The Study For The Mechanisms Of Anticancer Effect Of Resveratrol On Malignant Melanoma

Malignant melanoma (MM) is a cutaneous tumor originate from human melanocyte. Despite recent new insights into the pathogenesis of MM, until now, effective drugs and means were to seek as for the patients with transferred MM in middle and terminal term. New biologically based drugs and therapies are therefore urgently needed. Resveratrol has recently attracted considerable interest because of its inhibitory activity on biochemical events associated with many kinds of tumors development including MM. It was reported that resveratrol inhibts the growth of human malignant melanoma cells both cultured and fresh separated. However, the exact mechanism of the anti-tumor activity of resveratrol remains to be further determined. In the present study, we investigated the effect of resveratrol on the growth, the cell cycle and the apoptosis of human malignant melanoma cells in vitro and to explore the mechanisms involved. We further investigated the influence of the local renin-angiontein system on angiogenesis in malignant melanoma cells. And we also examined whether resveratrol affects the local renin-angiontein system expression and angiogenesis in malignant melanoma cells.PARTⅠThe Effect of Resveratrol on the Growth of Malignant Melanoma and Its MechanismsBackground and objective:Resveratrol, a kind of plant complement mainly derived from spermatophytes, belongs to nonflavanoid polyphenols. It has a variety of functions such as protecting heart and vessels, anti-inflammation, regulating the levels of blood lipids and inhibiting platelet aggregation. And recent researches have further confirmed that resveratrol have obvious inhibiting function on the initiation, promotion and progression of tumors. Therefore, resveratrol was considered as one of the most prospective natural chemo/anticancer. Earlier studies reported that resveratrol have significant inhibiting function on the growth of various kinds of tumor cells including malignant melanocytes. And more, recent investigation have showed that it can inhibit the propagation of cultured or newly-isolated malignant melanocytes, although the definite mechanism has yet to be elucidated. Consequently, our study aims to observe the influence of resveratrol on the propagation, apoptosis and cell cycle of malignant melanocytes, investigate its molecular mechanism of inhibiting the growth of malignant melanocytes and afford theoretical and laboratory evidence for the clinical application of resveratrol as a kind of anticarcinogen for malignant melonoma.Methods:The effect of resveratrol on the growth of human malignant melanoma cell line A375 and mice malignant melanoma cell line B16-F1 were studied through MTT assay, The effect of resveratrol on the apoptosis of A375 and B16-F1 cells were studied by annexin V-FITC, The effect of resveratrol on the cell cycle of A375 and B16-F1 cells were studied by a propidium iodide method, and the effect of resveratrol on the expression of Bcl-2, Bax, cyclinDl and p21 protein was studied by Western blot analysis.Results:Resveratrol inhibited proliferation of A375 and B16-F1 cells in a time- and dose-dependent manner. Treatment with 25μmol·L-1 resveratrol for 24h induced apoptosis of A375 cells. The rate of the apoptosis was (16.7±2.1)%. And the apoptosis rate of B16-F1 reached (39.6±3.3)% at concentration of 100μmol·L-1. And the apoptosis rate of A375 cells was(17.2±1.7)% and (52.3±4.1)% at concentration of 100μmol·L-1 for 12h and 72h respectively, while the apoptosis rate of B16-F1 was(18.4±1.6)% and (56.7±4.5) %. Flow cytometric analysis showed that A375 and B16-F1 cells treated with resveratrol were accumulated in G1 phase in the cell cycle.And the accumulated effect was increased in a dose-dependent manner:The percentage of G1 phase of A375 and B16-F1 was (40.51±3.97)% and (41.34±3.12)% when treated with 25μmol·L-1 resveratrol for 24h, while it was (55.64±4.95)% and (53.93±5.12)% when treated with 100μmol·L-1 resveratrol for the same time.The expression of Bcl-2 and cyclinDl protein was decreased in malignant melanoma cells treated with resveratrol, while Bax and p21 protein was increased.Conclusion:Resveratrol is able to inhibit the proliferation of malignant melanoma cells by regulating the cell cycle and inducing the cell apoptosis. In addition, regulating the expression of cyclinDl/p21 and Bcl-2/Bax may suggest an involvement in the resveratrol-induced apoptosis.PARTⅡThe Expression of AngiotensinⅡin Malignant Melanoma Cells And the Influence of AngiotensinⅡon Angiogenesis in Malignant Melanoma CellsBackground and objective:The main function of Renin-Angiotensin-System (RAS) was traditionally viewed as the regulation of blood pressure and balance of water and electrolyte. Interestingly, local RAS was recently found existing in many tissues and was evidenced to participate in a series of physiological and pathological processes such as inflammation, cellular propagation and apoptosis. In addition, the main effective peptide of RAS was angiotensinⅡ(AngⅡ), which has two specific receptors respectively named as AT1R and AT2R. And researches have evidenced that the biological activity of RAS was exerted by AngⅡ/ATI R signalling pathway. And moreover, in spite of the definite mechanism has yet to be