloma, preferred melanoma and breast carcinoma in embodiments of the invention;

Described arginase is by induction melanoma apoptosis and cell cycle arrest killing tumor cell; Described arginase is by inducing mammary cancer cell-apoptosis killing tumor cell.

Medicine of the present invention can be selected from following form further and carry out formula:

In medicinal composition of the present invention, be selected from following form further and prepare, comprising: tablet, solution, dispersant, micelle, Emulsion, liposome, Nano microsphere etc.

Invention has been cell experiment, experimental result shows, and when arginine desimidase (argininedeiminase) killing tumor cell, cell autophagy can protect tumor cell to avoid cell death, thus reduces the curative effect of medicine; People's recombinant arginase in described medicinal composition also significantly can induce melanoma cell and breast cancer cell generation cell autophagy, cell autophagy then can the antitumous effect of partial offset arginase, by using cell autophagy inhibitor T suppression cell autophagy can increase melanoma cell and breast cancer cell to the sensitivity of people's recombinant arginase, thus strengthen the curative effect of people's recombinant arginase

In the present invention, one or more autophagy inhibitors and people's recombinant arginase is adopted to make compound medicine, compositions administering drug combinations or sequential administration, be used for the treatment of malignant tumor, the lethal effect of people's recombinant arginase to tumor can be strengthened, strengthen the curative effect of people's recombinant arginase.

Outstanding beneficial effect of the present invention is: solve the activity of maintenance arginase while the Half-life in vivo extending arginase, reduce its immunogenic key technical problem, especially provide the arginase that polysialic acids is modified, the arginase that this polysialic acids is modified to be deposited at sodium cyanoborohydride by the polysialic acids activated and is reacted into conjugate with arginase in case; The present invention's autophagy inhibitor of the present invention and arginase medicinal composition can strengthen the lethal effect of arginase to tumor, strengthen the curative effect of arginase, especially treat the curative effect of melanoma and breast carcinoma.

Accompanying drawing explanation

Figure 1A, B: show cell subclones figure under A375 cell transmission electron microscope;

Fig. 1 C, D show A375 cell cell subclones figure after different time or the process of variable concentrations people recombinant arginase.

Fig. 2 A, 2B, 2C show, and arginase process 72h inhibits the propagation of A375, MDA-MB-231, MCF-7 cell significantly.

Fig. 3 A, B are the electrophoretograms of cell A375 and MDA-MB-231.

Fig. 4 shows: through the expression of the LC3II of the A375 cell of remarkable recombinant arginase process higher than matched group.

Fig. 5 A, B, C, D show: suppress autophagy to strengthen the reduction of the mitochondrial membrane potential that recombinant arginase causes in A375 cell.

Fig. 6 shows recombinant arginase induction A375 cell and produces active oxygen.

Fig. 7 is the gel electrophoresis analysis figure of the recombinant human arginase molecular weight through polysialic acids modification.

Detailed description of the invention

Embodiment 1: the preparation of people's recombinant arginase.

Adopt the method for PCR to obtain the coded sequence of encoding human arginase, this gene coded sequence total length 969bp, with restriction enzyme site XhoI and EcoRI; With XhoI and EcoRI restriction endonuclease double digestion PCR primer and empty plasmid vector respectively, ammonia benzyl resistant panel is transformed after being connected with carrier by genes of interest, several positive transformants of picking, get one of them and increase and extract plasmid, carry out the qualification of XhoI and EcoRI enzymes double zyme cutting; Extract recombiant plasmid, recombinant vector BglII enzyme action is made linearisation, electroporated Pichia sp.; Picking positive transformant; By RDB, MM and MD plate screening, obtain the clone that phenotype is his+muts, in shaking table level, carry out abduction delivering further, detected the expression of arginase in shaking table level by SDS-PAGE protein electrophoresis, thus the recon of screening secreting, expressing arginase; From some strains with the recon screening secreting, expressing arginase the recombination yeast of arginase gene; In Sample Purification on Single, after yeast is centrifugal, get supernatant.Sample, through ultrafiltration and concentration, adopts gel exclusion chromatography to refine; Select Sephacryl S-200 as the refining chromatographic column of sample;

Polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC is adopted to carry out purity detecting, result shows, obtain recombinant arginase purity be about 95%, people's recombinant arginase of acquisition is stored in 4 degrees Celsius of refrigerators, test before use with cell culture medium to working concentration.

