The Anti-Tumor Efficacy And Molecular Mechanisms Of Indole-3-Methanol In Esophageal Squamous Cell Carcinoma

Backgrounds and ObjectivesIncreasing studies have shown that indole-3-methanol(13 C)extracted from cruciferous vegetables(such as cauliflower,cabbage,turnip and Swedish food)has been proved to exert significant anti-tumor effect in liver cancer,colon cancer,melanoma,breast cancer and other tumors,and has the ability to suppress many oncogenic signaling pathways.I3C blocks cell growth and induces cell apoptosis by targeting cell proliferation,survival,apoptosis and cell cycle-related signaling pathways.However,the roles of I3C and molecular mechanisms in esophageal squamous cell carcinoma(ESCC)remain unknown.Therefore,the purpose of the current study was to elucidate the biological roles and possible molecular mechanisms of I3C in ESCC,which will provide the novel strategy for ESCC patients.PART I Anti-tumor efficacy and molecular mechanisms of indole-3-methanol in esophageal squamous cell carcinoma in vitroMethods1.Cell proliferation assay:The effects of different concentration I3C(0,100,200,300,400 and 500μM)on ESCC cell proliferation were detected by CCK-8 kit at different time points(24,48,72 and 96h).EdU staining and colony formation experiments were used to examine the effects of different concentration 13 C on cell proliferation and colony formation in a panel of ESCC cells including Eca109,EC9706,TE1 and KYSE450,respectively.2.Cell cycle,apoptosis,migration and invasion assay:Flow cytometry was utilized to investigate the effects of various concentration 13C on cell cycle and apoptosis in ESCC cells.Cell scratch and invasion experiments were employed to detect the effects of various concentration I3C on cell migration and invasion in ESCC cells.3.Cell apoptosis-related proteins and PTEN signaling pathway-related proteins were examined by Western blot in ESCC cells after treatment with different concentration I3C.Caspase-3 activity assay kit was used to detect the Caspase-3 activity after treatment with different concentration 13C.The expressions of PTEN and its related genes were analyzed using real-time quantitative PCR(qRT-PCR)and Western blot after treatment with I3C.4.EC9706 cells treated with 300 μM I3C were performed to complete whole transcriptome resequencing by Illumina high-throughput sequencing platform and 2×150 bp double terminal sequencing strategy.The results of mRNA differential expression were displayed by volcano map and heatmap,and the function and related signal pathway of differential expression genes were analyzed by GO and KEGG assay.5.Statistical assay:The experimental data were investigated by GraphPad Prism 8.0 statistical software.The data were expressed as mean 士 standard deviation(SD).The student’s t test was used to compare the sample means of two groups which obeyed normal distribution and homogeneity of variance,conversely,the Mann-Whitney U nonparametric test was performed to investigate these data.Three or more samples were analyzed by One-way ANOVA,LSD test was used for pairwise comparison among three samples with normal distribution and homogeneity of variance,and Bonferroni test was used for pairwise comparison among more than three samples.Kruskal-Wallis test was used for pairwise comparison among multiple samples without normal distribution and homogeneity of variance.AP value less than 0.05 was considered as statistical significance.Results1.The results of CCK-8 demonstrated that I3C at different doses significantly suppressed cell proliferation in ESCC cells(Eca109,EC9706,TE1,KYSE70,KYSE150,KYSE180 and KYSE450)in concentration and time-dependent manner.The values of IC50 gradually reduced with the elongation of time.2.The result of EdU staining revealed that cell proliferation was inhibited at 48h after treatment with various concentration I3C.Colony formation experiment showed that I3C displayed inhibitory ability on colony formation of ESCC cells.3.I3C arrested ESCC cell cycle in G0/G1 phase,induced cell apoptosis,coupled with more apoptotic cell numbers in higher I3C concentration.The result of Western blot demonstrated that I3C promoted the expressions of Bax and cleaved-Caspase-3 and suppressed the expression of Bcl-2.Caspase-3 activity assay revealed that I3C markedly elevated the activity of Caspase-3 in ESCC cells.4.Cell scratch experiment demonstrated that I3C dramatically suppressed migration of ESCC cells at 24h and 48h.Transwell experiment revealed that different concentration I3C markedly