CD147 silencing via RNA interference reduces tumor cell invasion, metastasis and increases chemosensitivity in pancreatic cancer cells

Article (PDF Available)inOncology Reports 27(6):2003-9 · March 2012with19 Reads
DOI: 10.3892/or.2012.1729 · Source: PubMed
Abstract
CD147, which belongs to the immunoglobulin superfamily, is a multifunctional glycoprotein that has been shown to increase tumor invasion, metastasis and multidrug resistance. To define the role of CD147 in invasion and metastasis more precisely, we utilized gene silencing to inhibit the expression of CD147 in pancreatic cancer cells. We observed that CD147 expression was significantly impeded at both the mRNA and protein levels and resulted in a decrease of MMP-2 and MMP-9 activities. There was also a decrease of MCT1 expression in the invasion and metastasis potential of pancreatic cancer cells, as well as increased chemosensitivity to gemcitabine in Panc-1 cells. Overall, these results suggest that CD147 plays an important role in the invasion, metastasis and chemosensitivity of the human pancreatic cancer cell line Panc-1, indicating that CD147 may be a promising therapeutic target for pancreatic cancer.
ONCOLOGY REPORTS 27: 2003-2009, 2012
Abstract. CD147, which belongs to the immunoglobulin
superfamily, is a multifunctional glycoprotein that has been
shown to increase tumor invasion, metastasis and multidrug
resistance. To dene the role of CD147 in invasion and metas-
tasis more precisely, we utilized gene silencing to inhibit the
expression of CD147 in pancreatic cancer cells. We observed
that CD147 expression was significantly impeded at both
the mRNA and protein levels and resulted in a decrease of
MMP-2 and MMP-9 activities. There was also a decrease of
MCT1 expression in the invasion and metastasis potential of
pancreatic cancer cells, as well as increased chemosensitivity
to gemcitabine in Panc-1 cells. Overall, these results suggest
that CD147 plays an important role in the invasion, metastasis
and chemosensitivity of the human pancreatic cancer cell line
Panc-1, indicating that CD147 may be a promising therapeutic
target for pancreatic cancer.
Introduction
Pancreatic cancer is a malignancy with an extremely poor
prognosis and is refractory to conventional chemotherapy
and radiotherapy. Despite efforts in the past years, conven-
tional treatment approaches, such as surgery, radiation,
chemotherapy, or combinations of these, the mortality rate of
pancreatic cancer still remains high (1-4). Therefore, it can be
effectively diagnosed, prevented, and treated only by devel-
oping a detailed understanding of the molecular biology of
underlying pancreatic cancer formation and progression.
CD147 (EMMPRIN) is a highly glycosylated cell surface
transmembrane protein that belongs to the immunoglobulin
superfamily (5), and it is thought to be involved in inamma-
tion, neural-glial interaction, and virus infection (6-9). CD147
is found to be highly expressed in a variety of malignant human
cancers, including malignancies of the pancreas (6,10,11), and
it induces tumor cell invasion by stimulating the production of
matrix metalloproteinases (MMPs), resulting in tumor inva-
sion and metastasis (12). In addition, CD147 plays a pivotal
role as a chaperone for the proper plasma membrane expres-
sion and the activity of monocarboxylate transporters (MCTs),
particularly MCT1 and MCT4 (13-15). MCTs are among
the most important cellular pH regulators likely involved in
cancer pH homeostasis (16-18). The MCT family has fourteen
members (19), six of which have been functionally character-
ized, but only MCT1-MCT4 have been shown to catalyze the
proton-coupled transport of lactate (20-24). Many studies
have demonstrated that CD147 acts as an essential chaperone
to take MCT1 and MCT4 to the plasma membrane where the
MCTs and CD147 are tightly associated (13,25).
CD147 is also involved in multidrug resistance of cancer
cells via hyaluronan-mediated activation of ErbB2 signaling
and cell survival pathway activities, but the mechanism of
CD147 in multidrug resistance of pancreatic cancer remains
elusive (26-28). We demonstrate here that CD147 silencing
inhibits pancreatic cancer cell invasion and metastasis and
increases chemosensitivity to gemcitabine. Our results
support the concept that CD147 expression is associated with
the malignant potential of cancer cells, since it sustains the
expression and function of MMPs and MCTs.
