Receptors For Clostridium Perfringens Enterotoxin Research Articles

High claudin-4 antigen expression in triple-negative breast cancer by the immunohistochemistry method.

Background:Triple-negative breast cancer is a heterogeneous subtype of breast cancer. Claudin is an epithelial tight junctional protein, and also it is a receptor for clostridium perfringens enterotoxin and shows impairment of expression in several cancers. The chief purpose of this study is to assess the claudin-4 expression in triple-negative breast cancer (TNBC) Iranian patients and evaluate its correlation with some clinicopathological factors.Materials and Methods:In this study, 81 TNBC patients were evaluated for the claudin-4 expression by immunohistochemistry. The slides’ staining intensity was examined and scored from 0 to 3. Then, slides were reviewed to assess the percentage of cells with membrane and cytoplasmic staining; the obtaining scores were 1–4. Finally, added the resulting two numbers from two stages, and the final number was a maximum of 7. Final scores of 0–3 were considered the low expression, and 4–7 were considered the high expression. Finally, the collected data were analyzed using the Chi-square test.Results:Eighty-one women with breast cancer and a mean age of 49 ± 12 years participated in the study. In 80% of the patients, there was a high expression of claudin-4 marker, and 20% had low expression. The expression level of the marker was not significantly correlated with age, tumor size, lymph node involvement, tumor grade, disease stage, Ki-67, and metastasis.Conclusion:The present study confirmed the high frequency of claudin-4 antigen expression in TNBC patients, and no significant correlation was observed between the expression of antigen and demographic or clinicopathological factors.

In silico prediction of amino acids involved in cCPE290-319 interaction with claudin 4.

Among the 26 human claudin proteins, the food-poisoning bacterium Clostridium perfringens produces an enterotoxin (~ 35.00 kDa) that specifically targets human claudin 4, causing diarrhea by fluid accumulation in the intestinal cavity. The Clostridium perfringens enterotoxin (CPE) C-terminal domain (cCPE ~ 15.00 kDa) tightly binds to claudin 4 and disrupts the tight junction barriers in the intestines. In this study, we aimed to determine the contribution and type of amino acid interactions involved in association between claudin 4 and the C-terminal CPE. First, the three-dimensional format of claudin 4 was downloaded from RCSB. Then, during 60.00 nanoseconds (nsec), molecular dynamics simulation was conducted using the GROMACS package on CPE of crystallographic structure. The results indicated that the simulations performed well during the simulation times and there were no noticeable problems or artifacts. We found that Coulombic (glycine 317, proline 311 and serine 313) and Lennard-Jones (tyrosine 310, leucine 315, serine 313 and glycine 317) interactions played a significant role in complex stability. This information localized the C-terminal of CPE as a linear sequence sufficient for recognition and binding to the eukaryotic CPE receptor. A detailed description of the dissociation process brings valuable insight into the interaction of the claudin 4-cCPE290-319 complexes, which could help in the future to design more potent drugs.

Targeting claudin-overexpressing thyroid and lung cancer by modified Clostridiumperfringens enterotoxin.

Clostridium perfringens enterotoxin (CPE) can be used to eliminate carcinoma cells that overexpress on their cell surface CPE receptors – a subset of claudins (e.g., Cldn3 and Cldn4). However, CPE cannot target tumors expressing solely CPE‐insensitive claudins (such as Cldn1 and Cldn5). To overcome this limitation, structure‐guided modifications were used to generate CPE variants that can strongly bind to Cldn1, Cldn2 and/or Cldn5, while maintaining the ability to bind Cldn3 and Cldn4. This enabled (a) targeting of the most frequent endocrine malignancy, namely, Cldn1‐overexpressing thyroid cancer, and (b) improved targeting of the most common cancer type worldwide, non‐small‐cell lung cancer (NSCLC), which is characterized by high expression of several claudins, including Cldn1 and Cldn5. Different CPE variants, including the novel mutant CPE‐Mut3 (S231R/S313H), were applied on thyroid cancer (K1 cells) and NSCLC (PC‐9 cells) models. In vitro, CPE‐Mut3, but not CPEwt, showed Cldn1‐dependent binding and cytotoxicity toward K1 cells. For PC‐9 cells, CPE‐Mut3 improved claudin‐dependent cytotoxic targeting, when compared to CPEwt. In vivo, intratumoral injection of CPE‐Mut3 in xenograft models bearing K1 or PC‐9 tumors induced necrosis and reduced the growth of both tumor types. Thus, directed modification of CPE enables eradication of tumor entities that cannot be targeted by CPEwt, for instance, Cldn1‐overexpressing thyroid cancer by using the novel CPE‐Mut3.

