Chemico-Biological Interactions 159 (2006) 90–103
The role of a novel copper complex in overcoming doxorubicin
resistance in Ehrlich ascites carcinoma cells in vivo
S. Majumder, P. Dutta, A. Mookerjee, S.K. Choudhuri ∗
Department of Environmental Carcinogenesis and Toxicology, Chittaranjan National Cancer Institute,
37, S.P. Mukherjee Road, Calcutta 700026, India
Received 27 August 2005; received in revised form 1 October 2005; accepted 4 October 2005
Available online 10 November 2005
Abstract
One of the important pathways of resistance to anthracyclines is governed by elevated levels of glutathione (GSH) in cancer
cells. Resistant cells having elevated levels of GSH show higher expression of multidrug-resistant protein (MRP); the activity of
glutathione S-transferases (GSTs) group of enzymes have also been found to be higher in some drug-resistant cells. The general mechanism in this type of resistance seems to be the formation of conjugates enzymatically by GSTs, and subsequent efflux
by active transport through MRP (MRP1–MRP9). MRPs act as drug efflux pump and can also co-transport drugs like doxorubicin (Dox) with GSH. Depletion of GSH in resistant neoplastic cells may possibly sensitize such cells, and thus overcome
multidrug resistance (MDR). A number of resistance modifying agents (RMA) like dl-buthionine (S, R) sulfoxamine (BSO) and
ethacrynic acid (EA) moderately modulate resistance by acting as a GSH-depleting agent. As most of the GSH-depleting agents
have dose-related toxicity, development of non-toxic GSH-depleting agent has immense importance in overcoming MDR. The
present study describes the resistance reversal potentiality of novel copper complex, viz., copper N-(2-hydroxy acetophenone)
glycinate (CuNG) developed by us in Dox-resistant Ehrlich ascites carcinoma (EAC/Dox) cells. CuNG depletes GSH in resistant (EAC/Dox) cells possibly by forming conjugate with it. Depletion of GSH results in higher Dox accumulation that may
lead to enhanced rate of apoptosis in EAC/Dox cells. In vivo studies with male Swiss albino mice bearing ascitic growth of
EAC/Dox showed tremendous increase in life span (treated/control, T/C = 453%) for the treated group with apparent regression
of tumor. Resistance to Dox in EAC/Dox cells is associated with over expression of GST-P1, GST-M1 (enzymes involved in
phase II detoxification) and MRP1 (a transmembrane ATPase efflux pump for monoglutathionyl conjugates of xenobiotics). CuNG
causes down regulation of all these three proteins in EAC/Dox cells. The effect of CuNG as RMA is better than BSO in many
aspects.
© 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Copper N-(2-hydroxy acetophenone) glycinate; Doxorubicin; Drug resistance; Resistance modifying agent; Glutathione
Abbreviations: ABC, ATP binding cassette; BSO, dl-buthionine (S,R) sulfoxamine; CuNG, copper N-(2-hydroxy acetophenone) glycinate;
Dox, doxorubicin; EAC, Ehrlich ascites carcinoma; MDR, multiple drug resistance; MRP, multidrug resistance protein
∗ Corresponding author. Tel.: +91 33 2476 5101/2/4x317; fax: +91 33 2475 7606.
E-mail addresses: majumder surajit@yahoo.co.in (S. Majumder), pranab76in@yahoo.co.uk (P. Dutta), anandamookerjee@yahoo.com
(A. Mookerjee), soumitra01@vsnl.net, soumitra01@yahoo.com (S.K. Choudhuri).
0009-2797/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2005.10.044
S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103
91
1. Introduction
The phenomenon of drug resistance is a major obstacle to successful application of cancer chemotherapy.
Drug resistance may be acquired or innate. In both
forms of resistance, neoplastic cells become refractory
to multiple drugs, different structurally and functionally. Multiple drug resistance (MDR) is a basic problem in cancer biology and sometimes a number of
mechanisms operate in a single drug-resistant system
[1]. In some systems of MDR, ATP-binding cassette
(ABC) membrane transporter proteins extrude a wide
range of drugs used in modern chemotherapy. Multidrug resistance protein (MRP), a plasma membrane
protein of 190 kDa ABC efflux pump is found to be over
expressed in numerous drug-resistant cell lines [2]. MRP
comprises a family of nine proteins (MRP1–MRP9)
[3,4] and is expressed in different tissues at low levels [5,6]. In normal physiology, MRPs play an important role in various secretory and transport process [7].
MRPs are involved in cellular detoxification process
together with the glutathione S-transferase (GST) group
of enzymes and reduced glutathione (GSH) the two
key members of the phase II detoxification machinery [8]. GSTs catalyze the process of conjugation of
many amphipathic drugs with GSH that renders them
into high affinity substrates for MRP. These monoglutathionyl conjugates are then transported out of the cell
in an ATP-dependant manner [9,10]. Most of the drugresistant cells that over express MRP and/or GST maintain an elevated intracellular pool of GSH and thereby
reduce the effective intracellular concentration of the
chemotherapeutic drug, leading to decreased cytotoxicity [11–13].
To overcome MRP/GST/GSH-mediated drug resistance, this molecular cascade needs to be disrupted. One
of the approaches to this end is to utilize compounds
that deplete cellular GSH levels leading to impaired drug
efflux machinery with concomitant rise in the sensitivity
of drug-resistant cells to antineoplastic agents [14,15].
Compounds that inhibit GST can also overcome MDR
as conjugation of xenobiotics with GSH is affected in the
process. Quite a few agents like dl-buthionine (S,R) sulfoxamine (BSO), ethacrynic acid (EA), sulphosalazine,
N-ethylmaleimide utilize either of the two routes [16].
However, the major drawback with these resistancemodifying agents (RMA) is that the doses required to
achieve a positive response in patients is fraught with
toxic side effects [17]. Under this perspective, we aimed
at developing novel RMA capable of overcoming MDR
in vivo. The manuscript describes the activity of a novel
RMA, viz., copper N-(2-hydroxy acetophenone) glyci-
Fig. 1. Structure of CuNG.
nate (CuNG) [18,19] to overcome MDR in vivo in ascitic
tumor model.
