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The Aberrant Expressions of Nuclear Matrix Proteins During the Apoptosis of Human Osteosarcoma Cells.

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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.
CONCLUSION
Our findings demonstrated that curcumin effectively
inhibits proliferation and induces apoptosis in MG-63
cells. An array of aberrantly expressed NMPs as well as
substantial morphological changes were observed in
MG-63 cells during apoptosis. In fact, our study has
identified several NMPs that associate with the apoptosis of OS cells. These findings will help to elucidate the
signaling pathways and the mechanism of OS cell apoptosis as well as carcinogenesis. Further study of the
proteins identified here will be useful for the development of clinical therapies targeting OS.
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expressions, aberrant, matrix, nuclear, apoptosis, osteosarcoma, protein, human, cells
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