Materials and methods
Plasmid constructs and generation of stable cell clones. The
vector pSilencer 3.1-H1 neo (Ambion) was used to generate
short hairpin RNA (shRNA) specically for CD147. Two pairs
CD147 silencing via RNA interference reduces
tumor cell invasion, metastasis and increases
chemosensitivity in pancreatic cancer cells
YUQIN PAN1*, BANGSHUN HE1*, GUOQI SONG1, QIAN BAO2,
ZHIPENG TANG1, FULIANG TIAN2 and SHUKUI WANG1
1Central Laboratory, Nanjing First Hospital, Nanjing Medical University, Nanjing, Jiangsu 210006;
2Department of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu 210046, P.R. China
Received February 1, 2012; Accepted March 5, 2012
DOI: 10.3892/or.2012.1729
Correspondence to: Dr Shukui Wang, Nanjing First Hospital,
Nanjing Medical University, Nanjing, Jiangsu 210006, P.R. China
E-mail: shukwang@163.com
*Contributed equally
Abbreviations: MMP, matrix metalloproteinase; MCT, mono-
carboxylate transporter; LDH, lactate dehydrogenase; shRNA, short
hairpin RNA; PPC, plasma peak concentrations
Key word s: CD147, invasion, metastasis, multidrug resistance,
pancreatic cancer, gemcitabine
PAN et al: TARGETED GENE THERA PY BY CD47 SILENCING
2004
of template oligonucleotides, each encoding one of the target
sequences, were designed and synthesized (designated as
shRNA1 and shRNA2, respectively), and the scrambled control
sequence (designated as shRNA-control) was also synthesized
(Table I). Subsequently, these oligonucleotides were cloned
into the plasmid pSilencer 3.1-H1 neo, using restriction endo-
nuclease BamHI and HindIII. These recombinant vectors
were designated as pH1-shRNA-control, pH1-shRNA1 and
pH1-shRNA2, respectively. The product was conrmed by
DNA sequenci ng.
The plasmids carrying the short hairpin RNA were
transfected into pancreatic cancer cells, using liposome
Lipofectamine 2000 (Invitrogen-Life Technologies, Carlsbad,
CA, USA), and subsequently cultured in the presence of
500 µg/ml G418 (Gibco-BRL, Grand Island, NY, USA) for
clonal selection.
Quantitative real-time PCR assays. Total RNA was extracted
with TRIzol (Invitrogen-Life Technologies), according to
the manufacturer's instructions. Following treatment with
DNase I (Takara Biotechnology Co., Ltd., Dalian, China) at
37˚C for 30 min, RNA quantication was performed using
spectrophotometry. The primers used for CD147, MCT1,
MCT4 and β-actin are listed in Table II. The mRNA levels
for CD147, MCT1, MCT4 and β-actin were analyzed by a one-
step real-time reverse transcriptase polymerase chain reaction
with RNA-direct™ SYBR-Green Real-time PCR Master Mix
(Toyobo Co., Ltd., Osaka, Japan), according to the manufac-
turer's instructions. The cycling conditions were as follows: 40
cycles consisting of denaturation at 95˚C for 5 sec, annealing at
60˚C for 5 sec, and extension at 72˚C for 30 sec. The Ct used in
the real-time PCR quantication was dened as the PCR cycle
number that crossed an arbitrarily chosen signal threshold in
the log phase of the amplication curve. To verify the fold
change of target gene expression, calculated Ct values were
normalized to Ct values of β-actin amplied from the same
sample (ΔCt = Ctt arget - Ctβ-act in), and the 2-ΔΔCt method was used
to calculate fold change (29). Each sample was prepared in
triplicate, and all reactions were triplicated independently to
ensure the reproducibility of the results.
Western blot analysis. The expression of CD147, MCT1
and MCT4 protein was evaluated by western blot analysis.
Total protein was separated by SDS-PAGE on 12% gels and
transferred to a polyvinylidene diuoride (PVDF) membrane.
Skim milk powder (5%) (soluble in TBST buffer solution) was
used at room temperature under sealed conditions for 1 h,
with mouse anti-CD147 primary antibodies (1:500), rabbit
anti-MCT1 primary antibodies (1:500), rabbit anti-MCT4
primary antibodies (1:500) and rabbit anti-human β-actin
primary antibodies (1:500) incubated at room temperature for
2 h, followed by incubation in a 1:2000 dilution of secondary
antibodies conjugated to horseradish peroxidase (Santa Cruz
Biotechnology, Santa Cruz, CA, USA) for 1 h at room temper-
ature. The protein was visualized by ECL. All of the western
blot analyses were performed at least three times.
Determination of intracellular lactate concentration. The
change of intracellular lactate concentration in Panc-1 cells
after CD147 silencing was assessed using a lactic acid assay
kit (KeyGen Biotech Co., Ltd., Nanjing, China). This assay is
based on the catalysis of lactate dehydrogenation by lactate
dehydrogenase (LDH) to generate pyruvate by NAD+ as
hydrogen acceptors. Subsequently, nitroblue tetrazolium
(NBT) is reduced to pur ple coloring when hydrogen is delivered
to it from phenazine methosulphate (PMS). There is a linear
relationship between the absorbance at 530 nm and the lactate
concentration. Cells (1x106) were harvested by centrifugation,
and cells were then ruptured by hypotonic salt solution for
1 h at room temperature. The supernatant was retained after
centrifuging. The optical density was read at 530 nm. Graphs
are representative of three separate experiments.