CuNG is copper coordinated Schiff’s base complex
(Fig. 1) and we had earlier reported the synthesis and
characterization of it [18,19]. It has low toxicity and
depletes GSH in time-dependent manner. This observation prompted us to investigate its potentiality as RMA
where drug resistance is due to elevated level of GST
and/or GSH. The present report describes that CuNG
indeed conforms to our hypothesis, as it reverses Dox
resistance in Ehrlich ascites carcinoma in vivo. The
resistance modifying properties of CuNG as RMA are
compared with respect to BSO, well known for its resistance reversal capability.
2. Materials and methods
2.1. Materials
dl-Buthionine (S,R) sulfoxamine, 1-chloro 2,4dinitro benzene (CDNB), dimethyl sulphoxide (DMSO),
doxorubicin hydrochloride (Dox), 5,5′ -dithio bis (2nitrobenzoic acid) (DTNB), May-Grunwald-Giemsa
(MGG), polyvinylidene difluoride (PVDF) membrane
were purchased from Sigma Chemical Company (St.
Louis, MO, USA). Nitroblue tetrazolium chloride (NBT)
and 5-bromo, 4-chloro 3-indolylphosphate (BCIP) were
purchased from SRL, India. Trichloro acetic acid (TCA)
and DPX mountant were from Merck, India. FITC conjugated active Caspase-3 antibody were from BD Phermingen (CA, USA). Polyclonal Rabbit Anti-Rat GST-P1
antibody purchased from Biotrend (Alpha Diagnostic
International, Germany). MRP1 (Goat polyclonal IgG,
N-19, sc-7774) and Rabbit Anti-Goat IgG-AP (sc-2771)
were from Santa Cruz Biotechnology (Europe). Rabbit
Anti-Goat IgG-FITC conjugate, Goat Anti-Rabbit IgGAP, Goat Anti-Rabbit IgG-FITC and Protein Molecular
Weight Marker (3–43 and 29–200 kDa) were from Bangalore Genei (Bangalore, India). Other chemicals used
were of highest purity available.
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S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103
2.2. Synthesis of the ligand
The ligand, potassium N-(2-hydroxy acetophenone)
glycinate was prepared according to the reported method
[20,21]. In brief, a cold aqueous solution of KOH (1.03 g
in 12 ml) was mixed with cold aqueous solution of
glycine (1.38 g in 12 ml) and held at 15–20 ◦ C in an ice
bath with continuous stirring. An ethanolic solution of 2(hydroxyl) acetophenone (2.5 g in 25 ml) was added drop
wise. Deep yellow color was developed and stirring was
continued for 1 h followed by 5 h at room temperature.
Rotary evaporator removed the solvent. The yellow mass
was washed with petroleum ether and precipitated with
methanol-diethyl ether mixture. The crude product was
recrystallized from methanol to yield potassium N-(2hydroxy acetophenone) glycinate. Yield 75%, melting
point 258–260 ◦ C.
2.3. Synthesis of CuNG
CuNG was synthesized by the reaction of potassium
N-(2-hydroxy acetophenone) glycinate and copper sulphate according to the reported method [18]. In brief,
0.68 g CuSO4 ·5H2 O was dissolved in 5 ml deionized
water. 0.785 g potassium N-(2-hydroxy acetophenone)
glycinate was dissolved in 25 ml ethanol. The solution of potassium N-(2-hydroxy acetophenone) glycinate
(yellow color) was slowly added to CuSO4 solution
(blue color) at room temperature with continuous stirring by magnetic stirrer for 1 h at 45–50 ◦ C. The color
of the mixture changed to deep green. The mixture was
cooled at room temperature and the green precipitate was
separated by filtration. The compound was dried and
recrystallized from DMSO. Yield 40%, melting point
242 ◦ C. Anal. Calc. C10 H15 O6 NCu: C, 39.0; H, 4.87; N,
4.54; Cu. 20.45. Found: C, 40.57; H, 5.04; N, 4.65; Cu,
21.24%.
2.4. Synthesis of CuNG–GSH conjugate
CuNG–GSH conjugate was synthesized by the reaction of CuNG and GSH [19]. In brief, 2 mg CuNG and
2 mg GSH were dissolved in 2 ml DMSO and 2 ml distilled water, respectively. The clear solution of CuNG
was slowly added to the solution of GSH. Gray colored
precipitate was formed. The mixture was made up to
the volume of 10 ml and rotated in a magnetic stirrer for
30 min and the mixture cooled to 4 ◦ C; the precipitate filtered, dried and recrystallized from water–alcohol, yield
80%, melting point 160 ◦ C (decomposed). Anal. Calc.
C20 H31 N4 O12 SCu: C, 29.15; H, 4.8; N, 9.3. Found: C,
28.82; H, 4.75; N, 8.99%. UV (water) λmax 226, 357,
600; IR (film) 4378, 4281, 4243, 4170, 4134, 4044 (sh),
3986 (sh), 3903 (sh), 3835 (sh), 3805, 3751, 3611, 3547,
3497, 3443, 3392, 3315, 3241, 3180 (b), 2998 (s), 2932
(b), 2803, 2039 (s), 1729 (sh), 1630 (sh, b), 1531 (b),
1410 (sh), 1229 (b), 1131 (s), 1096 (b), 1022 (b) cm−1 ;
1 H NMR (300 MHz, D O) δ 7.87–7.68 (m, 1H), 7.5
2
(s, 1H), 6.98–6.89 (m, 2H), 4.03 (s, 2H), 3.91 (s, 3H),
3.49 (s, 2H), 3.28–3.04 (m, 5H), 2.91–2.85 (m, 2H),
2.62–2.58 (d, 7H), 2.44 (s, 3H), 2.09 (s, 2H). LC–MS
calc. 613.5, found 613.3.
2.5. LC–MS studies
CuNG was dissolved in DMSO. GSH and
CuNG–GSH were dissolved in water. Two hundred
micromolar of each was employed for liquid chromatography–mass spectroscopy (LC–MS) analysis. LC–MS
analysis was performed on Shimadzu LC10 ADVP
series HPLC and Applied Biosystems Q trap system
with a Turbo ion spray as a source. Samples were
injected (20 l) into LC–MS system through LC10
ADVP series auto sampler. Separations were carried
out on 5 m YMC pro C18 column (4.6 mm × 50 mm)
at 35 ◦ C with a flow rate of 1 ml/min using HPLC
method. Aqueous samples were diluted in methanol,
water or mixture of both the solvents and analyzed by
flow injection analysis method in both positive and
negative mode techniques (Q1 multiple ion scan method
covering mass range of 50–800 Da) using acetonitrile
and water as mobile phase.
