The Effect Of Resveratrol On Proliferation And Apoptosis Of Human Colon Cancer Cells And Its Relationship With COX-2

Colon cancer is one of the major malignant tumors that are seriously detrimental to human health.The majority of patients are diagnosed with advanced cancer.The early stage patients receive radical operation combined with adjuvant therapy,however,approximately half of the patients die of metastasis and(or)recurrence after surgery.The effect of chemotherapy based on 5FU, irinotecan and oxaliplatin capecitabined reaches a plateau, and the chemotherapeutic drugs are naturally insensitive or tolerated quickly.What’s more,toxic effects arouse our attention.Therefore,finding low-poison and high-effect natural herb ingredients has becoming the hotpot of developing the anticancer drugs in colon cancers in recent years.RESV is a kind of significant floristic antitoxin,which exists widely in grapes,peanuts and mulberries.Many researches indicate that RESV has pharmacological functions such as anti-inflammatory,anti-oxidation anti-thrombus and anti- mutagen.Meanwhile people find that it also has notable anti-tumor functions,which mainly perform in suppression of cell proliferation,induction of cell apoptosis,intervention of signal transducers and activators,strengthening the activity of cancer suppressor gene as well as inhibiting the expression of cancer gene.An enzyme called cyclooxygenase(COX) is a kind of rate limiting enzyme,which catalyzes arachidonic acid to generate prostaglandin.Its main products are prostaglandin(PGs) such as PGE2, PGD2 and so on.Up to now, there are two subtypes of COX, namely COX-1 and COX-2.In recent years, the researches about COX-2 in the mechanism of tumor occurrence and development have made significant progress. It is found that special connections exist between cox-2 and human epithelial malignant tumors, especially in the digestive system tumors.Studies show that COX-2 is highly expressed in the colon cancer. In the researches of liver cancer and breast cancer, RESV has a strong pharmacological effect of inhibition of cox-2, and it can hinder the prompting of cancer by inhibiting the activity of epoxy synthase and hydrogen peroxidase.The anti-tumor effect of resveratrol has been confirmed in a variety of vitro experiments and animal models for tumors such as liver cancer, breast cancer, gastric cancer.However,the anti-tumor mechanism of resveratrol has not clarified yet.Therefore, it is necessary to study the anti-tumor activity and related mechanism of resveratrol in colon cancer.In this study human SW480 colon cancer cell lines are implied as experimental objects to study the effect and the mechanism of resveratrol on colon cancer cell proliferation and apoptosis, and to explore the relationship between its anti-tumor effect and cox-2 so as to provide a new theory foundation for the colon cancer therapy.Part one: The effect of resveratrol on SW480 colon cancer cellsObjective:To observe the influence of resveratrol on the proliferation and cell cycles of SW480 colon cancer cell lines in vitro, and to explore its possible related molecular mechanism in promoting apoptosis.Methods: SW480 colon cancer cell lines are cultivated under the condition of37 ℃ and 5% CO2.1、After a logarithmic growth, cells are inoculated in a 96-well culture plate.Three groups are set up respectively: the experimental group、the blank group and negative control group. After different concentration of resveratrol(0,10,20,30,50ug/ml)in different time(24 h,48 h,72h),MTT method is used to test the effects of resveratrol on cell proliferation. IC50 is also calculated.2、The cells are vaccinated in six-hole plate, being set up with blank control group, and different concentration dosage groups. Use flow cytometry to detect apoptosis cycle.3、After 48-hour influence of different concentration of resveratrol with the cells,RT-PCR is used to detect expression levels of the celluar apoptosis related genes,BCL – 2,bcaspas- 9 and caspase-3 m RNA.Results:1 、 MTT colorimetric results show that resveratrol can significantly inhibit SW480 colon cancer cell proliferation, as the drug concentrations(0 to 50 ug/ml)increasing and the effecting time gradually lengthening(24 h, 48 h and 72 h), its inhibition rate obviously prolongs, ranging from 17.4 to 57.1%,showing a significant concentration- time dependence. While in low concentration(10 ug/ml or less),tumor inhibition effect was not significant.As for resveratrol of the same concentration, the inhibition rates of 48 h and 72 h are higher than that of 24 h,with statistical significance(P < 0.05). The inhibition rates of 72 h are higher than that of 48 h,with no statistical significance(P >0.05),.The IC50 s of Resveratrol in people colon cancer cells SW480 acting 24 h, 48 h and 72 h are 91.19±0.017ug/ml 、 32.58±0.022ug/ml and 29.49±0.024ug/ml,respectively.2、 Flow cytometry analysis results indicate that: as resveratrol concentrations(0,20,30,50 ug/ml) increasing gradually, the early apoptosis rates range from 3.8 to15.7%, and the proportion of late apoptosis is 0.9-32.2%,the total ratio of apoptotic cells increases from 4.7 to 47.9%,manifesting a significant concentration dependence.With different concentrations of resveratrol acting on SW480 colon cancer cell lines, the proportion of S phase cell increases, while the proportion of G1 phase cell reduces, which indicates that inresveratrol can induce SW480 cells in S phase retardation.3、RT-PCR test results show that as the concentration of resveratrol(0,20,30,50ug/ml) gradually increasing,the ratio of BCL- 2 / GAPDH integral optical density value shows a trend of decreasing(p ﹤ 0.001).The ratio of Caspase-9 / GAPDH and caspase-3/GAPDH optical density value shows a trend of increasing(p ﹤ 0.001).Conclusion:(1) Certain range concentrations of resveratrol on human colon cancer SW480 cells inhibits proliferation and promotes apoptosis, and manifests a time-concentration dependence.