Molecular Mechanism Of Microcirculation Patterns In Choroidal Melanoma And The Effect Of Curcumin And Endostatin On Intraocular Melanoma Model Growth And Microcirculation Patterns.

Objective:1.To investigate the correlation between the different microcirculation patterns and clinicopathologic parameters and its prognosis;to evaluate thc cxprcssions of MMP-2, MMP-9,EphA2 and VEGF in different microcirculation of choroidal melanoma(CM) and its clinical significance.2.To study curcumin and endostatin effect on intraocular melanoma model growth and microcirculation patterns;to examine curcumin and endostatin influcnce on the expressions of MMP-2,MMP-9,EphA2 and VEGF,and thereby reveal the molecular mechanism in suppressing angiogenesis and proliferation of melanoma cells in order to provide new targets and experimental data for the treatment of malignant tumors.Methods:1.56 cases of CM were collected and HE stainings were studied to check CM microcirculation patterns and the transelectron microscopy was used to observe the vasculogenic mimicry(VM)ultrastructure.Then PAS staining and CD34/PAS double staining were adopted to confirm three microcirculation patterns of CM.According to the presence of VM,All cases were divided into two groups,and also microvessel density(MVD),mosaic vessels and VM were counted to explore the correlation between CM and clinicopathologic parameters.Univariate and multivariate survival analysis were adopted to analyze VM effect upon prognosis,immunohistochemical staining was used to study the expression level of MMP-2,MMP-9,EphA2 and VEGF,and compare the difference between the VM group and the control group and analyze its role in the formation of VM and their correlation.2.1×10~5B16 melanoma cell suspension was injected into the retinal subspace cavity of right eye for each of 72 C57/BL6 mice.72 mice wcrc randomly divided into 4 groups.3 days after inoculation,we administered intraperitoneally circumin at a dose of 100 mg/kg/day,endostatin at a dose of 10 mg/kg/day and circumin and endostatin at the same dose receptively in the treatment groups.We administered 0.9%NaCl solution to the control group.Injection continued 18 days.Animals were sacrificed on Day 21.30 minutes before the mice were sacrificed a diluted suspension of activated carbon was injected into the circulation of mice previously through the caudal veins to test the function of VM.2 models were chosen to observe the VM structure by the transelectron microscopy.At the same time,lung,liver,kidney,lien,brain were removed to observe side effect of the drug and the presence of metastasis.We measured tumor sizes with a vemier caliper and compared the sizes of the tumors. Immunohistochemical staining test was used to analyze the expression level of MMP-9,MMP-2,EphA2 and VEGF in the different groups.Real time RT-PCR test was used to detect MMP-2,MMP-9 and EphA2 mRNA level in the fresh tumor tissue in each group.Results1.(1)Statistical analysis found that VM was not related to gender,eye,and age(P=0.618,0.560,0.687),but was related to cell type,LTD and metastasis,and the differences were statistically significant.VM positive rate of the cases with epithelioid cells(91.67%)was higher compared with the control group (48.30%)(P=0.025);VM positive rate of the tumor with LTD＞12mm(58.5%)was higher than that of the tumor with LTD=12mm(47.8%)and the differences were statistically significant(P=0.021);VM positive rate of the patients with metastasis(81.80%)was higher compared with the control group(37.80%)(P=0.027).(2)Similarly,MVD was not related to gender,eye,and age(P=0.266,0.430,0.316) but was related to celltype,LTD and metastasis.There were more MVD(53.48±5.14) with epithelioid cells MVD(38.55±3.32)compared with the control group(P=0.017). There were more MVD(55.80±4.86)with LTD＞12mm compared with the cases of LTD=12mm MVD(40.16±3.88)and the difference was statistically significant (P=0.014).MVD(64.27±8.16)of patients with metastasis was higher than that of patients without tumor metastasis(41.18±3.08)(P=0.003).(3)The number of MVD in the VM group was slightly lower compared with that in the control group(P=0.082).