Identification of LZAP as a new candidate tumor suppressor in hepatocellular carcinoma.
ABSTRACT LZAP was isolated as a binding protein of the Cdk5 activator p35. LZAP has been highly conserved during evolution and has been shown to function as a tumor suppressor in various cancers. This study aimed to investigate LZAP expression and its prognostic value in hepatocellular carcinoma (HCC). Meanwhile, the function of LZAP in hepatocarcinogenesis was further investigated in cell culture models and mouse models.
Real-time quantitative PCR, western blot and immunohistochemistry were used to explore LZAP expression in HCC cell lines and primary HCC clinical specimens. The functions of LZAP in the proliferation, colony formation, cell cycle, migration, invasion and apoptosis of HCC cell lines were also analyzed by infecting cells with an adenovirus containing full-length LZAP. The effect of LZAP on tumorigenicity in nude mice was also investigated.
LZAP expression was significantly decreased in the tumor tissues and HCC cell lines. Clinicopathological analysis showed that LZAP expression was significantly correlated with tumor size, histopathological classification and serum α-fetoprotein (AFP). The Kaplan-Meier survival curves revealed that decreasing LZAP expression was associated with poor prognosis in HCC patients. LZAP expression was an independent prognostic marker of overall HCC patient survival in a multivariate analysis. The re-introduction of LZAP expression in the HepG2 and sk-Hep1 HCC cell lines significantly inhibited proliferation and colony formation in the HCC cells and induced G1 phase arrest and apoptosis of the HCC cells in vitro. Restoring LZAP expression in the HCC cell lines also inhibited migration and invasion. In addition, experiments with a mouse model revealed that LZAP overexpression could suppress HCC tumorigenicity in vivo.
Our data suggest that LZAP may play an important role in HCC progression and could be a potential molecular therapy target for HCC.
- [Show abstract] [Hide abstract]
ABSTRACT: NLBP (novel LZAP-binding protein) was recently shown to function as a tumor suppressor capable of inhibiting the NFκB signaling pathway. NLBP is also known as a negative regulator of cell invasion, and its expression is reduced in several cancer cell lines that have little invasive activity. Although these phenomena suggest that NLBP may be a potential tumor suppressor, its role as a tumor suppressor in human lung cancer is not well established. In contrast to our expectation, NLBP was highly expressed in the early stage of lung adenocarcinoma tissues, and overexpression of NLBP promoted proliferation of H1299 lung adenocarcinoma cells. We also found that p120 catenin (p120ctn) was a novel binding partner of NLBP, and that NLBP binds to the regulatory domain of p120ctn, and p120ctn associates with N-terminal region of NLBP, respectively. This binding leads to p120ctn stability to inhibit proteasomal degradation of p120ctn by inhibiting its ubiqutination. In addition, we also found that overexpression of NLBP and p120ctn in human lung cancer are closely related with adenocarcinoma compared with squamous cell carcinoma. Taken together, our findings reveal that NLBP is highly overexpressed in human lung adenocarcinoma, and that overexpression of NLBP promotes the cell proliferation of lung adenocarcinoma through interacting with p120ctn and suggest that NLBP may function as an oncogene in early stage carcinogenesis of lung adenocarcinoma.Cell cycle (Georgetown, Tex.) 06/2013; 12(15). · 5.24 Impact Factor
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ABSTRACT: BACKGROUND: We sought to investigate the expression levels and prognosis value of TCEAL7 in primary gastric cancer. METHODS AND RESULTS: We investigated TCEAL7 and other homologous five members of the TCEAL family expression in normal gastricepithelial cell line and gastric cancer cell lines using real-time quantitative PCR. Furthermore, we examined the expression of TCEAL7 in 39 paired cancerous and matched adjacent noncancerous gastric mucosa tissues by real-time quantitative PCR and western blotting. Moreover, we analyzed TCEAL7 expression in 406 gastric cancer patients using immunohistochemistry. The relationships between the TCEAL7 expression levels, the clinicopathological factors, and patient survival were investigated. RT- qPCR data showed that mRNA expression level of TCEAL7 was significantly lower in the gastric cancer cell lines comparing with the levels of other five members of the TCEAL family. Results also revealed decreased TCEAL7 mRNA (P = 0.025) and protein (P = 0.012) expression in tumor tissue samples compared with matched adjacent non-tumor tissue samples. Immunohistochemical staining data showed that TCEAL7 expression was significantly decreased in 43.3% of gastric adenocarcinoma cases. The result also showed that the low TCEAL7 expression was significantly correlated with female, larger tumor size, higher histological grade and worse nodal status. Kaplan-Meier survival curves revealed that the reduced expression of TCEAL7 was associated with a poor prognosis in gastric adenocarcinoma patients (P<0.001). Based on a univariate analysis that included all 406 patients, TCEAL7 expression was found to have statistically significant associations with overall survival (P<0.001). Multivariate analysis also demonstrated that TCEAL7 expression (P = 0.009), age, tumor size, histological grade, lymphovascular invasion, T stage, N stage and M stage were independent risk factors in the prognosis of gastric cancer patients. CONCLUSIONS: Our study suggests that TCEAL7 might serve as a candidate tumor suppressor and a potential prognostic biomarker in gastric carcinogenesis.PLoS ONE 01/2013; 8(1):e54671. · 3.53 Impact Factor
