Anti-hepatocarcinoma Mechanism Study Of New Sophoridinic Derivatives And The Regulation Mechanism Of GSK-3β On Hepatocarcinoma

Sophoridine is a kind of quinolizidine alkaloid isolated from Sophora alopecuroides L,which is one plant belongs to legume.And sophoridine has anti-tumor,anti-inflammatory and many other pharmacological activities.As one new drug belongs to 1.1 and developed by our Chinese,sophoridine was approved by the SFDA in 2005,mainly for choriocarcinoma,malignant hydatidiform mole and other malignant trophoblastic tumors.In the long history of finding anti-tumor active drugs from traditional Chinese medicine,we found that although sophoridine had good pharmacokinetic properties and significantly stable clinical anti-tumor effects,there were problems like narrow anti-tumor spectrum and neurotoxicity also existed.So far,there were few studies about the structure optimization and activity analyse of sophoridine.In our experiments,we designed the structure of sophoridine from tetracyclic core to tricyclic core,and analyzed the anti-tumor activity and mechanisms of the sophoridinic derivatives preliminarily.Firstly,human hepatocarcinoma HepG2 cell was selected as the target screening system.In the process of screening more than 200 sophoridinic derivatives synthetised by ourself in vitro,the derivative with high stability,high safety and good anti-tumor activity named IMB-6G was screened out finally.A few studies had shown that induction of apoptosis by activating endoplasmic reticulum stress is the anti-tumor mechanism of many drugs.In our experiment,we mainly investigated if IMB-6G played the role of anti-tumor through endoplasmic reticulum stress pathways.HepG2 and SMMC7721 were selected as the cell lines.By MTT,western blot,flow cytometry,confocal and many other methods,we confirmed that IMB-6G could induce the mitochondrial-dependent apoptosis in hepatocarcinoma cells,and then examined the expression of chaperone GRP78/Bip and pro-apoptotic factor CHOP which were important for endoplasmic reticulum stress pathways.WB results showed that the expressions of Bip and CHOP were all increased in time-dependent and concentration-dependent manners,and RT-PCR results showed that their mRNA levels were also significantly increased.Research suggested that the increasing CHOP expression was a direct result of activated PERK-eIF2α pathway.We saw significantly increased phosphorylation of PERK and eIF2a after IMB-6G’s treatment,thus proving that PERK was activated by IMB-6G.At the same time,the dual-luciferase gene reporter also detected that the CHOP promoter activity was increased.Then silencing the expression of PERK and CHOP by siRNA,the apoptosis induced by IMB-6G was significantly inhibited.So it was claimed that the activation of PERK and CHOP were indispensable for IMB-6G playing a role in apoptosis.In the IRE1α pathway,we detected IMB-6G could activate IRE1α firstly.As the formation of TRAF6-ASK1-JNK complex is the main cause of apoptosis,WB results showed the activation of ASK1 and JNK by IMB-6G also.Using siRNAs and special inhibitors to inhibit the activity of ASK1 and JNK,the anti-tumor effect of IMB-6G was reduced partially.At the same time,by using fluorescence microscopy,the specific XBP1 splice exists as the by-product of activated IRE1α pathway was detected.The ATF6 pathway,which plays a major role in cell protection,did not display any significant changes after IMB-6G.In conclusion,our results suggest that the sophoridinic derivative IMB-6G promoted apoptosis by activating both PERK and IRE1α pathways,and their activation of PERK-CHOP,IRE1α-ASK1-JNK signaling pathways played important roles in apoptosis.Autophagy is a self-digestive process that encapsulates cytoplasmic contents in bilayer structures and then transports to lysosome in order to degrade and recirculate.This is a kind of self-protection mechanism of cancer cells for resisting stress,and contributing to tumorigenesis and drug resistance.Inhibition of its activity may produce anti-tumor activity.We found one compound named 6b had a good anti-tumor activity,but if 6b through the autophagy pathway to play its anti-tumor activity was not clear.So we purposed to discuss the relationship between autophagy and its anti-tumor effect.Firstly,through the LC3 aggregation experiment,it was found that 6b could cause significant aggregation of LC3.At the same time,the level of LC3-Ⅱ was significantly increased showed by WB,but it was impossible to judge whether 6b enhanced autophagy.As a recognized marker of autophagy,p62 involves in the whole process of autophagy and eventually degrades in lysosomes.Our experiments demonstrated that 6b could increase the protein level of p62 in both concentration-dependent and time-dependent manners,which suggested that compound 