(4)Univariate survival analysis found that the survival rate for the patients with VM was significantly poorer than that for the patients without VM(P=0.000).Celltype, LTD,metastasis,MVD(average value 45.00),and VM had an effect on the prognosis(P=0.0193,0.0231,0.0328,0.0321,0.0076),while gender,age and eye, had no effect on it(P=0.662,0.278,0.885).(5)Immunohistochemical staining result:The positive rates of MMP-2 and MMP-9 (96.15%,100.0%,respectively)in the VM group were higher than those in the control group(66.67%,80.00%,respectively)(P=0.000,0.002).The expression level of MMP-2 and MMP-9 in the VM group were increased(57.16±6.39,55.96±7.67 respectively)compared with the control group(36.18±4.55,38.75±4.12, respectively)(P=0.000,0.000).The positive rate and expression level for EphA2 (92.31%,39.807±8.79)were higher compared with the control group(70.00%, 33.36±4.86)and the difference was statistically significant(P=0.002).The difference in positive rates and expression level of VEGF between the VM group and the control group was not statistically significant(P=0.089,0.419).2.(1)Immunohistochemical staining and transelectron microscopy showed that three blood supply patterns coexist in the intraocular malignant melanin transplanted tumor microcirculation patterns and the number of endothelium-dependent vessels,mosaic vessels and VM was obviously lower compared with the control group(P＜0.05).(2)Activated-carbon tracing showed that numerous carbon grains appeared in the central area of VM structure and it suggested that VM and endothelium-dependent vessels are interconnected and that VM together with endothelium-dependent vessels and mosaic vessels provided blood to tumor tissue.(3)Tumor size measurement showed that tumor growth in each treatment group was slower compared with the control group(F=8.30,P＜0.05).Tumors in curcumin group and in curcumin and endostatin group grew slower than in the control group(t =P＜0.05).Compared with the control group tumor metastasis in each treatment group was no significance(P＞0.05).(4)①All of the melanoma cells were positive for MMP-2,MMP-9 and EphA2 as detected by immunohistochemical staining,and the cytoplasm of positive cells was dark brown.The positive rates went from high to low as follows:the control group, endostatin group,curcumin group,curcumin and endostatin group.The positive rate for VEGF was higher than the control group.The positive rates of MMP-2,MMP-9, EphA2 and VEGF in each treatment group were statistically different from the contro 1 group(F=15.48,F=17.35,F=4.56,F=6.08,P＜0.05).②Real time PCR demonstrated that the expressions of MMP-9,MMP-2 and EphA2 in the treatment group were lower compared with the control group(F=19.35, F=19.43,F=8.13,P＜0.05).The expression level of MMP-2,MMP-9 mRNA in curcumin group and in curcumin and endostatin group was lower compared with the control group(t=7.17,4.53,P＜0.05).The expression of EphA2 mRNA was decreased in the curcumin and endostatin group compared with the control group (t=2.52,P＜0.05).Conclusion:1.(1)MVD and VM are related to LTD,tumor celltype and tumor metastasis rate. Moreover,the larger the tumor,the higher the MVD and VM and the more tumor metastasis.The positive rate for VM with epithelioid cell was increased.(2)VM can be used as an indicator of poor prognosis of CM patients.The survival rate for the patients with VM was poorer compared with the control group.Tumor celltype,LTD, VM are important and independently prognostic factors for CM patients.(3)The expression level of MMP-2,MMP-9 and EphA2 in the VM group was increased obviously while VEGF remained low.MMP-2,MMP-9,EphA2 are necessary for the formation of VM in CM and are important enhancing factors whereas VEGF is not a major factor.This shows that the mechanism of VM formation is different from that of endothelium-dependent vessels formation.2.(1)Three blood-supply patterns coexist in intraocular malignant melanin transplanted tumor microcirculation patterns.VM and host vessels are interconnected to supply tumor cells with nutrition.(2)Curcumin decreased MMP-2,MMP-9 and EphA2 expression,and inhibit the vasculogenic mimicry,mosaic vessel and endothelial dependent vessel formation.Endostatin decreased VEGF expression,and inhibit endothelial dependent vessel formation.The combination of curcumin and endostatin enhances the effect of anticancer.(3)Curcumin can decrease the activity of tumor cell proliferation and inhibit melanoma growth.