ONCOLOGY LETTERS 3: 1249-1254, 2012
Abstract. Acquisition of anoikis resistance is a prerequisite
for the metastasis of hepatocellular carcinoma (HCC) cells.
Activation of growth factor signaling pathways and rearrange-
ment of the cytoskeleton have been reported as vital steps
in this process. However, key molecules involved in anoikis
resistance remain to be determined. The aim of this study
was to investigate the effect of CD147 on HCC cells resistant
to anoikis. The human SMMC-7221 human HCC cell line
was used. Immunofluorescence was used to investigate the
expression levels of CD147. Anoikis-induced cell death was
assessed using trypan blue exclusion. In the present study,
the results showed that SMMC-7721 HCC cells exhibited
significant morphological changes when suspended in culture
medium supplemented with 1% methocel and a subpopulation
of cells resistant to anoikis was acquired with higher viability
and invasion ability. CD147 was identified to be significantly
increased in cells resistant to anoikis, when compared to the
parental cells. CD147 knockdown by siRNA notably induced
cell anoikis, partially through the inactivation of PI3K/Akt
pathway. All of these evidence provide a novel CD147-related
mechanism underlying the metastasis of HCC cells.
Metastasis is a multi-step biological process and becomes the
primary cause of cancer-related mortality in most solid tumors.
Therefore, preventing the metastatic spread of cancer cells is a
principal task in cancer therapy. During the metastatic process,
cells were required to detach from their primary site, and
then to migrate to the lymphatic and circulatory systems (1).
In metastasis, the majority of cells undergo anoikis, which
is induced by the detachment of anchorage-dependent cells
from the surrounding extracellular matrix (2). Acquisition of
anoikis-resistance is therefore a prerequisite for the metastasis
of cancer cells. Recently, several reports have confirmed
that the breakdown of anoikis contributes prominently to the
malignancy of breast (3), colon (4), lung (5) and head and
neck carcinomas (6). However, few studies have focused on
the role of anoikis-resistance in hepatocellular carcinoma
In recent years, accumulating studies indicate that anoikis
is mainly mediated by the death receptor pathway activated by
caspase (7,8). This process occurs in hepatocytes (9). Activation
of growth factor signaling pathways, which commonly occurs
in carcinoma cells, enables cells to acquire anoikis resistance
(10,11). Numerous kinase/phosphatase signaling molecules
are known to be involved in anoikis as central regulators,
such as Ras (12), Akt and raf (13). Other reports have also
confirmed that cell cytoskeletal rearrangement is critical for
the cells to survive anoikis resistance (14,15). Moreover, this
process is especially associated with cadherin family members,
which are the key elements in the complex network of survival
CD147, a member of the immunoglobulin superfamily,
was initially characterized as an inducer of matrix metal-
loproteinase (MMP) synthesis (17). CD147 is known to play
a vital role in tumor progression. High expression levels of
CD147 in epithelial carcinoma tissue are significantly associ-
ated with poor prognosis (18,19). Recently, CD147 has been
found to promote epithelial-mesenchymal transition (EMT)
through transforming growth factor (TGF)-β signaling (20)
and serve as an anoikis suppressor by the inhibition of Bim,
Acquisition of anoikis resistance through CD147 upregulation:
A new mechanism underlying metastasis
of hepatocellular carcinoma cells
XIA KE1, LING LI2, HONG-LIN DONG3 and ZHI-NAN CHEN1
1State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237;
2Cell Engineering Research Center and Department of Cell Biology, Fourth Military Medical University,
Xi'an, Shaanxi 710032; 3Department of General Surgery, Second Hospital,
Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
Received February 1, 2012; Accepted February 17, 2012
Correspondence to: Dr Zhi-Nan Chen, State Key Laboratory
of Bioreactor Engineering, East China University of Science and
Technology, Shanghai 200237, P.R. China
Dr Hong-Lin Dong, Department of General Surgery, Second Hospital,
Shanxi Medical University, Taiyuan, Shanxi 030001, P.R. China
Abbreviations: HCC, hepatocellular carcinoma; EMT, epithelial-
mesenchymal transition; PI3K, phosphoinositide-3 kinase; PI, propidium
iodide; siRNA, small interfering RNA; SNC, scrambled negative control
Key words: anoikis resistance, CD147, PI3K/Akt, hepatocellular
KE et al: CD147 PROMOTES HCC ANOIKIS RESISTANCE THROUGH PI3K
a pro-apoptotic protein interacting with Bcl-2 to mediate cell
death in breast cancer cells (21). In addition, a radio-immuno-
conjugate 131I-labeled antibody HAb18 F(ab')2 against CD147,
registered as Licartin, was used to treat primary HCC and
prevent tumor recurrence of post-orthotopic liver transplan-
tation in advanced HCC patients (22). However, the role of
CD147-induced anoikis resistance in HCC metastasis remains
The aim of the present study was to investigate the upregu-
lation of CD147-conferred HCC cell resistance to anoikis
partially through activation of the phosphoinositide-3 kinase
(PI3K)/Akt pathway, thereby providing a novel mechanism
supporting HCC metastasis.
Materials and methods
Cell culture and anoikis assay. The SMMC-7721 human
HCC cell line was purchased from the Shanghai Institute for
Biological Sciences (Shanghai, China) and routinely cultured
in RPMI-1640 medium (Hyclone Laboratories, Logan, UT,
USA) supplemented with 10% fetal bovine serum (Gibco,
Rockville, MD, USA). HEK293ar cells were obtained as
previously reported (23). The anoikis assay was essentially
performed as described by Frisch (24). Briefly, flasks were
pre-coated with 2% sterilized agar. Cells were trypsinized and
plated onto pre-coated flasks. Suspension medium consisted
of RPMI-1640 supplemented with 1% methocel and 10% FBS.
According to the process of anoikis adaptation for HEK293ar
cells (23), a subpopulation of SMMC-7721 cells resistant to
anoikis was obtained. Cultures were maintained routinely at
37˚C under a mixture of 95% air and 5% CO2.
Immunofluorescence. SMMC-7721 cells were suspended in
methocel medium for the indicated times. Cells were harvested
and dried on coverslips. Double-staining of Hoechst 33342 and
propidium iodide (PI) was developed to evaluate the pheno-
type and viability of treated cells. To examine the expression
levels of CD147 in treated cells, the cells were firstly fixed
and blocked, and then incubated with anti-CD147 monoclonal
antibody (1:200) (25). After washing, the cell samples were
incubated with FITC-conjugated goat anti-mouse IgG anti-
body (1:1,000; Pierce, Rockford, IL, USA) and the nuclei were
stained with DAPI (1:100; Biotium, Hayward, CA, USA). The
stained cells were examined with a laser scanning microscope
(Olympus, Tokyo, Japan).
Antibodies and immunoblot analysis. For the immunoblot
analysis, cells were lysed in RIPA buffer (Beyotime Inc.,
NanTong, China). Protein concentration was determined with
the Bradford reagent (Beyotime Inc.). Equal amounts of total
proteins were separated and transferred to polyvinylidene
difluoride membrane (Millipore, Bedford, MA, USA). The
membranes were subsequently immunoblotted with the appro-
priate primary antibody. The following primary antibodies
were used in this study: anti-CD147 antibody (25), anti-Akt
and anti-phosphorylated Akt (ser 473) antibody from Cell
Signaling Technology (Beverly, MA, USA). A secondary
horseradish peroxidase-conjugated goat anti-mouse antibody
(Pierce) was finally used for signal detection with an ECL kit
(Pierce) according to the manufacturer's instructions.
