Chemotherapy 2009;55:425-432 DOI: 10.1159/000264689
Received: April 9, 2009 Accepted: July 30, 2009 Published online: December 8, 2009
Inhibition of Adrenocortical Carcinoma by Diphtheria Toxin Mutant CRM197
Daniele Martarelli Pierluigi Pompei Giovanni Mazzoni
Department of Experimental Medicine and Public Health, University of Camerino, Camerino, Italy
Key Words
Adrenocortical carcinoma · CRM197 · Nude mice · Diphtheria toxin
Abstract
Background: In this study, we investigated the effect of CRM197 treatment in human adrenocortical carcinoma (AC) implanted in nude mice. CRM197 is a non-toxic mutant of diphtheria toxin that binds heparin-binding epidermal growth factor-like growth factor (HB-EGF) which is implicat- ed in the proliferative activity of several tumor cells. Meth- ods: HB-EGF expression in AC cells was evaluated by reverse transcription PCR and Western blot. AC tumors were im- planted in nude mice and then treated with CRM197. Effects of treatment on angiogenesis and apoptosis were investi- gated by immunohistochemistry and Western blot. The ef- fects on cell invasion and migration were investigated with a matrigel invasion assay. Results: We demonstrated that human AC cells express HB-EGF. A treatment with CRM197 blocked growth, reduced angiogenesis and induced apop- tosis in AC tumors implanted in nude mice. CRM197 also in- hibited invasion and migration of these tumor cells. Con- clusions: These data support the evidence for anticancer properties of CRM197 in AC tumors.
Copyright ® 2009 S. Karger AG, Basel
Introduction
Adrenocortical carcinoma (AC) is a tumor of the ad- renal gland with a poor diagnosis and with an incidence of approximately 2 persons per million people. In about 60% of patients, AC induces excessive hormone secretion, and 90% of tumors overexpress insulin-like growth fac- tor II and insulin-like growth factor-binding proteins [1]. In order to develop new and effective therapies for this disease, it is first necessary to understand the processes leading to AC initiation and growth. In this study, we in- vestigated the effect of CRM197 treatment in human AC tumors implanted in nude mice. As a nontoxic mutant of diphtheria toxin, CRM197 is a 58-kDa protein that binds the soluble form and the membrane-anchored form of heparin-binding epidermal growth factor-like growth factor (HB-EGF), thus inhibiting its action [2]. HB-EGF is a member of the EGF growth factor family and binds the ErbB1 and ErbB4 receptors, which belong to the ty- rosine kinase family and are expressed in various tissues of epithelial, mesenchymal and neuronal origin. HB-EGF has been shown to be highly expressed in some human cancers [3, 4], and several lines of evidence implicate HB- EGF in the proliferative activity of tumor cells. Recently, it has been shown that the expression of HB-EGF is es- sential for cell proliferation of human ovarian cancer
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cells in nude mice. Treatment with CRM197 completely suppresses tumor formation [3] as well as peritoneal dis- semination in nude mice. In addition, a reduction in vas- cular endothelial growth factor (VEGF), an angiogenic factor, was demonstrated to be related to HB-EGF expres- sion [5]. HB-EGF also plays a role in metastasis forma- tion, modulating the expression of metalloproteinases (MMPs). In a human bladder carcinoma cell line, HB- EGF induced the expression and activities of MMPs [6], whereas in human ovarian cancer cell lines, CRM197 treatment reduced MMP-9 expression [5]. CRM197 is currently the only inhibitor which can be used for cancer therapies in mice and humans [3, 7]. CRM197 also exerts a direct toxicity against cancer cells and stimulates an immunological response, which is supposed to be direct- ed against tumor cells [8-11]. Overall, these data and the minimal toxicity observed in humans [7] suggest that CRM197 is a potential drug for cancer therapy. Since no data on the involvement of HB-EGF in AC are available, we analyzed the expression of HB-EGF in AC cell lines and determined its role in tumor growth in nude mice. There are 2 AC human cell lines commercially available that have been mainly used in in vitro and in vivo studies. The human AC cell line H295R was selected for its abil- ity to grow in a monolayer from the H295 cell line. Im- planted in nude mice, the H295R cells preserve the abil- ity to produce and secrete steroids [12]. Another cancer cell line developed from the cortex of the human adrenal gland is SW-13, which develops tumors when implanted in nude mice [13-15]. We recently developed animal models to investigate the in vivo growth of both the H295R and SW-13 cell lines [16], which have been used here to test the effect of CRM197 on human AC growth in nude mice.
