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Introduction

As one of the most lethal gynecological cancers, ovarian cancer is the fifth leading cause of cancer-related mortality among women in Western countries. Most ovarian cancer cells originate from the ovarian surface epithelium and display a range of histological subtypes (1). Platinum is the first-line drug for the chemotherapy of ovarian cancer (2).

The TNF-related apoptosis-inducing ligand (TRAIL) is a TNF family member capable of inducing apoptosis through caspase-dependent mechanisms (3–6). TRAIL binds four major different receptors, two of which, death receptor (DR)4 and DR5, induce apoptosis; however, the other two, decoy receptor (DcR)1 and DcR2, do not have the intracytoplasmic death domain to transduce apoptotic death signals and, thus, they protect cells from TRAIL-mediated cell death by interfering with signaling through DR4 and DR5 (7,8). Another receptor, osteoprotegerin (OPG), is a soluble receptor that may play a more prominent role in bone and myeloid cell development (9). In tumor cells, the expression of DR5 which is rare or even absent in healthy cells predominates over that of DR4; therefore, DR5 plays a major role in TRAIL-induced apoptosis (4,10). In order to find an alternative for TRAIL for clinical treatment, monoclonal antibodies inducing the apoptosis of tumor cells via targeting DR5 have been developed, such as TRA-8, Apomab and CS-1008 (2,12–14).

The DR5 monoclonal antibody (D-6) is a type of murine monoclonal antibody directed againts DR5, produced by Santa Cruz Biotechnology Co. In this study, we explored the apoptosis-inducing effects of the DR5 monoclonal antibody (D-6), alone or in combination with the platinum-based drug, cisplatin, on the A2780 ovarian cancer cell line.

Materials and methods

Cell culture and reagents

All animal procedures were approved by the Animal Care and Scientific Committee of Sichuan University, Chengdu China. The A2780 cells were kindly provided by Kanghong Co. (Chengdu, China). The DR5 monoclonal antibody (D-6) was purchased from Santa Cruz Biotechnology Co. (Santa Cruz, CA, USA). Cisplatin was purchased from the West China Second Hospital, Sichuan University. The RPMI-1640 culture medium, dimethylsulfoxide (DMSO) and fetal bovine serum were purchased from Gibco (Carslbad, CA, USA). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay kit and trypsin were purchased from Sigma (Sigma, St. Louis, USA). The Annexin V-FITC apoptosis detection kit and protein extraction kit were purchased from KeyGen Biotech Co. (Nanjing, China). The BCA protein assay kit, SDS-PAGE protein sample buffer (5X), SDS-PAGE gel preparation kit and the BeyECL Plus chemiluminescence kit were purchased from Beyotime Co. (Jiangsu, China). The caspase 3, 8 and 9 precursor antibodies were purchased from Santa Cruz Biotechnology Co. Polyvinylidene difluoride (PVDF) membranes were purchased from Millipore (Billerica, MA, USA). The enhanced chemiluminescence (ECL) detection kit and X-ray films were purchased from Roche (Basel, Switzerland).

Apoptosis observation under a microscope

The A2780 cells were counted and cultured in 6-well plates at a concentration of 2×105 cells/well. they were then divided into four groups: the control group, where 2 ml culture medium was added to the wells; the cisplatin group, where 2 ml culture medium containing 2.5 μg/ml cisplatin was added; the DR5 (D-6) group, where 2 ml culture medium containing 2 μg/ml DR5 (D-6) was added; and the combination group, where 2 ml culture medium containing 2.5 μg/ml cisplatin and 2 μg/ml DR5 (D-6) was added. Twenty-four hours after the culture, the morphological changes of each group were observed under an inverted microscope.

