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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2004 May 19;130(7):423–428. doi: 10.1007/s00432-004-0556-9

Resistance to chemotherapy-induced apoptosis via decreased caspase-3 activity and overexpression of antiapoptotic proteins in ovarian cancer

Xiaokui Yang 1, Fang Zheng 2, Hui Xing 1, Qinglei Gao 1, Wang Wei 1, Yunping Lu 1, Shixuan Wang 1, Jianfeng Zhou 1, Wei Hu 3, Ding Ma 1,✉
PMCID: PMC12161855  PMID: 15156398

Abstract

Goals

Resistance to cisplatin is the main reason for treatment failure in ovarian cancer. Apoptosis is the main mechanism of action of most cancer chemotherapeutic agents. The apoptosis-associated proteins expressed in cisplatin-sensitive (A2780, COC1) and -resistant (A2780/DDP, COC1/DDP) ovarian cancer cell lines, as well as their effects on caspase-3 activity in these cells, were studied by reverse transcriptase polymerase chain reaction and Western blot analysis.

Methods

The apoptotic ratios of A2780, COC1, A2780/DDP, and COC1/DDP cells after treatment with cisplatin were measured by flow cytometry.

Results

Expression of Bcl-2 and Bcl-XL in A2780/DDP and COC1/DDP cells was significantly higher than that in A2780 and COC1 cells, respectively. Expression of Bax and Bcl-Xs did not differ in cisplatin-resistant and -sensitive cells. Caspase-3 activity was reduced markedly and apoptotic ratios were significantly lower in A2780/DDP and COC1/DDP cells than in A2780 and COC1 cells after treatment with cisplatin.

Conclusion

We conclude that overexpression of antiapoptotic proteins Bcl-2 and Bcl-XL and down-regulation of caspase-3 activity may be associated with cisplatin resistance in human ovarian cancer.

Keywords: Ovarian neoplasm, Cisplatin (cis-diamine-dichloro-platinum), Chemoresistance, Apoptosis, Caspase-3, Bcl-2

Introduction

Resistance to chemotherapeutic drugs is a frequently encountered problem in the treatment of human cancer. Cisplatin is an important drug in the treatment of ovarian cancer, but one of the major limits on its effectiveness is the acquisition of multidrug resistance (MDR). One of the mechanisms of MDR is inhibition of apoptosis (NicAmhlaoibh et al. 1999). Recent studies have shown that apoptosis is an important indicator of a tumor’s sensitivity to an anticancer drug (Ferreira et al. 2000). The roles of genes, such as p53, p21Waf1, Bcl-2, Bax, Bcl-XL, and Bcl-Xs in apoptosis have been characterized. Alterations of these genes and in signal transduction pathways leading to apoptosis have been reported in drug-resistant cells (Ohi et al. 2000).

The Bcl-2 family of proteins includes several homologous proteins that may be either antiapoptosis or proapoptosis. Bcl-2, Bcl-XL, and BAG-1 play antiapoptotic roles whereas Bax and Bcl-Xs play proapoptotic roles. These proteins regulate the sensitivity of cells to apoptosis stimuli such as cytotoxic chemotherapy. Bcl-2, first recognized as the protooncogene translocated to the immunoglobulin heavy-chain locus in human B-cell lymphoma cells, contributes to neoplastic progression by inhibiting apoptosis (Tsujimoto 1986). Bcl-2 overexpression is also associated with resistance to several cytotoxic chemotherapies (Ferreira et al. 2000). The Mr 25,600 Bcl-XL protein has been shown in a number of cell lines to be a potent protector of cellular apoptosis induced by anticancer agents. Anticancer effects induced by various chemotherapeutic agents are considered to be based on induction of apoptosis and to be mediated through a final common pathway: activation of caspase-3 and subsequent DNA fragmentation (Saporito et al. 2002, Yang et al. 2001, Blanc et al. 2000, Sasaki et al. 2002). Previous studies have shown that overexpression of antiapoptotic genes can affect the activity of caspase-3 (Ferreira et al. 2000, Ohi et al. 2000, Tsujimoto et al. 1986, Saporito et al. 2002, Yang et al. 2001, Blanc et al. 2000, Sasaki et al. 2002, Ding et al. 2000), and caspase-3 is a key caspase in the signaling cascade of apoptosis as well as an effector caspase in apoptosis induced by chemotherapeutic agents (Saporito et al. 2002, Yang et al. 2001, Ding et al. 2000 ). Poly (ADP-ribose) polymerase (PARP) is a substrate cleaved by caspase-3; induction of apoptosis is associated with caspase-3-mediated cleavage PARP and other proteins.

