Effect Of Cx43 Modified Bone Marrow Stromal Cells On Imatinib Effect Of K562 Cells

background:Chronic myeloid leukemia（Chronic Myelogenous Leukemia,CML）is a malignant hematological tumor marked by the chromosome of Philadelphia.Thanks to the recognition of BCR-ABL fusion genes and the development of tyrosine kinase inhibitors（Tyrosine Kinase Inhibitors,TKIs）such as imatinib（Imatinib Mesylate,IM）,the treatment of CML has made significant progress.However,the poor efficacy and drug resistance of imatinib still exist.Therefore,how to improve the efficacy of imatinib and reverse drug resistance is a problem that needs to be solved.In 1978,Schofield put forward the concept of niche of bone marrow hematopoietic stem cells.Niche refers to a microenvironment including blood vessels,nerves and a variety of cells,in which mesenchymal stem cells（MSCs）and endothelial cells（ECs）are the main components.These cells constitute the microenvironment of hematopoietic stem cells and maintain their ability of self-renewal,proliferation and differentiation by providing a variety of signals and molecules to interact with hematopoietic stem cells.The whole hematopoietic microenvironment plays an important role in maintaining and regulating the growth and differentiation of hematopoietic stem/progenitor cells and leukemic cells.As a key component of hematopoietic microenvironment,bone marrow stromal cells（BMSCs）participate in the maintenance of physiological function and homeostasis of bone marrow microenvironment through autocrine and paracrine activities of soluble factors.GJIC（Gapjunction Intercellular Communication）is a common way of direct communication between adjacent cells.Some studies have found that BMSCs participates in the regulation of hematopoietic cells through gap junction intercellular communication.Previous studies on drug sensitivity and MRD（Minimal Residual Disease）mainly focused on the changes of adhesion and secretion function of BMSCs.However,more and more evidence shows that GJIC in hematopoietic microenvironment plays a key role in leukemic cell proliferation,apoptosis and drug sensitivity.Connexin 43（Connexin43,Cx43）is an important cellular structural protein that widely exists on the cell surface and nuclear membrane.Cx43 can form a gap junction GJ（Gap Junctions）with similar molecules,enabling neighboring cells to transfer ions,small molecular metabolites,second messengers and Micro RNA directly.Our previous studies have shown that overexpression of Cx43 in human umbilical cord mesenchymal stem cells（h UCBSCs）can enhance the efficacy of chemotherapeutic drugs and eliminate a small number of residual L615 cells in animal models.However,the role and detailed mechanism of Cx43 in the drug sensitivity of imatinib in CML are not clear.In this study,K562 cells were used to study the effect of Cx43 knockdown or overexpression of BMSCs on imatinib sensitivity.This project is supported by National key Research and Development Program（2022YFA1103300）,National Natural Science Foundation of China（81873424,81570097）,Chongqing Natural Science Foundation Innovation Group Project（cstc2021jcjcxtt X0001）,Army military Medical University Clinical Medicine Research Program（2018XLC1006）,Chinese Academy of Sciences Transformation Research Grant Program（2020ZKZC02）.Objectives:The effect of Cx43 on the GJIC function of BMSCs was observed,and the Ca²⁺signal communication between BMSCs overexpressed by Cx43 and leukemic cells was significantly improved.By enhancing the downstream Ca²⁺signal pathway,it mediates the mitochondrial apoptosis pathway and Ca MK2-AKT-m TOR apoptosis pathway of K562 cells,thus enhancing the killing effect of imatinib on K562 cells.Methods:1.The expression of Cx43 in bone marrow biopsy specimens of 10 CML patients at initial diagnosis was measured by immunohistochemical method,and the expression of Cx43 in bone marrow biopsy specimens of 10 non-CML patients at initial diagnosis was detected in the control group.2.BMSCs were isolated from bone marrow blood of healthy donors,cultured and identified.BMSCs were induced into physiological hypoxia state by chemical hypoxia inducer Co Cl2（100μmol/L）,and the changes of m RNA and protein levels of Cx43 gene in BMSCs