The Potential Therapeutic Agent Mepacrine Protects Caco-2 Cells against Clostridium perfringens Enterotoxin Action.

Clostridium perfringens enterotoxin (CPE) causes the diarrhea associated with a common bacterial food poisoning and many antibiotic-associated diarrhea cases. The severity of some CPE-mediated disease cases warrants the development of potential therapeutics. A previous study showed that the presence of mepacrine inhibited CPE-induced electrophysiology effects in artificial lipid bilayers lacking CPE receptors. However, that study did not assess whether mepacrine inactivates CPE or, instead, inhibits a step in CPE action. Furthermore, CPE action in host cells is complex, involving the toxin binding to receptors, receptor-bound CPE oligomerizing into a prepore on the membrane surface, and β-hairpins in the CPE prepore inserting into the membrane to form a pore that induces cell death. Therefore, the current study evaluated the ability of mepacrine to protect cells from CPE. This drug was found to reduce CPE-induced cytotoxicity in Caco-2 cells. This protection did not involve mepacrine inactivation of CPE, indicating that mepacrine affects one or more steps in CPE action. Western blotting then demonstrated that mepacrine decreases CPE pore levels in Caco-2 cells. This mepacrine-induced reduction in CPE pore levels did not involve CPE binding inhibition but rather an increase in CPE monomer dissociation due to mepacrine interactions with Caco-2 membranes. In addition, mepacrine was also shown to inhibit CPE pores when already present in Caco-2 cells. These in vitro studies, which identified two mepacrine-sensitive steps in CPE-induced cytotoxicity, add support to further testing of the therapeutic potential of mepacrine against CPE-mediated disease. IMPORTANCEClostridium perfringens enterotoxin (CPE) causes the gastrointestinal (GI) symptoms of a common bacterial food poisoning and several nonfoodborne human GI diseases. A previous study showed that, via an undetermined mechanism, the presence of mepacrine blocks CPE-induced electrophysiologic activity in artificial membranes. The current study now demonstrates that mepacrine also inhibits CPE-induced cytotoxicity in human enterocyte-like Caco-2 cells and that mepacrine does not directly inactivate CPE. Instead, this drug reduces both CPE pore formation and CPE pore activity in Caco-2 cells. These results suggest mepacrine as a therapeutic candidate for treating CPE-mediated GI diseases.

Rapid eradication of colon carcinoma by Clostridium perfringens Enterotoxin suicidal gene therapy

BackgroundBacterial toxins have evolved to an effective therapeutic option for cancer therapy. The Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin with selective cytotoxicity. The transmembrane tight junction proteins claudin-3 and -4 are known high affinity CPE receptors. Their expression is highly upregulated in human cancers, including breast, ovarian and colon carcinoma. CPE binding to claudins triggers membrane pore complex formation, which leads to rapid cell death. Previous studies demonstrated the anti-tumoral effect of treatment with recombinant CPE-protein. Our approach aimed at evaluation of a selective and targeted cancer gene therapy of claudin-3- and/or claudin-4- expressing colon carcinoma in vitro and in vivo by using translation optimized CPE expressing vector.MethodsIn this study the recombinant CPE and a translation optimized CPE expressing vector (optCPE) was used for targeted gene therapy of claudin-3 and/or -4 overexpressing colon cancer cell lines. All experiments were performed in the human SW480, SW620, HCT116, CaCo-2 and HT-29 colon cancer and the isogenic Sk-Mel5 and Sk-Mel5 Cldn-3-YFP melanoma cell lines. Claudin expression analysis was done at protein and mRNA level, which was confirmed by immunohistochemistry. The CPE induced cytotoxicity was analyzed by the MTT cytotoxicity assay. In addition patient derived colon carcinoma xenografts (PDX) were characterized and used for the intratumoral in vivo gene transfer of the optCPE expressing vector in PDX bearing nude mice.ResultsClaudin-3 and -4 overexpressing colon carcinoma lines showed high sensitivity towards both recCPE application and optCPE gene transfer. The positive correlation between CPE cytotoxicity and level of claudin expression was demonstrated. Transfection of optCPE led to targeted, rapid cytotoxic effects such as membrane disruption and necrosis in claudin overexpressing cells. The intratumoral optCPE in vivo gene transfer led to tumor growth inhibition in colon carcinoma PDX bearing mice in association with massive necrosis due to the intratumoral optCPE expression.ConclusionsThis novel approach demonstrates that optCPE gene transfer represents a promising and efficient therapeutic option for a targeted suicide gene therapy of claudin-3 and/or claudin-4 overexpressing colon carcinomas, leading to rapid and effective tumor cell killing in vitro and in vivo.

