Salinomycin Inhibits Human Hepatocellular Carcinoma Cells (HepG2) In Vitro

Objectives To observe salinomycin affects Human Hepatocellular CarcinomaCells (HepG2) on proliferation and cell cycle in vitro, provide a theoretical evidenceof salinomycin anti-cancer mechanism.Methods①To observation Salinomycin Inhibits Proliferation of HepG2cellby MTT. salinomycin was added in six replicates of5、10、20、50、100、200μmol/Lfor each cell population, used DMSO for positive control, and blank control group.Detection each OD of salinomycin after24h、48h、72h working by ELISA, portrayingtime-chart which showed the relationship between concentration of salinomycin andrate of inhibition.②To observe the effects of salinomycin on HepG2cell for thecolony formation. Filtrating three effective concentration of Salinomycin (5、10、20μmol/L)HepG2was added in6-well plates,1000/well. Different concentration ofSalinomycin and DMSO were added into each plate. We will terminate cultivationafter14days. Cells were fixed by Methanol about10minutes and stained by Giemsaabout15minutes, count the number of colony which naked eye can see and count rateof colony.③To observe the effects of salinomycin on HepG2cell morphology,Sameconcentration cells was plated in6-well plates. Different concentration ofSalinomycin and DMSO were added into each plate after12h.The cells were stainedby PI and waiting24h. We can observe morphology of HepG2by Fluorescencemicroscope.④To observe the change of the cell division cycle percentage aftersalinomycin effect on cells. HepG2cells were plated in6-well plates. Waitingconcentration of cells was106, add different concentration of Salinomycin and DMSOto each plate. After24h, control cells, DMSO-treated cells, and Sal treated cells were collected. Then centrifuged and stained by DAPI. The cell cycle of each group wasanalyzed by flow cytometry.⑤To detect20μmol/L concentration group salinomycin’seffect with Bax and P53gene expression by Western Blot.Results①Sal can inhibit Proliferation of HepG2cells significantly in vitro.after Salinomycin affect HepG2cells24h, cell survival rates which in blank controlgroup, DMSO group and salinomycin concentration of5,10,20,50,100,200μmol/Lgroup were respectively100.00%,99.69%,91.79%,99.69%,64.07%,33.76%,25.40%,33.76%. after Salinomycin affect HepG2cells48h, cell survival rates whichin blank control group, DMSO group and salinomycin concentration of5,10,20,50,100,200μmol/L group were respectively100.00％、93.43％、74.60％、63.45％、48.32％、4.70％、2.16％、1.83％. after Salinomycin affect HepG2cells72h, cellsurvival rates which in blank control group, DMSO group and salinomycinconcentration of5,10,20,50,100,200μmol/L group were respectively100.00％、98.02％、54.35％、35.15％、14.74％、0.96％、0.93％、0.77％;②In colonyexperimental, colony formation rate which in blank control group, DMSO group andconcentration of salinomycin5,10and20μmol/L group of is respectively31.03%,30.71%,24.93%,21.90%and10.27%;③We used fluorescence microscope toinvestigate the morphological changes of apoptotic cell, we found that in10μmol/L,20μmol/L Salinomycin groups is the most significant.④In this study, we use flowcytometry instrument to detect the cell cycle, percent of G1phase cells which incontrol, DMSO group and concentration of salinomycin5,10and20μmol/L groupwere respectively71.40%,71.87%,71.40%,70.83%,79.20%, percent of G2phasecells which in control, DMSO group and concentration of salinomycin5,10and20μmol/L group were respectively10.83%,10.73%,10.50%,11.53%,10.50%,percent of S phase cells which in control, DMSO group and concentration ofsalinomycin5,10and20μmol/L group were respectively17.80%,17.40%,17.80%,17.63%and13.53%;⑤WB experiment showed that20μmol/L group’s P53and Baxprotein concentration increased significantly to the control group. Conclusion①Salinomycin can inhibit Proliferation of HepG2cellssignificantly in vitro in a concentration-and time-dependent manner.②Salinomycinis probably by increasing the expression of P53gene to cell cycle arrest in G1phaseto inhibit cancer cell proliferation;③Salinomycin is probably by increasing theexpression of P53and Bax genes promote cell apoptosis, which have inhibitory effecton tumor cells.