Molecular Mechanism Of Taxifolin Induced Cell Apoptosis In Melanoma A375 Cells

Background: Malignant melanoma is the most lethal type of skin cancer.Previous studies have shown that taxifolin has potent antitumor activity in a variety of cell lines.However,the anti-tumor effect of taxifolin on malignant melanoma remains unclear.Phosphatidylinositol 3-kinase(PI3K)is a key coordination molecule of intracellular signal response to extracellular stimulation.It is common that PI3 K / AKT signal cascade is over activated in the occurrence and development of cancer.Taxifolin can down regulate the AKT phosphorylation level of skin epidermal cells in JB6 P + mice,so as to play the role of chemoprevention.Taxifolin can down regulate the expression of AKT in osteosarcoma cell lines,resulting in the changes of c-myc and skp-2 expression.Taxifolin can down regulate the phosphorylation of PI3 K and AKT in colorectal cells.Taxifolin inhibits the development of scar cell carcinoma by inhibiting PI3 K / AKT / m TOR pathway,which leads to the down-regulation of MMP-2 and MMP-9 expression.Overall,taxifolin is likely to play an anti-cancer role by inhibiting PI3 K / AKT pathway,which is the most powerful evidence at present.In this study,we investigated whether taxifolin can inhibit the proliferation of melanoma cells and induce apoptosis of melanoma cells.In addition,we used A375 as a cell model to further explore the effect of taxifolin on PI3K/AKT under BRAF mutation and explained the role of taxifolin in melanoma cells.Objective: This study aimed to investigate the effect of taxifolin-induced cell apoptosis and the underlying molecular mechanisms.Method:The experiment was divided into control group and TAX group(low dose 100μM/high dose 200μM).(1)Use CCK8 assay to measure cell viability.The cell counting method determines the cell growth rate.Clone formation experiment,EDU proliferation staining to detect cell proliferation level.(2)PI staining to detect cell cycle distribution.Western Blot was used to detect the expression of cell cycle related proteins Cyclin B1 and Cyclin D1.(3)Woundhealingassay and Transwell assay explore the migration ability of A375 cells.(4)Annexin V-FITC/PI staining to detect cell apoptosis.Flow cytometry detects changes in the level of ROS.Mitochondrial membrane potential measurement,flow cytometry and fluorescence microscopy were used to detect cell apoptosis.(5)ROS reagent kit detects the level of ROS in cells.Flow cytometry analysis of the green fluorescence level of ROS probe DCF.(6)JC-10 Mitochondrial Membrane Potential Kit to detect mitochondrial damage.The fluorescence intensity of JC-10 was measured by flow cytometer and fluorescence microscope respectively.(7)Western Blot to detect the expression levels of apoptosis-related proteins Caspase8,Caspase3,Bcl-2,Bax and cytochrome C.(8)Western Blot to detect the expression of apoptosis-related signal pathways,and to determine the contents of PI3 K,AKT,phosphorylated AKT(p-AKT),and GSK-3β.(9)Use AKT agonist SC-79 to conduct reversionary verification experiments on the mechanism of taxifolin,and flow cytometry was used to detect the level of apoptosis.(10)Establish a nude mouse A375 xenograft tumor model and use taxifolin for treatment,divided into 30mg/kg and 60mg/kg high and low dose groups,and conduct a 14-day in vivo experiment.The treatment was administered every two days.After the treatment,the tumor mass and the heart,liver,spleen,lung and kidney of each group were taken out for HE staining.TUNEL assay was used to assess the level of apotosis and immunohistochemical staining experiments were used to detect the apoptosis-related proteins such as Bax,Bcl2,Cleaved Caspase 3 in each group of tumor masses.The expression of p-AKT and Ki-67.Result: Through CCK8 experiment,cell growth counting experiment and EDU staining experiment,it was found that taxifolin significantly inhibited the proliferation of BRAF mutant A375 cells in a concentration-dependent manner.After PI staining,flow cytometry found that taxifolin can induce cell G2/M phase arrest in A375 cells.Western Blot showed that taxifolin down-regulated Cyclin B1 and Cyclin D1 in A375 cells.Woundhealingassay and Transwell assayproved that taxifolin significantly inhibited the migration ability of A375 cells.In addition,reactive oxygen kits were used to detect ROS levels,JC-10 probes were used to detect mitochondrial membrane potential levels,and Annexin V/PI staining kits were used to detect apoptosis levels.Taxifolin not only induced an increase in ROS levels but also decreased mitochondrial membrane potentials.Theseresults eventually caused the increase in the level of apoptosis.Furthermore,Western Blot experiment was used to determine the expression level of apoptosis-related protein caused by taxifolin.The expression of Cleaved caspase 3,Cytochrome C,and Bax were significantly up-regulated,and the expression of Bcl2 was significantly down-regulated.Then the Western Blot results showed that the expressions of PI3 K,AKT,and p-AKT in A375 cells all decreased after taxifolin treatment,and the ratio of p-AKT/AKT decreased with the increase of the dose of taxifolin.GSK-3β,the inhibiting downstream of AKT up-regulated.Then,the AKT activator SC-79 was used to carry out a rescue experiment.First,the CCK8 experiment showed it can partially inhibit the cytotoxicity induced by taxifolin and maintain it to the inhibitory level acting on the L02 human normal liver cell line.Secondly,flow cytometry proved that SC-79 has a recovery effect on A375 cell apoptosis caused by taxifolin.Finally,we established a subcutaneous A375 xenograft tumor model in nude mice and gave high and low doses of taxfolin for treatment.We found that there was no significant difference in the body weight of the mice in each group,but the high and low dose treatment groups significantly suppressed the tumor volume and tumor mass are calculated to determine the effectiveness of the treatment by calculating the tumor growth inhibition rate.Through HE staining of various organs in mice,we can also see that taxifolin significantly inhibits the liver metastasis of tumors and has potential myocardial protection.TUNEL staining showed that taxifolin significantly increased the level of tumor cell apoptosis in vivo.Immunohistochemical staining further verified that Baxand Cleaved caspase 3 were significantly up-regulated,and Bcl2 was significantly down-regulated.Finally,its mechanism was verified.Immunohistochemical staining found that the p-AKT level was significantly downregulated in the high-dose taxifolin treatment group,and the expression of Ki-67,a marker of tumor malignancy,was significantly down-regulated.The taxifolintreatment group significantly promoted the expression of Cleaved caspase 3,cytochrome C and Bax,and inhibited the expression of Bcl-2,indicating that taxifolin can promote tumor progression by inhibiting the AKT signaling pathway.Conclusion: 1.Taxifolin can promote the production of ROS,induce the decrease of mitochondrial membrane potential level,and induce apoptosis.2.Taxifolin may induce cell apoptosis by inhibiting the PI3K/AKT signaling pathway to induce changes in the levels of apoptosis-related proteins.