In this study, we focused on the function of apoptosis-associated proteins and the activity of caspase-3 in cisplatin-sensitive and -resistant ovarian cancer cell lines. The results suggest that resistance to chemotherapy agents may be related to overexpression of antiapoptotic proteins and inhibition of caspase-3.

Materials and methods

Reagents

Cisplatin was obtained from the pharmaceutical manufacturer, QiLu (Shandong, China); RPMI 1640 medium, fetal bovine serum, and Lipofectamine 2000 were purchased from Life Technologies, Inc (Calsbad, CA, USA); rabbit polyclonal antihuman Bcl-2 antibody, rabbit polyclonal antihuman caspase-3 antibody, mouse monoclonal antihuman Bcl-XL antibody, mouse monoclonal antihuman Bax antibody, mouse monoclonal antihuman Bcl-Xs antibody, and mouse monoclonal antihuman PARP antibody were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA, USA); ApoAlert caspase-3 assay kit was purchased from Clontech Laboratories, Inc (Palo Alto, CA, USA).

Cell culture

Cisplatin-sensitive human ovarian epithelial carcinoma cell line A2780 was obtained from the American Type Culture Collection (ATCC); cisplatin-resistant cell line A2780/DDP was derived from its parental ovarian cancer cell line A2780 by applying stepwise increases in concentrations of cisplatin in our laboratory. The A2780/DDP cells were maintained in 30 μM of cisplatin for at least 6 months. The median inhibitory concentrations (IC50) of cisplatin for cisplatin-sensitive and -resistant ovarian cancer cells were determined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cisplatin-sensitive human ovarian adenocarcinoma cell line COC1 and cisplatin-resistant cell line COC1/DDP were obtained from Chinese Type Culture Collection.

The cells were cultured at 37°C in an atmosphere of 5% CO2 in RPMI 1640 medium with 10% fetal bovine serum (FBS), 50 IU/ml penicillin, 50 µg/ml streptomycin, and 0.3 µg/ml glutamine. For cisplatin concentration-response studies, cells were cultured for 24 h in the presence of different concentrations of cisplatin (0, 5, 10, 20 μM) with FBS-free medium harvested after being washed with phosphate-buffer saline (PBS) and cultured in 10% FBS medium (no cisplatin) for 12 h.

Cell-death measurement

Cells (2×106) treated with and without cisplatin at concentrations of 5, 10, or 20 μM for 24 h were fixed in ice-cold 70% ethanol and incubated overnight at −20°C. The analysis of apoptotic cells was performed by flow cytometry. Cells (2×106) were resuspended in PBS containing 10 µg/ml propidium iodide (PI) and 100 µg/ml RNase A, the percentage of apoptotic cells was determined on a FACScan flow cytometer, and the data were analyzed using Cell Fit software.

RNA isolation and reverse transcriptase polymerase chain reaction (PCR)

Total RNA was isolated from 1×107 cisplatin-sensitive or -resistant cells using TRIzol Reagent according to the manufacturer’s instructions. RNA (2 μg) was used for cDNA synthesis by reverse transcription. The RNA samples were incubated at 70°C for 5 min with 0.5 µg oligo deoxythymidine primers in a final volume of 10 μl and then at 37°C for 60 min in a 25 μl reaction volume containing 1.25 mM deoxynucleotide triphosphate, 200 U Muloney murine leukemia virus reverse transcriptase (RT), and Muloney murine leukemia virus RT buffer. A negative control included the same reaction system without Muloney murine leukemia virus RT. The cDNAs obtained were amplified by using specific primers (NicAmhlaoibh et al. 1999), as follows: Bcl-2 (306 bp), 5’-TCA TGT GTG TGG AGA GGG TCA A-3’ (sence) and 5’-CTA CTG CTT TAG TGA ACC TTT TGC-3’ (antisence); Bcl-XL (396 bp), 5’-CAA GCA GCA GTT TGG ATG C-3’ (sence) and 5’-CTC GGC TGC TGC ATT GTT C-3’ (antisence); Bcl-Xs (207 bp), 5’-CAC AGC AGC AGT TTG GAT GC-3’ (sence) and 5’-CTC GGC TGC TGC ATT GTT C-3’ (antisence); Bax (230 bp), 5’-GAC GAA CTG GAC AGT AAC ATG-3’ (sence) and 5’-AGG AAG TCC AAT GTC CAG CC-3’ (antisence); GADPH (142 bp), 5’-TGG ACA TCC GCA AAG ACC TGT AC-3’ (sence) and 5’-TCA GGA GGA GCA ATG ATC TTG A-3’ (antisence).

