Impacts of phillygenin on the malignant biological behaviors of colorectal cancer cells by regulating the Ras/Raf/MEK/ERK signal pathway
Objective To investigate the impacts and underlying mechanism of phillygenin(PG)on the proliferation,migration,invasion,apoptosis,and epithelial mesenchymal transition(EMT)of colorectal cancer cells.Methods HCT116 colorectal cancer cells were cultured and treated with 10～200μmol/L phillygenin.The cell survival rate was measured and the optimal experimental concentration was selected.HCT116 cells were induced with blank control,low-dose PG,medium-dose PG,high-dose PG,high-dose PG+transfection of negative control(NC)and high-dose PG+transfection of Ras lentivirus.Cell proliferation,migration and invasion were detected by colony formation assay,wound healing assay and Transwell assay,respectively.Cell apoptosis was detected by flow cytometry and Hoechst 33258 staining.Protein expressions of EMT markers,including the E-cadherin and Vimentin,and key proteins in the rat sarcoma virus(Ras)/rapidly accelerated fibrosarcoma(Raf)/mitogen-activated protein kinase(MEK)/extracellular signal-regulated kinase(ERK)signaling pathways,including p-Raf,p-MEK and p-ERK were detected by Western blot.A CRC-bearing nude mouse model was created to explore the influence of PG on in vivo growth of CRC.Results Treatment of 10-200pmol/L PG in HCT116 cells significantly inhibited cell survival,with the IC50 value of(140.4±2.147)μmol/L.Finally,10pmol/L,50μmol/L and 100μmol/L were selected as the low-dose,medium-dose and high-dose concentration of PG in the following experiments.Compared with those of blank control,PG cells induced with low-dose,medium-dose and high-dose PG presented with significantly lower colony formation rate,wound healing rate,invasive cell number and protein expressions of Vimentin,p-Raf,p-MEK and p-ERK,but significantly higher apoptotic rate and protein expression of E-cadherin in a dose-dependent manner(P＜0.05).Compared with those induced with high-dose PG+transfection of NC,HCT116 cells induced with high-dose PG+transfection of Ras lentivirus presented significantly higher colony formation rate,wound healing rate,invasive cell number and protein expressions of Vimentin,p-Raf,p-MEK and p-ERK,but significantly lower apoptotic rate and protein expression of E-cadherin(P＜0.05).PG induction significantly inhibited CRC growth in vivo.Conclusion Phillygenin can inhibit the proliferation,migration,invasion,apoptosis and epithelial mesenchymal transition of colorectal cancer cells by inhibiting the Ras/Raf/MEK/ERK signaling pathway.
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