Embodiment 2: the formula of autophagy inhibitor medicine

(1) preparation of chloroquine: get appropriate chloroquine and be dissolved in the storage liquid that pure water is made into 10mmol/L, be stored in 4 DEG C after degerming with the frit of 0.1 μm, experiment in vitro PRMI-1640 culture medium dilutes 500-1000 doubly for T suppression cell autophagy;

(2) preparation of ammonium chloride: get the water-soluble storage liquid being made into 0.4mol/L of appropriate ammonium chloride, be stored in 4 DEG C after degerming with the frit of 0.1 μm.50-80 is diluted doubly for T suppression cell autophagy during experiment in vitro;

(3) preparation of hydroxychloroquine: get appropriate hydroxychloroquine and be dissolved in the storage liquid that pure water is made into 10mmol/L, be stored in 4 DEG C after degerming with the frit of 0.1 μm, experiment in vitro room dilution 500-1000 is doubly for T suppression cell autophagy;

(4) preparation of 3-MA: get the storage liquid that appropriate 3-MA dry composition becomes 0.2mol/L, be stored in-20 DEG C after degerming with the frit of 0.1 μm.50-200 is diluted doubly for T suppression cell autophagy during experiment in vitro;

(5) preparation of LY294002: get the storage liquid that appropriate LY294002 dry composition becomes 0.2mol/L, be stored in-20 DEG C after degerming with the frit of 0.1 μm.50-100 is diluted doubly for T suppression cell autophagy during experiment in vitro;

(6) preparation of Bava Lip river mycin Al: get the storage liquid that appropriate Bava Lip river mycin Al dry composition becomes 0.5 μ g/ml, be stored in-20 DEG C after degerming with the frit of 0.1 μm, dilute 1000 times during experiment in vitro for T suppression cell autophagy;

(7) autophagy inhibitor and arginase combination drug are used in body or extracorporeal anti-tumor, the working concentration scope of autophagy inhibitor 3-MA is 0-1mol/L, the working concentration scope of autophagy inhibitor CQ is 0-50mol/L, the working concentration scope of hydroxychloroquine is 0-50mol/L, the working concentration scope of LY294002 is 0-1mol/L, the working concentration scope of Bava Lip river mycin Al is 0-10mol/L, and the working concentration scope of arginase is 0-100IU/ml.

Embodiment 3: people's recombinant arginase can induce melanoma A375 and breast carcinoma MDA-MB-231 and MCF-7 that cell autophagy occurs

After the arginase process 24h of A375 cell 3.2IU/ml, carry out paraffin embedding, section, dyeing, observation of cell ultrastructure under transmission electron microscope, result as shown in Figure 1A, have a large amount of typical double membrane structure autophagosomes in administration group cell, matched group does not then find; EGFP-LC3 plasmid is proceeded in melanoma A375, use the arginase process 24h of 3.2IU/ml again, see green fluorescence speckle subsequently under laser confocal microscope, result such as Figure 1B shows, have a large amount of fluorescence speckles in administration group cell, and matched group is less;

The melanoma A375 cell PBS collected is washed 1 time, with RIPA test kit cell lysis, and quantitatively after carry out protein electrophoresis according to each swimming lane 20 μ g after transferring film on pvdf membrane, close 1h with 5% skim milk, add corresponding antibodies, hatch 12h in 4 DEG C.TBST adds two anti-incubated at room 1.5h after washing film, develop the color with ECL nitrite ion, A375 cell is after different time or the process of variable concentrations people recombinant arginase, by the detection of Western Blot, result as shown in Figure 1 C and 1D, compared with matched group, the expression giving the LC3II of the A375 cell of people's recombinant arginase group strengthens.

Embodiment 4: people's recombinant arginase can suppress the propagation of A375 cell and breast cancer cell

By A375, MDA-MB-231, MCF-7 cell kind of debita spissitudo in 96 well culture plates, add the arginase of variable concentrations, system final volume is 100 μ L, cultivate 72h, add 10 μMs of TT liquid to every hole, in incubator, place 4h, dissolve with containing DMSO afterwards, measure optical density value (O.D.) under 570nm, result is as Fig. 2 A, 2B, 2C display, and arginase process 72h inhibits the propagation of cell significantly.