inhibited invasion of ESCC cells at 48h.5.The data derived from qRT-PCR showed that 13 C evidently upregulated PTEN mRNA level in ESCC cells at 48h,and the result of Western blot proved that I3C obviously promoted the expressions of PTEN and P53 proteins,but suppressed the expression of p-AKT.6.The results of whole transcriptome resequencing demonstrated that 3093 of differential expression genes were found in EC9706 cells after I3C treatment,in which 1169 genes were upregulated and 1924 genes was downregulated.These differential expression genes were implicated in the regulation of P53 pathway,MAPK pathway,ErbB pathway,VEGF pathway and PI3K-AKT pathway,etc.PART Ⅱ Reversal effect of VO-Ohpic trihydrate,a PTEN inhibitor,on indole-3-methanol against esophageal squamous cell carcinoma and its mechanismsMethods1.The experiments were splitted into four groups:control group,VO-Ohpic trihydrate(Abbreviated VO-Ohpic)inhibitor alone group(50nM),13C treatment group(200μM)and VO-Ohpic combined with I3C group.2.CCK-8 and EdU staining experiment was used to detect cell proliferative ability in ESCC cells(Eca109,EC9706,TE1 and KYSE450),Flow cytometry was employed to investigate apoptosis of ESCC cells in different treatment groups,and Transwell experiment was utilized to examine invasion of ESCC cells in different treatment groups.3.Western blot were used to investigate the gene expressions of PTEN and its related pathways.4.ESCC cells xenografted nude mice experiment:EC9706 cells was subcutaneously inoculated into nude mice,and the experiments was divided into four groups:corn oil control group,VO-Ohpic abdominal treatment group,I3C gavage group and VO-Ohpic combined with I3C group.Tumors were treated every two days,and tumor volume was measured every two days.Tumor growth curve was made using GraphPad Prism 8.0 software.5.Statistical assay:The experimental data were investigated by GraphPad Prism 8.0 statistical software.The data were expressed as mean ± standard deviation(SD).The student’s t test was used to compare the sample means of two groups which obeyed normal distribution and homogeneity of variance,conversely,the Mann-Whitney U nonparametric test was performed to investigate these data.Three or more samples were analyzed by One-way ANOVA,LSD test was used for pairwise comparison among three samples with normal distribution and homogeneity of variance,and Bonferroni test was used for pairwise comparison among more than three samples.Kruskal-Wallis test was used for pairwise comparison among multiple samples without normal distribution and homogeneity of variance.AP value less than 0.05 was considered as statistical significance.Results1.The results of CCK-8 and EdU staining demonstrated that PTEN inhibitor VO-Ohpic partially reversed the suppressive effect of I3C on ESCC cell proliferation in Ecal09,EC9706,TE1 and KYSE450 cells.2.The result of cell apoptosis showed that VO-Ohpic partially reversed the inductive effect of I3C on ESCC cell apoptosis,I3C triggered the high expression of Bax and cleaved-Caspase-3 and the low expression of Bcl-2.Transwell experiment demonstrated that VO-Ohpic partially reversed the inhibitory effect of I3C on ESCC cell invasion.3.The results of Western blot demonstrated that VO-Ohpic partially reversed the increased expression of PTEN and P53 and the inhibition of p-AKT evoked by I3C treatment in ESCC cells.4.Animal experiment showed that compared with control group,13 C treatment significantly suppressed tumor growth in EC9706 xenografted nude mice model,and VO-Ohpic partially reversed the growth suppression efficacy triggered by 13C treatment.The result of qRT-PCR demonstrated that 13 C treatment promoted PTEN expression,and the data from Western blot revealed that 13C treatment triggered the increased expressions of Bax,cleaved-Caspase-3,PTEN and P53 proteins and reduced expression of Bcl-2 and p-AKT proteins.However,VO-Ohpic could partially reversed the changes of cell phenotypes and gene expressions triggered by 13C treatment.Conclusion1.I3C suppresses ESCC cell proliferation and colony formation,blocks cell cycle in G0/G1 phase,induces apoptosis and inhibits cell migration and invasion and tumor growth in vivo,coupled with the changes of cell apoptosis-related proteins and PTEN-AKT signaling pathway key proteins,suggesting 13C may be an effective natural drug for clinical treatment of ESCC patients.2.The data from VO-Ohpic combined with I3C in vitro and in vivo suggest that I3C exerts anti-ESCC by targeting PTEN-AKT signaling pathway.