were observed.3.BMSCs were transfected with Ad-over Cx43-GFP,Ad-NC-GFP and Ad-sh Cx43-GFP,and Cx43 expression in BMSCs was divided into overexpression,control and knockdown.The GJIC function of three CX43-modified BMSCs was determined by gap fluorescence bleaching restoration assay（GAP-FRAP）.4.The co-culture system of K562 cells and several CX43-modified BMSCs was established under the condition of imatinib treatment,and the proliferation,cell cycle,apoptosis and other indicators of K562 cells in different groups were detected.To evaluate the effect of Cx43 modified BMSCs on the efficacy of imatinib.The changes of Cx43 expression before and after co-culture of BMSCs and K562 cells were detected by PCR and Western blot.The experiment was divided into five groups:（1）K562 group:K562 cells were cultured alone（2）IM group:K562 cells were treated with imatinib（1μmmol/L）（3）over Cx43 group:BMSCs-over Cx43 was co-cultured with K562 cells under imatinib treatment（1μmmol/L）.（4）NC group:BMSCs-NC was co-cultured with K562 cells under imatinib treatment（1μmmol/L）（5）Sh Cx43 group:BMSCs-sh Cx43 and K562 cells were co-cultured under imatinib treatment（1μmmol/L）5.BMSCs and K562 cells were separated using a Transwell plate with a Transwell penetration rate of 0.4μm（6-well）.Indirect contact co-culture system was established between K562 cells and several CX43-modified BMSCs under imatinib treatment to detect the apoptosis of K562 cells in different groups（groups were set up with direct contact co-culture models,Only the cultivation method is different）.6.The concentrations of ATP and Ca²⁺in K562 cells and different treatment conditions were determined and divided into four groups:K562 group,NC group,over Cx43 group and IM group.Mitochondrial membrane potential（MMP）depolarization and reactive oxygen species（ROS）of K562 cells were detected by flow cytometry.The signaling pathway of Ca²⁺induced apoptosis of K562 cells was detected and verified by Western-Blot method.7.A nude mouse transplanted tumor model was established to evaluate the synergistic effect of Cx43 on IM in K562 cells.The mice were randomly divided into Cx43 group,control group and K562 group（n=4）.The three groups of mice received 8×10⁶K562 cells+2×10⁶ BMSCs-over Cx43（Cx43 group）,respectively.Tumor volume was measured continuously after subcutaneous injection of 8×106 K562 cells+2×10⁶ BMSCs-NC（Control group）and 1×10⁷ K562 cells（K562 group）.Immunohistochemical staining was performed on the tumor to observe the staining results of tumor tissue proliferation（Ki67,BCL-2 and BCR-ABL）and apoptosis（BAX and TUNEL）,and to verify the K562 cell apoptosis pathway at the level of cell experiment.Results:1.The level of Cx43 in bone marrow of CML patients was relatively low:the mean integrated light intensity（IOD）of Cx43 in CML patients was significantly lower than that in control group（p<0.05）.Of the 10 patients with CML,4 were negative（IOD<2000was negative）.All bone marrow samples from the control group were positive,and Cx43expression was decreased in bone marrow biopsies of patients diagnosed with CML.2.BMSCs were successfully isolated from bone marrow and their phenotypes were identified,and differentiation of BMSCs towards osteogenesis and lipogenesis was successfully induced.The Cx43 expression of BMSCs decreased after the physiological hypoxia induced by Co Cl2 treatment（p<0.01）.3.Ad-over Cx43-GFP and Ad-sh Cx43-GFP successfully decreased or increased the expression of Cx43 in BMSCs（p<0.01）.The green fluorescent dye in BMSCs was quenched using laser confocal,and the fluorescence recovery time was observed.The fluorescence intensity of normal BMSCs recovered 22%at 300s and 30%at 500s.The fluorescence intensity of BMSCs-Over CX43 recovered 37%and 48%at 300s and 500s,respectively,while that of BMSCs-Shcx43 recovered 13%and 17%at 300s and 500s,respectively.After 1000s of observation,BMSCs-over Cx43,BMSCs-NC and BMSCs-sh Cx43 showed the highest fluorescence recovery rate with an average recovery percentage of 59%,43%and 23%respectively.4.The proliferation of K562 cells detected by CCK8 showed that imatinib treatment inhibited the proliferation of K562 cells（p<0.01）,but there was no significant difference in the proliferation ability