Materials and Methods
Human Cell Lines
H295R human AC cells from ATCC were maintained in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium containing 15 mM HEPES, 0.00625 mg/ml insulin, 0.00625 mg/ml transferrin, 6.25 ng/ml selenium, 1.25 mg/ml bo- vine serum albumin, and 0.00535 mg/ml linoleic acid, 97.5% (ITS+ Premix from BD Biosciences, Italy), and Nu-Serum I, 2.5%. SW-13 human adrenal carcinoma cells were maintained in Leibo- vitz’s L-15 medium with 2 mM L-glutamine and 10% fetal bovine serum. Cell cultures were maintained at 37°℃ in a humidified incubator in an atmosphere of 5% CO2 in air.
Animals
The animals were handled according to internationally ac- cepted principles for care of laboratory animals (European Com-
munity Council Directive 86/609, O.J. No. L358, December 18, 1986).
Athymic male nude mice Nu/Nu (Harlan, Italy) that were 6 weeks old were used. Mice were kept in laminar flow cages under standardized environmental conditions. Sterilized food (Harlan) and water were supplied ad libitum. Experiments were performed in accordance with the guidelines of the Italian Ministry of Health.
RNA Extraction and Reverse Transcription
Total RNA was isolated from H295R and SW-13 cell lines us- ing an RNeasy Mini Kit (Qiagen, Valencia, Calif., USA). Reverse transcription (RT) was performed using a High-Capacity cDNA Archive Kit (PE Applied Biosystems, Foster City, Calif., USA).
Reverse Transcription PCR
RT-PCR was performed using primers specific for HB-EGF (94℃ for 15 s, 60℃ for 30 s, 68℃ for 60 s, 30 cycles): sense 5’-GGTGGTGCTGAAGCTCTTTC-3’, antisense 5’-CCCATG- ACACCTCTCTCCAT-3’. The resulting product size was 418 bp.
Reagents
CRM197 was purchased from Calbiochem (San Diego, Calif., USA).
Western Blot Analysis
Lysates obtained from H295R and SW-13 cells were resus- pended in 0.2 ml of RIPA (0.1% Nonidet-P40, 1 mM CaCl2, 1 mM MgCl2, 0.1% sodium azide, 1 mM phenylmethylsulfonyl fluoride, 0.03 mg/ml aprotinin, and 1 mM NaVO4). Proteins were separated on 7 and 14% SDS polyacrylamide gels, transferred overnight at 20 V and incubated for 2 h at room temperature with the mouse monoclonal antibody followed by incubation with the secondary horseradish peroxidase-conjugated antibodies. Primary mouse monoclonal antibodies included anti-human HB-EGF anti- MMP-9, anti-MMP-2, anti-VEGF and anti-«-tubulin (Santa Cruz Biotechnology, Santa Cruz, Calif., USA). Immunoreactivity was detected using enhanced chemiluminescence (Amersham). Den- sitometric analysis was performed with a Chemidoc using the Quantity One software (BioRad).
Cell Proliferation Assays
H295R and SW-13 were plated in serum-supplemented growth medium at 5,000 cells/well in 96-well plates. After 24 h, cells were treated with CRM197 dissolved in DMSO at various concentra- tions: 0, 1, 10 and 100 µg/ml. Three days later, cells were counted after staining with trypan blue. Values were expressed as percent of control.
Cell Cytotoxic Assays
All tests were performed following the NCI guidelines. H295R and SW-13 were plated in serum-supplemented growth medium at 5,000 cells/well in 96-well plates. After 24 h, 2 plates of each cell line were used to measure the cell number for each cell line at the time of drug addition (Tz) by MTT assay. H295R and SW-13 cells in the other plates were treated with CRM197 dissolved in DMSO at var- ious concentrations: 0, 1, 10 and 100 µg/ml. After 3 days, cell num- bers were determined using an MTT assay (Ti). Briefly, after incu- bation with CRM197, the solutions were discarded and 20 ul of a solution of 5 mg/ml of MTT (Sigma, Italy) was added and the cul-
1
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SW-13
400 bp
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SW-13
HB-EGF
tures were incubated for an additional 3 h at 37℃. The supernatant was removed and the precipitate was dissolved in DMSO (100 pl/ well). After a few minutes, the plate was read on a microtiter plate reader at a wavelength of 570 nm. Percentage growth inhibition was calculated at each of the drug concentration levels as follows:
[(Ti-Tz)/(C-Tz)] × 100 for concentrations for which Ti ≥Tz [(Ti - Tz)/Tz] × 100 for concentrations for which Ti < Tz,
where C = control.
