Regulatory mechanism of prohibitin in human osteosarcoma MG-63 cells during apoptosis induced by curcumin

doi:10.16098/j.issn.0529-1356.2015.01.013

Abstract

Abstract:

Objective To investigate the expression and distribution of prohibitin in cellular nuclear matrix and the co-localization between prohibitin and the products of apoptosis-related genes in human osteosarcoma MG-63 cell before and after curcumin treatment. Methods Nuclear matrix proteins were selectively extracted and subjected to two-dimensional gel electrophoresis analysis, immunoblotting, and microscopy. Results Two-dimensional polyacrylamide Gel electrophoresis and mass spectrum analysis showed that prohibitin existed in the nuclear matrix protein components of the MG-63 cell and its expression were down-regulated by curcumin. The change of prohibitin was further confirmed by immunoblotting. The immunofluorescence microscopy observation demonstrated that the prohibitin located in the filaments of the nuclear matrix of MG-63 cell and its distribution and expression were altered after curcumin treatment. Laser scanning confocal microscopy revealed that there were co-localization between prohibitin and the products of Bax, Bcl-2, Fas, P53, c-Myc, and Rb genes in MG-63 cell, and the site of co-localization was altered by curcumin treatment. Couclusion Current research identifiys that prohibitin is a nuclear matrix protein existing in the nuclear matrix. The alteration of expression and distribution of prohibitin, and the relationship between prohibitin and multiple apoptosis-related genes may play a pivotal role in the process of human osteosarcoma MG-63 cell apoptosis. These results contribute to the elucidation of the regulatory mechanisms of tumor cellular apoptosis.

Key words: Prohibitin, Nuclear matrix, Human osteosarcoma MG-63 cell, Curcumin

SHI Song-lin ZHAO Zhen-li WANG Guo-hong LIU Fan LU Kun YANG Ling HAN Rong LI Xiao LI Qi-fu*. Regulatory mechanism of prohibitin in human osteosarcoma MG-63 cells during apoptosis induced by curcumin[J]. AAS, 2015, 46(1): 72-80.

