Effects of CRM197, a specifi c inhibitor of HB-EGF, in oral cancer
Abstract CRM197, a nontoxic mutant of diphtheria toxin, is a specifi c inhibitor of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), which belongs to the EGF family that has been implicated in the increased progression, proliferation, and metastasis of oral cancer. In this study, we analyzed the antitumor effects of CRM197, which represent possible chemotherapeutic agents for oral cancer. In the experiment, we used the oral squamous cell carcinoma cell lines HSC3 and SAS. Cells treated with CRM197 were analyzed based on cell viability, MTT assay, invasion assay, Western blot, and zymography. HSC3 cells were injected subcutaneously into female BALB/c nu/nu mice at 5 weeks of age. CRM197 and/or CDDP were injected intraperitoneally into tumor-bearing mice, and tumor volume was measured over time. HB-EGF expres-sion in HSC3 and SAS cells treated with CRM197 was signifi cantly reduced and cell proliferation was inhibited. The invasiveness of CRM197-treated cells was relatively low. MMP-9 and VEGF were suppressed in HSC3 treated with CRM197 on zymography and Western blot. Further, tumor growth in xenografted mice was suppressed by CRM197 or CDDP at 1 mg/kg/day. Also, the coadministra-tion of CDDP and CRM197 at 1 mg/kg/day completely inhibited tumor formation. These results suggest that HB-EGF is a target for oral cancer and that CRM197 is effective in oral cancer therapy.
S. Dateoka (*)Second Department of Oral and Maxillofacial Surgery, Graduate School of Dentistry, Osaka Dental University, 1-5-17 Otemae, Chuo-ku, Osaka 540-0008, JapanTel. +81-6-6910-1510; Fax +81-6-6910-1030e-mail: [email protected]
Y. Ohnishi · K. Kakudo Second Department of Oral and Maxillofacial Surgery, Osaka Dental University, Osaka, Japan
Introduction
Epidermal growth factor receptor (EGFR), which belongs to the ErbB family, plays a pivotal role in the diverse pro-cesses of proliferation and differentiation.1 EGFR is acti-vated by a number of ligands, termed epidermal growth factor (EGF)-related peptide growth factors.2 The ligands identifi ed to date include EGF, amphiregulin, epigen, and transforming growth factor-α (TGF-α), which bind specifi -cally to EGFR, and betacellulin, heparin-binding EGF-like growth factor (HB-EGF), and epiregulin, which exhibit dual specifi city for EGFR and ErbB4.3 HB-EGF is an EGFR ligand,4 which was initially identifi ed in the condi-tional medium of human macrophages.5 It is a potent mitogen and chemotactic factor for fi broblasts and smooth muscle cells.6–8
Several laboratories have described HB-EGF upregula-tion in response to oncogene expression and also in oncogene-transformed cells.9,10 Moreover, HB-EGF gene expression is signifi cantly elevated in many human cancers including hepatocarcinoma, melanoma, and colon, breast, myeloma, prostate, bladder, and oral tumors. It has also been implicated in increased proliferation and metastasis.11–19
CRM197 is a nontoxic mutant of diphtheria toxin that shares immunological properties with the native molecule. CRM197 binds to human HB-EGF and blocks its mitogenic activity by the inhibition of EGFR binding.20 CRM197 binds to the soluble form of HB-EGF, as well as to pro-HB-EGF, and inhibits the mitogenic action of HB-EGF by inhibiting its binding to ErbB receptors.20 Because CRM197 does not inhibit the mitogenic activity of other EGFR ligands, this protein has been utilized as a specifi c inhibitor of HB-EGF.20,21 HB-EGF also plays a role in metastasis forma-tion, modulating the expression of matrix metalloprotein-ases (MMPs). In a human bladder carcinoma cell line, HB-EGF induced the expression and activities of MMPs,22 whereas in human ovarian cancer cell lines, CRM197 treat-ment reduced MMP-9 expression.23 CRM197 is currently the only known inhibitor which can be used for cancer therapies in mice and humans.24,25 In addition, a reduction
Received: December 7, 2010 / Accepted: March 10, 2011
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in vascular endothelial growth factor (VEGF), an angio-genic factor, was demonstrated to be related to HB-EGF expression.26 CRM197 treatment reduced angiogenesis through the inhibition of VEGF expression in adrenocorti-cal carcinoma.27
In the present study, we analyzed the antitumor effects of CRM197, as a specifi c inhibitor of HB-EGF, in a cultured oral cancer cell line.
