| Cancer is one of the tough public health problems worldwide and it is also the leading cause of death of the global population.Lung cancer is the cancer with the highest morbidity and mortality in the world,while liver cancer is the cancer with the third highest mortality in the world.The apoptosis of cancer cells is one of the important ways to fight cancer at present.Drugs can be used to change the tumor cells and their living environment,and then induce programmed death of tumor cells to gradually eliminate the tumors.Cancer metastasis is the main cause of death in cancer patients,Epithelial-Mesenchymal Transition(EMT)is one of the biological process tumor cells,which is closely associated with invasion and metastasis of cancer.Transforming Growth Factor-?1(TGF-?1)can effectively induce EMT in cancer cells.Viceninll is a flavonoid C-glycoside extracted from Dendrobium officinale Kimura et Migo.The researches of Viceninll about tumor apoptosis are less,and the tumor metastasis mechanism research have even not been reported at present.Thus we explore the antitumor activities of Viceninll from this two aspects in this study.We investigate the effects and mechanism of Viceninll on the apoptotic activities in HepG2 cell and the inhibition of TGF-?1-induced EMT,migration and invasion in lung cancer A549 and H1299 cells.The results of this study will provide important experimental and supplementary basis for the quality evaluation and anti-tumor activities of the flavonoids in Dendrobium officinale.Objective:This study aims to investigate the effects and mechanism of Viceninll on the apoptotic activities in human hepatocellular carcinoma HepG2 cell and the inhibition of TGF-?1-induced EMT,migration and invasion in human lung cancer A549 and H1299 cells.Method:1.The apoptotic activities of Viceninll in HepG2 cell.(1)MTT assay was used to detect the cytotoxicity of Viceninll in different tumor cells.(2)The effect of Viceninll on the norphological changes of HepG2 cells was observed after staining with Hoechst 33258.(3)The apoptosis rate of Viceninll on HepG2 cell was analyzed by flow cytometer after staining with Annexin V-PI.(4)RT-qPCR was used to detect the effects of Viceninll on the expression of the relative gene about apoptosis and MAPK pathway in HepG2 cell.2.The mechanism of Viceninll on the inhibition of TGF-?1-induced EMT,migration and invasion in human lung cancer A549 and H1299 cells.(1)MTT assay was used to detect the cytotoxicity of Viceninll and TGF-?1 in A549 and H1299 cells.(2)The effect of Viceninll and TGF-?1 on the morphological changes of A549 and H1299 cells was observed using inverted microscope.(3)The ef-fect of Viceninll on the number of clones of A549 and H1299 cells induced by TGF-?1 was detected by clonogenic assay.(4)Wound healing assay was used to detect the effects of Viceninll on the migration of TGF-?1-induced A549 and H1299 cells.(5)Transwell assay was used to detect the effects of Viceninll on the invasion of TGF-?1-induced A549 and H1299 cells.(6)Immunofluorescence assay was used to observe the effects of Viceninll on the expression of an EMT epithelial marker protein E-cadherin and interstitial marker protein vimentin in TGF-?l-induced A549 and H1299 cells.(7)Western bolting assay was used to detect the effects of Viceninll and pathway inhibitors SB431542 and LY294002 on the expression of EMT marker protein,matrix metalloproteinase protein,and the pathway protein of PI3K/Akt/mTOR and TGF-?/Smad pathway in TGF-?1-induced A549 and H1299 cells.Results:1.The effects of Viceninll to induce apoptosis on HepG2 cell.(1)Different cancer cells were treated with 12.5-100 ?M Viceninll,the results of MTT assay shown that the proliferation inhibition effect in hepatocellular carcinoma HepG2 cell was the strongest,followed by the lung adenocarcinoma A549 cell,cervical cancer Hela cell,stomach cancer SGC7901 cell,colon cancer HT-29 cell,and malignant melanoma B16 cells,so the selection of cell model for the subsequent experiments was HepG2 cell.Vicenin? inhibited the growth of HepG2 cell in time-and concentration-dependent manners.The cell viability of 48 h was significantly lower than 24 h.When the concentration is less than 75?M,the cell viability concentration-dependent decrease,while was basically remain unchanged above 75?M.So we choose 75?M,48 h for subsequent experiments.IC 50 of Viceninll in 48 h was 50.35 ?M.