The Aberrant Expressions of Nuclear Matrix Proteins During the Apoptosis of Human Osteosarcoma Cells.код для вставкиСкачать
THE ANATOMICAL RECORD 293:813–820 (2010) The Aberrant Expressions of Nuclear Matrix Proteins During the Apoptosis of Human Osteosarcoma Cells ZHEN-LI ZHAO,1 QI-FU LI,1* YAN-BIN ZHENG,1 LAN-YING CHEN,2 SONG-LIN SHI,1 AND GUANG-JUN JING1 1 The Key Laboratory of Education Ministry for Cell Biology and Tumor Cell Engineering, School of Life Science, Xiamen University, Xiamen, China 2 Biomedical Department, Henan University of Urban Construction, Pingdingshan, China ABSTRACT The objective of this study was to investigate altered expressions of nuclear matrix proteins (NMPs) of human osteosarcoma (OS) MG-63 cells during curcumin-induced apoptosis of human OS MG-63 cells. MG-63 cells were cultured with curcumin (7.5 mg/L) for 72 hr. Morphological alterations of cells were captured using light microscopy and transmission electron microscopy, and cell cycle distribution was estimated by ﬂow cytometry. NMPs were selectively extracted and subjected to two-dimensional gel electrophoresis (2-DE) analysis. Western blots were performed to determine changes in the expression levels of speciﬁc NMPs. The results demonstrated that typical characteristics of apoptosis were observed. Cellular chromatin agglutinated, cell nuclei condensed, and apoptotic bodies were formed after treatment with curcumin. The 2-DE results displayed 27 NMPs, 21 of which were identiﬁed to have change in expression levels signiﬁcantly during apoptosis. The altered expressions of three of these NMPs (nucleophosmin, prohibitin, and vimentin) were further conﬁrmed by immunoblotting. These ﬁndings indicated that the apoptosis of MG-63 cells was accompanied by the expression alteration of NMPs. Our results might help to reveal the relationship between NMPs and the regulation of gene expression in the process of apoptosis, as well as provide the basic concepts for future studies on the mechanisms of apoptosis and the therapy for bone diseases. Anat Rec, 293:813–820, C 2010 Wiley-Liss, Inc. 2010. V Key words: human osteosarcoma cell; curcumin; apoptosis; cell morphology; cell cycle; nuclear matrix protein The investigation of apoptosis in tumor cells is one of the major areas of study in the current anticancer research ﬁeld, and it could reveal the molecular mechanism of tumor development (Evan and Littlewood, 1998). The nuclear matrix (NM), which is related to DNA replication, mRNA processing, and steroid hormone action, is the ﬁlamentous protein framework inside the nucleus. Through remodeling of the higher-order architecture of chromatin, it affects cell division, proliferation, and apoptosis (Tsutsui et al., 2005; Otake et al., 2006). The NM proteins (NMPs), which is a class of tissue-speciﬁc proteins, regulate signal transduction, gene expression, and apoptosis (Alvarez and Lokeshwar, 2007). Analyzing and C 2010 WILEY-LISS, INC. V Grant sponsor: National Science Foundation of China; Contract grant number: 30871241. *Correspondence to: Qi-Fu Li, Department of Biomedical Sciences, School of Life Sciences, Xiamen University, Xiamen 361005, China. Fax: þ86-592-2185363. E-mail: chifulee@xmu. edu.cn Received 21 August 2009; Accepted 1 October 2009 DOI 10.1002/ar.21074 Published online 25 March 2010 in Wiley InterScience (www. interscience.wiley.com). 814 ZHAO ET AL. identifying the alteration of NMPs in tumor cells preand postapoptosis and exploring the relationship between NMPs and apoptosis-related gene expression would bring important breakthroughs in the regulatory mechanisms of tumor cell apoptosis. Osteosarcoma (OS) is a highly malignant bone tumor that typically affects children and young adolescents between 10 and 20 years of age and is an extremely aggressive disease (Guo and Healey, 2002). In recent years, there has been some progress in the study of apoptosis of OS cells; however, the molecular mechanism of apoptosis is not well understood (Locklin et al., 2007). The human OS MG-63 cells, which have been widely used as model systems for elucidating osteogenic cell behavior on biomaterials, are typical examples of human OS. MG-63 cells possess the capacity to undergo osteoblastic differentiation in response to osteogenic chemical cues (Tabb et al., 2003). Curcumin (diferuloylmethane) is an yellow