EGCG And ECG Modulate Apoptosis And Autophagy In Human Melanoma Cells Via The PI3K/AKT/mTOR Pathway

Malignant melanoma is a skin tumor that caused by the malignant transformation of normal melanocytes.Melanoma has become a difficult public health problem because of high invasiveness and lethality.Now the treatment of melanoma is mainly based on surgical removal of the lesion,supplemented by radiotherapy and chemotherapy.This therapy has a significant effect on melanoma without metastasis,but it has a poor effect on melanoma that have already developed metastases,and new therapies are urgently needed in the clinic.Natural products have been widely used in the treatment of many diseases because of their wide source,high drug resistance and few side effects,and they have also become a research hotspot of anticancer drugs.Catechins,the main substances in tea that exert pharmacological activities,can inhibit the proliferation of cancer cells and have many biological activities and pharmacological effects on the body,such as antitumor formation and metastasis.However,the effects and mechanisms of action of catechins on melanoma suppression have not been elucidated.Therefore,in this study,we focused on four catechins,epigallocatechin-3-gallate(EGCG),epicatechin-3-gallate(ECG),epigallocatechin(EGC),epicatechin(EC)as research objects,combined with malignant melanoma models,to systematically evaluate their specific mechanisms of regulating melanoma cell proliferation through apoptotic and autophagic mechanisms,to provide experimental research evidence and theoretical support for catechin treatment of melanoma.The main research contents are as follows:(1)Inhibitory effects of different catechins on the proliferation of human melanoma A375 cells.Results showed that of the four catechins(EGCG,ECG,EGC,EC),only EGCG and ECG exhibited a significant inhibitory effect on the proliferation of human melanoma A375 cells in a time-and dose-dependent.(2)Effects of EGCG and ECG on apoptosis in human melanoma A375 cells.Flow cytometry experiments showed that compared with the control group,the proportion of apoptosis increased after treated with EGCG and ECG.EGCG and ECG could induce A375 cell cycle arrest in G0/G1 phase;meanwhile,EGCG can also arrest A375 cell cycle in S phase,and ECG can also arrest A375 cell cycle in G2/M phase.Mitochondrial membrane potential experiments showed that EGCG and ECG treatment decreased intracellular mitochondrial membrane potential.The results of Western blotting assay showed that treatment of cells with EGCG and ECG down-regulated the expression level of anti apoptotic protein B Cell Lymphoma-2 Gene(Bcl-2)and up-regulated the expression level of proapoptotic protein Caspase-3(Caspase-3),which promoted cell apoptosis.(3)Effects of EGCG and ECG on autophagy in human melanoma A375 cells.The results of RT-PCR and Western blotting experiments showed that EGCG and ECG decreased the expression levels of Sirt3,a silent information regulator,and two autophagy markers,microtubule associated protein 1 light chain 3 and autophagy effector protein-1(Beclin-1),at both the gene and protein levels.In addition,EGCG and ECG exerted apoptosis and autophagy regulation by inhibiting the expression of mammalian target of adenylate activated protein kinase/rapamycin(AMPK/mTOR)and elevating the expression of phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/mTOR)pathway related proteins,as detected by Western blotting assay.(4)Rapamycin,an autophagy activator,was added to detect the relationship between apoptosis and autophagy.The results showed that compared with the control group,after adding autophagy activator rapamycin,the level of autophagy marker protein LC3-Ⅱ/LC3-Ⅰ increased,the expression of Caspase-3 increased and the level of Bcl-2decreased.Rapamycin induced apoptosis and autophagy of A375 cells.Compared with treatment with EGCG and ECG alone,the expression of LC3-Ⅱ/LC3-Ⅰ was significant increased after co-treatment with EGCG,ECG and rapamycin,but the trend of cell apoptosis did not change.It shows that apoptosis and autophagy are independent of each other for human melanoma A375 cells.(5)EGCG suppresses malignant melanoma in nude mice.A xenogeneic melanoma model was constructed by axillary injection of human derived melanoma cells in nude mice,and the effects of EGCG on melanoma through apoptosis and autophagy were evaluated at the animal level.The experimental results showed that compared with the control group,EGCG could inhibit the enlargement of tumor volume,promote the expression of total Caspase-3 and activated-Caspase-3,downregulate the level of Bcl-2,inducing apoptosis in tumor tissues;EGCG downregulated the expression levels of Beclin-1 and LC3-Ⅱ/LC3-Ⅰ,thus decreased the level of autophagy within tumor tissues.In conclusion,catechin EGCG and ECG can inhibit melanoma cell proliferation,induce cell apoptosis,reduce the level of cell autophagy,and have a certain therapeutic effect on melanoma.