Research On The Mechanism For The Combination Of Vitamin C,PPM-18 And Vitamin K2 To Inhibit Bladder Cancer Cells

Biology and clinical studies show that the vitamin family and its analogs have an anti-tumor effect in a variety of tumor models.Although the vitamin family is natural and safe,it generally requires high doses and long treatment times to exert anti-tumor effects.Combination therapy of vitamins is a very potential anti-cancer strategy with significantly improving the anti-tumor effect and reducing the dosage of the drug.This thesis explores that the combination of vitamin C,vitamin K2 and vitamin K family analog PPM-18inhibits the proliferation of bladder cancer cells,and futher reseach its mechanism.The main work of this thesis is as follows:（1）It was found that the combination of vitamin C,PPM-18 and vitamin K2 at a lower dose could induce the death of bladder cancer cells,and the combination rate was optimized by orthogonal experiments.Through cell viability detection,cell morphology observation,crystal violet staining and cell plate cloning experiments,it was found that 4m M vitamin C+5μM PPM-18+50μM vitamin K2（called CPK for short）significantly inhibited the growth of bladder cancer cells EJ and T24,but no significanty effect on human normal liver cells L02.In addition,CPK had anti-cancer effect on human breast cancer cells MDA-MB-231,human lung cancer cells A549,human cervical cancer cells Hela,human prostate cancer cells PC3 and human glioma cells U87.（2）It was found that CPK induced the death of bladder cancer cells by caspase-independent apoptosis and autophagy.The Annexin V-FITC/PI staining showed that CPK induced apoptosis,but CPK could not induce the cleaved fragments of caspase 3and PARP by Western blot detecting.Futhermore,the caspase inhibitor Z-VAD-FMK could not rescue the CPK-induced cell death,indicating that CPK-induced apoptosis was not mediated by caspase.On the other hand,the transmission electron microscope was used to observe the structure of mitochondria and autophagosomes,and it was found that CPK induced mitochondria swollen and autophagosomes accumulated.Combined with Western blot,CPK gradually increased the autophagy marker protein LC3-II/I ratio and decreased the p62 content siginificantly,indicating that CPK induced autophagy flux in bladder cancer cells.In order to explore the relationship between autophagy and cell death,autophagy inhibitors 3-MA,CQ,Bafilomycin A1 and autophagy activators Rapamycin,HBSS starvation,serum starvation were treatmented with CPK.It was found that 3-MA,CQ,Bafilomycin A1 could partially compensate for cell death induced by CPK,while Rapamycin,HBSS starvation,and serum starvation could accelerate CPK-induced cell death,indicating that CPK induces cell death by causing autophagy.（3）To explore the mechanism of CPK inducing the death of bladder cancer cells,the levels of total reactive oxygen species（ROS）and mitochondrial ROS were detected by ROS detection probe DCFH-DA and the mitochondrial ROS detection probe Mito-SOX.It was found that CPK could increase the total ROS level in bladder cancer cells,but had no significant effect on mitochondrial ROS.Through the detection of extracellular H₂O₂content,it is found that CPK could form a redox cycle outside of cell,leading to produce H₂O₂,and then increased the level of intracellular ROS.In addition,it was found that the total ROS scavenger NAC could completely replenish the CPK-induced apoptosis,autophagy and cell death,indicating that CPK induced bladder cancer cell death by up-regulating the level of intracellular ROS.Furthermore,detecting the protein expression level of glucose transporter GLUT1 in bladder cancer cells by Western blot found that CPK up-regulated the content of GLUT1 in EJ cells.And glucose could effectively rescue the decrease in cell viability caused by CPK.Therefore,we speculate that the possible mechanism of CPK is:CPK forms a redox cycle outside of cell,oxidizing vitamin C to DHA,and producing the H₂O₂.After H₂O₂ enters the cell,it up-regulates the level of intracellular ROS.DHA and glucose competitively combine with GLUT1 to be transported into the cell,where it is reduced by glutathione to vitamin C and further promotes intracellular ROS,causing cell death.In summary,CPK induces bladder cancer cell apoptosis,autophagy and death by inducing ROS production.CPK significantly improves the anti-cancer activity of vitamins with reducing the dose of the drug,providing a new treatment strategy for bladder cancer and the combination of vitamins.