Experimental Study Of Vitamin E Succinate Inducing The Melanoma B16 Cells Differentiation In Vitro

Objective: This experiment aimed to study the effects of Vitamin E succinate on the proliferation, cell differentiation, distribution of cell cycle, expression of correlative protein and melanosome production of melanoma cells in vitro. Thus to provided new method and theoretical evidence for theatment of melanoma.Methods: Murine B16 cells were cultured in vitro. Effect of different dose of VES on the proliferation of B16 cells after 24h, 48h and 72h was measured by MTT colorimetric methods, and morphological changes of B16 cells was observed by light microscopy by Wright Giemsa stain. Distribution of cell cycle and apoptotic rate of B16 cells treated with VES at suitable drug concentration after 48h were examined by flow cytometry (FCM); melanosome was observed by the transmission electron microscopy. Melanin contents were determined by NaOH spallation;The expression of cyclinD1 and P21 proteins of B16 cells treated by VES after 48h were analyzed by flow cytometry and cell immunocytochemistry. Thus to initially investigated to support the mechanicsms of VES on induction differentiation of melanoma B16 cells.Results:1 VES inhibited the proliferation of B16 cells: after the administration of VES(5, 10, 20μg/ml) for 24h to 72h, the OD values of VES-treated groups decreased obviously. Compared with control group, there was statistically significant difference between control group and among every treatment group (P<0.01). Furthermore, with the increasing concentration of VES and prolong of treatment time, the OD values decreased gradually, the inhibitory rate increased, that was, VES inhibited the proliferation of B16 cells significantly in a dose and time dependent manner.2 VES could arrest cell cycle and induce apoptosis of B16 cells: After the administration of VES at the concentration of 5, 10, 20μg/ml for 48h, the number of cells in G0/G1 phase increased gradually and the number of cells in S phase decreased gradually, with the increasing concentration of VES, the proliferation index (PI) significantly decreased (P<0.01).It was demonstrated that, VES could induce an arrestment of cell cycle in G0/G1 phase in a dose-dependent manner. In addition, after the administration of VES, the apoptotic rate of every VES-treated group were 0.42±0.14%, 0.72±0.18%, 1.06±0.72%, respectively, and the control group was 0.25±0.04%; Because of the apoptotic rate of every VES-treated group all were smaller than 5% and without hypodiploid apoptotic peak, so 5-20μg/ml VES could not induce apoptosis to B16 cells.3 VES could cause morphological changes of B16 cells and observe melanosome by transmission electron microscope: After Wright Giemsa stain, the morphological changes of B16 cells were observed by light microscopy: The cells of normal B16 cells were adherence, multilayered and irregularly growth in vitro, round, oval and polygon. After treated with VES (10, 20μg/ml), cells show heteropolarity growth, parallel rank, without overlap; Following the time prolongation, the volume of the majority cells was gradually increasing and extent, and cellshad dendrite architecture, reticulate arrange, slow-growing, and cell population decreased obviously.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 round, karyoplasmic ratio larger, euchromatin more, heterochromatin less, intracytoplasm organelle junior except free ribosomes, without typical melanosome. However, after treatment with VES (10 and 20μg/ml), less microvilli on the surface of the cell membrane, cell nucleus smaller and irregular, heterochromatin more, karyoplasmic ratio smaller, more mitochondrion and rough endoplasmic reticulum and a great deal of melanosome in the cytoplasm, but without apoptosis cells could be seen, which were consisted with result of the flow cytometry above. 4 The determination of the melanin contents:Comparing the melanoma cells with the normal melanocytes, Melanin synthesis capability became lower, which increased obviously when B16 cells were induced differentiation by inductor. However, when melanin content was more than Two-fold, the differentiation is effective.The experiment result show that: after 5μg/ml, 10μg/ml, 20μg/ml VES affected B16 cells, compared the melanin content of every VES treated groupto the control group, about 1.0057±0.241-fold, 1.813±0.438-fold, 3.654±0.7-fold respectively. To sum up, there was statistically significant differrence between the negative control group and the groups that 10μg/ml, 20μg/ml (P<0.01), and the formation rate of melanin that 20μg/ml VES was 3.654±0.7 times than the normal group. Consequently, VES (20μg/ml) induce B16 cells differentiation effectually.5 Analysis on expressions of cyclinD1 and P21 by FCM: after the administration of VES at the concentrations of 5, 10 and 20μg/ml for 48h, the FI values of cyclinD1 decreased with the increasing concentration of VES, and the FI values of P21 increased. There was statistically significant difference between control group and among VES-treated group (P<0.01).6 The expressions of cyclinD1 and P21 protein of B16 cells were analyzed by immunocytochemical method, and scored in IHS value manner.CyclinD1 protein expressed both in the cytoplasm and nucleus of B16 cells which untreated with VES, especially in the cytoplasm, masculine intension. When after treatment with VES, following the concentration of VES increasing, cyclinD1 protein decreased gradually in the both cytoplasm and nucleus, especially in the nucleus. After scoring and statistics, expressive values of cyclinD1 (IHS value) decreased with the increasing concentration of VES. P21 proteinum expressed both in cytoplasm and nucleus that was extreme manipulus and strength weaker of B16 cells which untreated with VES and increased gradually in the both cytoplasm and nucleus, When after treated with VES, following the concentrateon of VES increasing, P21 proteinum increased gradually both in the cytoplasm and nucleus, especially in the nucleus. After scoring and statistics, expressive values of P21(IHS value) increased with the increasei-ng concentration of VES. To the expressive values of both proteins, there was statistically significant difference between the negative controlgroup and every VES treated group (P<0.01).Conclusions:1 VES could inhibit proliferation of B16 cells in a dose and time-dependent manner within a certain concentration and could not induce apoptosis.2 VES could arrest cell cycle in G0/G1 phase and induce them differentiation in a dose-dependent manner and especially after 48h.3 VES induced B16 cells differentiation obviously, on morphous respect, cells grow slowly, cell join reticular formation and a great deal of melanin granule in the cytoplasm. In the function, melanin content generate obviously increased, especially treated with VES (20μg/ml).4 VES could induce differentiation of melanoma cells and the mechanisms might be related with down-regulation the expression levels of cyclinD1 and up-regulation the expression levels of P21.5 The experiment demonstrated that: VES had the effect of inhibiting proliferation, inducing differentiation and arresting cell cycle, which provided new mind and theoretical evidence for curing of melanoma.