Rapamycin Combined With Imatinib For SHIP Gene Expression In K562Cells

Objective: Chronic myelogenous leukemia （CML） is a pluripotenthematopoietic stem cell malignant hyperplastic diseases, which is one of thetype of chronic leukemia,9and22chromosome translocation, form the BCR-ABL fusion gene,which has led to an increased tyrosine kinase activity, thisis the pathogenesis of chronic myelogenous leukemia[1-2]. Traditionaltreatment such as hydroxyurea, Sally, amine, interferon, although in acertain period can improve patients with symptoms, it can’t cure the disease,also cannot block the disease progress from the source. After a period ofchronic phase, the majority of patients enters the stage accelerated or snap andeventually die from complications such as infection or bone marrow failure.Imatinib is a kind of tyrosine kinase inhibitors （tyrosine kinase inhibitors,TKIs）, it to Bcr-abl kinase rise in leukemia cells inhibit proliferation andpromote apoptosis role, now as a treatment for chronic myelogenousleukaemia first-line drugs used in clinical. In the clinical, although imatinib forchronic myelogenous leukemia chronic phase has good treatment effect, manypatients can achieve Bcr-abl gene and Philadelphia chromosome overcast,there are still a part of patients with drug resistance. Study confirmed that theBcr/Abl kinase can activation of multiple signaling pathways in cells, affectthe cell cycle distribution and apoptosis, etc., lead to chronic myelocyticleukemia disease. Of rapamycin target protein （mTOR） signaling pathway inchronic myelogenous leukemia to TKIs resistance mechanisms play animportant role in inhibition of abnormal activation of mTOR signalingpathways may enhance the Bcr/Abl kinase positive cells sensitivity to theTKIs, treatment for chronic myelogenous leukemia. Rapamycin is a kind offrom the actinomycetales streptomycin in the extract were isolated by gogglessamples, is a kind of activity of small molecules called mTOR inhibitors, originally as an immunosuppressant used in clinical, with the deepening of theresearch on its, people discovered that it has dual efficacy ofimmunosuppression and antitumor, and has been used in clinic to treat somesolid tumors, resulting in malignant hematopoietic system with antitumor roleis being more and more people pay attention to and study.Physi CAL signal transduction mechanisms in long engaged in leukemiaresearch foundation, have found BCR-ABL fusion gene, SHIP geneexpression is restrained, prompt SHIP inhibition of gene deletion or functionis an important part of the onset of chronic myelogenous leukemia.Current clinical application of imatinib as first-line drugs for treatingchronic myelocytic leukemia chronic phase, its curative effect is distinct, butthe problem of drug resistance in patients with also nots allow to ignore.Studies have shown that PI3K/AKT/mTOR pathway is one of the importantpathways downstream of BCR/ABL fusion gene[3], this study intends todiscuss whether rapamycin can through the activity of the mTOR inhibitortargets to inhibit the growth of leukemia cell lines and explore the rapamycinwhich as mTOR pathway inhibitors in chronic myelogenous leukemia cell lineK562cell growth, proliferation, apoptosis of biological effects and theexpression of SHIP gene. Discuss mTOR pathway inhibitors rapamycin andtyrosine kinase inhibitor imatinib whether there is a synergism in the treatmentof chronic myelogenous leukemia, for the clinical treatment of chronicmyelogenous leukemia to explore new methods and strategies.Methods:1Determined by MTT method to detect different concentrations ofrapamycin and imatinib single medicine and the application effect on K562cell proliferation.2Flow cytometry analysis method to detect different concentrations ofrapamycin and imatinib single medicine and the application effect on theapoptosis of K562cells.3Western Blot method to detect the SHIP gene at the level of proteinexpression. 