pigment in turmeric (Curcuma longa) and is known to be a powerful antioxidant with strong antiinﬂammatory properties (Ammon and Wahl, 1991).Curcumin has been reported to effectively induce the apoptosis of several types of cancer cells in vitro (Samaha et al., 1997). Previously, we have demonstrated that apoptosis in human gastric adenocarcinoma (BGC-803), human esophageal carcinoma (EC-9706) and human epithelial cells (HaCaT) can be induced effectively by curcumin (Li et al., 2007; Chen et al., 2008; Yang et al., 2009). In the present study, we used human OS MG-63 cells to study the effects of curcumin-induced apoptosis and analyze expression changes of NMPs during the apoptotic process of MG-63 cells. In addition, we sought to identify differentially expressed NMPs related to MG-63 cells apoptosis, which may provide a foundation for future studies on the mechanisms of apoptosis and the therapy for bone diseases. MATERIALS AND METHODS Cell Culture and Treatment Human OS MG-63 cells, obtained from the China Center for Type Culture Collection (CCTCC), were propagated in RPMI-1640 medium supplemented with 15% heat-inactivated fetal calf serum, 100 U/mL penicillin, 100 lg/mL streptomycin, and 50 lg/mL kanamycin and incubated at 37 C with 5% CO2. Twenty-four hours after subculture, MG-63 cells were grown in a culture medium containing 7.5 mg/L curcumin (National Institute for the Control of Pharmaceutical and Biological Products) for 72 hr to induce apoptosis. MG-63 cells cultured in RPMI-1640 medium only acted as negative control. The concentrations of curcumin and treatment time were determined by preexperiment evaluation of the effects of apoptosis on MG-63 cells treated with curcumin. Determination of Cell Cycle MG-63 cells of the treated and control groups were digested, centrifuged at 1,800 rpm for 5 min, and then collected. All cells collected were rinsed in phosphate buffered saline (PBS), resuspended, ﬁxed in 75% precooled ethanol at 4 C overnight, centrifuged, and ﬁnally resuspended in 10 mg/mL RNase A at 37 C for 30 min. Then, 50 lg/mL propidum iodide (Sigma) was added to the suspended cells at 4 C in the dark for 30 min. The cells were ﬁltered through 300-mesh nylon nets to obtain single cell suspensions. Cell cycle analysis was performed by ﬂow cytometry (Beckman), and the data were analyzed using the Cell FIT cell cycle analysis software. Determination of DNA Fragmentation For qualitative analysis of DNA fragmentation, cells were harvested after 24-, 48-, and 72-hr incubation with curcumin (7.5 mg/L) by centrifugation and then lyzed in lysis buffer consisting of 10 mM Tris-HCl (pH 7.4), 10 mM EDTA, and 0.1% of Triton X-100. Afterward, the cell lysates were incubated with RNase A (500 U/mL) and proteinase K (20 mg/mL) at 37 C for 2 hr. After centrifugation, the soluble DNA fragments were precipitated by the addition of 0.5 volume of 7.5 M ammonium acetate and 2.5 volumes of ethanol. DNA pellets were dissolved in Tris þ EDTA (TE) buffer and loaded onto a 2.0% agarose gel and separated at 60 V for 90 min. DNA fragments were visualized after staining with ethidium bromide by transillumination under UV light. Sample Preparation for Fluorescence Microscopy MG-63 cells from the control group and the group treated with 7.5 mg/L curcumin for 48 hr were seeded in small penicillin bottles with coverslips and grown for 24 hr. Cells on the coverslips were rinsed with PBS at 37 C, ﬁxed with 4% paraformaldehyde at 4 C for 10 min, and then incubated with 5 g/L Hoechst 33258 at room temperature for 10 min in the dark. Cells were then rinsed with distilled water, mounted on glass microscopic slides in 90% glycerol, and examined under a ﬂuorescent microscope (Olympus DP-50). Sample Preparation for Light Microscopy MG-63 cells from the control group and the group treated with 7.5 mg/L curcumin for 48 hr were seeded in small penicillin bottles with coverslips and grown for 24 hr. Cells on the coverslips were rinsed with PBS at 37 C, ﬁxed overnight in Bouin-Hollande ﬁxative, stained with Hematoxylin–Eosin reagents, and observed under a light microscope (Olympus BH-2). Determination of Growth Rate of MG-63 MG-63 cells collected in the logarithmic phase of growth were grown at 5.0104 cells/mL. Twenty-four hours after subculture, the experimental groups were treated with 7.5 mg/L curcumin, while