Chinese Journal of Biochemistry and Molecular Biology

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1 ISSN CN ΠQ Chinese Journal of Biochemistry and Molecular Biology (2) : Rg1 MG263 Prohibitin 1), 1) 3, 1), 2), 1), 1) ( 1),, ; 2), ) Rg1 (RCT) MG263, prohibitin RCT MG263.,prohibitin MG263, RCT ; prohibitin MG263 RCT ; prohibitin, RCT ; prohibitin c2fos c2myc p53 Rb, RCT. prohibitin, RCT,. RCT prohibitin MG263, RCT. prohibitin ; ; MG263 ; ; Rg1 Q245 ; Q254 ; Q279 Differentiation Induced by Ginsenoside Rg1 Combined Formula Involved the Alteration of Prohibitin Localization and Expression in Human Osteosarcoma M G263 Cells SHI Song2Lin 1), LI Qi2Fu 1) 3, LIU Qing2Rong 1), XU Dong2Hui 2), TANGJian 1), LIANG Ying 1) ( 1) Key Laboratory, Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Xiamen , China ; 2) Department of Hepar Biliary Pancreas Vascular Surgery, Affiliated Xiamen First Hospital, Fujian Medical University, Xiamen , China) Abstract Prohibitin is a nuclear matrix protein and associates with various oncogene products. A combined formula with ginsenoside Rg1, cinnamic acid and tanshinone A (RCT) was used to treat human osteosarcoma MG263 cells in this study. We found that the prohibitin expression in the nuclear matrix was of decreased following RCT treatment by Western blot. The colocalization of prohibitin with the products of oncogenes or tumor suppressor genes, including c2fos, c2myc, p53 and Rb was evaluated by immunofluorescence staining and laser scanning confocal microscopy. We observed significant alternations of prohibiitin distribution and the degree of colocalization with the associated gene products after the cells exposed to RCT. The results suggested that prohibitin might be involved in the RCT2induced differentiation in MG263 cells. Our findings may provide : , : (No ) 3 Tel : ,E2mail : edu. cn Received : August 20, 2007 ;Accepted : November 6, 2007 Supported by National Natural Science Foundation of China, No Corresponding author Tel : ,E2mail : edu. cn

2 2 : Rg1 MG263 Prohibitin 181 information to expand the applications of Traditional Chinese Medicine formulas in cancer2related studies. Key words prohibitin ; nuclear matrix ; osteosarcoma MG263 cells ; cell differentiation ; ginsenoside Rg1 Prohibitin (phb),. Prohibitin [1 ],, [2 5 ]., prohibitin [6 8 ],.. MG263 MGc8023 prohibitin [9,10 ]., Rg1 ( ginsenoside Rg1) (cinnamic acid) A (tanshinone A) MG263 ( ), prohibitin MG263, prohibitin MG263, prohibitin, MG263 ( ) RPMI21640 ( GIBCO ) ( 15 % 100 UΠml 100 gπml 50 gπml,ph 712), CO Rg1 A. 24 h, RCT(33 gπml Rg1 + 2 mmolπl gπml A, ),, 2 3 d 1, 7 d. 112 [10 12 ]. MG263 PBS 2, CSK 100 (10 mmolπl PIPES, ph 618, 300 mmolπl,100 mmolπl NaCl,3 mmolπl MgCl 2, 5 % Triton X2100, 1 mmolπl EGTA,1 mmolπl PMSF),0 10 min,400 g,. CSK50 ( CSK mmolπl NaCl 50 mmolπl) 2,. 300 UΠml DNase ( CSK50 ) 30 min, 1 molπl (NH 4 ) 2 SO molπl, 15 min,600 g 5 min, DNA,, CSK50 1,, g 45 min,, 016 ml, (3 ),, PD Quest 810 (Bio2Rad) , DTT (iodoacetamide), MALDI2TOF (ReFlexTM,Bruke ) (PMF). MSDB Swiss2Prot NCBI,. Swiss2 Prot ( Western blot) SDS2PAGE 1215 %. PVDF (Millipore).,., PVDF 5 % 115 h ;TTBS(0105 % Tween220, 50 mmolπl Tris2HCl,150 mmolπl NaCl,pH 714) ; prohibitin (NeoMarkers ), 2 h ;TTBS 3 ; HRP, 1 h ;TTBS 3 ECL,X. 5 %. 116 Prohibitin MG263 RCT 5 d