Materials and methods
Cell culture
HSC3 and SAS cell lines were obtained from RIKEN Bio Resource Center (BRC, Ibaraki, Japan) and were main-tained in Dulbecco’s modifi ed Eagle’s medium (DMEM) (Shigma, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS), at 37°C in a humidifi ed atmosphere of 95% air and 5% CO2.
Reagents
CRM197 was purchased from Bio Academia (Osaka, Japan).
Cell proliferation assay
HSC3 and SAS cells were plated in serum-supplemented growth medium at 5,000 cells/well in 96-well plates. After cells were treated with CRM197 and/or cisplatin (CDDP) dissolved in dimethyl sulfoxide (DMSO), cells were counted after staining with trypan blue. Values are expressed as a percentage of the control.
Cell viability of HSC3 and SAS treated with CRM197 at various concentrations (0, 0.5, 1.0, 1.5, and 2.0 μg/ml) was assessed by the MTT assay after seeding. After 50 μl MTT reagent was added to each well of the plates, they were incubated for 1 h at 37°C. After the reaction, 500 μl isopro-panol with 0.04 N HCl was added to each well. Each reac-tion was transferred into a 96-well plate, and absorbance was measured at a test wavelength of 570 nm and reference wavelength of 630 nm with a model 680 Microplate Reader (Bio-Rad, Tokyo, Japan).
RNA isolation and quality measurement
Total RNA in each sample was isolated using the Qiaquick RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The quality of the RNA was verifi ed using an Agilent 2100 bio-analyzer (Agilent Tech-nologies, Palo Alto, CA, USA).
Real-time polymerase chain reaction (PCR)
Reverse transcription was performed with the TaqMan RT kit (Applied Biosystems, Foster City, CA, USA) according to
the manufacturer’s instructions. Briefl y, reverse transcription was carried out in a volume of 100 μl with 300 ng RNA sample, 15 pmol oligo deoxythymidine primers and random primers, 10 μl 10× TaqMan Buffer, 22 μl 25 mM MgCl2, 20 μl each 2.5 mM dNTP, RNase inhibitor, and reverse transcrip-tase. RT conditions were as follows: 10 min at 25°C, 30 min at 48°C, and 5 min at 95°C. Real-time PCR was performed in an ABI PRISM 7900HT machine (Applied Biosystems) using SYBR Green PCR Master Mix (Applied Biosystems), according to the manufacturer’s instructions, in a total volume of 25 μl. Cycling conditions were 2 min at 50°C, 10 min at 95°C, 40 cycles of 30 s at 95°C, 30 s at 58°C, and 1 min at 72°C. To correlate the threshold (Ct) values from the amplifi cation plots with the copy number, a standard curve was generated and a nontemplate con trol was run with every assay. The primers used for real-time PCR analysis were as follows: HB-EGF (forward) 5′-CTCAGCC TTTTGC TTTGCTAAT-3′, (reverse) 5′-GGAACTCACTTTCCCTTGTGTC-3′, GAPDH (forward) 5′-GGCATTGCTCTCAATGACAA-3′, (reverse) 5′-ATGTAGGCCAT GAGGTCC AC-3′.
Western blot analysis
Lysates obtained from 2.0 μg/ml CRN197-treated HSC3 cells incubated 24 h were resuspended in 0.2 ml RIPA (0.1% Nonidet-P40, 1 mM CaCl2, 1 mM MgCl2, 0.1% sodium azide, 1 mM phenylmethylsulfonyl fl uoride, 0.03 mg/ml aprotinin, and 1 mM NaVO4). Proteins were separated on 7% and 14% sodium dodecyl sulfate (SDS) polyacryl-amide gels, transferred overnight at 20 V, and incubated for 2 h at room temperature with the mouse monoclonal anti-body followed by incubation with the secondary horserad-ish peroxidase-conjugated antibodies. Primary mouse monoclonal antibodies included anti-human HB-EGF, anti-MMP-9, and anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoreactivity was detected using enhanced chemiluminescence. Densitometric analysis was performed with Chemidoc using Quantity One software (Bio-Rad, Tokyo, Japan).
