Study On The Inhibition Mechanism Of B16 Melanoma Cell Proliferation By Arsenic Trioxide

Objective: The mechanism of the proliferative inhibition effect of arsenic trioxide(As2O3)on melanoma cells remains still unclear.This work aims at using the pathways screened by reference transcriptomics and metabolomics to study the effects of As2O3 on apoptosis,autophagy and cycle of B16 cells,and to investigate the relevant molecular mechanisms.Methods:(1)MTT experiments of cell proliferation: Mouse melanoma B16 cells in the exponential growth period were selected for this study,which were inoculated into 96-well plates and co-cultured there with As2O3 for 48 h.As2O3 solutions with different concentrations(0,0.02,0.039,0.078,0.156,0.312,0.625,1.25,2.5,5and10 μM)were used to test the inhibition effect of different amounts of As2O3 on the growth activity of mouse B16 cells.(2)Flow cytometry experiments: Based on the results of the As2O3 effective concentration tests,the apoptosis and cell cycle changes of B16 cells induced by As2O3 in a specific concentration range were analyzed by flow cytometry.(3)Transmission electron microscopy(TEM)experiments: The apoptosis and autophagy phenomena of B16 cells induced by As2O3 were further observed by TEM.(4)Reference transcriptome sequencing and metabolomics analyses: B16 cells were co-cultured with As2O3(10 μM)for 48 h,and then the cell mass and supernatant were collected for referent transcriptome sequencing and metabolomics analyses.(5)Western blot analysis: The influences of As2O3 on the expression levels of apoptosis,autophagy,and cycle-related signaling pathway proteins in Cleaved Caspase-9,PINK1,Parkin,LC3-Ⅱ/Ⅰ,Beclin-1,and CDK1 were determined.Results:(1)After co-culturing mouse melanoma B16 cells with As2O3 for 48 h,it was found that the 1.25 μM of As2O3 solution significantly inhibited the proliferation of B16 cells(P<0.05)and this effect showed an obvious dose-dependent manner with IC50 of 5.68 μM.(2)As observed by flow cytometry,As2O3 significantly induced the apoptosis of B16 cells(P<0.05)and the apoptosis rate was positively correlated with the As2O3 concentration.In addition,the formation of apoptotic bodies in B16 cells was also confirmed with TEM measurements.(3)The formation of autophagic lysosomes in B16 cells induced by As2O3 was observed by TEM.(4)Flow cytometry analysis showed that the B16 cell cycle was stagnated in G1 phase (P<0.001)and the proportion of cells in S phase was significantly decreased(P<0.01).(5)In the analysis of reference transcriptome sequencing,it was found that 1,933 differentially expressed genes were significantly up-regulated and 1,181 were obviously down-regulated in different groups.In the As2O3 treatment group,PINK1 and Parkin genes were significantly up-regulated(P<0.05),and CDK1 gene in the treatment group was remarkably down-regulated(P<0.05).Besides,a strong correlation of DNA replication,cell cycle and lysosome pathways with the growth inhibition of B16 cells was observed.KEGG enrichment pathway mainly includes cell cycle,autophagy,DNA replication,metabolism,aging and oxidative phosphorylation.(6)Cluster analysis was conducted to investigate the related mechanisms involving apoptosis,autophagy and cycle.Furthermore,the changes in the related key genes were found as follows: Casp9,PINK1,Prkn,Becn1 and LC3 increased to different extents,but CDK1 decreased.(7)Metabolomics and cluster analyses revealed the following changes in metabolites that closely correlated to B16 cell proliferation: Significant increase in glutamic acid,dichlorobenzoic acid,piperidinic acid,lysine and other metabolites(P<0.05),and obvious decrease in thiamine(P<0.05).(8)Based on the transcriptomic results,the related proteins in B16 cells were subsequently verified by western blot,and it was found that As2O3 significantly increased the expression levels of Cleaved Caspase-9,PINK1,Parkin,LC3-Ⅱ/Ⅰ,and Beclin-1(P<0.05),and dramatically decreased the expression levels of CDK1(P<0.05).Conclusions: The experimental results indicate that As2O3 inhibits the proliferation of B16 cells in a dose-dependent manner.We proved it,the As2O3-induced autophagy of B16 cells through the PINK1/Parkin pathway is proven in this paper.It is assumed that the inhibition effect of As2O3 on B16 cells could be probably related to apoptosis,induction of autophagy,cell cycle arrest and the as-resulted metabolic changes.The inhibition of As2O3 on the proliferation of B16 cells might be a result of the synergistic effect of these mechanisms.