De Novo Ceramide Increased By Bisphenol A Promotes Epithelial-mesenchymal Transformation In Colorectal Cancer Cell

Background Bisphenol A（BPA）,widely used in the synthesis and manufacture of plastic products,is a group of environmental endocrine disruptors with typical estrogenic effects contributing to environmental exposure in humans.Studies have shown that BPA exposure is significantly associated with the development of tumors including breast cancer,prostate cancer and colorectal cancer（CRC）.Ceramide（Cer）not only sustains cellular integrity but also functions as a second messenger in intracellular signaling and performs an essential role in regulating biological processes relating to tumor cell proliferation,differentiation,migration and invasion.Objective This research was conducted to investigate the function and molecular mechanism of BPA exposure in promoting epithelial mesenchymal transformation in colorectal cancer cells by affecting the de novo synthesis of Cer,with the human colorectal cancer cell HCT-116 as a model,providing an experimental basis for elucidating the mechanism of environmental pollutants promoting CRC and a new target or biomarker for the treatment and diagnosis of CRC associated with environmental exposure.Methods In the first part,BPA exposure promoted epithelial-mesenchymal transition（EMT）in colorectal cancer through the TGF-β/Smad signaling pathway.The maximum dose and duration of BPA exposure to HCT-116 cells was firstly identified by the CCK8 assay.Subsequently,the effect of proliferation,invasion and migration ability was measured by plate colony formation assay,Transwell cell invasion assay and scratch assay.The expression levels of EMT and TGF-β-related marker molecules were identified via western blotting（WB）and real-time fluorescence quantitative PCR（RT-PCR）.And finally,the above process was validated using TGF-β inhibitors.In the second part,de novo synthesis of Cer was involved in the TGF-β/Smad signaling pathway to regulate epithelial mesenchymal transformation in CRC.Firstly,targeted quantitative LC/MS was performed to identify Cer in colorectal cancer cells,and WB and RT-PCR were applied to measure changes in the expression levels of various ceramide synthases.Subsequently,the expression levels of Cer,ceramide synthase,TGF-β/Smad signaling pathway,EMT and related molecules were observed in myriocin acting on the cells.Results 1.BPA exposure promoted the proliferation,invasion and migration of HCT-116 cells.The biological behavior of EMT in HCT-116 cells was markedly enhanced with increasing dose and duration of BPA exposure,and the expression of the EMT marker molecule E-cad decreased,the expression of Vimentin increased and the expression of the EMT transcription factor Snail was up-regulated in a dose-time dependent manner.The differences were statistically significant compared to solvent controls（P < 0.05）.2.BPA exposure promoted the expression levels of TGF-β/Smad signaling pathways molecules.With increasing dose and duration of BPA exposure,the expression levels of TGF-β1 and p-Smad2/3 molecules were clearly up-regulated and showed a dose-time dependence with BPA.The differences were statistically significant（P < 0.05）compared to solvent controls.3.BPA exposure modulated the development of interepithelial transformation through TGF-β/Smad signaling pathways.After pretreatment with TGF-β inhibitor,EMT marker molecule E-cad expression was increased,Vimentin expression was decreased,EMT transcription factor Snail expression was diminished,and EMT levels in HCT-116 cells were significantly inhibited.The differences were statistically significant（P < 0.05）compared to the dose group.4.BPA exposure remarkably elevated ceramide levels.With increasing dose and duration of BPA exposure,the concentrations of total ceramide,C16,and C18 ceramide in HCT-116 cells were continuously increased,and the expression levels of the associated ceramide synthase Sptlc1/2 and Cer S6 molecules were upregulated in a dose-time dependent manner with BPA.The difference was statistically significant（P < 0.05）compared to the solvent control group.5.Ceramide moderated the process of epithelial mesenchymal transition in HCT-116 cells.Following pretreatment with ceramide synthase inhibitor,the concentration of total ceramide,C16 and C18 ceramide in HCT-116 cells was decreased,and the expression levels of the related ceramide synthase Sptlc1/2 and Cer S6 molecules were downregulated.The proliferation,migration and invasion ability of HCT-116 cells were significantly inhibited,and the expression of EMT marker molecule E-cad was raised,the expression of Vimentin was declined,and the expression of EMT transcription factor Snail was decreased in HCT-116 cells.The differences were statistically significant（P <0.05）compared to the dose group.6.Ceramide is involved in the epithelial mesenchymal transition process of HCT-116 cells through the TGF-β/Smad signaling pathway.Pretreatment with ceramide synthase inhibitor obviously down-regulated the expression levels of TGF-β1 and p-Smad2/3molecules,inhibited the TGF-β/Smad signaling pathway,and de-regulated the expression of EMT-related molecules,while the proliferation,migration and invasion ability showed a decline.The difference was statistically significant（P < 0.05）compared to the dose group.Conclusion 1.BPA exposure contributed to the development of EMT in colorectal cancer through the TGF-β/Smad signaling pathway.2.Cer is involved in EMT of HCT-116 regulated by TGF-β/Smad signaling pathwayunder BPA exposure.