Subcutaneous Implantation of H295R and SW-13 Cells in Nude Mice
At near confluence, cells were harvested with trypsin/EDTA solution. Only cell suspensions of>90% viability (trypan blue ex- clusion) were used. For this, 1 × 106 H295R or SW-13 cells in 100 ul of matrigel (BD Biosciences) were injected subcutaneously on the left lateral chest wall close to the axilla in nude mice. For H295R cells, treatments started 5 weeks after tumor cell injection, whereas SW-13 cell treatments were started 3 weeks after tumor cell injection. CRM197 dissolved in 1 ml of 20 mM HEPES, and 0.15 M NaCl (pH 7.2; 1 mg/week) was injected intraperitoneally into tumor-bearing mice each week (3). Control groups were treated with 20 mM HEPES and 0.15 M NaCl (pH 7.2). Six animals per group were examined every week, and tumor volume was cal- culated using calipers with the formula V (mm3) = (D × d2)/2, where d (mm) and D (mm) are the smallest and largest perpen- dicular tumor diameter, respectively.
Matrigel Invasion Assays
Initially, 24 BioCoat Matrigel Invasion Chambers (BD Biosci- ences) were rehydrated with the addition of 2 ml of warm (37℃) serum-free culture medium for 2 h. Following rehydration, the medium was carefully removed, and 2.5 ml of medium contain- ing 10% FBS (chemoattractant) was then added to each well of the plate, and 2 ml of H295R cell suspension (without serum; 3 × 104 cells) was added in the presence or absence of CRM197 (0, 1, 10 and 100 µg/ml) to the matrigel-coated inserts. Plates were incu- bated for 48 h, after which time the medium was removed using a cotton swab, and the noninvading cells were removed from the upper surface of the membrane by scrubbing. Cells on the lower surface of the membrane were fixed in 70% ethanol for 30 min and then stained with hematoxylin. The numbers of invasive cells were counted in 5 random fields of the insert and a percent control was calculated (number of invasive cells treated with CRM197 x 100/number of invasive cells of the control group).
Apoptosis
DNA fragmentation assays were performed on subcutaneous tumors and tumor cells treated with CRM197 100 µg/ml for 24 h. According to the manufacturer’s protocol, DNA fragments were extracted using a Genomic Cell and Tissue Kit (Talent, Italy). DNA was then suspended in loading buffer (20% Ficoll 400, 0.1 M Na2-EDTA, pH 8.0, 1% SDS, 0.25% bromophenol blue; Sigma), heated for 10 min at 65℃ and electrophoresed on 1% agarose gel (Pharmacia, Biotech, Milano, Italy) and visualized by ethidium bromide staining on a UV transilluminator.
Angiogenesis Evaluation
Angiogenesis was evaluated in tumors implanted in nude mice. Tumors were placed in optimal cutting temperature com- pound after autopsy, snap frozen in liquid nitrogen and stored at -80°C. Frozen sections (10-20 pm) were fixed with cold acetone (5 min), acetone/chloroform 1/1 (5 min) and cold acetone (5 min). Samples were then rinsed with PBS/triton 1% and treated with 3% hydrogen peroxide in methanol (vol/vol). Treated slides were in- cubated in a blocking solution and set overnight at 4℃ in a hu- midified chamber with the endothelial cell marker rat anti-mouse CD31 monoclonal antibody. Slides were then rinsed with PBS and incubated, first with the blocking solution for 20 min and then with a biotin-conjugated goat anti-rat antibody for 1 h. Slides were rinsed again with PBS and incubated for 30 min with the Vector Vectastain ABC Kit (Vinci-Biochem, Vinci, Italy). After 3 washes with PBS, positive reactions were visualized by incubating the slides for about 5 min with stable DAB (Sigma, Italy). Slides were dried and mounted with Universal Mount.
Statistical Analysis
The significance of the in vitro data was determined by one- way analysis of variance. The significance of the in vivo data was determined by two-way analysis of variance with repeated mea- surements. Both tests were followed by two-tailed t test.