In vitro invasion assay. Transwell plates (Corning Costar,
Cambridge, MA, USA) were coated with basement membrane
Matrigel (20 mg/ml, Becton-Dickinson, Franklin Lakes, NJ,
USA) for 4 h at 37˚C. After the Matrigel solidied, 1x105
Table I. Sequences of the designed CD147 specic shRNAs.
shRNA Sequence
shRNA1 5'-ATCCGTCGTCAGAACACATCAACTTCAAGAGAGTTGATGTGTTCTGACGACTTTTTTGGAAA-3'
5'-AGCTTTTCCAAAAAAGTCGTCAGAACACATCAACTCTCTTGAAGTTGATGTGTTCTGACGACG-3'
shRNA2 5'-GATCCGTGACAAAGGCAAGAACGTCTTCAAGAGAGACGTTCTTGCCTTTGTCATTTTTTGGAAA-3'
5'-AGCTTTTCCAAAAAATGACAAAGGCAAGAACGTCTCTCTTGAAGACGTTCTTGCCTTTGTCACG-3'
shRNA-control 5'-GATCCACTACCGTTGTTATAGGTGTTCAAGAGACACCTATAACAACGGTAGTTTTTTTGGAAA-3'
5'-AGCTTTTCCAAAAAAACTACCGTTGTTATAGGTGTCTCTTGAACACCTATAACAACGGTAGTG-3'
Table II. Primers of CD147, MCT1, MCT4 and β-actin for
real-time PCR.
Target Primers
CD147 Sense: 5'-CCATGCTGGTCTGCAAGTCAG-3'
Antisense: 5'-CCGTTCATGAGGGCCTTGTC-3'
MCT1 Sense: 5'-CACTTAAAATGCCACCAGCA-3'
Antisense: 5'-AGAGAAGCCGATGGAAATGA-3'
MCT4 Sense: 5'-GTTGGGTTTGGCACTCAACT-3'
Antisense: 5'-GAAGACAGGGCTACCTGCTG-3'
β-actin Sense: 5'-CTGGAACGGTGAAGGTGACA-3'
Antisense: 5'-AAGGGACTTCCTGTAACAACGCA-3'
ONCOLOGY REPORTS 27: 2003-2009, 2012 2005
cells were seeded onto the Matrigel and incubated at 37˚C for
24 h. After 18 h, cells that migrated through the permeable
membrane were xed with 100% methanol for 10 min. The
membrane with cells were soaked in 0.1% crystal violet for
10 min and then washed with distilled water. The number of
cells attached to the lower surface of the polycarbonate lter
was counted at x400 magnication under light microscopy.
Each assay was carried out in triplicate and repeated three
times.
Drug sensitivity assay. To assess their multidrug chemo-
sensitivity, cells were plated in 96-well plates at a density of
1x104 cells/well and further incubated for 24 h. The medium
was then removed and replaced with fresh medium containing
gemcitabine, paclitaxel, and oxaliplatin, respectively, with
varying PPC (plasma peak concentrations, 0.1 PPC, 1.0 PPC
and 10.0 PPC) for another 48 h. After that, cells were stained
with 20 µl of sterile MTT dye [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide, 5 mg/ml; Sigma] at 37˚C
for 4 h, followed by removing the culture medium and mixing
150 µl of dimethylsulfoxide (DMSO) thoroughly for 10 min.
Spectrometric absorbance at 490 nm was measured with a
microplate reader. Each group was plated in three wells and
was repeated three times.
In vivo metastasis assay. We used 4-6-week-old male BALB/c
nude mice (Center for Comparative Medical Research of the
Yangzhou University, Yangzhou, China). Cells were washed
and resuspended in serum-free DMEM before inoculation.
In each of the nude mice (n=8), 2x105 cells in 200 µl culture
medium were inoculated into the tail vein. Six weeks after
inoculation, all animals were euthanized and the lungs were
removed. Harvested tissues were fixed in 10% buffered
formalin, embedded in parafn, sectioned at 4 µm, and stained
with H&E. The antitumor effect was evaluated by counting the
number of metastatic tumor clones on the surface of the lungs.
All experiments were performed in accordance with institu-
tional guidelines for the care and use of experimental animals.
Statistical analysis. Statistics were conducted by the SPSS
software. Experimental data are presented as the mean ± SD
(standard deviation) and assessed by Student's t-tests and
one-way ANOVA at a signicance level of P<0.05.
Results
shRNA targeting CD147 suppresses CD147 expression in
Panc-1 cells. To better understand the role of CD147 in tumor
cells, we established two recombinant vectors, including
pH1-shRNA1 and pH1-shRNA2. As demonstrated by quan-
titative reverse transcription PCR (qRT-PCR), pH1-shRNA2
effectively inhibited expression of CD147 in tumor cells
(P< 0.01) (Fig. 1A). In addition, western blot analysis conrmed
the downregulation of CD147 protein by pH1-shRNA2
(P<0.01) (Fig. 1D).
