The Effects And Mechanism Of An Anticancer Protein Extracted From Trichosanthes Kirilowii On Epithelial-mesenchymal Transition And Migration Of Colorectal Carcinoma Cells

Colorectal cancer(CRC)is a type of digestive tract malignancy caused by multiple factors.In concept,most colon cancers could be prevented by detection and removal of premalignant colon adenomas.Likewise,considerable benefit would be predicted by detecting colon cancers at early stages when the disease is amenable to cure by surgical excision.At present,the method commonly used to treat colorectal cancer is chemotherapy.However,in the course of clinical use,some chemotherapeutic drugs will bring varying degrees of toxic and side effects to the human body.While killing cancer cells,they may kill normal cells.At the same time,due to their toxic and side effects,they cause different complications.Studies have shown that plant-derived anti-tumor active proteins have unique mechanisms of action and significant anti-cancer effects,and are an anti-tumor drug worthy of study.Trichosanthes kirilowii is a kind of vine of the family Cucurbitaceae.T.kirilowii contains a variety of biologically active ingredients.Our research group has previously discovered an anti-tumor active protein TKP from the fruit of T.kirilowii.It was found that it can induce apoptosis of colorectal cancer cells and has good anti-tumor potential.This article intends to study the effect and mechanism of TKP on epithelial mesenchymal transformation and migration of colorectal cancer cells.The main research content includes the following sections:Part one: Effects of anti-tumor active protein TKP of T.kirilowii on EMT of colorectal cancer cells.After treatment of DLD1 and HCT116 colorectal cancer cells with TKP at the concentrations of 0,0.1,0.25 and 0.5 ?g/mL respectively for 24 h,the morphological changes of the cells were observed by inverted microscope.Furthermore,the expression of key EMT proteins was detected by qRT-RCR.Cell adhesion assay was used to detect the adhesion ability between cells and matrix.The effect of TKP on cell viability was detected by MTT assay.The results of morphological observation showed that TKP treatment could change cell morphology.Most of the cells in the control group were spindle-shaped and had multiple antennas,showing the characteristics of interstitial cells.After treatment with TKP,the antennas of the cells are reduced and the morphology is round,showing the characteristics of epithelial cells.After TKP treatment for 24 h,the expression of N-cadherin and Vimentin were down-regulated with the increase of TKP concentration.Cell adhesion experiments showed that the treatment of TKP resulted in a decrease in cell-matrix adhesion.MTT results showed that TKP treatment had no significant effect on cell survival.These results indicated that TKP treatment inhibits EMT in colorectal cancer cells.Part two: TKP regulates STAT3/Snail1 signaling through PKM2,which results into inhibition of EMT in colorectal cancer cells.The protein levels of STAT3 and p-STAT3 in the cells were detected by western blotting after treated with 0,0.1,0.25 and 0.5?g/mL TKP for 24 h.The results showed that TKP treatment reduced the protein levels of STAT3 and p-STAT3 in colorectal cancer cells,meanwhile the level of p-STAT3 in the nucleus and Snail1 mRNA also decreased.These results indicated that the STAT3/Snail1 signaling is inhibited.In addition,TKP treatment reduced PKM2 expression and inhibited its nuclear translocation.To further demonstrate the role of PKM2 in STAT3/Sanil1 signaling-mediated EMT,PKM2 was overexpressed using transfection techniques.The experimental results showed that over-expression of PKM2 reversed TKP-induced down-regulation of p-STAT3,and TKP-suppressed cell mesenchymal transition was significantly reversed.The above results indicated that TKP can inhibit the occurrence of EMT in colorectal cancer cells by PKM2 mediated STAT3/Sanil1 signalinginhibition.Part three: TKP inhibits the migration of colorectal cancer cells through wnt/?-catenin signaling.The colorectal cancer cells DLD1 and HCT116 were treated with TKP at concentrations of 0,0.1,0.25 and 0.5 ?g/mL for 24 h,respectively.The wound healing assay was used to detect the migration of colorectal cancer cells.The results showed that the colorectal cancer cells were treated with TKP,its migration ability decreased.Western blotting showed that the level of ?-catenin and p-GSK3? in cells were down-regulated,and the mRNA expression of MMP7 and MMP9 related to cell migration also decreased.After addition of the activator LiCl of Wnt pathway,the cell migration induced by TKP treatment was reversed,including the reversal of intracellular ?-catenin and p-GSK3? expression.In conclusion,the above results indicated that TKP inhibits the migration of colorectal cancer cells through the Wnt/?-catenin signaling.Taken together,TKP can inhibit the EMT of colorectal cancer cells by regulating STAT3/Snail1 signaling mediated by PKM2.In addition,TKP can inhibit the migration of colorectal cancer cells by regulating the Wnt/?-catenin pathway.