The Effects Of Sequential Treat With Bortezomib And Cytarabine On The Gene Expressions Of SHIP, MTOR And NF-κB Within K562Cells

Objective: chronic myeloid leukemia (CML) is a kind of clonalmalignant proliferative disease originated from pluripotent hematopoietic stemcell mutation. In our country, the morbidity rate of CML is0.36per100000population, second only to acute myelogenous leukemia and acutelymphocytic leukemia. With the development of molecular biologicaltechnology, the mechanism of chronic myelogenous leukemia has graduallybeen understood. There are approximately95%of CML patients with t(9,22)（q34;q11） karyotype, which is ph chromosome. BCR/ABL fusion gene canbe formed, and its encoded protein can enhance the activity of tyrosine kinase,thereby inhibiting cell apoptosis. Studies have shown that, PI3K is animportant signaling pathway in BCR/ABL downstream. SHIP (SH2domaincontaining inositol5’-phosphatase, SHIP, belonging to the PIPase family)gene is a novel inositol phosphatase expressed only in hematopoietic cellfollowing the discovery of PTEN gene, which can degrade PIP3, resulting innegative regulation of PI3K/Akt signal pathway. Previous studies have shownthat: there are SHIP gene mutations in patients with acute leukemia, and thelow expression of SHIP gene is related with human leukemia cell proliferation,and SHIP gene can induce apoptosis of K562cells. Gleevec can improve theexpression quantity of SHIP-2having high homology with SHIP gene.Although imatinib has a positive role, but almost20%of the patients in theuse of this drug do not have complete cytogenetic response, and some patientsmay have intolerable side effects or resistance1. So, it is necessary to seek onekind of new drug for CML. Bortezomib is the first proteasome inhibitorentering clinic, mainly used in refractory or relapsed multiple myeloma.Bortezomib combined with other antineoplastic drugs shows inhibitory effect in ariety of malignant tumors, with wide application prospect. At present, theaction mechanism of bortezomib is not fully understood. This study usedsingle drug and various combination of bortezomib with cytarabineto apply onK562cells, and inhibition rate and apoptosis rate were observed, and thechanges of SHIP gene, mTOR gene and NF-κB gene were detected, toinvestigate the mechanism of action, and to look for new treatment for CML.Methods: Study object is CML cell line K562. K562cell line wasdivided into6groups, which were1) non intervention group,2) bortezomibtreatment group,3) cytarabine treatment group,4) sequential application ofcytarabine after bortezomib treatment for6hours group,5) sequentialapplication of bortezomib after cytarabine treatment for6hours group,6)bortezomib and cytarabine simultaneous treatment group. MTT method wasapplied to check cell proliferation inhibition in each group, and flowcytometry was used to detect apoptosis rates, and polymerase chain reaction(PCR) was applied to detect related gene expression.Results:1Inhibition of MTT assay results showed that: monotherapy and combinationcan inhibit cell proliferation rate, positively associated with drug concentrationin a certain range. Combined effects were better than monotherapy, of which,cytarabine treatment followed by bortezomib (Ara-Câ†'BTZ) treatment hasstrongest inhibition, is86.46%,followed by simultaneous treatment group(Ara-C+BTZ) and bortezomib followed by cytarabine group(BTZâ†'Ara-C),both is62%,and monotherapy group is about46%, P<0.052apoptosis of Flow cytometry assay results showed that: monotherapy andcombination therapy can increase apoptosis rate.The effect of combinationwas superior to that of single drug, wherein (Ara-Câ†'BTZ) group was(84.26±.2.94）%.;(Ara-C+BTZ)group was （69.57±3.64）%;(BTZâ†'Ara-C)group was (62.21±.1.35)%; P<0.05. Monotherapy group was （24.01±1.84）%and（20.93±2.93）%，P>0.053PCR detection of SHIP， mTOR， NF-κB gene results showed that:bortezomib and cytarabine can upregulate expression of SHIP gene, combined effects were stronger than monotherapy(BTZ:1.1209±0.0776; Ara-C:0.9709±0.2747, no significant difference between monotherapy group;Ara-C+BTZ:2.5348±.13787; BTZâ†'Ara-C:2.2189±.0.3229;Ara-Câ†'BTZ:2.4019±.0.3067, there were no significant differenceamong the different combined groups.The control group:0.3141±.0.0822.there were significant differences among the three groups) anddecrease expression of mTOR gene(Control group,:2.1053±0.0967, Ara-C:1.3253±0.0448; BTZ:1.1863±.0247; Ara-C+BTZ:0.8623±.0725;BTZâ†'Ara-C:.7353±.0673; the Ara-Câ†'BTZ:0.8703±0.0556,there were no significant difference among the different combined groups.There was significant difference between the combination group,monotherapy group and the control group monotherapy group). Bortezomiband cytarabine can downregulate expression NF-κB gene expression,combined effects were stronger than monotherapy.The cytarabinepretreatment has strongest effect.(Followed by Ara-C:2.1386±0.01102;BTZ:1.8715±0.0.043; BTZâ†'Ara-C:1.2418±0.0429; Ara-C+BTZ:1.0170±.08411; Ara-Câ†'BTZ:.7104±.0235,(P <0.05).)Conclusion: Bortezomib and cytarabine could inhibit K562cellproliferation, and induce apoptosis. The effect of combination was superior tothat of single drug, in which,(Ara-Câ†'BTZ) treatment had the strongest effect,and the principle may be related to the effect on PI3K signal pathway byinfluencing the SHIP gene of mTOR geneand NF-κB gene expression levels.