Research On The Mechanism Of Death Induced By Combination Of PPM-18 And Vitamin C In Bladder Cancer Cell Lines In Vitro

Bladder cancer is one of the most common malignant tumors in the human urinary system.Recent years,the morbidity and mortality of bladder cancer have increased,which severely threat human survival and health.Traditional bladder cancer therapeutics,including radical cystectomy,chemotherapy,radiotherapy and immunotherapy,have made great breakthrough in bladder cancer treatment.However,the side of effect,such as high rate of relapse,long distant metastasis and cytotoxicity,remains effect on human survival and quality of lives.PPM-18(NQN1)is a novel histone deacetylase inhibitor with a naphthalene ring structure and has been reported to kill leukemia cells in vitro.Vitamin C is a natural and water-soluble vitamin.Previous studies have widely reported that high dose of Vitamin C exerts remarkable anticancer activity in vitro and in vivo.In this study,we investigated a new bladder cancer treatment strategy by combination of low-dose PPM-18 and low-dose Vitamin C to treat bladder cancer cell lines in vitro.As shown in MTS assay,the combination of 5 μM PPM-18 and 5 m M Vitamin C was able to significantly reduce bladder cancer T24 and EJ cell viability.Moreover,bright field photography,crystal violet and cell cloning assays showed that combination treatment not only repressed the proliferation of bladder cancer,but also triggered bladder cancer cells death,and similar results were also displayed in other six common cancer cell lines.Flow cytometry experiments showed that combination can induce the apoptosis of bladder cancer T24 and EJ cells.Through Western Blot,the combination did not activate the cleavage of the apoptosis indicator proteins Caspase-3,PARP.Intriguingly,western blot detected the upregulation of an autophagic indicator LC3 B II in bladder cancer cells,after treated with combination therapy.In addition,by transmission electron microscope,the accumulation of autophagosomes was observed in bladder cancer EJ cells,following the combination treatment.These results suggested that combination of PPM-18 and Vitamin C is able to induce autophagy in bladder cancer cells.Through flow cytometry,the reduction of the mitochondrial membrane potential of bladder cancer cells after combined treatment was detected,and the destruction of mitochondria was also observed through transmission electron microscopy.To further explore the mechanism of combineddrug-induced cell death,flow cytometry was utilized and intracellular ROS level was evaluated.The results showed that the combination induced a significant increased ROS levels in bladder cancer cells.The co-treatment of ROS scavenger NAC and combination drugs significantly restored the viability of bladder cancer cells and the above results were further verified by bright field photography,crystal violet,cell cloning.Western blot analysis showed that NAC decreased the up-regulation of LC3 BII induced by combination therapy.Besides,flow cytometry displayed that the co-treatment of NAC and combination rescued the disruption of mitochondrial membrane potential induced by combination.These results indicate that combination of PPM-18 and Vitamin C could kill bladder cancer cells via generating intracellular ROS levels.The safety assessment of the combination drug in human normal cell L02 found that the combination drug did not cause a significant decrease in the viability of L02 cells,and flow cytometry did not detect significant apoptosis in combination-treated L02 cells,which shows that the combination of PPM-18 and Vitamin C is safe for normal cells.In summary,this study found that the combination of PPM-18 and Vitamin C induces the destruction of mitochondrial membrane potential in bladder cancer cells by activating intracellular ROS,which ultimately leads to non-caspase-3 dependent apoptotic cell death,accompanied by the activation of autophagy,but it does not kill normal cells.Therefore,this combination has the potential to become a new anti-bladder cancer therapy.