Inhibition Of Human Melanoma Cells By Bufadienolides Based On AKT Signaling Pathway And Its Molecular Mechanism

Background and purpose: Melanoma is one of the most common skin malignancies in the clinic,and its morbidity and mortality are higher in all tumors in the world.The number of patients with malignant melanoma in the skin caused by ultraviolet radiation rapidly increased every year.For the treatment of melanoma,the current common treatment methods include medicine therapy,surgical resection,radiotherapy,chemotherapy,etc.However,these treatment strategies usually have incomplete treatment.Medicines,radiotherapy and chemotherapy cause adverse reactions such as nausea,vomiting,dizziness,etc.,and the disease is easy to recurrent.The clinical treatment effect is far from satisfactory,and it also increases the global public health burden to some extent.To this end,many medical scientific researchers are committed to finding medicines that are ideal for improving treatment,reducing side effects,and reducing melanoma mortality around the world.Especially in recent years,the field of cancer research is constantly exploring new therapies and concepts such as molecular targeted therapy and integrated Chinese and Western medicine treatment.Clinical studies have shown that Chinese medicine has unique advantages in the treatment of melanoma,including attenuation and synergism of radiotherapy and chemotherapy.Therefore,screening for medicine that can treat melanoma in many natural medicines and traditional Chinese medicines is one of the research hotspots at home and abroad.The bufadienolides is a natural mixture obtained by extracting and separating the sputum from the skin of the sputum and the venom of the parotid sputum.Traditionally,Chan Su is used for the treatment of various clinical diseases such as cardiotonic,analgesic,blood pressure,antibacterial,local anesthesia and anti-tumor in China.The separation and identification of chemical components and the pharmacological effects of the Chan Su found that bufadienolides is the main pharmacological component of Chan Su.In related reports,bufadienolides compounds have been shown to induce tumor cell cycle and apoptosis.In addition,bufadienolides can significantly inhibit the proliferation of cancer cells and induce autophagy invasion,and a significant anti-tumor effect through a series of pathways has been reported in the literature.Based on previous studies,this study selected the AKT signaling pathway commonly used in tumor mechanism research to investigate whether bufadienolides can reverse the activity of melanoma cells and increase apoptosis and autophagy by inhibiting AKT pathway,confirming its anti-tumor effect.Experimental approach:The melanoma A-375 cell line was cultured in vitro,CCK-8was used to detect cell viability,western blotting(WB)and flow cytometry(FCM)were used to detect autophagy,apoptosis levels and their effects on AKT signaling pathway-associated protein expression in A-375 cells.Key results: In melanoma A-375 cell line,CCK-8 results showed that different concentrations of bufadienolides had significant inhibitory effects on melanoma A-375 cells in a time and a dose dependent manner,with statistical difference(p<0.05).).Flow cytometry showed that the number of apoptosis in melanoma A-375 cells were increased with the increase of the concentration of bufadienolides.The results of western blot showed that the levels of apoptosis related proteins BAX and p53 were significant increased in A-375 cells treated with different concentrations of bufadienolides,while the levels of anti-apoptosis related proteins bcl-2 and caspase3 were decreased.The level of autophagy related protein LC3-II was significantly increased,and the p62 protein was inhibited by the bufadienolides.In addition,Western blot results showed that the AKT pathway and its downstream related proteins AKT,phospho-AKT(p-p AKT),m TOR,p70S6 K,phospho-p70S6K(p-p70S6K),GSK-3β,phospho-GSK-3β(p-GSK-3β)and cyclin D1 are involved in the regulation of this process.Conclusions and Implications: Bufadienolides has a significant inhibitory effect on melanoma cells,and its possible mechanism is that bufadienolides are able to impair the viability of melanoma cells and to increase the levels of apoptosis and autophagy via inhibition of the AKT signaling pathway.This study is a new natural drug for the treatment of melanoma cells,providing a potential therapeutic drug for the effective treatment of human melanoma.