低浓度双硫仑/铜抑制骨髓MSCs内NF-κB通路逆转急性B淋巴细胞白血病耐药机理研究

Chemotherapy resistance in acute lymphoblastic leukemia (ALL) may be mediated by increased interaction between leukemic blasts and the bone marrow mesenchymal stem cells (MSCs). Co-culture of MSCs with ALL cells activated nuclear factor (NF)- κB signaling pathway , the interaction between vascular cell adhesion molecule 1 (VCAM-1) and very late antigen-4 (VLA-4) played an integral role in the activation of NF-κB in MSCs and leukemic cells. And reciprocal NF-κB activation in MSCs and leukemia cells is essential for promoting chemoresistance in ALL cells. We recently found low dose of disulfiram/Cu could reverse MSCs induced chemoresistance of B-ALL cells to cytarabine, Yet the underlying mechanisms associated with this effect remain unclear. We have previously demonstrated disulfiram/Cu targets ALL cells in vitro and in vivo by inhibition of NF-κB pathway. We speculate disulfiram/Cu may reverse chemoresistance of ALL cells to Ara-C by inhibition of NF-κB pathway in MSCs and downregulate VCAM-1 expression. In the present study, we further investigate whether low dose of disulfiram/Cu could reverse MSCs induced chemoresistance in B-ALL cell lines, primary B-ALL cells and in patient-derived xenograft (PDX) humanized mouse models. Westernblot, EMSA, Immunohistochemistry as well as flow cytometry will also be employed to explore the underlying mechanisms.

骨髓微环境与白血病耐药相关，MSCs是骨髓微环境中重要的基质细胞。最近发现B-ALL细胞表面VLA-4和MSCs表面VCAM-1结合活化B-ALL细胞NF-κB通路诱导耐药；同时激活MSCs内NF-κB通路，上调MSCs的 VCAM-1，进一步增强MSCs和ALL细胞相互作用形成正反馈机制诱导耐药。我们最近研究发现低浓度双硫仑/铜能够逆转MSCs诱导的 B-ALL细胞对化疗药物耐药，但是具体机制不明。我们前期研究示双硫仑/铜能抑制白血病细胞NF-κB通路。因此，我们提出低浓度双硫仑/铜通过抑制MSCs内NF-κB通路下调其VCAM-1逆转B-ALL耐药的假说。本项目拟在细胞株、原代B-ALL细胞与MSCs共培养及人源化B-ALL移植鼠模型中，采用免疫印迹、流式细胞分析等方法从体内、外研究低浓度双硫仑/铜逆转耐药的作用及机理，为难治复发B-ALL治疗策略提供新的理论依据。

骨髓微环境异常与B-ALL耐药相关，MSCs是骨髓微环境中重要的基质细胞。MSCs可以通过其表面分子VCAM-1和B-ALL细胞表面VLA-4相互作用诱导耐药。本研究探讨低浓度双硫仑/铜（DS/Cu）逆转MSCs诱导B-ALL细胞耐药的潜能及其分子机制。CCK8分析发现低浓度DS/Cu（DS 0.4μM,Cu 1μM）联合阿糖胞苷（Ara-C）显著抑制共培养组RS4细胞和JM1细胞增殖，IC50分别从115±10.2nM下降至46.5±3.8nM(p=0.039)；189±15.3nM下降至50.8±4.6nM(p =0.012)。凋亡检测示低浓度DS/Cu联合 Ara-C作用72h后共培养组RS4细胞凋亡率从19.05±1.78%上升至60.38±2.34%（p=0.001）,JM-1细胞凋亡率从21.03±3.50%上升至74.73±5.35%（p=0.001）。我们分析了14例原代ALL细胞标本，DS/Cu+AraC组凋亡比例显（60.5±10.31%）显著高于Con组（21.5±5.45%），AraC单药组（26.5±7.35%）和DS/Cu单药组（27.1±6.56%）(p=0.035)。体内研究显示低浓度DS/Cu明显降低人源化NSI B-ALL 移植鼠外周血、骨髓及脾脏hCD45%比例，同时降低小鼠脾脏重量。Westernblot显示DS/Cu+Ara-C 下调MSCs内NF-κB-p65/RelB/NF-κB-p50蛋白表达，流式细胞分析显示DS/Cu+Ara-C组VCAM-1（9.53±1.05%）表达较对照组显著下降（28.57±3.06%）。此外，Westernblot显示DS/Cu+Ara-C抑制JM1细胞和RS4细胞内NF-κB-p65、RelB蛋白表达，抑制抗凋亡通路蛋白Bcl2/Bcl-xl表达，激活PARP，上调Bax/Casepase3表达。因此，低浓度DS/Cu能够从体内外逆转MSCs诱导的B-ALL细胞耐药，其分子机制同抑制MSCs内NF-κB通路下调其VCAM-1表达，同时抑制白血病细胞内NF-κB通路双重机制相关。本研究为难治复发性B-ALL治疗策略提供新的思路和方法，为将来临床应用奠定理论和实验基础。