2.6. Animals
Adult male Swiss albino mice weighing 18–20 g
were obtained from our animal-breeding colony. Animals were kept for a quarantine period of 1 week at a
temperature of 25 ± 2 ◦ C, relative humidity of 55 ± 2%
and with photo cycle of 12-h light/12-h dark. Water and
food pellets were provided ad libitum.
2.7. Effect of RMA singly and in combination with
Dox on hematological parameters
CuNG (at 8 and 10 mg/kg) and BSO (at 20 mg/kg)
were injected singly and in combination with Dox (at
2 mg/kg) to male Swiss albino mice. Blood was collected
from untreated as well as from treated mice 10 days
(10D) after treatment. Blood was obtained via closed
cardiac puncture with the help of a 22-guage hypodermic needle by subxiphoid approach. Blood from each
group (CuNG, BSO treated and untreated) was pooled
into separate glass tubes and treated with anticoagulant
S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103
(heparin). The average value of haemoglobin (Hb), total
count (T.C.) and differential count (D.C.) of three independent experiments were shown in Table 3.
2.8. Preparation of spleen cell suspension
Normal and drug (CuNG, BSO singly and in combination with Dox) treated male Swiss albino mice were
anaesthetized and 70% alcohol was sprayed on abdominal region. Spleen was removed aseptically and small
amount of PBS was injected to it. Spleen was rubbed
against the fine wire mesh of tissue grinder. The cell
suspension centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cells centrifuged in PBS
twice at room temperature for washing.
Cell viability was tested by trypan blue extrusion
method and cells were counted in a phase contrast microscope. The experiment was repeated thrice. The average
value of number of spleen cell of three independent
experiments is shown in Table 4.
2.9. Separation of bone marrow cells
Normal and drug (CuNG, BSO singly and in combination with Dox) treated mice were anaesthetized and
the femur bone was cut with the help of a vertebrate scissor. Bone marrow was flushed with 0.56% KCl solution
and centrifuged at 3000 rpm for 15 min at 37 ◦ C.
Drug treated and untreated cells were counted under
compound microscope. The experiment was repeated for
thrice. The average value of number of bone marrow cell
of three independent experiments is shown in Table 4.
2.10. Cell line, tumor implantation and
experimental protocol
EAC cell was maintained as an ascitic tumor in
male Swiss albino mice. A Dox-resistant subline
93
was developed by sequential transfer of Dox treated
EAC cells to the subsequent generation of host mice
with continuous Dox treatment [22,23]. Briefly, the
treatment regime consisted of 2 mg/kg/week Dox
intraperitoneally (i.p.). The daily treatment dose was
0.4 mg/kg for 5 days. Dox was started 24 h after
inoculation of 106 EAC cells i.p. into mice. The mean
survival time (MST in days) ± S.D. of untreated mice
bearing EAC cell was 19.4 ± 1.5 days (n = 20). The
MST ± S.D. of the host mice bearing EAC cell and
Dox was 33.4 ± 2.06 days (n = 22), whereas MST of
the 17th generation of host mice was came down to
24.8 ± 1.2 days (n = 20). After this degree of resistance
had been developed, the dose of Dox was increased
to 4 mg/kg/week (daily treatment dose was 0.8 mg/kg
for 5 days), which resulted in 36 ± 2.3 days MST
(n = 18). When this tumor subline was re-treated
with Dox after 17th passage the MSTs were sharply
decreased at every generation and came up to 19.2 ± 0.7
days (n = 25) at 25th generation. The animals with
drug-resistant cells (EAC/Dox) (25th generation mice)
had the survival (19.2 ± 0.7 days) close to that of mice
with drug-sensitive cells (EAC/S) (19.4 ± 1.5 days)
[24].
Eight sets of animals were taken for studies on animal
survival (Table 1). Each mouse was inoculated with
106 EAC/Dox cells i.p. Both CuNG (8, 10 mg/kg body
weight) and BSO (20 mg/kg body weight) were injected
i.p. 24 h later of EAC/Dox cell passage to appropriate
groups. Dox was injected i.p. after an hour of CuNG or
BSO treatment. Animals were checked daily for assessment of ascitic growth and body weights were measured.
Two weeks after the implantation of EAC cells, total
ascitic fluid (TAF) and packed cell volume (PCV) were
measured; the average value of three independent experiments of each set was presented in Table 6. Time of
death was recorded for calculation of mean survival time
(MST).
Table 1
Treatment schedule for studies on animal survival
Groupsa
Treatment
Group I (EAC/Dox)
Group II (EAC/Dox + Dox)
Group III (EAC/Dox + CuNG at 8)
Group IV (EAC/Dox + CuNG at 10)
Group V (EAC/Dox + BSO at 20)
Group VI (EAC/Dox + CuNG at 8 + Dox)
Group VII (EAC/Dox+CuNG at 10 + Dox)
Group VIII (EAC/Dox+BSO at 20 + Dox)
No treatment
Only Dox injected
Only CuNG injected at 8 mg/kg body weight
Only CuNG injected at 10 mg/kg body weight
Only BSO injected at 20 mg/kg body weight
Dox injected 1 h after CuNG injection at 8 mg/kg body weight
Dox injected 1 h after CuNG injection at 10 mg/kg body weight
Dox injected 1 h after BSO injection at 20 mg/kg body weight
a Each mouse was inoculated with 106 EAC/Dox cells intraperitoneally. Treatment was started 24 h after cell inoculation. For each of the groups,
Dox was administered at the dose of 2 mg/kg body weight.
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S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103
The MSTs were recorded following various drug
treatment protocols [23,25]. The statistical significance
of the survival data, i.e., survival of the drug treated
groups versus untreated groups was evaluated by Pvalues (Student’s t-test). Mouse survival times in different groups were also compared as treated/control (T/C)
ratio (percent), i.e., the ratio of the survival time (in days)
for treated mice to untreated control mice. As in standard
National Cancer Institute protocols for screening new
anticancer drugs, it was considered that the increase in
survival time corresponding to T/C ratios around 120%
to “marginal”, T/C ratios between 120 and 150% to be
“clear” and T/C ratios equal or superior to 150% to be
“marked”.