Trypan blue exclusion assay. The anoikis-induced cell
death was evaluated by trypan blue exclusion assay as previ-
ously described (26). In brief, the parental SMMC-7721
and SMMC-7721 cells resistant to anoikis were suspended
in methocel medium for the indicated times. Cells were
harvested, washed three times with PBS and resuspended in
100 µl PBS. After mixing with 100 µl of 0.8% trypan blue, the
cells were counted using a hemocytometer.
In vitro invasion assay. The migration ability was measured
by in vitro invasion assay as previously reported (27). The
parental SMMC-7721 and SMMC-7721 cells resistant to
anoikis were suspended in methocel medium for 48 h, resus-
pended in serum-free DMEM medium containing 0.5% FBS
and then added to the upper compartment of a chamber at
a total number of 2x105 cells per chamber. After incubation
for 24 h, the number of cells migrating through the filter was
counted by crystal violet staining and plotted as the mean
number of migrating cells per optic field in three independent
siRNA transfection. siRNA sequences corresponding to the
cDNA sequences of CD147 (Genebank accession NM_001728)
were designed (5'-GGUUCUUCGUGAGUUCCUCdTdT-3'
and 5'-GAGGAACUCACGAAGAACCdTdT-3'). Briefly, the
subpopulation of SMMC-7721 cells resistant to anoikis was trans-
fected with CD147 siRNA (100 nM) using Lipofectamine 2000
(Invitrogen, Carlsbad, CA, USA). Scrambled negative control
(SNC) siRNA was used as mock control. Transiently-transfected
cells were recovered for 24 h and re-plated on agar-coated
Apoptosis assay. SMMC-7721 cells resistant to anoikis trans-
fected with CD147 siRNA or control siRNA were suspended
in fresh methocel medium in an agar-coated plate for 24 h.
Cells were harvested and treated with 10 mmol/l EDTA to
disrupt the cell-cell contacts. The cells were then analyzed
by double-staining with Annexin V-FITC and PI using an
Apoptosis Detection kit (Calbiochem, San Diego, CA, USA)
followed by detection using a FACSCalibur flow cytometer.
Early apoptotic cells were labeled as Annexin+/PI- and necrotic
cells were labeled as PI+. The percentages of cells at early
apoptosis or necrotic stage were analyzed at least three times.
Statistical analysis. Statistical analyses were performed using
the SPSS 16.0 statistical software package (SPSS Inc., Chicago,
IL, USA). Statistical significance of the differences was
determined using Student's t-test. All P-values were based on
two-sided tests. P<0.05 was considered statistically significant.
Anoikis of suspended SMMC-7721 cells and formation of
multi-cellular spheroids. To characterize the morphological
change of SMMC-7721 cells after suspension in methocel
medium, typical images representing the morphology of cells
after treatment for 0, 24 and 48 h are shown in Fig. 1A. Cell-cell
contacts were spontaneously and gradually formed and multi-
cellular spheroids were generated in a time-dependent manner.
The volume of the aggregated spheroids was significantly
ONCOLOGY LETTERS 3: 1249-1254, 2012
increased at the time point of 48 h as indicated in Fig. 1B.
The viability of the SMMC-7721 cells was further analyzed by
double-staining of Hoechst 33342 and PI under a laser micro-
scope. At 0 h, the staining assay indicated that the treated
cells were alive (exclusion of PI). After cells were cultured for
24 h, anoikis with positive staining of PI was induced in some
of the cells and apoptotic bodies were also found along the
cytoplasmic membrane. Multi-cellular spheroids were found
at the time-point of 48 h, when the majority of cells underwent
anoikis with a strong staining of PI and a weak staining of
Hoechst 33342 (Fig. 3C).
CD147 was upregulated on the cell-cell contacts in
suspended multi-cellular spheroids. To explore the role of
CD147 in anoikis resistance, we examined the expression
levels of CD147 in HCC cells. Western blotting showed that
SMMC-7721 cells in suspended culture exhibited a notable
time-dependent accumulation of the CD147 protein (Fig. 2A).