elucidated, RAS is recently found to be involved in initiation and progression of some tumors. In our study, we preliminarily analyzed the expression of AngⅡin malignant melanocytes and further investigated the influence of AngⅡon the initiation and formation of vessels in malignant melanoma.Methods:Huan melanocyte and HUVEC were isolated and cultured successfully. The expression of angiotensin II in malignant melanoma cell lines A375 and human melanocytes were detected by radioimmunoassay. The effect of angiotensin II on the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro were studied by tube formation assay. The expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in A375 cells with or without angiotensinⅡor losartan, an inhibitor of AT1R, were studied by Reverse transcriptase polymerase chain reaction (RT-PCR) and Enzyme-Linked Immunosorbent Assay (ELISA).Results:In the case of the expression of angiotensinⅡ, A375 cells had a higher level than human melanocytes (F<0.05). Angiotensin II stimulated tube formation of HUVEC in vitro significantly. Using two-dimension Matrigel, the treatment of 1×10-6mol/L AngⅡled to formation of elongated and robust tube-like structures, which were organized by much larger number of cells compared with the control (P<0.05). After addition of angiotensinⅡ(1×10-6mol/L), the expression levels of mRNA and protein of VEGF and bFGF in A375 cells were greatly higher than those before the treatment whereas losartan (1×10-6mol/L), an inhibitor of AT1R, was contrary to angiotensinⅡ(P<0.05).Conclusion:These findings indicate that there exists abnormal expression of local renin-angiotensin system (AngⅡoverexpression). AngⅡcan promote malignant melanoma angiogenesis significantly, which might be one of the pathogenesis of that local renin-angiotensin system affects tumorigenesis of malignant melanoma.partⅢThe study for the down-regulation of local RAS and the inhibition of Angiogenesis in Malignant Melanoma by ResveratrolBackground and objective:Earlier studies carried out by others and us highly suggested that the overexpression of RAS existed in MM, which could obviously promote the initiation and formation of vessels in MM. In addition, other researches has also showed that angiotension converting enzyme inhibitors (ACEI) has the function of inhibiting the growth of partial tumor cells other than decreasing the blood pressure. And more, resveratrol was newly evidenced to have the bioactivity of ACEI, which highly suggested that some unknown anti-tumor machanisms different from inhibition of propagation and induction of apoptosis of tumor cells may exist in resveratrol. In our study, we preliminarily analyzed the influence of resveratrol on the expression of AngⅡ/AT1R in MM and further investigated whether resveratrol inhibited the initiation and formation of vessels in MM by the regulation of AngⅡ/ATIR, for sake of affording laboratory and theoretical evidences for the finding of some potential anti-tumor mechanisms exerted by resveratrol.Methods:The expression of angiotensin II in malignant melanoma cell lines A375 with or without resveratrol was detected by radioimmunoassay. The expression of angiotensin II type I receptor (AT1R) in A375 cells with or without resveratrol was studied by Reverse transcriptase polymerase chain reaction (RT-PCR) and Western-blot. The effect of resveratrol on the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro were studied by tube formation assay.The expression of VEGF and COX-2 in A375 cells with or without resveratrol was studied by Western-blot.Results:After addition of 100μmol·L-1 resveratrol for 24h,there was no difference between the expression levels of angiotensin II in A375 cells from the treatment group and the controls (P>0.05). However, Treatment with 100μmol·L-1 resveratrol for 24h, the expression levels of mRNA and protein of AT1R in A375 cells were greatly lower than those of the controls (P<0.05). Resveratrol suppressed tube formation of HUVEC in vitro significantly. Using two-dimension Matrigel, the treatment of 100μmol/L led to formation of incomplete and fluffy tube-like structures. The diameter of the tube and the area covered by the tube network of the resveratrol-treated group were significantly lower than that of the control group (P<0.05).Resveratrol significantly decreased the expression of VEGF and COX-2 in a dose- and time- dependent manner. After addition of 25μmol·L-1 resveratrol for 24h, the expression of VEGF and COX-2 in A375 cells was downregulated compared with the controls (P<0.05).Treatment with 100μmol·L-1 resveratrol for 24h, the expression of VEGF and COX-2 in A375 cells was greatly lower than those of the controls (P<0.01). And treatment with 100μmol·L-1 resveratrol for 6h, the expression of VEGF and COX-2 was decreased (P<0.05). After 24h, the expression of VEGF and COX-2 was much more lower than the controls (P<0.01).Conclusion:These findings indicate that resveratrol partly participates in the regulation of retin-angiotensin system of malignant melanoma, the mechanism of which was realized not by the ACEI effect, but by antagonism to AT1R. Resveratrol may inhibit malignant melanoma angiogenesis by regulating expression of VEGF and COX-2. The inhibition of AT1R may contribute, at least in part, to the resvertrol-induced anticancer effect of resveratrol.