Gecko Active Components Induce Apoptosis Via The ROS Pathway In Hepatocellular Carcinoma

OBJECTIVE To investigate the induction of apoptosis by gecko active components（GACs）in human human hepatocellular liver carcinoma HepG2 cell line is mediated through reactive oxygen species（ROS）generation,and to explore its molecular mechanism.METHODS Human hepatocellular carcinamo（HepG2）cells were routinely cultured in vitro and subsequent experiments were performed with HepG2 cells in logarithmic growth phase.The total experiment was divided into three parts.（1）The effect of GACs on apoptosis in HepG2 cells.After HepG2 cells were treated with a various rank of GACs concentrations（0.1,0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5mg/mL）respectively,the effect of GACs on HepG2 cell proliferation was examined by MTT.Then,according to MTT results,HepG2 cells were treated with different concentrations of GACs for 24h,4’,6-diamidino-2-phenylindole（DAPI）fluorescence staining was performed to observe nucleus morphological changes of HepG2 cells.Western blot were used to detect the levels of apoptosis-related proteins.The apoptosis rate was detected by flow cytometry.（2）The effects of GACs on the level of reactive oxygen species,calcium and mitochondrial membrane potential in HepG2 cells.The levels of ROS,calcium level and mitochondrial transmembrane potential（MTP）were measured by flow cytometry.In order to futher verify whether the GACs induce apoptosis of HepG2 cells by ROS,the experiment was divided into blank control group,N-acetylcysteine（NAC）experimental group,NAC and GACs combined experimental group,GACs experimental group.The levels of ROS and apoptosis were analyzed by flow cytometry.Western blot were used to detect the levels of apoptosis-related proteins.（3）The effects of GACs on PERK signaling pathway in HepG2 cells.Western blot and q-PCR were used to detect the protein and mRNA levels of PERK pathway related proteins.In order to futher verify whether the apoptosis of HepG2cells was induced by ROS activation of PERK signaling pathways.The expression level of PERK signaling pathway-related factors were performed by Western blot and q-PCR methods after pretreatment with NAC.RESUITS（1）The effect of GACs on apoptosis in HepG2 cells.The results showed that GPM could inhibit the proliferation of HepG2 cells.The IC₅₀ values of HepG2 cells were 0.26,0.21,0.19 mg/mL with treated GACs for 24h,48h,72h,respectively.According to MTT results,0.15,0.225,0.3375 mg/mL were selected as low,medium and high dose groups respectively.The morphology of nucleus was irregular under flurescence microscope after GACs treatment,showing nuclear pyknosis and nuclear fragmentation.The results of Western blot showed that the expression levels of apoptosis-related proteins in low,medium and high dose groups were significantly higher than that of blank control group.Flow cytometry showed that the early apoptosis rates of HepG2 cells in low,medium and high dose groups were6.46%±1.78%,11.36%±1.25%,24.29%±2.18%,respectively.Compared with the blank control group,the average early apoptotic rate was 1.28%±0.18%,the apoptotic rate was significantly increased（P<0.01）.（2）The effects of GACs on the level of reactive oxygen species,calcium and MTP in HepG2 cells.Flow cytometry showed that the average flurescence intensity of ROS in HepG2 cells treated with low,medium and high dose GACs were 85.81±2.56,268.91±2.34,1741.5±5.64,respectively,compared with the blank control group,the average flurescence intensity of ROS was16.61±1.12,the content of ROS in GACs treated group was significantly higher.The average fluorescence intensity of calcium in HepG2 cells were 9.67±0.98,12.30±1.07,94.80±2.75,910.99±5.31 in blank control group,low,medium and high dose groups,respectively,the calcium content in GACs treated group was significantly higher than in the blank control group.The MTP of HepG2 cells in different concentrations of GACs were（23.78±1.2）×10^-2,（18.27±1.5）×10^-2,（16.49±2.1）×10^-2,respectively,compared with in the blank control group（27.02±1.8）×10^-2,the level of MTP in the experimental group decreased significantly（P<0.01）.Compared with the GACs experimental group,the level of ROS,the expression of apoptosis-related protein and the apoptosis rate was significantly decreased in NAC and GACs combined experimental group.（3）The effect of GACs on PERK pathway in HepG2 cells.The Western blot and q-PCR results showed that GACs could significantly increased the expression of PERK pathway related proteins（P<0.05 or P<0.01）.Compared with the GACs experimental group,the expression of PERK pathway related was significantly decreased in NAC and GACs combined experimental group（P<0.05 or P<0.01）.CONCLUSION（1）GACs could inhibit proliferation of HepG2 cells and lead to the apoptosis of HepG2 cells.（2）GACs could increase the content of ROS in HepG2cells,while NAC could reduce the level of ROS in HepG2 cells and inhibit the apoptosis induced by GACs.The results suggested that GACs might induce the apoptosis of HepG2 cells through ROS.（3）GACs could activate the PERK signaling pathway in HepG2 cells.The results indicated that GACs might induce apoptosis of HepG2 cells through ROS activation of PERK signaling pathway.