| ObjectiveEffective cancer treatment has been limited by the emergence of intrinsic or acquired drug resistance of the tumor to chemotherapeutic agents. Multiple mechanisms are known to be involved in drug resistance, including increased drug efflux, enhancement of DNA repair and alterations of the genes and the proteins involved into the control of apoptosis. Although newly designed chemothrapeutic drugs and strategies have developed and applied, the outcomes are disappointed, which is mainly caused by the development of drug resistance in cancer cells upon drug treatment. Therefore, the investigation of the mechanism underlying the formation of drug resistance has instuctive importance not only to help us intervening the drug resistance but promoting the drug effectiveness.Methods1 In this study, we firstly establised two drug resistant cell lines:the doxorubicin-resistant human lung epithelial carcinoma cell line A549 (A549/DOX) and 5-fluorouracil-resistant human colon carcinoma cell line HCT-8 (HCT-8/5-FU), and expression level of Akt. pAkt were detected by Western blot analysis.2 We also found that the interaction between Akt and XRCC1 by immuniprecipitation and confocal microscopy.3 Cells were transiently transfected with pcDNA3.1(-)-Flag-XRCC1, pcDNA3.1-Flag-XRCC 1 (S518DT519DT523D).4 Resistant cells were pretreated with Akt inhibitor MK-2206 and BER inhibitor ABT-888. Apoptotic ratios were detected by flow cytometry.ResultsActivation of AKT not only supports tumor growth and progression but also contributes to tumor-cell evasion of the cytotoxic effects of cancer therapy through many avenues including the promotion of anti-apoptosis, proliferation, and migration and regulation of the cell cycle. We observed that Akt was highy activeted in these two cell lines. Additionally, we found that resistant cells have enhanced base excision repair and reduced DNA damages upon drug treatment.. We therefore studied the relationshp between Akt activation and enhanced BER.BER is a highly conserved system from bacteria to humans and is accomplished by the coordination of a series of enzymatic reactions. It has been demonstrated that BER enzymes must be tightly regulated because imbalances in BER lead to genomic instability. The X-ray repair cross-complement group 1 protein (XRCC1) has a crucial role as scaffolding protein associated with the enzymatic factors such as polyadenosine diphosphate (ADP)-ribose polymerase, DNA ligaseⅢ, DNA polymerase β and PARP. In contrast to the wild type cells, the resistant cells present a high basal level of XRCC1 and the phosphor-XRCCl. We also found that Akt interacts with XRCC1 by immuniprecipitation and confocal microscopy. Transient overexpression of XRCC1 or phosphor-XRCC1 ameliorated the genotoxic agents-elicited DNA damage and inhibited the drug-induced apoptosis. Finally, we demonstrated that the pretreatment of resistant cells with Akt inhibitor MK-2206 and BER inhibitor ABT-888 resulted in the improved anti-cancer agents-induced apoptosis. Taken together, our data highlight the role of Akt-mediatd BER in the drug resistance in cancer cells.ConclusionIn this study, using A549 derived DOX-resistant A549/DOX cells and HCT-8 derived 5-fluorouracil-resistant HCT-8/5-FU, we elucidate in both cell lines a mechanism of acquired resistance in both cell lines. Herein, we provide evidence, for the first time, Akt interacts wih BER scaffolding protein XRCC1, and promotes its phosphorylation and its protein level, which results in the enhanced BER and causes the development of drug resistance due to the inhibition of genotoxic drug induced apoptosis. Our study may help us to promote the efficacies of chemotherapeutic durgs and to design strategies to circumvent drug resistance. |
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