Immunofluorescence staining confirmed the elevated levels of
CD147 in SMMC-7721 cells after suspension for 48 h (Fig. 2B).
Additionally, in a large multi-cellular spheroid, the strong
CD147 staining areas were restricted to the cell-cell contacts
for both SMMC-7721 and HEK293ar cells as indicated by the
yellow arrows in Fig. 2B.
CD147 was significantly upregulated in anoikis-resistant
SMMC-7721 cells with an increase in both viability and inva-
sion ability. Based on a previous study (23), a subpopulation
of anoikis-resistant SMMC-7721 cells was obtained to explore
the correlation between CD147 expression levels and anoikis
resistance. Morphological changes occurred in cells during
the adaptation period for SMMC-7721 cells in suspension
culture (Fig. 3A). Western blotting indicated that CD147 was
significantly upregulated in the anoikis-resistant SMMC-7721
cells, compared to the parental SMMC-7721 cells (Fig. 3B).
The trypan blue exclusion assay demonstrated that a high
percentage of the anoikis-resistant SMMC-7721 cells remained
alive following suspension in culture medium for 6 days, while
almost all the parental SMMC-7721 cells underwent death
(Fig. 3C). In vitro invasion assay showed that the invasion ability
was significantly increased in SMMC-7721 cells resistant to
anoikis, compared to the parental SMMC-7721 cells (Fig. 3D).
Anoikis was induced by the knockdown of CD147 expression
in the subpopulation of SMMC-7721 cells resistant to anoikis
through inactivation of the PI3K/Akt pathway. To prove the
direct involvement of CD147 during the adaptation process of
Figure 1. Anoikis of suspended SMMC-7721 cells and formation of multi-
cellular spheroids. (A) Morphology of SMMC-7721 cells in agar suspension
culture at indicated time-points (magnification, x100). (B) Characterization
of the suspended spheroids during suspension culture of SMMC-7721 cells.
The amount of spheroids (3 cells/spheroid) per focus was counted under a
phase-contrast microscope. The volume of spheroids referred to the average
number of cells per multi-cellular spheroids (magnification, x100; n=8-10).
Each value is the mean ± SD of at least triplicate determinations. (C) Double
staining of Hoechst 33342 and PI SMMC-7721 cells after suspension at
different time-points by immunofluorescence under a laser scanning micro-
scope (magnification, x400).
Figure 2. CD147 was upregulated on the cell-cell contacts in suspended multi-
cellular spheroids. (A) Western blotting of CD147 expression in SMMC-7721
cells following suspension at the indicated time-points. Two major forms of
CD147 (45-66 and 35 kDa) were analyzed. Tubulin was used as a loading
control. (B) Expression levels of CD147 in SMMC-7721 and HEK293ar cells
following suspension at the indicated time-points as revealed by immunoflu-
orescence under a laser scanning microscope (n=6-10; magnification, x400).
Yellow arrow points to the enhanced expression levels of CD147 between
cell-cell contacts in a multi-cellular spheroid.
KE et al: CD147 PROMOTES HCC ANOIKIS RESISTANCE THROUGH PI3K
anoikis resistance, we knocked down the expression levels of
CD147 in the subpopulation of anoikis-resistant SMMC-7721
cells through transfection with specific small interfering RNA
(siRNA). Immunofluorescence assay results indicated that the
staining intensity of CD147 in the area of cell-cell contacts was
significantly reduced in anoikis-resistant SMMC-7721 cells
tansfected with CD147 siRNA, compared to those transfected
with SNC siRNA (Fig. 4A). Similarly, the cell nuclei tended to
be cracked in cells transfected with CD147 siRNA following
suspension in methocel medium for 24 h as indicated by the
white arrows (Fig. 4A). Flow cytometry showed that the inhi-
bition of CD147 expression significantly induced anoikis (both
early apoptosis and necrosis) in anoikis-resistant SMMC-7721
cells (Fig. 4B). To elucidate the possible signaling pathway
involved in the CD147-mediated anoikis resistance, western
blot analysis was performed to examine the expression levels
of CD147, phosphorylated Akt and Akt in anoikis-resistant
SMMC-7721 cells transfected with CD147 siRNA and SNC
siRNA, respectively. We found that the knockdown of CD147
resulted in a significant decrease of phosphorylated Akt levels,
whereas the expression of Akt was not affected (Fig. 4C). These
data suggest that activation of the PI3K-Akt signaling pathway
may be involved in CD147-mediated anoikis resistance.
