| Quinocetone(3-methyl-2-cinnamoylquinoxaline-1,4-dioxide,QCT)is a novel veterinary drug made in Lanzhou Institute of Husbandry and Pharmaceutical Science of CAAS.QCT is a new member of quinoxaline 1,4-dioxide(QdNO)family and has the antiseptic,antidiarrheal and growth promoting effects.Thus it has been approved and widely used as an animal feed additive in China since 2003.Carbadox and olaquindox,members of QdNOs,have been prohibited in Europe since 1998 owing to their carcinogenic and mutagenic effects.However,investigations indicate that QCT has potential toxicity due to the fact that it shows cytotoxicity,genotoxicity,hepatotoxicity,nephrotoxicity and immunotoxicity in vitro and in vivo.Although our previous investigations have showed that QCT induced mitochondrial apoptosis,the molecular mechanism remains unclear.This study was aimed to elucidate the potential molecular mechanism of QCT-induced mitochondrial apoptosis in HepG2 cells,which might provide insights in assessing the safety of QCT in animals and consumers.In order to investigate the role of VDAC1/2 oligomerization in QCT-induced mitochondrial apoptosis,we pretreated HepG2 cells with VDAC inhibitor 4,4-diisothiocyano stilbene-2,2-disulfonic acid(DIDS)and then treated cells with QCT.By MTT assay and flow cytometry analysis,we found that DIDS partly compromised QCT-induced cell viability decrease(from 34.1%to 68.5%)and mitochondrial apoptosis(from 34%to 9.8%)accompanied by abating VDAC 1/2 oligomerization,cytochrome c(Cyt C)release and the expression levels of cleaved caspase-9,-3 and poly(ADP-ribose)polymerase(PARP).Meanwhile,overexpression VDAC1/2 exacerbated QCT-induced VDAC1/2 oligomerization and Cyt C release.These date demonstrate that QCT promotes VDAC1/2 oligomerization and induces Cyt C release to cytoplasm,which may lead to mitochondrial apoptosis.To elucidate the role of Bak1 in QCT-induced mitochondrial apoptosis,firstly,we incubated HepG2 cells with QCT and then examined the protein expression level of Bakl.The result showed that QCT increased the protein expression level of Bakl in a dose-dependent manner.Further,we constructed the pCMV-Flag-Bak1 plasmid and overexpression Bak1 in HepG2 cells.By flow cytometry and western blot analysis,we found that overexpression Bakl aggravated QCT-induced mitochondrial apoptosis accompanied by decreasing the expression levels of caspase-9,-3 and increasing the expression level of cleaved-PARP.Consistently,when the expression level of Bakl was evidently inhibited by transfecting siRNA,QCT-induced mitochondrial apoptosis was reduced.Finally,we detected the formation of Bak1 oligomerization by EGS.The results showed that QCT promoted the formation of dimers,trimers and polymers of Bak1.In addition,when overexpression Bak1,it exacerbated QCT-induced formation of Bak1 oligomerization.These results indicate that QCT causes the formation of Bak1 oligomerization and then induces mitochondiral apoptosis.To explore the role of Wnt/?-catenin signaling pathway in QCT-induced mitochondrial apoptosis,we incubated cells with different concentration of QCT.The results demonstrated that QCT caused a dose-dependent reduction in the protein expression levels of Wntl and ?-catenin.In addition,QCT downregulated the protein content of ?-catenin both in nucleus and cytoplasm.In order to consolidate our observation,we pretreated cells with Wnt/?-catenin signaling activator LiCl.The results showed that the cell viability was raised(from 48.3%to 76.1%)and the rate of apoptotic cells were reduced(from 42%to 26%)accompanied by abating MMP decreasing and the expression levels of cleaved caspase-9,-3 and PARP by LiCl.Then we analyzed the mRNA expression level of ?-catenin by RT-PCR and result showed that the mRNA expression level of ?-catenin was increased 1.8 folds when comparing QCT treatment group to control group.Finally,we pretreated cells with MG132,a proteasome inhibitor and found that the protein expression level of ?-catenin was compromised comparing to QCT treatment group.These date suggeste that QCT-induced mitochondrial apoptosis by suppressing Wnt/?-catenin signaling.At the same time,QCT downregulated the protein expression level of ?-catenin through the proteasome dependent pathway.Next,we investigate whether ROS,which is produced by QCT,participates in regulation of QCT-induced VDAC1/2 and Bakl oligomerization and the downregulation of Wnt/?-catenin signaling pathway.We co-treated ROS scavenger NAC with QCT and then examined the level of intracellular ROS.By flow cytometry analysis,we found that NAC apparently abolished the production of ROS induced by QCT(from 46%to 9.6%).Meanwhile,NAC markedly blocked QCT-induced VDAC1/2 and Bakl oligomerization.NAC also partly recovered the expression levels of Wntl and ?-catenin which were suppressed by QCT.These results reveal that ROS,produced by QCT,mediates QCT-induced VDAC1/2 and Bakl oligomerization and the downregulation of Wnt/?-catenin signaling pathway.Lastly,we want to figure out the relationship among VDAC1,Bak1 and ?-catenin in QCT-induced mitochondrial apoptosis.Firstly,we built a Bakl-induced mitochondiral apoptosis model in 293T cells.In this model,we revealed that overexpression Bak1 hampered the protein expression level of ?-catenin and Wnt/?-catenin signaling pathway,while VDAC inhibitor DIDS absolutely abrogated the expression level of Bakl and Bakl-induced mitochondrial apoptosis.When knockdown VDAC1 and Bakl by siRNA separately in HepG2 cells,the protein expression level of P-catenin suppressed by QCT was raised.However,overexpression P-catenin has no effect on QCT-induced VDACI and Bakl oligomerization.These data indicate that VDAC1 and Bak1 may be in the upstream of ?-catenin in QCT-induced mitochondrial apoptosis.In conclusion,our results reveal that QCT mediates ROS-dependent promotion of VDAC 1/2 and Bakl oligomerization and suppression of Wnt/p-catenin signaling pathway,finally leads to mitochondrial apoptosis in HepG2 cells.In this process,VDAC1 and Bakl may be in the upstream of?-catenin. |
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