Comparative proteomics analysis of lanthanum citrate complex-induced apoptosis in HeLa cells

Abstract

In a previous study, the lanthanum citrate complex ([LaCit2]3−) has been found to induce apoptosis in the human HeLa cervical cancer cell line. To clarify the mechanism, we carried out
comparative proteomics analysis between treated and control cells. Differentially expressed proteins were separated electrophoretically
and identified by MALDI-TOF/TOF tandem mass spectrometry. There were profound changes in 14 proteins related to mitochondrial
function and oxidative stress, suggesting that mitochondrial dysfunction plays a key role in [LaCit2]3−-induced apoptosis. This was confirmed by a decrease in the mitochondrial transmembrane potential, and increases in cytochrome
c release and reactive oxygen species generation in [LaCit2]3−-treated cells. Western blotting analyses show that [LaCit2]3−-induced apoptosis was accompanied by the activation of caspase-9 and the specific proteolytic cleavage of PARP, leading to
an increase in the proapoptotic protein Bax and a decrease in the antiapoptotic protein Bcl-2. These results suggest that
[LaCit2]3− induced the apoptosis of HeLa cells through oxidative stress mediated pathway involving MT participation.

Full text

Page 1

Science in China Series B: Chemistry
© 2009 SCIENCE IN CHINA PRESS


Sci China Ser B-Chem | Nov. 2009 | vol. 52 | no. 11 | 1814-1820
www.scichina.com
chem.scichina.com
www.springerlink.com
Comparative proteomics analysis of lanthanum citrate
complex-induced apoptosis in HeLa cells
SHEN LiMing, LIU Qiong & NI JiaZuan†
College of Life Sciences, Shenzhen University, Shenzhen 518060, China
In a previous study, the lanthanum citrate complex ([LaCit2]3− −) has been found to induce apoptosis in
the human HeLa cervical cancer cell line. To clarify the mechanism, we carried out comparative pro-
teomics analysis between treated and control cells. Differentially expressed proteins were separated
electrophoretically and identified by MALDI-TOF/TOF tandem mass spectrometry. There were profound
changes in 14 proteins related to mitochondrial function and oxidative stress, suggesting that mito-
chondrial dysfunction plays a key role in [LaCit2]3− −-induced apoptosis. This was confirmed by a de-
crease in the mitochondrial transmembrane potential, and increases in cytochrome c release and re-
active oxygen species generation in [LaCit2]3− −-treated cells. Western blotting analyses show that
[LaCit2]3− −-induced apoptosis was accompanied by the activation of caspase-9 and the specific prote-
olytic cleavage of PARP, leading to an increase in the proapoptotic protein Bax and a decrease in the
antiapoptotic protein Bcl-2. These results suggest that [LaCit2]3− − induced the apoptosis of HeLa cells
through oxidative stress mediated pathway involving MT participation.
lanthanum citrate complex, HeLa cells, proteomics
1 Introduction
Effects of La3+ on cell proliferation and apoptosis have
been studied extensively[1―4]. Whether lanthanides pro-
mote cell proliferation or induce cell apoptosis depends
on their concentrations and the types of cancer cells be-
ing studied. Lanthanum citrate was reported to inhibit
the growth of human lung cancer PG cell line dose-
dependently, but it did not affect human gastric cancer
BGC-823 cells or human embryo diploid fibroblast cells.
It arrested PG cells in the G1 phase and inhibited DNA
synthesis, which is consistent with the inhibition of cell
proliferation[5]. Up to date, a redox-active gadolinium
texaphyrin complex has entered phase Ⅲ clinical trials
for the treatment of patients with brain metastases of
non-small cell lung carcinomas[6]. In addition, it was
also proposed that lanthanides might induce apoptosis
via mitochondrial (MT) pathways[7―9] and it appears
likely that reactive oxygen species (ROS) are involved
in the mechanism[8,9]. La3+ induced cell proliferation and
apoptosis in a manner compatible with a p53-related
mechanism in NIH 3T3 cells via an ERK-mediated signal-
ing cascade induced by a metal-sensing mechanism[2,10].
Lanthanum compounds also interact with DNA by
mechanisms involving intercalation or coordinative
binding[11,12].
In our previous work, a dose-dependent effect of
[LaCit2]3− was studied on the growth and viability of
different cancer cell lines[13]. [LaCit2]3− inhibited the
growth of different cancer cell lines at high concentra-
tions, but no significant inhibition was observed at low
concentrations. Additionally, the effect of [LaCit2]3− on
cell growth also depends on the type of cancer cells. The
most potent cytotoxicity of [LaCit2]3− was observed in