Results
Expression of HB-EGF in SW-13 and H295R Cells
We used RT-PCR and Western blots to determine the expression of HB-EGF in H295R and SW-13 cells. As shown in figure 1, the 418-bp PCR product demonstrated
120
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600
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350
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the expression of HB-EGF mRNA in both cell lines. Fig- ure 2 shows the Western blot results, which indicate the presence of the HB-EGF protein in H295R and SW-13 cells.
Effect of CRM197 on H295R Cell Growth in vitro
We tested the effect of CRM197 on H295R and SW-13 cell growth in vitro. As shown in figure 3a, the prolifera- tion test highlighted that CRM197 significantly inhibited the growth of cancer cells at a concentration higher than 1 µg/ml. The cell cytotoxic assays (fig. 3b) evidenced that at 100 µg/ml, cells were killed by CRM197.
Suppression of Tumor Growth by CRM197
We studied the effect of CRM197 on tumor growth. After subcutaneous injection of H95R and SW-13 cells, mice were given CRM197 (1 mg/week) every week by in-
traperitoneal injection. Tumor formation by H295R and SW-13 cells was significantly reduced by CRM197 treat- ment (fig. 4), which completely blocked the growth of the tumor mass. As such, these results confirm that HB-EGF is required for tumor formation of AC cancer cell lines in vivo. No side effects were evidenced in all the groups of animals.
Invasion and Migration of AC Cancer Cell Lines
To elucidate the role of HB-EGF expression in metas- tasis formation, we analyzed the effects of CRM197 on H295R cell migration and invasion. Cells were treated with increasing doses of CRM197 for 48 h in matrigel in- vasion chambers, cells on the lower surface of the mem- brane were fixed and were then stained with hematoxylin and counted. Data presented in figure 5 indicate that noncytotoxic doses of CRM197 significantly inhibited
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cell invasion. The total inhibitory effect attained at 100 ug/ml was also attributed to CRM197 cytotoxicity. These results suggest that HB-EGF plays a role in the invasive- ness of these cell lines.
Apoptosis
The DNA fragmentation assay demonstrated that CRM197 induced apoptosis in H295R and SW-13 cell lines. Figure 6 shows the typical ladder due to the apo- ptosis induction in cells and tumors treated with CRM197.
Angiogenesis
We also analyzed if treatment with CRM197 inhibited angiogenesis in subcutaneous tumors. As shown in fig- ure 7, CRM197 reduced blood vessel development. These results, together with the reduced expression of VEGF measured in AC cells treated with CRM197 (fig. 8), indi- cate that HB-EGF also modulates angiogenesis in H295R and SW-13 tumor formation.
Western Blot Analysis
To elucidate how migration, invasion and angiogene- sis were dependent on HB-EGF, the expression of VEGF, MMP-9 and MMP-2 was examined in H295R and SW-13
cells treated with CRM197 (1 µg/ml). Treatment reduced VEGF expression in agreement with reduced blood vessel development in CRM197-treated tumors. Because MMP- 9 and MMP-2 play significant roles in ovarian and blad- der tumor cell invasion and migration [5, 6], we analyzed their expression in SW-13 and H295R cell lines. As shown in figure 8, a significant reduction in band density cor- responding to MMP-2 and MMP-9 was found in treated cells.