4Rt-pcr detection between groups SHIPmRNA K562cells change.5Statistical processing.Results:1Rapamycin on K562cell proliferation and apoptosis of the influence ofdifferent concentrations of rapamycin in K562cells after24h, found thatrapamycin inhibition of K562cell proliferation, and dose dependence,25,50,100,200,500,1000nmol/L K562cell survival rate of treatment group weresignificantly lower than the solvent control group （P <0.05）. In25-1000nmol/L concentration range, with the increase of concentration of rapamycinK562cell survival rate were significantly lower. And the concentration of1,6.25,12.5nmol/L rapamycin treatment group had no significant effect onK562cell survival rate （P>0.05）, the IC₅₀ of186.7nmol/L.Rapamycin treatment after24h, found K562cells showed typicalapoptosis morphological observation, show the cell membrane integrity andnuclear pyknosis or karyorrhexis, apoptotic body. Flow cytometry analysisshowed that treatment group of G₀/G₁phase cells than the control group wassignificantly increased, while the proportion of S stage cells significantlydecreased （P <0.05）. Prompt rapamycin can significantly affect the K562cellcycle distribution, induction of cells in G₀/G₁phase retardation.2Imatinib inhibits proliferation induced the apoptosis of K562cells ofdifferent concentrations of imatinib in K562cells after24h, found thatimatinib inhibition of K562cell proliferation, and dose dependence,200,500,1000,500nmol/L K562cell survival rate of treatment group weresignificantly lower than the solvent control group （P <0.05）. In200²000nmol/L concentration range, with the increase of concentration of imatinibK562cell survival rate were significantly lower.3Rapamycin can enhance imatinib choice to enhance the apoptosis ofK562cells is lower than the IC₅₀ doses of50nmol/L rapamycin and differentconcentrations of imatinib combined effect on K562cells, and cell growthinhibition rate and the combination groups compare two single drug groupwere statistically significant （P﹤0.05）, shows that rapamycin and imatinib combined effect on K562cells, can produce effect.4Rapamycin for mTOR expression in K562cellsMTOR protein molecular weight of289kD, after sds-pageelectrophoresis, transfer film, blank control group, drug treatment group bothin289kD position fluorescence banding. Set for GAPDH internal genes,quantitative fluorescence banding, results show that rapamycin of mTORprotein expression of K562cell after treatment no significant influence （P>0.05）, but of mTOR inhibitors reduce the K562cell protein phosphorylationlevel obviously （P <0.05）.5Rapamycin affect SHIP gene protein in K562cellsSHIP, according to the results of Western leukemia cell line K562cellprotein expression, expression in K562cells treated with rapamycin SHIPproteins, and presents the concentration dependent, with the increase ofconcentration of rapamycin, protein expression increased.6Rapamycin SHIP gene expression of K562cell rt-pcr results show thatthe influence of different concentrations of rapamycin K562cells for24h, theSHIP gene expression level increased expression quantity than control group（P <0.05）, and increased with the increase of concentration of rapamycinexpression quantity increase.7Imatinib SHIP gene expression of K562cell rt-pcr results show that theinfluence of different concentrations of imatinib in K562cells after24h, theSHIP gene expression level increased expression quantity than control group（P <0.05）, and increased with the increase of concentration of imatinibexpression quantity increase.8Rapamycin combined imatinib SHIP gene expression of K562cellrt-pcr results show that the influence of two drug combination in K562cellsfor24h, the SHIP quantity of gene expression than single drug effect afterhigh expression （P <0.05）.Conclusion:1Rapamycin inhibition of K562cell proliferation, promote apoptosis,and increase SHIP gene expression in K562cells quantity, prompt rapamycin inhibits the proliferation of chronic myelogenous leukemia cells.2Imatinib inhibition of K562cell proliferation, promote apoptosis, SHIPand K562cell gene expression levels.3Rapamycin combined imatinib in curbing K56cell proliferation,promote apoptosis and gene expression level were significantly superior to thesingle SHIP medicine, showed that in treatment of chronic myelogenousleukemia can use imatinib as first-line treatment drugs at the same time try tocombined use of rapamycin, to reduce the patients with chronic myelogenousleukaemia of imatinib resistance, and also further study of mechanism of SHIPgene on the pathogenesis of chronic myelogenous leukemia, provide scientifictheory basis for leukemia gene therapy.