of K562 cells after co-culture with BMSCs modified with different Cx43 under Imatinib treatment（p>0.05）.The cycle and apoptosis of K562 cells were detected by flow cytometry after co-culture of BMSCs modified with different Cx43 under imatinib treatment.The cell cycle results showed that compared with the NC group,the proportion of G0/G1 phase was significantly increased in the sh Cx43 group（p<0.05）,while the proportion of G0/G1 phase was decreased in the over Cx43 group（p<0.05）.The results showed that compared with the IM group,the apoptosis rate of K562 cells in the NC group was lower（p<0.05）,compared with the IM and NC groups,the apoptosis rate of K562 cells in the sh Cx43 group was significantly decreased（p<0.01）,and the apoptosis rate in the over Cx43 group was similar to that in the IM group（p>0.05）.The expression of Cx43 in K562 cells was compared before and after co-culture with BMSCs.After co-culture with BMSCs-Over CX43,the expression of Cx43 in K562 cells was increased in both transcription and translation levels（p<0.05）.After co-culture,Cx43expression levels in BMSCs-over Cx43 and BMSCs-NC were higher than those before co-culture（p<0.05）.5.After 72 h Transwell co-culture,there was no significant difference in the apoptosis of K562 cells in four IM treatment groups:IM group（treated with IM alone）,BMSCs-sh Cx43group,BMSCs-over Cx43 group and BMSCs-NC group（p>0.05）.6.There was no significant difference in ATP concentration among the three IM treatment groups（p>0.05）.Imatinib combined with BMSCs increased Ca²⁺concentration in K562 cells,and the calcium fluorescence intensity in Over Cx43 group was the highest,exceeding that in any IM group（p<0.05）.The combination of imatinib and BMSCs could improve the MMP depolarization in K562 cells,and the MMP depolarization in over Cx43group was significantly increased,but there was no difference between over Cx43 group and IM group（p>0.05）and was higher than that in NC group（p<0.05）.Imatinib combined with BMSCs could increase ROS levels in K562 cells（p<0.05）,and ROS levels in over Cx43group increased the most.Western blot showed that compared with BMSCs-NC co-culture,the expression of BCL-2,BAX and Cyto C were increased in K562 cells co-cultured with BMSCs-over Cx43（p<0.05）.In addition,the expression levels of Cleaved-Caspase-9,Cleaved-Caspase-3 and PARP in Over Cx43 group were significantly higher than those in NC and K562 groups（p<0.05）.The levels of these apoptosis-related proteins in Over Cx43 group were close to those in IM group（p>0.05）.7.In the mouse tumor-bearing model,it was found that the tumor volume was always small and the growth rate was slow in the Over Cx43 group,while the tumor volume was larger in the control group（p>0.05）.In addition,the spleen was larger in the control group than in the Cx43 group.The K562 group had the largest tumor and spleen volume.There was no significant difference in average body weight among the three groups.Immunohistochemical staining showed that the number of tumor cells in Cx43 group was more and inflammatory infiltration was more obvious.Cx43 was strongly positive in tumor tissue in the Cx43 group,but almost none in the K562 group.Staining results of other indicators of proliferation（Ki67,BCL-2 and BCR-ABL）and apoptosis（BAX and TUNEL）also showed that Ki67 and BCL-2 levels were decreased,while BAX and TUNEL levels were increased in Cx43 group.Conclusions:The expression of Cx43 was decreased in bone marrow biopsies of CML patients,and the GJIC function of BMSCs was related to the expression of Cx43.High expression of Cx43 could improve the GJIC function of BMSCs.BMSCs can protect K562cells from IM induced apoptosis.Knockdown of Cx43 expression in BMSCs can enhance the protective effect of BMSCs on K562 cells,while high expression of Cx43 in BMSCs can reverse the protective effect of BMSCs on K562 cells.High expression of Cx43 in BMSCs can improve Ca²⁺flow between BMSCs and K562 cells,promote apoptosis of K562 cells through Caspase and Ca MKII-Akt-m TOR signaling pathways,and thus reverse the protective effect of BMSCs.Enhancing Cx43 expression and GJIC function in hematopoietic microenvironment may be a new strategy to improve imatinib sensitivity of leukemia cells.