Chinese Journal of Biochemistry and Molecular Biology MG-63. hnrnp A2 / B1 MG-63

ISSN 1007-7626 CN 11-3870 / Q 2010 6 Chinese Journal of Biochemistry and Molecular Biology 26 6 546 ~ 555 MG-63 hnrnp A2 /B1 1 2 1 * 1 2 1 1 1 1 361005 2 467044 curcumin MG-63 hnrnp A2 / B1 MG-63 hnrnp A2 / B1 MG-63 hnrnp A2 / B1 MG-63 hnrnp A2 / B1 MG-63 hnrnp A2 / B1 MG-63 Bax Bcl-2 Fas p53 hnrnp A2 / B1 MG-63 MG-63 hnrnp A2 / B1 MG-63 Q24 Q27 Curcumin-induced Apoptosis Affects the Expression and Localization of hnrnp A2 /B1 in Human Osteosarcoma MG-63 Cells ZHAO Zhen-Li 1 CHEN Lan-Ying 2 * LI Qi-Fu 1 * SHI Song-Lin 1 YANG Hai-Bo 2 JING Guang-Jun 1 WU Fu-Yun 1 KONG Hai-Yan 1 1 Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering Xiamen University Xiamen 361005 China 2 Henan University of Urban Construction Pingdingshan 467044 Henan China Abstract The morphology of human osteosarcoma MG-63 cells under light microscopy and transmission election microscopy was altered when treated with curcumin The distribution of heterogeneous nuclear ribonucleoprotein hnrnp A2 / B1 in the nuclear matrix and its colocalization with the apoptosis-related gene products were examined The results demonstrated that curcumin induced typical apoptotic responses such as chromatin agglutination nuclei condensation and apoptotic body formation Twodimentional PAGE and mass spectrum analyses showed that hnrnp A2 / B1 was complexed with the nuclear matrix Confirmed by Western blotting curcumin treatment resulted in a down-regulation of 2010-01-19 2010-03-29 No 30871241 * Tel 0592-2185363 E-mail chifulee@ xmu edu cn Received January 19 2010 Accepted March 29 2010 Supported by National Natural Science Foundation of China No 30871241 * Corresponding author Tel 0592-2185363 E-mail chifulee@ xmu edu cn

6 MG-63 547 hnrnp A2 / B1 expression An altered distribution of hnrnp A2 / B1 with the nuclear matrix was observed in immunofluorescence assays Laser confocal microscopy showed that hnrnp A2 / B1 colocalized with apoptosis-related molecules including Bax Bcl-2 Fas and p53 with different patterns before and after curcumin treatments Our study showed that expression level and the distribution of hnrnp A2 / B1 a nuclear matrix protein were altered by curcumin treatments and mought be involved in the process of apoptosis in MG-63 cells Key words curcumin hnrnp A2 / B1 nuclear matrix human osteosarcoma MG-63 cell apoptosis heterogeneous nuclear ribonucleoprotein hnrnp RNA 1 2 1 hnrnp A2 / B1 mrna hnrnp hnrnpa2 / B1 mrna 50 μg / ml ph 7 2 37 5% CO 2 DMSO DNA DNA 10 mg / ml - 20 2 ~ 4 hnrnp A2 / MG-63 24 h 7 5 B1 mg / L 72 h 5 6 hnrnp A2 / 1 3 Hoechst33258 B1 PBS 3 4% ph7 2 4 10 curcumin BGC-803 EC9706 Olympus BH-2 hnrnp A2 / B1 1 4 hnrnp A2 / B1 50 ml 7 ~ 9 24 h MG-63 10 7 5 μg / ml PBS hnrnp A2 / B1 MG-63 1 500 r / min hnrnp A2 / B1 MG-63 10 min 2 5% 4 2 h PBS 3 1% 4 hnrnpa2 / B1 2 h 823 JEM100-CXⅡ 1 5 1 CSK100 10 mmol / L PIPES 300 mmol / L 1 1 100 mmol / L NaCl 3 mmol / L MgCl 2 1 2 mmol / L MG-63 PMSF 0 5% TritonX-100 0 10 min 1 500 r / hnrnp A2 / B1 NeoMarkers IgG-FITC IgG-TRITC Bax Bcl-2 Fas P53 Santa Cruz RPMI-1640 MG-63 RPMI-1640 15% 100 U / ml 100 U / ml min PBS 5 mg / ml Hoechst33258 10 min PBS 90% / PBS min 5 min CSK50 10 mmol / L PIPES 300 mmol / L 50 mmol / L NaCl 3 mmol / L MgCl 2 1 2 mmol / L PMSF 0 5% TritonX-100 2 GIBCO 300 U / ml DNaseⅠ CSK50 30 min 1 mol / L 0 25 mol / L

