Imatinib resistance of K562 cells co-cultured with bone marrow stromal cells from patients with chronic myeloid leukemia

doi:10.3969/j.issn.2095-4344.2015.06.006

Abstract

Abstract:

BACKGROUND: Imatinib resistance is a key issue in treatment of chronic myeloid leukemia. It is confirmed that the leukemia cells can obtain drug resistance phenotype mediated by adhesion to the bone marrow stromal cells (BMSCs). But, the role of BMSCs in imatinib resistance is unclear because chronic myeloid leukemia is deficient in adhesion function.
OBJECTIVE: To in vitro simulate the bone marrow microenvironment of patients with chronic myeloid leukemia and to explore its influences on imatinib sensitivity as well as possible mechanisms.
METHODS: BMSCs isolated from patients with chronic myeloid leukemia but not undergoing treatment were co-cultured with K562 cells to construct the BMSCs-K562 cell co-culture model in chronic myeloid leukemia
patients, then exposed to 0.2-3.2 μmol/L imatinib for 48 hours, and the proliferation inhibition rate of K562 cells was studied by MTT assay. The apoptosis rate of K562 cells and the expression of the CXCR4 in K562 cells exposed to
0.5 μmol/L imatinib for 72 hours were detected by flow cytometry. The K562 cells were exposed to 0.5 μmol/L imatinib for 4 hours and labeled by Calcein-AM fluorescent labeling sytem, and then, the adhesion rate of the K562 cells was calculated based on fluorescence intensity.
RESULTS AND CONCLUSION: The suppressing effect of imatinib (0.2-3.2 μmol/L) on the proliferation of K562 cells was weakened significantly by co-culture with the bone marrow stromal cells from patients with chronic myeloid leukemia. The apoptosis rate of K562 cells exposed to 0.5 μmol/L imatinib for 72 hours in co-culture group was significantly lower than that in the suspension culture group (P=0.020). The positive rates of CXCR4 in the co-culture group and suspension culture groups were both increased after exposure to 0.5 μmol/L imatinib for 72 hours (P=0.001). The adhesion rate of the K562 cells to the BMSCs was elevated from (32.18±6.17)% to (68.97±11.08)% when the K562 cells were exposed to 0.5 μmol/L imatinib for 4 hours, and the difference had statistical significance (P=0.022). These findings indicate that the co-culture with the BMSCs from patients with chronic myeloid leukemia can mediate K562 cells resistance to imatinib, which may be related to that the co-culture with BMSCs and exposure to imatinib can induce the K562 cells to express CXCR4. But the mechanism needs in-depth studies.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: Leukemia, Myelogenous, Chronic, BCR-ABL Positive, K562 Cells, Coculture Techniques, Protein-Tyrosine Kinases

CLC Number:

R394.2

Wang Ji-gang, Zhang Hai-ting, Zhou Fan, Liu Yan-qin, Bai Ying, Liu Jing-hua, Li Min-yan. Imatinib resistance of K562 cells co-cultured with bone marrow stromal cells from patients with chronic myeloid leukemia[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(6): 849-853.

Figures/Tables 1

2.1 骨髓基质细胞-K562细胞共培养模型的形态学观察接种K562细胞后4 h，约30%的K562细胞黏附于基质细胞层，培养24 h黏附的K562细胞约40%，培养48 h黏附的K562细胞比例无升高，但可观察到少数K562细胞向基质细胞层迁移。扫描电镜观察发现K562细胞通过伪足龛于融合基质细胞层所形成的“网眼”中(图1A)。实验中观察到暴露于伊马替尼可诱导K562细胞黏附于基质细胞层，且向基质细胞层迁移现象更易见，部分K562细胞嵌合于基质细胞层，同时也观察到部分K562细胞发生固缩或呈碎块状(图1B)。 2.2 骨髓基质细胞共培养对伊马替尼抑制K562细胞增殖的影响表1所示，0-3.2 μmol/L伊马替尼梯度浓度处理48 h，共培养组与悬浮培养组K562细胞增殖抑制率比较差异均有显著性意义，提示骨髓基质细胞共培养明显减弱了伊马替尼对K562细胞的增殖抑制作用。 2.3 骨髓基质细胞共培养对伊马替尼诱导K562细胞凋亡的影响 K562细胞经0.5 μmol/L伊马替尼处理72 h，Annexin V-FITC/PI标记后流式细胞术检测Annexin V+/PI-的K562细胞百分比即凋亡率。基质细胞共培养组凋亡率为(15.48±4.17)%，而悬浮培养组为(32.01±6.83)%，两组比较差异有显著性意义(t=7.147，P=0.020)，提示骨髓基质细胞共培养显著抑制了伊马替尼对K562细胞的凋亡诱导效应。 2.4 流式细胞仪检测CXCR4表达 0.5 μmol/L伊马替尼作用K562细胞72 h，共培养组伊马替尼处理前和处理后K562细胞CXCR4表达阳性率分别为(20.31±3.76)%和(53.64±5.35)%，差异有显著性意义(t=10.262，P=0.001)；悬浮培养组伊马替尼处理前和处理后K562细胞CXCR4表达阳性率分别为(11.28±3.44)%和(25.34±3.21)%，差异有显著性意义(t=8.547，P=0.001)，提示伊马替尼能诱导K562细胞表达CXCR4。未经伊马替尼处理的共培养组K562细胞CXCR4表达阳性率高于未经伊马替尼处理的悬浮组，差异有显著性意义(t=5.761，P=0.022)；且伊马替尼处理后的共培养组K562细胞CXCR4表达阳性率也高于伊马替尼处理后的悬浮组，差异有显著性意义(t=6.278，P=0.019)，提示骨髓基质细胞共培养也能诱导K562细胞表达CXCR4 (图2，绿色实线为阴性对照，虚线为伊马替尼作用前，红色实线为伊马替尼作用72 h)。 2.5 伊马替尼对K562细胞-骨髓基质细胞黏附率的影响将经0.5 μmol/L伊马替尼作用4 h的K562细胞以Calcein-AM行荧光标记后接种于基质细胞层培养24 h，荧光显微镜观察发现，伊马替尼处理组荧光标记的K562细胞密度高于对照组，部分K562细胞聚集成簇(图3)。通过检测黏附于基质细胞层K562细胞的荧光强度计算黏附率，结果提示伊马替尼处理组黏附率为(68.97± 11.08)%，而对照组为(32.18±6.17)%，两组比较差异有显著性意义(t=6.247，P=0.022)，提示伊马替尼能诱导K562细胞黏附于骨髓基质细胞层。"