Dual-Targeting Nanoparticles for In Vivo Delivery of Suicide Genes to Chemotherapy-Resistant Ovarian Cancer Cells.

Ovarian cancer is the most lethal gynecologic cancer. Claudin-3 and -4, the receptors for Clostridium perfringens enterotoxin (CPE), are overexpressed in more than 70% of these tumors. Here, we synthesized and characterized poly(lactic-co-glycolic-acid) (PLGA) nanoparticles (NPs) modified with the carboxy-terminal-binding domain of CPE (c-CPE-NP) for the delivery of suicide gene therapy to chemotherapy-resistant ovarian cancer cells. As a therapeutic payload, we generated a plasmid encoding for the diphtheria toxin subunit-A (DT-A) under the transcriptional control of the p16 promoter, a gene highly differentially expressed in ovarian cancer cells. Flow cytometry and immunofluorescence demonstrated that c-CPE-NPs encapsulating the cytomegalovirus (CMV) GFP plasmid (CMV GFP c-CPE-NP) were significantly more efficient than control NPs modified with a scrambled peptide (CMV GFP scr-NP) in transfecting primary chemotherapy-resistant ovarian tumor cell lines in vitro (P = 0.03). Importantly, c-CPE-NPs encapsulating the p16 DT-A vector (p16 DT-A c-CPE-NP) were significantly more effective than control p16 DT-A scr-NP in inducing ovarian cancer cell death in vitro (% cytotoxicity: mean ± SD = 32.9 ± 0.15 and 7.45 ± 7.93, respectively, P = 0.03). In vivo biodistribution studies demonstrated efficient transfection of tumor cells within 12 hours after intraperitoneal injection of CMV GFP c-CPE-NP in mice harboring chemotherapy-resistant ovarian cancer xenografts. Finally, multiple intraperitoneal injections of p16 DT-A c-CPE-NP resulted in a significant inhibition of tumor growth compared with control NP in chemotherapy-resistant tumor-bearing mice (P = 0.041). p16 DT-A c-CPE-NP may represent a novel dual-targeting therapeutic approach for the selective delivery of gene therapy to chemotherapy-resistant ovarian cancer cells. Mol Cancer Ther; 16(2); 323-33. ©2016 AACR.

Distinct presence of the tight junction protein claudin-3 in olfactory bulb and fila olfactoria of the mouse.

The tight junction protein claudin-3 is overexpressed in diverse epithelial tumours and is associated with increased survival, progression and motility of tumour cells. Claudin-3 expression profiles are being increasingly used for diagnostic and prognostic tumour classification. Claudin-3 has been identified as a receptor for Clostridium perfringens enterotoxin, which is under consideration for selective lysis of claudin-3-expressing tumours, particularly brain metastases, and other translational medicine uses. However, the localization of claudin-3 in the brain has not been completely elucidated. While claudin-3 in brain tissue adjacent to claudin-3-expressing metastases had been excluded and low or undetectable levels proposed in the CNS, under physiological conditions, in adult human, rat and mouse brains, claudin-3 was exclusively found in choroid plexus epithelium where it is considered an integral component of the blood-cerebrospinal-fluid barrier. We report here the pronounced presence of claudin-3 not only in the nasal region (as described for rat), but also in the mouse olfactory bulb and nerve using immunohistochemistry and Western blot. Claudin-3 was present in the fila olfactoria from the epithelium to the olfactory nerve and in the main and accessory olfactory bulb. We propose that the abundant presence of claudin-3 in the olfactory system, particularly in nerve fibres and the olfactory bulb cone, which we present here, may play a role at the interface of the central and peripheral nervous system, both as barrier and for axonal growth and communication. Thus, claudin-3 should be considered and further explored with regards to treatment approaches addressing the olfactory bulb and nasal region.

