Effects of poly (ADP-ribosyl) polymerase (PARP) inhibitor on cisplatin resistance & proliferation of the ovarian cancer C13^* cells

Ovarian cancer is the second most common gynaecological malignancy and the leading cause of death of patients with gynaecological cancers1. Most of these cases lack early symptoms and are diagnosed at an advanced stage (approximately 70%), when the tumour has metastasized. The traditional treatment of advanced ovarian cancer is based on cytoreductive surgery, followed by platinum-based chemotherapy2. Unfortunately, despite the high response rate to the initial chemotherapy, there remains a significant risk for recurrence and resistance to the chemotherapy, and the long-term survival is relatively poor with only 10-15 per cent of advanced-disease patients surviving at 10 years3. Consequently, seeking promising novel agents for chemotherapy and elucidating drug resistance mechanisms are crucial for individualizing the treatment and prognosis of ovarian cancer patients.

Chemotherapy resistance is a significant problem and is the major obstacle in ovarian cancer treatment. The enhancement of DNA damage repair capability could be a critical mechanism for chemotherapy45. Poly(ADP-ribosyl)ation is a post-translational modification found in mammalian cells6. In humans, the conserved PARP [poly(ADP-ribose) polymerase] signature motif has been identified in 17 homologous genes, including parp-1, parp-2, parp-3, vault-parp, and tankyrases. PARP-1 is an essential member of the family of PARPs, which plays an important role in DNA repair, genomic stability, energy metabolism, transcriptional regulation, inflammation and cell death. DNA repair is a complex and multifaceted process, which is critical to cell survival7.

In our earlier study8, the expression levels of PARP-1 protein in cisplatin-resistant ovarian cancer C13^* cells were found to be higher than cisplatin-sensitive ovarian cancer OV2008 cells and PARP-1 RNA interference can significantly downregulated PARP-1 expression and effectively reversed resistance of C13^* cells to cisplastin. However, the RNA interference still has some limitations for clinical application. Therefore, in the present study PARP inhibitor 3-aminobenzamide (3-AB) was used to reduce the expression of PARP. The cisplatin-resistant ovarian cancer C13^* cells were treated with various concentrations of 3-AB, and the effect of decreased expression of the PARP-1 protein in C13^* cells was studied on proliferation activity and cisplatin sensitivity of cells.

Material & Methods

Cell culture: Cisplatin-resistant ovarian cancer C13^* cells, (kindly provided by Dr Du Meirong, Department of Gynecology, Fudan University), were cultured in RPMI1640 medium (Hyclone, Logan, UT, USA) supplemented with 10 per cent foetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1 per cent penicillin/streptomycin (Hyclone, Logan, UT, USA) at 37^° C in a 5 per cent CO₂ atmosphere.

Effect of 3-AB on the proliferation of C13^* cells: C13^* cells (4 × 10⁴/ml) were plated in each well of a 96-well plate containing 100 μl growth medium. On the following day, cells were treated with increasing concentration of 3-AB (0, 0.5, 1, 2.5, 5, 7.5 and 10 mmol/l) and cultured for 24 h, each group contained three samples. After 24 h, cells were added with 20 μl 3-4,5-demethylthylthiazol -2yl] -2,5 diphenyltetrazolium broide MTT (5 mg/ml; Sigma, St Louis, MO, USA) and incubated for 4 h. Cells were collected and centrifuged at 3000 g for 2 min, and the supernatant was discarded. The formazan crystals were dissolved in 150 μl Dimethyl sulfoxide (DMSO), and shaken for 15 min. The absorbance value (A) at 490 nm was read using an automatic multiwell spectrophotometer (Bio-Tek, USA). The inhibitory ratio was calculated as follows: Inhibitory ratio (%) = 1-(mean experimental absorbance mean control absorbance)× 100%.

Effect of 3-AB on the cell survival of C13^* cells: C13^* cells (3 × 10⁴/ml) were plated in each well of a 96-well plate containing 100 μl growth medium, and treated with 5 mmol/l 3-AB, each group contained six samples and plated six board. One board was taken out every day, the absorbency value was determined using the MTT assay and cell growth curve was drawn.

