Mechanisms Of Inhibiting Proliferation Of Phenylhexyle Isothiocyanate On Multiple Myeloma Cell Line U266

ObjectiveTo study the mechanisms of inhibiting proliferation of PHI , a kind of natural compounds, on multiple myeloma cell line U266 and illustrate effects of PHI on DNA methyltransferase;collect the data about PHI treated myeloma cells as a demethylation agent.Methodthe growth inhibition of U266 was observed by depicting cell growth curve and MTT ,the effect of apoptosis was observed by DAPI ,the cell cycle was analyzed by Flow cytometry,the expression of DNA methyltransferase 1(DNMT 1),DNMT3A and 3B gene mRNA were determined by RT-PCR,the activity of DNMTs was analyzed by like-ELISA, the methylation of p16 gene in U266 cell was detected by the nested-methylation specific PCR(n-MSP) after exposed by PHI.Result:1.The result of RT-PCR showed that DNMT1,3a,3b mRNA were highly expressed in U266 cell and lowly expressed in PBMC;2.Compared with untreated group,the proliferative capacity and activity of U266 were declined after treated by PHI. The rate of inhibition in different concentration of PHI(1.25umol/L,2.5umol/L,5umol/L,10umol/L)were 0.284±0.089,0.554±0.079,0.753±0.053,0.936±0.067(p<0.05).3 DAPI showed that U266 cells was apoptosised after treated with PHI,. IC50 calculated by MTT was 2.48.3. The G0-G1 cells were increased. 4.The mRNA level of DNA methyltransferase 1(DNMT 1),DNMT3A and 3B were down-regulated after exposed by PHI for 48h. Between the mRNA level and dose of PHI have a relationship of positive dependent manner.(p<0.05).5. The activity of DNMTs in U266 cells was decreased after treated by different concentrations PHI.6.Methylation specific PCR(MSP) showed that the p16 gene in U266 was hypermethylated. Having been treated by PHI for 6 days, the hypermethylation of p16 gene was released, the relationship between P 16 gene expression and drug concentration have positive.Conclusion1.The expression of DNMTs in U266 was very strong compared to normal people.2.PHI induced apoptosis and decreased the mRNA level of DNMT1,DNMT3a,DNMT3b in U266 cells. The activity of DNMTs was destroyed after treated with PHI.3.PHI released abnormal hypermethylation of P16 gene in U266 .4. DNMTs may be a new therapeutic target of multiple myeloma and PHI is a potential demymethylation agent .