548 26 15 min 1 500 r / min 5 min 0 2% 20 min CSK50 1-80 1 6 PBS 4 10 min PBS 3 10 min 5% BSA 1 h hnrnp A2 / B1 3 1 300 5% BSA 30 min 4 TBST 3 10 PDQuest 8 0 Bio-Rad 1 1 000 30 min 4 3 2 h TBST 2 10 min 2 1 7 10 min DTT 1 11 ReFlexTM III MALDI-TOF PBS 3 Bruke PMF 5 min Karnovsky s 20 min PBS flexanalysis 3 5 min 0 5% TritonX -100 MASCOT www matrixscience com 5 min 5% BSA 1 h 3% BSA 1 8 Western hnrnp A2 / B1 100 Bax Bcl-2 Fas p53 200 hnrnp 15% SDS-PAGE A2 / B1 / Bax hnrnp A2 / B1 / Bcl-2 hnrnp A2 / B1 / PVDF 4 3 % BSA Fas hnrnp A2 / B1 / p53 40 μl 1 2 000 30 min 4 PBS 2% BSA 1 5 h 3 5 min TBST 3 10 min 1 4 000 HRP 2% BSA 1 5 h TBST 3 10 TCS-SP2 MP min ECL β 3% BSA hnrnp A2 / B1 1 9 - - 2 2 d 2 d PBS 37 2 4 2 1 Hoechst33258 10 mmol / L PIPES ph 6 8 250 mmol / L NH 4 2 SO 4 300 mmol / L 3 mmol / L MgCl 2 1 2 mmol / L PMSF 0 5% Triton X-100 3 min 10 mmol / L PIPES ph6 8 50 mmol / L NaCl 300 mmol / L 3 mmol / L MgCl 2 1 2 mmol / L PMSF 0 5% Triton X-100 DNase I RNase A 400 μg / ml 23 20 min 23 5 - NM-IF 2 2 1 10 NM-IF 2% Fig 1C 4 30 min PBS NM-IF 4% min 5% BSA FITC PBS 20 min PBS 3 30 μl 30 min 4 3 h Hoechst33258 Fig 1A Fig 1B MG-63 MG-63

6 MG-63 549 2 3 MG-63 3 Bio- Fig 1D Fig 1 The effect of curcumin treatment on the morphology and ultrastructure of MG-63 cells Untreated MG-63 cells A and cells treated with curcumin B were observed after Hoechst33258 staining Bar = 40 μm Ultrastructure were visualized by transmission election microscopy in untreated MG-63 cells C and cells treated with curcumin D Bar = 1 μm Rad PDQuest 8 0 Fig 2A L3 hnrnp A2 / B1 Fig 2B hnrnp A2 / B1 Fig 2C hnrnp A2 / B1 MALDI-TOF L3 hnrnp A2 / B1 Table 1 2 4 SDS-PAGE PVDF Western ECL Fig 3 hnrnp 2 hnrnp B1 hnrnp A2 N 12 37 kd hnrnp A2 34 kd Western Western hnrnp A2 / B1 Table 1 Proteins identified by using MASCOT database www matrixscience com Spot No Protein Accession No Mass calculated pi calculated Coverage Matching peptides L3 hnrnp A2 / B1 gi 4504447 36 041 8 67 37% 8 2 5 hnrnp A2 / B1 MG-63 - Fig 4B MG-63 hnrnp A2 / B1 MG-63 NM-IF - hnrnp A2 / B1 Fig 4C 2 ~ 3 Fig 4A NM-IF hnrnp A2 / B1

