Clostridium perfringens enterotoxin carboxy-terminal fragment is a novel tumor-homing peptide for human ovarian cancer
- Emiliano Cocco1,
- Francesca Casagrande1,
- Stefania Bellone1,
- Christine E Richter1,
- Marta Bellone1,
- Paola Todeschini1,
- Jennie C Holmberg1,
- Han Hsuan Fu1,
- Michele K Montagna1,
- Gil Mor1,
- Peter E Schwartz1,
- Dan Arin-Silasi1,
- Masoud Azoudi1,
- Thomas J Rutherford1,
- Maysa Abu-Khalaf2,
- Sergio Pecorelli3 and
- Alessandro D Santin1Email author
© Cocco et al; licensee BioMed Central Ltd. 2010
Received: 21 May 2010
Accepted: 2 July 2010
Published: 2 July 2010
Development of innovative, effective therapies against recurrent/chemotherapy-resistant ovarian cancer remains a high priority. Using high-throughput technologies to analyze genetic fingerprints of ovarian cancer, we have discovered extremely high expression of the genes encoding the proteins claudin-3 and claudin-4.
Because claudin-3 and -4 are the epithelial receptors for Clostridium perfringens enterotoxin (CPE), and are sufficient to mediate CPE binding, in this study we evaluated the in vitro and in vivo bioactivity of the carboxy-terminal fragment of CPE (i.e., CPE290-319 binding peptide) as a carrier for tumor imaging agents and intracellular delivery of therapeutic drugs. Claudin-3 and -4 expression was examined with rt-PCR and flow cytometry in multiple primary ovarian carcinoma cell lines. Cell binding assays were used to assess the accuracy and specificity of the CPE peptide in vitro against primary chemotherapy-resistant ovarian carcinoma cell lines. Confocal microscopy and biodistribution assays were performed to evaluate the localization and uptake of the FITC-conjugated CPE peptide in established tumor tissue.
Using a FITC-conjugated CPE peptide we show specific in vitro and in vivo binding to multiple primary chemotherapy resistant ovarian cancer cell lines. Bio-distribution studies in SCID mice harboring clinically relevant animal models of chemotherapy resistant ovarian carcinoma showed higher uptake of the peptide in tumor cells than in normal organs. Imunofluorescence was detectable within discrete accumulations (i.e., tumor spheroids) or even single chemotherapy resistant ovarian cancer cells floating in the ascites of xenografted animals while a time-dependent internalization of the FITC-conjugated CPE peptide was consistently noted in chemotherapy-resistant ovarian tumor cells by confocal microscopy.
Based on the high levels of claudin-3 and -4 expression in chemotherapy-resistant ovarian cancer and other highly aggressive human epithelial tumors including breast, prostate and pancreatic cancers, CPE peptide holds promise as a lead peptide for the development of new diagnostic tracers or alternative anticancer agents.
Ovarian carcinoma remains the most lethal of gynecologic malignancies . Although many patients with ovarian cancer fully respond to the standard combination of surgery and chemotherapy, nearly 90% later develop recurrent chemotherapy-resistant cancer and inevitably succumb to their disease  Thus, development of novel, effective therapies against recurrent/chemotherapy-resistant ovarian cancer remains a high priority. As an early step toward our long-term goal to develop an innovative and highly effective therapeutic strategy for patients with chemotherapy-resistant ovarian cancer, we sought molecular targets to distinguish healthy cells from ovarian cancer cells. Using oligonucleotide microarrays to analyze global gene expression profiles of several highly purified primary ovarian serous papillary tumor (OSPC) cell lines, we and others have consistently identified the genes encoding tight junction (TJ) proteins claudin-3 and -4 as two of the most highly upregulated genes in primary ovarian carcinoma [3–13].
