The Molecular Mechanism Of Quinocetone-induced Programmed Cell Death In HepG2 Cell Line

Quinocetone is an I class new veterinary drugs developed by Lanzhou Institute of Animal Husbandry and Veterinary Drugs, Chinese Academy of Agricultural Sciences. It has altered olaquindox and carbadox to be the widely used animal feed additive during Animal husbandry and aquaculture owing to its non-toxic, non-mutagenic and non-carcinogenic features, and proving to be effective in improving the meat production of livestock and poultry.In the 90s of last century, a series of routine toxicity test proved the safety of quinocetone. Recently, the research from our lab found out the quinocetone-induced apoptosis and the changes of apoptotic pathways of itself, and clarified part, if not all, of the molecular mechanism of quinocetone-induced cell death.The result of MTT assay showed that when ML-7 at a non-toxicity concentration added to the quinocetone-incubated HepG2 cells, the cell death ratio increased. The flow cytometry assay of HepG2 cells stained by rhodamine 123 showed that, ML-7 enhanced the quinocetone-induced mitochondria outer membrane potential decrease. Western blot results showed that, ML-7 reinforced the quinocetone-induced apoptotic intrinsic and extrinsic pathways. These results suggest that ML-7 could enlarge the quinocetone-induced cell death. Moreover, the western blot results showed that ML-7 activated the quinocetone-induced MAPKs pathways, but inhibited the quinocetone-induced Akt pathway. To take a step forward, we used the MAPKs inhibitor and the Akt activator during quinocetone and ML-7 combined treatment. The western blot results showed that all the MAPKs and Akt pathways participated in the quinocetone-induced apoptosis. In conclusion, quinocetone induces cell death through MAPKs and Akt pathways modulated apoptosis. Specifically, quinocetone induces apoptosis through p38 MAPK pathway and ERK, JNK and Akt pathways inhibited the quinocetone-induced apoptosis.Autophagy also called type Ⅱ programed cell death. The GFP-LC3 puncta calculation assay and western blot of LC3 results showed that quinocetone induced autophagy in HepG2 cells.while, western blot results of BiP and CHOP, and RT-PCR result of sec24D, HerpUD, and BiP showed that quinocetone induced ER stress in HepG2 cells. Moreover, the western blot result of ATF6, DAPK1, and p-RLC, and the Immunofluorescence results of ATF6 and mAtg9a subcellular locations showed that quinocetone induced autophagy through ER stress. To confirm this result, shRNA of ATF6 and DAPK1 had been used to exam the change of the autophagic flux. The result showed that quinocetone-induced autophagy modulated by the activation of ATF6 and DAPK1.Mitophagy is a kind of selective autophagy that specifically sorting damaged mitochondria for degradation. The former experiment showed that quinocetone could induce mitochondria injury and the mitochondria injury also play central role in intrinsic apoptosis. While mitophagy is an important way to clean the damaged mitochondria, the relationship between mitophagy and quinocetone-induced mitochondria injury has been an important issue, when the quinocetone-induced autophagy has been proved. The western blot results showed that the core player of mitophagy parkin and PINK1 decreased in an time-and dose-dependent way in quinocetone-treated HepG2 cells. And the parkin and PINK1 changes in CCCP-treated HepG2 cells which used as positive control showed the same result. These results suggest that quinocetone-induced mitochondria injury caused the initiation of mitophagy. While the western bolt result of mitochondria outer membrane protein Tom20 and mitochondria inner membrane protein Tim23 and COX IV showed that during quinocetone or CCCP treatment the protein level of Tom20 and Tim23 had increased, the protein level of COX IV had decreased in HepG2 cells. The western blot results of various mitophagy receptors showed that the protein level of OPTN, TAX1BP1 and FUNDC2 had no significant changes, increased and decreased, respectively, in quinocetone-treated HepG2 cells. On the contrary, in the CCCP-treated HepG2 cells the protein level of OPTN, TAX1BP1 and FUNDC2 had increased, decreased and no significant changes, respectively. These results suggested that the quinocetone-induced mitophagy initiation is different from the classic situation which induced by the CCCP. Taking a step forward, parkin-Flag vector has been used to observe the quinocetone-induced mitophagy. The western blot results showed that the changes of mitochondria membrane protein and autophagy-related protein induced by quinocetone and CCCP had all been extended. The mitochondria membrane permeabilization (MOMP) detected by Rhodamine 123 staining showed that both quinocetone and CCCP treatment caused MOMP decrease, but the quinocetone-induced MOMP was much lighter than CCCP-induced MOMP. The mito-Red, which is a kind of mitochondria dye, staining showed that the CCCP caused an obvious decrease in mitochondria quantity, but the quinocetone-treat HepG2 cells showed no significant decrease. These results suggested that the quinocetone-induced mitophagy doesn’t sufficient to clear most of the mitochondria in HepG2 cells.In conclusion, the quinocetone induced apoptosis, the upregulation of autophagy, and the initiation of mitophagy in HepG2 cell line. And programmed cell death is essential for quinocetone-induced cytotoxicity.