Effection Of Proliferation And SHP-1Expression With Arsenic Tioxide And Valproic Acid In K562Cell Line

Background and objectiveArsenic trioxide (AS2O3) has recently been reported to induce complete remission in the patients with APL especially the one with relapsed or refractory APL. And without severe marrow suppression.But the mechanism of the antileukemic effect of As2O3has not been well understood.Datas have shown that AS2O3can destruct leukemia cells, induse apoptosis and differentiation. In addition, As2O3is related with the tumor suppressor gene which enables tumor suppressor gene to be demethylated and express. In K562cell line the SHP-1gene don’t expess,and the expression of SHP-1gene decreased in the ATL, B-ALL cell lines. MS-PCR (MSP) shows that in NK-YS, NK-TY2and K562cell lines, the promoter region of the CpG island in SHP-1gene is hypermethylation.Valproate acid (VPA), a traditional anti-epileptic drug,recently more and more reports show that it can not only inhibit malignant cell growth, induce differentiation and apoptosis,but can reduce histone deacetylation. Some datas show that DNA methylation and histone deacetylation are a kay mechanism of tumor suppressor gene expression. Recently, other studies show that inhibition of gene expression for DNA methylation not only interfere combination of the methylation-sensitive transcription factors with gene regulatory region,or directly inhibit RNA polymerase activity, but also the gene may express by histone acetylation.This discovery combined the two mechanisms-DNA methylation and histone acetylation. Studies of Arsenic trioxide combined with sodium valproate in leukemia K562cell line are few. In this study, AS2O3in different concentrations and combining with VPA act on leukemia K562cell line, to observe proliferation inhibition of the cell and expression of tumor suppressor gene in viro. Provide the experimental basis for the As2O3alone or combination with VPA used in leukemia treatment.Method 1. Cell culture:Recovery the cryopreserved K562cells, and inoculate in the RPMI1640medium which contains10%FBS, medium of cells was changed and every2-3days in a saturated humidity,37℃,5%CO2incubator. Until the cells grow well and emerge logarithmic growth, start to the experiments.2. WST-1(water soluble tetrazolium salt-ray absorption method) to detect cell proliferation. Groups as follows:1) Control group (adding normal saline).2) As2O3groups (final concentration is set to2.5μmol/L,5.0μmol/L,7.5μmol/L,10.0μmol/L).3) VPA groups (final concentration is4mmol/L).4) Combined treatment group (2.5μmol/L,5.0μmol/L,7.5μmol/L,10.0μmol/L As2O3joint for4mmol/LVPArespectively).Culture K562cell to logarithmic phase,and count cell to1-5×105/L,.cells were transplanted in96-well plates, add the four groups of drugs, and start timing.In24h,48h,72h read the cell absorbance by Universal microplate reader, and calculate the proliferation inhibition rate to find the optimal concentration and duration.3. RT-PCR (Reverse transcriptase polymerase chain reaction) to detect mRNA expression of SHP-1:The optimal concentration of AS2O3and joint VPA were added to the logarithmic phase of the K562cell line,and culture then for48hours, groups as follows:1) Control group (adding normal saline).2) Of As2O3group (7.5μmol/L).3) Combined treatment group (As2O37.5μmol/L+VPA4mmol/L).4) VPA group (4mmol/L).Design SHP-1gene primers, observed with1.5%agarose electrophoresis gel images and photographed, and analyze the gray value with related software.4. Detection of SHP-1protein expression by Western blot:Group ibid, collect K562cells, and cracking them to extract protein, the BCA method to measure the content of protein concentration, with SDS-PAGE electrophoresis, observe the protein in the gel imaging system andcamera, and analyze the gray value.5. Data analyzed by software SPSS17.0. The quantative data was presented as mean±standard difference(x±s). The mean of two groups was analyzed with the t test. Three or more groups was analyzed with the one-way analysis of variance after test of homogeneity of variance.Taking a=0.05as the significant standard of test, P<0.05indicate there is significant difference. P>0.05indicate there is no significant difference.Results1. cell culture:In cells of the logarithmic growth phase, adding drugs significant growth inhibition was observed under an inverted microscope,cell body become small, emerge irregular shape, and some dead. The cell morphology is varied in different time and different concentration.2. Cell proliferation effect by WST-1assay:Each concentration can inhibit the growth of K562cells, and significant dose-dependent and time-dependent inhibition of cell growth was observed used in this study.It was a significant difference between experimental and control groups (P<0.05).At the same time the concentration is higher as well as the increasing of growth inhibition.At the same concentration the time is longer,the inhibition rate is higher.There were significant difference between each experimental group and control group(P<0.05).Also there were significant differences between every experimental(P<0.05).The VPA(4mmol/L) and association with AS2O3(2.5μmol/L、5.0μmol/L、7.5μmol/L10.0μmol/L) can inhibite the growth of K562cells.These groups were compared with control group, there were significant difference (P<0.05). But the combination group (4mmol/L VPA+7.5μmol/L As2O3) and (4mmol/L VPA+10.0μmol/L As2O3) showed no significant difference (P>0.05).3. RT-PCR results showed:In the control group, there was no band of SHP-1mRNA.After being treated with drugs (AS2O3and/or VPA), mRNA of SHP-1expressed and increased gradually. Comparing the gray value of mRNA there were no significant difference between VPA group and control group(P>0.05).Comparing each two of other groups show significant differences (P<0.05).4. Western blot results showed:There was no expression of SHP-1protein,after adding drugs the protein expressed.In the group VPA (4mmol/L) there emerge a weak band,and the expression level in combination therapy group (As2O37.5μmol/L+VPA4mmol/L) was higher than other groups (P<0.05). Comparing As2O37.5μmol/L with VPA4mmol/L, there was a significant difference(P<0.05).Conclusion1. AS2O3can significantly inhibit the growth of K562cells, and its inhibitory effection dependent on concentration and time.Combining VPA and As2O3together, can make the inhibitory effection stronger than using single drug.2. There was no expression of SHP-1mRNA and protein in K562cells.3. As2O3and VPA can induce the expression of SHP-1protein and mRNA in K562cell.VPA also induce the expression of SHP-1,but the expression is lower.