3 182 24, 2 3 d, PBS 2,5 minπ, 2, [13 ]. NM2IF 4 % 10 min, R % Tween220 PBS(TPBS), 5 % BSA 1 h prohibitin, 30 min, h,ttbs 3 10 min. CY3 ( ),37 30 min,4 3 h. TPBS 2 10 min. 90 % ΠPBS. 5 % BSA prohibitin,.,. 117 MG263 RCT 5 d, 2 3 d, PBS 2, 5 min, 015 % Triton X2100 PBS min,pbs 2, 5 min, 4 % 10 min. TPBS 2. 5 % BSA 1 h. 5 %BSA :prohibitinπrb prohibitinπp53 prohibitinπc2myc prohibitinπc2fos, 40 l, 30 min, 4 ; TPBS 3 10 min.,, 30 min, 4 3 h, TPBS 3 10 min. 90 % 2PBS. TCS2SP2 MP,CY3 543 nm,fitc 488 nm. 2 Table 1 The results of prohibitin identification by MALDI2TOF2MS analysis 211 3,,prohibitin RCT. Fig11 P prohibitin,rct. Fig12 prohibitin, Fig13 prohibitin. Table 1 prohibitin P 1,,,. Protein name Accession number Molecular mass (kd) pi ScoreΠcoverage Matching peptides Prohibitin P Π44 % 8 Fig. 1 22D PAGE analysis of nuclear matrix proteins The proteins selectively extracted from MG263 cells (Control) and the cells treated with Ginsenoside Rg1, cinnamic acid, and tanshinone A ( RCT) were separated by 22D PAGE method. The gels were stained with silver nitrate protocol compatible with MS. The arrow indicated the differential expression of the protein spot (P) which was identified as prohibitin

4 2 : Rg1 MG263 Prohibitin 183 Fig. 2 Enlarged picture of 22D PAGE The spots P which marked with arrows respectively showed the differential expression of prohibitin before and after RCT treatment Fig. 4 The results of Western blot showed the expression of prohibitin in the nuclear matrix preparations from MG263 cells Control: the nuclear matrix protein sample of MG263 cell ; RCT: the nuclear matrix protein sample of MG263 cells treated by RCT; Marker : protein molecular weight marker was stained with Ponceau S Fig. 3 Relative abundance of the differential spot of pprohibitin The quantitative analysis of gel images was performed with PD Quest 810 software. The relative abundance of prohibitin in MG263 cell (control) is higher markedly than RCT treated cell 212 Western,,,,. prohibitin 2 phb 1 phb 2, phb1 32 kd, phb2 37 kd. Western 32 kd, MG263, RCT MG263 (Fig14).,. 213 Prohibitin MG263 2,,,, 2 2 (Fig15A B, 185 ).,MG , ;,., ( Fig15A). RCT MG263 2,,, ; ;,. (Fig15B). prohibitin MG , prohibitin MG263, ( Fig15C) ; RCT,prohibitin MG263 2,,,, prohibitin,prohibitin (Fig. 5D). 214 Prohibitin c2fos c2myc P53 Rb MG263 MG263, prohibitin CY3,, c2fos Myc P53 Rb FITC,

5 Prohibitin c2fos MG263 MG263,prohibitin, c2fos.,prohibitin c2fos ; RCT,MG263 prohibitin c2myc,,, (Fig. 6A F) Prohibitin c2myc MG263 MG263, Prohibitin,, ;c2myc, ; prohibitin c2myc. RCT MG263, prohibitin, ; c2myc,,, ; c2myc prohibitin (Fig16G L) Prohibitin P53 MG263 MG263,prohibitin,,,, ; p53, p53.,prohibitin p53,. RCT, prohibitin, ; p53,, p53 ; RCT MG263 prohibitin p53, (Fig. 6M R) Prohibitin Rb MG263 MG263,prohibitin, ; Rb,,,, ;,prohibitin Rb. RCT MG- 63, prohibitin, ; Rb,,.,Rb prohibitin, (Fig16S X) Prohibitin RCT MG263 Prohibitin,, [1 ],,., prohibitin, RCT, ;, MG263 prohibitin, RCT MG263 prohibitin,.,prohibitin,. prohibitin, prohibitin,. Prohibitin :, PHB, PHB, [14 ]. PHB [15 ]. PHB E2F, [16 ].,PHB,,. Prohibitin,, mrna 3 [17,18 ], prohibitin,,. prohibitin RCT