Embodiment 5: after T suppression cell autophagy, in cell, the expression of associated protein detects

Total protein of cell is extracted with the cracking of RIPA lysate, protein electrophoresis is carried out by every hole 20 μ g protein content loading and transferring film carries out Western blot quantitatively, chemiluminescence is carried out with ECL chemical luminescence reagent kit, result is as shown in Figure 4: through the expression of the LC3II of the A375 cell of 3.2IU/ml people's recombinant arginase process higher than matched group, the expression of the LC3II of the A375 cell after the process of 20 μMs of CQ and 3.2IU/ml people recombinant arginase is higher than the A375 cell after 3.2IU/ml people's recombinant arginase individual processing.And the expression of the LC3II of MDA-MB-231 cell after the 3-MA process of 3.2IU/ml people's recombinant arginase and 2mM is lower than the MDA-MB-231 cell after the process of 3.2IU/ml people's recombinant arginase, because 3-MA is a kind of PI3KIII inhibitor, PI3KIII plays an important role in the regulation and control of cell autophagy, and it can be combined with Beciln l equimolecular, participates in the transport of regulation and control autophagosome film, therefore suppress PI3KIII to check the formation of autophagosome, thus play the effect of T suppression cell autophagy, from phenomenon, because the formation of autophagosome film and transport are obstructed, cause ATG8/LC3 molecule cannot be located on autophagosome film by ubiquitination, so therefore the expression of LC3II molecule can decline, the result of Western Blot proves the autophagy that the 3-MA of 2mM can suppress the MDA-MB-231 cell of being induced by 3.2IU/ml people's recombinant arginase to bring out, and the CQ lysosomal inhibitor that is autophagy, CQ can cause lysosomal alkalization, lysosomal enzyme is lost activity, thus cause the accumulation of autophagosome, which results in autophagosome to degrade in lysosome, the amount that result in the LC3II molecule being in autophagosome film surface increases, the result of Western Blot proves that the CQ of 20 μMs can suppress the cell autophagy of the A375 cell of being induced by 3.2IU/ml people's recombinant arginase.

Embodiment 6: suppress autophagy can strengthen the A375 apoptosis of people's recombinant arginase induction

A375 cell through remarkable recombinant arginase and inhibitor process 72 hours through FCM analysis, the A375 cell PBS collected is washed 1 time, add 500 μ l afterwards in conjunction with liquid and 5 μ l Anexin V-FITC, lucifuge incubated at room 15min, carry out FCM analysis immediately, result as shown in Figure 4, through the apoptosis rate of the A375 cell of 3.2IU/ml people's recombinant arginase process 72h higher than matched group, and the apoptosis rate of the A375 cell of the CQ Combination intervention of 3.2IU/ml people's recombinant arginase and 20 μMs is significantly higher than the A375 cell of 3.2IU/ml people's recombinant arginase individual processing.

Embodiment 7: suppress autophagy to strengthen the reduction of the mitochondrial membrane potential that recombinant arginase causes in A375 cell

A375 cell processes 24h and 72h respectively through 3.2IU/ml people's recombinant arginase, collecting cell, dye through JC-1, carry out FCM analysis immediately, A375 cell after the process of 3.2IU/ml people's recombinant arginase is compared with matched group, mitochondrial membrane potential obviously reduces (Fig. 5 A), mitochondrial membrane potential inhibitor ciclosporin (CsA) weakens the cell autophagy (Fig. 5 B) that arginase is induced in A375 cell, strengthen arginase to the lethal effect (Fig. 5 C) of A375 cell, and 20 μMs of CQ strengthen the mitochondrial membrane potential that in A375,3.2IU/ml recombinant arginase causes and reduce (Fig. 5 D).

Embodiment 8: recombinant arginase induction A375 cell produces active oxygen

A375 cell is through 3.2IU/ml people's recombinant arginase process 72h, collecting cell, through DCFH-DA labelling, carry out FCM analysis immediately, A375 cell after the process of 3.2IU/ml people's recombinant arginase is compared with matched group, and the generation of active oxygen obviously increases (Fig. 6).

