Function Of Transcription Factor RUNX3in Leukemia Chemotherapy And MSC-induced Drug Resistance

ObjectiveIntensification of chemotherapy treatment has increased the responserate and survival duration of patients with leukaemia.However, the useof chemotherapy is complicated by the development of resistance orintolerance, which has caused researchers and clinicians to investigatenovel anticancer targets. Drug resistance is the main reason ofrecurrenceacute and failure in leukemia treatment.At present,mesenchymalstem cells (MSC) as an important role in drug resistance gets more andmore attention. As we learn more about the cellular mechanisms at workin leukaemia patients, the inactivation of genes that regulate growth andinduce cellular differentiation has been shown to be involved in thedevelopment of leukaemia. Alternatively, leukemia targeted therapy is themost promising treatment. Therefore, we investigated that RUNX3plays animportant role in leukemia cellular function and As2O3-induced apoptosis.Furthermore, we observed that MSC modulated the exprssion of RUNX3viaCXCL12/CXCR4signaling and the overexpression of RUNX3allows cells toovercome MSC-mediated drug resistance. Through the study of crucial genesin leukemia, we wish to find leukemia a new treatment strategy to supplement or even replace the existing treatments in order to achievethe optimum curative result.Method1. K562and Reh cells were treated with5-aza-CdR, TSA or bothcompounds. RT-PCR and Western blot analyses were used to examine theexpression of RUNX3at the mRNA and protein levels, respectively.Immunofluorescence microscopy was used to detect the cellular locationof RUNX3. Additionally, after K562cells were transfected with RUNX3,apoptosis and proliferation were studied using Annexin V staining and MTTassays.2. Treated NB4and K562with different concentrations of As2O3,Westernblot analyses were used to examine the expression of RUNX3proteinlevels.DCFDA staining was used to detect the level of ROS.Preincubatedwith NAC, As2O3-induced RUNX3expression was inhibited.Immunofluorescence microscopy was used to detect the cellular locationof RUNX3.Using RNA interference to reduce RUNX3mRNA, we established2stable clones expressing shRNA specific to RUNX3.MTT was used to examinethe cell growth.Apoptosis and proliferation were studied using AnnexinV staining and MTT assays.3. MSC was isolated from bone marrow.Treated K562cells with AS2O3,Annexin V and PI staining was used to examine cell apoptosis and cell cycle.Western blot was used to detect the total protein and the level of RUNX3in the nuclear fraction.K562were cultured with MSC and treated withAMD3100, AS2O3or a combination of both.The expression level of CXCR4compared with non-treated cells was analyzed by flow cytometry. Levelsof CXCL12produced by mesenchymal stem cells from patients were detectedby ELISA. To assess whether CXCR4/CXCL12could modulate the expressionof RUNX3, the expression of RUNX3was analyzed in the presence of CXCL12or AMD3100by western blot.PCDNA3.1/RUNX3expressing cells were cultured alone or in the presence of MSC. After24h, cells were treated with AS2O3at various concentrations, and annexin-V and PI-positive cells weredetected by FACS24h post-treatment.Results1．The expression of RUNX3in leukaemia cell lines was markedly lessthan that in the controls. Demethylating drug5-aza-CdR could induce RUNX3expression, but the combination of TSA and5-aza-CdR had a greater effectthan did treatment with a single compound. The combination of5-aza-CdRand TSA induced the translocation of RUNX3from the cytoplasm into thenucleus. TSA enhanced apoptosis induced by5-aza-CdR, and Annexin V andHoechst33258induced apoptosis but not necrosis. Furthermore, apoptosiswas dependent on the caspase-3pathway. RUNX3overexpression in K562cellsled to growth inhibition and apoptosis and potentiated the effects of5-aza-CdR induction.2. As2O3treatment resulted in the elevation of total RUNX3in K562and NB4cells in a dose-dependent manner. Protein levels of P21wereelevated in a dose-and time-dependent manner and corresponded to theelevation of RUNX3observed following As2O3treatment.Immunofluorescencestaining revealed that RUNX3was located mainly in the cytoplasm beforeAs2O3treatment and accumulated in the nucleus following As2O3treatmentThis ROS induction seems to be necessary for As2O3-dependent elevation ofRUNX3because pretreatment of cells with NAC，a scavenger of ROS，resultedin the inhibition of As2O3-induced expression of RUNX3.2stable clonesexpressing shRNA specific to RUNX3were estimated. As2O3inhibited thegrowth of SCR cells, but this growth inhibition was compromised in theRUNX3shRNA-transduced cells treated with As2O3apoptosis wassignificantly reduced in RUNX3knockdown clones reexpression of RUNX3enhanced sensitivity of K562cells to As2O3in a dose-dependent manner.Tuppression of RUNX3expression by RUNX3shRNA attenuated As2O3-induced cell cycle arrest.3. MSC could protect leukemia cells from As2O3-induced apoptosis.Up-regulation of Bax expression, as well as down-regulation of Bcl-2expression induced by As2O3, was blocked in the presence of MSC. Consistentwith the previous report, As2O3treatment induced the activation ofcaspase-8, where as this modulation was not noted in the presence of MSC.As2O3-induced death of cells is associated with strong activation ofcaspase-3, which was significantly inhibited when cells were treated inthe presence of MSC.Additionally, there were low levels of Mcl-1proteinin NB4and K562cells treated with As2O3.However, in the presence of MSC,the decrease in Mcl-1levels was compromised.The presence of MSC reducedRUNX3protein expression of cells treated with different concentrationsof As2O3compared to the cultures in which the leukemic cells were exposedto As2O3without MSC.The expression of P21induced by As2O3was alsoinhibited in the presence of MSC. In the presence of MSC, the level ofRUNX3in the nuclear fraction was significantly reducedat the indicatedtime in NB4and K562cells.CXCL12/CXCR4signaling modulated theexpression of RUNX3. Over-expression of RUNX3restores the effect of As2O3on K562cells.Conclusion1. RUNX3plays an important role in leukaemia cellular functions, andthe induction of RUNX3-mediated effects may contribute to the therapeuticvalue of combination TSA and5-aza-CdR treatment.2. As2O3induces expression of RUNX3and translocation of RUNX3intothe nucleus.suppression of RUNX3expression by RUNX3shRNA attenuatedAs2O3-induced cell cycle arrest and cell apotosis.RUNX3plays an importantrole in As2O3-induced apoptosis.3. Mesenchymal stromal stem cells protect leukemia cells fromAs2O3-induced apoptosis. MSC reduce the expression of RUNX3induced by As2O3 via CXCL12/CXCR4signaling. Expression of RUNX3overcomes MSC-mediatedAs2O3resistance. RUNX3maybe serve as a new treatment strategy tosupplement or even replace the existing treatments in order to achievethe optimum curative result.