2.11. Treatment schedule for enzyme assay,
immunoblot and flow cytometry
For enzyme assay, immunoblot and flow cytometry,
ascitic growth in mice was allowed for a period of 10
days. Mice with similar ascitic growth were randomized into different groups and treated with RMAs and/or
Dox. RMAs used in the experiments are BSO and CuNG.
Whenever treatment schedule included both RMA and
Dox, Dox was administered 1 h after RMA injection.
At different time intervals, cells were collected from
the intraperitoneal cavity and were washed in phosphate
buffer saline (PBS). Cells were then processed accordingly as described in the following sections. CuNG at
a dose of 10 mg/kg body weight showed the highest
activity as RMA, and hence applied in the experiments
(Table 2).
2.12. In vitro assay for cell cytotoxicity
One-dimensional titrations were performed to
determine the IC50 value of individual drugs in drug
sensitive (EAC/S) and drug-resistant (EAC/Dox) cells
following the method of Choudhuri and Chatterjee
[23]. Briefly, ascitic fluid was extracted from the mice
within 10–15 days of inoculation of tumor cells. No
treatment was given during the last passage before
an in vitro experiment. Cells (2 × 104 ) were collected
and washed thrice in PBS, and then plated in each
well of flat bottomed 96-well plate with RPMI 1640
medium (Gibco BRL), containing HEPES (Sigma),
penicillin–streptomycin and 10% FCS (Gibco BRL).
Cells were incubated without drug for 24 h. EAC/Dox
cells were treated with 3.07 ng/ml of CuNG. After
an hour, different concentrations of Dox [effective
concentration (EC) 0.15–5.8 g/ml] were added. The
cells were then incubated for an additional 4 days.
Cells were then fixed with TCA (EC 16%), followed by
washing with double distilled water. Fixed cells were
stained with crystal violet. Cell-associated dye was then
solubilized with 10% acetic acid and the viable cell
number was assayed by measuring the concentration of
stain in the well with an ELISA reader at 570 nm.
2.13. Assessment of apoptotic cells by MGG
staining
Morphological assessment of the apoptotic cells was
performed using the May-Grunwald-Giemsa (MGG)
staining method [26]. Briefly, drug treated EAC/Dox
cells were collected in cold PBS. After washing, a cell
suspension (106 cells/ml PBS) was prepared and 30 l
of it was spread over grease free glass slides. The slides
were dried overnight and stained with MGG stain for
5 min followed by intense PBS washing. Then, the slides
were again stained with Giemsa for 20 min followed by
intense wash in deionized water. After air-drying the
slides were mounted with DPX. The morphology of cells
was examined under light microscope. Apoptotic cells
were identified on the basis of nuclear fragmentation
[27]. Apoptotic index (AI) was determined as the percentage of apoptotic cells from at least 400 counted cells.
2.14. Measurement of cellular glutathione
Table 2
Treatment schedule for evaluation of in vivo GST-P1, GST-M1 and
MRP1 modulatory activity of CuNG and BSO
Groupsa
Treatment
Group I (EAC/S)
Group II (EAC/Dox)
Group III (EAC/Dox + CuNG)
Group IV (EAC/Dox + BSO)
No treatment
Dox injected i.p. at 4 mg/kg
CuNG injected i.p. at 10 mg/kg
BSO injected i.p. at 20 mg/kg
a All animals received 106 EAC cells (EAC/S and EAC/Dox, respectively) and ascitic growth was allowed for a week before being subjected to experiments. Cells were aseptically collected after 24 h of
treatment from the intraperitoneal cavity of mice.
GSH was measured following the method of Sedlack and Lindsay [28] briefly, 106 cells homogenate in
0.1 ml PBS was mixed with 2.4 ml EDTA (0.02 M) and
kept on ice bath for 10 min. Then, 2 ml deionized water
and 0.5 ml 50% TCA were added. The mixture was
again kept on ice bath for 10–15 min and centrifuged
at 3000 rpm for 15 min at 4 ◦ C. Two milliliters of supernatant was mixed with 2 ml 0.4 M Tris buffer (pH 8.9).
Fifty microliters of DTNB (0.01 M) was added to the
mixture and vortexed. Cell lysate from each RMA and/or
Dox treated animal was used to prepare a corresponding
S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103
sample blank. The sample blank was prepared following the same protocol where the mixture was lacking
DTNB; only the solvent methanol was added to the
mixture. Within 2–3 min of addition of DTNB, optical
density (O.D.) was measured at 412 nm against reagent
blank. The reading obtained for sample blank was subtracted from that obtained for samples with DTNB.
Cell protein was measured according to Lowry et al.
[29].
2.15. Measurement of glutathione S-transferase
activity
GST activity was measured following the method
of Habig et al. [30]. Briefly, 106 cells homogenate in
0.1 ml PBS was mixed with 0.5 ml 0.2 M sodium phosphate buffer (pH 6.5), 50 l 20 mM GSH and 0.35 ml
deionized water. The mixture was kept at room temperature for few minutes. Then, 50 l CDNB was added and
the samples vortexed. Increase in absorbance at 340 nm
was measured for a period of 5 m with an interval of
30 s. GST activity was expressed as a rate of change in
absorbance/min/mg of protein. Total protein was measured as mentioned before.
2.16. Immunoblotting for GST-P1, GST-M1 and
MRP1
95
2.17. Determination of Dox penetration/retention in
drug-resistant (EAC/Dox) cells
Cells (106 ) were collected from treated animal at different time points in chilled PBS at dark and washed
thrice with cold PBS. Cell samples collected at different
time points were sent for data acquisition by using a FACSCalibur and analyzed by Cell quest software (Becton
Dickinson) for determination of Dox penetration [32].
The excitation and emission wavelengths were 495 and
590 nm, respectively.
2.18. Flow cytometry analysis of Caspase-3
activation in EAC/Dox cells
Cells (106 ) were collected from treated animal and
were washed twice with PBS. Cells were then incubated
with PFCS (10% FCS in PBS) at room temperature for
30 min. Cells were then labeled with FITC conjugated
anti active Caspase-3 and incubated at 4 ◦ C for 1 h at
dark. Cells were washed twice and resuspended in 0.5 ml
PFCS and sent for data acquisition by using a FACSCalibur and analyzed by Cell quest software (Becton
Dickinson).