Embodiment 9: the activation of polysialic acids

(1) polysialic acids is pressed 10mg/ml polysialic acids to mix with freshly prepared 100mM sodium metaperiodate, be placed in 20 DEG C of dark places and stir 5min;

(2) add sodium metaperiodate in the ethylene glycol of two volumes He excessive, be placed in 20 DEG C of dark places and continue to stir 30min;

(3) polysialic acids of oxidation is placed in 0.01% ammonium carbonate buffer (pH7.4) 4 DEG C dialysis 24h, bag filter should be able to retain the molecule of 3.5KDa;

(4) be that the dry Polyethylene Glycol of 8KDa covers anti-phase dialysis and concentrates polysialic acids solution with molecular weight;

(5) by dialysis solution lyophilizing, be placed in-40 DEG C and save backup.

Embodiment 10: polysialic acids modifies the reaction of recombinant arginase

(1) buffer system of people's recombinant arginase is replaced into pH7.4Tris-Hcl buffer;

(2) deposit in case at 4mg/ml sodium cyanoborohydride, the polysialic acids of activation mixes by 50: 1 with people's recombinant arginase mol ratio;

(3) reaction vessel is placed in the airtight stirring reaction 24h of environment of 35 DEG C;

(4) reaction terminates to use 70% ammonium sulfate precipitated protein afterwards, and 4 DEG C are stirred centrifugal collecting precipitation after 1h;

(5) by the phosphate buffer of precipitation pH7.410mM dissolve and in identical buffer 4 DEG C dialysis 24h;

The arginase getting the polysialic acids of activation and purification to react in molar ratio at 50: 1, and the consumption controlling sodium cyanoborohydride is 4mg/ml, and protein concentration is 1mg/ml, and reaction system is 5ml, 35 DEG C of airtight stirring reaction 48h; To the 24h that dialyses in reacted product and 10mM PBS pH7.4 solution, concentration response product also carries out SDS-PAGE gel electrophoresis analysis, experimental result is as shown in Figure 7: compared with unreacted recombinant human arginase, the recombinant human arginase molecular weight modified through polysialic acids obviously increases, the molecular weight of the polysialic acids of one unit is 30KDa, the molecular weight of reaction afterproduct is about 65KDa, with expection molecular size range consistent (Fig. 7).

Claims (10)

1. cell autophagy inhibitor and an arginase medicinal composition, is characterized in that, is made up of one or more autophagy inhibitors and arginase;

Described cell autophagy inhibitor is selected from 3-MA, wortmannin, LY294002, cycloheximide, Ba Faluo mycin A1, NH4C1, chloroquine or hydroxychloroquine;

Described arginase is selected from the arginase that Mus source arginase, people's recombinant arginase or long-actingization are modified.

2., by cell autophagy inhibitor according to claim 1 and arginase medicinal composition, it is characterized in that, described cell autophagy inhibitor be selected from 3-MA, chloroquine and hydroxychloroquine one or more.

3. by cell autophagy inhibitor according to claim 1 and arginase medicinal composition, it is characterized in that, described arginase is selected from people's recombinant arginase of polyethyleneglycol modified people's recombinant arginase or polysialic acids modification.

4. by cell autophagy inhibitor according to claim 1 and arginase medicinal composition, it is characterized in that, the arginase that described long-actingization is modified adopts single aldehyde radical of polysialic acids end the 7th of activation and the amino of arginase to carry out reaction to obtain the long-acting arginase of chemical modification.

5. by cell autophagy inhibitor according to claim 1 and arginase medicinal composition, it is characterized in that, described cell autophagy inhibitor and arginase make the compound medicine of following dosage form: tablet, solution, dispersant, micelle, Emulsion, liposome or Nano microsphere.

6., by cell autophagy inhibitor according to claim 1 and arginase medicinal composition, it is characterized in that, in described medicine, cell autophagy inhibitor and the sequential use of arginase.

7. cell autophagy inhibitor according to claim 1 and arginase medicinal composition are preparing the purposes in tumor.

8., by purposes according to claim 7, it is characterized in that, described tumor is selected from melanoma, breast carcinoma, lymphoma, leukemia, hepatocarcinoma, pulmonary carcinoma and myeloma.

9., by purposes according to claim 7, it is characterized in that, described tumor is selected from melanoma or breast carcinoma.

10. by purposes according to claim 7, it is characterized in that, described cell autophagy inhibitor suppresses the cell autophagy of arginase induction, offsets the antagonism to arginase treatment that tumor causes due to cell autophagy.