CD147 silencing inhibits MCT1 and MCT4 expression. Many
studies have demonstrated that the functionality of MCT1
and MCT4, natural transporters of lactate, on mitochondrial
membranes depends on the association with the mature,
C D
Figure 1. Expression levels of CD147, MCT1 and MCT4 in Panc-1 cells after CD147 silencing. Relative mRNA levels of (A) CD147 (B) MCT1 (C) MCT4
were analyzed by quantitative RT-PCR. β-actin was used as the norma lization control. *P<0.01 compared with the control group. Graphs are representative
of three separate exper iments. (D) Western blot analysis of CD147, MCT1 and MCT4 protein expression levels. β-actin was used as the loading control. The
results show that the protein expression levels of CD147 and MCT1 were signicantly downregulated by pH1-shRNA2 in Panc-1 cells. There was no signicant
change of MCT4 protein levels. The data were obtained from three independent experiments.
PAN et al: TARGETED GENE THERA PY BY CD47 SILENCING
2006
glycosylated form of CD147. We thus examined whether
CD147 silencing could reduce the expression of MCT1 and
MCT4. As demonstrated by qRT-PCR, the mRNA expression
of MCT1 was downregulated by pH1-shRNA2 in the Panc-1
cell line (P<0.01), but the expression of MCT4 was not signi-
cantly altered (P>0.05) (Fig. 1B and C). In addition, western
blot analysis conrmed the downregulation of MCT1 protein
by pH1-shRNA2 in the Panc-1 cell line. Furthermore, there
was no signicant change of the MCT4 protein (Fig. 1D).
CD147 silencing reduces MMP-2 and MMP-9 activities.
CD147 has been suggested to induce MMP in tumor-associated
mesenchymal cells, so we examined whether CD147 silencing
could reduce the activities of MMP-2 and MMP-9, using
gelatin zymography. The activities of MMP-2 and MMP-9
were reduced signicantly by pH1-shRNA2 in the Panc-1 cell
line, compared with the control group (P<0.01); and there was
no significant difference between the pH1-shRNA-control
group and the corresponding controls (P>0.05) (Fig. 2).
CD147 silencing inhibits the function of lactate transporters.
We then examined whether CD147 silencing inhibited the func-
tion of lactate transporters. To conrm that the downregulation
of MCT1 expression by pH1-shRNA2 inhibits the function of
these transporters we assessed the intracellular lactate concen-
tration in Panc-1 cells. As shown in Fig. 3, the CD147 silencing
induced an increase of the intracellular lactate concentration
in Panc-1 cells (P<0.01). This demonstrates that the CD147
silencing-induced decrease in MCT1 expression is associated
with an increase in intracellular lactate concentration.
Inhibition of CD147 alters tumor cell invasion in vitro. To
examine whether the downregulation of CD147 in Panc-1
cells affected its invasive ability, we performed an in vitro
Matrigel transwell analysis. The results showed that silencing
of CD147 signicantly reduced invasion activities in Panc-1
cells when compared with corresponding controls (P<0.05)
(Fig. 4).
CD147 silencing increases the sensitivity to chemotherapeutic
drugs. CD147 has been found to be overexpressed in multidrug
resistance tumor cells and could confer resistance to some
antitumor drugs. To examine whether the downregulation of
CD147 in Panc-1 cells affected its sensitivity to chemothera-
peutic drugs, we assessed whether CD147 silencing induced
an alteration in the chemosensitivity of Panc-1 cells to various
agents by the MTT assay. As shown in Fig. 5, CD147 silencing
signicantly increased the chemosensitivity of Panc-1 cells
to gemcitabine at 1.0 PPC and 10.0 PPC but not at 0.1 PPC
compared with the control groups (P<0.05). The results also
show that there was no signicant change of the chemosensi-
tivity induced by CD147 silencing to paclitaxel and oxaliplatin
in Panc-1 cells (P>0.05).
CD147 silencing inhibited the metastatic potential of Panc-1
cells. To investigate the effect of CD147 silencing on Panc-1-
cell metastasis, we injected cells (2x105⁄200 µl) into the tail
vein of nude mice and evaluated the presence of metastatic
nodes in the lungs after 6 weeks. We observed a signicant
reduction in the number of metastatic nodes in the mice
that received Panc-1 cells stably transfected with CD147, as
compared with the corresponding controls (P<0.01) (Fig. 6).
Figure 2. Activities of MMP-2 and MM P-9 in Panc-1 cells after CD147
silencing. Cells were incubated for 24 h and conditioned media were used for
the measur ement of MMP-2 and MMP-9 protein levels by gelatin zy mography.
(A) Photographs of the MMP-2 and MMP-9 bands, which are representative
of three independent experiments. Quantitative analysis of the ( B) MMP-2
bands and (C) the MMP-9 bands. *P<0.01 compared with the control. #P<0.01
compared with the control. Graphs are representative of three separate experi-
ments.
Figure 3. Intracellular lact ate in Panc-1 cells after CD147 silencing. Panc-1
cells were transfected with pH1-shRNA2-CD147. CD147 silencing increased
the intracellular lactate concentration (P<0.01). Transfection with pH1-
shRNA2- control ser ved as a negative control. The graph is representative of
three separate experiments.