6 2 : Rg1 MG263 Prohibitin 185 Fig. 5 Expression of pprohibitin on the NM - IF system of MG263 cell NThe nuclear matrix of MG263 cells were selectively extracted and stained with coomassie brilliant blue or antibody labeled CY3 (A) Light microscopy shows the NM2IF system in MG263 cells (CBB stain). (B) Light microscopy shows the NM2IF system in MG263 cells after RCT treatment (CBB stain). (C) Immunofluorescence microscopy shows the expression of Prohibitin on the NM2IF system of MG263 cell. (D) Immunofluorescence microscopy shows the expression of Prohibitin on the NM2IF system of MG263 cell after RCT treatment Fig. 6 The co2localizational relationship between prohibitin and the products of some interrelated genes MG263 cells treated with or without RCT were stained with antibody labeled with CY3 or FITC and were observed under laser scanning confocal microscopy. The overlay showed the yellow colocalizational fluorescence. A F : The localizationa of prohibitin and c2fosp53 in MG263 cells ; G L : The localizationa of prohibitin and c2myc in MG263 cells ; M R : The localizationa of prohibitin and P53 in MG263 cells ; S X: The localizationa of prohibitin and Rb in MG263 cells

7 186 24, RCT, MG Prohibitin RCT MG263,prohibitin,. 2, prohibitin c2fos c2myc p53 Rb RCT. c2fos c2myc,,prohibitin c2 Fos c2myc MG263, MG263,prohibitin c2fos, prohibitin c2myc,., prohibitin c2fos c2myc, prohibitin c2fos Rb, Rb prohibitin c2fos [19 ] ; prohibitin c2myc, c2myc prohibitin [20,21 ],. prohibitin c2myc c2fos, RCT, prohibitin c2 Myc c2fos MG263.,MG263 p53 prohibitin, RCT,,,prohibitin MG263 p53.,prohibitin p53. Fusaro [22,23 ],prohibitin p53 E2F1, prohibitin p53 p53 ;,p532prohibitin., p53 prohibitin, MG263 p53.,prohibitin p53, p53,.,prohibitin Rb,prohibitin Rb, E2F, E2F, [24 26 ]., RCT MG263,prohibitin Rb,. DNA,,prohibitin Rb, E2F,.,prohibitin, MG263. prohibitin,, prohibitin,.,. prohibitin. RCT MG263 prohibitin,.. ( References) [ 1 ] Nijtmans L G, de Jong L, Artal Sanz M, et al. Prohibitins act as a membrane2bound chaperone for the stabilization of mitochondrial proteins[j ]. EMBO J,2000, 19 (11) : [ 2 ] Artal2Sanz M, Tsang W Y, Willems E M, et al. The mitochondrial prohibitin complex is essential for embryonic viability and germline function in Caenorhabditis elegans[j ].J Biol Chem, 2003,278(34) : [ 3 ] Nijtmans L G, Artal S M, Grivell L A, et al. The mitochondrial PHB complex : roles in mitochondrial respiratory complex assembly, ageing and degenerative disease[j ]. Cell Mol Life Sci, 2002,59(1) : [ 4 ] Piper P W, Jones G W, Bringloe D, et al. The shortened replicative life span of prohibitin mutants of yeast appears to be due to defective