A typical PCR system consisted of 5 µl of cDNA or negative control sample, 0.2 mM deoxynucleotide triphosphate, 1.25 mM MgCl2, 2.5 U Taq polymerase, 1× buffer, and 10 μM primers. The PCR profile was 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min for 25 cycles, followed by extension for 8 min at 72°C. The PCR products were subjected to electrophoresis on 1.5% agarose gels that included 0.1 µg/ml ethidium bromide. The amounts of mRNA were semiquantitated by comparing relative intensities of the amplified GADPH to an equal amount of cDNA.

Protein extraction and Western blot analysis

Cisplatin-sensitive and -resistant cells were washed three times with cold PBS and then subjected to lysis in buffer containing 12.5 mM HEPES (pH 8.0), 200 mM KCl, 5 mM EDTA, 50 mM NaF, 0.5% NP-40, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 5 μg/ml leupeptin, 5 μg/ml Aprotinin, and 2 mM Na3VO4. Cell lyses were centrifuged at 21,000×g for 10 min, and supernatants were analyzed. Extracts were immediately stored at −80°C until use. Protein concentrations of extracts were determined using the Bio-Rad protein assay system with bovine serum albumin (BSA) as the standards. A total of 50 μg of protein from each cell-free extract was denatured in SDS sample buffer, heated to 100°C for 5 min, and loaded onto SDS-polyacrylamide gel. Electrophoresis was initially carried out at 100 V through the stacking gel and then at 150 V through the separation gel. After electrophoresis, the proteins were transferred to nitrocellulose membranes. The membranes were blocked for 2 h with a blocking buffer containing 5% nonfat dry milk and 0.1% v/v Tween 20 in Tris-buffered saline (TBST) at room temperature, incubated at 4°C with the relevant primary antibodies [anti-Bcl-2 antibody, anti-Bcl-XL antibody, anti-Bcl-Xs antibody, anti-Bax antibody, anti-caspase-3 antibody, or anti-PARP antibody (1:1000)] overnight, and washed three times (5 min each time) with TBST. Primary antibodies were detected using peroxide-conjugated secondary antibody (1:1000) incubated for 2 h at room temperature and visualized with enhanced chemiluminescence detection system. Protein loading equivalence was assessed by the expression of β-actin.

Colorimetric assay of caspase-3 activity

Colorimetric assay of proteolytic activity was carried out by using the substrates Asp-Glu-Val-Asp-p-nitroanilide (DEVD-pNA) to measure caspase-3 activity. Experiments were performed according to the manufacturer’s protocols. Colorimetric detection was finished in a spectrophotometer at 405 nm. To confirm that substrate cleavage was due to caspase-3 activity, we incubated extracts in the presence of caspase-3-specific Asp-Glu-Val-Asp-FMK (DEVD-fmk) for 30 min at 37°C before adding substrate.

Statistical analysis

All experiments were repeated at least three times. Student’s t test was used to evaluate the differences between experimental groups and between experimental and control groups. All P values represent two-sided tests and were considered significant when <0.05.

Results

Apoptotic cells

After treatment with different concentrations of cisplatin for 24 h, apoptotic A2780/DDP and COC1/DDP cells were analyzed by flow cytometry. The numbers of apoptotic A2780/DDP and COC1/DDP cells were significantly lower than those of A2780 (P=0.033) and COC1 cells (P=0.026) at a higher concentration of cisplatin (10 μM). Apoptotic ratio increased as the concentration of cisplatin increased (Fig. 1).

Fig. 1.

Fig. 1

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The apoptotic ratios of cisplatin-resistant A2780/DDP and COC1/DDP cells were significantly lower than those of cisplatin-sensitive A2780 and COC1 cells

Changes in apoptosis-associated genes in COC1/DDP and A2780/DDP cells

To measure expression of mRNAs of apoptosis-associated genes, semiquantitative RT-PCR was performed. Variations in the loaded amounts of RNA and cDNA were normalized by comparing band densities of the GADPH controls. Increased expression of Bcl-2 and Bcl-XL mRNA was observed in cisplatin-resistant cell lines (P<0.05 for both when compared with cisplatin-sensitive cell lines, Fig. 2A,B). However, expression of proapoptosis gene Bax mRNA did not differ significantly between cisplatin-resistant cell lines and cisplatin-sensitive cells (P>0.05 for both, Fig. 2C). Bcl-Xs was not detectable in these four cell lines by RT-PCR (Fig. 2D).