MTT analysis

The A2780 ovarian cancer cells were first counted and incubated in two 96-well plates at a concentration of 5×103 cells/well and a volume of 0.1 ml/well in 30 wells of each plate. After culture in the RPMI-1640 culture medium containing 10% fetal bovine serum, the 30 wells of one plate were randomized into six groups (five wells/group) and 0.1 ml culture medium containing 0, 0.125, 0.25, 0.5, 1 and 2 μg/ml DR5 (D-6) was added, after drying out the supernatant. The other plate was also divided into six groups (five wells/group) and 0.1 ml culture medium containing 2.5 μg/ml cisplatin and 0.125, 0.25, 0.5, 1 and 2 μg/ml DR5 (D-6) was added, after drying out the supernatant, except for one group (the contol) in which 0.1 ml culture medium was added. After 48 h of culture in the incubator, 10 μl MTT was added to each well. The plates were then incubated at 37°C for an additional 4 h to allow MTT to form formazan crystals by reacting with metabolically active cells. The formazan crystals were solubilized in 0.1 ml DMSO per well at 37°C for 10 min. The absorbance values of the solution in each well were measured at 450 nm using a multiscanner autoreader. The cell growth inhibition rate was calculated as follows: [1 - (OD test - OD blank)/(OD control - OD blank) ×100% (11).

Flow cytometry

The A2780 ovarian cancer cells were counted and cultured in four wells of a six-well plate at a concentration of 2×105 cells/well and a volume of 2 ml/well. After culture in the incubator for 24 h, the supernatant was dried out and the four wells were randomized into four groups: the control group, where 2 ml culture medium was added; the DR5 (D-6) group, where 2 ml culture medium containing 1 μg/ml DR5 (D-6) was added; the cisplatin group, where 2 ml culture medium containing 2.5 μg/ml cisplatin was added; and the combination group, where 2 ml culture medium containing 1 μg/ml DR5 (D-6) and 2.5 μg/ml cisplatin was added. After 24 h in the incubator, the cells were digested with EDTA-free trypsin and washed twice with PBS. For each group, 1–5×105 cells were collected. The cells were then suspended in binding buffer (500 μl), and then 5 μl Annexin V-FITC and 5 μl PI were added. After 10 min of reaction in a dark environment, the cell apoptotic rate was evaluated by flow cytometry.

Western blot analysis

The A2780 ovarian cancer cells were counted and cultured in the four culture dishes at a concentration of 106 cells/well and a volume of 5 ml/well. After culture in the incubator for 24 h, the supernatant was dried out and the four wells were randomized into four groups: the control group, where 5 ml culture medium was added; the DR5 (D-6) group, where 5 ml culture medium containing 2 μg/ml DR5 (D-6) was added; the cisplatin group, where 5 ml culture medium containing 2.5 μg/ml cisplatin was added; and the combination group, where 5 ml culture medium containing 2 μg/ml DR5 (D-6) and 2.5 μg/ml cisplatin was added. After 48 h in the incubator, the cells from each group were collected and protein extraction was carried out using the protein extraction kit. Protein (35 μg) extracted from the cells of each group was subjected to SDS-PAGE electrophoresis and then the gel was electroblotted on a PVDF membrane for 60 min. The membrane was incubated with 5% non-fat dry milk in TBS for 1 h to block non-specific binding sites, and then incubated with the appropriate primary antibody concentration (1:200 dilution for caspase 3, 8 and 9 precursors) overnight. The membrane was subsequently rinsed in TBS-T and incubated for 1 h at 37°C with secondary antibody. After incubation, the membrane was finally rinsed and visualized with ECL detection reagents.

Statistical analysis

The cell growth inhibition rate and apoptotic rate were analyzed by one-way ANOVA and the Student's t-test. All statistical analyses were performed using the SPSS 17.0 software package. All P-values were two-sided and P<0.05 was considered to indicate a statistically significant difference.

Results

Morphological changes observed under a microscope

As shown in Fig. 1, 24 h after the treatment, the cells of both the DR5 (D-6) and cisplatin groups showed evident apoptosis; these effects were more evident in the DR5 (D-6) and cisplatin combination group. In the combination group, there were many floating cells and only a few cells grew along the wall. The cells underwent significant changes in morphology; their original shape was completely altered, the cytoplasm became rougher, the nucleus became pycnotic and the refractive index in the cells decreased, demonstrating significant cellular damages. By contrast, A2780 cells in the control group did not demonstrate significant morphological changes.

