| Research Background and ObjectivesMalignant melanoma(MM) causes the greatest number of worldwide skin cancers related to deaths.It may lead to rapid invasion and blood vessel and lymph metastasis.Proangiogenesis factor vascular endothelial growth factor(VEGF) is the core of angiogenesis mechanism.Researches have shown that MM angiogenesis is so active that tumor cells stimulate the proliferation,migration and tubulogensis of endothelial cells by producing large amount of VEGF and thus lead to angiogenesis of malignant tumor.Therefore,VEGF has been one of the frontiers in MM research in the mechanism of antiangiogenesis.There are at least two types of cannabinoid receptor,cannabinoid receptor 1 (CB-R1) and cannabinoid receptor 2(CB-R2) both G- protein coupled receptors(GPCRs).GPCRs plays an important role in the development and angiogenesis of malignant tumors.Researches have shown that CB-R2 agonist have effect on apoptosis in vitro and inhibit the growth of tumor in vivo and downregulation expression in VEGF.Our previous studies have shown that CB-R2 has high significance expression in MM,but it is yet unknown whether CB-R2 contributes to migration,invasion and angiogenesis in MM.The study is to investigate the effect of CB-R2 agonist JWH-133 and WIN55,212-2 in human malignant melanoma cell line A375 and xenografted malignant melanoma in nude mice,trying to find whether they have the effect of antiangiogenesis by regulating the expression of VEGF.Thus,it is to provide the novel targets for the treatment of MM. Methods1.Study on human malignant melanoma cell line A375 in vitroFirstly,we detect the expression and location of CB-R2 in A375 cell line by immunofluorescence,finding the effect of CB-R2 agonist JWH-133 and WIN55,212-2 on the expression of CB-R2 mRNA and protein levels by Real-time PCR,immunohistochemistry and Western blot.Then,we explore the inhibitory effect of CB-R2 agonist in antiproliferation,migration and invasion by MTT, wound-healing and Transwell chamber.Furthermore,we detecte the relationship between tumor cells growth and the production of angiogenic factors VEGF by ELISA and Real-time PCR.Finally,we study the effect on apoptosis of CB-R2 agonist by TUNEL.2.Study on xenografting malignant melanoma nude mice model in vivoEstablish the xenografting malignant melanoma nude mice model to observe the growth of the tumor,detecting the expression and location of CB-R2 in tumor tissue by immunofluorescence.And then we study the effect of CB-R2 agonist JWH-133 and WIN55,212-2 on the expression of CB-R2 mRNA and protein in tumor tissue by Real-time PCR,immunohistochemistry and Western blot. Furhermore we detecte the relationship between tumor cells growth and the production of angiogenic factors VEGF by Western blot and Real-time PCR.Finally, we study the effect on apoptosis of CB-R2 agonist by TUNEL.Results1.in vitro:(1)A375 cells have expressed the CB-R2 in mRNA and protein levels.The expression is located in kytoplasmand membrane,no dyeing on nucleus.(2)JWH-133 and WIN55,212-2 have effect on the inhibition of A375 cells at different concentrations(0.01/0.1/1/10/100umol/L) with the peak time on 48h (p＜0.05).No significantly difference between JWH-133 and WIN55,212-2 on inhibition of proliferation of A375 cells(p＞0.05).(3) JWH-133 and WIN55,212-2 inhibit the migration and invasion of A375 cells. JWH-133 and WIN55,212-2 of 1umol/L concentration has significantly smaller area than that of control group(p＜0.05).No significantly difference between JWH-133 and WIN55,212-2 on corr-migration area and trans-memberance quantity(p＞0.05).(4) Different concentration with JWH-133 and WIN55,212-2 (1umol/5umol/10umol/L) stimulate A375 cells after 24h,48h and 72h,with mRNA and protein levels significantly(p＜0.05) downregulating levels of VEGF.The inhibition effect has shown time and dose-dependent relationship.(5) JWH-133 and WIN55,212-2 of 1umol/L concentration stimulate A375 cells after 24h,48h and 72h and induce apoptosis.Apoptosis index(AI) of JWH-133 is 7.22%,9.17%and 11.45%;WIN55,212-2 is 6.45%,8.12%and 10.34%;Control group is 6.35%,6.09%and 6.39%respectively.The AI is significantly higher than that of control group(p＜0.05) and no significantly difference between JWH-133 and WIN55,212-2(p＞0.05).2.in vivo(1)Treatment of JWH-133 and WIN55,212-2 inhibit the tumor growth and the inhibition ratio is 43.14%and 39.47%respectively.(2)Treatment of JWH-133 and WIN55,212-2 significantly upregulates CB-R2 expression in mRNA and protein levels in tumor tissue(p＜0.05) and no significantly difference between JWH-133 and WIN55,212-2(p＞0.05).(3) Treatment of JWH-133 and WIN55,212-2 significantly downregulates levels of VEGF in mRNA and protein levels in tumor tissue(p＜0.05) and no significantly difference between JWH-133 and WIN55,212-2(p＞0.05).(4) Treatment of JWH-133 and WIN55,212-2 induces apoptosis in tissue levels.AI of JWH-133,WIN55,212-2 and Control was 12.84%,11.57%and 7.33% respectively.The AI is significantly higher than that of control group(p＜0.05) and no significantly difference between JWH-133 and WIN55,212-2(p＞0.05). Conclusions1.Cannabinoid receptor 2 participate the onset process of MM.2.JWH-133 and WIN55,212-2 significantly inhibit A375 cells proliferation, migration and invasion.3.JWH-133 and WIN55,212-2 induce A375 cells apoptosis in vitro and vivo.4.JWH-133 and WIN55,212-2 can inhibit angiogenesis and control the growth of tumor by downregulating levels of VEGF.5.JWH-133 and WIN55,212-2 have no significant difference in inhibiting A375 cell proliferation,migration,invasion and induction of apoptosis in vitro and vivo.
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