Fig. 2A–D.

Fig. 2A–D

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RT-PCR analysis of expression of apoptosis-associated genes in human ovarian cancer cell lines: RNA samples isolated from cisplatin-sensitive cell lines A2780 and COC1 and cisplatin-resistant cell lines A2780/DDP and COC1/DDP. The expression of mRNA levels of Bcl-2 (A), Bcl-XL (B), Bax (C), and Bcl-Xs (D). All PCRs were determined and coamplified with GADPH as an internal control. Lane 1 contains polymerase chain reaction (PCR) marker; lane 2 contains H2O as negative control

Changes in apoptosis-associated proteins in cisplatin-resistant cells

Expression of these apoptosis-associated proteins was determined by Western blot in A2780, COC1, A2780/DDP, and COC1/DDP cell lines. Expression of Bcl-2 was significantly higher in cisplatin-resistant cell lines A2780/DDP and COC1/DDP than in cisplatin-sensitive cell lines A2780 (P=0.038) and COC1 (P=0.003). Expression of Bcl-XL, another important member of the Bcl-2 family known to inhibit apoptosis in chemotherapy, was also significantly enhanced in A2780/DDP (P=0.027) and COC1/DDP cells (P=0.032). The proapoptotic protein Bax has been shown to affect chemotherapy-induced apoptosis. In this experiment, Bax protein was detected in similar quantities in these four cell lines, and little Bcl-Xs expression was noted (Fig. 3).

Fig. 3.

Fig. 3

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Western blot of Bcl-2, Bax, Bcl-XL, and Bcl-Xs expression in cisplatin-sensitive (A2780 and COC1) and -resistant (A2780/DDP and COC1/DDP) ovarian cancer cell lines. Blots were reprobed for β-actin to confirm equal protein loading

Decreased activation of caspase-3 and PARP in cisplatin-resistant cells

To determine whether inhibition of cisplatin-induced apoptosis in cisplatin-resistant cell lines A2780/DDP and COC1/DDP was due to decreased caspase-3 activity, we tested PARP degraded by active caspase-3. Activation of caspase-3 by cleavage was represented on the Western blot by decrease in the intensity of procaspase-3 (32 kDa) protein band and appearance of its active subunit p17 (17 kDa). The quantity of p17 at a low concentration of cisplatin (5 μM) differed significantly between cisplatin-resistant and -sensitive cell lines (P<0.05 for both). The higher the concentration of cisplatin applied, the more subunit p17 appeared, but expression of subunit p17 in A2780/DDP and COC1/DDP cells was lower than that in A2780 and COC1 cells (Fig. 4A,B).

Fig. 4.

Fig. 4

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Activation of caspase-3 and poly (ADP-ribose) polymerase (PARP) by cleavage after treatment with various concentrations of cisplatin. Caspase-3 activation (A,B) and PARP cleavage (C,D) were indicated by Western blot after administration of cisplatin (0, 5, 10, and 20 μM) in A2780/DDP, COC1/DDP, A2780, and COC1 cells

PARP degradation was represented in the Western blot by decrease in intensity of the 116-KDa PARP protein band and appearance of an 85-KDa PARP cleavage product. The significant decrease in caspase-3 active subunit p17 observed in this study was mirrored in the 85-KDa PARP cleavage product in A2780/DDP and COC1/DDP cells, which showed dose-dependent activation of caspase-3 and PARP (Fig. 4C,D).

Activity of caspase-3

Caspase-3 activity was significantly greater in A2780 and COC1 cells than in A2780/DDP (P=0.041) and COC1/DDP cells (P=0.035) at a higher concentration of cisplatin (10 μM), and the increases were dose dependent. In A2780/DDP and COC1/DDP cells, decrease of proteolytic activity resulted in decrease in the cleavage of DEVD-pNA to pNA (Fig. 5A,B).

Fig. 5.

