Study On The Molecular Mechanism And Regulation Of Quinocetone-induced Cytotoxicity And Apoptosis In HepG2Cells

Quinocetone, a new quinoxaline1,4-dioxide derivative with antimicrobial, antidiarrhea and growth-promoting efficacy, has been classified as China’s first class of new veterinary drug and widely used in swine industry. Similar drugs, such as carbadox and olaquindox, due to their genotoxic and potentially carcinogenic effects, have been banned or restricted to use as feed additives in many countries around the world. Having the same core chemical structure, quinocetone showed genetic toxicity in vivo and in vitro, but its mechanism of toxicity was still unclear. In this study, human hepatoma HepG2cells with the metabolic activity in vitro were used as a model to study quinocetone’s toxicity and its mechanism of action, analyze quinocetone’impact on the genome-wide expression profiling of HepG2cells and investigate the molecular mechanisms regulating quinocetone-induced apoptosis in HepG2cells, in order to provide a theoretical basis for assessing the safety of quinocetone for food animals and human.MTT results showed quinocetone inhibited the proliferation of HepG2cells. Its IC50values for24h and48h were9.2μg/ml and6.7μg/ml, respectively. LDH release assay indicated quinocetone could induce the release of lactate dehydrogenase from HepG2cells. Flow cytometry found quinocetone significantly arrested cell cycle and low concentrations of quinocetone arrested cells in S phase, while high concentrations of quinocetone arrested cells in G0/G1phase. Quinocetone-induced apoptosis in HepG2cells were confirmed using optical microscope observation, Hoechst33342/PI staining and Annexin V-FITC/PI double staining methods. The results of MDC staining, transmission electron microscopy and flow cytometry proved quinocetone induced autophagy in HepG2cells. All the above results suggested that quinocetone was cytotoxic.Using specific fluorescent dyes DCFH-DA and DHE to detect intracellular ROS level changes, the results showed quinocetone increased the intracellular ROS levels in a dose-dependent manner. Simultaneously, the increased levels of8-OHdG, an intracellular oxidative DNA damage marker, were detected using immunocytochemistry after quinocetone exposure. NAC and GSH reduced ROS levels, improved the vitality of the cells, eased the disruption of mitochondrial membrane potential and inhibited quinocetone-induced apoptosis. Western blotting results showed NAC inhibited the cleavage of Caspase-8, Caspase-3, Bid and PARP-1precursor proteins, the elevation of Bax/Bcl-2ratio, as well as the phosphorylation levels of JNK and p38MAPK. These results showed quinocetone’s cytotoxicity might be related to oxidative stress in HepG2cells.Utilizing the whole human genome microarray of Agilent company to detect genome-wide expression profiling changes of HepG2cells treated with quinocetone, data showed there were6202differentially expressed genes, including3909upregulated genes and2293downregulated genes. These genes belonged to4877GO entries and903pathway entries. The results showed that quinocetone’s cytotoxicity involved a variety of metabolic and signaling pathways, including cell metabolism, cell cycle, DNA replication and repair, apoptosis, oxidative stress, MAPK pathway, p53signaling pathway, mTOR signaling pathway, et al.. Among them, the apoptosis-related differentially expressed genes were categorized into the extrinsic and intrinsic apoptosis pathways, and also involved in the regulation of DNA damage and repair as well as cell proliferation-related gene transcription.Finally, the molecular regulation mechanisms of apoptosis in quinocetone-treated HepG2cells were studied. TUNEL assay results showed quinocetone caused DNA fragmentation. Activation of Caspase-8,-9and-3in quinocetone-induced apoptosis were detected by Western blotting, RT-PCR and caspase activity assay. Flow cytometry results also showed pan caspase inhibitor and Caspase-3,-8and-9specific inhibitors significantly inhibited quinocetone-induced apoptosis. The mitochondrial apoptotic pathway relevant assay results found quinocetone disrupted mitochondrial membrane potential, promoted the release of cytochrome C, upregulated the mRNA and protein levels of Bax, downregulated the mRNA and protein levels of Bcl-2, and cleaved PARP-1protein. Western blotting results showed that TNFR1, TNF-a, Fas and FADD protein contents were increased and Bid was cleaved. The elevated mRNA levels of TNFR1, TNF-a and Fas were determined by RT-PCR. TNFRl/Fc chimera inhibited quinocetone-induced apoptosis, indicating that the death receptor pathway was also involved in the regulation of quinocetone-induced apoptosis. The protein levels of p53, p21, p-p38and p-JNK were increased, indicating their involvement in the regulation of quinocetone-induced apoptosis. Taken together, quinocetone induced apoptosis in HepG2cells via activation of caspase, interaction of TNF-a and TNFR1, modulation of the protein levels of Bid, Bax and Bcl-2, and involvement of the regulation of p53, p38and JNK.In summary, quinocetone was cytotoxic to HepG2cells. The mechanism of quinocetone-induced cytotoxicity might be associated with oxidative stress. Quinocetone affected a variety of metabolic pathways within HepG2cells and induced apoptosis through mitochondrial and death receptor pathway.