(2)The morphological changes of HepG2 cell after the staining with Hoechst 33258 showed that 75?M treatment with Viceninll induced typical apoptotic morphological features,including number decrese,nuclear condensation and fragmentation,chromatin condensation,and emit dense granular blue fluorescence.(3)The flow cytometric analysis results showed that 14.57±0.91%apoptotic rate was detected in response to 75?M Viceninll treatment in HepG2 cell,which was significant differences with control group.(4)RT-qPCR assay showed that 75?M Viceninll could significant up-regulate pro-apoptotic gene expression of Bax and caspase 8,down-regulate anti-apoptotic gene Bcl-2 and increased the Bax/Bcl-2 ratio.Meanwhile,it can also significant increase the key gene expression of MAPK pathway liked ERK?JNK?P38 and NF?B,which could induce apoptosis of HepG2 cell.2.The effects and mechanism of Viceninll on the inhibition of TGF-?1-induced EMT,migration and invasion in human lung cancer A549 and H1299 cells.(1)MTT results showed that low concentration of Viceninll(?10 ?M)and TGF-?1(?5ng/mL)had no cytotoxic effect on human lung cancer A549 and H1299 cells.While high concentration of Viceninll(>10 ?M)and TGF-?1(>5ng/mL)was sinificant inhibited cell proliferation in a time-and concentration-dependent manners.Thus we choose the no cytotoxicity concentration of TGF-?1(5ng/mL)to induce EMT A549 and H1299 cells as well as Viceninll(2.5,5,10 ?M)for subsequent EMT experiments.(2)Morphology changes showed that 5 ng/mL TGF-?1 treatment successfully promoted EMT by remarkably inducing the disappearance of intercellular junctions and spindle-like appearance of A549 and H1299 cells.,instead inducing a fibroblast-like appearance with a long shape and central nucleus.These morphological changes were obviously reversed by the cotreatment of TGF-?1 with Viceninll(2.5,5,and 10?M).(3)The results of clonogenic formation asssay showed that TGF-?1 treatment significantly increased colony amounts compared with the untreated cells,while significant dose?dependent decreases in the number of colonies were observed for Viceninll(2.5,5,10 ?M)treatments in A549 and H1299 cells.(4)Wound-healing and transwell assay showed that TGF-?1-treated cells exhibited highly enhanced migration and invasion potential,which was significantly suppressed by co-treatment with Vicenin?(2.5,5 and 10 ?M)in a dose-dependent manner.These results prove that Vicenin? effectively inhibited the TGF-?1-induced migration and invasion of A549 and H1299 cells.(5)Immunofluorescence assay showed that 10 ?M Vicenin? significantly increased the green fluorescence expression of EMT epithelial marker E-cadherin weakened by TGF-?1,and decreased the expression of mesenchymal marker Vimentin enhanced by TGF-?1 in A549 and H1299 cells.(6)Western bolting results showed that the TGF-?1-enhanced protein expression of EMT mesenchymal marker N-cadherin and Vimentin,matrix metalloproteinase protein MMP-2,transcription factor snail and slug was significantly inhibited,whereas the TGF-?1-weakened protein of epithelial marker E-cadherin,tight junction protein Claudin-1 and ZO-1 was increased after treatment with Vicenin?(2.5,5 and 10 ?M)in a dose-dependent manner.These results demonstrate that Vicenin? could effectively reverse TGF-?1-induced alterations in the protein expression of EMT biomarkers in A549 and H1299 cells.(7)Western boltting assay was used to explore EMT mechanism of Viceninll.The results showed that the TGF-?1-enhanced TGF-?1/Smad and PI3K/Akt signaling pathway-related proteins p-Smad2,p-Smad3,p-mTOR,p-Akt and p-P70S6K were prominently down-regulated in a dose-dependent manner when treated with Vicenin?(2.5,5 and 10 ?M).PI3K/Akt inhibitors LY294002 and TGF-?/Smad inhibitors SB431542 significantly enhanced the expression of E-cadherin and ZO-1 weakened by TGF-?1,and significantly decreased the expression of N-cadherin,MMP-2,Snail and Slug enhanced by TGF-?1,which has the same effect with Vicenin?.These results indicated that Viceninll inactivate TGF-?/Smad and PI3K/Akt/mTOR signaling pathways to inhibit TGF-?1-induced EMT in A549 and H1299 cells.Conclution:This study demonstrate that Vicenin? could induce human hepatocellular carcinoma HepG2 cell apoptosis via MAPK signaling pathway.In addition,for the first time,we demonstrate that Viceninll targets the TGF-?/S mad and PI3K/Akt/mTOR pathways to inhibit TGF-?1-induced EMT,migration and invasion in lung adenocarcinoma A549 and H1299 cells.Furthermore,these results provide evidence that Viceninll could be a promising repressor against the metastasis of lung adenocarcinoma by affecting TGF-?1-induced EMT. |
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