Thalidomide Influence The Growth And Angiogenesis Patterns Of Melanoma

OBJECTIVE: The aim of this research was to examine the inhibition of thalidomide on the microcirculation patterns and proliferative activity of melanoma in mouse B16 melanoma transplanted tumor model. The effects and primary mechanism of thalidomide on melanoma microcirculation and tumor cell growth would be elucidated. These results would be hoped to elicit a novel approach to the clinical treatment of melanoma.METHODS: Thirty-five C57 mice were randomly divided into experiment group and control group (experiment, 20; control, 15). The melanoma transplanted model was established by subcutaneous inoculated 1×10~6 /ml murine melanoma cell line B16 cells 0.2ml into the mice lower left flank. Thalidomide dissolved in 0.5% sodium carboxylmethyl cellulose (CMC) suspension due to its poor solution. And thalidomide was lavage administered once a day at the dose of 200 mg/kg in experiment group from the fifth day of inoculation, and an equivalent volume of 0.5% CMC in the control group by the same way. The diameters of the tumors were measured from the fifth day of inoculation. Mice were sacrificed on the seventeenth day. These tumors were peeled off and fixed in 10% formalin. The morphological characteristics of slides of tumor specimens stained with H&E were examined in microscope. The vasculogenic mimicry, mosaic vessel, endothelial dependent vessel and necrosis ratio of tumor were measured byH&gstaining. Counting methods of microcirculation patterns: The vasculogenic mimicry, mosaic vessel and endothelial dependent vessel were counted in ten high fields in each slide. Necrosis ratio count: Observe ten representative high visual fields, count the necrosis cells among 100 tumor cells in every visual field; the mean value of the necrosis cells in 10 visual fields was the necrosis ratio. The expression level of MMP-2, MMP-9, VEGF, NF-κB and PCNA were examined by immunohistochemistry. Methods of counting: ten representative high visual fields were selected in each slide avoiding of putrescence areas,and the number of positive staining tumor cells were counted in 100 tumor cells in each field. Staining index was used to evaluate the entire staining status of each slide. Statistical significance of differences between the experiment group and the control group was calculated by using one-way ANOVA and unpaired t test. Statistical software SPSS 10.0(Chicago, Illinois) was used in the analysis. Pvalue less than 0.05 was considered as statistically significant.RESULTS: The tumor volume of experiment group was statistically significantly decreased compared with the control group (P＜0.05). The vasculogenic mimicry, mosaic vessel and endothelial dependent vessel count in experiment group were statistically significantly decreased compared with the control group (P＜0.05). The necrosis ratio of tumor was increased compared with the control group (P＜0.05). The expression level of MMP-2, MMP-9, VEGF, NF-κB and PCNA of experiment group tumor tissues were statistically significantly decreased compared with the control group (P＜0.05).CONCLUSIONS: The mechanism of inhibition B16 melanoma growth by thalidomide could be as these: (1) Thalidomide could decrease VEGF, NF-κB, MMP-2 and MMP-9 expression, and inhibit the vasculogenic mimicry, mosaic vessel and endothelial dependent vessel information. VEGF, NF-κB, MMP-2 and MMP-9 are inducible vasculogenic factors of tumor. Thalidomide decreases the expression of VEGF, NF-κB, MMP-2 and MMP-9 in order to inhibit the angiogenesis of melanoma. ROS (reactive oxygen species), which induced by thalidomide, could inhibit tumor cells proliferation especially endothelial cells. It maybe another factor which thalidomide produces could inhibit melanoma microcirculation. (2) Thalidomide could decrease the expression of PCNA, so could inhibit the proliferation of melanoma cells. Thalidomide could induce necrosis of melanoma cells and this also related to ROS because ROS could impair the DNA and protein of tumor ceils. In brief, thalidomide could inhibit melanoma growth through regulating the vasculogenic factors (VEGF, NF-κB, MMP-2 and MMP-9), decreasing proliferation and inducing necrosis of melanoma cells by producing ROS.