the control group was continuously cultured in fresh medium. During the ﬁrst 7 days, untreated or treated cells were harvested from three culture ﬂasks (75 mL/ﬂask) everyday. The number of viable cells was counted three times via the trypan blue dye exclusion test to obtain an average value. Sample Preparation for Transmission Electron Microscopy Untreated MG-63 cells and cells treated with 7.5 mg/L curcumin for 72 hr were rinsed with PBS at 37 C, detached with plastic scraper, and transferred into centrifuge tubes. Cells were centrifuged at 1,800 rpm for 15 min, and the supernatants were removed. The cell pellets were preﬁxed in 2.5% glutaraldehyde for 2 hr and postﬁxed in 1% osmium tetroxide for 2 hr, ABERRANT EXPRESSIONS OF NUCLEAR MATRIX PROTEINS 815 Fig. 1. The effect of curcumin on the proliferation of MG-63 cells. A: Viable cells were counted by the trypan blue dye exclusion test to obtain average values. B: The cell cycles of untreated or treated MG63 cells were analyzed by ﬂow cytometry, and the data were analyzed with the Cell FIT cell cycle analysis software. Results are obtained from three independent experiments. *denotes a statistically signiﬁcant difference (P < 0.05). C: After different treatment times, a DNA ladder was shown in MG-63 cells treated with 7.5mg/L curcumin. M: Marker; 1: control group; 2: 24 hr; 3: 48 hr; 4: 72 hr. dehydrated in ethanol, embedded in epoxy resin 823, stained with uranyl acetate (3%) and lead citrate (0.4%), and observed under a transmission electron microscope (JEM-100CX ||). Western Blot Analysis 2-DE PAGE Analysis The NM lysates were prepared according to the methods described by Liang et al. (2009). Two-dimensional gel electrophoresis (2-DE PAGE) was performed using standard methods. The gels were stained using a silver nitrate protocol compatible with mass spectrometry. Image scanning (UMAX Power Look |||) and analyses (PD Quest 8.0 software, Bio-Rad) of the three triplicate sets of silver-stained 2-DE gels were performed. After background subtraction, spot detection, and matching of spots from one gel with spots from another gel, spot intensities were obtained by the integration of the Gaussian function with units of intensity calculated as intensity area as parts per million (INT Area PPM). The intensity of each protein spot was normalized to the total intensity of the entire gel. The spots of protein whose intensity changed at least twofold were deﬁned as differentially expressed NMPs. MALDI-TOF-MS Analysis and Protein Identiﬁcation Spots containing differentially expressed proteins were cut from the gels. After a series of steps including silver removal, reduction with DL-dithiothreitol, alkylation with iodacetamide, and in-gel digestion with trypsin (Peng et al., 2004; Zhang et al., 2004), peptide mass ﬁngerprints (PMFs) were generated using Bruker ||| matrix-assisted laser desorption/ionization time-of-ﬂight mass spectrometry (MALDI-TOF-MS). The Flex Analysis software was used to analyze the PMF data to calibrate and remove polluted peaks. The data were searched against the NCBInr and Swiss-Prot protein databases using the Mascot tool from Matrix Science. The NMPs were separated by sodium dodecyl sulfate polyacrylamide gel eletrophoresis (SDS-PAGE) using 15% polyacrylamide gels (15 lg protein/lane) and then transferred onto pylidene ﬂuoride (PVDF) membranes (Millipore). Nonspeciﬁc reactivity was blocked by incubating the membranes at 37 C for 2 hr in 5% BSA in tris-buffered saline tween-20 (TBST). The blots were then incubated with primary antibodies against nucleophosmin, prohibitin, and vimentin (1:2,000 dilutions, Santa Cruz) overnight at 4 C. Upon three washes of 10 min each with TBST, the membranes were incubated at 37 C for 1 hr with the appropriate secondary antibody (1:4,000 dilution, NeoMarkers) and washed three times for 10 min each with TBST. Immunoreactivity was detected by using an enhanced chemiluminescence (ECL) detection system (Pierce). b-Actin was also detected as an internal control. RESULTS Apoptosis of Human OS MG-63 cells Induced by Curcumin Effects of curcumin on the proliferation and death of MG-63 cells. The cell growth curve showed that the proliferation of MG-63 cells is extremely fast. On the seventh day, the number of cells in the control and treated groups increased from 5.0 104 cells/mL