Inhibitory Effects And Mechanisms Of Vitamin E Succinate On Experimental Melanoma B16 In Mice

Objective: This experiment aimed to study the effects of vitamin E succinate on the cell proliferation, cell differentiation and apoptosis in the murine melanoma cell B16 in vivo. To study the inhibitory effects and mechanisms of VES on the treatment of melanoma, through expression of correlative protein. Thus to provided new method and theoretical evidence for theatment of melanoma.Methods: Forty healthy male BALB/c mice were randomized into five groups, eight mice in each group. A suspension of murine melanoma cell B16 was inoculated into right back of each mouse,10~6 cells in each mouse ,to establish murine B16 metastatic tumor model. Beginning to administer and receive intraperitoneal injection respectively when tumors were touched on most mice. The control group received intraperit-oneal injection of seasame oil(0.05ml per mouse)once per day; the mice in VES groups received intraperitoneal injection of low-dose VES(12.5mg/kg),middle-dose VES(25mg/kg),high-dose VES(50mg/kg)once per day respectively, in 2 cycles of 5 consecutive daily injections followed by 2 days of rest. The Dacarbazine(DTIC)group received intraperitoneal injection of Dacarbazine 0.2ml (80mg/kg )per mouse once per day for five days. Two weeks later, all mice were killed,the tumor were taken and weighed,then the inhibitory rate of tumor growth could be calculated. The morphological changes of B16 cells was observed by light microscopy. The ultrastructure changes of B16 cells was observed by transmission electric microscope. At the same time,distribution of cell cycle and apoptotic rate of tumor cells of every group were examined by FCM; the expression of S-100,Survivin and Caspase-3 protein of tumor cells was analyzed by immunohistochemistry. Results:1 VES could inhibit the growth of mice tumors: the inhibitory rate of B16 metastatic tumor treated with12.5mg/kg,25mg/kg and 50mg/kg VES were 13.72%,31.22%,45.58% respectively,and it was in a dose-dependent manner. The inhibitory rate of DTIC group was 55.64%%,higher than every VES-treated group. Comparing murine weight of every group before being killed:there was no statistically difference between the control group and every VES-treated group(p>0.05).The weight of Dacarbazine group was obviously lower than the control group,there was statistically significant difference(p <0.01).2 VES could arrest cell cycle , inducing differentiation and apoptosis of mice tumor cells: the distribution of cell cycle of B16 metastatic tumor cell treated with 12.5mg/kg,25mg/kg ,50mg/kg VES were analyzed by flow cytometry:the number of cells in S phase increased gradually,in a dose-dependent manner , there was significant difference comparing to the control group( p<0.05 or p<0.01). But the distribution of cell cycle of B16 metastatic tumor cell treated with 12.5mg/kg,25mg/kg VES also were analyzed by flow cytometry:the number of cells in G0/G1 phase increased,there was statistically significant difference comparing to the control group(P<0.05 or P<0.01). The apoptotic rate of every VES-treated group were 20.88±0.58%,22.71±0.55%, 27.22±0.59% respectively, the control group was 6.73±0.97%,there was statistically significant difference between the control group and every VES-treated group(p<0.01).The apoptotic rate of DTIC group was 30.95±0.52%,which was higher than every VES-treated group(p<0.01).3 VES could cause morphological changes of B16 metastatic tumor cell:3.1 After HE staining, the morphological changes of B16 cells were observed by light microscopy: The control group tumor tissue boundary is not clear,and cells were pleomorphism ,in a dense array ; the VES-treated group tumor tissue center and brim saw different degree of a large patch or focal necrosis, and melanin particle released, scatterring in between the cells.3.2 The ultramicrostructure changes of B16 cells were observed by transmission electron microscope: the B16 cells without treatment with VES, cell membrane complete, more microvilli on the surface, cell nucleus bigger and irregular , karyoplasmic ratio larger, euchromatin more, heterochromatin less, intracytoplasm organelle junior except free ribosomes, without typical melanosome. However, after treatment with VES (12.5mg/kg,25mg/kg), there were less microvilli on the surface of the cell membrane, and cell nucleus got smaller, heterochromatin more, karyoplasmic ratio smaller, more mitochondrion and rough endoplasmic reticulum and a great deal of melanosome in the cytoplasm; after treatment with VES (50mg/kg), the cells presented apoptotic changes of different levels: cellular volume was smaller, vacuolus could be observed in the kytoplasm of some cells, nucelus overshoot in angle outwards, nuclear chromatin concentrated highly,electron density raised and they were enriched as crescent below the nuclear membrane,which was the characterized morphologic changes of apoptotic cells.But there wae not typical apoptotic body.4 The expressions of S-100,Survivin and Caspase-3 protein of tumor cells were analyzed by immunohistochemistry , and scored in IHS value manner.Expressive values of Survivin and S-100 (IHS value) decreased with the increasing concentration of VES, caspase-3 increased with the increasing concentration of VES.To the expressive values of every protein,there was statistically significant difference between the control group and every VES-treated group(p <0.05 or p <0.01). There was a close correlation between the expression of Survivin and the expression of S-100, Caspase-3 in the mouse melanoma cells.Conclusions:1 VES can inhibit obviously the growth of B16 transplantation tumor, in a dose-dependent relationship.2 VES has duple function: 12.5mg/kg and 25mg/kg VES could arrest cell cycle in G0/G1 phase, inducing part of cells differentiation ,and a great deal of melanin granule could be observed in the cytoplasm by transmission electron microscope . 50mg/kg VES could arrest cell cycle in S phase, inducing cells apoptosis, then inhibit B16 cells proliferation.3 VES has the effects of inhibiting malignant melanoma proliferation, decreasing the expressiong of S-100 protein,and reducing malignant degrees. The mechanisms might be related with down-regulation the expression levels of Survivin and up-regulation the expression levels of caspase-3.4 VES has a significant inhibitory effect on B16 metastatic tumor growth. Small doses of VES induce cell differentiation, and high doses of VES induce cell apoptosis,for the clinical treatment of malignant melanoma and chemical prevention provides new ideas and theoretical basis.