6aR,12aR,12a-羟基紫穗槐苷元上调Bim诱导肝癌细胞SMMC-7721发生凋亡的机制
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摘要
肝细胞癌(HCC)是一种高度恶性的肿瘤。化疗是临床上一种重要的治疗方法,但由于化疗药物的毒副作用和耐药效应,现有的化疗药物或多或少有一定的局限性。因此,寻找毒副作用小且有效的化疗药物一直是肝癌患者的迫切需求。本研究从紫穗槐种子中分离纯化出一种天然产物6aR,12aR,12a-羟基紫穗槐苷元,采用CCK-8法检测了其对人肝癌SMMC-7721细胞的杀伤作用,应用流式细胞术检测了其对肝癌细胞的凋亡诱导情况,通过免疫印迹法检测了6aR,12aR,12a-羟基紫穗槐苷元上调Bim蛋白表达进而诱导肝癌细胞凋亡的作用机制。结果表明,6aR,12aR,12a-羟基紫穗槐苷元可呈浓度依赖性和时间依赖性抑制人肝癌SMMC-7721细胞的活力,24 h、48 h和72 h的半数抑制浓度(IC50)分别为21.77μmol/L、5.65μmol/L、5.0μmol/L;同时可浓度依赖性地提高细胞凋亡率;可通过下调抑凋亡蛋白Survivin、Mcl-1、Bcl-XL、Bcl-2,上调促凋亡蛋白Bak、Bim、Bax、Bid的表达,进而促进PARP和caspase-3的活化,从而诱导细胞发生凋亡;进一步地研究发现:6aR,12aR,12a-羟基紫穗槐苷元通过延长Bim的半衰期而增加Bim的积累,且具有和MG132类似的生物学功能,能够阻断蛋白的降解途径来抑制Bim降解。上述研究表明,6aR,12aR,12a-羟基紫穗槐苷元可通过抑制蛋白降解从而上调细胞中Bim蛋白的水平,进而诱导肝癌细胞SMMC-7721发生凋亡。
Hepatocellular carcinoma(HCC) is a highly malignant tumor. Chemotherapy is an important treatment in the treatment of HCC patients, however because their side effects and drug resistance, there are more or less limitation for the present clinical drugs. Therefore, the search for new chemotherapy drugs is still the requirement of patients with liver cancer. In this study, we focused on investigating the mechanisms of natural compound named 6 aR,12 aR,12 a-dalbinol on apoptosis of human HCC cells SMMC-7721. Specifically, the viability of SMMC-7721 cells was evaluated by CCK-8 assay, apoptosis was analyzed by flow cytometry, the expressions of apoptotic related proteins were measured by Western blotting. The results showed that 6 aR, 12 aR, 12 a-dalbinol not only repressed the growth of SMMC-7721 in dose-dependent and time-dependently with IC5021.77 μmol/L,5.65 μmol/L and 5.0 μmol/L at 24 h, 48 h and 72 h, respectively, but also induced cell apoptosis, decreased anti-apoptotic protein levels such as Survivin, Mcl-1, Bcl-XL and Bcl-2 and increased pro-apoptotic protein levels such as Bak, Bim, Bax and Bid. Further, it enhanced the accumulation of Bim by extending the half-life of Bim and blocking proteasome degradation pathway. To summarize, 6 aR,12 aR,12 a-dalbinol increased Bim level by blocking proteasome degradation pathway, thus induced cell apoptosis in HCC SMMC-7721 cells.
Hepatocellular carcinoma(HCC) is a highly malignant tumor. Chemotherapy is an important treatment in the treatment of HCC patients, however because their side effects and drug resistance, there are more or less limitation for the present clinical drugs. Therefore, the search for new chemotherapy drugs is still the requirement of patients with liver cancer. In this study, we focused on investigating the mechanisms of natural compound named 6 aR,12 aR,12 a-dalbinol on apoptosis of human HCC cells SMMC-7721. Specifically, the viability of SMMC-7721 cells was evaluated by CCK-8 assay, apoptosis was analyzed by flow cytometry, the expressions of apoptotic related proteins were measured by Western blotting. The results showed that 6 aR, 12 aR, 12 a-dalbinol not only repressed the growth of SMMC-7721 in dose-dependent and time-dependently with IC5021.77 μmol/L,5.65 μmol/L and 5.0 μmol/L at 24 h, 48 h and 72 h, respectively, but also induced cell apoptosis, decreased anti-apoptotic protein levels such as Survivin, Mcl-1, Bcl-XL and Bcl-2 and increased pro-apoptotic protein levels such as Bak, Bim, Bax and Bid. Further, it enhanced the accumulation of Bim by extending the half-life of Bim and blocking proteasome degradation pathway. To summarize, 6 aR,12 aR,12 a-dalbinol increased Bim level by blocking proteasome degradation pathway, thus induced cell apoptosis in HCC SMMC-7721 cells.