to 81.76 104, and 20.07 104 cells/mL, respectively; this presents 16.4- and 4.0-fold increases in growth rates for each group. The doubling time of the control group was 10.28 hr, whereas that of the treated group was 41.90 hr. Thus, the growth in the treated group was slowed by 75.45% (Fig. 1A). The cell cycle of MG-63 cells was analyzed by ﬂow cytometry. The results revealed that the cell cycle distribution of MG-63 cells changed signiﬁcantly when the cells were treated with curcumin. The effect of curcumin-induced apoptosis was more obvious than control, and an apoptotic peak (sub-G1) appeared. The proportion 816 ZHAO ET AL. Fig. 2. The effect of curcumin 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 ¼ 40 lm). Untreated MG-63 cells (C) and cells treated with curcumin (D) were observed after H&E staining (Bar ¼ 40 lm). Ultrastructure was visualized by transmission electron microscopy in untreated MG-63 cells (E) and cells treated with curcumin (F) (Bar ¼ 1 lm). of untreated cells in the G0/G1 phase was 48.2%, 25.2% in the S phase, and 25.8% in the G2/M phase. However, the proportion of cells at G0/G1 phase was 41.3%, 10.0% at S phase, and 21.9% at G2/M phase after treatment with curcumin (Fig. 1B). nuclei had become pyknotic (shrunken and dark); and the nuclei had undergone karorrhexis and karyolysis (Fig. 2B). Under the light microscope, MG-63 cells showed typical malignant morphological characteristics similar to other human OS cells. The overall volume of the MG-63 cells was relatively large, cells were irregular with epithelioid, round, fusiform, or triangular shape, and so forth; the nuclei were large and irregular with several nucleoli per nucleus, and the nucleoplasmic ratio was relatively large (Fig. 2C). In contrast to untreated cells, cells treated with curcumin underwent a signiﬁcant morphological change and appeared as apoptotic cells: the volume of the cells was reduced, the amount of acidophillic cells was increased, and the shape of the cells became round or oval-shaped. The nuclei became smaller, appeared karyopyknotic, and were deeply stained (Fig. 2D). In the control group, it was revealed by transmission electron microscopy that the nucleoplasmic ratio of MG-63 cells was relatively large, the nuclear membrane was invaginated, the shape of the nucleus was irregular, and most of the heterochromatin in the nuclei and nucleoli was large and with some nucleolus vacuoles. The rough endoplasmic reticulum was not well developed. Golgi vesicles were few, arranged irregularly, and the Golgi cisterna were signiﬁcantly swollen. The mitochondria were arranged irregularly and the cristae within the mitochondria arranged Detection of DNA fragment in curcumininduced apoptotic MG-63 cells. The results of agarose gel electrophoresis demonstrated that there was no DNA degradation in the control group, and a bright band of high molecular weight was observed; whereas DNA laddering was observed as an evidence of DNA fragmentation (200 bp) in the treated group. A comparison with molecular weight markers indicated that the fragments were multiples of 200 bp (Fig. 1C). Effects of curcumin on the morphology and ultrastructure of MG-63 cells. The results of Hoechst33258 staining showed that the nuclei of untreated MG-63 cells emitted a low-intensity ﬂuorescence of homogeneous dispersion, and the integrity of the nuclear structure was maintained (Fig. 2A). In the treated group, the MG-63 cells displayed typical morphological features of apoptosis: the chromatin had condensed and begun to form a block; the nuclear membrane had gradually disintegrated; the shape of the ABERRANT EXPRESSIONS OF NUCLEAR MATRIX PROTEINS 817 Fig. 3. 2-DE protein proﬁles from the nuclear matrix of 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 are shown as circular symbols on the gels. L indicates down-regulated NMPs, and H indicates up-regulated NMPs in MG-63 cells treated with curcumin. B,C: Relative abundance of upregulated and down-regulated NMPs in untreated MG-63 cells and cells treated with curcumin. Results are obtained from three independent experiments. *denotes signiﬁcant difference (P < 0.05). irregularly, the polyribosomes were abundant while free ribosomes were few (Fig. 2E). However, after being treated with curcumin, the ultrastructure of MG-63 cells had also undergone a