| Resveratrol belongs to polyphenols and is a kind of important active ingredients in wine. Resveratrol has many physiological functions such as anti-tumor, anti-cardiovascular diseases, regulating immune and so on. Resveratrol has inhibitory effect on many types of tumor cells, and its mechanisms includes inducing tumor cell apoptosis, inhibiting the proliferation of tumor cells and promoting tumor cell metastasis, but the molecular mechanism is not very clear, especially the molecular mechanism of inhibiting of tumor metastasis is not clear. In this study, the colorectal cancer cell HCT116 was used to study the role of resveratrol on tumor metastasis. From the view of mobility of cancer cells, we evaluated the immigration ability and invasion ability of resveratrol on cancer metastasis. On this basis, the molecular mechanism of cancer cell mobility affected by resveratrol was investigated in the project.Resveratrol has inhibitory effect on colorectal cancer cells. In this study, MTS cell survival experiment was used to analysis the effects of resveratrol on proliferation of colorectal cancer cells HCT116. After treatments of resveratrol with the final concentrations of 0 μmol/L,6.25 μmol/L,12.5 μmol/L,25 μmol/L,50 μmol/L,100 μmol/L,150 μmol/L for 12 h,24 h and 48 h, we found that high concentrations of resveratrol can obviously inhibit the proliferation of HCT116 cells, and lower concentrations of resveratrol 0-50μmol/L for 12 h did not show obvious inhibition effect.Resveratrol inhibited the cell cycle of colorectal cancer cells. After treatments of resveratrol with final concentrations of 0 μmol/L,6.25 μmol/L,12.5 μmol/L,25 μmol/L,50 μmol/L,100 μmol/L for 12h, flow cytometry was used to analyze the effect of resveratrol on cell cycle. Our data showed that more than 100 μmol/L resveratrol has obvious blocking effect on colorectal cancer (cell cycle arrest), but at low concentrations 0-50μmol/L has not obvious change. According to above data, our results showed that 0-50 μmol/L resveratrol treatment has not obiviously affect the proliferation ability of colorectal cancer cell HCT116.Resveratrol promotes the apoptosis of colorectal cancer cells. To observe the effect of resveratrol on the process of colorectal cancer apoptosis, fluorescence staining was used to detect apoptosis. After treatments of resveratrol with the final concentrations of 0 μmol/L,6.25 μmol/L,12.5 μmol/L,25 μmol/L,50 μmol/L,100 μmol/L,150 μmol/L for 0 h,12 h,24 h and 48 h respectively, our data showed that the dose of less than HCT116 cell morphology did not obvious change after treatment of 50 μmol/L resveratrol for 24 h and showed full nucleus, uniform color, no nuclear condensation and apoptotic bodies appeared, but high concentration of resveratrol treatments results in apoptosis. Flow cytometry was used to analyze the effect of rersveratrol on cell apoptosis. Our data showed that more than 0-50 μmol/Lgroups had not obvious apoptosis during 12 h. According to the results of cell proliferation and apoptosis, we selected 0-50 μmol/L concentration of resveratrol treatment for 12 h to analyze the effect of resveratrol on the motility of colorectal cancer cells.Tumor metastasis is the final cause of death in patients. Tumor metastasis is a very complex process. The motility of cancer cells is an important decision factors for tumor metastasis. The migration and invasion of cancer cells are the two main indexes as determined cell motility. In this study, we evaluated the effects of resveratrol on the motility of colorectal cancer cells HCT116. In the scratch test,50 μmol/L resveratrol treatment group has less narrow scratch than 25 μmol/L and 12.5 μmol/L treatment group, there is no visible difference between the 6.25 μmol/L group and 0 μmol/L group. Resveratrol treatment can inhibited the migration of colorectal cancer cells and with dosage dependent manner. In the Transwell experiment, compared with Control group, different dosages of resveratrol treated HCT116 cells, the cell number of invasion significantly reduced in the other side and the reduction is related with the dosages. The elution of stained cells was detected OD changes, and found resveratrol also inhibit the immigration of cancer cells and match count cell data.High motility is related with a variety of gene abnormal expression of cancer cells, and recent studies showed higher expression of CD44 is closely associated with cell adhesion and cell migration. In order to explore the molecular mechanism of resveratrol inhibiting cancer cells motility, the expression level of CD44 was analyzed colorectal cancer cells after treatments of resveratrol. In the experiment, resveratrol treated cells for 12h and quantitative real-time PCR (RT-qPCR) was used to analyze the mRNA expression levels of CD44 in the HCT116 cells, and the high concentration treatments result in 0.3 fold (comparing with Control group) and with a dose-dependent manner. Further Western blot analysis found that resveratrol can significantly inhibit the protein expression levels of CD44 in colorectal cancer cells, and the result is consistent with the data of RT-qPCR analysis. Through the analysis of the transcription and translation level of CD44, we confirmed that resveratrol can inhibit the expression of CD44 in colorectal cancer cells.In the promoter region of CD44 gene, it exists the binding sites of transcription factors such as AP-1, NFκB, SP1 and so on, and those transcription factors regulate the transcription of CD44 gene. To investigate the signal pathway which is involved in resveratrol inhibiting CD44 expression, we analyzed the effect of resveratrol on transcription factor activities. In this experiment, AP-1, NFκB reporter gene with firefly luciferase was transfected into colorectal cancer cells HCT116, resveratrol treatment can inhibit both activaties of AP-1 and NFκB. It suggested that resveratrol inhibit CD44 expression may be mediated by reducing AP-1 and NFκB transcription activities.Based on the above experimental research, the project approved that inhibit the motility of colorectal cancer cells, and analyzed the molecular mechanism: resveratrol treatment-*AP-1 and NFκB activitiesâ†'â†'CD44 expression level↓â†' motility of cancer cell↓。... |
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