引文
A hmed-Belkacem A.,Macalou S.,Borrelli F.,Capasso R.,Fattorusso E.,Taglialatela-Scafati O.,and Di Pietro A.,2007,Nonprenylated rotenoids,a new class of potent breast cancer resistance protein inhibitors,Journal of Medicinal Chemistry,50(8):1933-1938
Biswas S.C.,and Greene L.A.,2002,Nerve growth factor(NGF)down-regulates the Bcl-2 homology 3(BH3)domain-only protein Bim and suppresses its proapoptotic activity by phosphorylation,The Journal of Biological Chemistry,277(51):49511-49516
Chaveli-López B.,2014,Oral toxicity produced by chemotherapy:A systematic review,J.Clin.Exp.Dent.,6(1):e81-e90
Chen W.,Zheng R.,Baade P.D.,Zhang S.,Zeng H.,Bray F.,Jemal A.,Yu X.Q.,and He J.,2016,Cancer statistics in China,2015,CA Cancer J.Clin.,66(2):115-132
de Lope C.R.,Tremosini S.,Forner A.,Reig M.,and Bruix J.,2012,Management of HCC,Journal of Hepatology,56(1):S75-S87
Delbridge A.R.,Grabow S.,Strasser A.,and Vaux D.L.,2016,Thirty years of BCL-2:translating cell death discoveries into novel cancer therapies,Nat.Rev.Cancer,16(2):99-109
Essafi M.,Baudot A.D.,Mouska X.,Cassuto J.P.,Ticchioni M.,and Deckert M.,2011,Cell-penetrating TAT-FOXO3 fusion proteins induce apoptotic cell death in leukemic cells,Mol.Cancer Ther.,10(1):37-46
Forner A.,Llovet J.M.,and Bruix J.,2012,Hepatocellular carcinoma,Lancet,379(9822):1245-1255
Hail N.J.,and Lotan R.,2004,Apoptosis induction by the natural product cancer chemopreventive agent deguelin is mediated through the inhibition of mitochondrial bioenergetics,Apoptosis,9(4):437-447
Ki m J.K.,Jung K.H.,Noh J.H.,Eun J.W.,Bae H.J.,Xie H.J.,Ahn Y.M.,Ryu J.C.,Park W.S.,Lee J.Y.,and Nam S.W.,2009,Targeted disruption of S100P suppresses tumor cell growth by down-regulation of cyclin D1 and CDK2in human hepatocellular carcinoma,Int.J.Oncol.,35(6):1257-1264
Ley R.,Ewings K.E.,Hadfield K.,and Cook S.J.,2005,Regulatory phosphorylation of Bim:sorting out the ERK from the JNK,Cell Death and Differentiation,12(8):1008-1014
Mehta R.,Katta H.,Alimirah F.,Patel R.,Murillo G.,Peng X.,Muzzio M.,and Mehta R.G.,2013,Deguelin action involves c-Met and EGFR signaling pathways in triple negative breast cancer cells,PLo S One,8(6):e65113
Moriguchi M.,Umemura A.,and Itoh Y.,2016,Current status and future prospects of chemotherapy for advanced hepatocellular carcinoma,Clin.J.Gastroenterol.,9(4):184-190
Parkin D.M.,Bray F.,Ferlay J.,and Pisani P.,2005,Global cancer statistics,2002,CA Cancer J.Clin.,55(2):74-108
Puthalakath H.,Huang D.C.S.,O'reilly L.A.,King S.M.,and Strasser A.,1999,The proapoptotic activity of the Bcl-2family member Bim is regulated by in teraction with the dynein motor complex,Molecular Cell,3(3):287-296
Shamas-Din A.,Brahmbhatt H.,Leber B.,and Andrew D.W.,2011,BH3-only proteins:orchestrators of apoptosis,Biochim.Biophys.Acta,1813(4):508-520
Suh Y.A.,Kim J.H.,Sung M.A.,Boo H.J.,Yun H.J.,Lee S.H.,Lee H.J.,Suh Y.G.,Kim K.W,and Lee H.Y.,2013,A novel antitumor activity of deguelin targeting the insulin-like growth factor(IGF)receptor pathway via up-regulation of IGF-binding protein-3 expression in breast cancer,Cancer Lett.,332(1):102-109
Trojan J.,Zangos S.,and Schnitzbauer A.A.,2016,Diagnostics and treatment of hepatocellular carcinoma in 2016:standards and developments,Visc.Med.,32(2):116-120
Vervliet T.,Parys J.B.,and Bultynck G.,2016,Bcl-2 proteins and calcium signaling:complexity beneath the surface,Oncogene,35(39):5079-5092