signiﬁcant change. The volume of the cells was reduced, the nucleoplasmic ratio had decreased, the cytoplasm was vacuolated and heavy in electron density; the nuclei contained transparent regions and the densities of the nuclei were more pronounced; the mitochondria were clearly swollen and the chamber of the endoplasmic reticulum was expanded. Apoptotic bodies were also observed by transmission electron microscopy (Fig. 2F). sis of the images for three triplicate sets of silver-stained 2-DE gels was performed using the PDQuest 8.0 software (Bio-Rad). The analysis of proteins was based on the evaluation of at least two gels. A total of 27 protein spots changed appreciably. Most spots corresponding to NMPs from apoptotic MG-63 cells showed similar intensities to those in the control group (Fig. 3A). The ranges of pI and molecular weight of most spots distributed in the area corresponded to pI 4–9 and 10–100 kDa, respectively. The expression of 27 NMPs was changed during the apoptotic process, 7 of which were increased (H1– H7) and 14 were decreased (L1–L14). The other six spots were not identiﬁed because of low protein abundance or a lack of matches to the protein database. The relative expression levels of the 21 proteins were shown using the Melanie Viewer|| software (Fig. 3B,C). The relative abundance (ppm) of spots was used to correct the data for artiﬁcial intensity differences between gels due, for Changes of NMPs During Apoptosis of MG-63 Cells Induced by Curcumin 2-DE PAGE and image analysis. The NPMs extracted from MG-63 cells and cells treated with curcumin were subjected to 2-DE PAGE. Quantitative analy- 818 ZHAO ET AL. TABLE 1. Differential expression of nuclear matrix proteins were identiﬁed by MS Spot no. Protein name Upregulated proteins H1 DNA-Damage-inducible transcript H2 Discoidin H3 DNA polymerase zeta H4 HCV-2 H5 Bullous pemphigoid antigen 1 isoforms H6 growth-regulating protein H7 Cyclic nucleotide hosphodiesterase Down -regulated protein L1 Mannose 6-phosphate receptor L2 ARMC8 protein L3 HERV-K_1q23.3 L4 GTPase activating Rap L5 Nucleophosmin L6 Glutamate dehydrogenase L7 TCP-1-beta L8 Pyrophosphatase 1 L9 TER ATPase L10 Vimentin L11 Prohibitin L12 Mutant beta-globin L13 PROM2 protein L14 Heat-shock 70 Accession Mol. mass calc (kDa) pI (calc) Coverage (%) Matching peptides Q96D03 Q5H994_HUMAN Q5TF36_HUMAN AAC37555 BPA1_HUMAN A56008 PDE1A_HUMAN 22,248 9,336 37,663 13,569 374,544 6,703 61,314 7.49 8.75 9.54 5.12 6.38 7.85 5.73 13 46 26 36 5 61 26 4 3 6 3 11 2 7 A32700 Q8IUR7_HUMAN VPK7_HUMAN Q5JS19_HUMAN gi|145580402 DEHUE TCPB_HUMAN Q2M348_HUMAN TERA_HUMAN VIME_HUMAN I52690 Q9UP81_HUMAN Q2HIX6_HUMAN Q5IST7_MACFA 31,495 75,049 17,206 30,539 13,631 61,707 57,669 33,095 89,819 53,546 29,844 9,741 40,990 66,972 5.57 6.27 7.96 6.01 5.82 7.66 6.02 5.54 5.14 5.06 5.57 7.93 6.02 5.22 11 12 42 27 66 20 17 39 31 29 44 58 13 21 4 6 4 4 5 8 5 8 22 8 8 3 5 12 example, to differences in protein loading or staining. These proteins were identiﬁed via searching in the Swiss-Prot database (Table 1). Additionally, NMP samples were obtained from ﬁve different apoptotic cell groups, and there was no signiﬁcant difference in the level of variation in NMP proﬁles from one apoptotic cell group to another (P < 0.05). Immunoblotting of the identiﬁed NMPs. Bands for nucleophosmin (NPM, 38 kDa), prohibitin (PHB, 32 kDa), and vimentin (55 kDa) were observed in the NMP samples of control and curcumin-treated cells (Fig. 4). The nucleophosmin, prohibitin, and vimentin bands detected in control cells were at higher levels than those from curcumin-treated cells. These ﬁndings reveal that curcumin treatment downregulated the expression of nucleophosmin, prohibitin, and vimentin, which are consistent with the results of the 2-DE PAGE analysis. DISCUSSION Effects of Curcumin on the Apoptosis of Human OS MG-63 Cells Inhibition of cell proliferation is the most important characteristic that differentiates normal cells from apoptotic cells. The study of the proliferation rate and the cell cycle provides a signiﬁcant index for identifying exogenous inducers of apoptosis (Squires et al., 2003). In this study, the cell growth curve and cell cycle analysis indicated that MG-63 cells could proliferate vigorously. The proliferation of the group treated with curcumin was inhibited as early as the day