| Background:Glioma is the most common primary malignant brain tumor. In recent years, there is considerable progress in the field of neurological surgery, but even with the most modern neurosurgical techniques, it is difficult to achieve completely pathological resection of glioma, local spread and distant metastasis is the malignant biological behavior of gliomas, which makes glioma recurrence frequently. There is an urgent need for an effective adjuvant therapy to improve the prognosis of malignant gliomas. Resveratrol (Res) is extracted from Polygonum cuspidatum, veratridine, grapes and other natural plant,it is a kind of polyphenol compounds, with the effect of regulation the lipid metabolism, inhibition platelet aggregation, protect cardiovascular system, anti-inflammatory effect and other biological activity. In recent years, several studies have found that resveratrol can inhibit the growth of some tumor cells, eg:breast cancer cells, prostate cancer cells, skin cancer cells and gastric cancer cells. there is a good blood-brain barrier permeability for resveratrol, so it is a very promising natural anticancer drugs.The first part Objective:To study the inhibition effect of resveratrol (Res) on U87and U251cell growth and to investigate the possible machanisam. Methods: Different concentrations of resveratrol (0,25,50,100?M) were used to treat glioma U87cells and U251cells, inverted microscope was used to observe the effects of resveratrol on the morphology change of glioma cells, trypan blue staining and MTT proliferation inhibition assay were uesd to detect the growth inhibition effect of resveratrol on the growth of glioma cells, RT-PCR and Western blot were used to detect the mRNA and protein expression of EGFR, LRIG1, phosphorylated Akt and phosphorylated Ert. Results:The experimental results showed that after treated with different concentrations of resveratrol, the shape of U87cells and U251cells began to become round, cell viability decreased, and cell volume became smaller. Trypan blue staining and MTT proliferation tests showed that resveratrol can inhibit the growth of U251cells and U87cells in a time and dose dependent. RT-PCR and western blot results found that after treated with resveratrol, there is decreased expression of EGFR and increased expression of LRIG1in both mRNA and protein level, resveratrol can inhibit the phosphorylation of Akt but there is no change on the expression of phosphorylated ERK. Conclusion:Resveratrol can inhibit the proliferation of glioma cells through inhibition the PI3K/Akt signal transduction pathway. The second part Objective:To study the apoptosis induction effect of resveratrol on Glioma U87cells and to explore the possible apoptosis singal transduction pathway. Methods:DNA agarose gel electrophoresis and Annexin V-FITC apoptosis kit were used to detect the apoptosis-inducing effect of resveratrol on glioma cells. Flow cytometry was used to detect the mitochondrial membrane potential. Western blot were used to detect the expression of apoptosis releated protein Cyto C and caspase-9. Results:After treated with different concentrations of resveratrol (0,25,50,100?M), there is typical apoptotical DNA ladder on the DNA agarose gel electrophoresis, Annexin V-FITC apoptosis detection kit also found that resveratrol can induce apoptosis of U87cells. Further studies shew that after stained with Rhodamine123, flow cytometry detected that there is significantly reduction of the mitochondrial membrane potential of U87cells. Western blot analysis shew that resveratrol can enhance the expression Cyto C and caspase-9in a dose-dependent manner. Conclusion:Resveratrol can induce apotosis of glioma U87cells, the activation of endogenous apoptosis pathway may be involed in this process.The third part Objective:To study the autophagy induction effect of resveratrol on Glioma U87cells and to explore the possible machanisam. Methods: U87glioma cells were treated with different concentrations of resveratrol for24h, electronic microscope was uesd to observe the typical autophagic vacuoles. RT-PCR, Realtime PCR and Western blot analysis were used to detect the mRNA and protein expression level of LC-3, Beclin-land VPS34gene. Results:Typical appearance of autophagic vacuoles were observed with electronic microscope, Semi-quantitative RT-PCR and Realtime PCR analysis shew that resveratrol can increase the expression of LC-3, Beclin-land VPS34gene expression. Western blot analysis also shew that LC-3, Beclin-1, VPS34protein expression was significantly enhanced after treated wirh resveratrol. Conclusion:Autophagy is involved in Res induced U87cell death.The up expression of autophagy related gene LC-3?Beclin-land VPS34may mediate this process.The fourth part Objective:to study the cell invasion and metastasis inhibion effect of resveratrol and explore the possible mechanisms. Methods:Wound healing assay and Transwell invasion assay were used to dectect the movement ability and the effect on invasion and metastasis of glioma cells after treated with different concentration of resveratrol, RT-PCR and Western Blot were used to detected the mRNA expression and protein expression levels of MMP-2, MMP-9, TIMP-land TIMP-2after treated with resveratrol. Results:Wound healing assay and Transwell invasion assay shew that resveratrol can signifcantly inhibit the movement and invasion ability of glioma cells. RT-PCR and Western Blot results shew that resveratrol could reduce MMP-2and MMP-9expression, and increase the expression of TIMP-land TIMP-2. Conclusion:Resveratrol may inhibit the migration and invasion of glioma cells by inhibit MMP-2and MMP-9expression and regulate the homeostasis of TIMP-2/MMP-2and TIMP-1/MMP-9.Taken together, in this study, set the glioma cells as experimental model, we evaluated the proliferation inhibition effect, the apoptosis and autophagy induction effect and the cell invasion and metastasis inhibion effect of resveratrol and explore the possible mechanisms, which provide a scientific basis for the clinical use of resveratrol on the treatment of glioma. |
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