目的: 探讨去甲斑蝥素(norcantharidin,NCTD)抗骨髓瘤作用及其相关机制。方法: MTT法检测NCTD对人骨髓瘤细胞株RPMI 8226增殖的抑制作用, 并观察其对荷瘤小鼠肿瘤生长的抑制作用; 流式细胞术检测细胞凋亡率, Western blotting检测核因子-κB(NF-κB)p65、磷酸化NF-κB p65(p-NF-κB p65)、NF-κB抑制因子IkBα、磷酸化IkBα(p-IkBα)、IkB激酶α(IKKα)以及Survivin、Bcl-2、Bax蛋白表达水平, 分光光度法检测Caspase-3活性, RT-PCR检测NF-κB mRNA表达。结果: NCTD抑制RPMI 8226细胞增殖, 促进其凋亡;其剂量为20、40 mg/kg时对荷瘤小鼠的抑瘤率分别为29.8%、53.5%。NCTD抑制胞核NF-κB p65、p-NF-κB p65以及胞浆p-IkBα、IKKα的表达, 增加胞浆IkBα的表达, 对胞浆NF-κB p65的表达无显著影响。NCTD还抑制Survivin、Bcl-2蛋白的表达, 促进Bax的表达和RPMI 8226细胞Caspase-3的活化, Caspase-3抑制剂能部分拮抗NCTD引起的RPMI 8226细胞凋亡(P<0.05)。结论: NCTD对RPMI 8226细胞有抗增殖和促凋亡作用, 其机制可能与NF-κB信号通路有关。

Objective: To explore the anti-myeloma effect of norcantharidin (NCTD) and the possible mechanism. Methods: Human multiple myeloma (MM) cell line RPMI 8226 was treated with different concentration of NCTD. MTT was used to detect cell proliferation. The inhibition of NCTD on the growth of BALB/C nu tumor-bearing mice was observed. Flow cytometry was used to determine apoptosis. The protein expression of nuclear factor-κB (NF-κB) p65, phosphorylated NF-κB p65 (p-NF-κB p65), and its inhibitor IkBα, phosphorylated IκBα (p-IκBα), IκB kinase α (IKKα) and the protein expression of Survivin, Bcl-2, and Bax were determined by Western blotting. Spectrophotometry was used to examine the activity of Caspase-3. RT-PCR was used to determine the mRNA expression of NF-κB. Results: NCTD could inhibit the proliferation and induce apoptosis of RPMI 8226 cell in vitro. The inhibition rates to tumor-bearing mice were 29.8% and 53.3% at the dose of 20 and 40 mg/kg, respectively. NCTD inhibited the growth of transplanted tumor in mice in a dose-dependent manner. The nuclear protein expression of NF-κB p65 and p-NF-κB p65 and the cytoplasm expression of p-IkBα and IKKα were inhibited by NCTD in a time-dose dependent manner. The cytoplasm expression of IkBα was increased while the cytoplasm expression of NF-κB p65 was unchanged. NCTD inhibited the protein expression of Survivin and Bcl-2, while the expression of Bax and the activiation of Caspase-3 were improved in a dose dependent manner. The inhibitor of Caspase-3 z-DEVD-fmk could inhibit the RPMI 8226 apoptosis induced by NCTD (P<0.05). Conclusion: Our results suggest that NCTD could inhibit the proliferation and induce apoptosis in RPMI 8226 via NF-κB/IKKα signaling pathway, for which the mechanism may be related to NF-κB-regulated gene products including survivin, Bcl-2, Bax and Caspase-3.

河北省中医药管理局资助项目(2007136)