A BIOCHEMICAL STUDY TO EVALUATE THE ROLE OF APURINIC/APYRIMIDINIC ENDONUCLEASE 1 (APE1) IN LUNG CANCER PROGRESSION
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Date
2018
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Central University of Punjab
Abstract
Globally, lung cancer has the highest incidence and mortality rate. Environmental factors such as chlorpyrifos (CP) and monocrotophos (MCP), widely used organophosphate pesticides (OPPs), and are reported to be involved in the process of lung carcinogenesis. Present study attempts to investigate the genotoxic potential of CP and MCP, and investigates the oxidative DNA base damage response evoked by CP and MCP in non-small cell lung carcinoma (NSCLC) A549 and NCI-H1299 cells. A549 and NCI-H1299 cells were exposed to a range of concentration of CP and MCP at different time points; cell viability and reactive oxygen species (ROS) generation were measured to select the non-toxic dose. In order to establish whether CP and MCP treatment can initiate the DNA repair and cell survival signalling pathways in A549 cells, semi-quantitative RTPCR, qRT-PCR and Western blotting techniques were used to investigate the mRNA and protein expression levels of DNA base excision repair (BER)-pathway enzymes, transcription factors (TFs) involved in pro-survival mechanisms and apoptosis-related factors. Significant increase in ROS generation was observed when cells were exposed to low and moderate doses of CP and MCP. A549 cells displayed a dose-dependent accumulation of DNA damage apurinic/apyrimidinic (AP) sites after MCP treatment. Cellular responses to CP and MCP-induced oxidative stress resulted in alterations in the mRNA and protein expression of BER-pathway enzymes viz. PARP1, APE1, XRCC1, DNA pol β and DNA ligase III α at different time points, which indicates imbalanced BER-pathway. OPPs treatment resulted in alterations in TFs viz. Nrf2, c-jun, phospho-c-jun (p-c-jun); inducible nitric oxide synthase (NOS2) and PCNA, which may lead to cell proliferation and promotion of carcinogenic events. Possible protein-protein interactions of BER-pathway’s key enzyme APE1 were also analyzed using STRING database, and intrinsically disordered (ID) regions of APE1 and TFs AP- v 1, NF-ĸB and Nrf2 were determined using PONDR analysis. PONDR prediction score and presence of ID regions in N-terminal segment of APE1, middle and Cterminal segments of AP-1, NF-ĸB and Nrf2 advocates for the possible direct protein-protein interactions, to be required for redox-regulatory function of APE1. Altered subcellular localization of APE1 and colocalization between APE1 and cjun; APE1 and p-c-jun; and APE1 and NF-ĸB were analyzed in A549 cells and; between APE1 and c-jun and, APE1 and Nrf2 were analyzed in NCI-H1299 cells following CP and MCP exposure using immunofluorescent confocal laser scanning microscopy. The present study results indicate that CP and MCP induces translocation of APE1 within the cytoplasm at an early time point of 6 hr; whereas it promotes nuclear localization at 24 hr, which suggests that APE1 subcellular localization is dynamically regulated in response to the oxidative stress. Furthermore, nuclear colocalization of APE1 and TF c-jun, APE1 and NF-ĸB and, APE1 and Nrf2 was also observed in response to CP and MCP treatment in A549 and NCI-H1299 cells, which indicates for the APE1-mediated redox regulation of TFs c-jun, NF-ĸB and Nrf2. However, no significant nuclear colocalization was observed for APE1 and p-c-jun indicating the role of phosphorylation on c-jun on Ser63 (p-c-jun) to act as a “switch off” form from the redox complex after the regulation. Immunoprecipitation (IP) was also performed to identify the interacting protein partners of APE1 under the influence of CP and MCP exposure; result indicated that MCP could moderately induce the interaction between APE1 and Nrf2, and APE1 and c-jun. Therefore, the present study suggest that CP and MCP-induced oxidative stress alters BER-pathway and mediates cell-survival signalling mechanisms via APE1 regulation thereby promoting lung cancer cell survival and proliferation.
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Keywords
Lung Cancer; Monocrotophos; Chlorpyrifos; Oxidative stress; Base excision repair (BER); APE1; Nrf2; c-jun; NF-ĸB.
Citation
Thakur, Shweta, Mantha, Anil K. and Dhiman, Monisha (2018) A BIOCHEMICAL STUDY TO EVALUATE THE ROLE OF APURINIC/APYRIMIDINIC ENDONUCLEASE 1 (APE1) IN LUNG CANCER PROGRESSION