Claudin-3 and -4 are the epithelial receptors for Clostridium perfringens enterotoxin (CPE), a potent cytolytic toxin [14–16]. CPE triggers lysis of mammalian epithelial cells through interactions with claudin-3 and -4 receptors while cells that do not express CPE receptors (i.e., mesothelial cells and most healthy human tissues) are protected from the lethal effects of CPE because they fail to bind the toxin. Our previous studies suggest that chemotherapy-resistant ovarian tumors express extremely high levels of claudin-3 and -4 and that these tumors are highly sensitive to CPE in vitro . Unfortunately, the systemic (i.v.) administration of CPE in animals is toxic, limiting this approach to regional/local therapy . Importantly, functional domain mapping of the full length 319-amino acid CPE protein demonstrated that the carboxy-terminal fragment (C-CPE290-319 peptide) is sufficient for high-affinity binding to target cell receptors and small complex formation, although this fragment is not toxic because it is incapable of initiating large complex formation and subsequent cytolysis . Because peptides are promising molecules to deliver radionuclides or chemotherapeutic drugs into tumors, we hypothesized that using the C- CPE290-319 peptide to target ovarian cancer cells based on their high levels of claudin-3 and -4 may represents a novel, potentially highly effective diagnostic and therapeutic approach to identify and treat chemotherapy-resistant ovarian cancer.
In this study we have carefully assessed the accuracy and specificity of the CPE peptide in vitro in multiple cell binding assays against primary chemotherapy-resistant ovarian carcinoma cell lines. Confocal microscopy experiments showed tumor internalization of the CPE peptide in a time-dependent manner. Biodistribution experiments in highly relevant clinical models of chemotherapy-resistant freshly explanted human ovarian cancer in SCID mouse xenografts were done demonstrating high uptake of the FITC-conjugated CPE peptide in established tumor tissue as well as tumor spheroids floating in the ascitic fluid, recommending this peptide as a promising lead structure for improved targeting of chemotherapy-resistant ovarian cancer.
The Clostridium Perfringens carboxy-terminal fragment peptide (CPE290-319 peptide) was synthesized by InVitrogen Corporation (Invitrogen, Grand Island, N.Y.), with the sequence SLD AGQ YVL VMK ANS SYS GNY PYS ILF QKF and a purity level >95%. The CPE peptide and its FITC-conjugated form were purified by high performance liquid chromatography and their sequence and structure were confirmed by mass spectrometry.
Primary Chemotherapy-Resistant Ovarian Carcinoma Cell Lines
Claudin-3 and -4 mRNA and protein expression in primary ovarian cancer cell lines
MFI* + SD
mRNA copy #
mRNA copy #
11.2 ± 3.4
86.8 ± 10.5
118.8 ± 12.5
58.7 ± 6.1
89.8 ± 8.7
45.9 ± 16.2
102.7 ± 11.3
108.3 ± 8.7
105.7 ± 10.2
RNA Extraction and Quantitative Real-time PCR
RNA isolation from primary cell lines was performed using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. q-PCR was performed with an ABI Prism 7000 Sequence Analyzer using the manufacturer's recommended protocol (Applied Biosystems, Foster City, CA) to evaluate expression of claudin-3 and claudin-4 in all the samples. Each reaction was run in triplicate. Briefly, five μg of total RNA from each sample was reverse transcribed using SuperScript III first-strand cDNA synthesis (Invitrogen, Carlsbad, CA). Five μl of reverse transcribed RNA samples (from 500 μl of total volume) were amplified by using the TaqMan Universal PCR Master Mix (Applied Biosystems) to produce PCR products specific for claudin-3 and claudin-4. The primers for claudin-3 and claudin-4 were obtained from Applied Biosystems as Assay-on-Demand products. Assays ID were Hs00265816_s1 (claudin-3) and Hs00433616_s1 (claudin-4). The comparative threshold cycle (C T) method (PE Applied Biosystems) was used to determine gene expression in each sample relative to the value observed in the nonmalignant ovarian epithelial cells, using GAPDH (Assay-on-Demand Hs99999905_m1) RNA as internal controls.