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ABSTRACT: GTP binding protein overexpressed in skeletal muscle (Gem) is a Ras-related protein whose expression is induced in several cell types upon activation by extracellular stimuli. To investigate the potential roles of Gem in hepatocellular carcinoma (HCC), expression of Gem was examined in human HCC samples. Western blot analysis showed that compared with primary human hepatocytes and adjacent noncancerous tissue, significant down-regulation of Gem was found in HCC cells and tumor tissues. In addition, immunohistochemical analysis of Gem expression was investigated in 108 specimens of HCC tissues. Clinicopathological data were collected to analyze the association with Gem expression. Expression of Gem was significantly negatively correlated with histological grade (P = 0.001), tumor size (P = 0.020), and vascular invasion (P = 0.005), and Gem was also negatively correlated with proliferation marker Ki-67 (P < 0.01). More importantly, the Kaplan-Meier survival curves analysis revealed that low expression of Gem was associated with poor prognosis in HCC patients. Univariate analysis showed that Gem expression was associated with poor prognosis (P = 0.006). Multivariate analysis indicated that Gem expression was an independent prognostic marker for HCC (P = 0.007). Finally, serum starvation and release experiments showed that Gem expression was negatively related with cell proliferation. In the conclusion, our results suggested that down regulation of Gem expression was involved in the pathogenesis of hepatocellular carcinoma, and it might be a favorable independent prognostic parameter for HCC.Pathology - Research and Practice 01/2014; · 1.21 Impact Factor
Identification of LZAP as a New Candidate Tumor
Suppressor in Hepatocellular Carcinoma
Jing-jing Zhao1, Ke Pan1, Jian-jun Li1, Yi-bing Chen1, Ju-gao Chen1, Lin Lv1, Dan-dan Wang1, Qiu-zhong
Pan1, Min-shan Chen1,2, Jian-chuan Xia1*
1State Key Laboratory of Oncology in Southern China and Department of Experimental Research, Sun Yat-sen University Cancer Center, Guangzhou, People’s Republic of
China, 2Department of Hepatobiliary Oncology, Sun Yat-sen University Cancer Center, Guangzhou, People’s Republic of China
Background: LZAP was isolated as a binding protein of the Cdk5 activator p35. LZAP has been highly conserved during
evolution and has been shown to function as a tumor suppressor in various cancers. This study aimed to investigate LZAP
expression and its prognostic value in hepatocellular carcinoma (HCC). Meanwhile, the function of LZAP in
hepatocarcinogenesis was further investigated in cell culture models and mouse models.
Methods: Real-time quantitative PCR, western blot and immunohistochemistry were used to explore LZAP expression in
HCC cell lines and primary HCC clinical specimens. The functions of LZAP in the proliferation, colony formation, cell cycle,
migration, invasion and apoptosis of HCC cell lines were also analyzed by infecting cells with an adenovirus containing full-
length LZAP. The effect of LZAP on tumorigenicity in nude mice was also investigated.
Results: LZAP expression was significantly decreased in the tumor tissues and HCC cell lines. Clinicopathological analysis
showed that LZAP expression was significantly correlated with tumor size, histopathological classification and serum a-
fetoprotein (AFP). The Kaplan–Meier survival curves revealed that decreasing LZAP expression was associated with poor
prognosis in HCC patients. LZAP expression was an independent prognostic marker of overall HCC patient survival in a
multivariate analysis. The re-introduction of LZAP expression in the HepG2 and sk-Hep1 HCC cell lines significantly inhibited
proliferation and colony formation in the HCC cells and induced G1 phase arrest and apoptosis of the HCC cells in vitro.
Restoring LZAP expression in the HCC cell lines also inhibited migration and invasion. In addition, experiments with a mouse
model revealed that LZAP overexpression could suppress HCC tumorigenicity in vivo.
Conclusions: Our data suggest that LZAP may play an important role in HCC progression and could be a potential
molecular therapy target for HCC.
Citation: Zhao J-j, Pan K, Li J-j, Chen Y-b, Chen J-g, et al. (2011) Identification of LZAP as a New Candidate Tumor Suppressor in Hepatocellular Carcinoma. PLoS
ONE 6(10): e26608. doi:10.1371/journal.pone.0026608
Editor: Andrei L. Gartel, University of Illinois at Chicago, United States of America
Received May 11, 2011; Accepted September 29, 2011; Published October 19, 2011
Copyright: ? 2011 Zhao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the National Natural Science Foundation of China (u0772002, 30700985, 30973398) and Guangdong Natural Science
Foundation (925100890). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Hepatocellular carcinoma (HCC) is currently the fifth most
common solid tumor worldwide and the fourth leading cause of
cancer-related death in many countries, especially in East Asia
[1–3]. Even with aggressive treatment, HCC usually has a poor
prognosis, with a 5-year survival rate as low as 25%–39%
after common treatments, such as surgery, chemotherapy, and
radiotherapy . The poor prognosis of HCC is also caused by its
poorly differentiated phenotype, portal venous invasion, and
intrahepatic metastasis . To improve patient outcomes, it is
clinically important to find efficient new targets for the early
diagnosis and effective treatment of HCC . Hepatocarcinogen-
esis is a multifactorial and multistep process that involves
activating oncogenes and inactivating tumor suppressor genes in
different stages of HCC progression [7–9]. Clarifying and
investigating the roles of the genes involved in HCC development
will contribute to our understanding of the mechanisms of
LZAP, also known as the Cdk5rap3 or C53 protein, has been
highly conserved throughout evolution and was originally isolated
as a binding protein of the Cdk5 activator p35. Human LZAP
consists of 506 amino-acid residues without well-defined domains,
except for a small leucine zipper region . Recently, LZAP has
been identified as an ARF-binding protein and was found to have
numerous tumor-suppressor functions . LZAP overexpression
potentiates DNA damage-induced cell death by etoposide and x-
ray irradiation and renders cells susceptible to multiple genotoxins
by modulating the G2/M checkpoint . In addition, it has been
shown that LZAP acts as a novel tumor suppressor in primary
head and neck cancers by specifically inhibiting NF-kB signaling
and that decreased LZAP expression promotes cellular transfor-
mation, xenograft tumor growth, and xenograft tumor vascularity
. It has also been shown that the tumor suppressor protein C53
PLoS ONE | www.plosone.org1 October 2011 | Volume 6 | Issue 10 | e26608
antagonizes checkpoint kinases to promote cyclin-dependent
kinase 1 activation . Recently, LZAP has been shown to
inhibit cell invasion through binding to NLBP, another tumor
suppressor . Until now, however, LZAP expression and its
prognostic value in HCC patients have not been examined, and
the functional role of LZAP in the pathogenesis and tumorigenic-
ity of HCC have not been determined.
