| Cancer is a major public health problem worldwide.It is the leading cause of death and the most important obstacle to improving life expectancy in most countries of the world in the twenty-first century.The incidence and mortality rates of cancer are increasing rapidly all over the world.Although significant progress has been made in the diagnosis and treatment of cancer,metastasis remains a major obstacle to good clinical outcomes,as more than 90%of cancer-related deaths are caused by metastatic diseases.Epithelial-Mesenchymal Transition(EMT)is closely related to the occurrence,invasion,metastasis and resistance to cancer treatment in cancer.Many studies have shown that transforming growth factor-β1(TGF-β1)is one of the most effective inducers of EMT,which can promote the formation of EMT through Smad and non-Smad pathways.Isoviolanthin is a flavonoid glycoside in Dendrobium officinale Kimura et Migo,a precious traditional Chinese medicine.Its anti-metastasis mechanism has not been reported so far.In this study,two kinds of human hepatocellular carcinoma HepG2 and Bel-7402 cells were induced by TGF-β1 to investigate the effects and the mechanisms of isoviolanthin on the migration,invasion and EMT of hepatocellular carcinoma cells enhanced by TGF-β1.The results of this study will provide the basis for the pharmacological activities of flavonoids in Dendrobium officinale.Objective:Based on TGF-β/Smad and PI3K/Akt/mTOR signaling pathways,the regulatory effects of isoviolanthin on the EMT of two kinds of human hepatocellular carcinoma HepG2 and Bel-7402 cells induced by TGF-β1 were discussed.Methods:1.MTT assay was used to detect the effect of 0-100 μM isoviolanthin on the activity of human normal LO2 cells and human hepatocellular carcinoma HepG2 and Bel-7402 cells,in order to determine the appropriate concentration of to determine for subsequent EMT experiments.2.The effect of isoviolanthin(2.5 μM,5 μM,10 μM)on the number of clones of human hepatocellular carcinoma HepG2 and Bel-7402 cells induced by TGF-β1 was detected by clonogenic assay.3.Scratch assay was used to detect the effects of isoviolanthin(2.5 μM,5 μM,10 μM)on the migration of human hepatocellular carcinoma HepG2 and Bel-7402 cells enhanced by TGF-β1.4.Transwell lab assay was used to detect the effect of isoviolanthin(2.5 μM,5 μM,10μM)on the invasion of human hepatocellular carcinoma HepG2 and Bel-7402 cells enhanced by TGF-β1.5.Enzyme-linked Immunosorbent assay(ELISA)was used to detect the effects of isoviolanthin(2.5 μM,5 μM,10 μM)on matrix metalloproteinases(MMPs)MMP-2 and MMP-9 in human hepatocellular carcinoma HepG2 and Bel-7402 cells induced by TGF-β1.6.Reverse transcription-quantitative Real-time PCR(RT－qPCR)was used to detect the effects of isoviolanthin(2.5 μM,5 μM,10 μM)on the expression of E-cadherin and ZO-1,N-cadherin and vimentin,transcription factors Snail and Slug in human hepatocellular carcinoma HepG2 and Bel-7402 cells induced by TGF-β1.7.Western bolting assay was used to detect the effects of isoviolanthin(2.5 μM,5 μM,10 μM)on the expression of EMT epithelial marker protein E-cadherin and ZO-1,N-cadherin and vimentin,transcription factor protein Snail and Slug,matrix metalloproteinase protein MMP-2 and MMP-9 induced by TGF-β1 in human hepatocellular carcinoma HepG2 and Bel-7402 cells,and the effects of TGF-β1/Smad and PI3K/Akt signaling pathway-related proteins.8.Immunofluorescence assay was used to observe the effects of 10 μM isoviolanthin on the expression of E-cadherin,an EMT epithelial marker protein,and vimentin,an