550 26 Fig 2 2-D PAGE gels of nuclear matrix proteins from MG-63 cells A Proteins were separated on the basis of pi X-axis and molecular mass Y-axis and visualized by silver staining The differentially expressed proteins were showed as circular symbols on the gels L indicated down-regulated NMPs and H indicated up-regulated NMPs in MG-63 cells treated with curcumin B Enlarged maps of changed expression of hnrnp A2 / B1 from MG-63 cells C Relative abundance of the expression level of hnrnp A2 / B1 in untreated MG-63 cells and cells treated with curcumin Fig 4D 2 6 A2 / B1 Bax MG-63 MG-63 hnrnp A2 / B1 Bax Bcl-2 Fas mtp53 MG-63 MG-63 hnrnp A2 / B1 TRITC FITC Fig 5 hnrnp Bax hnrnp A2 / B1 hnrnp A2 / B1 Bax 2 6 1 hnrnp A2 / B1 Bax MG-63 Fig 5 1 ~ 6 MG-63 hnrnp A2 / B1 2 6 2 hnnrnp A2 / B1 Bcl-2 MG- 63 Bax MG-63 hnrnp

6 MG-63 551 Fig 3 Confirmation of the differential expression of hnrnp A2 / B1 from NMPs samples by Western boltting β-actin was used as a protein loading control The bands were detected by an enhanced chemiluminescence ECL detection system The control was the samples from MG-63 cells and curcumin was the samples from curcumin-treated MG-63 cells M was the marker Fig 4 Light microscope LM or fluorescent microscope observation of the nuclear matrixintermediate filament system of MG-63 cells Observation of NM-IF system in MG-63 cells A and treated cells B by light microscopy dyed by CBB G-250 Bar = 30 μm Distribution of NPM in the NM-IF system of MG-63 cells C and treated cells D by fluorescence microscopy labeled with fluorescence dye FITC Bar = 30 μm hnrnp A2 / B1 Bcl-2 MG- 63 Bcl-2 hnrnp A2 / B1 Bcl-2 Fig 5 7 ~ 12 2 6 3 hnrnp A2 / B1 Fas MG-63 MG-63 hnrnp A2 / B1 Fas hnrnp A2 / B1 Fas MG-63 Fas hnrnp A2 / B1 Fas Fig 5 13 ~ 18 2 6 4 hnrnp A2 / B1 mtp53 MG- 63 MG-63 hnrnp A2 / B1 mtp53 hnrnp A2 / B1 mtp53 MG-63 mtp53 hnrnp A2 / B1 mtp53 A2 / B1 Bcl-2 Fig 5 19 ~ 24

552 26 Fig 5 The co-localizational relationship between hnrnp A2 / B1 and the products of some apoptosisrelated genes MG-63 cells treated with or without curcumin were stained with antibody labeled with TRITC or FITC and observed under laser scanning confocal microscopy The overlay showed the yellow co-localization fluorescence 1 ~ 6 The localization of hnrnp A2 / B1 and Bax in MG-63 cells Bar = 30 μm 7 ~ 12 The localization of hnrnp A2 / B1 and Bcl-2 in MG-63 cells Bar = 30 μm 13 ~ 18 The localization of hnrnp A2 / B1 and Fas in MG-63 cells Bar = 30 μm 19 ~ 24 The localization of hnrnp A2 / B1 and mtp53 in MG- 63 cells Bar = 30 μm

6 MG-63 553 3 3 1 hnrnp A2 / B1 B1 Bax Bcl-2 hnrnp A2 / B1 MG-63 Fas MG-63 hnrnp A2 / B1 hnrnp A2 / B1 p53 hnrnp A2 / B1 MG-63 hnrnp A2 / B1 Bax Bcl- 2 hnrnp 11 A2 / B1 Fas hnrnp A2 / B1 p53 hnrnp A2 / B1 hnrnp A2 / B1 MG-63 MG-63 hnrnp A2 / B1 hnrnp Bax Bcl-2 Fas p53 MG-63 A2 / B1 MG-63 - hnrnp A2 / B1 hnrnp A2 / B1 MG-63 - hnrnp A2 / B1 13 hnrnp A2 / B1 hnrnp B1 hnrnp DNA-PK G 1 S A2 / B1 hnrnp B1 G 1 / S hnrnp A2 / B1 - Bax 9 14 Bax NPM hnrnp A2 / B1 Bak Mcl-1 hnrnp A2 / B1 15 ~ 18 Bcl-2 hnrnp A2 / B1 MG-63 19 hnrnp A2 / B1 Bcl-2 MG-63 hnrnp A2 / B1 MG-63 hnrnp hnrnp A2 / B1 Fas 3 2 hnrnp A2 / B1 20 Abe 21 MG-63 H596 hnrnp A2 / B1 RNA mrna 12 hnrnp A2 / Bcl-2 A2 / B1 Bcl-2 Fas Fas RNA pre-mrna MG-63 Fas hnrnp A2 / B1

