importazole and Multiple-Myeloma

Multiple myeloma (MM) is a plasma cell neoplasm that is currently incurable. The activation of nuclear factor-κB (NF-κB) signalling plays a crucial role in the immortalisation of MM cells. As the most important transcription factor of the canonical NF-κB pathway, the p50/p65 heterodimer requires transportation into the nucleus for its successful signal transduction. Importin β1 is the key transport receptor that mediates p50/p65 nuclear import. Currently, it remains unclear whether the regulation of importin β1 function affects the biological behaviour of MM cells. In the present study, we investigated the changes in p65 translocation and the proliferation and apoptosis of MM cells after treatment with small interfering RNA (siRNA) or an importin β1 inhibitor. The underlying mechanisms were also investigated. We found importin β1 over-expression and the excessive nuclear transport of p65 in myeloma cells. Confocal laser scanning microscopy and Western blot analysis results indicated that p65 nuclear transport was blocked after inhibiting importin β1 expression with siRNA and the importin β1-specific inhibitor importazole (IPZ). Importantly, electronic mobility shift assay results also verified that p65 nuclear transport was dramatically reduced. Moreover, the expression of the NF-κB signalling target genes involved in MM cell apoptosis, such as BCL-2, c-IAP1 and XIAP, were markedly reduced, as demonstrated by the RT-PCR results. Furthermore, the proliferation of MM cells was inhibited, as demonstrated by MTT assay results, and the MM cell apoptosis rate was higher, as demonstrated by the annexin V/propidium iodide (PI) double-staining assay results. Additionally, the percentage of S phase cells in the myeloma cell lines treated with IPZ was dramatically reduced. In conclusion, our results clearly show that importin β1 mediates the translocation of NF-κB into the nuclei of myeloma cells, thereby regulating proliferation and blocking apoptosis, which provides new insights for targeted myeloma therapies.

Topics: Active Transport, Cell Nucleus; Apoptosis; beta Karyopherins; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Humans; Multiple Myeloma; NF-kappa B; Quinazolines; RNA, Small Interfering; Signal Transduction; Transcription Factor RelA

To investigate the effect of nuclear receptor inhibitor importazole (IPZ) on cell cycle and apoptosis of multiple myeloma (MM) cells and its regulatory mechanisms.. MM cell lines RPMI 8226 and NCI-H929 cells were treated with different concentrations of IPZ. Cell viability was detected through MTT method. Cell cycle and apoptosis were measured by flow cytometry (FCM). Nuclear NF-κBprotein expression was tested by Western blot. Electrophoretic mobility shift assay (EMSA) was used to analyze the DNA binding activity.. IPZ induced a dose- and time- dependent inhibition of myeloma cells growth. And the IC50 values of IPZ on RPMI 8226 and NCI-H929 after 48 hours incubation were (4.43±0.41) and (4.78±0.35) μmol/L, respectively, and the percentages of S phase cells decreased from (54.95±4.34)％ and (51.38±2.43)％ to (42.77±3.19)％ and (40.98±6.46)％, respectively. After treatment with IPZ at 8, 12 and 16 μmol/L, the apoptosis rate significantly increased from (2.47±0.60)％ of the control group to (14.53±0.90)%, (32.57±1.80)% and (58.3±1.9)% (P<0.05) in RPMI 8226 and from (2.37±0.70)％ of the control group to (19.46±0.70) %, (46.02±1.10) % and (60.63±1.60)% in NCI-H929, respectively. Treatment of RPMI 8226 and NCI-H929 cells with 8 μmol/L IPZ for 24 h could inhibit NF-κB import to nucleus and reduce its DNA binding activity.. The nuclear receptor inhibitor importazole inhibits proliferation and induces apoptosis of multiple myeloma cells by blocking the NF-κB signal pathway in vitro.. 目的 探讨入核受体抑制剂Importazole（IPZ）对多发性骨髓瘤（MM）细胞周期、凋亡的影响及其可能的作用机制。方法 用不同浓度IPZ处理骨髓瘤细胞系RPMI 8226和NCI-H929细胞，用四甲基偶氮唑盐比色(MTT)法检测细胞活性，用流式细胞术测定细胞周期和细胞凋亡，Western blot法和凝胶迁移实验（EMSA）检测核内NF-κB的蛋白表达和DNA结合活性。结果 IPZ以时间浓度依赖方式抑制RPMI 8226和NCI-H929细胞增殖及诱导凋亡，作用48 h的半数抑制浓度（IC50）分别为（4.43±0.41）、（4.78±0.35）μmol/L。4 μmol/L IPZ分别作用RPMI 8226和NCI-H929细胞48 h，诱导S期细胞分别由（54.95±4.34）％、（51.38±2.43）％减少至（42.77±3.19）％、（40.98±6.46）％。以8、12、16 μmol/L的IPZ分别处理细胞48 h，RPMI 8226细胞凋亡率[（14.53±0.91）％、（32.57±1.80）％、（58.30±1.90）％]较空白对照组[（2.47±0.60）％]明显增加（P<0.05），NCI-H929细胞凋亡率依次为（19.46±0.70）％、（46.02±1.10）％及（60.63±1.60）％，较空白对照组[（2.37±0.70）％]明显增加（P<0.05）。8 μmol/L IPZ作用RPMI 8226和NCI-H929细胞24 h均能明显抑制NF-κB入核进而影响其与DNA的结合活性。结论 IPZ通过阻断NF-κB信号通路抑制多发性骨髓瘤细胞增殖并诱导凋亡。

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Humans; Multiple Myeloma; NF-kappa B; Quinazolines; Signal Transduction