Research On The Apoptosis Induced By Combination Of Vitamin K2 And Vitamin C In Bladder Cancer Cell Lines In Vitro

Bladder cancer is a malignant tumor of the human urinary system.Previous studies have shown that Vitamin K2 was used as auxiliary chemotherapy drugs to treat cancer,with high dose and low anticancer efficiency in short-term treatment.This study aims to explore a novel therapeutics for bladder cancer through treating bladder cancer cell lines in vitro by combination of Vitamin K2 and Vitamin C.As shown in MTT results,the cell viability of bladder cancer T24 and EJ cells was markedly decreased after treatment with 50μM Vitamin K2 and 5m M Vitamin C in combination.Crystal violet assay showed that combination treatment significantly suppress the growth of bladder cancer cells.Moreover,Flow cytometry demonstrated the combination treatment notably induces apoptosis in bladder cancer cells.To determine the remarkable apoptotic effect of combination treatment in bladder cancer cells,western blot was utilized in this study.Our data showed that Caspase-3,an apoptotic protein,was activated in combination-treated bladder cancer cells,which results in the cleavage of PARP protein.To unveil the exact apoptotic mechanism in combination-treated bladder cancer cells,intracellular ROS level was assessed in this study.Flow cytometry showed that combination treatment markedly increased intracellular ROS level in bladder cancer cells.Furthermore,NAC,a typical ROS scavenger,remarkably revered combination treatment-reduced cell viability and inhibited apoptosis in bladder cancer cells.To investigate whether mitochondrial pathway is implicated in combination treatmentinduced apoptosis in bladder cancer cells,the membrane potential and morphology of mitochondria were respectively detected in this study.As shown in Flow cytometry assay,mitochondria membrane potential was significantly reduced in bladder cancer cell following combination treatment.Besides,Transmission electron microscopy showed that combination treatment markedly disrupted the morphology of mitochondria in bladder cancer cells.In addition,the inhibitory effect of combination treatment on cell viability in multiple cancer cell lines including human breast cancer was validated in this study.Additionally,the safety of the combination treatment was also evaluated in human normal liver cell L02.As shown in our results,neither the decreased cell viability nor apoptosis was found in combination-treated normal cells.Collectively,this study reveals that the combined treatment with Vitamin K2 and Vitamin C is able to induce Caspase-3-mediated apoptosis in bladder cancer cells via intracellular ROS generation and reduction of mitochondria membrane potentials,whereas it had no effect on normal cells.Therefore,this combination treatment program has the potential to be a novel anti-bladder cancer therapeutics.