Figure 1 Morphological changes in the A2780 ovarian cancer cells under an inverted microscope after 24 h of treatment. (A) Control group; (B) cisplatin group; (C) DR5 (D-6) group; (D) DR5 (D-6) combined with cisplatin group.

Cell growth inhibition rate

The cell growth inhibition rates were analyzed by MTT assays. As shown in Fig. 2, the cell growth inhibition rates increased following the increasing DR5 (D-6) concentrations >0.25 μg/ml (P<0.05) in both groups, and in the DR5 (D-6) and cisplatin combination group these rates were more significant than in the DR5 (D-6) group (P<0.05). However, the cell inhibition rates decreased following treatment with DR5 (D-6) concentrations of <0.25 μg/ml in both groups, and there were evident cell growth-promoting effects in the DR5 (D-6) and cisplatin combination group.

Figure 2 Cell growth inhibition rate of the A2780 ovarian cancer cells analyzed by MTT assay. (Bottom panel) DR5 (D-6) group: the cells were treated with various concentrations (0.125, 0.25, 0.5, 1 and 2 μg/ml) of DR5 (D-6). (Top panel) DR5 (D-6) and cisplatin group: the cells were treated with various concentrations (0.125, 0.25, 0.5, 1 and 2 μg/ml) of DR5 (D-6) and cisplatin at a constant concentration (2.5 μg/ml).

Apoptosis inducing effects of cisplatin and DR5 (D-6) on the A2780 human ovarian cancer cell line

The apoptotic rates were measured by flow cytometry. The apoptotic rates were 1.1±0.1, 12.90±0.70, 12.70±1.35 and 16.77±0.37%, in the control, DR5 (D-6), cisplatin and combination groups, respectively (Fig. 3). The apoptotic rate of the DR5 (D-6) and cisplatin combination group was significantly higher than that of the other three groups (P<0.05); it was higher in the DR5 (D-6) and cisplatin groups than in the control group (P<0.05). However, there was no significant difference between the DR5 (D-6) group and the cisplatin group (P>0.05).

Figure 3 Apoptotic rate of the A2780 ovarian cancer cells analyzed by flow cytometry after treatment. (A) Control group; (B) cisplatin group; (C) DR5 (D-6) group; (D) DR5 (D-6) combined with cisplatin group.

Western blot analysis of intracellular protein changes

As shown in Figs. 4 and 5, 48 h after treatment, the caspase 3, 8 and 9 precursors decreased in the DR5 (D-6), cisplatin and DR5 (D-6) and cisplatin combination groups compared to the control group; this decrease was more significant in the combination group.

Figure 4 Western blot analysis of caspase 3 precursor after treatment. (A) Control group, treated with 5 ml culture medium. (B) DR5 (D-6) group, treated with 5 ml culture medium containing 2 μg/ml DR5 (D-6). (C) Cisplatin group, treated with 5 ml culture medium containing 2.5 μg/ml cisplatin. (D) Combined group, treated with 5 ml culture medium containing 2 μg/ml DR5 (D-6) and 2.5 μg/ml cisplatin.

Figure 5 Western blot analysis of caspase 8 and 9 precursors after treatment. (A) Control group, treated with 5 ml culture medium. (B) DR5 (D-6) group, treated with 5 ml culture medium containing 2 μg/ml DR5 (D-6). (C) Cisplatin group, treated with 5 ml culture medium containing 2.5 μg/ml cisplatin. (D) Combined group, treated with 5 ml culture medium containing 2 μg/ml DR5 (D-6) and 2.5 μg/ml cisplatin.