References

［1］Nijtmans LG, Artal SM, Grivell LA, et al. The mitochondrial PHB complex: roles in mitochondrial respiratory complex assembly, ageing and degenerative disease ［J］. Cell Mol L ife Sci, 2002, 59 (1) :143-155.
［2］Luan Z, He Y, Alattar M, et al. Targeting the prohibitin scaffold-CRAF kinase interaction in RAS-ERK-driven pancreatic ductal adenocarcinoma［J］. Molec Cancer, 2014, 13: 38.
［3］Chen YW, Chou HC, Lyu PC, et al. Mitochondrial proteomics analysis of tumorigenic and metastatic breast cancer markers ［J］. Funct Integr Genomics, 2011, 11(2): 225-239.
［4］Yang HB, Song W, Chen LY, et al.Differential expression and regulation of prohibitin during curcumin-induced apoptosis of immortalized human epidermal HaCaT cells［J］. Int J Mol Med, 2014,33(3):507-514.
［5］Chen LY, Yang HB, Li QF, et al. Apoptosis of human esophageal carcinoma cell line EC9706 induced by curcumin［J］. Progress in Modern Biomedicine, 2008, 8(9):1601-1604. (in Chinese)
陈兰英, 杨海波, 李祺福, 等. 姜黄素对人食管癌EC9706 细胞凋亡的诱导作用［J］. 现代生物医学进展, 2008, 8(9):1601-1604.
［6］Yang HB,, Li QF, Shi SL,et al. Curcumin induces apoptosis of immortalized human epithelial cell line HaCaT［J］.Acta Anatomica Sinica, 2009, 40(2):64-68. (in Chinese)
杨海波, 李祺福, 石松林, 等. 姜黄素对人永生化上皮细胞HaCaT细胞凋亡的诱导作用［J］. 解剖学报, 2009, 40(2):64-68.
［7］Zhao ZhL, Zheng YB, Chen LY, et al. Effects of curcumin on anti-proliferation and expression of apoptosis interrelated gene of human osteosarcoma MG-63 Cells［J］. Progress in Modern Biomedicine, 2009, 9(12):2201-2205. (in Chinese)
赵振利，郑燕彬，陈兰英，等. 姜黄素对人成骨肉瘤MG-63细胞增殖抑制和凋亡相关基因表达的影响［J］. 现代生物医学进展, 2009, 9(12):2201-2205.
［8］Kolonin MG, Saha PK, Chan L, et al. Reversal of obesity by targeted ablation of adipose tissue［J］. Nat Med, 2004, 10(6): 625-632.
［9］Theiss AL, Sitaraman SV. The role and therapeutic potential of prohibitin in disease ［J］. Biochim Biophys Acta, 2011, 1813(6): 1137-1143.
［10］Pan TL, Wang PW. Explore the molecular mechanism of apoptosis induced by tanshinone IIA on activated rat hepatic stellate cells ［J］. Evid Based Complement Alternat Med, 2012, 2012:734987.
［11］Savulescu D, Feng J, Ping YS, et al. Gonadotropin-releasing hormone-regulated prohibitin mediates apoptosis of the gonadotrope cells ［J］. Mol Endocrinol, 2013, 27(11): 1856-1870.
［12］Chowdhury I, Thompson W E, Welch C, et al. Prohibitin (PHB) inhibits apoptosis in rat granulosa cells (GCs) through the extracellular signal-regulated kinase 1/2 (ERK1/2) and the Bcl family of proteins ［J］. Apoptosis, 2013, 18(12): 1513-1525.
［13］Merkwirth C, Dargazanli S, Tatsuta T, et al. Prohibitins control cell proliferation and apoptosis by regulating OPA1-dependent cristae morphogenesis in mitochondria ［J］. Genes Dev, 2008, 22(4): 476-488.
［14］Artal-Sanz M, Tsang WY, Willems EM, et al. The mitochondrial prohibitin complex is essential for embryonic viability and germline function in Caenorhabditis elegans［J］. J Biol Chem, 2003, 278(34):32091-32099.
［15］Rastogi S, Joshi B, Fusaro G, et al. Camptothecin induces nuclear export of prohibitin preferentially in transformed cells through a CRM-1-dependent mechanism ［J］. J Biol Chem, 2006, 281(5): 2951-2959.
［16］Ding J, Mooers BHM, Zhang Z, et al. After embedding in membranes antiapoptotic Bcl-XL protein binds both Bcl-2 homology region 3 and helix 1 of proapoptotic Bax protein to inhibit apoptotic mitochondrial permeabilization［J］. J Biol Chem, 2014, 289(17): 11873-11896.
［17］Walensky LD, Gavathiotis E. BAX unleashed: the biochemical transformation of an inactive cytosolic monomer into a toxic mitochondrial pore ［J］. Trends Biochemical Sci, 2011, 36(12): 642-652.
［18］Upreti M, Chu R, Galitovskaya E, et al. Key role for Bak activation and Bak-Bax interaction in the apoptotic response to vinblastine［J］. Mol Cancer Ther, 2008, 7(7): 2224-2232.
［19］Kirsch DG, Doseff A, Chau BN, et al. Caspase-3-dependent cleavage of Bcl-2 promotes release of cytochrome c［J］. J Biol Chem, 1999, 274(30):21155-21161.
［20］Kathiria AS, Neumann WL, Rhees J, et al. Prohibitin attenuates colitis-associated tumorigenesis in mice by modulating p53 and STAT3 apoptotic responses ［J］. Cancer Res, 2012, 72(22): 5778-5789.
［21］Qiu QC, Hu B, He XP, et al. STGC3 inhibits xenograft tumor growth of nasopharyngeal carcinoma cells by altering the expression of proteins associated with apoptosis ［J］. Genetics Mol Biol, 2012, 35(1): 18-26.
［22］Fridman JS, Lowe SW. Control of apoptosis by p53 ［J］. Oncogene, 2003, 22(56): 9030-9040.
［23］Fusaro G, Dasgupta P, Rastogi S, et al. Prohibitin induces the transcriptional activity of p53 and is exported from the nucleus upon apoptotic signaling ［J］. J Biol Chem, 2003, 278(48): 47853-47861.
［24］Wahlstr?m T, Arsenian Henriksson M. Impact of MYC in regulation of tumor cell metabolism［J］. Biochim Biophys Acta, 2014 Jul 17. pii: S1874-9399(14)00192-00198. 
［25］Kummetha Venkata J, An N, Stuart R, et al. Inhibition of sphingosine kinase 2 down-regulates the expression of c-Myc and Mcl-1 and induces apoptosis in multiple myeloma［J］. Blood, 2014 Aug 13. pii: blood-2014-03-559385.
［26］Cao X, Bennett RL, May WS. c-Myc and caspase-2 are involved in activating Bax during cytotoxic drug-induced apoptosis ［J］. J Biol Chem, 2008, 283(21): 14490-14496.
［27］Wang S, Fusaro G, Padmanabhan J, et al. Prohibitin co-localizes with Rb in the nucleus and recruits N-CoR and HDAC1 for transcriptional repression ［J］. Oncogene, 2002, 21(55): 8388-8396.
［28］Fusaro G, Sheng W, Chellappan S. Differential regulation of Rb family proteins and prohibitin during camptothecin-induced apoptosis ［J］. Oncogene, 2002, 21(29): 4539-4548.

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[5]	. Changes in expression and localization of heat shock protein 70 during curcumin-induced apoptisis of human esophageal cancer cell line EC9706 [J]. , 2012, 43(4): 490-499.
[6]	. Influence of laminin in cell growth inhibition of curcumin on human hepatocellular carcinoma HepG-2 cells [J]. , 2011, 42(1): 38-44.
[7]	. Curcumin induces apoptosis of immortalized human epithelial cell line HaCaT [J]. , 2009, 40(2): 223-227.
[8]	. Effects of the combination of ginsenoside Rg1,cinnamic acid and tanshinone IIA on the morphology and terminal differentiation of MG-63 cells EM>in vitro/EM> [J]. , 2009, 40(1): 13-19.
[9]	. THE EFFECTS OF COMBINATION OF CHINESE MEDICINE EFFECTIVE CONSTITUENTS ON CONFIGURATION AND COMPONENTS OF NUCLEAR MATRIX OF THE OSTEOSARCOMA MG-63 CELLS [J]. , 2008, 39(5): 670-676.