Yang Y.L.,Ji C.,Bi Z.G.,Lu C.C.,Wang R.,Gu B.,and Cheng L.,2013,Deguelin induces both apoptosis and autophagy in cultured head and neck squamous cell carcinoma cells,PLo S One,8(1):e54736
Biswas S.C.,and Greene L.A.,2002,Nerve growth factor(NGF)down-regulates the Bcl-2 homology 3(BH3)domain-only protein Bim and suppresses its proapoptotic activity by phosphorylation,The Journal of Biological Chemistry,277(51):49511-49516
Chaveli-López B.,2014,Oral toxicity produced by chemotherapy:A systematic review,J.Clin.Exp.Dent.,6(1):e81-e90
Chen W.,Zheng R.,Baade P.D.,Zhang S.,Zeng H.,Bray F.,Jemal A.,Yu X.Q.,and He J.,2016,Cancer statistics in China,2015,CA Cancer J.Clin.,66(2):115-132
de Lope C.R.,Tremosini S.,Forner A.,Reig M.,and Bruix J.,2012,Management of HCC,Journal of Hepatology,56(1):S75-S87
Delbridge A.R.,Grabow S.,Strasser A.,and Vaux D.L.,2016,Thirty years of BCL-2:translating cell death discoveries into novel cancer therapies,Nat.Rev.Cancer,16(2):99-109
Essafi M.,Baudot A.D.,Mouska X.,Cassuto J.P.,Ticchioni M.,and Deckert M.,2011,Cell-penetrating TAT-FOXO3 fusion proteins induce apoptotic cell death in leukemic cells,Mol.Cancer Ther.,10(1):37-46
Forner A.,Llovet J.M.,and Bruix J.,2012,Hepatocellular carcinoma,Lancet,379(9822):1245-1255
Hail N.J.,and Lotan R.,2004,Apoptosis induction by the natural product cancer chemopreventive agent deguelin is mediated through the inhibition of mitochondrial bioenergetics,Apoptosis,9(4):437-447
Ki m J.K.,Jung K.H.,Noh J.H.,Eun J.W.,Bae H.J.,Xie H.J.,Ahn Y.M.,Ryu J.C.,Park W.S.,Lee J.Y.,and Nam S.W.,2009,Targeted disruption of S100P suppresses tumor cell growth by down-regulation of cyclin D1 and CDK2in human hepatocellular carcinoma,Int.J.Oncol.,35(6):1257-1264
Ley R.,Ewings K.E.,Hadfield K.,and Cook S.J.,2005,Regulatory phosphorylation of Bim:sorting out the ERK from the JNK,Cell Death and Differentiation,12(8):1008-1014
Mehta R.,Katta H.,Alimirah F.,Patel R.,Murillo G.,Peng X.,Muzzio M.,and Mehta R.G.,2013,Deguelin action involves c-Met and EGFR signaling pathways in triple negative breast cancer cells,PLo S One,8(6):e65113
Moriguchi M.,Umemura A.,and Itoh Y.,2016,Current status and future prospects of chemotherapy for advanced hepatocellular carcinoma,Clin.J.Gastroenterol.,9(4):184-190
Parkin D.M.,Bray F.,Ferlay J.,and Pisani P.,2005,Global cancer statistics,2002,CA Cancer J.Clin.,55(2):74-108
Puthalakath H.,Huang D.C.S.,O'reilly L.A.,King S.M.,and Strasser A.,1999,The proapoptotic activity of the Bcl-2family member Bim is regulated by in teraction with the dynein motor complex,Molecular Cell,3(3):287-296
Shamas-Din A.,Brahmbhatt H.,Leber B.,and Andrew D.W.,2011,BH3-only proteins:orchestrators of apoptosis,Biochim.Biophys.Acta,1813(4):508-520
Suh Y.A.,Kim J.H.,Sung M.A.,Boo H.J.,Yun H.J.,Lee S.H.,Lee H.J.,Suh Y.G.,Kim K.W,and Lee H.Y.,2013,A novel antitumor activity of deguelin targeting the insulin-like growth factor(IGF)receptor pathway via up-regulation of IGF-binding protein-3 expression in breast cancer,Cancer Lett.,332(1):102-109
Trojan J.,Zangos S.,and Schnitzbauer A.A.,2016,Diagnostics and treatment of hepatocellular carcinoma in 2016:standards and developments,Visc.Med.,32(2):116-120
Vervliet T.,Parys J.B.,and Bultynck G.,2016,Bcl-2 proteins and calcium signaling:complexity beneath the surface,Oncogene,35(39):5079-5092
Yang Y.L.,Ji C.,Bi Z.G.,Lu C.C.,Wang R.,Gu B.,and Cheng L.,2013,Deguelin induces both apoptosis and autophagy in cultured head and neck squamous cell carcinoma cells,PLo S One,8(1):e54736
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