Matrigel invasion assay
Assays were performed according to the manufacturer’s instructions. Briefl y, cells (2 × 104/well) were seeded in a 6-well BioCoat Matrigel Invasion Chamber (Becton Dick-inson, Bedford, MA, USA) in DMEM containing 10% (v/v) heat-inactivated fetal calf serum. After 48 h incubation with CRM197 (2.0 μg/ml), noninvading cells were removed from the upper surface of the membrane by scrubbing, and the membrane was stained with a Diff-Quik stain kit. Subse-quently, invading cells were counted using a microscope.
Zymograms
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with gels containing gelatin (0.1%) and 12.5%
93
polyacrylamide. CRM197 (2.0 μg/ml)-treated HSC3 cells were incubated in serum-free medium for 48 h; the medium supernatants were aspirated and subjected to zymograms. Samples were diluted in SDS-PAGE sample buffer. Electro-phoresis was carried out at 60 mA and 4°C for 2 h, and then samples were incubated overnight in 0.05 M Tris/HCL (pH 7.5) buffer containing 10 M CaCl2. Gels were stained with Coomassie blue (0.25%) and destained in methanol/acetic acid/water (50 : 10 : 40).
Cell apoptosis assays
Cells (0.5–1.0 × 104) were treated as aforementioned and seeded on 6-cm polylysine-coated 6-cm dishes. Thereafter, the medium was replaced with DMEM containing 10% fetal bovine serum at 37°C for 48 h in the presence of CDDP (5.0 μg/ml) and/or CRM197 (2.0 μg/ml), with or without each pharmacological agent, after assuring the com-plete adherence of the cells to the 6-cm polylysine-coated dishes. Cells were incubated with TdT reaction reagent for 1 h at 37°C, according to the manufacturer’s recommended protocol (MEBSTAIN Apoptosis Kit Direct; MBL, Nagoya, Japan). The proportions of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells were calculated as the apoptotic cell rate, and the mean is a representative value for each experiment. Each experi-ment was conducted in triplicate.
Tumor growth in nude mice
A total volume of 250 μl containing 5.0 × 106 HSC3 cells suspended in DMEM was injected subcutaneously into female BALB/c nu/nu mice at 5 weeks of age (Shimizu Laboratory Supplies, Kyoto, Japan). The tumor volume was calculated as L × W2 × 0.5236 (L, longest dimension of tumor; W, dimension perpendicular to L). To assess the antitumor effects of CDDP and/or CRM197, CRM197 or CDDP was dissolved in 20 mM HEPES and 150 mM NaCl (pH 7.2), or in equal volumes of absolute ethanol and
Cremophor EL (n = 5). The indicated amount of CRM197 and/or CDDP was injected intraperitoneally into tumor-bearing mice for 4 weeks. All experimental procedures using animals complied with the Guidelines of Animal Care of Osaka Dental University.
Statistical analysis
The Mann–Whitney U test was used to assess the signifi -cance of differences between samples on real-time PCR and Matrigel invasion assay. P values less than 0.05 were con-sidered to indicate signifi cance by real-time PCR and Matri-gel invasion assay.
Results
Effect of CRM197 treatment on HB-EGF expression
To determine whether CRM197 reduced the levels of HB-EGF expression, total RNA from HSC3 and CRM197-treated HSC3 cells was analyzed by real-time PCR. More-over, Western blot analysis was performed. The amount of HB-EGF mRNA was signifi cantly reduced in the CRM197-treated HSC3 cells when compared with parental HSC3 (Fig. 1A). Moreover, the amount of HB-EGF protein was signifi -cantly reduced in the CRM197-treated HSC3 cells (Fig. 1B).
Effect of CRM197 on HSC3 cell growth and apoptosis in vitro
We tested the effect of CRM197 on HSC3 cell growth in vitro. The proliferation test showed that CRM197 inhibited the growth of cancer cells at a concentration higher than 0.5 μg/ml. The MTT assay showed inhibition of the growth of CRM197-treated HSC3 cells compared with parental HSC3. The cell proliferation rate in the presence of both CRM197 and CDDP was markedly inhibited compared with the other groups (Fig. 2B). Moreover, both results
1000
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400
200
0CRM-HSC3 HSC3
(P < 0.05)
CRM-HSC3HSC3
b-Actin
HB-EGF
(A) (B)
∗
278
809
( HB-EGF expression level )
Fig. 1. Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) expression in HSC3-treated CRM197. A Expression of HB-EGF mRNA in HSC3-treated incubated with 2.0 μg/ml CRM197 for 48 h shown by real-time polymerase chain reaction (PCR). The amount of HB-EGF mRNA was signifi cantly reduced in HSC3 treated with CRM197 under the same conditions of real-time PCR when compared to parental HSC3. B HB-EGF expression as determined by Western blot analysis. The amount of HB-EGF was signifi cantly reduced in CRM197-treated HSC3 when compared to parental HSC3 cells
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showed that cell growth inhibition was dependent on the CRM197 dose.