In the present study, we investigated the expression of LZAP in
primary HCC using real-time quantitative RT-PCR, western
blotting and immunohistochemistry. We also identified the
relationship between LZAP expression and several clinicopatho-
logical features of HCC and evaluated the prognostic value of
LZAP expression for the survival of HCC patients. Furthermore,
we explored the role of LZAP in HCC tumor progression using
cell proliferation, colony formation, cell cycle, migration, invasion,
and apoptosis assays in vitro. Finally, we examined the role of
LZAP on HCC tumorigenicity in injectable mouse models.
LZAP mRNA and protein expression in primary HCC
tissue samples and HCC cell lines
A real-time quantitative PCR was performed on 57 paired
clinical samples from HCC patients (tumor tissues and matched
adjacent non-tumor liver tissues) and hepatocellular carcinoma cell
lines to determine their LZAP mRNA levels. In the clinical
samples, the LZAP expression was lower in the tumor tissues than
in the matched adjacent non-tumor liver tissues (p,0.01, Fig. 1A).
Furthermore, the HepG2, Hep3B, Huh7, Bel7402 and sk-Hep1
HCC cell lines showed decreased LZAP transcript levels relative to
the LO2 normal liver cell line. Additionally, LZAP expression was
significantly lower in the HepG2 and sk-Hep1 cells (Fig. 1B).
To investigate whether LZAP was also reduced at the protein
level, western blotting was performed on 24 HCC clinical samples,
the corresponding adjacent non-tumorous liver tissues and the
HCC cell lines. As shown in Figure 1C, LZAP protein expression
was lower in the tumors (p,0.01), consistent with the results of
real-time quantitative PCR. Likewise, LZAP protein expression
was decreased in the HepG2 and sk-Hep1 cells compared to the
LO2 cells (Fig. 1D).
Immunohistochemical analysis of LZAP expression in
HCC clinical samples and its relationship to
LZAP expression was investigated in 126 HCC surgical
specimens using immunohistochemical staining; 76 (60.3%) cases
showed low LZAP expression (LZAP2 or LZAP+), and 50
(39.7%) cases exhibited high LZAP expression (LZAP++ or
LZAP+++) (Table 1). In the positive cases, LZAP was detected
in the cytoplasm of the cells (Fig. 2G, H and I). LZAP expression
was also observed in normal liver tissues distant from the tumors
(Fig. 2B and G). In cases with adjacent hyperplastic tissue, we often
observed a sharp contrast between the infiltrative areas of negative
staining representing the tumor and the adjacent, positively
stained non-tumor tissue (Fig. 2A and F). The relationship
between LZAP expression and various clinicopathological param-
eters is described in Table 1. LZAP expression was significantly
correlated with tumor size (p=0.040), histological differentiation
(p=0.001), and serum AFP (P=0.006). Well-differentiated cases
showed strongly positive LZAP expression (Fig. 2C and H),
moderately-differentiated cases showed weakly positive expression
(Fig. 2D and I), and the most poorly differentiated cases often
showed no detectable LZAP expression (Fig. 2E and J). There was
no statistically significant difference in LZAP expression by age,
gender, liver cirrhosis, HBV, recurrence, or distant metastasis.
LZAP expression and patient survival
The prognostic value of LZAP for overall survival in HCC
patients was evaluated by comparing the patients with high and
Figure 1. The expression of LZAP mRNA and protein in the human primary HCC surgical specimens and HCC cell lines, as evaluated
by real-time quantitative PCR and western blotting. (A) The relative mRNA expression of LZAP was lower in 57 HCC tumor tissues than in
matched adjacent non-tumorous tissues (p,0.01). (B) The LZAP mRNA expression in human hepatocellular carcinoma cell lines was down-regulated
in the HepG2, Hep3B, Huh7, Bel7402 and sk-Hep1 cells, particularly in the HepG2 and sk-Hep1cells, compared with the normal liver cell line LO2. (C)
The LZAP protein expression was lower in the tumor tissues than in matched adjacent non-tumorous tissues (p,0.01). (D) The LZAP protein levels
were significantly lower in the HepG2 and sk-Hep1 cells than in the normal liver cell line LO2.
LZAP and Hepatocellular Carcinoma
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low LZAP expression. According to a Kaplan–Meier survival
analysis, low LZAP expression was significantly associated with
poor prognosis. The HCC patients with low LZAP expression had
obviously lower overall survival rates than did those with high
LZAP expression (Fig. 3, p=0.008 for the log rank test).
Univariate and multivariate analyses of prognostic
variables in HCC patients
Further univariate and multivariate analyses were conducted
using a Cox proportional-hazards model to examine the impact of
LZAP expression and other clinical pathological parameters in
HCC patients. LZAP expression, histological grade, serum AFP,
and recurrence were significant prognostic factors in the univariate
analysis (Table 2). Multivariate Cox regression analyses showed
that LZAP was an independent predictor. Thus, LZAP expression
may be useful for predicting the overall survival of HCC patients
(p=0.043, Table 2).
