Study On The The Effect And Mechanism Of Proliferation Inhibition Of Pseudomonas Aeruginosa-Mannose Sensitive Hemagglutinin Bacteria(PA-MSHA)on Oral Tongue Squamous Cell Carcinoma Cell Lines

Backgrounds:Bacterial treatment of cancer began in the 19 th century,Dr.Coley observed some sarcoma patients infected with erysipelas remission.Because antibiotics did not appear at the time,erysipelas is not easy to control,and is not easy to induce,Dr.Coley uses inactivated bacterial products to treat Patient instead of viral bacteria,however,the effectiveness of this method is limited to a small number of patients,ie sarcoma patients who cannot be treated by other methods.Later,with the advent of radiotherapy and chemotherapy,the method of bacterial treatment with great limitations was gradually ignored.A few years later,people found that the long-term survival rate and control rate of cancer patients did not improve,so gradually ignited hope for bacterial treatment of cancer.In order to expand the indications for bacterial therapeutic vaccines,bacterial vaccines have been gradually modified,for example by genetically engineering bacteria to have specific traits,or to use attenuated live bacterial vaccines.Improved bacterial therapy for cancer has the advantage that other treatments cannot match: specific targeting of tumors.Pseudomonas aeruginosa-mannose-sensitive blood coagulum(PA-MSHA)is a modified Pseudomonas aeruginosa established by Mou Xiya,which is surrounded with type I pili.It can improve cellular and humoral immunity in vivo by means of immunogenic I type pili: including enhancing the killing of NK cells,increasing the number of T lymphocytes and changing the proportion of T lymphocyte subsets,and increasing anti-PA-MSHA antibody.PA-MSHA is mainly used as an immunomodulator in clinical practice and as an adjuvant treatment for cancer patients,it activates the immune system to kill tumors.PA-MSHA has strong cytotoxicity to tumor cells.It was found that the cause of PA-MSHA cytotoxicity is achieved by inhibiting the EGFR signaling pathway,and the literature found that PA-MSHA could enter the cell under electron microscope,Therefore,this experiment explored the killing effect and mechanism of PA-MSHA on tongue squamous cell carcinoma,and autophagy flux level changes of tongue squamous cell carcinoma affected by PA-MSHA.Objectives:1.To study the killing effect and mechanism of PA-MSHA on tongue squamous cell carcinoma cell lines CAL-27 and SCC-15.2.To investigate the effect of PA-MSHA on autophagy of tongue squamous cell carcinoma cell lines CAL-27 and SCC-15.3.To investigate the effect of PA-MSHA on the TLR pathway of tongue squamous cell carcinoma cell lines CAL-27 and SCC-15.Methods:1.The tongue squamous cell carcinoma cell line was processed with PA-MSHA,and the cell morphology was observed.The effects of CCK8 and LDH on the activity and toxicity of tongue squamous cell carcinoma were detected.2.PA-MSHA was used to treat tongue squamous cell carcinoma cells,and nuclear morphology was detected by Hoechst 33258 staining.3.Annexin-V/PI staining,and flow cytometry were used to detect the apoptosis cells of tongue squamous cell carcinoma cell lines treated by PA-MSHA.4.RT-qPCR detected mRNA expression of Caspase-associated protease in tongue squamous cell carcinoma cells treated with PA-MSHA5.western blot was used to detect the expression of Caspase-associated protease in tongue squamous cell carcinoma cells treated with PA-MSHA.6.The squamous cell carcinoma cell line was treated with chloroquine and bafilomycin A1,and the safe dose of CCK8 was detected.7.RT-qPCR was used to detect the expression changes of TLR9 and IFR7 mRNA in tongue squamous cell carcinoma cells treated with PA-MSHA.8.western blot was used to detect the expression of LC3 and p62-related proteins in tongue squamous cell carcinoma cells treated with PA-MSHA.Results:1.PA-MSHA kills tongue cancer cell lines in a dose-dependent manner.2.PA-MSHA induces apoptosis in tongue squamous cell carcinoma.3.PA-MSHA promotes the increase of expression of Bax,one of the Bcl-2 family,,and increases the level of cleved Caspase9 in tongue squamous cell carcinoma cells.4.For the tongue squamous cell carcinoma cell line in this experiment,the saturation working concentration of BAF used to inhibit the autolysosome was 25 nM.5.PA-MSHA can affect autophagy of tongue squamous cell carcinoma.6.Tongue squamous cell carcinoma strains CAL-27 and SCC-15 express TLR-9,and PA-MSHA can activate TLR9 receptor through MyD88-dependent pathway of SCC-15 cell line,which produces interferon.PA-MSHA can promote the expression of EGFR mRNA in tongue squamous cell carcinoma cells.Conclusions:PA-MSHA killed tongue squamous cell carcinoma cells CAL-27 and SCC-15 through mitochondria-Caspase mechanism,and could change the level of autophagy and activate TLR9 receptor.