The TUNEL assay demonstrated that CRM197 induced the apoptosis of HSC3 cells the same as did CDDP (Fig. 3).
Invasive activity of CRM197-treated HSC3 and SAS cells
The invasive activity of CRM197-treated and nontreated HSC3 and SAS cells was assayed in vitro using a Matrigel invasion chamber. Parental HSC3 cells were invasive. However, the invasiveness of CRM197-treated cells was relatively low level compared to parental HSC3 and SAS cells (Fig. 4).
Reduction of MMP-9 and VEGF expression in CRM197-treated HSC3 cells
We examined the effects of CRM197 treatment of HSC3 cells on MMP-9 and VEGF expression using zymograms
and Western blot analysis. The amount of MMP-9 was sig-nifi cantly reduced in CRM197-treated HSC3 cells when compared with parental HSC3. CRM197-treated HSC3 cells showed less 92-kDa collagenolytic activity than paren-tal HSC3 (Fig. 5A). Next, we examined VEGF and MMP-9 production with Western blot analysis. CRM197-treated HSC3 VEGF and MMP-9 cells showed lower-level activity than parental HSC3 (Fig. 5B).
Suppression of tumor growth by CRM197
The administration of CDDP (1 mg/kg/day) or CRM197 (1 mg/kg/day) suppressed tumor formation by HSC3 cells. However, the coadministration of 1.0 mg/kg CDDP and 1.0 mg/kg CRM197 completely inhibited tumor formation (Fig. 6). No side effects were noted in any of the groups of animals. When CRM197 or CDDP was administered alone, tumor growth in xenografted mice was suppressed in a dose-dependent manner.
(CRM197 Concentration: µg/ml)
Cell proliferation rate
(h)
(h)
MTT assay
(Absorbance)
(Absorbance)
(HSC3) (HSC3)
(SAS)(SAS)
1.90
1.85
1.80
1.75
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0.0 0.5 1.0 1.5 2.0
0 2 4 8 12 24 48 72
140%
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0 2 4 8 12 24 48 72
140%
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20%
0%
a
b
c
d
e
f
3
2.5
2
1.5
1
0.5
0
(A) (B)
Fig. 2. Cell proliferation rate and apoptosis in CRM197-treated HSC3 and SAS cells. A Effect of CRM197 depended on a dose of more than 0.5 μg/ml. B Cells were treated with CRM197 at 2.0 μg/ml and CDDP at 5.0 μg/ml (a), CRM197 at 1.0 μg/ml and CDDP at 5.0 μg/ml (b),
CRM197 at 2.0 μg/ml (c), CRM197 at 1.0 μg/ml (d), CDDP at 5.0 μg/ml (e), and the absence of CRM197 or CDDP as a control (f). The cell proliferation rate in the presence of both CRM197 and CDDP was strongly inhibited compared with the other groups in both cases
95
CDDP (5.0 μg/ml) CRM197 (2.0 μg/ml)
TUNEL assay (HSC3)
Control
Fig. 3. Apoptosis in CRM197-treated oral cancer cells by TUNEL assay. The proportions of TUNEL-positive cells was similar on CRM197 and CDDP treatment
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0.2
0CRM-HSC3 HSC3
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Invasion index
∗(P < 0.05)
∗
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0.80
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MMP9
CRM-HSC3HSC3
(B)(A)CRM-HSC3HSC3
VEGF
MMP9
b-Actin
Fig. 4. The invasive activity of HSC3 and SAS was determined employing a Matrigel invasion chamber. Parental cells were highly invasive, but the invasiveness of CRM197-treated cells was relatively low level
Fig. 5. MMP-9 expression as determined by gelatin-zymography, and vascular endothelial growth factor (VEGF) and matric metalloprotein-ase (MMP)-9 expression as determined by Western blot analysis. A HSC3 cells were treated with CRM197 at 2.0 μg/ml and incubated for 48 h. The amount of MMP-9 was signifi cantly reduced in CRM197-treated HSC3 when compared with parental HSC3 cells. B The amount of VEGF and MMP-9 was signifi cantly reduced in CRM197-treated compared to parental HSC3
Discussion
In this study, we demonstrated that treatment with CRM197 signifi cantly reduced growth, angiogenesis, and induced apoptosis in HSC3 cells. We also demonstrated that CRM197 inhibited the invasive activity of HSC3 cells. Moreover, we demonstrated that the coadministration of CDDP and CRM197 synergistically blocked tumor formation in mice. CRM197 is a potent inhibitor of HB-EGF, but its anticancer properties also depend on the toxicity of the molecule and its infl ammatory and immunological properties. The use of diphtheria toxin as an anticancer molecule was proposed several years ago, on the supposition that it could induce the activation of the immune system against tumor cells.28,29 In 2007, Kageyama et al.30 demonstrated that CRM197 exhibits a weak toxicity, which is mediated through diphthe-ria toxin receptors. In this study, an antidiphtheria toxin monoclonal antibody administered with CRM197 reduced the antitumorigenic effect of CRM197, thus indicating that the antitumorigenic effect of CRM197 is caused by its