We used FACS analysis to evaluate binding of the FITC-labeled C-CPE peptide to the surface of primary chemotherapy-resistant ovarian carcinoma cell lines overexpressing claudin-3 and -4 receptors. As positive control, we used a single chain fragment variable (scFv) specific against claudin-3 recently developed and characterized in our laboratory . Negative controls included normal cells (i.e., peripheral blood lymphocytes (PBL), Epstein-Barr transformed B lymphocytes (LCL) and human fibroblasts) and tumor cell with low and/or negligible claudin-3 and -4 receptor expression (i.e., primary squamous and adenocarcinoma cervical cancer cell lines). Briefly, approximately 2.5 × 105 cells were resuspended with 50 μL PBS 1% bovine serum albumin (BSA) that contained 4 μg of FITC-labeled C-CPE peptide or primary scFv antibody (positive control) and cells were then incubated for 1 hour at room temperature. For scFv antibody staining, after further washing, cells were resuspended with 100 μL PBS 1% BSA solution that contain 0.7 μg of an anti-myc tag antibody 9E10 (Roche Diagnostics) for 1 hour at 4°C, followed by washing and incubation with 2 μg of fluorescein isothiocyanate (FITC)-conjugated goat antimouse immunoglobulin G (Jackson Immuno Research, West Grove, PA) for 30 minutes at 4°C. Cells stained with FITC-labeled C-CPE peptide or scFv and unstained control cells were then washed with PBS, resuspended in 500 μL PBS, and immediately analyzed by FACScalibur (BD Biosciences, San Diego, CA).
The surface binding localization of FITC-conjugated-C-CPE290-319 peptide on chemotherapy resistant ovarian tumors was further evaluated by fluorescence microscopy. Briefly, unfixed ovarian cancer cells or control cells in suspension or adherent to plastic were incubated with different concentrations of FITC-conjugated-C-CPE290-319 peptide for 30 to 60 minutes at 37°C and binding to cell surface expressed claudin-3 and claudin-4 was analyzed by fluorescent microscopy using a Nikon fluorescence microscope Eclipse E400. Images were captured digitally and analyzed using Metamorph 3.6a version software.
Internalization experiments and Confocal laser scanning microscopy
We used confocal microscopy to evaluate whether the FITC-conjugated-C-CPE 290-319 peptide was able to internalize after binding to claudin-3 and claudin-4 receptors on the surface of chemotherapy-resistant ovarian cancer cells. Briefly, 30,000 ovarian cancer cells were seeded in 3 cm petri dishes. After 24 hours of cultivation, the medium was replaced by fresh medium and FITC-conjugated CPE 290-319 peptide (10 μg/ml) was added to the cells. The FITC-labeled peptide was incubated with the tumor cells for the indicated times (i.e., from 30 to 60 minutes at 37°C). The medium was then removed and the cells were washed thrice with 1 mL PBS before being analyzed by confocal imaging on an inverted microscope (Leica DM IRBE) with a confocal laser scanning unit (Leica SP2 MP). Some of the experiments were also carried out using the unlabeled CPE 290-319 peptide as competitor for the binding of FITC-conjugated CPE290-319 peptide while samples without FITC-conjugated CPE 290-319 peptide were analyzed to determine autofluorescence of tumor cells. Processed serial sections were constructed into three-dimensional images using VoxelView (Vital Images Ltd., Fairfield, IA) on a Silicon Graphics Indy workstation (Mountain View, CA).
Tumor targeting in SCID Mouse Tumor Xenografts
C.B-17/SCID female mice 5-7 weeks old were obtained from Harlan Sprague-Dawley (Indianapolis, IN) and housed in a pathogen-free environment at Yale University. They were given commercial basal diet and water ad libitum. The experimental protocol for the use of the animals for these studies was approved by the Institutional Animal Care and Use Committee. Animals were used to generate ovarian tumor xenografts as previously described . Briefly, OVA-ARK-1 cancer cell line was injected intraperitoneally (i.p.) at a dose of 5 × 106 into C.B-17/SCID mice in groups of three to five. Six weeks after i.p. tumor injection when carcinomatosis nodules had grown to 0.5 to 0.8 cm, 100 μg FITC-CPE peptide was injected into the tail vein and allowed to circulate for 12, 24 or 48 hours. In some experiments mice were perfused at the time of sacrifice with PBS through the left ventricle to remove blood and unbound peptide. Tumors and control organs were excised after the injection of the fluorescent peptide and examined for fluorescence using a whole-body optical imaging system. Selective excitation of FITC was produced through a D455/70 nm band-pass excitation filter (HB Optical). Emitted fluorescence was collected through a long-pass filter (520 nm; HB Optical) on a 3.3 MP digital camera (DC290; Kodak).
Statistical comparisons between groups were done by the unpaired Student's t test using SPSS version 15 (SPSS, Chicago, IL). P ≤ 0.05 was considered statistically significant.
