硼替佐米通过阻断自噬溶酶体途径抑制多发性骨髓瘤细胞的增殖及其作用机制  

Mechanism of bortezomib inhibits the proliferation of multiple myeloma cells by blocking autophagosome pathway

作　　者：石小敏 王玮[1] 马铭帅 张荣娟[1] 郝长来[1] 杨新宏[1] 邢恩鸿[1] 王丽红[1] 张志华[1] SHI Xiaomin;WANG Wei;MA Mingshuai;ZHANG Rongjuan;HAO Changlai;YANG Xinhong;XING Enhong;WANG Lihong;ZHANG Zhihua(Department of Hematology,Affiliated Hospital of Chengde Medical College,Chengde 067000,China)

机构地区：[1]承德医学院附属医院血液科,承德067000

出　　处：《生命的化学》2024年第8期1487-1496,共10页Chemistry of Life

基　　金：河北省自然科学基金项目(H2022406041)。

摘　　要：本文探讨了硼替佐米对多发性骨髓瘤细胞系增殖的抑制作用及自噬溶酶体途径在其中可能发挥的作用机制。采用CCK-8法检测了体外培养多发性骨髓瘤细胞株RPMI8226及U266细胞的增殖情况。采用Western blot和RT-PCR法检测自噬相关因子LC3,溶酶体相关因子LAMP1,自噬体溶酶体融合相关因子STX17、SNAP29、VAMP8的蛋白质及RNA表达量变化。采用免疫荧光法检测LC3的荧光强度变化。采用溶酶体探针法观察溶酶体数量变化。采用吖啶橙染色法观察酸性小体的数量。结果发现,硼替佐米对RPMI8226及U266细胞株具有增殖抑制作用,且随时间的延长和浓度的增大其抑制作用更明显(P<0.05);硼替佐米可以使LC3蛋白、RNA及荧光表达增强、LC3-Ⅱ/LC3-Ⅰ蛋白表达水平降低(P<0.05),使LAMP1的蛋白质表达上调(P<0.05)、溶酶体数量增加,使STX17、SNAP29、VAMP8的蛋白质及RNA表达减少(P<0.05)。吖啶橙检测结果显示,硼替佐米抑制的是自噬体溶酶体的融合过程。结果表明,硼替佐米对多发性骨髓瘤细胞具有增殖抑制作用,自噬通路的阻断可能是其发挥抗肿瘤功能的作用机制之一;硼替佐米可以阻断自噬通路引起自噬体及溶酶体的累积增加。这可能是通过其阻断自噬体溶酶体融合实现的。To investigate the inhibitory effect of bortezomib on the proliferation of multiple myeloma cell lines and the possible mechanism of autophagic lysosomal pathway, multiple myeloma cell lines RPMI8226and U266 were cultured in vitro, and cell proliferation was detected by CCK-8 method. Western blot and RTPCR were used to detect the protein and RNA expression levels of autophagy related factors LC3, lysosome related factors LAMP1, and autophagy lysosome fusion related factors STX17, SNAP29 and VAMP8. The fluorescence intensity of LC3 was detected by immunofluorescence method. The quantity of lysosome was observed by lysosome probe. Acridine orange staining was used to observe the number of acidic bodies.Bortezomib had proliferation inhibition on RPMI8226 and U266 cell lines, and for inhibition increased with the increase of time and concentration(P<0.05). Bortezomib enhanced the expression of LC3 protein, RNA and fluorescence, decreased the expression level of LC3-Ⅱ/LC3-Ⅰ protein(P<0.05), increased the protein expression of LAMP1(P<0.05), and increased the number of lysosomes. The protein and RNA expressions of STX17, SNAP29 and VAMP8 were decreased(P<0.05). Acridine orange results showed that bortezomib inhibited the fusion process of autophagosomal lysosomes. Bortezomib can inhibit the proliferation of multiple myeloma cells, and the blocking of autophagy pathway may be one of the mechanisms of its anti-tumor action.Bortezomib can block the autophagy pathway and cause the accumulation of autophagosome and lysosome,which may be caused by blocking the fusion of autophagosome lysosome.

关 键 词：多发性骨髓瘤 自噬 溶酶体 硼替佐米 

分 类 号：R733.3[医药卫生—肿瘤]