Anti-tumor Efficacy And Molecular Mechanisms Of Glucose Transporter Inhibitor In Esophageal Squamous Cell Carcinoma By Suppressing Glycolysis

Backgrounds and ObjectivesEsophageal squamous cell carcinoma(ESCC)is one of the most malignant and lethal digestive tract tumors.At present,the pathogenic mechanisms of ESCC remains to be further elucidated.Glucose transporter 1(Glut1)is a kind of membrane transporters responsible for the transport of glucose from extracellular to intracellular,which is involved in the glycolysis of many tumors.Many studies have shown that Glut1 is overexpressed in multiple different tumors,and may become a potential molecular marker for diagnosis and prognosis of tumors,and thus inhibiting Glut1 expression may become a new strategy for tumor treatment.Many studies have discovered that Glut1 inhibitors including WZB117,BAY-876 and Phloretin elicited beneficial anti-tumor activity in a variety of tumors,but their roles in ESCC needs to be further explored.The aim of this study was to investigate the anti-tumor effects of Glut1 inhibitors(WZB117,BAY-876 and phloretin)in ESCC in vivo and in vitro,and to preliminarily dissect their possible molecular mechanisms,which will provide experimental evidence for the targeted therapy of ESCC patients.PART Ⅰ Anti-tumor efficacy and molecular mechanisms of glucose transporter inhibitor in ESCC Methods1.CCK-8,Ed U and colony formation experiments were used to detect the effects of different concentration of WZB117,BAY-876 and Phloretin on cell proliferation of ESCC cells(Eca109,EC9706 and KYSE70)at different time points(24 h,48 h,72 h and 96 h).2.Flow cytometry was used to detect cell apoptosis at 72 h after treatment with different concentrations of WZB117,BAY-876 and Phloretin,and cell scratch and Transwell experiment were used to investigate cell migration and invasion at 72 h after treatment with different concentrations of WZB117,BAY-876 and Phloretin.3.Glucose and lactate measurement kits were employed to detect the glucose consumption and lactate production at 72 h after treatment with different concentrations of WZB117,BAY-876 and Phloretin,and Western blot was used to detect the glycolysis-related protein expressions at 72 h after treatment with different concentrations of WZB117,BAY-876 and Phloretin.4.EC9706 cells treated with 0.5 μM BAY-876 or 110 μM Phloretin were performed to complete whole transcriptome resequencing by Illumina high-throughput sequencing platform and 2 × 150 bp double terminal sequencing strategy.The results of m RNA 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 Graph Pad Prism8.0 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.A P value less than 0.05 was considered as statistical significance.Results1.The data from CCK-8 revealed that WZB117,BAY-876 and Phloretin all suppressed cell proliferation,which displayed in time and dose-dependent manner,and inhibitory effect was maxium at 72 h.The results of Ed U staining and colony formation revealed that the proliferative ability of ESCC cells was markedly inhibited with the elevation of concentration.2.The results of cell apoptosis,cell scratch and Transwell experiment demonstrated that WZB117,BAY-876 and Phloretin at different concentration induced apoptosis and suppressed migration and invasion of ESCC cells at 72 h.3.Glucose and lactate measurement demonstrated that WZB117,BAY-876 and Phloretin suppressed glucose consumption and lactate production of ESCC cells.Western blot assay showed that WZB117,BAY-876 and Phloretin elicited the reduced expressions of Glut1,HK2,PFKM,PKM2,LDHA,PDK1,HIF-1α and c-Myc of ESCC cells.4.The whole transcriptome resequencing demonstrated that 223 of differential expression genes were found in EC9706 cells after BAY-876 treatment,in which 63 genes were upregulated and 160 genes was downregulated.These differential expression genes were implicated in the regulation of TNF pathway,TGFβ pathway,PI3K-AKT pathway,etc.Besides,507 of differential expression genes were found in EC9706 cells after Phloretin treatment,in which 241 genes were upregulated and 266 genes was downregulated.These differential expression genes participated in the regulation of PI3K-AKT pathway,Erb B pathway,Ras pathway,Wnt pathway,JAK-STAT pathway,etc.PART Ⅱ The antitumor effect of BAY-876 combined with DMOG in ESCC and its molecular mechanism Methods1.The experiments were splitted into four groups: Control group,BAY-876 alone group,DMOG alone group and DMOG combined with BAY-876 group.2.CCK-8 experiment was used to detect cell proliferative ability in ESCC cells(Eca109,EC9706 and KYSE70)at different treatment group.3.q RT-PCR and Western blot were employed to investigate the expressions of Glut1,SEMA5 B and HIF-1α m RNA and protein in different treatment group.4.ESCC cells xenografted nude mice experiment: EC9706 cells was subcutaneously inoculated into nude mice,and the experiments was divided into four groups: physiological saline group,BAY-876 treatment group,DMOG treatment group and BAY-876 combined with DMOG treatment group.Tumors were treated every two days,and tumor volume was measured every two days.Tumor growth curve was made using Graph Pad Prism 6.0 software.After treatment,nude mice was euthanatized,and tumor tissues were peeled off.q RT-PCR and Western blot were used to detect the expressions of Glut1,SEMA5 B and HIF-1α m RNA and protein.5.Statistical assay: The experimental data were investigated by Graph Pad Prism8.0 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.A P value less than 0.05 was considered as statistical significance.Results1.The data from CCK-8 revealed that BAY-876 significantly suppressed cell proliferation of ESCC cells,and there was no differnence in cell proliferative ability between blank group and DMOG treatment.However,BAY-876 combined DMOG displayed most inhibitory effect,and statistical difference was found(P<0.05).2.The results of q RT-PCR and Western blot showed that compared with control group,BAY-876 treatment alone significantly reduced the expressions of Glut1,SEMA5 B and HIF-1α m RNA and protein,but DMOG treatment alone triggered no difference of these m RNA and proteins above.BAY-876 combined with DMOG treatment reached most efficacy on the suppressive degree of Glut1,SEMA5 B and HIF-1α expression.3.The result of animal experiment demonstrated that there was no difference between control treatment group and DMOG treatment group,but BAY-876 alone treatment group substantially induced tumor shrinkage in EC9706 cells xenografted nude mice,and BAY-876 combined with DMOG displayed the most treatment efficacy.The results of q RT-PCR and Western blot revealed that compared with control group,BAY-876 signficantly reduced the expressions of Glut1,SEMA5 B and HIF-1α m RNA and protein,DMOG treatment signicantly promted the expressions of Glut1,SEMA5 B and HIF-1α m RNA and proteins.However,BAY-876 combined with DMOG treatment resulted in the most suppressive efficacy on the expressions of Glut1,SEMA5 B and HIF-1α m RNA and proteins.Conclusion1.WZB117,BAY-876 and Phloretin suppresses cell proliferation,migration and invasion,and induces cell apoptosis,coupled with the downregulation of glycolysis-related proteins,suggesting that targeting Glut1 may be a novel therapeutic strategy.2.The whole transcriptome resequencing and in vitro and in vivo experiments suggest that BAY-876 exerts antitumor efficacy by targeting SEMA5 B in ESCC.