LZAP inhibits the viability of HepG2 and sk-Hep1 cell
To evaluate the effect of LZAP on cell viability, the HepG2 and
sk-Hep1 cells with low LZAP expression were infected with Ad-
LZAP and Ad-control. LZAP expression in the infected cells was
confirmed by immunofluorescence (Fig. 4) and western blotting
(Fig. S1). A colony formation assay was used to explore the ability
of LZAP to inhibit tumor cell growth. Compared with the Ad-
control-infected cells, the HepG2 and sk-Hep1 cells infected with
Ad-LZAP showed a significant reduction in colony formation
ability (Fig. 5A). Cell proliferation assays revealed that the growth
rate of the HepG2 and sk-Hep1 cells infected with Ad-LZAP was
significantly lower than that of the HepG2 and sk-Hep1 cells
infected with the Ad-control (Fig. 5B). We then examined the
possible effects of LZAP expression on the cell cycle by flow
cytometric analysis. We found that overexpressing LZAP in the
HCC cell lines induced G1 phase arrest (Fig. 5C). Furthermore,
we investigated the role of LZAP in the apoptosis of HCC cells.
More apoptotic cells were found in the LZAP-overexpressing
HCC lines than in control lines (Fig. 5D).
To further elucidate the biological functions of LZAP in HCC,
we used siRNA to knockout LZAP expression in the HepG2 and
sk-Hep1 cells infected with Ad-LZAP. Western blotting showed
that siLZAP-3# had the highest knockout efficiency of the three
siRNAs tested (Fig. S2). Therefore, siLZAP-3# was used for all the
subsequent experiments. Silencing LZAP expression in the HepG2
and sk-Hep1 cells infected with Ad-LZAP increased cell
proliferation and colony formation to levels similar to those of
the HepG2 and sk-Hep1 infected with the Ad-control (Fig. S3).
LZAP inhibits cell migration and invasion in the HepG2
and sk-Hep1 cell lines
We employed a transwell assay to evaluate the effects of LZAP
expression on cell migration and invasion. HepG2 and sk-Hep1
cells infected with Ad-LZAP migrated into the lower compartment
of the migration chamber significantly less frequently than did cells
infected with the Ad-control (Fig. 6A). Consistent with the
migration assay results, LZAP also significantly inhibited cell
invasion through a Matrigel-coated membrane (Fig. 6B).
LZAP suppresses tumorigenicity of HCC in vivo
To assess the role of LZAP in tumor growth in vivo, the HepG2
or sk-Hep1 cells infected with Ad-control and Ad-LZAP were
injected subcutaneously into nude mice. The results showed that
LZAP overexpression in the HCC cells significantly delayed tumor
growth in the mice (Fig. 7A). Furthermore, the mean tumor
volume in the LZAP overexpressed group at the end of
observation was significantly smaller than that of the control
group (49.37 mm3vs. 743.57 mm3for HepG2, 74.48 mm3vs.
464.81 mm3for sk-Hep1; Fig. 7A and B). Accordingly, the mean
tumor weight in the LZAP overexpressed group was markedly
lower than in the control group (0.037 g vs. 0.646 g for HepG2,
0.062 g vs. 0.329 g for sk-Hep1; Fig. 7C).
In the present study, we used a relatively large series of clinical
tissue samples to explore the role of LZAP in HCC for the first
time. We examined LZAP mRNA and protein expression in
paired primary HCC samples and HCC cell lines using real-time
quantitative PCR and western blotting. We found that LZAP
expression was down-regulated at both the transcriptional and
translational levels in most primary HCC tumor tissues and
HCC cell lines. Consistent with these observations, immunohis-
Table 1. Relationship between LZAP expression and
clinicopathological features of 126 patients with
All cases 12676 50
,25 ug/l38 1622
Yes 18 108
LZAP and Hepatocellular Carcinoma
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tochemical analyses also showed that LZAP expression was
decreased in most HCC tumor tissues compared with the
corresponding non-tumorous liver tissues. These results indicat-
ed that down-regulated LZAP expression may play a role in
In the immunohistochemical analysis, decreased LZAP expres-
sion in HCC was significantly associated with tumor size,
histological differentiation, and serum AFP. The relationship
between low LZAP expression and larger tumor size suggested
that the decline in LZAP expression may help facilitate the rapid
expansion of the tumor. Additionally, most of the well-differen-
tiated HCC samples were positive for LZAP expression, but LZAP
expression was profoundly weaker in the moderately and poorly
differentiated tumor samples. Thus, decreased LZAP expression is
correlated with poor differentiation in HCC cells and may further
promote HCC progression.
Figure 2. Immunohistochemical analysis of the LZAP protein expression in the primary hepatocellular carcinoma surgical
specimens. (A) and (F) Immunostaining of an HCC tumor and the adjacent non-tumorous area. (B) and (G) Normal liver tissue distant from the
tumor, scored as LZAP (+++). (C) and (H) Well-differentiated HCC, scored as LZAP (++). (D) and (I) Moderately differentiated HCC, scored as LZAP (+).
(E) and (J) poorly differentiated HCC, scored as LZAP (2). N: non-tumor tissue; T: tumor tissue (A–E with 2006 magnification; F–J with 4006
Figure 3. The Kaplan–Meier survival analysis of the primary HCC patients (n=126) with high LZAP expression (n=50) and low LZAP
expression (n=76) after surgical resection. Based on their LZAP immunostaining scores, the HCC patients were divided into low-LZAP
expression (LZAP2 or LZAP+) and high-LZAP expression (LZAP++ or LZAP+++) groups. The survival rate of the patients in the low-LZAP group was
significantly lower than that of the patients in the high-LZAP group (p=0.008 for the log-rank test).
LZAP and Hepatocellular Carcinoma
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Our Kaplan-Meier survival analysis revealed that low LZAP
expression was significantly linked to poor prognosis after surgical
resection in the HCC patients (p=0.008). Furthermore, LZAP
expression was an independent prognostic factor for overall
survival in the multivariate analysis. These results suggest that
LZAP can serve as a new predictor of prognosis in HCC patients
after surgical resection.
Based on the low LZAP expression in the HepG2 and sk-Hep1
HCC cell lines, we transfected full-length LZAP into these cells to
further examine the mechanism by which it suppresses HCC
progression. Restoring LZAP expression significantly suppressed
cell proliferation and colony formation. In parallel experiments,
we also found that LZAP overexpression suppressed tumor growth
in injectable mouse models. Consistent with our results, a previous
Table 2. Univariate and multivariate analysis of overall survival in hepatocellular carcinoma.
