Study On Induction Of Apoptosis And Molecular Mechanisms By Gambogenic Acid In Melanoma Cell Line B16

Cancer mortality has become the first leading reason in various causes of death inthe world. Malignant melanoma is referred to as MM (malignant melanoma). It’s ahighly malignant melanoma cells to tumor. Malignant melanoma mostly occurs in theskin, it can also be found in the digestive tract, reproductive system, mucousmembranes, eye, choroidal and meningeal places prone to metastasis. The research ofGNA on mouse melanoma cell line B16proliferation and its possible anti-tumormechanism for the development of GNA anticancer drugs provide the theoretical basis.Aim: Research GNA inhibits the proliferation of melanoma B16cells, and to explorethe role of the mechanism of cell apoptosis.Methods: The inhibitory effect of GNA on the proliferation of B16cells was measuredby methyl thiazolyl tetrazolium (MTT) assay; Alternation of ultra structure was detectedby AO/EB staining under fluorescent microscope; Annexin V-FITC/PI double stainingwas used to detect cell apoptosis rate by flow cytometry. Using flow cytometryintracellular reactive oxygen species (ROS) generated GNA treatment of B16cells.Western blotting to investigate intracellular the expression of Caspase-3, PI3K,p-PI3K, Akt, p-Akt, p-mTOR、PTEN proteins changes.Results：(1) MTT results showed that the GNA at a certain time and concentration significantlysuppressed the proliferation of B16cells and in a time-and concentration-dependent.(2) Morphological changes were observed by fluorescence microscope on B16cellsafter GNA treatment. AO/EB staining showed that normal cells stained with normal cellmorphology, cell density was relatively large; apoptotic cells or damaged cells showedorange fluorescence, the major cell morphological also changed, cell density decreased.GNA treated cells showed obvious apoptotic status. (3) Hoechst33258staining detected by Fluorescence Microscopy showed that thenuclei were stained into bright blue after treated with GNA, compared with the nuclei inthe control group.(4) After GNA treated with cells, in a short period of time, intracellular ROS levels canincrease dramatically compared with the control group (P<0.01), and the mitochondrialmembrane, which had a low potential consistently.(5) Flow cytometry (FCM) results showed that after GNA treatment, the apoptosis rateof B16cells could be significantly increased dependent accompanied withconcentrations of GNA.(6) Western blotting to detect changes of intracellular proteins expression in the releaseof Caspase-3and PTEN proteins expression levels were increased after GNA treatment;and proteins expression of p-PI3K, p-Akt and p-mTOR proteins had certain changesalso detected by western blotting. The trend of PI3K and Akt proteins changes is notobvious.Conclusion: GNA could inhibit malignant melanoma B16cells growth andproliferation and induce apoptosis within a certain time and the concentration, theinduction of apoptotic mechanism may be related to PI3K/Akt/mTOR signalingpathways.