Cellular sensitivity to cisplatin after treatment with 3-AB: C13^* cells (4 × 10⁴/ml) were plated in each well of a 96-well plate containing 100 μl growth medium. Cells were incubated for 16 h to allow adhesion, and then treated with combination of 3-AB (0, 2.5, 5 or 7.5 mmol/l) plus cisplatin (0, 1.25, 2.5, 5, or 10 μl /ml) (Qilu Parmaceutical Co. Ltd, Jinan, China). Each group contained three samples. After cells were cultured for 24 h, the inhibitory ratio was determined using the MTT assay.

Reverse transcription-polymerase chain reaction (RT-PCR): Total RNA was extracted using Trizol (Takara, Japan) according to the manufacturer's protocol. The first strand cDNA was generated by reverse transcription reagent kit (Takara, Japan). After a sufficient amount of cDNA was obtained, DNA was amplified by performing PCR with primer of PARP-1; forward, 5’-AAGGCGAATGCCAGCGTTAC-3’; reverse, 5’-GGCACTCTTGGAGACCATGTCA-3’ (Takara, Japan). Amplification was carried out for 30 cycles with 30 sec of incubation at 94^°C, 30 sec at 55^°C and 1 min at 72^°C. The products were analyzed on a 2 per cent agarose gel and examined by staining with ethidium bromide. Their amount was normalized to the value of β-actin as the standard value.

Western blot: Control and treated cells were lysed, and protein concentration was measured by using the BCA protein assay kit (Source?). Cell lysates (60 μg/lane) were separated by SDS-PAGE (10% polyacrylamide gel), transferred to an nitrocellulose (NC) membrane. After blocking with 5 per cent non-fat milk in Tris-buffered saline, the blots were incubated with human anti-rabbit parp-1 antibody (1:1000; Cell Signaling Technology, Beverly, MA, USA) at 4^°C overnight. Mouse anti-beta-actin monoclonal antibody (1:1000; Cell Signaling Technology, Beverly, MA, USA) served as a control for protein loading in each lane. Membranes were incubated with horse radish peroxidase (HRP)-conjugated secondary antibodies (1:8000; Santa Cruz, CA, USA), for 1 h at room temperature. The protein bands were visualized by ECL detection system, and the density of the bands was quatified by Alpha Imager 2200 (Alpha Innotech, San Leandro CA, USA).

Immunofluorescence: Control and treated cells were washed with PBS, fixed in 4 per cent paraformaldehyde, and permeabilized with 0.2 per cent Triton-X 100 for 20 min in a humidified tissue culture incubator at 37^° C, then processed using standard immunofluorescence staining procedures9. Cells were incubated with a human anti-rabbit parp-1 antibody (1:200; Cell Signaling Technology, Beverly, MA, USA) at 4^°C overnight. One well was incubated with PBS instead of the primary antibody and thus was used as the negative control. Fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:300; Santa Cruz, CA, USA), for 30 min at 37^°C. The nuclei were stained with 4’6-diamidino-2-phenylindole (DAPI) solution for 2 min. The cells were observed using fluorescence microscopy (Thermo Hybaid, USA) and photographed with a CCD camera (Olympus, Japan).

Statistical analysis: Each experiment was repeated three times. All statistical analyses were performed using SPSS 18.0 software (SPSS, Inc., Chicago, IL, USA). Differences among various treatment groups were determined by analysis of variance followed by Dunnett's t test.

Results

The effects of 3-AB on the proliferation of C13^* cells: The inhibitory ratios of the C13^* cells treated with 3-AB at 0.5, 1, 2.5, 5, 7.5 or 10 mmol/l were 5 ± 1.4, 13 ± 1.2, 21 ± 1.3, 30 ± 1.5, 41 ± 1.8, and 59 ± 1.0 per cent, respectively. Hence, the inhibitory ratio was associated with the concentration of 3-AB (Fig. 1; P< 0.05).