554 26 p53 Cell 2006 17 8 3521-3533 P53 22 P53 P53 MDM2 23 24 Dogu 25 P53 Bcl-2 26 HeLa HPV18 E6 P53 hnrnp A2 / B1 p53 MG-63 hnrnp A2 / B1 P53 2 Fas p53 hnrnp A2 / B1 J hnrnp A2 / B1 Mod Biomed 2008 8 9 1601-1604 9 HaCaT J hnrnp A2 / B1 Sin 2009 40 2 64-68 10 MG-63 J hnrnp A2 / B1 hnrnp A2 / B1 11 Zhou J Nong L Wloch M et al Cancer 2001 34 3 341-350 References 1 Zhou J Mulshine J L Unsworth E J et al Purification and characterization of a protein that permits early detection of lung cancer identification of heterogeneous nuclear ribonucleoprotein A2 / B1 as the antigen for monoclonal antibody 703D4 J J Biol Chem 1996 271 18 10760-10766 2 Kosturko L D Maggipinto M J Korza G et al Heterogeneous nuclear ribonucleoprotein hnrnp E1 binds to hnrnp A2 and inhibits translation of A2 response element mrnas J Mol Biol 3 Moran-Jones K Wayman L Kennedy D D et al hnrnp A2 a potential ssdna / RNA molecular adapter at the telomere J Nucleic Acids Res 2005 33 2 486-496 4 Griffin M E Hamilton B J Roy K M et al Post-transcriptional regulation of glucose transporter-1 by an AU-rich element in the 3 'UTR and by hnrnp A2 J Biochem Biophys Res Commun 2004 318 4 977-982 5 Satoh H Kamma H Ishikawa H et al Expression of hnrnp A2 / B1 proteins in human cancer cell lines J Int J Oncol 2000 16 4 763-767 6 Lee C H Lum J H Cheung B P et al Identification of the heterogeneous nuclear ribonucleoprotein A2 / B1 as the antigen for the gastrointestinal cancer specific monoclonal antibody MG7 J Proteomics 2005 5 4 1160-1166 hnrnp A2 / B1 Bax Bcl- 7 BGC- 823 J Li Peng Li Qi-Fu Shi Song-Lin et al Regulation of bioactive peptides of oyster BPO on the cell cycle and gene expression of human gastric adenocarcinoma cell line BGC-823 J Chin J Mar Drugs 2007 26 3 1-8 8 EC 9706 Chen Lan-Ying Yang Hai-Bo Li Qi-Fu et al Apoptosis of human esophageal carcinoma cell line EC 9706 induced by curcumin J Prog Qi-Fu Li Zhong-Zhu et al Yang Hai-Bo Li Apoptosis of immortalized human epithelial cell line HaCaT induced by curcumin J Acta Anat MG-63 Zhao Zhen-Li Zheng Yan-Bin Chen Lan-Ying et al Effects of curcumin on anti-proliferation and expression of apoptosis interrelated gene of human osteosarcoma MG-63 cells J Prog Mod Biomed 2009 9 12 2201-2205 Expression of early lung cancer detection marker hnrnp-a2 / B1 and its relation to microsatellite alteration in non-small cell lung cancer J Lung 12 Pino I Pio R Toledo G et al Altered patterns of expression of members of the heterogeneous nuclear ribonucleoprotein hnrnp family in lung cancer J Lung Cancer 2003 41 2 131-143 13 hnrnp B1 A549 DNA-PK J Han Juan Li Wei-Min Tang Feng-Ming et al Effects of hnrnp B1 on DNA-PK activity cell cycle and apoptosis in human lung adenocarcinoma cell line A549 J J Sichuan Univ Med Sci Ed 2008 39 5 815-818 14 Wolter K G Hsu Y T Smith C L et al Movement of Bax from

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