ONCOLOGY REPORTS 27: 2003-2009, 2012 2007
Discussion
CD147 is a multifunctional glycoprotein that has been shown
to increase tumor invasion. It plays an important role in cancer
progression, such as promoting invasiveness via the stimulation
of matrix metalloproteinase production (MMPs), interacting
with certain lactate transporters (MCT1 and MCT4) and
facilitating their expression on the cell surface, and mediating
multidrug resistance via the hyaluronan-mediated upregula-
tion of ErbB2 signaling and cell survival pathway activities
(13,26,30). CD147 is highly expressed on the surface of various
tumors, including pancreatic cancer (31,32); however, the
molecular mechanisms involved and the role of CD147 in
pancreatic cancer remain poorly understood. In the present
study, we constructed the CD147 shRNA expression vector to
inhibit the expression of CD147 in the pancreatic cancer cell
Figure 4. Invasive ability of Panc-1 cells after CD147 silencing. Using a
Matrigel polycarbonate lter, 1x105 cells were seeded in the Millicell upper
chamber (A) Representative images of cells invading the Matrigel (x400). (B)
The number of cells that invaded through the chamber was averaged. The
invading cells were counted as a sum of 10 elds of vision under a micro-
scope. *P<0.01 compared with control group. The graph is representative of
three separate experiments.
Figure 5. Multidrug chemosensitivity of Panc-1 cells after CD147 silencing. (A) Cells were treated with gemcitabine with var ying plasma peak concentrations
(PPC; 0.1, 1.0 and 10.0 PPC) for 48 h. Cell viability was determined by the MTT assay. CD147 silencing signicantly increased the chemosensitivity of Panc-1
cells to gemcitabine at 1.0 PPC and 10.0 PPC compared with the control groups (*P<0.05, #P<0.05). The graphs are representative of three separate experi-
ments. Cells were treated with (B) paclitaxel or (C) oxaliplatin with var ying PPC (0.1, 1.0 and 10.0 PPC) for 48 h. Cell viability was determined by the MTT
assay. The results show that there was no signicant change of the chemosensitivity induced by CD147 silencing to paclitaxel or oxaliplatin in Panc-1 cells
(P>0.05). Graphs are representative of three separate experiments.
PAN et al: TARGETED GENE THERA PY BY CD47 SILENCING
2008
line in order to investigate the role of CD147 silencing in inva-
sion, metastasis, and multidrug resistance of pancreatic cancer.
Tumor cell invasion and metastasis are the main causes
of treatment failure and mortality in patients. CD147 can
stimulate the production of MMPs, thereby leading to extra-
cellular matrix degradation and increased tumor invasion and
metastasis. It has been reported that the expression of MMP-2
and MMP-9 is correlated with the invasion and local recur-
rence rate in pancreatic cancer cells (33-35). Transfection of
CD147 cDNA into human MDA-MB-436 breast cancer cells
resulted in an enhancement of tumor growth and an increase in
metastatic incidences, both of which were directly correlated
with high levels of tumor-derived MMP-2 and MMP-9 (36).
In the present study, the results showed that CD147 silencing
in human pancreatic cancer cells reduced the secretion of
MMP-2 and MMP-9 and inhibited the invasion and metastasis
ability of pancreatic cancer cells in vitro. This was consistent
with previous studies (33-35).
CD147, by its close association with MCT1 and MCT4, plays
a pivotal role in the glycolysis reected by the transmembrane
transport of lactate and the regulation of cell proliferation.
Tumor cell expression of MCT1 and MCT4 has been reported
to be regulated by CD147, which facilitates their cell surface
expression, so CD147 plays a pivotal role in the glycolysis
reected by the transmembrane transport of lactate and the
regulation of cell proliferation (37). MCT1 inhibition has also
been shown to have antitumor potential against in vivo models
of lung carcinoma, colorectal carcinoma, and a squamous
carcinoma cell line after a cyano-4-hydroxycinnamate-medi-
ated MCT1 inhibition (38). The present results showed that
CD147 silencing resulted in a signicant reduction of MCT1,
but not MCT4 expression, which supports the concept that
CD147 is an ancillary protein required for the expression of
MCT1. The results also showed that CD147 silencing resulted
in an increase of the intracellular lactate concentration in
pancreatic cancer cells. The increase of lactate concentration
may inhibit the cell growth, since lactate has been demon-
strated to decrease pyruvate reduction to lactate by inhibition
of (LDH) (39). However, the present results did not show that
the increase of lactate concentration inhibited the proliferation
in Panc-1 cells (data not shown), and we will investigate the
mechanism in the future.
Multidrug resistance is a major obstacle in the treatment
of pancreatic cancer, and upregulation of CD147 has been
reported in multidrug resistant cancer cells. The relationship
between tumor metastasis and multidr ug resistance is not ful ly
dened in pancreatic cancer, although indirect evidence in
the advanced disease suggests a functional link between these
processes. In the present study, CD147 silencing increased
the chemosensitivity to gemcitabine, but not to paclitaxel and
oxaliplatin in Panc-1 cells, suggesting that the expression of
CD147 is closely related to multidrug resistance in pancreatic
cancer. Gemcitabine is the rst-line chemotherapeutic agent
for advanced adenocarcinoma of pancreatic cancer; however,
chemoresistance to gemcitabine remains a major cause of
failure for the clinical treatment of this disease. Studies
have indicated that resistance to gemcitabine is dependent
on mitochondria-mediated apoptosis, but various mediators
of gemcitabine-mediated apoptosis have been described
(40-43). The precise mechanism is not fully understood.
