The Inhibitory Effect Of Acitretin And Thalidomide On Proliferation And The Expression Of VEGF In Mouse Melanoma Cell Line B16

Objective: Malignant melanoma is one of the skin cancers which lead to death. With the development of the basic research on oncologic fields, though the different available multi-modalities of therapies, including surgical operation, radiotherapy, chemotherapy, endocrinotherapy and intervention therapy and so on, have currently been used in the treatment for malignant melanoma, but the prognosis of malignant melanoma has not been markedly improved. To study the inhibitory effect and mechanisms of acitretin and thalidomide on B16 cell line.Methods: The mouse melanoma cell line B16 were treated with acitretin and thalidomide by different concentration.1 The prolifer inhibition was examed by MTT assay at different time.2 The morphological changes of cells were observed by reverse microscopy and HE staining method.3 Flow cytometry(FCM) were used to detect apoptosis and the changes of cell cycle.4 The expression of vascular endothelial growth factor(VEGF) was detected by immunocytochemical method.5 The FCM was used to detect the changes of VEGF in different groups.Results: 1 The results of MTT indicated that acitretin and thalidomide inhibited the proliferation of B16 cells. The B16 cells were treated with different concentration of acitretin (0.1μmol/L, 1.0μmol/L, 10μmol/L, 100μmol/L) after 24 hours, the cicytotoxic index (CI) was 20.53%, 25.08%, 29.31%, 31.89% respectively. The CI was 27.59%, 33.30%, 44.07%, 48.98% respectively after 48 hours. And the CI was 56.10%, 64.05%, 69.88%, 71.83% after 72 hours. Acitretin of 100μmol/L being the most efficient concentration after 72 hours, the CI was 71.83%. Acitretin of 10μmol/L had the same effects as 100μmol/L (P>0.05). These effects were in dose-dependent and time-dependent manner in the range from 0.1μmol/L to 100μmol/L; Thalidomide inhibited the proliferation of B16 cells slightly, The B16 cells were treated with different concentration of thalidomide (10μg/ml, 50μg/ml, 100μg/ml, 200μg/ml) after 24 hours, the cicytotoxic index (CI) was 4.86%, 8.67%, 10.42%, 9.64% respectively .The CI was 8.25%, 12.54%, 16.82%, 13.00% respectively after 48 hours. And the CI was 12.25%, 16.87%, 25.96%, 18.36% after 72 hours. Thalidomide of 100μg/ml being the most efficient concentration after 72 hours, the CI was 25.96%. The effects of 200μg/ml thalidomide were lower than that of 100μg/ml thalidomide, the CI was 18.36%. These effects were in dose-dependent and time-dependent manner in the range from 10μg/ml to 100μg/ml.2 More B16 cells became rounded and bad refraction when be treated by acitretin. The clump of B16 cell were found by HE staining method in the group of acitretin. The cells of the negative control group and thalidomide group proliferated actively with good refraction.3 The B16 cells were treated by different acitretin and thalidomide after 72 hours were detected by FCM, indicating the increase of proportion of G₀/G₁ period and the reduce of S period; Apoptosis was not observed by FCM in both experimental group and control group.4 Results of immunocytochemistry: The expression of VEGF in the acitretin groups (1.0μmol/L, 10μmol/L) was significantly lower than that in the control group. There were no difference between the thalidomide group and the control group.5 Results of FCM: VEGF expression of B16 cells in the acitretin group (10μmol/L) was significantly lower than that in the control group. There was no difference between the control group and the tholidomide group (100μg/ml).6 All data were analysized by SPSS12.0 for Windows.Conclusions: 1 Acitretin and thalidomide inhibited the proliferation of B16 cells in vitro. All these effects were in dose-dependent and time-dependent manner within the appropriate extent.2 Acitretin and 100μg/ml thalidomide inhibited the increase of proportion of G0/G1 period, but the apoptosis of B16 cells was not observed.3 Acitretin could inhibit the expression of VEGF in B16 cells, but the action of thalidomide was poor.4 The sensitivity of acitretin was higher than that of thalidomide in B16 cells.These results suggested that acitretin was supper to thalidomide in inhibiting proliferation and metastasis of B16 cells. The use of acitretin in conbination with thalidomide increased curative effect, meanwhile decreased side effect.