The Mechanism Of An Anticancer Protein Extracted From Trichosanthes Kirilowii On Migration And Invasion Of Colorectal Carcinoma Cells

Colorectal cancer(CRC)refers to malignant lesions of the large intestinal mucosa epithelium under the influence of environmental or genetic carcinogenic factors.It has a high mortality rate and is one of the common malignant tumors in China.At present,the treatment of colorectal cancer mostly adopts comprehensive treatment methods,including surgical treatment,radiotherapy,chemotherapy,and targeted therapy.Due to the metastatic properties of tumor cells in CRC,the prognosis is still poor,and long-term use of these therapies can cause serious side effects and reduce the quality of life.An antitumor active protein has been extracted from the fruits of Trichosanthes kirilowii in our laboratory.Studies have found that TKP induces apoptosis in human colorectal cancer cells through the mitochondrial pathway and PI3K/AKT signaling.By regulating PKM2,STAT3/Snail1 signaling can be inhibited,thereby inhibiting epithelial-mesenchymal transition in colorectal cancer cells.In addition,preliminary studies have found that TKP can inhibit colorectal cancer cell migration by regulating the Wnt/β-catenin signaling.This research further studied the mechanism of TKP on colon cancer cell migration and invasion.The main research includes the following parts:Part I : The effects of anti-tumor active protein TKP on Colorectal cancer cell invasion and expression of proteins related to migration and invasion.Colorectal cancer cells DLD1 and HCT116 were treated with different concentrations of TKP(final concentrations of 0,0.1,0.25 and 0.5 μg/m L)for 24 h,and then the transwell invasion assay was used to detect the invasion abilities of the two cells.The invasive abilities of the two cells treated with TKP were significantly lower than that of the control group.MMP2 m RNA expression in DLD1 and HCT116 cells was detected by q RT-PCR.The results showed that MMP2 m RNA expression in both cells was down-regulated along with TKP concentration increasing.Western blot analysis showed that the expression of MMP2 and MMP9 proteins decreased compared with the control group.MMP2 and MMP9 activities were detected in the two cells by gelatin zymography.The results showed that TKP inhibited the activities of MMP2 and MMP9.Therefore,these results indicated that TKP treatment can inhibit the expression of proteins related to invasion and migration of colorectal cancer cells.Part II : TKP inhibited the invasion of colorectal cancer cells and the expression of proteins related to migration and invasion through the Wnt/β-catenin pathway.Li Cl was used to upregulate the Wnt/β-catenin pathway.DLD1 and HCT116 cells were treated with 10 m M Li Cl for 24 h,and treated with 0 and 0.5 μg/m L TKP.QRT-PCR was used to detect the relative m RNA expression level of MMP2 in DLD1 and HCT116 cells.The results showed that TKP inhibited MMP2 m RNA expression in cells through the Wnt/β-catenin pathway.Western blot assay was used to detect the effect of TKP on the expression of MMP2 and MMP9 proteins in DLD1 and HCT116 cells.We found that the TKP and Li Cl combined treatment groups had higher protein expression levels of MMP2 and MMP9 compared with the TKP single treatment group.Using gelatin zymography to detect MMP2 and MMP9 activities in colorectal cancer cells,we found that the TKP and Li Cl combined treatment group had an increased ability to digest gelatin compared to the TKP single treatment group.The transwell invasion assay was used to test the invasion ability.The results showed that the decreased invasion ability of colorectal cancer cells by TKP after Li Cl treatment was significantly reversed.The above results indicated that TKP inhibits the invasion of colorectal cancer cells and the expression of proteins related to migration and invasion through the Wnt/β-catenin pathway.Part III : TKP inhibited the migration and invasion of colorectal cancer cells through the Hedgehog/Gli1 pathway.Colorectal cancer cells DLD1 and HCT116 were treated with different concentrations of TKP(final concentrations of 0,0.1,0.25 and 0.5 μg/m L)for 24 h,and then q RT-PCR was used to detect the expression levels of Gli1 and Patched1.The results showed that the expression of Gli1 and Patched1 m RNA were down-regulated after TKP treatment.We used western blot to detect Gli1 levels in the cytoplasm and nucleus of colorectal cancer cells DLD1 and HCT116.The results showed that Gli1 protein levels in the cytoplasm and nucleus were significantly down-regulated.Using the Gli1 overexpression method to upregulate the Hedgehog/Gli1 pathway,we found that TKP-induced inhibition of m RNA expression,protein expression,and activity of MMP2 and MMP9 was reversed after overexpression of Gli1.Cell wound healing assay showed that the weakened cell migration ability changed by TKP treatment was significantly reversed after Gli1 overexpression.Transwell invasion assay results showed that Gli1 overexpression reversed the inhibitory effect of TKP on cell invasion.The above results indicated that TKP inhibits the migration and invasion of colorectal cancer cells by regulating the Hedgehog/Gli1 pathway.In summary,TKP inhibits the Wnt/β-catenin and Hedgehog/Gli1 pathways,and then down-regulates the expression of MMP2 and MMP9,which ultimately inhibits the colorectal cancer cell migration and invasion.