| Objectives:Bladder cancer(BC for short)is the fourth most common male galignant tumor after prostate cancer,lung-bronchial cancer,and colorectal cancer,and the incidence of bladder cancer in the United States is estimated to be approximately 7%in the past 5 years.When talk about China,prostate cancer and bladder cancer are the most common urological tumor,of which urinary urothelial tumors could account for 95%of bladder cancer,and non-muscular invasive bladder cancers(NMIBC for short)could account for about 75%.Recently,transurethral resection of bladder tumor,(TRBR for short)is main treatment,what’s more,adding adjunctive bladder perfusion therapy could significantly reduce the recurrence rate of BC,five-year survival rate could reach as high as 88%.Unfortunately,there are still 50-70%would relapse,one thirds in which would develop higher-level bladder cancer.At present,the commonly used bladder perfusion drugs have obvious systemic and local side effects,at same time,it cannot completely solve the problem of postoperative recurrence.BCG-perfusion is recommended for the patients with high-grade papillary urinary tract bladder cancer or carcinoma in situ whose pathology is confirmed after transurethral cystectomy,however,there is no effective second-line treatment for NMIBC with BCG failure.For muscle-invasive bladder cancer(MIBC for short),radical resection combined with neoadjuvant chemotherapy with cisplatin as the critical is standard treatment.The disadvantages for anti-tumor drug are long courses of treatment and obvious toxic side effects,both in bladder perfusion of chemotherapy drugs and neoadjuvant chemotherapy with systemic using.Therefore,the treatment of both non-muscle invasive bladder cancer and muscle invasive bladder cancer still needs be improved continuously.In recent years,a number of clinical studies of immunotherapy and targeted therapy for muscle-invasive bladder cancer have achieved certain results in a small range.However,the cost of the new method is high,both safety and long-term tolerability need to be further studied.Therefore,the search for a postoperative perfusion drug or systemic drug with low toxic side effects that takes into account both efficacy and safety is regarded as a hot spot and research topic in the treatment of bladder cancer,and it is also an urgent clinical practical problem that needs to be solved.Cobra venom membrane toxin is one of the main toxic components of cobra venom,with cardiotoxicity and cytotoxicity.In recent years,studies on the toxic effect of membrane toxin in tumor cells have shown that MT occupies a very critical position in the study of the anti-tumor effect of snake venom.MT-12 is a membrane toxin monomer extracted from the Chinese cobra,with special "hydrophobic and hydrophilic finger-like structures",which is beneficial to bind to the cell membrane and destroy the cell membrane,thereby having a selective killing effect on tumors in vivo and in vitro.This project aims to confirm that MT-12 can inhibit the proliferation of bladder cancer cells through in vivo and in vitro,and to explore the mechanism of MT-12 exerting anti-tumor effects by inducing apoptosis,inducing autophagy and mediating oxygen radical formation,etc.,so as to provide new ideas for clinical search for effective and safe treatment for bladder cancer.Methods:The first part of this project was to observe the inhibition effect of MT-12 on the proliferation of bladder cancer cells cultured in vitro by CCK-8 and soft agar clonal formation experiments,and to verify that MT-12 had the effect of inducing apoptosis on bladder cancer cells cultured in vitro via transmission microscopy observation and flow cytometry detection.At last,to detect the production of apoptotic proteins caspase3 and caspase8 by western blot.In the second part of this project,the apoptosis inhibitor Z-VAD-FMK and autophagy-specific inhibitor 3-MA were used separately or in combination to weaken the inhibition ability of MT-12 on the proliferation of bladder cancer cells cultured in vitro.And then their effects on the subcutaneous tumorigenesis of MT-12 tumor-bearing mice were observed and compared,and the autophagy-related proteins were measured by immunofluorescence and western blot,thus confirming that MT-12 can also inhibit the development of bladder cancer through autophagy.The third part of this project is to detect the transmembrane potential,ROS production,ATP and mtDNA content determination of bladder cancer cells pretreated with the mitochondrial damage agent TTFA,mitochondrial protector CsA and strong antioxidant NAC in advance.And through autophagy-related proteins,it was confirmed that MT-12 produced a large number of reactive oxygen radical ROS by damaging mitochondria,and enriched in cells,by inducing cancer cells to develop autophagy,exerting its anti-bladder cancer effect,in order to discuss preliminarily signaling pathway for inducing autophagy by MT-12.Results:In the first part of this project,the proliferation inhibition effect of MT-12 on bladder cancer cells cultured in vitro was found to be concentration-dependent and time-dependent.MT-12 acting on T24 cells,24 hours and 48 hours,50%Inhibitory Concentration was 0.468 μg/mL and 0.386 μg/mL and on RT4 cells,24 hours and 48 hours,50%Inhibitory Concentration was 0.527 μg/mL and 0.399 μg/mL.And after the detection of apoptosis by flow cytometry and the detection of apoptosis protein tested by western blot,it was confirmed that MT-12 can play an anti-bladder cancer cell proliferation effect by activating the caspase-dependent apoptosis pathway.In the second part of this project,it was confirmed that either the use of the apoptotic inhibitor Z-VAD-FMK and the autophagy-specific inhibitor 3-MA,respectively,or a combination of both,can weaken the ability of MT-12 to inhibit the proliferation of bladder cancer cells,and the weakening effect is most significant when used in combination,and the same results are reflected in nude mice in the subcutaneous tumorigenesis model,indicating that MT-12 can inhibit the proliferation of bladder cancer cells through autophagy or apoptosis.The detection of autophagy-related proteins by immunofluorescence and western blot further shows that MT-12 can exert anti-tumor effects by inducing autophagy of bladder cancer cells.In the third part of this project,by detecting the ratio of MT-12 to bladder cancer cells pretreated with the mitochondrial damage agent TTFA and mitochondrial protector CsA,mt-12 can reduce the mitochondrial transmembrane potential,increase the accumulation of ROS,reduce the production of mtDNA and ATP,and this effect can be weakened by the mitochondrial protector CsA and enhanced by the mitochondrial damage agent TTFA.This shows that MT-12 can induce autophagy in bladder cancer cells by damaging mitochondria.By detecting its autophagy-related proteins AMPK,mTOR,ULK1,JNK and p53,IT was found that MT-12 can enhance the phosphorylation of AMPK and ULK1,while inhibiting the phosphorylation of mTOR.Snake venom acts on NAC-treated bladder cancer cells,which strongly blocks the phosphorylation of AMPK and ULK1,indicating that its MT-12 acts on bladder cancer cells,which can produce a large number of ROS and induce autophagy of bladder cancer cells by activating the AMPK/mTOR/ULK1 pathway;however,when MT-12 acts on the mitochondrial damage agent TTFA and mitochondrial protective agent CsA,the impact on AMPK and ULK1 is not large.It is explained that this pathway may not be related to mitochondrial function damage caused by MT-12 acting on bladder cancer cells.Conclusions:MT-12 can exert its antitumor effect by inducing apoptosis and autophagy of bladder cancer cells,and this effect presents time-dependent and concentration-dependent;MT-12 can induce autophagy of bladder cancer cells by injuring mitochondria causing ROS accumulation and thus activating the AMPK/mTOR/ULK1 pathway. |
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