Claudin-3 and Claudin-4 Transcript Levels in Chemotherapy-Resistant Ovarian Tumors
We used q-RT-PCR assays to get highly sensitive measurements of claudin-3 and claudin-4 CPE receptor expression in normal tissue cells and primary human tumors derived from patients harboring chemotherapy-resistant ovarian carcinomas. Both claudin-3 and/or claudin-4 genes were found highly expressed in all primary ovarian cancer studied including five high grade serous papillary tumors and two high grade clear cell carcinomas when compared to normal ovarian epithelial cells (NOVA) as well as other normal cells or other gynecologic tumors (i.e., mean claudin-3 expression = 15,119 folds and mean claudin-4 expression = 1,800 folds, respectively, when compared to NOVA, Table 1, p < 0.05, and data not shown). These results are consistent with previous reports from us as well as others, showing high expression of claudin-3 and claudin-4 in ovarian carcinomas by gene expression profiling [3–13], and confirm high expression of the CPE receptor transcripts in chemotherapy-resistant ovarian tumors.
Flow Cytometry experiments
FITC-labeled CPE peptide binds and compartmentalizes on the surface of chemotherapy-resistant ovarian tumor cells in vitro
FITC-labeled CPE peptide specifically binds and internalizes in chemotherapy-resistant ovarian tumor cells in vitro
Determination of FITC-conjugated-CPE peptide binding in biodistribution studies in vivo
Biodistribution of FITC-CPE peptide in SCID mice with OSPC-ARK-1 tumor xenografts
Claudin-3 and claudin-4 are tight junction proteins overexpressed in ovarian carcinomas [3–13], the most lethal gynecologic malignancy in the United States [1, 2]. Because claudin-3 and claudin-4 have been recognized as the receptors for Clostridium perfringens enterotoxin (CPE), these findings imply that ovarian cancer refractory to standard treatment modalities may be susceptible to CPE-based therapeutic approaches. Consistent with this view, in the last few years CPE structure/function relationship has been extensively investigated, mainly by characterizing the functional properties of enterotoxin fragments and point mutants [14–16]. In this regard, it has been demonstrated that the CPE290-319 COOH-terminus fragment is sufficient for high affinity binding to target cell receptor and small complex formation, although this fragment is incapable of initiating large complex formation and cytolysis [14–16].
In this study, we have taken advantage of the functional properties of enterotoxin fragments and we have first confirmed at both RNA and protein levels a high expression of the CPE receptors in multiple primary ovarian carcinomas characterized by high in vitro and in vivo resistance to chemotherapy. Secondly, using these freshly established biologically aggressive ovarian tumor cell lines, we have carefully assessed the accuracy and specificity of the binding of the carboxy-terminal fragment of CPE conjugated to a fluorescent tag (i.e., FITC-CPE peptide) as well as its internalization properties in vitro as well as in in vivo experiments using a highly relevant clinical model of chemotherapy resistant freshly explanted human ovarian cancer in SCID mouse xenografts.
Our results established, to our knowledge for the first time, that the CPE peptide is capable of specifically targeting chemotherapy resistant ovarian carcinoma cells overexpressing the CPE receptors claudin-3 and claudin-4. However, it did not bind to normal tissue cells or tumors not expressing significant levels of claudin-3 and/or claudin-4. These findings clearly indicate that the CPE peptide possesses specific targeting and/or homing properties for chemotherapy-resistant ovarian tumors. The remarkable tumor-homing efficiency of the peptide may be due to its propensity to be internalized by cells. Indeed, cells that bind CPE peptide due to the overexpression of the claudin-3 and claudin-4 receptors may transport it across the cell membrane and into the cytoplasm. Thus, peptide internalization is likely to contribute to its effectiveness in becoming concentrated in the targeted tumors. In this regard, it is worth noting that the CPE peptide used in our experiments was conjugated to fluorescin, a complex organic molecule with 44% of the molecular mass of paclitaxel . While we found the fluorescence intensity to be time- and dose-dependent, all tumor cell lines tested exhibited internal FITC, thus demonstrating efficient accumulation of the fluorescent compound. These results, obtained in fresh unfixed tumor cells exclude the possibility of the peptide being artificially internalized during fixing. Furthermore, none of the cell lines exhibit significant auto-fluorescence. Of interest, under high magnification, a punctate fuorescent pattern was observed, which suggest, similarly to the data we have recently reported using an anti human claudin-3 scFv , that the CPE peptide may be compartmentalized after entry into cells. Interestingly, this punctuate pattern is similar to that previously observed with internalized epidermal growth factor, which enters via receptor-mediated endocytosis .
