苦瓜蛋白MAP30通过AKT/mTOR通路促进多发性骨髓瘤细胞的自噬和凋亡
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摘要
目的:探讨苦瓜蛋白MAP30在多发性骨髓瘤中的作用及其机制。方法:将人骨髓瘤RPMI-8226、NCI-H929和U266细胞为靶细胞,以不同浓度(1～10μmol/L)的MAP30处理后,采用CCK-8法检测多发骨髓瘤细胞株RPMI-8226、NCI-H929和U266的增殖能力,Annexin V/PI流式细胞术检测骨髓瘤细胞的凋亡情况,Wb实验检测骨髓瘤细胞凋亡相关蛋白(PARP)、自噬相关蛋白(LC3II、P62)及AKT/mTOR通路相关蛋白的表达水平,CCK-8法、流式细胞术和Wb实验检测MAP30联合自噬激动剂雷帕霉素(Rap)或自噬抑制剂巴弗洛霉素(Baf)作用后骨髓瘤细胞增殖、凋亡和自噬的变化。结果:MAP30(1～10μmol/L)抑制骨髓瘤细胞的增殖,且其对增殖的抑制呈时间和剂量依赖关系(P<0.05或P<0.01)。MAP30单独作用骨髓瘤细胞后,细胞凋亡和细胞自噬增加,PARP裂解增多,LC3II表达水平增加,P62表达明显下降(均P<0.05或P<0.01);MAP30+Rap作用骨髓瘤细胞后,较单用MAP30细胞增殖率增加、细胞凋亡率减少、细胞自噬减少、PARP裂解降低、LC3II表达降低及P62表达增加(均P<0.05或P<0.01);相反,MAP30+Baf作用骨髓瘤细胞较单用MAP30,细胞增殖率减少、细胞凋亡率增加、细胞自噬增加、PARP裂解增加、LC3II表达增加、P62表达降低(均P<0.05或P<0.01);MAP30作用骨髓瘤细胞后,AKT/mTOR通路相关蛋白p-AKT和p-mTOR表达水平明显降低(均P<0.05)。结论:MAP30可通过AKT/mTOR通路促进骨髓瘤细胞的凋亡和自噬,可能为骨髓瘤的治疗提供新的治疗策略。
Objective: To investigate the role of momordica protein MAP30 in multiple myeloma(MM) and the possible mechanism.Methods: Human myeloma RPMI-8226, NCI-H929 and U266 cells were treated with MAP30 at different concentration(1-10μmol/L)and then the proliferation rates of cells were detected by CCK-8 assay. Annexin V/PI flow cytometry was used to evaluate the apoptosis rate of myeloma cells, and the expressions of apoptosis-related protein(PARP), autophagy-related proteins(LC3 II, P62) and Akt/mTOR pathway-related proteins in multiple myeloma cells were also detected via Wb. The changes in cell proliferation, apoptosis and autophagy after the treatment of MAP30 combined with autophagy agonist rapamycin(Rap) or autophagy inhibitor bafilomycin(Baf) were observed by CCK-8, flow cytometry and Wb, respectively. Results: MAP30(1-10μmol/L) inhibited the proliferation of myeloma cells in a time-and dose-dependent manner(P<0.05 or P<0.01). With MAP30 acting on myeloma cells alone, the apoptosis and autophagy of MM cells, as well as the expression of PARP cleavage and LC3 II increased while the expression of P62 decreased significantly(all P<0.05 or P<0.01). After being treated with MAP30+Baf, compared with MAP30 treatment alone, the cell proliferation was remarkably enhanced while cell apoptosis and cell autophagy were suppressed, besides, the expression of PARP cleavage and LC3 II were decreased and P62 level was augmented(all P<0.05 or P<0.01). Conversely, after being treated with MAP30+Baf, compared with MAP30 treatment or Baf treatment alone, cell proliferation and P62 level were reduced, while apoptosis and autophagy as well as the expressions of PARP cleavage and LC3II level were increased(all P<0.05 or P<0.01). The expressions of p-AKT and p-mTOR were significantly reduced with the effect of MAP30 on myeloma cells(all P<0.05). Conclusion: MAP30 can promote the apoptosis and autophagy of myeloma cells through AKT/mTOR pathway, which may provide a new therapeutic strategy for treatment of multiple myeloma.