Collectively, these observations identify CD147 as a key
regulator of the invasion, metastasis, and multidrug resistance
in pancreatic cancer cells and suggest that patients with this
malignancy may benefit from targeted therapies blocking
effectors of this signaling pathway.
Acknowledgements
This project was supported by grants from the National Nature
Science Foundation of China (no. 81172141), Nanjing Science and
Technology Committee project (no. 201108025), and Nanjing
Medical Technology Development Project (no. ZKX11025).
Figure 6. CD147 silencing inhibits the metastatic nodes in the lungs. A
volume of 2x105 cells⁄ 200 µl was injected into each of the nude mice through
the tail vein. (A) Microscope images show that CD147 silencing signicantly
inhibits the lung metastatic nodes after inoculation with Panc-1 cells (H&E
staining). Magnication, x200. (B) The number of lung metastatic nodes.
*P<0.01 compared with the control group.
ONCOLOGY REPORTS 27: 2003-2009, 2012 2009
References
1. Li D, Xie K, Wolff R and Abbruzzese JL: Pancreatic cancer.
Lancet 363: 1049-1057, 2004.
2. Vernejoul F, Faure P, Benali N, et al: Antitumor effect of in vivo
somatostatin receptor subtype 2 gene transfer in primary and
metastatic pancreatic cancer models. Cancer Res 62: 6124-6131,
2002.
3. Torrisani J and Buscail L: Molecular pathways of pancreatic
carcinogenesis. Ann Pathol 22: 349-355, 2002.
4. Korc M: Pathways for aberrant angiogenesis in pancreatic cancer.
Mol Cancer 2: 8, 2003.
5. Suzuki S, Sato M, Senoo H and Ishikawa K: Direct cell-cell
interaction enhances pro-MMP-2 production and activation in
co-culture of laryngeal cancer cells and broblasts: involvement
of EMMPRIN and MT1-MMP. Exp Cell Res 293: 259-266,
2004.
6. Riethdorf S, Reimers N, Assmann V, Kornfeld JW, Terracciano L,
Sauter G and Pantel K: High incidence of EMMPRIN expression
in human tumors. Int J Cancer 119: 1800-1810, 2006.
7. Muramatsu T and Miyauchi T: Basigin (CD147): a multifunc-
tional transmembrane protein involved in reproduction, neural
function, inammation and tumor invasion. Histol Histopathol
18: 981-987, 2003.
8. Kaname T, Miyauchi T, Kuwano A, Matsuda Y, Muramatsu T
and Kajii T: Mapping basigin (BSG), a member of the immu-
noglobulin superfamily, to 19p13.3. Cytogenet Cell Genet 64:
195-197, 1993.
9. Fadool JM and Linser PJ: 5A11 antigen is a cell recognition
molecule which is involved in neuronal-glial interactions in
avian neural retina. Dev Dyn 196: 252-262, 1993.
10. Biswas C, Zhang Y, DeCastro R, Guo H, Nakamura T, Kataoka H
and Nabeshima K: The human tumor cell-derived collagenase
stimulatory factor (renamed EMMPRIN) is a member of the
immunoglobulin superfamily. Cancer Res 55: 434-439, 1995.
11. Schneiderhan W, Diaz F, Fundel M, et al: Pancreatic stellate
cells are an important source of MMP-2 in human pancreatic
cancer and accelerate tumor progression in a murine xenograft
model and CAM assay. J Cell Sci 120: 512-519, 2007.
12. Kanekura T, Chen X and Kanzaki T: Basigin (CD147) is
expressed on melanoma cells and induces tumor cell invasion
by stimulating production of matrix metalloproteinases by bro-
blasts. Int J Cancer 99: 520-528, 2002.
13. Kirk P, Wilson MC, Heddle C, Brown MH, Barclay AN and
Halestrap AP: CD147 is tightly associated with lactate trans-
porters MCT1 and MCT4 and facilitates their cell surface
expression. EMBO J 19: 3896-3904, 2000.
14. Wilson MC, Meredith D, Fox JE, Manoharan C, Davies AJ and
Halestrap AP: Basigin (CD147) is the target for organomercurial
inhibition of monocarboxylate transporter isoforms 1 and 4: the
ancillary protein for the insensitive MCT2 is EMBIGIN (gp70). J
Biol Chem 280: 27213-27221, 2005.
15. Philp NJ, Ochrietor JD, Rudoy C, Muramatsu T and Linser PJ:
Loss of MCT1, MCT3, and MCT4 expression in the retinal
pigment epithelium and neural retina of the 5A11/basigin-null
mouse. Invest Ophthalmol Vis Sci 44: 1305-1311, 2003.
16. Izumi H, Torigoe T, Ishiguchi H, et al: Cellular pH regulators:
potentially promising molecular targets for cancer chemotherapy.