8 2 : Rg1 MG263 Prohibitin 187 mitochondrial segregation in old mother cells[j ]. Aging Cell, 2002, 1(2) : [ 5 ] 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) : [ 6 ] Jang J S, Cho H Y, Lee YJ, et al. The differential proteome profile of stomach cancer : identification of the biomarker candidates [J ]. Oncol Res,2004, 14(10) : [ 7 ] Tsai H W, Chow N H, Lin C P, et al. The significance of prohibitin and c2metπhepatocyte growth factor receptor in the progression of cervical adenocarcinoma[j ]. Hum Pathol, 2006, 37 (2) : [ 8 ] Qi Y, Chiu J F, Wang L, et al. Comparative proteomic analysis of esophageal squamous cell carcinoma[j ]. Proteomics, 2005, 5 (11) : [ 9 ] Zhao C H, Li Q F, Zhao Y, et al. Changes of nuclear matrix proteins following the differentiation of human osteosarcoma MG263 cells [J ]. Genomics Proteomics Bioinformatics, 2006,4 (1) :1027 [10 ] Zhao C H, Li Q F. Altered profiles of nuclear matrix proteins during the differentiation of human gastric mucous adenocarcinoma MGc8023 cells [J ]. World J Gastroenterol, 2005, 11 (30) : [11 ] Michishita E, Kurahashi T, Suzuki T, et al. Changes in nuclear matrix proteins during the senescence2like phenomenon induced by 52 chlorodeoxyuridine in HeLa cells [J ]. Exp Gerontol, 2002, 37(7) : [12 ] Nickerson J A, Krockmalnic G, Wan KM, et al. The nuclear matrix revealed by eluting chromatin from a cross2linked nucleus[j ]. Proc Natl Acad Sci USA, 1997, 94(9) : [13 ] Li Q F. Effect of retinoic acid on the changes of nuclear matrix in termediate filament system in gastric carcinoma cells [J ]. World J Gastroenterol 1999,5(5) : [14 ] DixitV D, Sridaran R, Edmonsond M A, et al. Gonadotropin2 releasing hormone attenuates pregnancy2associated thymic involution and modulates the expression of antiproliferative gene product prohibitin [J ]. Endocrinology,2003, 144(4) : [15 ] Sun L,Liu L,Yang X J, et al. Akt binds prohibitin2 and relieves its repression of MyoD and muscle differentiation[j ]. J Cell Sci,200 4, 117(14) : [16 ] Joshi B,Ko D,Ordonez2Ercan D, et al. A putative coiled2coil domain of prohibitin is sufficient to repress E2F12mediated transcription and induce apoptosis [J ].Biol Biophys Res Commun, 2003,312(2) : [17 ] Campbell I G, Allen J, Eccles D M. Prohibitin 3 untranslated region polymorphism and breast cancer risk [J ]. Cancer Epidemiol Biomarkers Prev, 2003, 12(11 Pt 1) : [18 ] Manjeshwar S, Branam DE, Lerner MR, et al. Tumor suppression by the prohibitin gene 3 untranslated region RNA in human breast cancer[j ]. Cancer Res, 2003,63(17) : [19 ] Buchmann A M, Swaminathan S, Thimmapaya B. Regulation of cellular genes in a chromosomal context by the retinoblastoma tumor suppressor protein[j ]. Mol Cell Biol, 1998, 18(8) : [20 ] Menssen A, Hermeking H. Characterization of the c2myc2regulated transcriptome by SAGE: identification and analysis of c2myc target genes [J ]. Proc Natl Acad Sci USA, 2002, 99(9) : [21 ] Haggerty T J, Zeller K I, Osthus R C, et al. A strategy for identifying transcription factor binding sites reveals two classes of genomic c2myc target sites[j ]. Proc Natl Acad Sci USA, 2003,100 (9) : [22 ] Fusaro G, Dasgupta P, Rastogi S, et al. Prohibitin induces the transcriptional activity of p53 and exported from the nucleus upon apoptotic signaling[j ]. J Biol Chem, 2003,278(48) : [23 ] Rastogi S, Joshi B, Fusaro G, et al. Camptothecin induces nuclear export of prohibitin preferentially in transformed cells through a CRM2 12dependent mechanism[j ].J Biol Chem, 2006,281(5) : [24 ] Wang S, Fusaro G, Padmanabhan J, et al. Prohibitin co2localizes with Rb in the nucleus and recruits N2CoR and HDAC1 for transcriptional repression[j ]. Oncogene, 2002, 21 (55) : [25 ] Fusaro G, Wang S, Chellappan S. Differential regulation of Rb family proteins and prohibitin during camptothecin2induced apoptosis [J ]. Oncogene, 2002, 21(29) : [26 ] Wang S, Zhang B, Faller D V. Prohibitin requires Brg21 and Brm for the repression of E2F and cell growth [J ]. EMBO J, 2002,21(12) :