Role Of ROS And DNA-PKcs/Akt Signaling Pathway In Hydroquinone-induced Cell Damage And Cell Apoptosis

It was generally recognized that benzene metabolites rather than benzene itself mediate the multiple biological effects of benzene on the hematopoietic cells to give rise to bone marrow toxicity. Hydroquinone, a quinone metabolite of benzene, can induce cells damage and apotosis.Recent studies show that, DNA-PKcs is an important damage repair gene, play a key role in DNA double-strand breaks in non-homologous end joining (NHEJ), V(D)J recombination and the maintenance of telomere structure, involved in transcription and cell apoptosis process; is also involved in radiotherapy and chemotherapy tolerance in tumor cells. Akt signaling pathway has been implicated in a wide range of cellular functions involving cell growth, proliferation, apoptosis, metabolism, cell migration and so forth. However, whether DNA-PKcs/Akt signalling pathway plays a role in hydroquinone-induced cell damage and cell apoptosis in K562cells is unknown. The present study investigated the role of DNA-PKcs/Akt and its downstream pathways in hydroquinone-induced damage and apoptosis in K562cells.Part1The Role of ROS and DNA-PKcs/Akt Signaling Pathway in Hydroquinone-Induced Damage and Apoptosis in K562CellsObject:The aim of the present study is to elucidate the role of ROS and DNA-PKcs/Akt in hydroquinone-induced K562cells damage and apoptosis. Methods:CCK-8assay were carried out to measure cell growth induced by hydroquinone. The AnnexinV-FITC/Propidium Iodine assays for apoptosis were used to measure cell apoptosis induced by hydroquinone treatment by flow cytometry. Molecular probes of CM-H2DCFDA and DHE were applied to determine ROS production by hydroquinone treatment using flow cytometry. DNA-PKcs and Akt mRNA levels were monitored by real time PCR. Immunofluorescent staining was used to detect protein expression of DNA-PKcs. Western Blotting Assay were performed to measure protein expression of DNA-PKcs, Akt, phospho-Akt Ser473. Results:CCK-8assay revealed that hydroquinone has strong inhibition on K562cells in concentration-dependent and time-dependent manners. In hydroquinone-induced cell damage and apoptosis, with the concentration varying from0to 50μM, ROS generation and cells apoptosis rate also increased in a concentration-dependent manner; there was a positive correlation between them; When it reached100μM, ROS production decreased, the highest rate of apoptosis was observed. K562cells treated0、3、6、12and24h by50μM hydroquinone, with the time varying from0to12h, ROS generation increased in a time-dependent manner; When it reached24h, ROS production obviously decreased. From0to100μM, mRNA expression of DNA-PKcs and Aktl induced by hydroquinone were upgraded in concentration-dependent manners in K562cells, and protein expression of DNA-PKcs and P-Akt were also the same; however, Akt did not alter. Conclusions:These results demonstrate that hydroquinone-induced ROS and DNA-PKcs/Akt signaling pathways are associated with cell damage and cell apoptosis in K562cells. Part2Inhibition of DNA-PKcs/Akt Signaling Pathways by NU7026Sensitizes Human Leukemic K562Cells to Hydroquinone-Induced ApoptosisObject:To investigate the role of NU7026in DNA-PKcs/Akt signalling pathway in hydroquinone-induced cell apotosis. Methods:Direct viable cell counting assay were carried out to measure cell growth induced by hydroquinone. The AnnexinV-FITC/Propidium Iodine assays for apoptosis were used to measure cell apoptosis induced by hydroquinone treatment by flow cytometry. Molecular probes of CM-H2DCFDA and DHE were applied to determine ROS production by hydroquinone treatment using flow cytometry. DNA-PKcs mRNA levels were monitored by real time PCR. Immunofluorescent staining were used to detect protein expression of DNA-PKcs. Western Blotting Assay were performed to measure protein expression of DNA-PKcs, Akt, phospho-Akt (phospho-Akt Ser473), Bax, Bcl-2and caspase3. Results:In this study, we used a selective inhibitor of DNA-PKcs,2-(morpholin-4-yl)-benzo[h]chomen-4-one (NU7026), to co-treat with0,10,25,50and100μM hydroquinone to analyze the molecular events and pathways in hydroquinone-induced cells apoptosis. Inhibition of DNA-PKcs by NU7026markedly potentiated the growth inhibition and apoptotic effects of hydroquinone in K562cells in a dose-dependent manner. Treatment with NU7026did not alter the production of reactive oxygen species and oxidative stress by hydroquinone, but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phosphorylation of Akt to activate Akt, whereas co-treatment with NU7026prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026exhibited synergistic effects on promoting apoptosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone hematotoxicity in which it coordinates pathways of DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage. Conclusions:NU7026sensitizes human leukemic K562cells to hydroquinone-induced apoptosis, by inhibition of DNA-PKcs/Akt signaling pathways, leading to Bcl-2downregulation, Bax and caspase3upregulation.