(2) Resveratrol induces SW480 cells retardation in S phase and induces cell apoptosis.(3) One of the mechanism of inducing cell apoptosis may be that the resveratrol acts in colorectal cancer SW480 cells by downregulating the expression of Bcl-2gene, upraising the expression of caspase-9, caspase-3 gene, thus triggering the activation of cytochrome C-Apaf-1- caspase-9-caspase-3 apoptosis pathway.Part two:The relationship between the anti-tumor effect of resveratrol and COX-2Objective: To explore the relationship between the proliferation inhibition effect of resveratrol and COX-2.1 、Methods: Three COX-2 si-RNA primers are designed to transfect SW480 colon cancer cells, use RT-PCR to screen the best targeting cox-2 si-RNA for subsequent transfection, use the MTT method to detect the cell proliferation after the expression of cox-2 being inhibited.2、Western-Blotting experiment:Cultivate the CCD- 18 co of normal colon cells and SW480、HT29 colon cancer cell lines under the condition of 37 ℃ and 5% CO2.After a logarithmic growth, these cell lines are inoculated in the 96- well plates,using Western Blotting to test expression differences of cox-2 protein between normal intestinal cells and colon cancer cell. Cultivate SW480 human colon cancer cells in24 h under the same conditions, later add different concentrations of resveratrol(10,20, 30 and 50 ug/ml) for 48 h, set up blank control group. Using Western Blotting to test the influence of different concentrations of resveratrol on cox-2 and PGE2 protein expression in colon cancer cell. Targeting cox-2 si RNA transfected SW480 cells after72 h, add resveratrol with concentration of 30 ug/ml for 48 h.Set up negative control group, the drug effect group, pure transfection group and drug effect group after transfection. Use Western Blotting to detect COX-2 protein expression changes of transfection SW480 cells after resveratrol.Results:1、The optical density scanning analysis show that: after transfection of cox-2si RNA for 72 h, compared with blank control and negative control group,cox-2 /GAPDH integral optical density value of NO.1, NO.2 and NO.3 COX-2 si RNA is significantly lower(P ﹤ 0.05), indicating that after cox-2 si RNA transfection,expression of cox-2 m RNA is restrained. For NO.2 group—the inhibition effect of cox-2 si RNA on cox-2 m RNA expression is stronger, therefor, NO.2 sequence is listed as subsequent transcription experiment design for cox-2 RNA.2、MTT test results indicate that:Compared with the blank control group and negative si RNA transfection control group, 48 hours later after cox-2 si RNA transfection cells SW480 cell,celluar proliferation begins to be restrained;72 hours later there is a significant cell proliferation inhibition(P < 0.05),which indicate that SW480 cell proliferation is restrained after transfection of COX-2 si RNA.3、 Western Blotting experimental results indicate that :(1) Cox-2 protein is expressed in the normal colon cells CCD- 18 co as well as human colon cancer cells SW480, HT29. The cox-2 / beta-actin integral optical density value ratio of CCD- 18 co in normal colon cells is lower than that of human colon cancer cells SW480, HT29, with a statistically significant difference(P <0.05),indicating that the cox-2 protein expression is higher than that of normal colon cells.(2) With the increasing concentration of resveratrol(0 to 50 ug/ml), cox-2 and PGE2 and beta-actin integral optical density value ratio are gradually reduced, with a statistically significant difference(P < 0.05).It is indicated that with the increasing of resveratrol concentration,the expression levels of COX-2 and PGE2 in SW480 cell are gradually decreased, and they are concentration-dependent.(3) The ratio of integral optical density value in COX-2 si RNA transfection group and beta-actin is lower than that of the negative comparative group, with a statistical difference(P < 0.05),indicating that the cox-2 si RNA transfection can reduce the expression of cox-2 protein.Resveratrol of 30 ug/ml on cox-2 si RNA transfected group, its COX- 2 and beta-actin integral optical density value of ratio compared with pure transfection group, decreased more significantly(P < 0.05),which can further suggest that resveratrol effect can reduce cox-2 protein expression of SW480 cell.Conclusion:(1) The cox-2 protein expression in colon cancer is higher than that of normal colon cells.(2) The cox-2 is closely related to the growth of colon cancer cells, and inhibition of cox-2 expression in colon cancer cells can hinder the growth of colon cancer cells.(3) The growth inhibition effect of resveratrol on colon cancer SW480 cell may be related to its inhibition of cox-2 and PGE2 expression.