Figure 3. CD147 was significantly upregulated in anoikis-resistant
SMMC-7721 cells with an increase in both viability and invasion ability.
(A) The process of suspension culture adaptation under a phase-contrast
microscope (magnification, x200). The left panel shows the morphology of
SMMC-7721 cells when suspended in methocel medium. The right panel
shows the morphology of SMMC-7721 cells following reattachment to the
extracellular matrix. The subpopulation of anoikis-resistant SMMC-7721
cells was obtained by sequential cycles of suspension and adhesion culture.
(B) Findings of the immunoblot assay with regard to the expression levels of
CD147 in wild-type SMMC-7721 cells and the subpopulation of SMMC-7721
cells resistant to anoikis. Tubulin was used as a loading control. (C) The
roportion of viable cells under suspension culture of wild-type SMMC-
7721 and SMMC-7721 cells resistant to anoikis at the indicated time-points
were detected by trypan blue exclusion assay. Data are the means ± SD for
eight replicates. (D) Images of invading SMMC-7721 or anoikis-resistant
SMMC-7721 cells in a transwell invasion assay after suspension for 48 h, and
column graph for three independent experiments. Data were normalized to
wild-type SMMC-7721 cells and are the means ± SD (*p<0.05).
Figure 4. Anoikis was induced by knocking down CD147 expression in
the subpopulation of SMMC-7721 cells resistant to anoikis by inactivating
the PI3K/Akt pathway. (A) Immunofluorescence of CD147 in SMMC-7721
cells resistant to anoikis transfected with CD147 siRNA and control siRNA
(n=6-10; magnification, x400). Arrows point to the different morphologies of
cell-cell contacts and nuclei after the inhibition of CD147. (B) Flow cytometry
for apoptosis and necrosis of anoikis-resistant SMMC-7721 cells through
double staining of Annexin V-FITC and PI after culture was suspended
for 24 h. Each value is the means ± SD of at least triplicate determinations.
(C) Western blotting was performed to examine the expression levels of
CD147, phosphorylated Akt and Akt in the subpopulation of SMMC-7721
cells resistant to anoikis transfected with CD147 siRNA and control siRNA.
ONCOLOGY LETTERS 3: 1249-1254, 2012
This study provides evidence that an elevated endogenous
expression of CD147 contributes to anoikis resistance in
SMMC-7721 cells partially through activation of the PI3K/ Akt
survival pathway, which has been considered an important
process for the metastasis of HCC cells.
We first established a model for cell suspension culture to
characterize the morphology of SMMC-7721 cells following
suspension in methocel medium. A number of studies have
validated that anoikis can be inhibited despite loss of cell-
matrix adhesion when cell-cell adhesion is preserved among
various epithelial carcinoma cell lines (16,28,29). Our research
model also revealed a multi-cellular spheroid formation in a
time-dependent manner. Furthermore, we observed a signifi-
cant increase of CD147 expression in SMMC-7721 cells under
suspension culture, especially restricted to the region along the
cytoplasmic membrane of cell-cell contacts, which indicates a
potential role of CD147 in mediating the formation of multi-
cellular spheroids. Earlier evidence has shown that CD147
promotes breast cancer cell survival by regulating intercellular
contacts and inhibiting anoikis through BIM (21). Besides,
our previous study has verified a vital role of CD147 involved
in cell-cell contacts in an E-cadherin-dependent manner in
HEK293 cells (23). These data suggest that CD147 may also be
involved in anoikis resistance in HCC when cells are exposed
to a condition of extracellular matrix-detached culture.