Univariate analysisMultivariate analysis
p valueHR 95% CIp value
LZAP 0.538 0.337–0.858 0.009a
Age 0.886 0.574–1.3660.583
Gender 0.702 0.305–1.6130.404
Tumor size1.413 0.906–2.2040.127
Histologic grade1.772 1.272–2.4700.001a
Liver Cirrhosis 0.7000.454–1.081 0.108
Serum AFP 2.271 1.329–3.8810.003a
Distant Metastasis1.504 0.858–2.6380.154
HR Hazard ratio, CI confidence interval.
Figure 4. LZAP protein expression in the HepG2 and sk-Hep1 cells infected with Ad-control and Ad-LZAP. (A) The LZAP expression
(green, mainly in the cytoplasm) in the HepG2 cells infected with Ad-LZAP was strikingly higher than that that in the cells infected with the Ad-
control. (B) The LZAP expression (green, in the cytoplasm) was also higher in the sk-Hep1 cells infected with Ad-LZAP than in the sk-Hep1 cells
infected with the Ad-control. The cell nuclei (blue) were stained with DAPI. 406magnification. Inset: 1006magnification.
LZAP and Hepatocellular Carcinoma
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Figure 5. Overexpression of LZAP decreases HCC cell viability. (A) LZAP inhibited colony formation in the HepG2 and sk-Hep1 cells. The
images are shown on the left and on the right, and the mean 6 SD of the foci for each group are shown. The experiments were performed in
triplicate. The p values were calculated using the Student’s t-test. (B) The MTS assay, showing that LZAP suppressed the proliferation of the HepG2
and sk-Hep1 cells. (C) The effect of LZAP overexpression on the cell cycle. LZAP overexpression caused G1 arrest in the HepG2 and sk-Hep1 cells. (D)
The effect of LZAP overexpression on apoptosis in the HepG2 and sk-Hep1 cells at 48 hours, 72 hours, and 96 hours after the adenovirus infection.
LZAP induced cell apoptosis, especially at 72 hours and 96 hours. *p,0.05 versus Ad-control; **,0.01 versus Ad-control.
Figure 6. Transwell migration assays and Matrigel invasion assays of HepG2 and sk-Hep1 cells infected with Ad-LZAP and Ad-
control. Images are shown on the left (magnification: 1006), and the quantification of 10 randomly selected fields is shown on the right. The values
shown are expressed as the mean 6 SD of three independent experiments. The p values were calculated using Student’s t-test. (A) LZAP inhibited
cell migration in the HepG2 and sk-Hep1 cells. (B) LZAP also significantly inhibited cell invasion by the HepG2 and sk-Hep1 cells. *p,0.05 versus Ad-
control; **,0.01 versus Ad-control.
LZAP and Hepatocellular Carcinoma
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study has shown that decreased LZAP expression in human head
and neck squamous cell carcinomas promotes cellular transfor-
mation and xenograft tumor growth . LZAP overexpression
also led to G1 phase cell cycle arrest and induced apoptosis of
HCC cells, indicating that LZAP suppresses HCC tumorigenicity
by inducing cell cycle arrest and apoptosis. Wang et al. have also
confirmed that LZAP expression, in addition to ARF, increases the
percentage of cells in the G1 phase of the cell cycle . In our
study, we also found that LZAP overexpression inhibited the
migration and invasion of the HepG2 and sk-Hep1 cells,
suggesting that LZAP may suppress tumor metastasis. Consistent
with our results, Wang et al. have found that losing LZAP
increases cellular invasion . This finding strengthens the
hypothesis that LZAP acts as an HCC tumor suppressor.
In conclusion, our study found that LZAP expression was down-
regulated in the majority of the HCC clinical tissue specimens at
both mRNA and protein levels and that low LZAP expression may
be correlated with unfavorable prognosis in HCC patients. Cell
culture studies confirmed that LZAP overexpression inhibited cell
proliferation, colony formation, migration/invasion and induced
cell cycle arrest, apoptosis in HCC cell lines. The mouse model
experiments revealed that LZAP overexpression significantly
inhibited the tumor growth. Our findings provide evidence that
LZAP could be a novel prognostic biomarker and a new molecular
therapy target for HCC.
Materials and Methods
Cell lines and culture conditions
The HepG2, Hep3B and sk-Hep1 HCC cell lines were obtained
from the American Type Culture Collection (ATCC). The LO2,
Bel7402 and HEK293 cell lines were obtained from the
Committee of Type Culture Collection of the Chinese Academy
of Sciences (Shanghai, China). The Huh7 cell line was obtained
from the RIKEN cell bank (Ibaraki, Japan). The LO2, Bel7402
and sk-Hep1 cells were cultured in RPMI 1640 supplemented with
10% heat-inactivated FBS (fetal bovine serum) and 1% penicillin-
streptomycin. The HepG2, Hep3B, Huh7 and HEK293 cells were
cultured in DMEM (Dulbecco modified Eagle medium) supple-
mented with 10% heat-inactivated FBS (fetal bovine serum) and
1% penicillin-streptomycin. All cells were incubated at 37uC in a
humidified chamber containing 5% CO2.
Patients and tumor tissue samples
A total of 126 human primary HCC tissues and matched
control tissues were obtained from patients who underwent
hepatectomy at the Sun Yat-sen University Cancer Center
between 2001 and 2004. None of these patients had received
preoperative chemotherapy or radiotherapy. The follow-up data
from the HCC patients in this study were available and complete.