Objective: To investigate the role of momordica protein MAP30 in multiple myeloma(MM) and the possible mechanism.Methods: Human myeloma RPMI-8226, NCI-H929 and U266 cells were treated with MAP30 at different concentration(1-10μmol/L)and then the proliferation rates of cells were detected by CCK-8 assay. Annexin V/PI flow cytometry was used to evaluate the apoptosis rate of myeloma cells, and the expressions of apoptosis-related protein(PARP), autophagy-related proteins(LC3 II, P62) and Akt/mTOR pathway-related proteins in multiple myeloma cells were also detected via Wb. The changes in cell proliferation, apoptosis and autophagy after the treatment of MAP30 combined with autophagy agonist rapamycin(Rap) or autophagy inhibitor bafilomycin(Baf) were observed by CCK-8, flow cytometry and Wb, respectively. Results: MAP30(1-10μmol/L) inhibited the proliferation of myeloma cells in a time-and dose-dependent manner(P<0.05 or P<0.01). With MAP30 acting on myeloma cells alone, the apoptosis and autophagy of MM cells, as well as the expression of PARP cleavage and LC3 II increased while the expression of P62 decreased significantly(all P<0.05 or P<0.01). After being treated with MAP30+Baf, compared with MAP30 treatment alone, the cell proliferation was remarkably enhanced while cell apoptosis and cell autophagy were suppressed, besides, the expression of PARP cleavage and LC3 II were decreased and P62 level was augmented(all P<0.05 or P<0.01). Conversely, after being treated with MAP30+Baf, compared with MAP30 treatment or Baf treatment alone, cell proliferation and P62 level were reduced, while apoptosis and autophagy as well as the expressions of PARP cleavage and LC3II level were increased(all P<0.05 or P<0.01). The expressions of p-AKT and p-mTOR were significantly reduced with the effect of MAP30 on myeloma cells(all P<0.05). Conclusion: MAP30 can promote the apoptosis and autophagy of myeloma cells through AKT/mTOR pathway, which may provide a new therapeutic strategy for treatment of multiple myeloma.
引文
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[3] COOPER D L. Treatment of relapsed multiple myeloma[J]. N Engl J Med, 2015, 372(18):1774-1777. DOI:10.1056/NEJMc1501348.
[4] ZENG W, WANG X, XU P, et al. Molecular imaging of apoptosis:from micro to macro[J]. Theranostics, 2015, 5(6):559-562. DOI:10.7150/thno.11548.
[5] BOYA P, REGGIORI F, CODOGNO P. Emerging regulation and functions of autophagy[J]. Nat Cell Biol, 2013, 15(7):713-720.DOI:10.1038/ncb2788.
[6] DORIA A, GATTO M, PUNZI L. Autophagy in human health and disease[J]. N Engl J Med, 2013, 368(19):1845-1851. DOI:10.1056/NEJMc1303158.
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[8] BOURINBAIAR A S, LEE-HUANG S. The activity of plant-derived antiretroviral proteins MAP30 and GAP31 against herpes simplex virus in vitro[J]. Biochem Biophys Res Commun, 1996, 219(3):923-929. DOI:10.4161/auto.7.3.14072.
[9] WANG Y X, JACOB J, WINGFIELD P T, et al. Anti-HIV and antitumor protein MAP30, a 30 KD single-strand type-I RIP, shares similar secondary structure and beta-sheet topology with the a chain of ricin, a type-II RIP[J]. Protein SC, 2000, 9(1):138-144. DOI:10.1110/ps.9.1.138.
[10] FANG E F, ZHANG C Z, WONG J H, et al. The MAP30 protein from bitter gourd(Momordica charantia)seeds promotes apoptosis in liver cancer cells in vitro and in vivo[J]. Cancer lett, 2012, 324(1):66-74. DOI:10.1016/j.canlet.2012.05.005.