The direct role of CD147 in the adaptation of anoikis
resistance was investigated. The SMMC-7721 cell line has
been extensively studied in the anoikis resistance of HCC
(30,31). Therefore, we established an anoikis-resistant cell
model (SMMC-7721 cells resistant to anoikis) to investigate
the mechanism of anoikis suppression. The adaptation process
in this cell model is closer to the natural in vivo status of the
physiological conditions compared to earlier research models
that directly modified the expression levels of target gene by
manual intervention (21). The significantly high expression
levels of CD147 led to anoikis-resistant SMMC-7721 cells
exhibiting a notably higher viability and invasion ability
compared to their parental SMMC-7721 cells. To provide
direct evidence that CD147 is involved in the process of
anoikis resistance in the subpopulation of SMMC-7721 cells
resistant to anoikis, we inhibited CD147 through transfection
of specific targeting siRNA in anoikis-resistant SMMC-7721
cells. The proportion of the apoptotic and necrotic cells was
greatly increased compared to the mock control after the cells
were suspended in methocel medium for 24 h. Accordingly,
the morphology of nuclei tended to split after the inhibition of
CD147. These results strongly suggest that the upregulation of
CD147 is important in rendering cell anoikis resistance, which
is a premise for the metastasis of primary cells.
The present study also provided a potential molecular
mechanism supporting the hypothesis that the upregulation
of CD147 induces anoikis resistance in SMMC-7721 cells. A
number of published observations have demonstrated the prin-
cipal role of PI3K/Akt pathway activation in anoikis resistance
(32-34). Activation of PI3K/Akt is essential for the integrity of
adherent junctions and is associated with the rearrangement of
the cytoskeleton (35). Our previous studies have demonstrated a
vital role of CD147 in the activation of the PI3K/Akt pathway to
protect cell survival under the different stress exposition (26,36).
Therefore, we explored the correlation between the upregulation
of CD147 expression and activation of the PI3K/Akt pathway
in our anoikis-resistant cell model. Our results have shown that
the knockdown of CD147 significantly decreased the levels of
pAkt, suggesting that activation of the PI3K/Akt pathway is
involved in anoikis resistance by the upregulation of CD147.
However, our preliminary data only provide a potential molec-
ular mechanism of CD147-induced anoikis resistance in HCC
cells. The possible involvement of other signaling molecules
cannot be excluded. Moreover, a thorough evaluation of other
related signaling pathways would shed light on the mechanisms
of CD147-mediated anoikis resistant regulation.
In conclusion, the upregulation of CD147 significantly
contributes to anoikis resistance in SMMC-7721 cells following
suspension in methocel medium. Spontaneous upregulation of
the expression levels of CD147 in SMMC-7721 cells resistant
to anoikis may promote the cell-cell contacts and then activate
the PI3K/Akt pathway. Thus, our findings provide a new
mechanism underlying the metastasis of HCC cells, which
emphasizes an important role of CD147 in anoikis resistance
regulation (Fig. 5).
The authors are thankful to Mr. Kai Qu for his technical
Figure 5. Schematic image of the underlying mechanism of the upregulation
of CD147 for anoikis resistance in HCC metastasis. Enhanced expression of
CD147 enables HCC cells to form multi-cellular spheroids and to activate
the PI3K/Akt pathway, protecting cells from anoikis through a synoikis-like
survival style. Thus, metastasis from the primary tumor was eventually
induced in the liver. In comparison, during the process of metastatic circula-
tion, the cell-cell contacts were destroyed when CD147 was inhibited and
the disassociated cells ultimately underwent anoikis. HCC metastasis was
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