Roles of the first-generation claudin binder, Clostridium perfringens enterotoxin, in the diagnosis and claudin-targeted treatment of epithelium-derived cancers.

Given that most malignant tumors are derived from epithelium, developing a strategy for treatment of epithelium-derived cancers (i.e., carcinomas) is a pivotal issue in cancer therapy. Carcinomas, including ovarian, breast, prostate, and pancreatic cancers, are known to overexpress various claudins (CLDNs); in particular, CLDN-3 and -4 are frequently overexpressed in malignant case. The generation of CLDN binders is a key for expanding CLDN-targeted cancer therapy but has been delayed due to the small size of CLDN extracellular domains (approximately 50 amino acids for the first domain and 15 amino acids for the second) and their high homology among species. Interestingly, however, the receptors for Clostridium perfringens enterotoxin (CPE), a foodborne toxin in humans, happen to be identical to CLDN-3 and -4. Thus, the first CLDN binder, CPE, has provided us CLDN-targeted cancer therapy from a concept into a potential reality. In this review, we describe roles of CPE technology in cancer therapy and discuss future directions in the CLDN-targeting concept-to-therapy process.

MP61-15 CLOSTRIDIUM PERFRINGES ENTEROTOXIN AS A POTENTIAL THERAPEUTIC FOR INTRAVESICAL TREATMENT OF BLADDER CANCER

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II1 Apr 2016MP61-15 CLOSTRIDIUM PERFRINGES ENTEROTOXIN AS A POTENTIAL THERAPEUTIC FOR INTRAVESICAL TREATMENT OF BLADDER CANCER Victor Romanov, Wayne Waltzer, Terry Whyard, Olga Povcher, April Szafran, and Theodore Gabig Victor RomanovVictor Romanov More articles by this author , Wayne WaltzerWayne Waltzer More articles by this author , Terry WhyardTerry Whyard More articles by this author , Olga PovcherOlga Povcher More articles by this author , April SzafranApril Szafran More articles by this author , and Theodore GabigTheodore Gabig More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.889AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The current intravesical treatment of bladder cancer (BC) is limited to a few chemotherapeutic drugs that shows imperfect effectiveness and are associated with some serious clinical complications. Thus, there is an urgent need for identification of alternative therapies, especially for patients with high-risk non-muscle invasive BC. Clostridium perfringens enterotoxin (CPE), cytolytic protein binds to its receptors: claudin 3 and 4 that expressed in epithelial cells. This binding is followed by pore formation in cell membranes of targeted cells that in turn causes rapid cell death. Claudin 4 is present in bladder urothelium and this expression is substantially elevated in some forms of BC. In addition, binding of CPE to claudins increases membrane permeability that creates conditions for drugs penetration of urothelium and better accession of the tumor. Therefore, we evaluated CPE as a potential candidate for intravesical treatment of BC using an in vitro cellular model. METHODS We examined the cytotoxic efficiency of CPE against BC cells using an array of established cell lines and primary 2 and.3D cultures of cells derived from the surgical samples using in vitro cell viability assay. To better elucidate cellular mechanisms, activated by CPE we synthesized the receptor-binding domain of the enterotoxin (C-CPE), compared its cytotoxic activity with full CPE and examined claudin 4 expression and intracellular localization after that treatment using immunofluorescence and Western blot techniques RESULTS CPE induced cell death after 1 h in low aggressive RT4 cells, in moderately aggressive 5637 cells and in the primary 3D cultures of BC cells derived from non-muscle invasive tumor. Conversely, non-transformed urothelial cells and cells derived from highly aggressive tumor (T24) survived this treatment. The reason for this resistance to CPE might be the lower expression of CLDNs in these cells or inaccessibility of the receptors for CPE. Indeed, claudin 4 was abundantly expressed in cells that can be destroyed by CPE and was present at much lower level in CPE resistant cells. C-CPE treatment for 48 h did not induce cell death, but declined expression of CLDN4 in RT4 cells. In addition, translocation of claudin 4 from cell periphery was observed at these conditions. CONCLUSIONS These observations shows that after additional preclinical studies CPE can be considered as a potential drug for intravesical treatment of BC because of its ability highly effectively destroy BC cells expressing claudin 4 and low toxicity against normal urothelium. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e809 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Victor Romanov More articles by this author Wayne Waltzer More articles by this author Terry Whyard More articles by this author Olga Povcher More articles by this author April Szafran More articles by this author Theodore Gabig More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 4937: Fluorescence imaging using Clostridium Perfringens Enterotoxin carboxi-terminal fragment (c-CPE) to target metastatic chemotherapy-resistant human ovarian cancer in xenograft mice

