c—Myc在骨髓基质细胞介导的急性髓细胞白血病耐药中作用

Role of c-Myc in mesenchymal stromal cell-mediated drug resistance in acute leukemia cells

目的探讨c—Myc住骨髓基质细胞介导的急性髓细胞白血病（AML）细胞系耐药中的作用，从肿瘤微环境的角度探索AML耐药的分子机制。方法将AML细胞系U937、KGla与造血干细胞移植健康供者的骨髓基质细胞（间允质干细胞，MSC）苁培养，以细胞单独培养作为对照。流式细胞术及膜联蛋V（AnnexinV）／碘化丙啶（PI）双染色法和DAPI染色法比较两种培养条件下AML细胞埘米托总醌诱导的凋亡的差异；Western印迹检测两种培养条件下AML细胞c—Myc蛋白的表达；在培养体系中加入c～Myc抑制剂10058-F4，检测AML细胞对米托蒽醌诱导的凋广的变化。结果U937、KGIa两种AMI．细胞系与MSC共培养后，对米托蒽醌诱导的凋亡均低于对照组（9．88％±1．53％比42．83％±2．03％，P=0．004；20．60％±2．87％比42．53％±5．29％，P=0．030），共培养促进了AML细胞对化疗药物的耐药性。AML细胞系与MSC共培养后，Western印迹检测c—Myc蛋白的表达明显高于对照组。c—Myc抑制剂10058-F4可诱导AMI．细胞的凋亡10058-F4加人共培养体系后，KGla细胞系对米托蒽醌诱导的凋亡率从23．87％±1．55％碌著升高到57．23％±3．88％（P=0．009），存U937细胞共培养体系中同样观察到细胞凋亡率从16．07％±2．11％显著提高到53．47％±4．08％（P=0．004），从而克服耐药。结论AML细胞与MSC共培养可导致c—Myc蛋白表达的上凋，从而介导AML细胞对化疗药物的耐药；针对c—Myc的靶向治疗将为AML的治疗提供新思路。

Objective To explore the role of c-Myc in mesenchymal stromal cell-mediated drug resistance and elucidate the molecular mechanism of acute myeloid leukemia （AML） from the version of tmnor mieroenvironment. Methods AML cell lines U937 and KGla were eo-euhured with mesenchymal stromal cells （MSC） from bone marrow of healthy donors between January to March 2012. The AML cell lines plated alone was euhured as controls. Apoptosis induced by mitoxantrone was measured by flow cytometry and Annexin V/PI double and 4＇45-diamidino-2-phenylindole （DAPI） staining. And c-Mye protein was detected by Western blot under both culturing conditions. After a pre-treatment of c-Myc inhibitor 10058-F4, the apoptosis of AML cell was also evaluated. Results Apoptosis of AML cells （ U937 and KG1 a） significantly decreased during co-culturing with MSC （9.88%± 1.53% vs 42.83% ± 2. 03% , P =0. 004;20. 60% ±2.87% vs 42.53% ±5.29% ,P =0. 030）. Drug resistance was implicated. The co- culturing of AML cells with MSC significantly induced an up-regulation of e-Mye. The inhibition of c-Myc with 10058-F4 could induce apoptosis of AML cells. After an addition of 10058-F4 into the co-culture system, the apoptotie rate of KGla cells significantly increased from 23.87% ±1.55% to 57.23% ±3.88% （P =0. 009）. Similarly the apoptotie rates spiked from 16. 07% ±2. 11% to 53.47% ±4.08% in U937 cells （P = 0. 004） to overcome the stromal cell-mediated drug resistance. Conclusions The co-culturing of AML cells and MSC induces an up-regulation of e-Myc protein so as to cause the emergence of chemoresistance. Therefore targeting c-Myc protein may provide a novel therapeutic strategy of AML.