after treatment, and these results demonstrate that curcumin inhibits the proliferation of MG-63 cells, induces apoptosis, and an apoptotic peak appears in cell cycle analysis. The effects of curcumin on cell growth and cell cycle are consistent with its antiproliferative effects through cell cycle arrest, which was reported in human lung cancer and human Fig. 4. Conﬁrmation of the differential expression of speciﬁc NMPs from NMP samples by Western blotting. b-Actin was used as a protein loading control. Control is the samples from untreated MG-63 cells, and curcumin is the samples from curcumin-treated MG-63 cells (NPM, nucleophosmin; PHB, prohibitin). leukemia k562 cells (Radhakrishna Pillai et al., 2004; Xie et al., 2009). These data show that curcumin can effectively inhibit the proliferation of human OS MG-63 cells. Changes of cell morphology play a role in determining apoptosis, which involves cell shrinkage, chromatin agglutination, marginalization, nuclear fragmentation, and apoptotic body formation. The changes of cell morphology are primary indicators of apoptosis (Hunot and Flavell, 2001; Danial and Korsmeyer, 2004). The results from light microscopy and electron microscopy showed that MG-63 cells had a typical malignant phenotypical characteristic of the morphology and ultrastructure in tumor cells. The arrangement of cell populations was irregular and cell morphology varied, and there were fewer organelles in the cytoplasm. The nucleoplasmic ratio was relatively large, the nuclei were larger and irregular in shape with masses of heterochromatin and large nucleoli in them. After being treated with curcumin, the cells were homogenous and the volume of the cells was ABERRANT EXPRESSIONS OF NUCLEAR MATRIX PROTEINS smaller; the nuclei was hyperchromatic and the chromatin was condensed around the nuclear membrane. Transparent regions appeared in the nucleus, and the dense nuclei were observed clearly. Cell membranes shrunk, but the morphology was maintained and vesicles were formed; the mitochondria were obviously swollen, the cavity of endoplasmic reticulum expanded, and apoptotic bodies were observed under electron microscopy. These results agree with previous reports regarding the changes of apoptotic cell morphology of human gastric adenocarcinoma, human esophageal carcinoma, and human epithelial cell lines (Li et al., 2007; Chen et al., 2008; Yang et al., 2009) and are consistent with the results of the apoptosis of B cell lymphoma induced by curcumin (Han et al., 1999). These results suggest that curcumin is capable of inducing human OS MG-63 cell apoptosis. Changes of NMPs Composition During Apoptosis of MG-63 Cells Induced by Curcumin Abnormalities of the NM system are closely associated with apoptosis (Martelli et al., 1999). Previous studies showed that the NMPs of apoptotic cells have a distinctive composition different from that of normal cells (Alvarez and Lokeshwar, 2007). Therefore, discovering and identifying speciﬁc NMPs that are related to apoptosis is relevant to exploring and clarifying the mechanism of apoptosis. In the present study, we further analyzed the alterations of NMPs to identify the effects of curcumin on the apoptosis of MG-63 cells. The 2-DE electrophoresis identiﬁed 21 NMPs that were differentially expressed during the apoptotic process. Among the spots whose intensities changed during apoptosis, seven of which increased and 14 of which decreased in expression levels in the apoptotic cells. Speciﬁc NMPs could be divided into the following categories according to their functions: DNA breakage-related proteins, such as DNAdamage-inducible transcript 4-like and REV3-like; NM structure proteins, such as vimentin; apoptosis-related regulatory proteins, such as prohibitin; cell cycle-associated proteins, such as T-complex protein 1 subunit beta (TCP-1-beta); cell proliferation, apoptosis-related phosphorylase categories, such as transitional endoplasmic reticulum ATPase (TER ATPase), heat shock protein 70 (Hsp70), and so forth. In addition to vimentin, Hsp70, prohibitin, and nucleophosmin, the rest of the proteins such as DNA-damage-inducible transcript, discoidin, DNA polymerase zeta, HCV-2, bullous pemphigoid antigen 1 isoforms, growth-regulating protein, cyclic nucleotide phosphodiesterase, mannose 6-phosphate receptor, ARMC8 