Abstract Epithelial ovarian cancer is the most lethal gynecologic malignancy. It is treated through up front surgery followed by chemotherapy or with neoadjuvant chemotherapy before surgical debulking. Achieving complete or optimal cytoreduction improves both progression free and overall survival. In the operating room, no matter the care taken, there is the potential for areas of macroscopic, microscopic and occult metastases to remain unvisualized. The development of a sensitive and specific intraoperative system for the visualization and removal or destruction of metastatic disease may improve patient outcome. Fresh ovarian cancer samples were recently analyzed by our group with genetic fingerprinting. This analysis revealed high expression of claudin-3 and -4, the epithelial receptors for Clostridium Perfringens Enterotoxin (CPE). Although the administration of the full length CPE in mice is toxic, the injection of the only carboxi-terminal fragment (c-CPE) avoids toxicity while preserving the binding to the receptors. Our previous data showed specific binding of FITC conjugated c-CPE (FITC-c-CPE) to primary ovarian cancer cell lines in vitro as well as preferential accumulation of the labeled peptide into ovarian cancer xenografts in vivo. This study evaluates the in vivo distribution of FITC-c-CPE after intraperitoneal (IP) injection of the peptide as well as the kinetics and tumor binding capacity of c-CPE conjugated to the NearInfraRed Dye CW800 (CW800-c-CPE), focusing on the ability of CW800-c-CPE to identify metastases of chemotherapy-resistant ovarian cancer overexpressing claudin-3 and -4 in vivo. We found that fluorescence uptake by the tumor starts 30 minutes after FITC-c-CPE injection with negligible staining of healthy organs. When the abdominal cavity of FITC-c-CPE injected mice was visualized using a fluorescence microscope, strong signal was detected in near microscopic metastatic nodules and in malignant tumor spheroids isolated from the ascites. Similarly, CW800-c-CPE also accumulated in tumors in vivo following IP or systemic (IV) injection. Ex vivo distribution analysis demonstrated a significantly higher mean fluorescence intensity (MFI) in tumor compared to healthy organs (MFI: mean ± STDV: 156.55 ± 23.73, 95.72 ± 18.19, 30.68 ± 5.88, 23.33 ± 4.05, 34.71 ± 12.71, 28.16 ± 6.1413.46 ± 1.35, 19.78 ± 5.43 in the tumor, kidney, liver, spleen, bowel, lungs and brain, respectively; p<0.01). The accumulation of CW800-c-CPE was also noted in small size tumor implants in the abdomen. These data suggest that c-CPE has tremendous specificity for targeting metastatic chemotherapy-resistant ovarian cancer in vivo. Furthermore, the c-CPE peptide has the potential to be implemented into the operative setting to allow for improved detection of residual tumor during staging and cytoreductive surgery for ovarian cancer patients. Citation Format: Emiliano Cocco, Sara Gasparrini, Erik M. Shapiro, Carlton Schwab, Stefania Bellone, Ileana Bortolomai, Salvatore Lopez, Natalia J. Sumi, Elena Bonazzoli, Roberta Nicoletti, Yang Deng, William M. Saltzman, Caroline J. Zeiss, Dan-Arin Silasi, Thomas J. Rutherford, Peter E. Schwartz, Alessandro D. Santin. Fluorescence imaging using Clostridium Perfringens Enterotoxin carboxi-terminal fragment (c-CPE) to target metastatic chemotherapy-resistant human ovarian cancer in xenograft mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4937. doi:10.1158/1538-7445.AM2014-4937

Abstract 732: Iron oxide nanoparticles functionalized with the Clostridium Perfringens Enterotoxin carboxi-terminal fragment peptide: A novel diagnostic and therapeutic approach to specifically target ovarian cancer.