Discussion

Various cancer therapies using TRAIL-induce apoptosis have been developed over the past few years, and many DR5 monoclonal antibodies, including murine or humanized, have been produced, such as TRA-8, Apomab and CS-1008 (2,12–14). The DR5 monoclonal antibodies have been used to a greater extent than TRAIL, since they have a longer half-life in the circulatory system and have been shown to have significant anti-tumor effects (7,15,16).

A number of studies have focused on the mechanism of TRAIL-induced apoptosis, and it is generally accepted that the caspase-dependent pathway is involved (5,6,10). Upon the binding of TRAIL to either DR4 or DR5 on the cell membrane, DR4 and DR5 promote the formation of the death-inducing signaling complex (DISC) via the intracellular death domain (DD), recruiting certain adaptor proteins (5). After the formation of DISC, caspase 8 and 10 are recruited to DISC, and are activated automatically by protein hydration and, thereafter, downstream effectors, such as caspase 3, 6 and 7 are activated, through a series of cascade reactions to trigger biological reactions of apoptosis (13). Exposure to chemotherapy may stimulate the intrinsic pathway which causes the destabilization of the mitochondrial membrane with the release of cytochrome c, leading to caspase 3 activation. During the cascade reaction, caspase 9 has also been shown to be highly activated (6,17).

In this study, we show that DR5 (D-6), a novel DR5 monoclonal antibody, is effective in inducing apoptosis of the A2780 ovarian cancer cell line. The combination of DR5 (D-6) and cisplatin was even more effective. In the experiment under the inverted microscope, although both the DR5 (D-6) and cisplatin groups showed apoptosis-inducing effects, the effects in the combination group were more significant. In the combination group, most of the cells were changed into round-shaped and floated on the surface. Therefore, we hypthesize that they had a synergistic function in inducing apoptosis.

The results of MTT revealed that the A2780 ovarian cancer cells were sensitive to DR5 (D-6) in a dose-dependent manner, and this effect was enhanced by cisplatin. A number of studies have investigated the effect of other DR5 monoclonal antibodies on other tumor cells in combination with chemotherapy or radiotherapy (18–21). It is thought that chemotherapy sensitizes the apoptosis-inducing effect of the DR5 monoclonal antibody (20,22). A number of studies have provided evidence of a correlation between cisplatin and DR5 monoclonal antibodies. Chemotherapy not only sensitizes the target cells to the DR5 monoclonal antibody, but DR5 monoclonal antibody may also enhance the effects of chemotherapy (14,22). This is very important for the platinum-resistant types of cancer, such as ovarian cancer. Most ovarian cancers are sensitive to platinum-based chemotherapy at the time of diagnosis; however, recurrence of the disease is frequent and, ultimately, platinum-resistant disease develops in all patients (1). Thus, the DR5 monoclonal antibody may play a role in the treatment of ovarian caner. In this study, we found that the A2780 ovarian cancer cell line did not undergo apoptosis under the concentration of 0.25 μg/ml. This phenomenon occurred after many repeated examinations. The reason is unknown and requires further study.

The results of flow cytometry showed that the treatment of the A2780 ovarian cancer cells with DR5 (D-6), cisplatin or the combination of both produced apoptosis-inducing effects; these effects were more significant in the combination group. In order to observe the internal reaction of the ovarian cells after treatment, we examined the expression of caspase 3, 8 and 9 precursors, which are usually used as reaction proteins of apoptotic pathways (8). Western blot analysis showed that the caspase 3, 8 and 9 precursors decreased evidently in the treatment group. Therefore, we believe that the apoptosis of the ovarian cancer cells in this study occured in a caspase-dependent manner.

In conclusion, the monoclonal antibody (D-6) directed against DR5 was capable of inducing the apoptosis of ovarian cancer cells in a dose-dependent manner, and this effect was enhanced by cisplatin. The combined treatment of cells with the DR5 monoclonal antibody and cisplatin may be a promising treatment for ovarian cancer.

Acknowledgements

This study was supported by grants from the National Natural Scientific Foundation of China (no. 30973192).

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References