Discussion
In this study, we demonstrated that treatment with CRM197 significantly blocked growth, reduced angio- genesis and induced apoptosis in AC tumors. Moreover, we demonstrated that CRM197 inhibited invasion and migration of these tumor cells. CRM197 is a potent in- hibitor of the HB-EGF, but its anticancer properties also depend on the toxicity of the molecule and its inflamma- tory-immunological properties. The use of diphtheria toxin as an anticancer molecule was proposed several years ago, on the supposition that it could induce activa- tion of the immune system against tumor cells [10, 11]. In 2004, Buzzi et al. [7] demonstrated in humans that CRM197 injected subcutaneously elicited an inflamma- tory-immunological reaction, with an increase in circu- lating neutrophils and tumor necrosis factor-«. A posi- tive cancer response to the therapy was also reported. In 2007, Kageyama et al. [8] demonstrated that CRM197 has a weak toxicity, which is mediated through diphtheria toxin receptors. In this work, an antidiphtheria toxin monoclonal antibody administered with CRM197 re- duced the antitumorigenic effect of CRM197, thus indi- cating that the antitumorigenic effect of CRM197 is due to its inhibitory activity towards HB-EGF and its cyto- toxicity. Miyamoto et al. [3] found that the administra- tion of CRM197 into the peritoneal cavity of nude mice completely suppressed subcutaneous ovarian tumor growth without any inflammatory reaction. However, CRM197 does not bind to mouse HB-EGF [2], so nude mice are not an appropriate model to asses the toxicity and the consequence of an immunological activation in- duced by CRM197, also because these animals have a compromised immune system. However, the majority of CRM197 anticancer effects are due to the inhibition of HB-EGF functions. In fact, a key role of HB-EGF in can- cer progression has been demonstrated in several kinds of tumors, with increased expression of this ligand in liv- er, melanoma, bladder, colon, gastric, ovarian and pan-
creatic tumors [3, 17-20]. In addition, extensive data con- cerning the relationship between HB-EGF expression and cancer have been reported by Miyamoto et al. [3] an- alyzing ovarian cancer. These authors demonstrated that HB-EGF expression, as well as its levels in ascitic fluid, are higher in malignant tumors compared with benign tumors. There are several lines of evidence which relate HB-EGF to proliferation of tumor cells. Among these and similarly to the results presented here is the inhibition of HB-EGF with CRM197 or specific antibody suppression of myeloma cell proliferation and induction of apoptosis [21]. HB-EGF has been shown to be implicated in metas- tasis formation, as it modulates cancer cell migration and invasion through the expression of MMPs [5, 6]. Based on these findings, HB-EGF has been targeted for cancer therapy. Administration of CRM197 completely inhibit- ed ovarian cancer implanted in nude mice [3, 5], but promising results were also obtained in human clinical trials. CRM197 injected subcutaneously in patients with tumors refractory to standard therapies reduced or stabi- lized cancer growth with minimal toxicity [7]. Different from the results obtained by Miyamoto et al. [3] in ovar- ian cancer, in our study, the CRM197 treatment was not able to completely suppress AC tumors implanted subcu- taneously in nude mice. This difference can be partially explained considering that we implant the cells with matrigel, a mix of extracellular matrix proteins. H295R and SW-13 are the most used and well-known AC cell lines, but they do not grow easily in vivo as previously demonstrated by our [16] and other research groups [12]. The use of matrigel is necessary to obtain similar and therefore comparable tumor masses in animals, but can alter tumor properties. The extracellular matrix, in fact, recreates an adequate environment, facilitating cell im- plantation, but also influences survival and can affect an anticancer treatment. Nevertheless, CRM197 was able to completely stop AC tumor progression, thus confirming in vitro data where CRM197 inhibited cell proliferation and induced apoptosis as demonstrated by Western blot- ting. In addition, apoptosis was also detected in subcuta- neous tumors. CRM197 treatment reduced angiogenesis through the inhibition of VEGF expression. Angiogene- sis is a crucial process for cancer growth and dissemina- tion. Emerging evidence indicates that a successful can- cer treatment should derive from the synergic effects of antiangiogenic, cytotoxic and conventional therapies. In this context, an anti-HB-EGF therapy could be integrat- ed in standard protocols to directly block VEGF produc- tion in endothelial as well as in AC cells. We also demon- strated that CRM197 treatment inhibited cell migration
and invasion, 2 processes responsible for metastasis for- mation. Analysis of the cellular mechanisms revealed that CRM197 reduced the expression of MMP-2 and MMP-9, which is known to be implicated in the invasive- ness of several tumors, of which AC is included [22-24]. Overall, these data support the evidence for the antican- cer properties of CRM197 in AC tumors. The blocking of HB-EGF could affect tumor development in different ways, reducing cell proliferation and inducing apoptosis. Since HB-EGF directly stimulates the production of VEGF in endothelial cells [25], the antiangiogenic effect of CRM197 is due to its direct action on tumor blood ves- sels and reduces VEGF expression in AC cells. Moreover, CRM197 inhibits the invasiveness and motility of AC cells, thus reducing the risk of metastasis formation. Last- ly, CRM197 may be administered as immunotherapy to suitable cancer patients. The molecule may preferentially bind to malignant cells overexpressing HB-EGF. The link to this strong immunogen may render the cells heterolo- gous so that they become a clear target for the immune system. In conclusion, we have demonstrated that HB- EGF can be a target in AC tumors. Similar studies on pri- mary tumor cell cultures would elucidate the role of HB- EGF on AC progression and the opportunity to integrate CRM197 into clinical treatment.
Acknowledgment
This work was supported by a grant from Rete 7 S.p.A., Bolo- gna, Italy.
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