Received August 15, 2009; accepted September 3, 2009
doi: 10.1007/s11426-009-0272-z
†Corresponding author (email: jzni@szu.edu.cn)
Supported by the National Natural Science Foundation of China (Grant No.
20637010) and the Shenzhen Bureau of Science, Technology and Information

Page 2

SHEN LiMing et al. Sci China Ser B-Chem | Nov. 2009 | vol. 52 | no. 11 | 1814-1820 1815
HeLa cells, while it had relatively lower cytotoxicity
toward human breast cancer (MCF 7), prostate cancer
(PC-3), lung cancer (PG) and hepatocellular carcinoma
(HepG2) cell lines.
In this study, [LaCit2]3−-induced apoptosis in HeLa
cells was explored using a proteomic approach. Using
two-dimensional gel electrophoresis (2-DE) on proteins
extracted from control and [LaCit2]3−-treated cells, dif-
ferentially expressed proteins were observed and identi-
fied by matrix-assisted laser desorption/ionization time-
of-flight tandem mass spectrometry (MALDI-TOF/TOF
MS). Confirmed by the results of Western blotting
analysis, measurements of ΔΨ m, cytochrome c (cyt-c)
release and intracellular H2O2 level, we conclude that
[LaCit2]3− induces apoptosis by activating intrinsic apop-
totic pathways.
2 Materials and methods
2.1 Materials
The human HeLa cervical epithelial cell line was pur-
chased from the Shanghai Institute of Biochemistry and
Cell Biology, Chinese Academy of Sciences. [LaCit2]3−
solution was prepared as described previously[13] from
lanthanum oxide (purity >99.9%, Yuelong New Materi-
als Co., Ltd., Shanghai, China) and citric acid (BBI,
Canada). All other reagents were obtained from GE
Healthcare (Yian Plaza, Guangzhou, China), unless oth-
erwise noted.
2.2 Cell culture
The cells were cultured in RPMI-1640 medium plus
10% fetal bovine serum (FBS) (Hyclone, Logan, UT,
USA), 100 U·mL penicillin and 100 μg·mL streptomycin
(Merck & Co., Inc, Whitehouse Station, NJ, USA). The
cells were maintained in a humidified incubator in 95%
air and 5% CO2 at 37℃. When the cells reached 80%
confluence, they were harvested and plated for either
subsequent passages or drug treatments. For the treated
samples, the medium was removed and fresh FBS-free
RPMI-1640 medium containing 0.15 mmol·L−1 [LaCit2]3−
was added.
2.3 Proteome analysis
After treatment with 0.15 mmol·L−1 [LaCit2]3− for 24 h,
the cells were harvested and the proteins were extracted
as described previously[14]. 2-DE was performed using
IPGphor isoelectric focusing (IEF) and electrophoresis
units (GE Healthcare)[14]. Briefly, whole cell protein
lysates (150 μg for analysis gels and 800 μg for prepara-
tive gels) were mixed with rehydration solution to a final
volume of 450 μL. Precast 24 cm immobilized pH gra-
dient (IPG) strips were rehydrated for 12 h at 30 V, and
IEF was performed for a total of 75000 Vh. After the
first dimensional run, the proteins were reduced in
equilibration buffer and alkylated for 15 min in the same
buffer containing 135 mmol·L−1 iodoacetamide instead of
DTT. Proteins were then separated in the second dimen-
sional gel made from 12.5% SDS-polyacrylamide using
the Ettan DALTsix Electrophoresis Unit. The second
dimensional gels for analysis were stained with silver
nitrate and the gels for mass spectrometry were stained
with Coomassie brilliant blue G-250. The gels were
scanned with ProXPRESS 2D imaging system (Perkin-
Elmer Inc., Waltham, MA, USA) and analyzed with
Image Master 2D Elite software (GE Healthcare). Only
those spots that changed consistently in three replicates
and significantly (more than 2.0-fold) were selected for
the analysis with MS. These spots were excised and the
gel pieces were subjected to in-gel digestion with trypsin
followed by polypeptide analysis using MALDI-MS and
PMF[14]. MASCOT was used in database searching for
protein identification by MS data in the NCBI database
(http://www.ncbi.nlm.nih.gov/).
2.4 Measurement of MT transmembrane potential
and oxidative stress
Flow cytometry was used for the analysis of ΔΨ m and
generation of H2O2. Briefly, after [LaCit2]3− treatment,
the cells were incubated with 5 mg·L JC-1 (Beyotime
Biotech, Jiangsu, China) or 10 μmol·L−1 DCFH-DA
(Beyotime Biotech). Flow cytometry was performed
using a Beckman Coulter Altra flow cytometer equipped
with a single 488 nm argon laser. A minimum of 10,000
cells per sample was acquired and analyzed using
WinMDI V2.8 software.
2.5 Detection of cyt-c release from the mitochondria
Cells were grown on 35 mm glass bottom culture
dish (MatTek Corporation, German) and treated with
0.15 mmol·L−1 [LaCit2]3− for 6 or 16 h as described
above. Immunocytochemistry was performed using pri-
mary antibody to cyt-c and a secondary anti-mouse IgG
antibody conjugated with Cy3 (Beyotime Biotech), and
processed as described in the manufacturer’s protocol
and ref. [8].

