The Effect Of Oxaliplatin On The Proliferation And Apoptosis Of Human Retinoblastoma Cells Y79in Vitro

Objective1.To study the effects of oxaliplatin on the proliferation and apoptosis of human retinoblastoma cells Y79, and to be expected to provide a new basis for clinical treatments of retinoblastoma.2.To investigate the effect of small interfering RNA-mediated myeloid cell leukemia-1(Mcl-1) knockdown enhanced sensitivity to oxaliplatin on apoptosis of human retinoblastoma cells Y79.Methods1.MTT method was used to detect the influence of human retinoblastoma cells Y79proliferation inhibition of the cells treated with oxaliplatin of different concentration (0,0.125,0.25,0.5,1,2μmol/L) acting on the cells at the end of24,48,72h, to clear the dosage-effect relationship.2.Apoptosis of the human retinoblastoma cells Y79was analyzed using flow cytometry with propidium iodide staining after0.25μmol/L oxaliplatin acting on the cells for24h.3.Caspase-3active detection kit was used to detect drug effect within24h for Caspase-3activity change after0.25μmol/L oxaliplatin acting on the cells.4..PI/Annexin V staining was used to observe apoptosis of human retinoblastoma cells Y79transfected with pSH1Si-Mcl-1after treating with oxaliplatin (0.25μmol/L), and no transfection, negative siRNA transfection were used as blank and negative control. 5.Western blot was used to detect Mcl-1expression on human retinoblastoma cells Y79treated with0.25μmol/L oxaliplatin and the cells transfected with pSHl Si-Mcl-1.Results1.Oxaliplatin inhibits proliferation of the human retinoblastoma cells Y79.It showed the effect of proliferation inhibition of the human retinoblastoma cells Y79treated with different concentration of oxaliplatin by MTT. In the exam, different concentration of oxaliplatin(0,0.125,0.25,0.5,1,2μmol/L) treated the cells Y79, MTT method checked the cell survival rate. It showed that the cell survival rate was reduced along with the higher concentration and longer time of the drug acting on the cells.0.25μmol/L oxaliplatin treated on retinoblastoma cells Y79for24,48,72h, the cell survival rate was (83.59±0.76)%、(77.30±.24)%and (61.41±1.39)%respectively, statistical significance was definite compared with control group (P<0.05)2. Oxaliplatin enhances the apoptosis of the human retinoblastoma cells Y79.After0.25μmol/L oxaliplatin treated on the human retinoblastoma cells Y79for24h, the apoptosis rate was (36.23±0.25)%using flow cytometry with propidium iodide staining (P<0.05)3.Oxaliplatin enhances the activation of Caspase-3of the human retinoblastoma cells Y79.0.25μmol/L Oxaliplatin enhanced the activation of Caspae-3of the human retinoblastoma cell Y79after it stimulated the cells, it was (5.76±0.25)%, higher than the control group (1.96±0.15)%(P＜0.01)4.Mcl-1expression of the human retinoblastoma cells Y79after oxaliplatin stimulatingIn the exam, the cells Y79were collected and extracted the protein after being cultured for6,16,24h after oxaliplatin stimulating the cells and check the expression of Mcl-1using Western blot. The expression of Mcl-1of the cells Y79stimulated by oxaliplatin was enhanced.5.The inhibitory effect of pSH1Si-Mcl-1on Mcl-1expression of the human retinoblastoma cells Y79.For the purpose of observing Mcl-1expression of the human retinoblastoma cells Y79after knockdown of Mcl-1, transfected with pSH1Si-Mcl-1to the cell Y79, compared with groups of no transfection and negative siRNA transfection. Change with fresh culture media6h after transfection, collect and extract the protein from the cells the next day. Western blot assay checked Mcl-1expression. It showed that Mcl-l-homo-991inhibited the expression of Mcl-1significantly.6.The pSH1Si-Mcl-1expression up-regulates the apoptosis of the human retinoblastoma cells Y79.For the purpose of observing apoptosis the effect of oxaliplatin on retinoblastoma cells Y79after Mcl-1knockdown, PI/Annexin V staining was used to the cells treated differently and check using flow cytometry, observing effect of up-regulating apoptosis of the cells Y79after knockdown Mcl-1the early stage. The rate of apoptosis of RB cells Y79is10.8%after being treating with0.25μmol/L oxaliplatin, no statistical significance compared with negative control group(5.8%)(P>0.05). The rate of apoptosis is21.0%and48.2%respectively when Mcl-1knockdown and treating with0.25μmol/L oxaliplatin after Mcl-1knockdown, the difference is statistically significant compared with the negative control group and oxaliplatin group(P<0.05). It showed that oxaliplatin itself couldnot up-regulate the apoptosis of RB cells Y79, but up-regulate that when knockdown of Mcl-1.Conclusions1.Oxaliplatin has inhibitory effect on proliferation of human retinoblastoma cells Y79, the cell viability decreased with the dose concentration increased and prolonged duration of action, but little effect at low concentrations, the mechanism of which maybe relate to anti-apoptotic effects of the apoptosic key protein Mcl-1.2.Mcl-1expression of human retinoblastoma cells Y79increases when treated with oxaliplatin, the mechanism of action may relate to the effect of resistance to apoptosis and lead to drug resistance of Mcl-1.3.The survival rate is down and apoptosis rate is up significanty when knockdown Mcl-1of the human retinoblastoma cells Y79, it proves that Mcl-1has protective effect on RB cells Y79.