3. Results
3.1. Effect of CuNG on hematological parameters
Cells (106 ) were collected from animals and were
lysed by adding 100 l 2× sample buffer (4% SDS, 4%
-marcaptoethanol, 20% glycerol, 0.01% bromophenol
blue, 2 M urea, 0.01 M Na-EDTA, 0.15 M Tris–HCl) to
106 cells [31]. Cells were sheared by pipetting up and
down for 3 min at room temperature and cell suspension
was boiled at 95 ◦ C for 15 min, followed by centrifugation at 13,000 rpm for 10 min. The supernatant was
then loaded onto 12% SDS-PAGE pre-cast gel (Biotech,
India). Proteins were wet-transferred to PVDF membrane (0.1 m pore size) at 20 mA for 14 h. Blocking was
done [3% BSA in Tris buffer saline, TBS] at 37 ◦ C for an
hour. Then, the membranes were washed with PBS followed by primary antibody reaction [Polyclonal Rabbit
Anti-Rat GST-P1, Polyclonal Rabbit Anti-Rat GST-M1
and Polyclonal Goat MRP1] separately in 1:500 dilutions for 3 h with continuous shaking. Membranes were
washed thrice for 10 min each in buffer (0.1% Tween-20
in TBS) followed by secondary antibody [Goat AntiRabbit IgG-AP, Rabbit Anti-Goat IgG-AP], respectively,
in 1:1000 dilutions for 1 h at the same temperature. Again
the membranes were washed thrice with Tween-TBS followed by washing in TBS. Reactive bands were detected
by NBT/BCIP substrate.
Hematological parameters showed that CuNG (at 8
and 10 mg/kg) singly and in combination with Dox had
no toxic effect, but BSO (at 20 mg/kg) slightly reduce the
level of Hb and RBC (Table 3). There was no change in
spleen and bone marrow cell count when CuNG (at 8 and
10 mg/kg), BSO (at 20 mg/kg) singly and in combination
with Dox was injected (Table 4).
3.2. Effect of CuNG on drug sensitivity
The sensitivity of the resistant EAC/Dox to various
chemotherapeutics is given in Table 5. The degree of
resistance developed was greatest for Dox, almost 39folds as compared to the sensitive parental cell line, i.e.,
EAC/S. Prior treatment with CuNG reversed drug resistance in vitro and enhances the sensitivity of EAC/Dox
cells towards Dox as indicated by the resistance index
(RI) value of 1.13 (Table 5).
3.3. Effect of CuNG on survival of animal
To determine whether CuNG elicits similar response
in vivo, male Swiss albino mice were inoculated with the
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S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103
Table 3
Effect of CuNG and BSO singly and in combination with Dox on hematological parameters of male Swiss albino mice
RMA (mg/kg)
Dox (mg/kg)
Hba (g/dl)
WBCa (×103 l−1 )
RBCa (×106 l−1 )
Lymphocytea (%)
Neutrophila (%)
–
CuNG 8
CuNG 10
BSO 20
CuNG 8
CuNG 10
BSO 20
–
–
–
–
2
2
2
11.89
11.92
11.28
09.57
10.73
10.92
09.12
2.93
2.84
2.79
2.39
2.36
2.26
2.01
5.62
5.67
5.52
4.98
5.21
5.11
4.37
59.23
59.26
58.64
51.53
57.23
56.46
50.45
38.07
41.07
40.10
36.07
40.07
39.19
36.45
±
±
±
±
±
±
±
0.35
0.96
0.13
0.49
0.31
0.52
0.46
±
±
±
±
±
±
±
0.12
0.21
0.13
0.46
0.26
0.45
0.35
±
±
±
±
±
±
±
0.21
0.08
0.11
0.56
0.09
0.36
0.42
±
±
±
±
±
±
±
0.83
2.83
2.13
1.06
2.83
1.35
2.95
±
±
±
±
±
±
±
0.33
0.83
0.72
0.39
0.24
0.64
0.85
The data are mean ± S.D. of three independent experiments. CuNG (8 and 10 mg/kg) singly or in combination with Dox (2 mg/kg) showed no
hematological toxicity when compared with untreated control (P > 0.05). But BSO (20 mg/kg) slightly deplete the Hb as well as reduces the RBC
level.
a 10D.
Table 4
Effect of CuNG and BSO singly and in combination with Dox on
spleen and bone marrow of male Swiss albino mice
RMA (mg/kg)
Dox (mg/kg)
Spleen cella
(×103 )
Bone marrow
cella (×103 )
–
CuNG 8
CuNG 10
BSO 20
CuNG 8
CuNG 10
BSO 20
–
–
–
–
2
2
2
4.73
4.70
4.61
3.95
4.36
4.32
4.13
1.07
1.03
0.97
0.88
0.94
0.98
0.82
±
±
±
±
±
±
±
0.82
0.62
0.31
0.45
0.25
0.28
0.35
±
±
±
±
±
±
±
0.06
0.14
0.08
0.09
0.09
0.02
0.11
The data are mean ± S.D. of three independent experiments. CuNG (8
and 10 mg/kg) and BSO (20 mg/kg) alone or in combination with Dox
(2 mg/kg) were non-toxic to spleen and bone marrow when compared
to untreated control (P > 0.05).
a 10D.
resistance subline and treated with 8 and 10 mg/kg body
weight of CuNG 1 h before Dox administration. This
treatment regimen was determined from the observation that CuNG (10 mg/kg) caused maximum depletion
of GSH at 1 h post treatment [23] (Fig. 2). We wanted
to evaluate its effect on EAC/Dox bearing mice with
10 mg/kg as the highest dose. The results presented
in Table 6 shows that CuNG is able to enhance the
survival significantly compared to untreated controls.
Treatment with CuNG only did not show any significant
change in the life span of the EAC/Dox bearing animals with respect to control. At a dose below 8 mg/kg
body weight, it showed no significant effect (data not
Table 5
Drug sensitivity of EAC/S and EAC/Dox cells
Drugs
Doxorubicin
CuNG
CuNGa + Doxorubicin
RIb
IC50 (g/ml)
EAC/S
EAC/Dox
0.15 ± 0.02
0.05 ± 0.001
0.15 ± 0.05
5.80 ± 0.39
0.06 ± 0.003
0.17 ± 0.04
38.67
–
1.13
Cells were grown in RPMI 1640 medium, containing HEPES,
penicillin–streptomycin and 10% FCS. Cells were incubated without
drug for 24 h and then different concentrations of Doxorubicin (EC
0.15–5.8 g/ml) were added.
a EAC/Dox cells were treated with 3.07 ng/ml CuNG, after an hour
Dox was added. The cells were then incubated for an additional 4 days.
