| Background and ObjectiveOvarian cancer is the leading cause of death of patients with gynecological cancers. Its incidence is rising slowly. Most of these cases lack early symptoms and are diagnosed at an advanced stage (approximately70%), when the tumor has metastasized. The traditional treatment of advanced ovarian cancer is based on optimal cytoreductive surgery, followed by platinum-based combination chemotherapies. Although the platinum is first line anti-cancer drug, Inherent and therapy-induced resistance to the Platinum-based therapies has emerged as a major obstacle in the treatment of ovarian cancers. As a result, the5-year survival rate for advanced-stage patients rarely exceeds30%.At present, identification of the potential molecular pathways involved in cisplatin-resistant chemotherapy has become one of the most significant topics in the research of tumor treatment. Platinum is a non-specific anti-tumor medicine, and its cytotoxicity against tumor cells has been thought to be mediated through several mechanisms including formatting and enhancing the platinum-DNA adduct repair capacity, decreasing platinum accumulation, and increasing drug inactivation and its ability to tolerate platinum-DNA damage. The DNA repair capability is a rate-limiting factor for cells genetic basis stability, and the enhancement of DNA repair capability may be responsible for the drug resistance of the tumor cells, and poly-(ADP-ribosyl) ation plays an important role in DNA repair. Poly-(ADP-ribosyl) ation is a post-translational modification found in mammalian cells, which plays an important role in DNA repair, genomic stability, energy metabolism, transcriptional regulation, inflammation and cell death. PARP-1is responsible for sensing the signals of DNA damage. DNA strand breaks can be caused by exposures to ionizing radiation, UV light, chemotherapy, or products of cellular metabolism. Activation of PARP-1can lead to the addition of poly-(ADP-ribose) branched chains onto the damaged DNA, as well as the recruitment of proteins involved in DNA repair. In addition to its role in DNA repair, poly (ADP-ribosyl) ation can also protect the naked DNA from being degraded by nuclease.In our study, the cisplatin-resistant ovarian cancer C13cells were treated with various concentrations of PJ34, and investigated the effects of PJ34on growth activity and cisplatin resistance of the C13cells, and provided theoretical guidance for the clinical application of PARP-1inhibitors.Methods1. MTT assay was used to determine the influence of PJ34on the cisplatin sensitivity and proliferation of C13*cells.2. Flow cytometry were used to determine the influence of PJ34on apoptosis and cell cycle of C13*cells, separately.3. The expression levels of PARP-1protein in the C13*cells were examined using Immunofluorescence microscopy and Western blot, and changes caused by PJ34treatment were investigated.Results1. Cellular sensitivity to cisplatin after treatment with PJ34:When the Pt concentration was fixed, the inhibitory ratio of C13*cells exhibited a significant increase in response to higher PJ34concentrations (P<0.05). Similarly, when the PJ34concentration was fixed, the inhibitory ratio of C13*cells was also increased by higher concentrations of Pt (P<0.05).2. Effects of PJ34on the cell apoptosis and cell cycle:The apoptosis ratio of C13*cells significantly increased with the increase of the concentration of PJ34(P<0.05). The proportion of cells in G0/G1phases in the high concentration PJ34-treated group was more than that in the low concentration PJ34-treated group.3. The location of PARP-1proteins detected by Immunofluorescence:The immunofluorescence staining method showed that the PARP-1proteins were largely located in the nuclei, and a small fraction of the proteins appeared in the cytoplasm. After PJ34treatment, the expression level of PARP-1in the C13cells was obviously decreased.4. The Western blot analysis revealed that after treating with PJ34(0,1,5μmol/l) or2.5μg/ml Pt plus5μmol/l PJ34for24h, the expression levels of PARP-1in C13*cells were0.98±0.03,0.78±0.01,0.43±0.04and0.12±0.01, respectively. Our data indicated that the expression of PARP-1protein was significantly decreased in response to higher PJ34concentrations, and was remarkably decreased when Pt and PJ34were combined.Conclusion:1. All these results indicated that the PARP-1inhibitor PJ34was able to remarkably inhibit the proliferation activity of C13*cells, and increase the cellular sensitivity to cisplatin.2. PJ34promoted apoptosis, and the apoptosis ratio of C13*cells significantly increased with the increase of the concentration of PJ34. The proportion of cells in GO/Gl phases in the high concentration PJ34-treated group was more than that in the low concentration PJ34-treated group.3. The Western blot and immunofluorescent microscopy analyses revealed that the expression of PARP-1in the C13*cells was significantly attenuated by higher concentrations of PJ34, indicating that the over-expression of PARP-1protein in ovarian cancer cells was involved in cellular sensitivity to cisplatin. |
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