Page 3

1816 SHEN LiMing et al. Sci China Ser B-Chem | Nov. 2009 | vol. 52 | no. 11 | 1814-1820
2.6 Western blotting
Western blotting was performed as described previ-
ously[14] using primary antibodies (Abs) against Bcl-2
(from Santa Cruz Biotechnology, Santa Cruz, CA, USA),
Bax (Santa Cruz), caspase-9 (Santa Cruz), PARP (Be-
yotime Biotech), SOD1 (BIOS, Beijing, China),
VDAC1 (Santa Cruz), VDAC2 (Santa Cruz), Nm23
(BIOS) and eEF2 (from Abzoom, Dallas, TX, USA) at
optimized dilutions. GAPDH (Santa Cruz) was used for
the normalization of each protein to ensure equal protein
loading.
3 Results and discussion
3.1 MT dysfunction is a key event in the apoptosis
induced by [LaCit2]3− −
The proteomes of control and [LaCit2]3−-treated cells
were analyzed by 2-DE. Representative gel images are
shown in Figure 1. By applying a threshold of 2-fold
variation, 14 spots were identified as differentially ex-
pressed after [LaCit2]3− treatment. Table 1 lists the dif-
ferentially expressed proteins identified by peptide mass
fingerprinting (PMF). The first group was proteins in-
volved in apoptosis and cell proliferation. Expressions
of human nucleoside diphosphate kinase B, nucleo-
phosmin (NPM), S100 calcium binding protein A11
(S100-A11) were downregulated, whereas the prohibitin
(PHB) was upregulated. The second group of proteins
was related to stress response and redox activity. Heat
shock 70-kDa protein 9 precursor, heat shock protein
beta-8 (HspB8) and superoxide dismutase 1 (SOD1)
were decreased, whereas the level of probable bifunc-
tional methylenetetrahydrofolate (MTHFD2) was in-
creased. Another group of proteins was related to trans-
lation and protein degradation, including the downregu-
lation of eukaryotic translation elongation factor 2
(eFF2), ribosomal protein (RPLP0), calreticulin precur-
sor variant and far upstream element (FUSE) binding
protein 1, isoform CRA_b and the upregulation of pro-
teasome beta 3 subunit. Several representative proteins
were selected from each group to be further confirmed
by western blot analysis, while others remained to be
confirmed later.
The ΔΨ m and H2O2 levels were measured in relation
to the proteomic changes of MT proteins and oxidative
stress. The experimental results show that [LaCit2]3−
treatment resulted in a decrease in ΔΨ m (Figure 2(a)) and
the generation of H2O2 (Figure 2(b)). In addition, apop-
totic stimuli generally induce the opening of the mem-
brane permeability transition (MPT) pores and swelling
followed by release of proapoptotic proteins from the
MT intermembranous space[15,16]. We observed the cyt-c

Figure 1 Two-dimensional gel electrophoresis (2D-GE) images of control and [LaCit2]3−-treated cells. The arrows indicate those proteins whose expres-
sions were altered and identified by peptide mass fingerprinting (PMF). The numbers are correlated with that spot number listed in Table 1.

Page 4

SHEN LiMing et al. Sci China Ser B-Chem | Nov. 2009 | vol. 52 | no. 11 | 1814-1820 1817
Table 1 Proteins identified by mass spectrometry and their alterations in the proteome after [LaCit2]3− treatment (0.15 mmol·L−1 for 24 h)
NCBI accession
Spot no. Protein ID
no.
MW
(kDa)/pI
95.3/6.41

73.6/5.87

68.7/7.18

23.2/6.14
Reported
function
Expression
level a)
−

−

−

+
Peptides
matched b)
15
1 Eukaryotic translation elongation factor 2 (eEF2)
4503483

24234688

119626762

22538465
Translation

response to stress

RNA folding

protein degradation

4 Heat shock 70 kDa protein 9 precursor
6

10

9
12
Far upstream element (FUSE) binding protein 1,
isoform CRA_b
Proteasome beta 3 subunit 14
16 Superoxide dismutase 1, soluble (SOD1) 4507149
18314408