The IC50 value was determined by plotting the logarithm of the drug
concentration against the number of dead cells. Standard deviations
(S.D.) were usually within 10% of the mean.
b Resistance index (RI), which is a measure of degree of resistance, was determined by dividing the IC50 value for the drug-resistant
EAC/Dox by the IC50 for the drug sensitive EAC/S cells.
Fig. 2. Intracellular levels of GSH at different time intervals in EAC/S
and EAC/Dox cells in vivo after different resistance modifying agents
(RMA, such as BSO and CuNG) treatment. The figure indicates that,
cellular glutathione depletion by CuNG (at 10 mg/kg body weight) is
far more drastic than that of BSO (at 20 mg/kg body weight) even at
half the treatment dose and within the same time interval.
97
S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103
Table 6
Effect of CuNG and BSO as RMA on survivality of EAC/Dox cell bearing mice
Groups (vide Table 1)
I
II
III
IV
V
VI
VII
VIII
RMA (mg/kg)
–
–
CuNG at 8
CuNG at 10
BSO at 20
CuNG at 8
CuNG at 10
BSO at 20
Dox (mg/kg)
–
2
–
–
–
2
2
2
EAC/Dox
MST (days)
T/C (%)
TAF (ml)
PCV (×107 ml−1 )
19.2
22.6
21.7
20.9
22.5
78.3
87.0
27.8
100
118
113
109
117
408
453
145
13.5
13.9
12.3
12.7
9.2
5.2
1.6
6.9
3.67
3.25
3.65
3.43
2.35
0.89
0.72
1.48
±
±
±
±
±
±
±
±
0.7
0.9
0.5
0.6
0.3
0.8
0.8
0.3
±
±
±
±
±
±
±
±
0.7
0.5
0.4
0.6
0.5
0.3
0.1
0.2
±
±
±
±
±
±
±
±
0.3
0.2
0.5
0.3
0.2
0.2
0.2
0.1
In EAC/Dox cell bearing mice, Dox resistance was reversed with CuNG treatment. CuNG works as resistance modifying agent (RMA). Like BSO,
CuNG also depletes cellular glutathione level of EAC/Dox cells 1 h before Dox administration, CuNG and BSO were injected i.p. The combined
effect of CuNG (at 10 mg/kg) and Dox (at 2 mg/kg) increased the mean survival time (MST) of EAC/Dox cells bearing mice from 19 to 87 days. T/C
value (expressed in percentage) was calculated by dividing the MST value for mice receiving treatment by MST value for the control group, which
comprised of EAC/Dox cells bearing mice receiving no treatment. The total ascitic fluid (TAF) as well as the packed cell volume (PCV) decreased
much more than EAC/Dox cells bearing mice. Values represent mean ± standard deviation (S.D.) for 20 mice in each group.
shown). Treatment with CuNG at the doses of 8 and
10 mg/kg body weight showed significant improvement
for EAC/Dox bearing animals. The T/C values for CuNG
treated groups were 408 and 453%, respectively. These
values were 4–4.5 times the T/C values observed for
control (i.e., only EAC/Dox cell bearing mice). The average volume of ascitic fluid collected from 8 mg/kg body
weight treated group was 5.2 ml and for 10 mg/kg body
weight treated group was 1.6 ml. The corresponding values for EAC/Dox bearing mice (no drug treatment) and
for mice receiving only Dox treatments were 13.5 and
13.9 ml, respectively. Hence, there was significant reduction in ascitic growth in treated groups. In this animal
model, prognostic value for CuNG treated mice emulates that of the group treated with BSO (at 20 mg/kg
body weight). BSO treated group showed a T/C value
of 145%, only 1.4 times the control. The values of total
ascitic fluid and packed cell volume commensurate well
with life span and are optimal for the dose of 10 mg/kg
body weight.
3.4. Effect of CuNG on cellular GSH level and GST
activity
The intracellular GSH concentration in resistant
EAC/Dox cells was nearly 13 times higher than that
of the parental sensitive EAC/S cells. CuNG depleted
GSH in EAC/Dox cells drastically after 1 h following treatment. GSH level was found to increase albeit
slowly thereafter (Fig. 2). After an hour following CuNG
injection, intracellular GSH concentration in resistant
EAC/Dox cells dropped down to two-times the concentration found in EAC/S; even after 3 h, level of GSH
in resistant cells was only 2.6 times the value obtained
in sensitive cells. On the other hand, BSO showed the
highest depletion of GSH in EAC/Dox cells after 3 h of
administration. Although, BSO treated EAC/Dox cells
showed 4.6 times the level of GSH in sensitive EAC/S
cells. The difference in the pattern of GSH depletion by
CuNG and BSO could possibly be attributed to different
modes of action. CuNG might form monoglutathionyl
conjugate by directly combining with GSH. This caused
depletion of intracellular GSH. BSO, on the contrary, is
an inhibitor of GSH biosynthesis. It seemed that, BSO
did not affect the already present intracellular pool of
GSH. This might be the cause that we found BSO to
deplete GSH in a steady manner whereas CuNG caused
a steep fall (Fig. 2).
The effect of CuNG on GST activity was also determined and the result is shown in Fig. 3. EAC/Dox cells
showed nearly 3.6 times higher level of GST activity
compared to EAC/S cells. CuNG treated EAC/Dox cells
had GST activity fallen to the level of EAC/S cells. The
lowest GST activity was obtained after the first hour of
treatment. Thereafter, a slight gain in GST activity was
observed in EAC/Dox cells. But, this was far less than the
high level of GST activity observed in EAC/Dox cells.
BSO treated EAC/Dox-resistant cells showed minimal
change in GST activity after an hour following the treatment, 3 h after BSO treatment GST activity dropped to
almost half the value found in untreated EAC/Dox.
