The Effect Of Demethylation Preparation Decitabine On Proliferation, Apoptosis And WAVE1Expression In K562Cells

Background and PurposeBlastic phase（BP）is the terminal phase of chronic myeloid leukemia (CML),andhardly to be cured. In the CML patients,95%of them have be detected theabnormality chromosome.The molecular basis associated with bcr/abl abnormal meltgene. Recently years, more and more researches show that the existence anddevelopment of CML have high relevant with DNA’s abnormal methylation. In which,are mainly expressed in tumor gene’s demethylation, anti-tumor gene’shypermethylation and fusion-gene’s hypermethylation, these three aspects.High methylation of tumor suppressor genes in development of tumor oftenplays an important role in the process, which makes the application to methylatedtumor drug treatment known as possible.The medicine for demethylation whichcalled Decitabine(DAC)is a kind of special methyltransferase (DNMT) suppressant.Previous studies about DAC are focused on the effect of treatment leukemia bydemethylation. But the last studies found DAC also take part in acticating ofJAK-STAT Signal transduction pathway.these studies show that DAC may play apromoting apoptosis effect by other means. But through other ways about the possiblerole of genes, is less studied.The drug resistance treatment of CML is still problem. Verprolin CHP1(WAVE1) is a member of WSP family, which is mainly participate in the multidrug resistancegene and other relevant genes control and express. It is a new kind of anti-apoptosisprotein, and had been proved that it has expressed wildly in the leukemia. WAVE1’sabnormal expression has high relevant with the tumors, tumor-ingiltration, andanti-tumor drug resistance. Wave can control the cell cycle, participate JAK-STATsignal transfer tunnel, cell apoptosis trials to anti-apoptosis. Currently, it is hardly tofind that report about DAC has effect in CML treatment whether or not. Inconsequence, in this, different consentration DAC will be taken in the leukemia K562cell therapy, and observe how K562effect the cell prolifation inhibition and multidrug resistance gene expression.Method1. WST-1inhibition of cell proliferation measured: Using final concentrations0.5μmol/L,1μmol/L,5μmol/L,10μmol/L, respectively, of decitabine K562cells12h,24h,48h,72h, determine the role of decitabine in a suitable cell concentration andtime.2. Apoptosis by flow cytometry: With5μmol/L of decitabine role logarithmicphase of K562cells for48hours, flow cytometry AnnexinV FITC/7ADD doublestaining with different concentrations of decitabine role K562cell apoptosis.3. Flow cytometry cell cycle: With5μmol/L of decitabine role in the logarithmicphase of K562cells for48hours, changing flow cytometry cell cycle.4. RT-PCR was used to detect the expression wave1mRNA: With5μmol/L ofdecitabine role logarithmic phase K562cells for48hours, was detected by RT-PCRunder different drug concentrations wave1mRNA expression levels.5. Western blot assay WAVE1protein expression: With5μmol/L of decitabinerole logarithmic phase of K562cells for48hours, with the Western blot assayWAVE1protein expression changes.6.flow cytometry cell differentiation:1μmol after/L of decitabine rolelogarithmic phase K562cells24hours,48hours and72hours, application of flowcytometry cell surface antigens CD71and CD33expression of.7. Morphological changes: Respectively0.5μmol,1μmol/L and5μmol/decitabine role in the logarithmic phase of K562cells for24hours and48hours afterwright stain cells were observed in the oil morphological changes.8. Statistical analysis: All experimental data were applied SPSS17.0softwarefor statistical analysis, measurement data were analyzed using t methods, statisticaldata expressed as (x±s). With α=0.05significance level set up to P <0.05indicatessignificant difference. Results1. WST-1result show：Different concentrations of decitabine to K562cellproliferation have obvious inhibiting effect. Use