Occult micro-metastatic chemotherapy-resistant ovarian disease, by definition, is not detected by even the most sophisticated imaging techniques such as standard MRI and positron emission tomography (PET). To determine whether CPE peptide localizes to tumor, SCID mice harboring established peritoneal carcinomatosis of OSPC-ARK-1-derived tumor xenografts were injected i.v. with FITC-conjugated CPE peptide. Importantly, tumor explanted 12, 24 and 48 hrs after peptide injection showed fluorescing tumor cells while untreated tumor cells did not significantly autofluoresce. Moreover, brain, heart, lungs, kidney, spleen and liver from FITC-conjugated CPE peptide injected tumor bearing mice showed little labeling. A similar result was also obtained with FITC-conjugated CPE peptide injected tumor-free mice, which suggested that the inefficient labeling was not attributable to peptide depletion by the tumor. Remarkably, in our in vivo experiments, we found even discrete accumulation (i.e., tumor spheroids) or single chemotherapy-resistant ovarian cancer cells floating in the ascites of OSPC-ARK-1 xenografted animals to be specifically targeted by the FITC-conjugated-C-CPE peptide. These experiments suggest that CPE peptide may have a remarkable ability to target very early stage of occult metastasis foci and/or recurrence disease by detecting concealed tumor cells before they become clinically evident in vivo.
Eradication of occult micrometastases of chemotherapy-resistant disease by coupling radioactive isotopes or anticancer agents to the CPE peptide would be greatly beneficial in protecting patients with microscopic residual disease or small-volume macroscopic cancer resistant to standard chemotherapeutic agents. Consistent with this view, the potential of the CPE peptide as a carrier to specifically deliver therapeutic drugs in vivo in patients harboring incurable chemotherapy-resistant ovarian disease or other human malignancies overxpressing the CPE receptors such as breast, prostate, lung, endometrial, thyroid and pancreatic cancer [20, 23–25] is further strengthened by the fact that it is nontoxic (no histological evidence of organotoxicity was observed in in vitro experiments or in CPE-peptide injected mice), while is able to behave as a shuttle during transit, and accumulates efficiently within the tumor in a few hours. As a result of our experiments, efforts to conjugate the CPE peptide to diagnostic and therapeutic radionuclides are currently in progress in our laboratory.
Our results demonstrate for the first time that the CPE peptide binds and internalizes in ovarian tumor cells and therefore possesses excellent targeting or homing properties for the early diagnosis and/or treatment of chemotherapy-resistant ovarian carcinoma. These data combined with the high expression of claudin-3 and -4 receptors recently reported in other biologically aggressive epithelial human tumors, such breast, prostate, lung, endometrial, thyroid and pancreatic cancer [20, 23–25] provide further evidence to suggest that CPE peptide may have great potential for diagnostic and/or radiometabolic therapy of ovarian carcinomas as well as other aggressive human tumors after labeling with suitable chemotherapeutics, radionuclides or toxins.
ovarian serous-papillary carcinoma
Clostridium Perfringens Enterotoxin
fetal bovine serum
single chain fragment variable
peripheral blood lymphocytes
quantitative real-time polymerase chain reaction
Supported in part by grants from the Angelo Nocivelli, the Berlucchi and the Camillo Golgi Foundation, Brescia, Italy, NIH R01 CA122728-01A2 to AS, and grants 501/A3/3 and 0027557 from the Italian Institute of Health (ISS) to AS. This investigation was also supported by NIH Research Grant CA-16359 from the National Cancer Institute. We are grateful to Dr. Xiao-Bing Gao for his technical assistance and expertise in the confocal microscopy experiments.
- Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ: Cancer Statistics, 2009. CA Cancer J Clin. 2009, 59: 225-249. 10.3322/caac.20006.View ArticlePubMedGoogle Scholar
- DiSaia PJ, Creasman WT: Epithelial Ovarian Cancer. Clinical Gynecologic Oncology. 2007, St. Louis: Mosby Year Book, Inc, 313-368. 7Google Scholar
- Hough CD, Sherman-Baust CA, Pizer ES, Montz FJ, Im DD, Rosenshein NB, Cho KR, Riggins GJ, Morin PJ: Large-scale serial analysis of gene expression reveals genes differentially expressed in ovarian cancer. Cancer Res. 2000, 60: 6281-6287.PubMedGoogle Scholar
- Rangel LB, Agarwal R, D'Souza T, Pizer ES, Alò PL, Lancaster WD, Gregoire L, Schwartz DR, Cho KR, Morin PJ: Tight junction proteins claudin-3 and claudin-4 are frequently overexpressed in ovarian cancer but not in ovarian cystadenomas. Clin Cancer Res. 2003, 9: 2567-2575.PubMedGoogle Scholar
- Santin AD, Zhan F, Bellone S, Palmieri M, Cane S, Bignotti E, Anfossi S, Gokden M, Dunn D, Roman JJ, O'Brien TJ, Tian E, Cannon MJ, Shaughnessy J, Pecorelli S: Gene expression profiles in primary ovarian serous papillary tumors and normal epithelium: identification of candidate molecular markers for ovarian cancer diagnosis and therapy. Int J Cancer. 2004, 112: 14-25. 10.1002/ijc.20408.View ArticlePubMedGoogle Scholar
- Ismail RS, Baldwin RL, Fang J, Browning D, Karlan BY, Gasson JC, Chang DD: Differential gene expression between normal and tumor-derived ovarian epithelial cells. Cancer Res. 2000, 60: 6744-6749.PubMedGoogle Scholar
- Welsh JB, Zarrinkar PP, Sapinoso LM, Kern SG, Behling CA, Monk BJ, Lockhart DJ, Burger RA, Hampton GM: Analysis of gene expression profiles in normal and neoplastic ovarian tissue samples identifies candidate molecular markers of epithelial ovarian cancer. Proc Natl Acad Sci USA. 2001, 98: 1176-1181. 10.1073/pnas.98.3.1176.View ArticlePubMedPubMed CentralGoogle Scholar
- Schwartz DR, Kardia SL, Shedden KA, Kuick R, Michailidis G, Taylor JM, Misek DE, Wu R, Zhai Y, Darrah DM, Reed H, Ellenson LH, Giordano TJ, Fearon ER, Hanash SM, Cho KR: Gene expression in ovarian cancer reflects both morphology and biological behavior, distinguishing clear cell from other poor-prognosis ovarian carcinomas. Cancer Res. 2002, 62: 4722-4729.PubMedGoogle Scholar
- Ono K, Tanaka T, Tsunoda T, Kitahara O, Kihara C, Okamoto A, Ochiai K, Takagi T, Nakamura Y: Identification by cDNA microarray of genes involved in ovarian carcinogenesis. Cancer Res. 2000, 60: 5007-5011.PubMedGoogle Scholar
- Shridhar V, Lee J, Pandita A, Iturria S, Avula R, Staub J, Morrissey M, Calhoun E, Sen A, Kalli K, Keeney G, Roche P, Cliby W, Lu K, Schmandt R, Mills GB, Bast RC, James CD, Couch FJ, Hartmann LC, Lillie J, Smith DI: Genetic analysis of early- versus late-stage ovarian tumors. Cancer Res. 2001, 61: 5895-5904.PubMedGoogle Scholar
- Hough CD, Cho KR, Zonderman AB, Schwartz DR, Morin PJ: Coordinately up-regulated genes in ovarian cancer. Cancer Res. 2001, 6: 3869-3876.Google Scholar
- Shridhar V, Sen A, Chien J, Staub J, Avula R, Kovats S, Lee J, Lillie J, Smith DI: Identification of underexpressed genes in early- and late-stage primary ovarian tumors by suppression subtraction hybridization. Cancer Res. 2002, 62: 262-270.PubMedGoogle Scholar
- Jazaeri AA, Lu K, Schmandt R, Harris CP, Rao PH, Sotiriou C, Chandramouli GV, Gershenson DM, Liu ET: Molecular determinants of tumor differentiation in papillary serous ovarian carcinoma. Mol Carcinogenesis. 2003, 36: 53-59. 10.1002/mc.10098.View ArticleGoogle Scholar
