Umbilical cord mesenchymal stem cells enhance imatinib-induced apoptosis in chronic myeloid leukemia

doi:10.3969/j.issn.2095-4344.2017.25.016

Abstract

Abstract:

BACKGROUND: Imatinib has a significant pro-apoptosis effect on chronic myelogenous leukemia (CML), but there are still some patients being resistant to it. Human umbilical cord mesenchymal stem cells (hUC-MSCs) affect the apoptosis of a variety of hematologic malignancies. However, the impacts of hUC-MSCs on the apoptosis of CML cells induced by imatinib remain unclear.
OBJECTIVE: To investigate whether hUC-MSCs have an influence on the apoptosis of K562 cells induced by imatinib and to reveal the possible underlying mechanism.
METHODS: K562 cells were cultured with hUC-MSCs or/and imatinib. Cellular apoptosis was measured with Annexin-V and PI staining by flow cytometry analysis. The protein expressions of Bax, Bcl-2, caspase-3, caspase-9 and cleaved-PARP in K562 cells were detected by western blot assay. Pan-caspase inhibitor Z-VAD-FMK was used to block apoptosis in each group, and during this process the effect of caspase apoptosis signaling pathway was detected.
RESULTS AND CONCLUSION: The apoptosis of K562 cells was enhanced, when imatinib was combined with hUC-MSCs. Western blot analysis showed that the expression of pro-apoptotic protein Bax was enhenced and the expression of anti-apoptotic protein Bcl-2 was suppressed. Furthermore, the cleaved forms of caspase-9, caspase-3 and PARP in K562 cell were higher in the hUC-MSCs+imatinib group than in the imatinib group. The apoptosis of K562 cells induced by the hUC-MSCs combined with imatinib was significantly inhibited by Z-VAD-FMK. In conclusion, these findings indicate that hUC-MSCs can enhance imatinib-induced apoptosis of K562 cells by activating caspase apoptosis signaling pathway.

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

Key words: Umbilical Cord, Apoptosis, Tissue Engineering

CLC Number:

R394.2

Liu Ying, Song Bao-quan, Wei Yi-meng, Fan Hui-fang, Yu Yi, Dong Shu-xu, Han Zhong-chao, Ma Feng-xia. Umbilical cord mesenchymal stem cells enhance imatinib-induced apoptosis in chronic myeloid leukemia[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(25): 4032-4037.

Figures/Tables 2

2.1 脐带间充质干细胞的鉴定胶原酶与胰酶联合消化法分离人脐带间充质干细胞，接种后2周左右可形成80%融合的单层贴壁梭形细胞，呈平行排列或旋涡状生长(图1A)；成脂诱导14 d后，油红O染色可见红色脂滴形成(图1B)；成骨诱导21 d后，经Von Kossa染色可见黑色钙盐沉积(图1C)；流式细胞仪检测人脐带间充质干细胞表面标志物显示HLA-ABC，CD44，CD73，CD90和CD105呈阳性表达，HLA-DR，CD11b，CD14，CD34和CD45呈阴性表达(图1D)。 2.2 人脐带间充质干细胞对K562细胞凋亡的影响采用Annexin V-FITC/PI双标法检测各组细胞凋亡情况，结果表明人脐带间充质干细胞和伊马替尼联合应用与单用伊马替尼相比，K562细胞凋亡显著增加(P < 0.01)；单独使用人脐带间充质干细胞不能增加K562细胞凋亡(P > 0.05；图2)。 2.3 人脐带间充质干细胞联合伊马替尼对K562细胞凋亡相关蛋白及caspase凋亡信号通路相关蛋白表达的影响为检测caspase凋亡信号通路在人脐带间充质干细胞对伊马替尼诱导的K562细胞凋亡的影响，Western blot检测各实验组中凋亡相关蛋白及caspase凋亡信号通路相关蛋白表达。与伊马替尼组相比，伊马替尼联合人脐带间充质干细胞组K562细胞中促凋亡蛋白Bax表达升高，抑凋亡蛋白Bcl-2表达降低。伊马替尼联合人脐带间充质干细胞组K562细胞中caspase凋亡信号通路相关蛋白的酶原形式，即非活化形式，caspase-9，caspase-3和PARP的表达明显低于伊马替尼组，酶原形式caspase-9可被上游信号通路分子，如细胞色素c，激活剪切为cleaved-caspase-9，cleaved-caspase-9又可将下游信号分子caspase-3剪切为具有酶活性的cleaved-caspase-3，进而将PARP剪切为cleaved-PARP [10-13]。结果表明，与伊马替尼组相比，伊马替尼联合人脐带间充质干细胞组K562细胞中上述蛋白的剪切体形式cleaved-caspase-9、cleaved-caspase-3、cleaved-PRAP的表达明显增高(图3)。 2.4 caspase泛抑制剂Z-VAD-FMK逆转人脐带间充质干细胞联合伊马替尼所致的K562细胞凋亡的增强为进一步检测caspase凋亡信号通路在人脐带间充质干细胞联合伊马替尼增强K562细胞凋亡中的作用机制，采用caspase泛抑制剂Z-VAD-FMK(30 μM)预处理各实验组4 h。结果发现，伊马替尼联合人脐带间充质干细胞组中K562细胞凋亡可被caspase泛抑制剂Z-VAD-FMK显著抑制(P < 0.01；图4)。"

