Research On The Mechanism Of Vitamin K2-Induced Apoptosis In Human Bladder Cancer Cells

Vitamin K2 belongs to Vitamin K family which is fat-soluble and greatly required for maintaining human normal metabolism,prevention and treatment for a variety of disease.It was indicated from the original studies that Vitamin K2 functions as a cofactor of carboxyglutamyl carboxylase and plays an important role in blood coagulation.Subsequent studies have shown that Vitamin K2 was implicated in the regulation of osteoblast differentiation,promotion of the metabolism of bone cells and improvement of bone health.In addition,Vitamin K2 is able to prevent the vascular calcification and reduce the risk of cardiovascular disease occurrence.It was suggested from the recent studies that vitamin K2 functions as anticancer activity in the various human cancer cells,including liver,lung,colon,leukemia,prostate,gastric,ovarian and pancreas cancer,via the inhibition of cell growth and induction of cell apoptotic death.However,to date,it has not been reported that Vitamin K2 repressed the cell growth and triggered the cell apoptosis in human bladder cancer cells.Furthermore,the precise mechanism that Vitamin K2 inhibits the cell proliferation and induces the apoptosis in cancer cells remains unclear.Based on the anticancer property of Vitamin K2,the anticancer activity of Vitamin K2 in human bladder cancer cells,including inhibiting the cell growth and inducing the cell apoptosis,was evaluated in our studies.Meanwhile,the exact mechanism of the anticancer effect of vitamin K2 on human bladder cancer cells was also been investigated.It was shown in our study that Vitamin K2 significantly reduced the viability of human bladder cancer T24,J82 and EJ cells in a dose-dependent manner.Moreover,the T24,J82 and EJ cells viability was also remarkably decreased after the prolonged treatment with 100 μM Vitamin K2.These results indicated that Vitamin K2 exhibited inhibitory growth effect on human bladder cancer T24,J82 and EJ cells.Subsequently,we evaluated whether Vitamin K2 induced the apoptosis in T24,J82 and EJ cells.As shown in Flow cytometry and Tunel immune-fluorescence assay,Vitamin K2 was able to trigger significant apoptosis in T24,J82 and EJ cells.To further investigate the relative mechanism the Vitamin K2 triggers the apoptosis in human bladder cancer cells,the changes of mitochondria,MAPK and ROS was evaluated in our studies.It was displayed from the Flow cytometry assay,the mitochondria membrane potential was remarkably decreased in Vitamin K2-treated T24,J82 and EJ cells.On the contrary,ROS level was significantly increased in T24,J82 and EJ cells.By western blot,Both JNK and p38(MAPK kinase)were significantly phosphorylated in T24,J82 and EJ cells after exposure to Vitamin K2.In order to investigate whether mitochondria,MAPK and ROS were involved in Vitamin K2-induced apoptosis in human bladder cancer cells,the exact inhibitors were utilized in our study.As shown in our study,SP600125(a JNK inhibitor)as well as SB203580(a p38 inhibitor)were able to remarkably abolished the Vitamin K2-induced apoptosis and mitochondria membrane potential decrease in human bladder cancer cells.In addition,NAC(a scavenger of ROS)also significantly reversed the apoptosis,the disruption of mitochondria membrane potential and the phosphorylation of JNK and p38 in Vitamin K2-treated bladder cancer cells.These results suggested that Vitamin K2 could trigger the apoptosis in human bladder cancer cells via ROS-MAPKmitochondria signal pathway.To investigate whether Vitamin K2 was able to trigger the apoptosis in human bladder cancer cells in vivo,the nude mice bearing human bladder cancer EJ cells were used in our study.It was indicated in our study,Vitamin K2 had an ability to repress the cell growth of human bladder cancer EJ cells in the nude mice.Furthermore,it was shown in the Tunel,caspase-3 activity and HE staining the sections from the mice,Vitamin K2 was able to induce the apoptosis in human bladder cancer EJ cells in the nude mice.In order to enhance the apoptotic effect of Vitamin K2 on bladder cancer cells,UBIAD1,a tumor suppressor protein,was utilized in this study.It was shown that the inhibitory viability and apoptotic content in bladder cancer T24 cells were enhanced after treatment with combination of UBIAD1 over-expression and Vitamin K2,comparing with the significant inhibitory viability and induction of apoptosis in only Vitamin K2-treated T24 cells.Collectively,in our study,Vitamin K2 indeed suppressed the cell propagation and triggered the apoptosis in human bladder cancer cells via ROS-MAPK-mitochondria signal pathway.Consequently,our study will provide the theory insight to explore the mechanism of anticancer activity of Vitamin K2 and search the approaches for curing human bladder cancer.