Abstract Ovarian carcinoma remains the cancer with the highest mortality among gynecological tumors. Development of novel strategies for the diagnosis and treatment of chemotherapy resistant ovarian disease remains a high priority. Magnetic iron oxide nanoparticles (NP) consist of an iron oxide core and hydrophilic coating. NP can establish magnetic field gradients that alter proton relaxation properties, thus providing an excellent source of contrast for magnetic resonance imaging (MRI). Moreover, since they can be heated in alternating magnetic fields (AMF), they may also provide an effective form of hyperthermia for cancer therapy. Unfortunately, currently available iron oxide NP, although potentially able to allow MRI detection of single cells in vivo, are not tumor specific. Our group has recently analyzed the genetic fingerprints of ovarian cancer identifying extremely high expression of the genes encoding the proteins claudin-3 and -4, the epithelial receptors for Clostridium perfringens enterotoxin (CPE). Since the C-terminus domain (C-CPE) of the toxin is sufficient for CPE binding and it is not toxic to cells, we used iron oxide NP complexed to C-CPE peptide to target chemotherapy resistant ovarian cancer cells overexpressing claudin-3 and -4. We report that NP functionalized by the fluorescent C-CPE peptide effectively bind and rapidly internalize into ovarian cancer cells using flow cytometry,confocal microscopy and Prussian blue staining. A 5-fold increase in iron cellular uptake after incubation of tumor cells with C-CPE NP was consistently detected when compared to tumor cells exposed to non specific control NP [i.e., iron concentration of (mean ± STDV) 20.3 ± 0.5 vs 4.1 ± 1.3 nmoles/5*10 cells respectively; p<0.01]. Importantly, only tumor cells exposed to C-CPE NP were detectable in MRI in vitro experiments using a Bruker 4.7Tmagnet. Finally, to assess the potential of C-CPE NP to be used for hyperthermia therapy against chemotherapy resistant ovarian cancer, we characterized the magnetic heating capacity of C-CPE NP using an induction heating system (Ambrell, Easy Heat). Our results show a temperature increase of 7.85 ± 0.6 °C (mean ± STDV) when C-CPE NP were subjected to an AMF of 343 kHz of frequency and 2 KW of power for 10 minutes. Our data clearly suggest that C-CPE NP may represent a novel theranostic tool to specifically target chemotherapy resistant ovarian cancer. Citation Format: Emiliano Cocco, Ileana Bortolomai, Stefania Bellone, Sara Gasparrini, Diana English, Erik Shapiro, Sergio Pecorelli, Dan-Arin Silasi, Masoud Azodi, Elena Ratner, Thomas Rutherford, Peter Schwartz, Alessandro Santin. Iron oxide nanoparticles functionalized with the Clostridium Perfringens Enterotoxin carboxi-terminal fragment peptide: A novel diagnostic and therapeutic approach to specifically target ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 732. doi:10.1158/1538-7445.AM2013-732

Claudin-6: a novel receptor for CPE-mediated cytotoxicity in ovarian cancer

Claudins are integral tight junction proteins that are responsible for maintaining the integrity of epithelial cell architecture and cell polarity. Claudin-3 and -4 are overexpressed in several cancers and have been shown to act as receptors for the Clostridium perfringens enterotoxin (CPE), a toxin that causes rapid cell lysis. CPE has demonstrated effectiveness in treating several different cancers in mouse models, provided that these cancers express claudin-3 or claudin-4. Here, we show that claudin-3/4 expression is not an absolute requirement for CPE action and, through overexpression and knockdown experiments, we identify claudin-6 as a novel functional receptor for CPE. Indeed, UCI-101, an ovarian cancer cell line highly sensitive to CPE, does not express claudin-3/4 and knockdown of claudin-6 in these cells decreases CPE sensitivity. Moreover, two different ovarian cell lines that are resistant to the effects of CPE can be made sensitive through claudin-6 overexpression. Binding assays show that CPE can indeed bind claudin-6 in cells and that this binding is associated with CPE cytotoxicity. Multicellular tumor spheroids experiments demonstrate that claudin-6 can also be a target of CPE in three-dimensional cultures. Our data establish claudin-6 as a novel receptor for CPE and introduces the possibility of a novel targeted therapeutic for ovarian and other cancers that express claudin-6.