Fig. 5

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Caspase-3 activity of A2780, A2780/DDP, COC1, and COC1/DDP cells after exposure to various concentration of cisplatin (0, 5, 10, 20 μM)

Discussion

Ovarian cancer is a disease with poor prognosis. Chemotherapeutic drugs kill ovarian cancer cells by inducing apoptosis or programmed cell death. Although platinum-based anticancer agents such as cisplatin and carboplatin play critical roles in the treatment of ovarian cancer, chemoresistance remains a major problem in control of this disease. The Bcl-2 family of proteins are important factors in regulating apoptosis. Overexpression of Bcl-2 or Bcl-XL has been shown to protect many different cancer cell lines from apoptosis induced by a wide variety of chemotherapeutic agents such as etoposide, methotrexate, cisplatin, and cyclophosphamide (Grobholz et al. 2002, Granville et al. 1999, Frankel et al. 2001). In the experiments presented here, which used cisplatin-resistant ovarian cancer cell lines A2780/DDP and COC1/DDP as a cell model, we compared expression of Bcl-2, Bcl-XL, Bax, and Bcl-Xs in these cells and in their cisplatin-sensitive parental cells. Just as Ding et al, observed, overexpression of antiapoptotic proteins in human multidrug-resistant cervical cells (Ding et al. 2000), we found that Bcl-2 and Bcl-XL were overexpressed in cisplatin-resistant ovarian cancer cell lines on both mRNA and protein levels. We conclude, therefore, that chemoresistance may be associated with enhanced expression of antiapoptosis genes Bcl-2 and Bcl-XL.

Recent studies suggest that caspase-3 plays an important role in several key events during apoptosis, such as nuclear fragmentation and DNA fragmentation (Blanc et al. 2000, Taylor et al. 1999, Porter et al. 1999). Caspase family members have been located in the cytosol as zymogens, and cleavage of procaspase-3 (Mr 32,000) to generate Mr 17,000 and Mr 12,000 fragments is considered indicative of commitment to apoptosis. The cleavage of procaspase-3 is an early event in apoptosis induced by chemotherapeutic agents (Henkels et al. 1999). Further insight regarding caspase-3 activation was obtained by reference to a known substrate, PARP, which was cleaved directly by caspase-3 and served as a “death substrate” (Henkels et al. 1999, Sleiman et al. 2000, Donepudi et al. 2002, Soldatenkov et al. 2000, Liang et al. 2001). In the investigation presented here, A2780, A2780/DDP, COC1, and COC1/DDP cells were treated with cisplatin at various concentrations. Cleavage of caspase-3 and PARP was found after cisplatin treatment in cisplatin-sensitive cells. With the increase of cisplatin concentration, caspase-3 activation and PARP fragmentation increased in a dose-dependent fashion. In cisplatin-resistant cells, caspase-3 activation and PARP fragmentation were significantly lower. We suggest that cisplatin-resistant human ovarian cancer cells overexpressing Bcl-2 and Bcl-XL may block caspase-3 activity and PARP cleavage and that cisplatin can induce apoptosis through a caspase-3-dependent signal transduction pathway.

Members of the Bcl-2 family such as Bax and Bcl-Xs can accelerate cell death mediated by several chemotherapeutic agents (Liang et al. 2001, Kobayashi et al. 2000, Choi et al. 2002). Previous studies had demonstrated that Bax overexpression enhances apoptosis induced by paclitaxel, vincristine, or doxorubincin but not that induced by carboplatin, etoposide, or hydroxyurea in ovarian cancer cells. In this study, we tested Bax and Bcl-Xs in cisplatin-resistant and -sensitive ovarian cancer cells. Unlike Bcl-2 and Bcl-XL, which were expressed at high levels in cisplatin-resistant ovarian cancer cells, Bax expression was no different between cisplatin-resistant and -sensitive cells. We found little Bcl-Xs expression in these four cell lines. It is well known that overexpression of Bax enhances many forms of apoptosis, but the observed similarity in Bax expression in cisplatin-resistant and -sensitive ovarian cancer cell lines in this study suggested that cisplatin resistance may be independent of Bax expression.

In conclusion, our results suggest that overexpression of antiapoptosis proteins Bcl-2 and Bcl-XL and reduction of caspase-3 activity may be reasons for cisplatin resistance in human ovarian cancers.

Acknowledgement

The authors are grateful to Fily Gao for technical support. This work was supported by grants from the National Science Foundation of China (No. 30025017) and “973” Program of China (No. 2002CB513107).

Footnotes

This paper reports on an experimental study.

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