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Fig. 1

Influence of 3-AB on the cellular proliferation (inhibitory ratio) detected by MTT. The inhibitory ratios of the C13^* cells were associated with the concentration of 3-AB (0, 0.5, 1, 2.5, 5, 7.5 and 10 mmol/l).

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Effect of 3-AB on the cell survival of C13^* cells: After treated with 5 mmol/l 3-AB, the proliferation capacity of C13^* cells was inhibited, and cell growth slowed down. The difference was significant at 2, 3, 4, 5, and 6 days compared to C13^* group (Fig. 2, P< 0.05).

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Fig. 2

Effect of 3-AB on the cell survival of C13^* cells detected by MTT. The difference of cell growth was significant at 3, 4, 5 and 6 days compared to control group.

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Cellular sensitivity to cisplatin after treatment with 3-AB: When the Pt concentration was fixed (2.5 μg/ml), the inhibitory ratio of C13^* cells exhibited a significant increase in response to higher 3-AB (5 mmol/l) concentrations (P< 0.05). Similarly, when the 3-AB concentration was fixed, the inhibitory ratio of C13^* cells was also increased by higher concentrations of Pt (P<0.05). These results indicated that the PARP inhibitor 3-AB inhibited the proliferation activity of C13^* cells, and increased the cellular sensitivity to cisplatin (Table).

Table Influence of 3-AB on the cellular sensitivity to cisplatin (inhibitory ratio) detected by MTT

RT-PCR analysis: The RT-PCR analysis revealed that after cells were treated with 2.5 μg/ml Pt, 5 mmol/l 3-AB, or 2.5 μg/ml Pt plus 5 mmol/l 3-AB, the expression levels of PARP-1 mRNA in the C13^* cells were 0.6188 ± 0.03, 0.4348 ± 0.01, and 0.2219 ± 0.02, respectively, compared to the blank control (0.6844 ± 0.06). Our data indicated that the expression of PARP-1 mRNA was unaffected by Pt treatment, but was significantly decreased when Pt and 3-AB were combined (P< 0.05).

Western blot analysis: The Western blot analsyis revealed that after C13^* cells were treated with 3-AB (0, 2.5, 5 or 7.5 mmol/l) for 24 h, the expression levels of PARP-1 in C13^* cells were 1.69 ± 0.08, 1.30 ± 0.04, 0.69 ± 0.04 and 0.45 ± 0.07, respectively. Compared to the blank control (0 mmol/l 3-AB), the PARP-1 expression levels of 3-AB treated cells were significantly decreased by 23.15 ± 2.53 per cent for the 2.5 mmol/l 3-AB group, 59.11 ± 2.23 per cent for the 5 mmol/l 3-AB group, and 73.24 ± 3.88 per cent for the 7.5 mmol/l 3-AB group (P< 0.05). These results suggested that 3-AB significantly downregulated the PARP-1 expression at the translational level, and further implicated that the overexpression of PARP-1 protein in the ovarian cancer C13^* cells was related to the cellular sensitivity to cisplatin.

Immunofluorescence: PARP-1 proteins were largely located in the nuclei, and a small fraction of the proteins appeared in the kytoplasm (Fig. 3). After 3-AB treatment, the expression level of PARP-1 in the C13^* cells was decreased. These data demonstrated that 3-AB significantly downregulated the expression of PARP-1.

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Fig. 3

Detection of the PARP-1 protein expression in C13^* cells by Immunofluorescent microscopy. (A) PARP-1 protein expression and localization in C13^* cells (red). (B) PARP-1 protein expression and localization in C13^* cells after treated with 5 mmol/l 3-AB (red) for 24 h. Nuclei stained with DAPI (blue). Original magnification, × 400.