[11] HLIN H, ZHI G Z, CONG H H, et al. Expression of momordica charantia MAP30 and its antitumor effect on bladder cancer cells[J]. Minerva Urol Nefrol, 2016, 68(3):275-281.
[12] QIAN S, SUN L, LI J, et al. MAP30 inhibits autophagy through enhancing acetyltransferase P300 and induces apoptosis in acute myeloid leukemia cells[J]. Oncol Rep, 2016, 35(6):3705-3713. DOI:10.3892/or.2016.4705.
[13] FAN G, YU J, ASARE P F, et al. Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling[J]. J Cell Mol Med, 2016, 20(10):1908-1919. DOI:10.1111/jcmm.12883.
[14] LI J, ZHOU J, ZHANG D, et al. Bone marrow-derived mesenchymal stem cells enhance autophagy via PI3K/AKT signalling to reduce the severity of ischaemia/reperfusion-induced lung injury[J]. J Cell Mol Med, 2015, 19(10):2341-2351. DOI:10.1111/jcmm.12638.
[15] PALUMBO A, ANDERSON K. Multiple myeloma[J]. N Engl J Med, 2011, 364(11):1046-1060. DOI:10.1056/NEJMra1011442.
[16] LEVINE B, KROEMER G. Autophagy in the pathogenesis of disease[J]. Cell, 2008, 132(1):27-42. DOI:10.1016/j.cell.2007.12.018.
[17] WHITE E. Deconvoluting the context-dependent role for autophagy in cancer[J]. Nat Rev Cancer, 2012, 12(6):401-410. DOI:10.1038/nrc3262.
[18] ARONSON L I, DAVIES F E. DangER:protein ovERload. Targeting protein degradation to treat myeloma[J]. Haematologica, 2012,97(8):1119-1130. DOI:10.3324/haematol.2012.064923.
[19] ELGENDY M, SHERIDAN C, BRUMATTI G, et al. Oncogenic Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death and limits clonogenic survival[J]. Mol Cell, 2011, 42(1):23-35. DOI:10.1016/j.molcel.2011.02.009.
[20] PATTINGRE S, BAUVY C, CARPENTIER S, et al. Role of JNK1-dependent Bcl-2 phosphorylation in ceramide-induced macroautophagy[J]. J Biol Chem, 2009, 284(5):2719-2728. DOI:10.1074/jbc.M805920200.
[21]赵家义,韩一平. PI3K-AKT-mTOR信号通路抑制剂与肿瘤免疫治疗[J].中国肿瘤生物治疗杂志, 2017, 24(12):1424-1430. DOI:10. 3872/j.issn.1007-385X.2017.12.015.
[22] CAO B, LI J, ZHOU X, et al. Clioquinol induces pro-death autophagy in leukemia and myeloma cells by disrupting the mTOR signaling pathway[J/OL]. Sci Rep, 2014, 4:5749[2019-01-11]. https://www.nature.com/articles/srep05749.DOI:10.1038/srep05749.
[2] KYLE R A, RAJKUMAR S V. Multiple myeloma[J]. N Engl J Med,2004, 351(18):1860-1873. DOI:10.1056/NEJMra041875.
[3] COOPER D L. Treatment of relapsed multiple myeloma[J]. N Engl J Med, 2015, 372(18):1774-1777. DOI:10.1056/NEJMc1501348.
[4] ZENG W, WANG X, XU P, et al. Molecular imaging of apoptosis:from micro to macro[J]. Theranostics, 2015, 5(6):559-562. DOI:10.7150/thno.11548.
[5] BOYA P, REGGIORI F, CODOGNO P. Emerging regulation and functions of autophagy[J]. Nat Cell Biol, 2013, 15(7):713-720.DOI:10.1038/ncb2788.
[6] DORIA A, GATTO M, PUNZI L. Autophagy in human health and disease[J]. N Engl J Med, 2013, 368(19):1845-1851. DOI:10.1056/NEJMc1303158.
[7] JOHANSEN T, LAMARK T. Selective autophagy mediated by autophagic adapter proteins[J]. Autophagy, 2011, 7(3):279-296. DOI:10.4161/auto.7.3.14072.
