Anti-tumor Effect And Mechanism Of Vitamin K Analog PPM-18 On Bladder Cancer

Bladder cancer is one of the most common malignant tumors in the urinary system.Although traditional bladder cancer treatments,such as radical cystectomy,radiotherapy,chemotherapy and immunotherapy,had made substantial progress,the defect of the above treatments,including distant metastasis,high recurrence rate and toxic effect on normal tissues,still remains,which makes exploration of novel drugs against bladder cancer become urgent.PPM-18,a novel analog of Vitamin K,was reported to suppress seizures in zebrafish and reduce the viability of leukemia cells in vitro.However,whether PPM-18,like Vitamin K,exerts remarkable anti-cancer function remains to be elucidated.Therefore,this study aims to explore the therapeutic effect of PPM-18 on bladder cancer.The main research content and results are as follows:(1)PPM-18 inhibits the proliferation of bladder cancer cells in vitroPPM-18 decreased the viability of human bladder cancer cells in a dose-or timedependent manner by analyzing cell viability.Meanwhile,PPM-18 could significantly inhibit the proliferation of bladder cancer cells by Brd U and cellular colony assays.In addition,PPM-18 showed a broad spectrum of anti-cancer effects and no obvious inhibitory effect on normal cells in vitro experiments.(2)PPM-18 induces apoptosis and autophagy in bladder cancer cellsPPM-18 could effectively induce the apoptosis of bladder cancer cells by flow cytometry and Western blot experiments,accompanied by a decrease in mitochondrial membrane potential,suggesting that PPM-18-induced apoptosis of bladder cancer cells was mediated by mitochondrial pathway.PPM-18 could stimulate autophagy in bladder cancer cells by Western blot and transmission electron microscopy.Following treatment with 3-methyladenine(3-MA)and Chloroquine(CQ),two autophagy inhibitors,PPM-18-induced autophagy and apoptosis were notably suppressed.In contrast,the apoptotic effect of PPM-18 on bladder cancer cells was further enhanced,upon the activation of autophagy by treatment with Rapamycin,an autophagy agonist.These results suggest that PPM-18-activated autophagy promotes apoptosis in bladder cancer cells.However,PPM-18,at a certain concentration,neither inhibited the viability,nor exerted autophagic and apoptotic effect on human normal cells,implying that normal cells are insensitive to PPM-18 treatment.(3)PPM-18 promotes apoptosis through activating AMPK-dependent autophagy in bladder cancer cellsPPM-18 could significantly activate AMPK signaling pathway in bladder cancer cells by Western blot asaay.On the contrary,PI3K/AKT/m TORC1 signaling pathway antagonized by AMPK was significantly inhibited after PPM-18 treatment.PPM-18-induced autophagy and apoptosis of bladder cancer cells were significantly reduced after AMPK inhibition,indicating that PPM-18-induced autophagy and apoptosis of bladder cancer cells were AMPK dependent.(4)PPM-18 regulates apoptosis and autophagy in bladder cancer cells by activating ROS/AMPK signaling pathwayThe levels of reactive oxygen species(ROS)in bladder cancer cells were significantly upregulated after PPM-18 treatment by flow cytometry.However,treatment with N-acetylL-Cysteine(NAC),Glutathione(GSH)and Vitamin E(VE),three ROS scavengers,significantly inhibited PPM-18-induced autophagy and apoptosis in bladder cancer cells.Moreover,PPM-18 activated AMPK,whereas suppressed PI3K/AKT/m TORC1 signaling pathway was notably reversed following ROS elimination.These results suggest that PPM-18-increased ROS levels could cause intracellular oxidative stress,thus resulting in AMPK activation and PI3K/AKT/m TORC1 signaling pathway suppression to induce autophagy and apoptosis in bladder cancer cells.Through exploring the mechanism of PPM-18-upregulated intracellular ROS levels in bladder cancer cells,we found that the ability of consuming the glucose in medium by bladder cancer cells was notably inhibited,following treatment with PPM-18,accompanied by the significant decrease in the content of lactate(the final product of glycolysis)released into medium,suggesting that PPM-18 could effectively inhibit the glycolysis in bladder cancer cells.On the contrary,PPM-18 could promote the generation of ATP in bladder cancer cells,and cause the rapid elevation of intracellular mitochondrial ROS,indicating that PPM-18 could activate the mitochondria oxidative phosphorylation in bladder cancer cells.(5)PPM-18 inhibits bladder cancer growth in vivoPPM-18 could inhibit the growth of subcutaneous human bladder cancer xenografts in nude mice by measuring tumor volume.PPM-18 was capable of inducing autophagy and apoptosis in bladder cancer cells in nude mice by immunohistochemistry(IHC)and TUNEL assays.Meanwhile,the phenomenons that PPM-18 activated AMPK and elevated ROS levels in bladder cancer cells were also confirmed in tumor-bearing nude mice.In addition,PPM-18 had no significant toxicity to the major organs,including heart,liver,spleen,lung and kidney in nude mice by H&E staining.Moreover,PPM-18 did not significantly affect the body weight of tumor-bearing nude mice during the entire administration period,showing the good biosafety of PPM-18.In conclusion,this study found that PPM-18,as a novel Vitamin K analog,could effectively inhibit the proliferation of bladder cancer cells in vitro and in vivo,and induce ROS/AMPK pathway-dependent autophagy and apoptosis of bladder cancer cells with good biosafety.This study provides a new insight into the exploration of novel drugs against bladder cancer,and also renders a feasible and necessary theoretical basis for the clinical application of PPM-18 in the treatment of bladder cancer.