6b inhibited cell autophagic activity.Subsequently,LC3 turnover experiment was conducted using the inhibitor of CQ and Rapamycin.The LC3-Ⅱprotein level was not further increased after the combination of CQ,indicating the LC3-Ⅱthat degraded by autophagy was rarely.When combined with Rapamycin,LC3-Ⅱ was higher than that of 6b only.So it was further confirmed that compound 6b might inhibit the degradation of autophagosomes.Furthermore,we used the dual-fluorescence plasmid mCherry-EGFP-LC3 to determine the change of autophagic activity.Taking the GFP’s advantage that it is unstable and easy to quench in the lysosomal acidic environment,we tried to find the difference between red fluorescence of mCherry and green fluorescence of GFP.The confocal microscopy result showed that the red fluorescence and green fluorescence in the cells were increased both after 6b,and the changes were basically synchronized.So it seemed that even though the autophagosomes increased significantly,the autolysosomes did not strengthen.In the final experiments,we used the lysosomal dye LysoTracker to mark lysosomal activity.It was obviously weakened after 6b,indicating that 6b inhibited the acidity of lysosomes and reduced the degrading ability.The conclusion had no difference with the increasing protein level of LC3-II and p62 that we observed early.Meanwhile,the results of mCherry-GFP-LC3 fluorescence also speculated that lysosomal activity may be inhibited by 6b.Summarying our study,compound 6b destroyed the degradation of lysosomes by inhibiting the acidity.Then the function of autophagy supplying for small moleculars and energy was disrupted,thereby inhibiting cancer cells’ proliferation.China is one of the highest regions of hepatocarcinoma in the world.New cases of hepatocarcinoma accounted for about 55%of the world,and the death toll accounted for 51%of the global death.Deep studies of hepatocarcinoma are helpful for early detection and treatment.GSK-3β as a Ser/Thr kinase is involved in the cell cycle,cell differentiation,apoptosis and many other biologic processes.At the same time,a number of studies had shown that in many tumors,GSK-3β showed abnormal expression.But how to promote the occurrence and development of GSK-3β is not clear.So our goal is to clarify its role in hepatocarcinoma.As a member of MAP3K family,ASK1 is a pro-apoptotic factor in cells,and playing an important role in stress response such as ROS.Our experiment selected H2O2 as the stimulating factor and human hepatocarcinoma cells HepG2 and SMMC7721 as the cell lines.Firstly,GSK-3β siRNA enhanced H2O2-induced cell death detected by flow cytometry.Simultaneously,H2O2-induced cell death was significantly weakened after overexpression of GSK-3β.So the protection effect of GSK-3β on cancer cells was proved.In Eukaryotes,the phosphorylation of Thr845 was crucial for ASK1 activity.Through overexpression of HA-GSK-3β WT and the mutated type HA-GSK-3β K85A in HepG2 cells,we found that HA-GSK-3P WT reduced H2O2-induced p-ASK1(Thr845),whereas HA-GSK-3β K85A increased H202-induced p-ASK1(Thr845).Similarly,p-ASK1(Thr845)was also increased after expression of GSK-3β siRNA in HepG2 cells,once again demonstrating that GSK-3β inhibited the phosphorylation of ASK1.In order to explain how GSK-3β reduced the level of p-ASK1(Thr845),we detected the basic level of ASK1 itself,and found that overexpression of GSK-3β could decrease the expression of ASK1.Subsequently,the proteasome inhibitor MG 132 and the post-transcriptional inhibitor CHX were used.The decrease effect of GSK-3β on ASK1 expression was inhibited after addition of MG 132,and overexpression of GSK-3β significantly accelerated the degradation rate of ASK1 after CHX was added.These results indicated that GSK-3β regulated ASK1 at post-transcriptional levels,and the degradation of ASK1 was dependent on the proteasome pathway.Then,by ubiquitination’s experiment,we detected that HA-GSK-3β WT increased the ubiquitination of ASK1,whereas HA-GSK-3β K85A did not affect,suggesting that GSK-3β might regulate ubiquitin pathway of ASK1 which affected the degradation process.According to other studies,Roquin-2 and CHIP ubiquitination ligase and deubiquitination enzyme USP9X may be involved in ASK1 degradation.Through the combination of siRNA and inhibitors,it was proved that GSK-3β might affect ubiquitination of ASK1 through USP9X,independent of Roquin-2 and CHIP.Summarying this part of study,our results showed that GSK-3β had a protective effect on hepatocarcinoma via inhibiting the pro-apoptotic protein ASK 1.Specific mechanism studies showed that GSK-3β increased the ubiquitination pathway of ASK1 by inhibiting the role of deubiquitinase USP9X,thereby reducing ASK1 expression,thus played its role in promoting the survival of hepatocarcinoma cells.