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Discussion

Platinum-based chemotherapy has been used as primary treatment for recurrent ovarian epithelial cancer. However, the median time to recurrence is not more than 2 years, with a 5-years survival rate of 20-25 per cent10. Acquired resistance to chemotherapy is the primary cause of failure in the treatment of cancers. Therefore, identification of the potential molecular pathways involved in cisplatin-resistant chemotherapy has become one of the significant areas in the research of tumour treatment.

Platinum is a non-specific anti-tumour medicine, and its cytotoxicity against tumour cells has been thought to be mediated through several mechanisms11, including formatting and enhancing the platinum-DNA adduct repair capacity, decreasing platinum accumulation, and increasing drug inactivation and its ability to tolerate platinum-DNA damage12. The DNA repair capability of cells may be a rate-limiting factor for cisplatin resistance and sensitivity in cisplatin resistant ovarian cancer cells13.

Poly(ADP-ribosyl)ation is post-translational type of protein modification involved in the regulation of cell cycle, transcription, initiation of the DNA repair and apoptosis1415. The poly(ADP-ribosyl)ation process can regulate a series of molecular events through regulating enzymatic activities and macromolecular interactions between proteins, DNA and RNA. PARP-1 is responsible for sensing the signals of DNA damage16. DNA strand breaks can be caused by exposures to ionizing radiation, UV light, chemotherapy, or products of cellular metabolism17. Activation of PARP-1 can lead to the addition of poly-(ADP-ribose) branched chains onto the damaged DNA, as well as the assembling proteins involved in DNA repair18. In addition to its role in DNA repair, poly(ADP-ribosyl)ation can also protect the naked DNA from being degraded by nuclease.

It has been suggested that PARP-1 facilitates diverse inflammatory responses through upregulating the expression of inflammation-relevant genes that encode oxidation-reduction-related enzymes, cytokines, and adhesion molecules19. Further, excessive activation of PARP-1 can induce arteriosclerosis, myocardial infarction, hypertension and cardiovascular complications of diabetes2021. However, the relationship between PARP and tumours has been rarely reported. The overexpression of PARP in breast cancer22, glioblastoma23, liver cancer24, prostatic carcinoma25 and pancreatic cancer26 can facilitate the proliferation of tumour cells. Gurung et al27 suggested that the inhibition of PARP-1 and telomerase in MEFs rendered cells is more susceptible to DNA-damaging agents. Other studies1723 demonstrated that inhibiting the activation of PARP could enhance glioblastoma and lung cancer sensitivity to radiation. The PARP-1 inhibitor ANI and the suppression of PARP-1 expression by siRNA can significantly sensitize human liver cancer cells to doxorubicin treatment24.

The inhibitory effects of 3-AB on PARP-1 expression has been well established, Valenzuela et al28 showed that a concentration of 5mM of 3-AB after 2 h-exposure effciently blocks PARP activity avoiding PARP-polymer formation in response to to DNA damage in primary mouse embryonic firoblast. Wang et al29 reported that 3-AB was able to reduce PARP-1 activity in the millimole range in the repair proficient human glioma M059K cells. Zheng et al30 indicated that 3-AB suppressed cell growth, cell invasion and enhanced the suppressive effects of cisplatin in vitro in U2OS cells. They suggested that 3-AB may be developed into an effective agent for the treatment of human osteosarcoma.

In summary, our study indicated that the PARP inhibitor 3-AB inhibited the proliferation activity of C13^* cells, and increased the cellular sensitivity to cisplatin. We thus proposed that combined treatment of PARP inhibitor with cisplatin can reverse the drug resistance of ovarian cancer cells, reduce the dosage and side effects of cisplatin chemotherapy, and improve the prognosis of patients suffering from ovarian cancer. However, the PARP protein family includes 17 members, and chemical inhibitors may have nonspecific suppression effects. Systemic long-term administration of PARP inhibitors may harm the DNA repair and genomic stability in normal cells, which can lead to secondary tumours years after3132. Therefore, novel PARP-1 inhibitor with low toxicity and high specificity need to be tested in future. With the progress in the PARP research and combination therapy, targeted therapy using PARP inhibitors may play a vital role in the prevention of tumours.