interstitial marker protein,in human hepatocellular carcinoma HepG2 and Bel-7402 cells induced by TGF-β1.9.Scratch and Transwell lab tests were performed to examine the effects of 20 μM TGF-β/Smad inhibitors SB431542 and 20 μM PI3K/Akt inhibitors LY294002 on the migration and invasion of human hepatocellular carcinoma HepG2 and Bel-7402 cells induced by TGF-β1.The effects of SB431542 and LY294002 on E-cadherin,N-cadherin,Snail and MMP-2 in human hepatocellular carcinoma HepG2 and Bel-7402 cells induced by TGF-β1 were detected by Western bolting assay.Results:1.MTT results showed that 100 μM isoviolanthin had no cytotoxic effect on human normal hepatocyte LO2 cells.Low concentration(<10 μM)isoviolanthin had no cytotoxic effect on human hepatocellular carcinoma HepG2 and Bel-7402 cells,but.high concentration(>10 μM)isoviolanthin inhibited the activity of HepG2 and Bel-7402 cells in a dose-time dependent manner.2.The results of clone formation experiment showed that isoviolanthin(2.5 μM,5 μM,10 μM)inhibited the increase of HepG2 and Bel-7402 cell clones induced by TGF-β1 in a dose dependent manner.3.Scratch assay showed that isoviolanthin(2.5 μM,5 μM,10 μM)inhibited the TGF-β1-enhanced migration of HepG2 and Bel-7402 cells in a dose dependent manner.4.Transwell lab experiment showed that isoviolanthin(2.5 μM,5 μM,10 μM)inhibited the TGF-β1-enhanced invasion ability of HepG2 and Bel-7402 cells in a dose dependent manner.5.ELISA results showed that isoviolanthin(2.5 μM,5μM,10μM)dose dependent attenuated the TGF-β1-enhanced ability of HPG2 and Bel-7402 cells to secrete MMP-2 and MMP-9.6.RT-qPCR assay results showed that isoviolanthin(2.5 μM,5 μM,10 μM)dose dependent attenuated the TGF-pl-enhanced expression of N-cadherin,vimentin,Snail and Slug in HepG2 and Bel-7402 cells,and enhanced the reduced expression of E-cadherin and ZO-1 induced by TGF-β1.7.Western bolting experiment showed that isoviolanthin(2.5 μM,5 μM,10 μM)dose dependent attenuated the expression levels of N-cadherin,vimentin,Snail,Slug,MMP-2 and MMP-9 in HepG2 and Bel-7402 cells enhanced by TGF-β1,and enhanced the expression levels of E-cadherin and ZO-1 protein attenuated by TGF-β1;The expression levels of p-Smad2,p-Smad3,p-Akt.p-mTOR and p-P70S6K proteins related to TGF-p/Smad and PI3K/Akt signaling pathways induced by TGF-β1 in HepG2 and Bel-7402 cells were significantly decreased by isoviolanthin.8.Immunofluorescence assay showed that 10 μM isoviolanthin significantly increased the expression of E-cadherin in HepG2 and Bel-7402 cells weakened by TGF-β1,and decreased the expression of vimentin enhanced by TGF-β1.9.Scratch and Transwell lab experiments showed that inhibitors SB431542 and LY294002 significantly reduced the migration and invasion ability of HepG2 and Bel-7402 cells enhanced by TGF-β1.Western bolting experiments showed that SB431542 and LY294002 significantly enhanced the expression level of E-cadherin protein weakened by TGF-β1,and significantly decreased the expression levels of N-cadherin,Snail and MMP-2 enhanced by TGF-β1.The results were consistent with those of 10 μM isoviolanthin.Conclusion:This study demonstrated for the first time that isoviolanthin can inhibit the migration,invasion and EMT of HepG2 and Bel-7402 cells by inhibiting TGF-β/Smad and PI3K/Akt/mTOR signaling pathways induced by TGF-β1.In addition,these data suggest that isoviolanthin can be used as one of the active ingredients of Dendrobium officinale for anti-metastasis of hepatocellular carcinoma cells. |
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