Cancer Treat Rev 29: 541-549, 2003.
17. Fang JS, Gillies RD and Gatenby RA: Adaptation to hypoxia and
acidosis in carcinogenesis and tumor progression. Semin Cancer
Biol 18: 330-337, 2008.
18. Wahl ML, Owen JA, Burd R, et al: Regulation of intracellular
pH in human melanoma: potential therapeutic implications. Mol
Cancer Ther 1: 617-628, 2002.
19. Halestrap AP and Meredith D: The SLC16 gene family-from
monocarboxylate transporters (MCTs) to aromatic amino acid
transporters and beyond. Pugers Arch 447: 619-628, 2004.
20. Halestrap AP and Price NT: The proton-linked monocarboxylate
transporter (MCT) family: structure, function and regulation.
Biochem J 343: 281-299, 1999.
21. Bröer S, Bröer A, Schneider HP, Stegen C, Halestrap AP and
Deitmer JW: Characterization of the high-afnity monocarbox-
ylate transporter MCT2 in Xenopus laevis oocytes. Biochem J
341: 529 -535, 19 99.
22. Grollman EF, Philp NJ, McPhie P, Ward RD and Sauer B:
Determination of transport kinetics of chick MCT3 mono-
carboxylate transporter from retinal pigment epithelium by
expression in genetically modified yeast. Biochemistry 39:
9351-9357, 2000.
23. Dimmer KS, Friedrich B, Lang F, Deitmer JW and Bröer S: The
low-afnity monocarboxylate transporter MCT4 is adapted to
the export of lactate in highly glycolytic cells. Biochem J 350:
219-227, 2000.
24. Manning Fox JE, Meredith D and Halestrap AP: Characterisation
of human monocarboxylate transporter 4 substantiates its role in
lactic acid efux from skeletal muscle. J Physiol 529: 285-293,
2000.
25. Poole RC and Halestrap AP: Interaction of the erythrocyte
lactate transporter (monocarboxylate transporter 1) with an
integral 70-kDa membrane glycoprotein of the immunoglobulin
superfamily. J Biol Chem 272: 14624-14628, 1997.
26. Misra S, Ghatak S, Zoltan-Jones A and Toole BP: Regulation of
multidrug resistance in cancer cells by hyaluronan. J Biol Chem
278: 2 5285-25288, 2003.
27. Yang JM, Xu Z, Wu H, Zhu H, Wu X and Hait WN: Overexpression
of extracellular matrix metalloproteinase inducer in multidrug
resistant cancer cells. Mol Cancer Res 1: 420-427, 2003.
28. Marieb EA, Zoltan-Jones A, Li R, et al: Emmprin promotes
anchorage-independent growth in human mammary carcinoma
cells by stimulating hyaluronan production. Cancer Res 64:
1229-1232, 2004.
29. Livak KJ and Schmittgen TD: Analysis of relative gene expres-
sion data using real-time quantitative PCR and the 2(-Delta Delta
C(T)) method. Methods 25: 402-408, 2001.
30. Toole BP: Hyaluronan: from extracellular glue to pericellular
cue. Nat Rev Cancer 4: 528-539, 2004.
31. Li M, Wang H, Li F, Fisher WE, Chen C and Yao Q: Effect of
cyclophilin A on gene expression in human pancreatic cancer
cells. Am J Surg 190: 739-745, 2005.
32. Li M, Zhai Q, Bharadwaj U, et al: Cyclophilin A is overexpressed
in human pancreatic cancer cells and stimulates cell proliferation
through CD147. Cancer 106: 2284-2294, 2006.
33. Zhang W, Erkan M, Abiatari I, et al: Expression of extracel-
lular matrix metalloproteinase inducer (EMMPRIN/CD147) in
pancreatic neoplasm and pancreatic stellate cells. Cancer Biol
Ther 6: 218-227, 2007.
34. Yang X, Staren ED, Howard JM, Iwamura T, Bartsch JE and
Appert HE: Invasiveness and MMP expression in pancreatic
carcinoma. J Surg Res 98: 33-39, 2001.
35. Koshiba T, Hosotani R, Wada M, et al: Involvement of matrix
metalloproteinase-2 activity in invasion and metastasis of
pancreatic carcinoma. Cancer 82: 642-650, 1998.
36. Zucker S, Hymowitz M, Rollo EE, et al: Tumorigenic potential of
extracellular matrix metalloproteinase inducer. Am J Pathol 158:
1921-1928, 2 001.
37. Su J, Chen X and Kanekura T: A CD147-targeting siRNA
inhibits the proliferation, invasiveness, and VEGF production of
human malignant melanoma cells by down-regulating glycolysis.
Cancer Lett 273: 140-277, 2009.
38. Sonveaux P, Végran F, Schroeder T, et al: Targeting lactate-
fueled respiration selectively kills hypoxic tumor cells in mice. J
Clin Invest 118: 3930-3942, 2008.
39. Omasa T, Higashiyama K, Shioya S and Suga K: Effects of
lactate concentration on hybridoma culture in lactate-controlled
fed-batch operation. Biotechnol Bioeng 39: 556-564, 1992.