3.5. Study of conjugation of CuNG with GSH
We were able to synthesize CuNG–GSH conjugate
in vitro as mentioned in Section 2. Mass spectral studies
98
S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103
Fig. 3. Level of GST activity in EAC/S and EAC/Dox cells at different
time intervals after RMA treatment in vivo. Like GSH depletion, CuNG
also inhibits the elevated GST activity of EAC/Dox cell and the degree
of inhibition is more pronounced than that of BSO at each time point.
revealed that the molecular weight of CuNG was 308
(observed 308, calculated 307) [18]. GSH showed a
peak at 307 and the conjugate had peak at 613.3 in the
positive mode of LC–MS spectra. Based on elemental
analysis, LC–MS (Fig. 4b) and other spectral data
like UV, IR, 1 H NMR the structure of the proposed
conjugate was drawn (Fig. 4a). LC–MS spectrum
of the conjugate is shown in Fig. 4b. Probably the
conjugation of CuNG to GSH took place by replacing
one H-atom of GSH and forming sulphur–nitrogen bond
(Fig. 4a).
Fig. 5. Effect of CuNG and BSO on Dox uptake by EAC/Dox
cells with EAC/S serving as control. Mice bearing 10 days of
ascitic growth were injected with the RMAs (either CuNG or BSO)
i.p. 2 h prior to Dox administration. After a period of 1 h, cells
were collected from the intraperitoneal cavity and drug uptake
was measured using flow cytometry. Values represent mean ± S.D.
(P < 0.05).
3.6. Effect of CuNG on Dox accumulation in vivo
Increased level of GSH helps in the efflux of Dox
from EAC/Dox bearing animals. This results in reduced
accumulation of the drug within the cells. Hence, depletion of GSH may enhance intracellular accumulation of
Dox by reducing its efflux. To explore the possibility,
EAC/Dox bearing mice were treated with CuNG before
Dox administration. Cells were collected from these
mice 1 h after Dox administration and were examined by
flow cytometry. CuNG was able to enhance Dox accumulation in EAC/Dox cells (Fig. 5). This increase in Dox
concentration resulted in almost equal accumulation to
that of the parental EAC/S cells. In the next 2 h, there was
slow decrease in the amount of intracellular Dox. The
retention of Dox after 3 h of administration within CuNG
treated group was much higher than that of untreated
EAC/Dox and slightly lower than the sensitive parental
cells. Effect of CuNG was also more pronounced than
that achieved by BSO, under similar experimental condition.
3.7. Effect of CuNG on caspase-3 activation
Fig. 4. (a) Structure of CuNG–GSH conjugate. (b) LC–MS spectrum
of CuNG–GSH conjugate.
Enhanced accumulation of Dox in EAC/Dox
with prior exposure to CuNG resulted in high
of caspase-3 activation (Fig. 6), when probed
anti-caspase-3 antibody. Normally, EAC/Dox
cells
level
with
cells
S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103
99
3.8. Effect of CuNG on apoptotic morphology
Fig. 6. Percentage of caspase-3 activation in EAC/Dox. EAC/Dox was
treated with different RMAs (either CuNG or BSO) 1 h before Dox
administration. Activation of caspase-3 in EAC/S after Dox treatment
was taken as control. The data were obtained using flow cytometry as described in Section 2. The figure implies that CuNG-induced
Dox uptake in EAC/Dox correlates well with Dox mediated caspase3 activation; an indicator of apoptosis induced by this anthracycline
antibiotic.
exposed only to Dox showed less degree of activated
caspase-3 compared to EAC/S cells. But EAC/Dox
cells treated with CuNG and Dox both showed nearly
double the level of caspase-3 activation with respect
to the untreated control. Activation of caspase-3 is one
of the important early events in Dox-induced apoptotic
cascade. We primarily studied this aspect to ensure that
reduced efflux of Dox in EAC/Dox cells translated well
into higher cytotoxicity. In some drug-resistant cell
lines, chemotherapeutics are sequestered into intracellular organelles rendering those less effective [33]. No
similar mechanism seemed to be functional in EAC/Dox
cells.
We examined the late apoptotic stage in EAC/Dox
by MGG staining technique as described in Section 2.
The apoptotic index (AI) was determined as the ratio
of number of apoptotic cells with total number of cells
counted and was expressed as percentage. The appearance of apoptotic morphology (Fig. 10) in EAC/Dox
cells treated with 4 mg/kg body weight of Dox was significantly less (AI: 17.83%) than that of EAC/S cells
treated with 2 mg/kg Dox (AI: 43.54%). Incidentally,
EAC/S cells were treated with half the dose used for its
resistant counterpart. When EAC/Dox cells were treated
with CuNG, the number of apoptotic cells was much
higher upon subsequent Dox treatment (AI: 39.59%).
This was comparable to the result obtained from EAC/S
cells, as doses of Dox were equal (i.e., 2 mg/kg of body
weight) in both cases. This observation indicated that
CuNG was able to induce reversal of Dox-resistant phenotype.
3.9. Immunoblot analysis of GST-P1, GST-M1 and
MRP1 expression
GST-P1 (Fig. 7) and GST-M1 (Fig. 8) are associated
with phase II detoxification system. In EAC/Dox cells,
these two classes of enzyme were over expressed. Also,
acquired resistance to Dox in EAC/Dox cells was found
to induce over expression of MRP1 (Fig. 9). MRP1, a
trans membrane efflux pump, belongs to the so-called
phase III detoxification system. The substrates for MRP1
include many GSH-conjugated xenobiotics. We examined whether CuNG could exert any effect upon the
expression of these three proteins. Twenty-four hours
Fig. 7. Immunoblot analysis of GST-P1 expression in resistant (EAC/Dox) cells compared to that of Dox sensitive (EAC/S) [control] cells and the
status of GST-P1 expression after 24 h of CuNG and BSO administration. The figure is representative of three similar experiments.
100
S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103
Fig. 8. Immunoblot analysis of GST-M1 expression in resistant (EAC/Dox) cells than in Dox sensitive (EAC/S) [control] cells and the status of
GST-M1 expression after 24 h of CuNG and BSO administration. The figure is representative of three similar experiments.
Fig. 9. Immunoblot analysis of MRP1 expression in Dox treated resistant (EAC/Dox) cells compared to that of Dox sensitive (EAC/S) [control]
cells and the effect of CuNG on the same after 24 h of administration. The figure is representative of three similar experiments.
after CuNG treatment expression of all three proteins
were down regulated in EAC/Dox cells compared to
the untreated control (i.e., only EAC/Dox cells). BSO
showed similar response though on a smaller scale. Interestingly, the levels of expression for each of the three
proteins, viz., MRP1, GST-P1 and GST-M1 in EAC/Dox
cells were lower than that of the sensitive parental cell
line only 24 h after CuNG exposure.
