蛋白酶体抑制剂诱导多发性骨髓瘤RPMI8226细胞凋亡及对Notch1和NF-κB表达的影响

Proteasome Inhibitor Induces Apoptosis and Influences the Expression of Notch 1 and NF-κB in Multiple Myeloma RPMI8226 Cells

多发性骨髓瘤(multiple myeloma,MM)的发生、发展与骨髓微环境关系密切,有多种信号通路参与。Notch信号是造血微环境调节造血细胞增殖分化的重要信号通路,其中Notch1与血液肿瘤的发生、发展较为密切。骨髓瘤是高度依赖NF-κB的恶性肿瘤,后者与肿瘤细胞的发生和转移、增殖和凋亡、耐药相关。已证实NF-κB2家族是Notch信号通路的一个靶基因。蛋白酶体抑制剂Bortezomib已获美国FDA批准用于治疗难治及复发多发性骨髓瘤,但其诱导骨髓瘤细胞凋亡的机制尚未完全明确。本研究探讨bortezomib诱导多发性骨髓瘤细胞凋亡和对Notch1、NF-κB表达水平的影响,采用透射电子显微镜检、流式细胞术及原位缺口末端标记技术观察药物对MM细胞凋亡的影响,用RT-PCR法和免疫荧光细胞化学染色分析分别检测细胞凋亡时Notch1及NF-κB的表达情况。结果表明:RPMI8226细胞高表达Notch1和NF-κB;随着bortezomib作用浓度的增高,RPMI8226细胞出现典型的凋亡改变,Notch1的表达逐渐降低,NF-κB在胞核表达减少,胞浆表达增多。这种改变在浓度升高到一定数值时与对照相比具有显著性差异(p<0.05)。结论:bortezomib治疗多发性骨髓瘤机制中有Notch1和NF-κB两条通路的参与,二者可能存在一定的联系,提示Notch1信号可能作为MM治疗的潜在靶点,为相关新药的研发提供一定实验基础。

The occurrence and development of Multiple myeloma （MM） are closely related to bone marrow microenvironment in which many signal pathways were involved. Notch signal plays an important role in regulating hematopoietic cells proliferation and differentiation in hemopoietic microenvironment, especially Notchl has close touch with the occurrence and development of neoplastic hematologic disorders. MM deeply depends on NF-κB, and the latter has something to do with oncogenesis, metastasis, proliferation, apoptosis and drug resistance of tumor cells. NF-κB2 family has been confirmed as one of target genes of Notch signal. Bortezomib as representative of proteasome inhibitors has been licensed by FDA in treating refractory and relapsing MM, but the mechanism of bortezomib inducing cells apoptosis is not yet clear. This study was purposed to investigate the effects of bortezomib inducing cell apoptosis and influencing the erpression of Notchl and NF-κB in MM RPMI 8226 cells. The effect of bortezomib on apoptosis of MM RPMI8226 cells was assayed by transmission electron microscopy, flow cytometry and TUNEL; the expressions of Notch 1 and NF-KB in apoptotic RPMI8226 cells were detected by RT-PCR and immunofluorescence cytochemical staining respectively. The results showed that Notchl and NF-κB highly expressed in RPMI 8226 cells. With the increase of bortezomib concentration, the typical apoptosis features of RPMI8226 cells were observed, the expressions of Notchl and the expression of NF-κB decreased in nuclei and increased in cytoplasm. These changes had significant difference from control after concentration of bortezomib was raised to a certain degree. It is concluded that Notchl and NF-κB signaling pathways participate in bortezomib-inducing RPMI8226 cell apoptosis and there may be some correlation between the Notch 1 and NF-κB signaling pathways, indicating that Notch 1 signal may be a latent target in treating MM.This study provides a certain experimental basis for research and development of new drugs.