References

[1] Kang ZJ, Liu YF, Xu LZ, et al. The Philadelphia chromosome in leukemogenesis. Chin J Cancer. 2016;35:48.

[2] Mughal TI, Radich JP, Deininger MW, et al. Chronic myeloid leukemia: reminiscences and dreams. Haematologica. 2016; 101(5):541-558.

[3] Jabbour E. Chronic myeloid leukemia: First-line drug of choice. Am J Hematol. 2016;91(1):59-66.

[4] Polillo M, Galimberti S, Baratè C, et al. Pharmacogenetics of BCR/ABL Inhibitors in Chronic Myeloid Leukemia. Int J Mol Sci. 2015;16(9):22811-22829.

[5] Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tissues. Science. 1997;276(5309):71-74.

[6] Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999;284(5411):143-147.

[7] Tian K, Yang S, Ren Q, et al. p38 MAPK contributes to the growth inhibition of leukemic tumor cells mediated by human umbilical cord mesenchymal stem cells. Cell Physiol Biochem. 2010;26(6):799-808.

[8] Chen F, Zhou K, Zhang L, et al. Mesenchymal stem cells induce granulocytic differentiation of acute promyelocytic leukemic cells via IL-6 and MEK/ERK pathways. Stem Cells Dev. 2013;22(13):1955-1967.

[9] Smith JR, Cromer A, Weiss ML. Human Umbilical Cord Mesenchymal Stromal Cell Isolation, Expansion, Cryopreservation, and Characterization. Curr Protoc Stem Cell Biol. 2017;41:1F.18.1-1F.18.23.

[10] Maes ME, Schlamp CL, Nickells RW. BAX to basics: How the BCL2 gene family controls the death of retinal ganglion cells. Prog Retin Eye Res. 2017;57:1-25.

[11] Cheng CH, Cheng YP, Chang IL, et al. Dodecyl gallate induces apoptosis by upregulating the caspase-dependent apoptotic pathway and inhibiting the expression of anti-apoptotic Bcl-2 family proteins in human osteosarcoma cells. Mol Med Rep. 2016;13(2):1495-1500.

[12] Yuan J, Najafov A, Py BF. Roles of Caspases in Necrotic Cell Death. Cell. 2016;167(7):1693-1704.

[13] Rascón-Valenzuela LA, Velázquez C, Garibay-Escobar A, et al. Apoptotic activities of cardenolide glycosides from Asclepias subulata. J Ethnopharmacol. 2016;193:303-311.