4. Discussion
Of the several molecular events leading to crossresistance to multiple drugs in neoplastic cells, higher
level of GSH is a very important one. Drug-resistant phenotypes with elevated GSH levels show a concomitant
rise in the expression of GSTs, viz., GST-P1, GST-M1,
etc. The detoxification of chemotherapeutics involves
GST-catalyzed monoglutathionyl conjugate formation;
MRP1 complements this phase II detoxification by
actively extruding the conjugate. Resistance to anthracyclines has been reported to be associated with the abovementioned mechanism [34]. In drug-resistant EAC/Dox,
we have found that both MRP1 and GSTs have been over
expressed along with the increase in the level of intracellular GSH compared to sensitive (EAC/S) cells. This
suggests that the resistance in EAC/Dox cells may occur
by active efflux of drugs through MRP1. The present
study reports that CuNG, a novel copper complex, can
reverse drug resistance of EAC/Dox cells grown as
ascitic growth in male Swiss albino mice.
Hematological parameters show that CuNG alone
(8 and 10 mg/kg) and in combination with Dox (i.e.,
CuNG + Dox) has no toxic effect but BSO (20 mg/kg)
causes slight reduction in the level of Hb and RBC
S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103
(Table 3). No significant change in spleen and bone marrow cell count has been observed with the application
of CuNG and BSO alone or in combination with Dox
(Table 4). Moreover, the body weight of treated mice
remains unchanged when compared with untreated
group (control) (data not shown).
CuNG is noted to be a strong GSH-depleting agent.
We have assumed that the metallic center would potentiate conjugation of CuNG with GSH. Previous work
from our laboratory had disclosed that CuNG was able to
deplete GSH in various organs when injected into normal
Swiss mice [23]. We have first evaluated CuNG for its
MDR modulation potency on drug-resistant cells where
resistance was due to elevated level of GSH. In vitro cytotoxicity assays (IC50 ) indicate the significant reversal of
Dox resistance in EAC/Dox cells (Table 5) by CuNG.
CuNG showed promising results in reversing Dox resistance in vivo. Mean survival times (MST) for EAC/Dox
bearing mice treated with CuNG before Dox administration have been noted to be significantly higher compared to control. CuNG at the range of 8–10 mg/kg body
weight causes tremendous increase in survival (Table 6).
101
The increase of life span has been found to be highest
(T/C = 453%) when CuNG (10 mg/kg body weight) is
applied along with Dox in EAC/Dox cell bearing mice.
The exact fate of Dox in phase II detoxification is
still unknown. Controversies still remain regarding the
formation of monoglutathionyl conjugate of Dox [35].
On the other hand, the efflux of Dox by MRP1 has
been reported by number of workers [36–39]. This active
efflux process does require the presence of GSH, if not
for conjugation but for co-transport [36–39].
It has been reported by a number of workers that
compounds which share the same transport route with
Dox is able to reverse Dox resistance in EAC in its
ascitic form of growth [40]. According to our hypothesis, CuNG belongs to this category of compounds
and have the potential to restrict the drug efflux traffic.
This may ultimately result in impaired efflux machinery
causing Dox treatment to be highly effective. In this
regard, our data clearly demonstrate that by depleting
GSH, CuNG is able to increase Dox accumulation and
retention within the first 3 h of exposure in EAC/Dox
cells. Increased accumulation proved to be effective as
Fig. 10. Apoptotic morphology (nuclear fragmentation) observed upon MGG staining. Apoptotic index (AI) percentage indicates the percentage of
apoptotic cells from at least 400 counted cells. Where as EAC/S cells, treated with Dox (at 2 mg/kg body weight) showed a maximum (43.54%) AI
value after 96 h of Dox treatment. EAC/Dox cells itself and with Dox (at 4 mg/kg) never showed impressive result (AI within 16–18%). But CuNG
(at 10 mg/kg) with Dox (at 2 mg/kg) was able to accelerate the AI up to ∼40% after 96 h of drug treatment.
102
S. Majumder et al. / Chemico-Biological Interactions 159 (2006) 90–103
it translated into greater cytotoxicity (Fig. 5). High level
of activated caspase-3 in drug-resistant cells (Fig. 6) as
well as the rise in the number of apoptotic cells found by
MGG staining (Fig. 10) confirmed greater cytotoxicity
for CuNG treated EAC/Dox cells. We have studied the
chemical reaction of CuNG and GSH and have observed
that CuNG forms conjugate with GSH at N.T.P. Elemental analysis and other spectral studies like UV, IR, 1 H
NMR and LC–MS (Fig. 4b) also support the formation
of CuNG–GSH conjugate. The steep fall in the amount
of GSH in CuNG treated EAC/Dox cells (Fig. 2)
indicates the formation of conjugate in vivo. In our
experimental condition, the maximum depletion of GSH
occurs after 1 h of CuNG treatment. Direct interaction
of CuNG with GSH perhaps results in the rapid fall in
GSH level.
To summarize, the immediate effect of CuNG is GSH
depletion that is highest at 1 h of treatment. Apart from
GSH depletion, CuNG administration has far reaching
consequences. It is able to alter the protein expression
status of the resistance subline. MRP1, GST-P1 and GSTM1 have all been down regulated after 24 h of CuNG
treatment. Interestingly, the expression levels of these
three proteins in EAC/Dox cells are lower than that of
EAC/S cells. Further work is necessary to decipher the
signaling cascade that CuNG possibly triggers.
Nevertheless, our work clearly shows that CuNG renders the Dox-resistant EAC/Dox cells susceptible to
doxorubicin. Moreover, CuNG shows better results in
every aspect as resistance modifier in comparison to
well-recognized GSH depletory drug like BSO. Another
very important property that CuNG exhibits is absence of
toxicity at the dose(s) required to reverse Dox resistance.
We may cautiously expect CuNG to be one significant
addition to our arsenal to fight drug resistance in cancer
and probably in other fields where resistance is related
with elevated level of glutathione.
Acknowledgements
This investigation received financial support from
Indian Council of Medical Research (ICMR), New
Delhi, no. 5/13/17/2001-NCD-III. We acknowledge the
help of Mr. Arup Sarkar, Senior Research Fellow,
Department of Pharmacology, Institute of Post Graduate
Medical Education & Research, Calcutta 20, for providing technical support.
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