concentration of0.5μmol/L、1μmol/L、5μmol/L、10μmol/L decitabine respectively dispose K562cell48h，caninhibit the proliferation of K562cells. proliferation inhibition rate was (21.4±2.28)%,(30.4±1.06)%,(55.7±1.22)%,(63.2±1.84)%, while the control group (3.8±0.92)%, each experimental group compared with the control group, there werestatistically differences (P <0.01). Compare between the dfferent concentrations ofdecitabine group two groups, there were statistically differences (P <0.05).5μmol/LDAC respectively deal with K562cells for24h,48h,72h proliferation inhibitionrate, respectively（40.2±2.01）%，（55.7±1.22）%，（58.1±2.17）%，there werestatistically differences (P <0.05), Groups of inhibition rate compared with the sametime control group respectively(（1.7±0.75）%，（3.8±0.92）%，（6.2±2.16）%), thedifferences were statistically significant (P <0.01). But comparison between groups of5μmol/L DAC effect K562cells for24h and48h, on K562cell proliferationinhibition rate（（40.2±2.01）%vs （55.7±1.22）%）, the differences were notstatistically significant (P>0.05).Thus calculated decitabine treatment of K562cells50%inhibitory concentration (IC50value) was4.82umol/L. Choose from the50%inhibitory concentration5umol/L DAC for48h subsequent experiments conducted inthe experimental group.2. flow cytometry apoptosis Show:5umol/L of decitabine in K562cells after48h,apoptosis rate (43.80±3.77)%. compared with the control group ((2.29±0.41)%),apoptosis was significantly higher, there was statistically differences (P <0.01).3. changes in the cell cycle:5umol/l of decitabine in K562cells after48h, G0/G1phase cells in the experimental group (59.83±5.47)%compared with the controlgroup (51.29±2.75)%increase, S phase cells in the experimental group (31.27±7.11)%compared with the control group (38.81±6.31)%increase, there werestatistically differences (P <0.05, P <0.05), the G2/M phase cells did not changesignificantly(respectively (8.29±2.36)%,(7.93±2.15)%, P>0.05)), indicate after decitabine affect K562cells, cell cycle arrest in G0/G1phase.4. PCR amplification showed:5umol/L of decitabine in K562cells after48h,WAVE1mRNA expression was significantly lower (0.17±0.051), Compared withthe control group (0.37±0.033), there was statistically differences (P <0.05).5. Western blot results showed that: DAC on K562cells WAVE1protein5umol/L of decitabine in K562cells after48h, WAVE1expression (0.48±0.049) wassignificantly reduced, compared with the control group (0.92±0.052), there wasstatistically differences (P <0.01)..Bcl-2expression (0.36±0.039) was significantlyreduced, compared with the control group (0.87±0.061), there was statisticallydifferences (P <0.01).6. Cell surface differentiation results: Flow cytometric analysis of cell surfacedifferentiation antigen test results show that Concentration of1μmol/L decitabine inK562cells24h,48h and72h, CD71（+CD33+and CD33-）expression levels were (93.0±2.16)%,(92.2±0.63)%,(91.6±1.27)%.Compared with the control group (62.9±1.03%), the expression was significantly increased, there were statistically differences(P <0.01), The CD71+expression in different time groups was not statisticallysignificant (P>0.05).7. K562cell morphological changes: DAC effect after24h, cell blank controlgroup was rounding, nuclear, less cytoplasm. After1umol/L decitabine processedK562cell appears cytoplasm increased multiple nuclei to one side, nucleuskidney-shaped or irregular in shape cell differentiation performance. And by5umol/Ldecitabine role cells appeared smaller cell size, karyopyknosis apoptosis or apoptoticbody performance.Conclusion1. Decitabine can inhibit proliferation of K562cells, and the effect was time-andconcentration-dependent manner.2. Small dose decitabine can induce K562cells into myeloid direction.High-dose decitabine can induce apoptosis of K562cells.3. Decitabine significantly downregulated in K562cells WAVE1mRNA and protein. and the phenonmen maybe associate with the Bcl-2protein change.