[14] Baksh D, Yao R, Tuan RS. Comparison of proliferative and multilineage differentiation potential of human mesenchymal stem cells derived from umbilical cord and bone marrow. Stem Cells. 2007;25(6):1384-1392.

[15] Hoffmann A, Floerkemeier T, Melzer C, et al. Comparison of in vitro-cultivation of human mesenchymal stroma/stem cells derived from bone marrow and umbilical cord. J Tissue Eng Regen Med. 2016. in press.

[16] Pappa KI, Anagnou NP. Novel sources of fetal stem cells: where do they fit on the developmental continuum? Regen Med. 2009;4(3):423-433.

[17] Nagamura-Inoue T, Mukai T. Umbilical Cord is a Rich Source of Mesenchymal Stromal Cells for Cell Therapy. Curr Stem Cell Res Ther. 2016;11(8):634-642.

[18] Arutyunyan I, Elchaninov A, Makarov A, et al. Umbilical Cord as Prospective Source for Mesenchymal Stem Cell-Based Therapy. Stem Cells Int. 2016;2016:6901286.

[19] Aristizábal JA, Chandia M, Del Cañizo MC, et al. Bone marrow microenvironment in chronic myeloid leukemia: implications for disease physiopathology and response to treatment. Rev Med Chil. 2014;142(5):599-605.

[20] Wang B, Hu Y, Liu L, et al. Phenotypical and functional characterization of bone marrow mesenchymal stem cells in patients with chronic graft-versus-host disease. Biol Blood Marrow Transplant. 2015;21(6):1020-1028.

[21] Han Y, Wang Y, Xu Z, et al. Effect of bone marrow mesenchymal stem cells from blastic phase chronic myelogenous leukemia on the growth and apoptosis of leukemia cells. Oncol Rep. 2013;30(2):1007-1013.

[22] 张海梅,张连生.人骨髓间充质干细胞对慢性粒细胞白血病细胞增殖的影响及其机制探讨[J].癌症,2009,28(1):39-42.

[23] 朱雅姝,孙昭,李振亚,等.脂肪MSCs分泌的DKK-1抑制慢性粒细胞白血病细胞K562的增殖[J].基础医学与临床,2009,29(5):479- 483.

[24] 庞一琳,张斌,陈虎,等.间充质干细胞与肿瘤细胞相互作用:促进还是抑制[J].中华细胞与干细胞杂志(电子版),2013,3(1):31-38.

[25] Chen JR, Jia XH, Wang H, et al. Timosaponin A-III reverses multi-drug resistance in human chronic myelogenous leukemia K562/ADM cells via downregulation of MDR1 and MRP1 expression by inhibiting PI3K/Akt signaling pathway. Int J Oncol. 2016;48(5):2063-2070.

[26] Zhang X, Dong W, Zhou H, et al. α-2,8-Sialyltransferase Is Involved in the Development of Multidrug Resistance via PI3K/Akt Pathway in Human Chronic Myeloid Leukemia. IUBMB Life. 2015;67(2):77-87.

[27] Guerra B, Martín-Rodríguez P, Díaz-Chico JC, et al. CM363, a novel naphthoquinone derivative which acts as multikinase modulator and overcomes imatinib resistance in chronic myelogenous leukemia. Oncotarget. 2017;8(18):29679- 29698.

[28] Shi X, Chen X, Li X, et al. Gambogic acid induces apoptosis in imatinib-resistant chronic myeloid leukemia cells via inducing proteasome inhibition and caspase-dependent Bcr-Abl downregulation. Clin Cancer Res. 2014;20(1): 151-163.

[29] Vida A, Márton J, Mikó E, et al. Metabolic roles of poly(ADP-ribose) polymerases. Semin Cell Dev Biol. 2017;63: 135-143.

[30] Beneke S, Bürkle A. Poly(ADP-ribosyl)ation, PARP, and aging. Sci Aging Knowledge Environ. 2004;2004(49):re9.