青蒿琥酯对骨髓瘤细胞SP2／0的作用观察及其机制的初步探讨

Effects of artesunate on myeloma cell line SP2/0 and its mechanism

目的观察青蒿琥酯对骨髓瘤细胞SP2／0的增殖抑制及促凋亡作用，并对其机制进行初步探讨。方法利用二苯基溴化四氮唑蓝（MTT）法检测不同浓度青蒿琥酯对SP2／0细胞作用24、48和72h后的增殖抑制作用，并观察其对SP2／0小鼠移植瘤的抑瘤效应。常规Giemsa染色、DAPI荧光染色以及透射电镜下观察细胞凋亡的形态学变化，琼脂糖凝胶电泳观察凋亡细胞的DNA梯带，利用AnnexinV／PI双染和流式细胞术检测青蒿琥酯作用后24、48hSP2／0细胞的凋亡率和细胞周期。Westernblot检测核因子κB（NF—κB）065及其抑制蛋白（IKB）d的水平，酶联免疫吸附（ELISA）法检测NF-κB065的转录活性。结果2μg／ml青蒿琥酯作用24h，对SP2／0细胞增殖抑制率即达33．0％；40μg/ml青蒿琥酯作用72h，细胞增殖抑制率达91.9％，呈现出明显的时间、剂量依赖性。50、100和200mg·kg^-1·d^-1的青蒿琥酯对SP2／0小鼠移植瘤的抑瘤率分别为17.8％、36.5％和48．0％。2μg/ml青蒿琥酯作用24h，G0／G1期细胞数目增多（50．6％±0．8％），凋亡率为7．7％；40μg/ml青蒿琥酯作用48h，G0／G1期细胞达75．3％±7.0％，凋亡率为78.7％，呈时间、剂量依赖性。胞核中NF—κBp65蛋白减少，胞浆中IκBα蛋白的表达增加，NF—κBp65的转录活性降低。结论青蒿琥酯对骨髓瘤细胞SP2／0有显著的增殖抑制和诱导凋亡作用，是一种潜在的抗骨髓瘤药物，其作用机制与抑制NF—κB p65的转录活性有关。

Objective The aim of this study was to explore the effects and the mechanism of Artesunate on mouse myeloma cell line SP2/0. Methods The proliferation of SP2/0 cells was assessed by MTT method after the cells were treated with artesunate at different concentrations for 24 - 72 hours and observe the inhibitory effect of artesunate on the transplanted carcinoma in BALB/C mice. The rate of apoptotic cells and the change of cell cycle was analyzed by flow cytometry and annexin V/PI double staining after artesunate treatment for 24 and 48 hours. The morphological changes of apoptotc cells was observed under microscope with conventional Giemsa staining, DAPI counterstaining and under transmission electron microscope. DNA ladder of apoptosis was checked on regular agarose gel. The expression of NF-κB p65 protein in nucleus and IκBα in cytalplasm was determined by Western blotting. ELISA method was used to measure NF-κB p65 transcription activity. Results Artesunate significantly inhibited the proliferation and induced apoptosis in SP2/0 cells （ from 2 μg/ml to 40 μg/ml） and the effect was dose- and time-dependent. Artesunate inhibited the growth of transplanted tumor in mice （from 50 mg ·kg^- 1 ·d^- 1 to 200 mg·kg^- 1 ·d^- 1 ） in a dose-dependent manner. Artesunate increased the portion of cells in G0/G1 phase and decreased the portion of cells in G2/M or S phase. Artesunate decreased the expression of NF-κB p65 protein in nucleus and increased the expression of IκBα protein in cytalplasm. It inhibited NF-κB p65 transcription activity. Conclusion Artesunate can significantly inhibit the proliferation of SP2/0 cells ,induce apoptosis of SP2/0 cells and its mechanism is related to inhibition of NF-κB p65 transcription activity. It is hopeful to apply artesunate in the treatment of patients with multiple myeloma.