Screening Of Probiotics Inhibiting Growth Of Colorectal Cancer Cells And Its Mechanism Of Action

Colorectal cancer(CRC)is a type of cancer that occurs in the lower digestive tract of the human body,including colon cancer and rectal cancer.The incidence of CRC is high in the world and ranks third in cancer incidence.In China,with the improvement of living standards and the westernization of living habits,the incidence of colorectal cancer is also increasing year by year.The traditional surgical treatment supplemented by chemical drug therapy and radiation therapy has great toxic and side effects on patients.Although it improves the five-year survival rate,it seriously affects the quality of life of patients.There is a natural or acquired resistance to targeted therapy.Immunotherapy may also contain some unpredictable complications.However,gene therapy has some problems such as low introduction rate and high cost.So what a safe and reliable treatment type with small side effects is fund has become the main research direction.Probiotics are a type of live microbes that inhabit the human intestine and reproductive system and are good for human health.Lactic acid bacteria(LAB)is the main member of probiotics.In recent years,LAB has been found to not only improve the flavor of the material,but also provide natural antiseptic and antibacterial functions.It can also maintain the microecological balance in the host and protect the host's intestinal health.Stimulate host immunity and prevent host cancer.Therefore,the use of probiotics to prevent or assist in the treatment of CRC may be one of the safe and reliable side effects that we are seeking.In this study,lactic acid bacteria were isolated from Yunnan traditional fermented food,and 10 strains were selected to screen for the inhibition of proliferation of colorectal cancer cell HT-29.After the metabolites of probiotics(fermentation supernatant)were prepared into freeze-dried powder solution,they were used to treat HT-29 cells,and MTT method was used to detect cell viability,calculate cell survival rate,and evaluate the anti-proliferation ability of lactic acid bacteria.Finally,two strains with good inhibitory effect were screened.The two strains were further screened by other colorectal cancer cell lines.After the screened lactic acid bacteria were treated with HT-29 cells,the cell cycle and apoptosis of HT-29 cells were detected by flow cytometry.Finally,a lactic acid bacteria with the best anti-proliferation effect was selected,and its anti-proliferation mechanism was further studied.Study.Transcriptionomic methods can understand the overall situation of gene expression in HT-29 cells at the molecular level,and predict the molecular mechanism of its anti-proliferation effect.The main research contents and experimental results are as follows:1.Ten strains were randomly selected from the laboratory for the isolation and storage of the strains.They were Lactobacillus fermentum JSL8-2,Pediococcus pentosus 2-4L,Lactobacillus plantarum AY01,QB3-1,QB3-3,YM4-3,Lactococcus latics BZ06,Lactobacillus casei AS02,Lactobacillus brevis 3M,Lactobacillus curvatus 6M.The fermentation of these lactic acid bacteria was made into freeze-dried powder,which was diluted into different solutions after re-suspension in cell culture medium.HT-29 cells were treated with different concentrations,and MTT assay was used to detect cell viability.Finally,two strains AY01 and JSL8-2 with good anti-proliferation effect were screened.2.After treatment of HT-29 cells with different concentrations of AY01 and JSL8-2,the results showed that the higher the concentration of freeze-dried powder solution,the better anti-proliferation effect of the strain,and the best inhibition effect was at the concentration of 64 mg/mL and 128 mg/mL.In addition,with the increase of treatment time,the anti-proliferation effect was better,and the inhibition effect reached the best at 48 h and 60 h,but there was no significant difference between the two time points.3.CRC cell lines Caco-2,HCT116 and SW620 were treated with freeze-dried powder solutions of different concentrations of AY01 and JSL8-2,respectively.Cell viability was measured by MTT method.AY01 and JSL8-2 also had anti-proliferation effect on other CRC cell lines.The best inhibitory effect was found at the concentration of 64 mg/mL and 128 mg/mL,and at 48 h and 60 h.4.The cell cycle and apoptosis of HT-29 cells treated with AY01 and JSL8-2 were detected by flow cytometry,and the anti-proliferation ability of these two strains was evaluated at the cellular level.It was found that both AY01 and JSL8-2 had anti-proliferation effect by inducing apoptosis,not by blocking cell cycle.In addition,we found that AY01 had stronger ability of inducing apoptosis than JSL8-2,so the final selected strain was AY01.5.According to the results of flow cytometry,HT-29 cells were treated with 32 mg/mL AY01 freeze-dried powder solution,and the gene expression in cells was investigated by transcriptome.The results showed that there were significant differences in gene expression related to cell apoptosis,which coincided with the results of flow cytometry.6.The transcriptome data analysis showed that AY01 inhibited the proliferation of HT-29 cells mainly through two signaling pathways,TGF-beta/smad signaling pathway and Ferroptosis death pathway,to achieve cell cycle arrest,induce apoptosis,autophagy and Ferroptosis death.