| In medicine, cancer refers to the malignant tumor originated from the epithelial tissue, which is the most common type of malignant tumors. Corresponding, originated from mesenchymal tissue are collectively named as sarcoma. There are a fraction of malignant tumors not named according to the above principles, such as renal tumor,malignant teratoma. Generally, what is called the "cancer" customarily denotes all malignant tumors. Skin melanoma is a kind of skin cancer, which is originated from melanin cell with highly malignant degree. Because of its features of easy transfer and little sensitive to traditional chemotherapy, as well as the yearly increasing incidence and the relatively younger trend in recent years, it has become hot issues in the skin cancer research field at homeland and abroad at present, but drugs with good effects have not been found for the treatment of melanoma.Gamboge, as a kind of traditional Chinese medicines in our country, is the dry resin secreted from gambogic trees of gambogic branch. Its detoxification, anti-inflammatory and desinsectization and some other biological functions were recorded in ancient medicine books. In modern TCM clinic, it is widely used to treat cancer, ulcers,folliculitis, etc. Lv Guibao isolated a kind of compounds similar to gambogic acid in molecular structure,which was named gambogic acid(GNA). Preliminary research results show that the gambogic acid can selectively inhibit a variety of tumor cell proliferation through multiple pathways and targets, while it has more extensive antitumor activity and lower toxicity compared with gambogic acid. However, the mechanism on the antitumor of GNA is not very clear. Early experiments in the laboratory have confirmed that the GNA of specific concentration can inhibit melanoma B16 cells in vitro, and induce their apoptosis, but the study about whether GNA can inducel autophagy and even death of melanoma B16 cells is rare. This topic on the basis of previous research probes into the influence of GNA inhibiting mouse melanoma B16 cells proliferation and its possible mechanisms of antitumor, thus, provide certaintheoretical basis for developing GNA to be clinical effective anti-tumor drugs or anti-tumor synergistic agent.1 Objective Investigate the influence of the GNA inhibiting B16 cells proliferation and discuss the role of autophagy mechanism in it.2 Methods In vitro culture B16 cells, adopt MTT assay to detect the change of the cell survival rate after the disposal of GNA, observe the form change of B16 cells after the action of the GNA in the inverted microscope; observe the change of B16 cell ultrastructure in transmission electron microscope; detect the change of B16 cells membrane potential and ROS induced by GNA by FCM; detect the change of autophagy related proteins,such as, LC- 3, m TOR, AMPK and SIRT3 by Western blot assay; investigate the molecular mechanism of B16 cells autophagy induced by GNA.3 Results(1) MTT results showed that the GNA has obvious inhibitory effect on the growth and proliferation of melanoma B16 cells in vitro, and the cell viability decreased obviously with the increase of the GNA concentration and the extension of time;(2) The observation of B16 cells in the inverted microscope showed cell structure was obvious damaged and cell death increased with the increase of GNA concentration;(3) Cell morphological changes of autophagy for B16 cells under the influence of GNA occurs obviously, which was observed in transmission electron microscope;(4) Western blot assay tests showed protein expressions of AMPK and SIRT3 rised by time dependence; the protein expression of LC3-I had reverse by time dependence, the protein expression of LC3-II rised by time dependence; the protein expression of m TOR declined over time.4 Conclusion GNA in a certain time and the concentration range can inhibit B16 cells proliferation and induce the occurrance of cell autophagy. The mechanism of inducing cells autophagy may be related to the regulation of ROS/SIRT3 / AMPK signaling pathway. |
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