- Katahira J, Inoue N, Horiguchi Y, Matsuda M, Sugimoto N: Molecular cloning and functional characterization of the receptor for Clostridium perfringens enterotoxin. J Cell Biol. 1997, 136: 1239-1247. 10.1083/jcb.136.6.1239.View ArticlePubMedPubMed CentralGoogle Scholar
- McClane BA: An overview of Clostridium Perfringens Enterotoxin. Toxicon. 1996, 34: 1335-1343. 10.1016/S0041-0101(96)00101-8.View ArticlePubMedGoogle Scholar
- Niilo L: Measurament of biological activities of purified and crude enterotoxin of Clostridium perfringens. Infect Immun. 1975, 12: 440-442.Google Scholar
- Santin AD, Cané S, Bellone S, Palmieri M, Siegel ER, Thomas M, Roman JJ, Burnett A, Cannon MJ, Pecorelli S: Treatment of chemotherapy-resistant human ovarian cancer xenografts in C.B-17/SCID mice by intraperitoneal administration of Clostridium perfringens enterotoxin. Cancer Res. 2005, 65: 4334-4342. 10.1158/0008-5472.CAN-04-3472.View ArticlePubMedGoogle Scholar
- Holloway RW, Mehta RS, Finkler NJ, Li KT, McLaren CE, Parker RJ, Fruehauf JP: Association between in vitro platinum resistance in the EDR assay and clinical outcomes for ovarian cancer patients. Gynecol Oncol. 2002, 87: 8-16. 10.1006/gyno.2002.6797.View ArticlePubMedGoogle Scholar
- Romani C, Comper F, Bandiera E, Ravaggi A, Bignotti E, Tassi RA, Pecorelli S, Santin AD: Development and characterization of a human single-chain antibody fragment against claudin-3: a novel therapeutic target in ovarian and uterine carcinomas. Am J Obstet Gynecol. 2009, 201 (70): e1-9.PubMedGoogle Scholar
- Long H, Crean CD, Lee WH, Cummings OW, Gabig TG: Expression of Clostridium perfringens enterotoxin receptors claudin-3 and claudin-4 in prostate cancer epithelium. Cancer Res. 2001, 61: 7878-7881.PubMedGoogle Scholar
- Nicolaou KC, Yang Z, Liu JJ, Ueno H, Nantermet PG, Guy RK, Claiborne CF, Renaud J, Couladouros EA, Paulvannan K, Sorensen EJ: Total synthesis of taxol. Nature. 1994, 367 (6464): 630-634. 10.1038/367630a0.View ArticlePubMedGoogle Scholar
- Beguinot L, Werth D, Ito S, Richert N, Willingham MC, Pastan I: Functional studies on the EGF receptor with an antibody that recognizes the intracellular portion of the receptor. J Biol Chem. 1986, 261: 1801-1807.PubMedGoogle Scholar
- Michl P, Buchholz M, Rolke M, Kunsch S, Löhr M, McClane B, Tsukita S, Leder G, Adler G, Gress TM: Claudin-4: a new target for pancreatic cancer treatment using Clostridium perfringens enterotoxin. Gastroenterology. 2001, 121: 678-684. 10.1053/gast.2001.27124.View ArticlePubMedGoogle Scholar
- Kominsky SL, Tyler B, Sosnowski J, Brady K, Doucet M, Nell D, Smedley JG, McClane B, Brem H, Sukumar S: Clostridium perfringens enterotoxin as a novel-targeted therapeutic for brain metastasis. Cancer Res. 2007, 67 (17): 7977-7982. 10.1158/0008-5472.CAN-07-1314.View ArticlePubMedGoogle Scholar
- Facchetti F, Lonardi S, Gentili F, Bercich L, Falchetti M, Tardanico R, Baronchelli C, Lucini L, Santin A, Murer B: Claudin 4 identifies a wide spectrum of epithelial neoplasms and represents a very useful marker for carcinoma versus mesothelioma diagnosis in pleural and peritoneal biopsies and effusions. Virchows Archiv. 2007, 451 (3): 669-80. 10.1007/s00428-007-0448-x.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/349/prepub
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