The Effect Of Gemcitabine On Proliferation, Apoptosis And Expression Of Mrna Of Bcr-abl Fusion Gene Of K562 Cells

Aim:Chronic myelogenous leukemia(CML) is a myeloproliferative disorder originated from early pluripotent hematopoietic stem cells,accounting for 15%of adult leukemia.It is characterized by Ph chromosome and Bcr-Abl fusion gene,The resulting Bcr-Abl fusion gene encodes a chimeric protein with strong tyrosine kinase activity,which leads to the development of the CML phenotype.Median survival of CML is 3-5 years,while the blastic phase(BP) is the end-stage of CML,which clinical manifestations is similar to acute leukemia and prognosis is extremely poor.There are not effective methods to control blastic phase of CML,patient with CML BP often dies in a few months.K-562 is a erythroleukemia cell line derived from a chronic myeloid leukemia patient in blastic crisis,which plays an important role in the basic research about blastic crisis.Gemcitabine(2′-deoxy-2′,2′-difluorocytidine monohydrochloride(β-isomer))(gemcitabine GEM) is a nucleoside analogue that exhibits antitumor activity.Gemcitabine exhibits cell phase specificity,primarily killing cells undergoing DNA synthesis(S-phase) and also blocking the progression of cells through the G1/S-phase boundary.The structural formula,property and Pharmacokinetics of gemcibine is similar to cytarabine,but phosphorylation efficiency is powerful than cytarabine.First,gemcitabine diphosphate inhibits ribonucleotide reductase,which causes a reduction in the concentrations of deoxynucleotides,including dCTP.Second,gemcitabine triphosphate competes with dCTP for incorporation into DNA.After the gemcitabine nucleotide is incorporated into DNA,there is inhibition of further DNA synthesis.DNA polymerase epsilon is unable to remove the gemcitabine nucleotide and repair the growing DNA,which induce cell apoptosis.At present,it has been widely applied in the treatment of patients with solid tumors.It' s therapeutic effect was identificated and side reactions could be tolerated.So the FDA approved gemcitabine as the first line chemotherapeutic drug in the treatment of non-small cell lung cancer,pancreatic cancer and metastatic breast cancer.Recently,relevant researches about nucleoside analogues treat chronic myeloid leukemia in alone or joint are ongoing,for which imatinib resistance or in advanced phase of CML.In this experiment,proliferation and apoptosis of the K-562 cells were observed after cells treated with gemcitabine. And the expression of Bcr-Abl mRNA in K-562 cells was assayed.To explore the efficacy that patient in chronic myeloid leukemia blastic phase is treated with gemcitabine.Methods:1.WST-1 method:K-562 cells were cultured and treated with 0.1μg/ml,1.0μg/ml, 10μg/ml gemcitabine for 12h,24h,48h and 72h.The activities and proliferation of cells were examined by WST-1 assay.Then the growth inhibitory rates were counted and the optimal concentration and time was found.2.The cell apoptosis was analyzed by TUNEL(Terminal deoxynucleotidyl transferase dUTP nick end labeling) staining when the K-562 cells were treated with 10μg/ml GEM for 48h.The positive cell was defined as brown pellet in cytoplasm.300 cells in each slide were randomly counted with positive and negative cells for differential count.Positive rate of cells was calculated based on formula:positive rate=positive cells/300×100%.3.RT-PCR technique was used to evaluate the difference of Bcr-Abl mRNA expression in K-562 cells treated with and without 10μg/ml GEM for 48h,and compared with group treated by 10μg/ml Ara-c.4.The data were analyzed using software SPSS 13.0.The counting data were analyzed with theχ~2 test.The quantative data were presented as mean±standard difference.Taking a=0.05 as the significant standard of test. Results:1.Growth inhibitory effect of GEM on K-562 cells:After the K-562 cells were treated with 0.1μg/ml,1.0μg/ml,10μg/ml,GEM for 48h,cells growth were significantly inhibited with inhibition rates at18.7%,23.9%,29.3%,respectively. Compared with Ara-c group,the differences had statistical significance(P＜0.05). Data analysis revealed that gemcitabine could suppress the proliferation of K562 cells in dose-dependent manners,The optimal concentration was 10μg/ml of GEM; and suppress the proliferation of K562 cells in time- dependent manners,but there was no statistical difference between 48h group and 72h group,the optimal time was 48h.Conclusively,GEM was capable of inhibiting the K-562 cells proliferation with optimal concentration at 10μg/ml and optimal time at 48h.2.Apoptosis of K-562 cells induced by GEM:After the K-562 cells were treated with 10μg/ml GEM for 48h,the apoptotic cells increased significantly as detected by TUNEL staining.The positive rate was 15.3%in GEM group,compared with Ara-c group(positive rate 3.7%) and control group(positive rate 2.0%).The differences between GEM group and Ara-c group had statistical significance (P＜0.05);the differences between Ara-c group and control group had no statistical significance(P＞0.05).3.Expression of Bcr-Abl mRNA:After the K-562 cells were treated with 10μg/ml GEM for 48h,Bcr-Abl mRNA significantly down-regulated as observed by RT-PCR assay.Gray value of GEM group was 0.12,Gray value of Ara-c group was 0.15 and Gray value of control group was 0.40.The differences between GEM group and control group had statistical significance(P＜0.05);but the differences between GEM group and Ara-c group had no statistical significance (P＞0.05).Conclusion:1 Gemcitabine could suppress the proliferation of K562.2 Gemcitabine could induce apoptosis of K562.3 Gemcitabine at 10mg/L can obviously down-regulated the mRNA expression of Bcr-Abl fusion gent of K562 cells.