XPA Promotes Autophagy To Facilitate Cisplatin Resistance In Melanoma Cells Through The Activation Of PARP1

BackgroundMelanoma, a malignancy deriving from melanocytes, has the highest mortality among skin cancers. In addition to early excision, there is currently no effective treatments to advanced melanoma. As the classic chemotherapy, cisplatin improves the clinical responses when they are used in combination with other type of chemotherapy agents or after immunotherapy and targeted therapy, especially in the treatment of refractory, progressive, and relapsed melanoma. However, the drug resistance of melanoma cells limits clinical application of cisplatin. Thus, clarifying the mechanisms underlying the resistance of melanoma cells to chemotherapeutic drugs such as cisplatin is clinically beneficial to melanoma patients, especially for those in advanced stages.Treatment with cisplatin is cytotoxic to cells, resulting from DNA crosslink formation and inhibition of both replication and transcription. Therefore, adequate and prompt repair of cisplatin-elicited DNA lesions can render tumor cells less susceptible to cisplatin. As a key subunit involved in nucleotide excision repair(NER) system, Xeroderma pigmentosum group A(XPA) protein acts as a central organizer in NER pathway by recognizing DNA damage and recruiting other NER proteins to DNA lesions. XPA has been reported to promote the resistance of cancer cells to platinum-based drugs by facilitating cell-protective NER pathway in malignancies such as nasopharyngeal carcinoma, lung adenocarcinoma and prostate cancer. However, the role of XPA in the resistance of melanoma cells to cisplatin treatment has not been investigated.Autophagy is a cellular process that leads to the degeneration of cytoplasmic components and the recycle of cellular contents and energetic substances. Accumulating evidences suggest that autophagy can protect tumor cells from chemotherapy-caused stress by providing energy and necessary cellular materials. However, the mechanism of autophagy activation after chemotherapy has not yet been fully elucidated. It has been sugguested that autophagy could be activated during DNA repair process, for which consumes much energy. Besides, according to previous studies, DNA repair proteins can directly influence the autophagy process. More importantly, Fang et al. found that there was defective mitophagy in XPA deficient cells, which indicated the involvement of XPA in regulating autophagy. On the basis of these premises, we postulate our hypothesis. Besides the role of XPA in NER repair, XPA may also promote cisplatin resistance through regulating autophagy in melanoma cells.Objectives 1. To analyse and verify the correlation between XPA expression and IC50 of cisplatin;2. To discuss the molecular mechanism of cisplatin resistance mediated by XPA.Methods1. q RT-PCR was performed to testify the m RNA expression of XPA in melanocytes and melanoma cells; Cell counting kit-8 assay(CCK8) was performed to testify the cell viability after cisplatin treatment and IC50 was calculated in melanocytes and melanoma cells; The correlation between XPA expression and IC50 of cisplatin was analyzed;2. CCK8 assay was performed to testify the effect of XPA to the resistance of cisplatin in melanoma cells after XPA transient knockdown; Western blot and flow cytometry were performed to testify the effect of XPA to apoptosis in melanoma cells treated with cisplatin after XPA transient knockdown;3. Western blot, transmission electron microscopy and immunofluorescence were performed to testify the effect of cisplatin treatment to autophagy in melanoma cells; 4. Western blot and immunofluorescence were performed to testify the effect of XPA to autophagy in melanoma cells treated with cisplatin after XPA transient knockdown; 5. Western blot was performed to testify the effect of XPA to the activation of PARP1 in melanoma cells treated with cisplatin after XPA transient knockdown;6. Western blot and immunofluorescence were performed to testify the effect of PARP1 to autophagy in melanoma cells treated with cisplatin after PARP1 transient knockdown or pretreated with PARP1 inhibitor; 7. Western blot was performed to testify the effect of PARP1 to the activation of m TOR, the total expression and the cytoplasmic content of HMGB1 in melanoma cells treated with cisplatin after PARP1 transient knockdown; 8. Colony formation assay was performed to testify the effect of PARP1 or ATG12 to cisplatin-induced cytotoxicity in melanoma cells treated with cisplatin after PARP1 or ATG12 transient knockdown.Results1. The m RNA level of XPA in all the melanoma cell lines was obviously increased compared with that in melanoma cell line and was correlated with resistance to cisplatin; Hs294 T and A2058, two cell lines with high m RNA level of XPA and high resistant to cisplatin were chose for next experiment. 2. The result of CCK8 assay demonstrated that IC50 of cisplatin in Hs294 T and A2058 cells decreased after XPA knockdown; 3. The result of western blot and flow cytometry demonstrated that cleaved caspase-3 and proportion of early and late apoptotic cells enhanced in Hs294 T and A2058 cells treated with cisplatin after XPA knockdown; 4. The result of western blot, transmission electron microscopy and immunofluorescence demonstrated that autophagy was induced in Hs294 T and A2058 cells after cisplatin treatment; 5. Western blot and immunofluorescence assay showed that autophagy augment was impaired in Hs294 T and A2058 cells treated with cisplatin after XPA knockdown; 6. The result of western blot demonstrated that PARP1 activation wes impaired in Hs294 T and A2058 cells treated with cisplatin after XPA knockdown; 7. Western blot and immunofluorescence assay showed that autophagy augment was impaired in Hs294 T and A2058 cells treated with cisplatin after PARP1 knockdown or pretreated with PARP1 inhibitor; 8. The result of colony formation assay demonstrated that cisplatin-induced cytotoxicity was enhanced in Hs294 T and A2058 cells treated with cisplatin after PARP1 or ATG12 knockdown.Conclusion In conclusion, we demonstrated that the relatively higher expression of XPA in cisplatin-resistant melanoma cells promotes cisplatin-induced autophagy through the activation of PARP1, which facilitates the resistance to cisplatin in melanoma cells. To our knowledge, the role of XPA in facilitating cell-protective autophagy induced by cisplatin has not been reported previously, which indicates that the inhibition on the XPA-PARP1-mediate autophagy can be exploited to sensitize resistant melanoma cells to platinum-based drugs.