40. Schniewind B, Christgen M, Kurdow R, Haye S, Kremer B,
Kalthoff H and Ungefroren H: Resistance of pancreatic cancer to
gemcitabine treatment is dependent on mitochondria-mediated
apoptosis. Int J Cancer 109: 182-188, 2004.
41. Yang C, Kaushal V, Shah SV and Kaushal GP: Mcl-1 is downreg-
ulated in cisplatin-induced apoptosis, and proteasome inhibitors
restore Mcl-1 and promote survival in renal tubular epithelial
cells. Am J Physiol Renal Physiol 292: F1710-F1717, 2007.
42. Cascalló M, Calbó J, Capellà G, Fillat C, Pastor-Anglada M
and Mazo A: Enhancement of gemcitabine-induced apoptosis
by restoration of p53 function in human pancreatic tumors.
Oncology 68: 179-189, 2005.
43. Koizumi K, Tanno S, Nakano Y, et al: Activation of p38 mitogen-
activated protein kinase is necessary for gemcitabine-induced
cytotoxicity in human pancreatic cancer cells. Anticancer Res
25: 3347-3353, 20 05.
    • " For most of the genes in the focal aberrations (85 of 94) expression data in a set of 31 osteosarcoma samples were available. This allowed us to identify the genes of which the expression status (over-or underexpression) in these tumors was Analysis of Focal Aberrations in Osteosarcoma PLOS ONE | DOI:10.1371/journal.pone.0115835 December 31, 2014frequently (.35%) in accordance to their copy number status (gain or loss), as deduced from the focal aberration analyses. These were, in addition to the known tumor suppressor genes PTEN and DOCK5 and the known oncogene MYC, in total 8 candidate tumor suppressor genes and 4 candidate oncogenes as potential new driver genes in osteosarco"
    [Show abstract] [Hide abstract] ABSTRACT: Osteosarcoma is an aggressive bone tumor that preferentially develops in adolescents. The tumor is characterized by an abundance of genomic aberrations, which hampers the identification of the driver genes involved in osteosarcoma tumorigenesis. Our study aims to identify these genes by the investigation of focal copy number aberrations (CNAs, <3 Mb). For this purpose, we subjected 26 primary tumors of osteosarcoma patients to high-resolution single nucleotide polymorphism array analyses and identified 139 somatic focal CNAs. Of these, 72 had at least one gene located within or overlapping the focal CNA, with a total of 94 genes. For 84 of these genes, the expression status in 31 osteosarcoma samples was determined by expression microarray analysis. This enabled us to identify the genes of which the over- or underexpression was in more than 35% of cases in accordance to their copy number status (gain or loss). These candidate genes were subsequently validated in an independent set and furthermore corroborated as driver genes by verifying their role in other tumor types. We identified CMTM8 as a new candidate tumor suppressor gene and GPR177 as a new candidate oncogene in osteosarcoma. In osteosarcoma, CMTM8 has been shown to suppress EGFR signaling. In other tumor types, CMTM8 is known to suppress the activity of the oncogenic protein c-Met and GPR177 is known as an overexpressed upstream regulator of the Wnt-pathway. Further studies are needed to determine whether these proteins also exert the latter functions in osteosarcoma tumorigenesis.
    Full-text · Article · Dec 2014
    • "Recently, HAb18G/CD147 was shown to be highly expressed in pancreatic cancer cells; and HAb18G/CD147 is widely involved in metastasis and chemoresistance and correlates with cellular stress responses [24, 26]. Therefore, we explored the role of HAb18G/CD147 in gemcitabineenhanced migration/invasion by exposing pancreatic cancer cells to 0-10 μM gemcitabine for 24 hours. "
    Full-text · Article · Nov 2014
    • "CD147 plays a critical role in tumor progression and metastasis [7]. Recent studies have revealed that knockdown of CD147 reduced cell proliferation and improved chemosensitivity in many cancer cells [8, 9] . HAb18 is a singlechain monoclonal antibody fragment (scFv). "
    [Show abstract] [Hide abstract] ABSTRACT: Hepatocellular carcinoma (HCC) is characterized by alterations in multiple genes. High expression of CD147 on the surface of HCC cells promotes proliferation. The monoclonal antibody HAb18 recognizes CD147. We constructed an oncolytic adenoviral vector to express HAb18 (ZD55-HAb18) in HCC cells. Interleukin 24 (IL24) was co-expressed through the use of an F2A linker. ZD55-HAb18-IL24 decreased HCC cell viability to a greater degree than either ZD55-HAb18 or ZD55-IL24 alone. ZD55-HAb18-IL24 also induced apoptosis and autophagy in PLC/PRF/5 HCC cells. Intratumoral injection of ZD55-HAb18-IL24 repressed tumor growth in a PLC/PRF/5 xenograft model. Our results suggest that antibody-antitumor gene conjugation elicited a stronger antitumor effect than the antibody alone, and that this strategy could broaden the applications of antibody-based therapies in HCC.
    Article · Nov 2014
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