protein, HERV-K_1q23.3, GTPase activating Rap, glutamate dehydrogenase, TCP-1-beta, pyrophosphatase 1, TER ATPase, mutant beta-globin, and the PROM2 protein are newly discovered NMPs. Previous studies have shown that NMPs of the cancer cells would change during apoptosis, conﬁrming that there are signiﬁcant changes in the composition of NMPs during the process of apoptosis of MG-63 cells (Dynlacht et al., 2000; Gotzmann et al., 2000). Further studies on speciﬁc NMPs of apoptotic cells are important in clarifying the mechanism of apoptosis. Hsp70 is one of the stress-induced proteins and widely exists in the cytoskeleton and nuclear skeleton, and it 819 has antiapoptotic properties in tumor cells. The other 17 proteins identiﬁed are also NMPs that are associated with apoptosis. Previous studies demonstrate that poly ADP-ribose polymerase (PARP) is a content-rich NMbinding protein of mammalian cells, and PARP-1 is a member of this family and negatively regulates the process of apoptosis (Tang and Li, 2004). Protein kinase CK2 is a Ser/Thr kinase and widely exists in the cytoplasm and nucleus. It is a NM-binding protein and plays a role in preventing apoptosis (Guo et al., 2001). NuMA (nuclear mitotic apparatus protein) is an important NMP that is part of the mitotic spindle. It performs a speciﬁc function in interphase cells, which is illustrated in a RNA interference gene silencing experiment that results in the apoptosis of HeLa cells (Taimen and Kallajoki, 2003). Our results reveal that there is a close relationship between NMPs and apoptosis of the human OS MG-63 cells. More and more research has shown that these identiﬁed NMPs are signiﬁcant not only for their binding to the NM but also because they may inﬂuence cell proliferation and differentiation or apoptosis by regulating gene expression at the transcriptional, mRNA processing and posttranscriptional levels. The vimentin, prohibitin, and nucleophosmin were important functional proteins and played important roles in cell proliferation, differentiation, and apoptosis. What’s more, in our previous studies we demonstrated that these three kinds of proteins were conﬁrmed to be common differential NMPs during the induced-apoptosis of various tumor cell lines. Therefore, Western blotting was performed to investigate these three proteins, which were used as an entry point for in-depth study of their functions in the regulation of cell apoptosis. The vimentin protein identiﬁed in this study was one of the structural proteins of the NM, and it is associated with cell proliferation, differentiation, and apoptosis (Belichenko et al., 2001). The results of the 2-DE and immunoblotting showed that the expression of vimentin was downregulated during the apoptosis of the MG-63 cells; this may be because it was degraded in the process of apoptosis, which resulted in the change of its expression level. This observation is consistent with previous reports in other cancer cell lines (van Engeland et al., 1997; Morishima, 1999). Prohibitin is a highly conserved protein and is vital to cell development (Sato et al., 1992). Recent studies have shown that it plays an important role in apoptosis (Fusaro et al., 2003). In the present study, we found that prohibitin inhibited cell apoptosis, illustrated by the reduced expression level of prohibitin observed in the 2DE and immunoblotting. The effects of prohibitin on MG-63 cells are consistent with the antiapoptotic ability of this protein reported in OS cells (Fellenberg et al., 2003). Nucleophosmin is a nucleolar phosphor protein, which is reported to be related to a variety of signaling pathways that regulate cell proliferation and apoptosis (Grisendi et al., 2006). The results of the 2-DE and immunoblotting demonstrated that the expression of nucleophosmin was downregulated, which likely contributed to the cancer-speciﬁc apoptotic effect of curcumin on MG-63 cells. Similar results were obtained from the studies on the human leukemia HL-60 cells (Hsu and Yung, 2000). Further characterizations of these NMPs and their involvement in apoptosis may offer a new direction in the study of apoptosis-speciﬁc target 820 ZHAO ET AL. proteins, which could, in turn, provide a gateway to the understanding of apoptosis in tumor cells and the mechanism of carcinogenesis. 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