Paper One: A Case Of Erythrodermic Psoriasis Inward With Staphylococcus Aureus Septicemia Paper Two: Encorafenib(LGX818),A Potent BRAF Inhibitor,Induces Senescence Accompanied By Autophagy In Brafv600e Melanoma Cells

Objective:In the present study,we investigated the anticancer activities and the mechanism of Encorafenib(LGX818)on BRAFV600 E melanoma cells.Methods:1.Human melanoma A375 and G361 cells were used in the present studies.The cells were treated with LGX818 or DMSO(control conditions)respectively.2.Cell viability was measured by MTT assay and Colony Formation assay in melanoma cells.3.Cell cycle and Apoptosis rate were analyzed by propidium iodide(PI)flow cytometry and Annexin V-FITC/PI double staining flow cytometry respectively in melanoma cells.4.Cellular senescence was detected by SA-?-gal staining assay.5.Formation of autophagosomes in the cytoplasm was detected by TEM(transmission electron microscopy)in melanoma cells.6.The expressions of apoptosis-related proteins PARP ?Caspase-3 and p53,autophagy-related proteins LC3?p62?p-m TOR?pp70S6K,related proteins of senescence:p27?p21?p Rb,related proteins of cell cycle:Cyclin D1?CDC6?CDK2 and ERK/MAPK pathway in melanoma cells were detected by Western blot.7.Stable cell lines : Lentiviral particles were used to directly infect A375 and G361 cells,and then stable clones were selected using puromycin.The selected cell populations were subjected to immunoblotting to determine the silencing efficiency.Results:1.MTT assay and Colony Formation show that LGX818 exhibits antiproliferative activity in BRAFV600 E melanoma cells.2.Flow cytometry shows that LGX818 induces cell cycle arrest at the G1 phase in BRAFV600 E melanoma cells.3.Flow cytometry shows that apoptosis is not involved in LGX818-mediated melanoma cell growth4.The expression of senescence-associated ?-galactosidase(SA-?-gal)serves as readouts for cellular senescence.5.TEM analysis shows that LGX818 increases the accumulation of autophagosomes in the cytoplasm in BRAFV600 E melanoma cells.6.Western blot analysis shows that LGX818 can reduce p-Erk1/2 protein levels to suppress the ERK/MAPK pathway,decrease related proteins of cell cycle:Cyclin D1?CDC6,increase p27KIP1 expression and retinoblastoma protein levels,trigger autophagy through inhibition of the m TOR/70S6 K pathway in BRAFV600 E melanoma cells,but can not increase the levels of cleaved PARP?cleaved Caspase-3 and p53.7.A375/sh LC3 and G361/sh LC3 cells exhibite decreased p27KIP1 expression and down-regulated p Rb activation in the presence of LGX818 compared to A375/sh Control and G361/sh Control cells,respectively,and display decreased ?-gal staining after a 48 h LGX818 treatment compared with cells stably infected with lentivirus targeting control sh RNA(A375/sh Control or G361/sh Control).Conclusions: Collectively,we demonstrated that LGX818 exerts its anti-melanoma effects via inducing cellular senescence accompanied by autophagy in BRAFV600 E melanoma cells.Therefore,our study reveals a mechanism by which LGX818 displays its anti-tumor activity in BRAFV600 E melanoma cells and supports further exploration of LGX818 as a candidate drug for patients with BRAFV600 E melanoma.