119602242
16.1/5.7
32.5/4.59

10.8/11.3121.
oxidation reduction
anti-apoptosis

unclear
−
−

−
6
9

6
19 Nucleolar phospho-protein B23, numatrin (NPM)
25 HCG1785879
32 Heat shock protein beta-8 (HspB8)
Human nucleoside diphosphate kinase B com-
plexed (Nm23)
5901655
1421609

5032057

4505773
5/5.0
17.1/8.55

11.8/6.56

29.8/5.57
response to stress
apoptosis

cell proliferation

apoptosis
−
−

−

+
5
8

6

7
34
35 S100 calcium binding protein A11 (S100-A11)
41 Prohibitin (PHB)
88 Ribosomal protein, large, P0 (RPLP0)
Probable bifunctional methylenetetra
-hydrofolate dehydrogenase/cyclohy
-drolase2( MTHFD2L)
Calreticulin precursor variant
a) Expression levels in [LaCit2]3−-treated HeLa cells at 24 h compared with the control (+, increase; −, decrease); b) peptides matched: unique peptides
identified by peptide mass fingerprinting.
12654583
10434969


62897681
34.2/5.42
25.8/8.54


46.9/4.3
translation
oxidation reduction


protein folding
−
+


−
9
6


10
94
123

Figure 2 Mitochondrial membrane potential changes (ΔΨ m) and
[LaCit2]3−-induced generation of H2O2 in HeLa cells. (a) Percentages of
cells with high ΔΨ m. Each value represents the mean ± S.D. of three sepa-
rate determinations (*P < 0.05 compared with the control.) (b) HeLa cells
were pretreated with 0.15 mmol·L−1 [LaCit2]3− in FBS-free medium for 24 h
and incubated with 10 mmol·L−1 DCFH-DA for 30 min at 37℃. The fluo-
rescence intensity was measured using flow cytometry (*P < 0.05).
release from the MT (Figure 3), the activation of caspase-
9 and the 89 kDa cleaved form of PARP (Figure 4(a)).
Moreover, the expression level of the proapoptotic pro-
tein Bax was increased whereas the antiapoptotic protein
Bcl-2 was decreased (Figure 4(a)). Most signals that
control the survival of mammalian cells modulate the
activity of the Bcl-2 family of proteins, which are
well-characterized regulators of the MT pathway of
apoptosis. The intracellular balance of Bax and Bcl-2
could decide the cell fate: death or survival[17]. In addi-
tion, here we found that caspases acted as downstream
activators of [LaCit2]3−-induced apoptosis and that MT
permeabilization was mediated by the regulation of the
Bcl-2 family proteins in [LaCit2]3−-treated HeLa cells.
These results suggest that [LaCit2]3−-induced stress leads
to MT dysfunction, which in turn induces cell apoptosis
as well as inhibits protein synthesis.
As described above, the mitochondria respond to an
apoptotic signal by opening the PTP. This is composed
of the VDAC, OMM (Outer Mitochondrial Membrane),
the adenine nucleotide translocase (ANT) and cyclo-
philin D. Recent studies involving both down- and
overexpression clearly indicate that the expression level
of VDAC is a critical element in cell life and death and

Page 5

1818 SHEN LiMing et al. Sci China Ser B-Chem | Nov. 2009 | vol. 52 | no. 11 | 1814-1820

Figure 3 Effects of [LaCit2]3− on the release of cyt-c in HeLa cells. (a) In situ distribution of cytochrome c in HeLa cells analyzed by laser scanning
confocal microscopy. The white arrow indicates the representative control cell with a non-homogeneous (or clumpy) cyt-c staining pattern. The yellow
arrows indicate the representative [LaCit2]3−-treated cells with the release of cyt-c to cytosol. (b) The fluorescent intensity of cyt-c in the cytosol quantified
with confocal software. Each value represents the mean ± S.D. of three separate determinations (*P < 0.05 compared with the control.)

Figure 4 The [LaCit2]3−-sensitive proteins in HeLa cells identified by western blotting analysis. (a) Apoptosis-regulating proteins, including Bcl-2, Bax,
the 37-kDa cleaved form of caspase-9 and 89-kDa cleaved PARP. (b) Western blot analysis of the altered proteins in [LaCit2]3−-treated HeLa cells. This is
a representative result from three independent experiments. GAPDH was used as an internal control.
that the VDAC plays a key role in the regulation of
MT-mediated apoptosis. In the present study, the protein
VDAC2 was not detected (result not shown) and VDAC1
showed no changes in expression level (Figure 4(b)).
These results suggest that VDAC expression levels
might not play a key role in the process of [LaCit2]3−-