The postoperative follow-up occurred at our outpatient depart-
ment and included clinical and laboratory examinations every 3
months for the first 2 years, every 6 months during the third to
fifth years, and annually for an additional 5 years or until patient
death, whichever occurred first. Overall survival, which was
defined as the time from the operation to patient death or the last
follow-up, was used as a measure of prognosis. Both the tumor
and the corresponding non-tumor tissues not less than 2 cm away
from the HCC were sampled, and the diagnosis was confirmed
by pathological examination. After surgical resection, the
matched fresh tissues were immediately immersed in RNAlater
(Ambion, Inc., USA), kept at 4uC overnight, then stored at
280uC until the RNA isolation. All the tissue samples were fixed
in 10% formalin and embedded in paraffin, and consecutive
2 mm sections were cut. Histological types were assigned
according to the WHO classification criteria. This study was
approved by the Ethics Committee of the Sun Yat-sen University
Cancer Center, and written informed consent was obtained from
Figure 7. LZAP suppresses the tumorigenicity of HCC in vivo. HepG2 or sk-Hep1 cells infected with Ad-control and Ad-LZAP were injected
into nude mice, as described in the Materials and Methods section. The tumor volumes were measured every 3 days. At the end of the experiment,
the animals were sacrificed and the tumors were excised for volume and weight measurement. (A) The tumor growth curves for each group. The
tumor growth rate was reduced in the tumors that overexpressed LZAP. (B) Photographs of dissected tumors from the nude mice. The final tumor
volumes were smaller in the HepG2-LZAP and sk-Hep1-LZAP groups than in the control group. (C) The tumor weights of each group. The final tumor
weights were reduced in the tumors that overexpressed LZAP. The data are presented as mean 6 SD. *p,0.05 versus Ad-control; **,0.01 versus Ad-
LZAP and Hepatocellular Carcinoma
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RNA extraction and real-time quantitative PCR
Total RNA was extracted using TRIzol solution (Invitrogen,
USA) according to the manufacturer’s instruction. RNase-free
DNase I was used to remove DNA contamination. The total RNA
concentration and quantity were assessed by absorbency at
260 nm using a Nanodrop spectrophotometer (ND-1000, Thermo
Scientific, USA). The first-strand cDNA synthesis was performed
using 2 mg of total RNA and M-MLV reverse transcriptase
according to the manufacturer’s instructions (Promega, USA). The
resulting cDNAs were subjected to real-time PCR analysis to
evaluate the relative expression levels of LZAP and GAPDH (an
internal control) using the following primers: 59- TCTGGG-
TCCTACATTCACTACTTTC-39 (F) and 59-CTCCTGCCAA-
TCCTTCATCC-39 for LZAP; and 59-CTCCTCCTGTTCGA-
CAGTCAGC-39 (F) and 59- CCCAATACGACCAAATCCGTT-
39 for GAPDH. Each 15 ml of reaction volume contained 0.5 ml of
cDNA that was synthesized as above, 7.5 ml of 26SYBR Green
master mix (Invitrogen, USA), and 200 nM of each pair of
oligonucleotide primers described above. The cycling parameters
began with 95uC for 10 minutes, followed by 45 cycles of 90uC for
30 seconds and 60uC for 60 seconds, followed by a melting curve
analysis. Ct was measured during the exponential amplification
phase, and the amplification plots were analyzed using the
software provided with the instrument (SDS 2.0). The relative
expression levels of the target gene were normalized to that of the
internal control gene, GAPDH. The data were analyzed using the
comparative threshold cycle (22DDCT) method.
Protein extraction and western blotting analysis
The frozen HCC samples, including the tumor tissue, non-
tumor control tissue and cells from the HCC cell lines (LO2,
HepG2, Hep3B, Huh7, Bel7402 and sk-Hep1), were homogenized
in a RIPA lysis buffer, and the lysates were cleared by
centrifugation (14,000 rpm) at 4uC for 30 minutes. Approximately
40 mg of protein sample were run on a 12% SDS-PAGE gel and
transferred to PVDF membranes. After blocking the non-specific
binding sites for 60 minutes with 5% non-fat milk, the membranes
were incubated with primary monoclonal antibodies against LZAP
(at a 1:1000 dilution) or GAPDH (at a 1:10000 dilution) overnight
at 4uC. Next, the membranes were subjected to three 15 minute
washes with TBST and then incubated with HRP-conjugated
secondary antibody (at a 1:2000 dilution) for 45 minutes at room
temperature. The membrane was washed three more times with
TBST and developed using an enhanced chemiluminescence
system (ECL, Cell Signaling Technologies).
Immunohistochemistry and immunofluorescence
Paraffin-embedded tissue blocks were sectioned for immuno-
histochemistry. The sections were deparaffinized and rehydrated
with graded ethanol. For the antigen retrieval, the slides were
immersed in EDTA (1 mmol/L, pH 8.0) and boiled for
15 minutes in a microwave oven. After rinsing with PBS, the
endogenous peroxidase was blocked with 0.3% hydrogen peroxide
for 15 minutes at room temperature. The slides were incubated
with the primary antibody (mouse anti-LZAP monoclonal
antibody, Santa Cruz Biotechnology, USA, at a 1:500 dilution)
overnight in a humidified chamber at 4uC. The sections were
washed three times with PBS, incubated with horseradish
peroxidase-conjugated secondary antibody (EnvisionTMDetection
Kit, GK500705, Gene Tech) at 37uC for 30 minutes, and then
washed three more times with PBS. Finally, 3, 39-diaminobenzi-
dine tetrahydrochloride (DAB) was used for signal development,
and the sections were counterstained with 20% hematoxylin. The
slides were dehydrated, cleared and evaluated. Each sample was
incubated with an isotypic antibody dilution under the same
experimental conditions as the negative control.
The HepG2 or sk-Hep1 cells infected with the Ad-control or
Ad-LZAP were plated on glass coverslips. At 48 hours post-
infection, the coverslips were washed extensively in PBS and fixed
with 4% paraformaldehyde. After rinsing with PBS, the cells were
permeabilized with 0.2% Triton X-100 in PBS for 10 minutes.