96
(mm )3
(mm )3
(mm )3 (mm )3
(Day)
(Day)
(Day) (Day)
Fig. 6. Tumor volume was calculated as L × W2 × 0.5236 (L, longest dimension of tumor; W, dimension perpendicular to L). Groups were divided into control (1), CRM197 1 mg/kg/day (2), CDDP 1 mg/kg/day
(3), and CRM197 and CDDP 1 mg/kg/day (4). Coadministration of CRM197 and CDDP completely inhibited tumor proliferation com-pared with the other groups
inhibitory activity toward HB-EGF and its cytotoxicity. Moreover, CRM197 restrain the expression of HB-EGF. Miyamoto et al.24 demonstrated that the administration of CRM197 into the peritoneal cavity of nude mice completely inhibited subcutaneous ovarian cancer growth without any infl ammatory reaction. Moreover, extensive data concern-ing the relationship between HB-EGF expression and cancer have been reported on analyzing ovarian cancer. These authors demonstrated that HB-EGF expression, as well as its levels in ascitic fl uid, are higher in malignant compared with benign tumors. Several lines of evidence relate HB-EGF to the proliferation of tumor cells. Among these results presented here is the inhibition of HB-EGF by CRM197, or the specifi c antibody suppressed the prolifera-tion of myeloma cells and induced apoptosis.31 HB-EGF has been shown to be implicated in metastasis formation, as it modulates cancer cell invasion and migration through the expression of MMPs.22,23 Based on these results, HB-EGF
has been targeted for cancer therapy. The administration of CRM197 completely inhibited ovarian cancer implanted in nude mice,23,24 but promising results were also obtained in human clinical trials. A clinical trial of CRM197 was per-formed involving 25 patients with advanced human cancer.32 In this trial, 2 patients showed a complete response, 1 a partial response, and 6 showed stable disease. This study also reported that toxicities were minimal, as only 1 patient had irritating skin reactions in the injection sites and a fl u-like syndrome with fever, suggesting that CRM197 can be recognized as a useful and safe agent for cancer therapy. In this study, the treatment with CRM197 in conjunction with CDDP resulted in a marked synergistic antitumor effect in oral cancer cells in vivo, suggesting a novel combination therapy for oral cancer patients including those showing chemoresistance.
CRM197 treatment reduced angiogenesis through the inhibition of VEGF expression. Angiogenesis is a crucial
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process for cancer growth and dissemination. Emerging evi-dence indicates that successful cancer treatment should be derived from the synergic effects of antiangiogenic, cyto-toxic, and conventional therapies. In this context, anti-HB-EGF therapy could be integrated in standard protocols to directly block VEGF production in endothelial as well as oral SCC cells. We also demonstrated that CRM197 treat-ment inhibited cell invasion, the process responsible for metastasis development. Analysis of the cellular mecha-nisms revealed that CRM197 reduced the expression of MMP-9, which is known to be implicated in the invasiveness of several tumors, in which oral SCC is included. Overall, these data support evidence for the anticancer properties of CRM197 in oral cancer. The blocking of HB-EGF could affect cancer development in different ways, reducing cell proliferation and inducing apoptosis.
Because HB-EGF directly stimulates the production of VEGF in endothelial cells,33 the antiangiogenic effect of CRM197 is caused by its direct action on tumor blood vessels and reduction of VEGF expression in SCC cells. Moreover, CRM197 inhibits the invasiveness and motility of SCC cells, thus reducing the risk of metastasis development.
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