[8] BOURINBAIAR A S, LEE-HUANG S. The activity of plant-derived antiretroviral proteins MAP30 and GAP31 against herpes simplex virus in vitro[J]. Biochem Biophys Res Commun, 1996, 219(3):923-929. DOI:10.4161/auto.7.3.14072.
[9] WANG Y X, JACOB J, WINGFIELD P T, et al. Anti-HIV and antitumor protein MAP30, a 30 KD single-strand type-I RIP, shares similar secondary structure and beta-sheet topology with the a chain of ricin, a type-II RIP[J]. Protein SC, 2000, 9(1):138-144. DOI:10.1110/ps.9.1.138.
[10] FANG E F, ZHANG C Z, WONG J H, et al. The MAP30 protein from bitter gourd(Momordica charantia)seeds promotes apoptosis in liver cancer cells in vitro and in vivo[J]. Cancer lett, 2012, 324(1):66-74. DOI:10.1016/j.canlet.2012.05.005.
[11] HLIN H, ZHI G Z, CONG H H, et al. Expression of momordica charantia MAP30 and its antitumor effect on bladder cancer cells[J]. Minerva Urol Nefrol, 2016, 68(3):275-281.
[12] QIAN S, SUN L, LI J, et al. MAP30 inhibits autophagy through enhancing acetyltransferase P300 and induces apoptosis in acute myeloid leukemia cells[J]. Oncol Rep, 2016, 35(6):3705-3713. DOI:10.3892/or.2016.4705.
[13] FAN G, YU J, ASARE P F, et al. Danshensu alleviates cardiac ischaemia/reperfusion injury by inhibiting autophagy and apoptosis via activation of mTOR signalling[J]. J Cell Mol Med, 2016, 20(10):1908-1919. DOI:10.1111/jcmm.12883.
[14] LI J, ZHOU J, ZHANG D, et al. Bone marrow-derived mesenchymal stem cells enhance autophagy via PI3K/AKT signalling to reduce the severity of ischaemia/reperfusion-induced lung injury[J]. J Cell Mol Med, 2015, 19(10):2341-2351. DOI:10.1111/jcmm.12638.
[15] PALUMBO A, ANDERSON K. Multiple myeloma[J]. N Engl J Med, 2011, 364(11):1046-1060. DOI:10.1056/NEJMra1011442.
[16] LEVINE B, KROEMER G. Autophagy in the pathogenesis of disease[J]. Cell, 2008, 132(1):27-42. DOI:10.1016/j.cell.2007.12.018.
[17] WHITE E. Deconvoluting the context-dependent role for autophagy in cancer[J]. Nat Rev Cancer, 2012, 12(6):401-410. DOI:10.1038/nrc3262.
[18] ARONSON L I, DAVIES F E. DangER:protein ovERload. Targeting protein degradation to treat myeloma[J]. Haematologica, 2012,97(8):1119-1130. DOI:10.3324/haematol.2012.064923.
[19] ELGENDY M, SHERIDAN C, BRUMATTI G, et al. Oncogenic Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death and limits clonogenic survival[J]. Mol Cell, 2011, 42(1):23-35. DOI:10.1016/j.molcel.2011.02.009.
[20] PATTINGRE S, BAUVY C, CARPENTIER S, et al. Role of JNK1-dependent Bcl-2 phosphorylation in ceramide-induced macroautophagy[J]. J Biol Chem, 2009, 284(5):2719-2728. DOI:10.1074/jbc.M805920200.
[21]赵家义,韩一平. PI3K-AKT-mTOR信号通路抑制剂与肿瘤免疫治疗[J].中国肿瘤生物治疗杂志, 2017, 24(12):1424-1430. DOI:10. 3872/j.issn.1007-385X.2017.12.015.
[22] CAO B, LI J, ZHOU X, et al. Clioquinol induces pro-death autophagy in leukemia and myeloma cells by disrupting the mTOR signaling pathway[J/OL]. Sci Rep, 2014, 4:5749[2019-01-11]. https://www.nature.com/articles/srep05749.DOI:10.1038/srep05749.