The coverslips were then washed and blocked with 1% BSA in
PBS for 60 minutes. The slides were incubated with mouse anti-
LZAP monoclonal antibody (Santa Cruz Biotechnology, USA, at a
1:100 dilution) in PBS with 0.2% Triton X-100 and 0.1% BSA at
room temperature for 1 hour. The slides were then washed
extensively with PBS and treated with fluorescein (FITC)-
conjugated goat anti-mouse secondary antibody (Santa Cruz
Biotechnology, USA) for 30 minutes. After further washing, the
slides were labeled with DAPI and images were acquired using a
Laser Scanning Confocal Microscope.
The total LZAP immunostaining score was calculated as both
the percentage of positively stained tumor cells and the staining
intensity. The percent positivity was scored as ‘‘0’’ (,5%,
negative), ‘‘1’’ (5%–25%, sporadic), ‘‘2’’ (25%–50%, focal), or
‘‘3’’ (.50%, diffuse). The staining intensity was scored as ‘‘0’’ (no
staining), ‘‘1’’ (weakly stained), ‘‘2’’ (moderately stained), or ‘‘3’’
(strongly stained). Both the percentage of positive cells and the
staining intensity were evaluated under double-blind conditions.
The LZAP immunostaining score was calculated as the percentage
positive score 6the staining intensity score and ranged from 0 to
9. We defined the LZAP expression levels as follows: ‘2’ (score 0–
1), ‘+’ (score 2–3), ‘++’ (score 4–6) and‘+++’ (score .6). Based on
the LZAP expression levels, the HCC patients were divided into
two groups: the low LZAP expression group (LZAP2 or LZAP+)
and the high LZAP expression group (LZAP++ or LZAP+++).
RNA oligonucleotides and cell transfections
The siRNAs for the LZAP knockouts were synthesized by
GenePharma (Shanghai, China). The siRNA sequences were as
follows: siLZAP-1#, sense=59- GGAGAUUAUAGCUCUGU-
AUTT-39 and antisense=59- AUACAGAGCUAUAAUCUCC-
TT- 39; siLZAP-2#, sense=59- GAGAUCCCCUCACUGAA-
GATT -39 and antisense=59- UCUUCAGUGAGGGGAUCU-
CTT -39; siLZAP-3#, sense=59- CCCUGACACUGCUUGA-
AUATT- 39 and antisense=59- UAUUCAAGCAGUGUCAG-
GGTT -39; and negative control (NC), sense=59- UUCUCC-
GAACGUGUCACGUTT-39and antisense=59- ACGUGACA-
The HepG2 and sk-Hep1 cells were infected with Ad-LZAP
and transfected with 20 mM siLZAP or NC 48 hours later. The
transfection was performed using the Lipofectamine RNAi MAX
reagent (Invitrogen, USA) according to the manufacturer’s
Recombinant adenovirus construction and tumor cell
The LZAP-recombined adenoviral expression vector and the
control vector were constructed by the rapid BP/LR reaction in
the Gateway cloning system (Invitrogen, USA) according to the
manufacturer’s instructions. PacI enzyme-linearized adenoviral
vectors were transfected into the HEK293 cells using Lipofecta-
mine 2000 (Invitrogen, USA). At 10–13 days after the transfection,
when an approximately 80% cytopathic effect (CPE) was observed
in the HEK293 cells, the adenovirus-containing HEK293 cells and
LZAP and Hepatocellular Carcinoma
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media were collected. Three freeze/thaw cycles followed by
centrifugation were used to prepare the viral lysates. The Ad-LZAP
and Ad-control titers were measured with an Adenovirus Titer
Immunoassay Kit (Innogent, China). The recombinant adenovi-
ruses were stored at 280uC for use. The HepG2 and sk-Hep1 cells
were cultured in 6-well plates and infected with adenovirus (Ad-
LZAP and Ad-control) at a multiplicity of infection (MOI) of 200.
Colony formation assay
The cells infected with Ad-LZAP and Ad-control (16103) were
plated in each well of a 6-well plate. The surviving colonies (.50
cells) were counted with crystal violet staining after two weeks of
culture. Colony-forming efficiency (CFE %) was defined as the
ratio of the number of colonies formed in culture to the number of
cells inoculated. This experiment was performed in triplicate.
The MTS cell proliferation assay was used to evaluate the growth
rate of the cells infected with Ad-LZAP and Ad-control. The cells
were seeded in 96-well plate at a density of 56103per well. The
growth rate was detected using the MTS cell proliferation kit
according to the manufacturer’s instructions (Promega, USA).
Three independent experiments were performed.
Cell cycle assay
The cell cycle analysis was performed at 48 hours after the cells
were infected. The HepG2 or sk-Hep1 cells infected with Ad-
LZAP and Ad-control were washed twice with ice-cold PBS and
fixed with ice-cold 75% ethyl alcohol at 220uC for one hour. After
two PBS washes, the cells were resuspended in 400 ml of ice-cold
PBS and incubated with RNase in a 37uC water bath for
30 minutes. The cells were subsequently incubated with propi-
dium iodide at 4uC in the dark for 30–60 minutes and analyzed
using a flow cytometer (Beckman, USA).
The apoptosis assays were performed at 48 hours, 72 hours and
96 hours after the cells were infected with the adenovirus. The
HepG2 and sk-Hep1 cells infected with Ad-LZAP or Ad-control
were washed twice in ice-cold PBS, resuspended in 400 ml of 16
Binding Buffer and incubated with Annexin V-FITC (Bestbio,
China) for 15 minutes at 4uC in the dark, according to the
manufacturer’s instructions. After staining, the cells were incubat-
ed with propidium iodide for 5 minutes at 4uC in the dark and
then analyzed using a flow cytometer (Beckman, USA).