References

[1] Jabbour E, Kantarjian H.Chronic myeloid leukemia: 2014 update on diagnosis, monitoring, and management.Am J Hematol. 2014;89(5):547-556.
[2] Waller CF.Imatinib mesylate.Recent Results Cancer Res. 2014; 201:1-25.
[3] Castagnetti F, Palandri F, Amabile M,et al.Results of high-dose imatinib mesylate in intermediate Sokal risk chronic myeloid leukemia patients in early chronic phase: a phase 2 trial of the GIMEMA CML Working Party.Blood. 2009;113(15): 3428-3434.
[4] Lee F, Fandi A, Voi M.Overcoming kinase resistance in chronic myeloid leukemia.Int J Biochem Cell Biol. 2008;40(3): 334-343.
[5] Weisberg E, Liu Q, Zhang X,et al.Selective Akt inhibitors synergize with tyrosine kinase inhibitors and effectively override stroma-associated cytoprotection of mutant FLT3-positive AML cells.PLoS One. 2013;8(2):e56473.
[6] Anderson KC.Targeted therapy of multiple myeloma based upon tumor-microenvironmental interactions.Exp Hematol. 2007;35(4 Suppl 1):155-162.
[7] Schraufstatter IU, Discipio RG, Khaldoyanidi S.Mesenchymal stem cells and their microenvironment.Front Biosci (Landmark Ed). 2011;16:2271-2288.
[8] Tabe Y, Konopleva M.Advances in understanding the leukaemia microenvironment.Br J Haematol. 2014;164(6): 767-778.
[9] 王吉刚,陈幸华,周凡,等.TRAIL逆转基质细胞黏附介导Jurkat细胞耐药的研究[J].中华肿瘤防治杂志,2009,16(8):621-624,632.
[10] 赵兴海,孙京杰,刘爱武,等．丙戊酸钠调节组蛋白乙酰化抑制胃癌细胞增殖的实验研究[J].中华肿瘤防治杂志,2009,16(8): 587-591.
[11] Jacamo R, Chen Y, Wang Z,et al.Reciprocal leukemia-stroma VCAM-1/VLA-4-dependent activation of NF-κB mediates chemoresistance.Blood. 2014 4;123(17):2691-2702.
[12] Bewry NN, Nair RR, Emmons MF,et al.Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance.Mol Cancer Ther. 2008;7(10):3169-3175.
[13] 刘志,刘晓力,杜庆锋,等.纤维连接蛋白黏附共培养对伊马替尼诱导K562细胞凋亡影响的研究[J].中华血液学杂志,2009,30(12): 839-840.
[14] Seke Etet PF, Vecchio L, Nwabo Kamdje AH.Signaling pathways in chronic myeloid leukemia and leukemic stem cell maintenance: key role of stromal microenvironment.Cell Signal. 2012;24(9):1883-1888.
[15] Azizidoost S, Babashah S, Rahim F,et al.Bone marrow neoplastic niche in leukemia.Hematology. 2014;19(4):232-238.
[16] Lundell BI, McCarthy JB, Kovach NL,et al. Activation- dependent alpha5beta1 integrin-mediated adhesion to fibronectin decreases proliferation of chronic myelogenous leukemia progenitors and K562 cells.Blood. 1996; 87(6): 2450-2458.
[17] Zhang B, Li M, McDonald T,et al.Microenvironmental protection of CML stem and progenitor cells from tyrosine kinase inhibitors through N-cadherin and Wnt-β-catenin signaling.Blood. 2013;121(10):1824-1838.
[18] Krause DS, Lazarides K, Lewis JB,et al.Selectins and their ligands are required for homing and engraftment of BCR-ABL1+ leukemic stem cells in the bone marrow niche. Blood. 2014;123(9):1361-1371.
[19] Martín-Henao GA, Quiroga R, Sureda A,et al. L-selectin expression is low on CD34+ cells from patients with chronic myeloid leukemia and interferon-a up-regulates this expression. Haematologica. 2000;85(2):139-146.
[20] Peled A, Tavor S. Role of CXCR4 in the pathogenesis of acute myeloid leukemia. Theranostics. 2013;3(1):34-39.
[21] Sison EA, McIntyre E, Magoon D, et al. Dynamic chemotherapy-induced upregulation of CXCR4 expression: a mechanism of therapeutic resistance in pediatric AML. Mol Cancer Res. 2013;11(9):1004-1016.