Molecular Mechanism Of Isoquercitrin Inhibiting Bladder Cancer Cells Growth

Many functional ingredients,such as polysaccharide,lipids,peptides,flavonoids,saponins,sterols,monoterpenes and alkaloids,are widely presented in dietary plants,like fruits and vegetables.These compounds can not only regard as natural antioxidants in food industry but also display beneficial health and diseases preventing effcts in organisms.Among these compounds,the flavonoids have become research hotspot since they are rich in content and are also heat stable in dietary plants.It was estimated that 95% flavonoids existed in the form of flavonoid-C-glycosides or flavonoid-O-glycosides,but most of studies have focused on flavonoid-O-glycosides because of its acid and enzymolysis resistant properities that are responsible for its good bioavailability.Isoquercitrin(ISO,C21H20O12)is the quercetin glycoside derivatives,which widely occurred in Chinese herbal medicines,vegetables and fruits.It has been proved in vitro and vivo experiments that ISO exhibited remarkable biological activities including antioxidant,antiallergic,antidiabetes and prevention of cardiovascular and cerebrovascular diseases and its activities are better than quercetin.However,the current studies are mainly focusing on quercetin and the comprehensive investigations on ISO are insufficient.Therefore,this paper took ISO as target to simulate body environment at the cellular and molecular level,and revealed the molecular mechanism of ISO inhibiting bladder cancer proliferation.This study is expected to provide theoretical for the efficient utilization of ISO in food industry and improve the guidance for the prevention of chronic deiseases such as bladder cancer.1.The erythrocyte hemolysis assay indicated that ISO can significantly improve the activities of antioxidant enzymes-superoxide dismutase(SOD)and glutathione peroxidase(GPx),thereby it reduced the production of harmful substances and protected the cells from oxidative damage,finaly prevented cancer cells occurence.Subsequently,MTT assay showed the antiproliferation effects of ISO on human bladder cancer cell lines and normal bladder cells.The results indicated that the IC50 of ISO on EJ and T24 were 28.8 ?M and 34.4 ?M,respectively,which had no obvious difference from quercetin in pH7.2 condition.However,when bladder cancer cells were exposed to sub-acid environment(pH6.2),the IC50 of ISO on EJ and T24 cells were 62.32 ?M and 55.7?M,respectively,which indicated that ISO displayed higher stability in terms of inhibiting bladder cancer growth than quercetin.On the other hand,ISO also displayed lower toxicity on normal bladder cells SV-HUC-1 than positive control group(paclitaxel).Furthermore,ISO can significantly inhibit the migration of bladder cancer EJ and T24 cells.All these results indicated that ISO possessed significant anti-bladder cancer cells activity and stability as well as had no toxicity in bladder normal SV-HUC-1 cells.2.The investigation on the action mechanisms by flow cytometric analysis showed that SubG1 peaks and G2/M phase ratios of EJ and T24 cells in a dose-dependent increased.The results indicated that ISO induced EJ and T24 cells growth inhibition was by the means of apoptosis and G2/M cell cycle arrest.Meanwhile,the intracellular ROS generation was investigated by DHE assay,which revealed the inhibition of ISO on T24 cells was related with its ability to excessively produce intracellular ROS.Further research on protein signaling pathway of ISO on T24 cells was conducted by western blotting.The results showed that ISO could promote T24 cells to produce excess ROS to initiate p53,AKT and MAPKs signaling pathways,which activated the downstream signaling receptor-mediated extrinsic and mitochondria-mediated intrinsic pathways that caused apoptosis.Furthermore,ISO also down-regulated the expression of CyclinB1 and CDK4/6 as well as up-regulated p15,p21 and p27 cell cycle inhibitory factors and promoted phosphorylation of JNK,which could induce G2/M cell ctcle arrest in T24 cells.3.The metabolomics was conducted by UHPLC-QTOF-MS to explain the changes of protein expression in T24 cells.The results showed that when T24 cells were pretreated with 80 ?M ISO,784 metabolites were significantly changed(fold change >2)respectively in the positive and negative mode,which mainly involved in 7 metabolic pathways.AMPK-mTOR signaling pathway played important role in metabolism,especially in lipid synthesis and glycolysis,which can also effect the expression of p53 and AKT proteins and lead to apoptosis and cycle attest in T24 cells.