Cell migration assay
The cell migration assays were performed in a chamber system
consisting of polycarbonate membrane inserts with an 8-mm pore
size (Corning, USA) placed in 24-well cell culture insert
companion plates. The migration assay was conducted at 48 hours
after the HepG2 and sk-Hep1 cells were infected with Ad-control
or Ad-LZAP. The cells (56104in 100 ml of growth medium
without FBS) were placed in the upper chamber and 500 ml of
growth medium with 10% FBS was placed in the lower chamber.
The cells were incubated at 37uC for 12 hours. Following the
incubation, the insert membranes were fixed with 75% methanol
for 30 minutes. The cells on the upper surface were removed with
cotton-tipped swabs, and the migrated cells on the lower surface
were stained with 0.5% crystal violet containing 20% methanol for
60 minutes. The stained cells were counted under an inverted
microscope (10 fields per membrane). Each experiment was
performed in triplicate.
Matrigel invasion assay
The Matrigel invasion assay was performed in a chamber system
consisting polycarbonate membrane inserts with 8-mm pores
(Corning, USA) placed in 24-well cell culture insert companion
plates. The inserts were coated with a thin layer of 0.5 mg/ml
Matrigel Basement Membrane Matrix (BD Biosciences, Bedford,
MA). The invasion assay was conducted at 48 hours after the
HepG2 and sk-Hep1 cells were infected with Ad-control or Ad-
LZAP. The cells (16105in 200 ml of growth medium without FBS)
were placed in the upper chamber, and 0.5 ml of growth medium
containing 20% FBS was placed in the lower chamber. The cells
were incubated at 37uC and allowed to invade through the Matrigel
layer for 48 hours. After incubation, the insert membranes were
fixed with 75% methanol for 30 hours. The cells on the upper
surface were removed with cotton-tipped swabs, and the invading
cells on the lower surface were stained with 0.5% crystal violet
containing 20% methanol for 60 minutes. The stained cells were
counted under an inverted microscope (10 fields per membrane).
Each experiment was performed in triplicate.
Tumorigenicity assays in nude mice
Female BALB/c athymic nude mice (4–5 weeks old) were
obtained from the Medical Experimental Animal Center of
Guangdong Province. The mice were randomly divided into 4
groups of 8 mice each. Group 1 was injected with HepG2 cells that
had been infected with Ad-control; Group 2 was injected with
HepG2 cells that had been infected with Ad-LZAP; group 3 was
injected with sk-Hep1 cells that had been infected with Ad-control;
group 4 was injected with sk-Hep1 cells that had been infected
with Ad-LZAP. For the injections, 86106tumor cells were
suspended in 200 ml PBS and then subcutaneously injected into
the posterior flank of the mice. The tumor size was monitored
every 3 days by measuring the length (L) and width (W) of the
tumor with calipers. The tumor volume was calculated according
to the following formula: (L6W2)/2. At 4–5 weeks after
inoculation, all the mice were sacrificed, and the tumors were
harvested and photographed. The weight of the tumors was also
measured. All the experimental procedures involving animals were
performed in accordance with the Guide for the Care and Use of
Laboratory Animals (NIH publications Nos. 80–23, revised 1996)
and the institutional ethical guidelines for animal experiments.
The statistical analyses were performed using the Statistical
Package for the Social Sciences, version 16.0 (SPSS Inc., Chicago,
IL, USA). A paired-samples t-test was used to compare LZAP
mRNA and protein expression in the HCC tumors with that of
their paired adjacent non-cancerous tissue samples. The correla-
tion between tumor LZAP expression and the clinical and
pathological features was performed using x2 tests. Overall
survival curves were calculated with the Kaplan-Meier method
and were analyzed with the log-rank test. A Cox proportional-
hazards analysis was used in univariate and multivariate analyses
to explore the effects of LZAP expression and HCC clinicopath-
ological variables on survival. The results were expressed as mean
6 SD and analyzed using the Student’s t-test. Differences were
considered significant at p,0.05.
(LO2) and the HepG2 and sk-Hep1 cells infected with
Ad-LZAP at different titers. Western blotting showed that the
LZAP expression in the HepG2 and sk-Hep1 cells infected with
LZAP protein expression in normal liver cells
LZAP and Hepatocellular Carcinoma
PLoS ONE | www.plosone.org9October 2011 | Volume 6 | Issue 10 | e26608
Ad-LZAP at MOIs of 50, 100, 200 and 400 was significantly
higher than that of the HepG2 or sk-Hep1 cells infected with the
Ad-control. The LZAP expression in the HepG2 and sk-Hep1
cells infected with Ad-LZAP at a MOI of 200 was higher than that
in normal liver cells.
cells infected with Ad-LZAP at MOI 200. Western blotting
showed that siLZAP-3# had the highest knockout efficiency of the
three siRNAs tested. Therefore, siLZAP-3# was used for all the
LZAP silencing in the HepG2 and sk-Hep1
cells infected with Ad-LZAP at a MOI of 200 and its effect
on cell viability. (A) The silencing of LZAP expression in the
LZAP knockout in the HepG2 and sk-Hep1
HepG2 and sk-Hep1 cells infected with Ad-LZAP significantly
increased cell proliferation, as assessed by the MTS assay. The
proliferation levels were similar in the knockout cells and in the
cells infected with the Ad-control. (B) The colony formation assays
revealed a marked increase in colony number and size after the
LZAP knockout in the HepG2 and sk-Hep1 cells infected with Ad-
LZAP. Similar values were found for the HepG2 and sk-Hep1
cells infected with the Ad-control. *p,0.05; NS: not significant.
Conceived and designed the experiments: JCX KP. Performed the
experiments: JJZ KP JJL YBC. Analyzed the data: JJZ JGC LL DDW
QZP. Contributed reagents/materials/analysis tools: MSC. Wrote the
paper: JJZ KP.
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