Abstract 364: In vivo targeting of CD44+ ovarian cancer stem cells (CSC) by Clostridium Perfringens Enterotoxin binding domain (CPE-peptide) conjugated to poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP)

Abstract Recent evidence strongly supports the hypothesis that the capability to sustain tumor formation, growth and resistance to chemotherapy in ovarian cancer exclusively resides in a small proportion of tumor cells termed cancer stem cells (CSC) overexpressing the CD44 marker. If this hypothesis is correct, development of novel, more specific approaches to target and eradicate the CD44+ ovarian CSC population remains a high priority. Our group has recently reported high expression of claudin-4 tight junction protein in CD44+ ovarian CSC. Because this protein is the natural high affinity receptor for Clostridium Perfringens Enterotoxin (CPE), we have evaluated the in vitro targeting potential of fluorescent (i.e. Coumarin 6 loaded) poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) conjugated with the C-terminus binding domain of CPE (CPE-peptide) against ovarian CSC and the in vivo biodistribution of these NP after intravenous (IV) injection in SCID mice harboring intraperitoneal CD44+ ovarian CSC tumor. Using flow cytometry, we report a higher time and dose-dependent in vitro uptake of fluorescent PLGA NP conjugated to CPE peptide (CPE-peptide NP) in CD44+ ovarian CSC when compared to unconjugated NP (i.e., control NP, Mean-Fluorescence-Intensity = MFI of 37.1 and 15.0 respectively; p=0.037). Of interest, confocal microcopy images clearly show internalization of CPE-peptide NP within tumor cells after NP exposure. More importantly, when SCID mice harboring claudin-4 overexpressing CD44+ ovarian CSC tumors were injected via the tail vein (IV) with 25 μg/g of CPE-peptide NP or the same concentration of unconjugated NP, a 4 fold increase in fluorescence was detected in tumors excised from mice injected with CPE-peptide NP compared to controls 48 hours after NP administration (mean ± STDV 33159.6 2707 and 6655.8 ±1391 respectively; p<0.01). Finally, non specific NP uptake in healthy organs calculated as the ratio between organs and tumor fluorescence was found to be significantly lower after injection of CPE-peptide NP than the unconjugated control NP. Taken together these results suggest that the CPE-peptide, when conjugated to NP, may confer high specificity in targeting claudin-4 overexpressing CD44+ ovarian CSC tumors in vivo. CPE-peptide NP may represent an unconventional, potentially highly effective strategy to specifically target chemotherapy-resistant CD44+ ovarian CSC for diagnostic and/or therapeutic purposes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 364. doi:1538-7445.AM2012-364

Novel Clostridium perfringens enterotoxin suicide gene therapy for selective treatment of claudin-3- and -4-overexpressing tumors

Bacterial toxins are known to be effective for cancer therapy. Clostridium perfringens enterotoxin (CPE) is produced by the bacterial Clostridium type A strain. The transmembrane proteins claudin-3 and -4, often overexpressed in numerous human epithelial tumors (for example, colon, breast, pancreas, prostate and ovarian), are the targeted receptors for CPE. CPE binding to them triggers formation of membrane pore complexes leading to rapid cell death. In this study, we aimed at selective tumor cell killing by CPE gene transfer. We generated expression vectors bearing the bacterial wild-type CPE cDNA (wtCPE) or translation-optimized CPE (optCPE) cDNA for in vitro and in vivo gene therapy of claudin-3- and -4-overexpressing tumors. The CPE expression analysis at messenger RNA and protein level revealed more efficient expression of optCPE compared with wtCPE. Expression of optCPE showed rapid cytotoxic activity, hightened by CPE release as bystander effect. Cytotoxicity of up to 100% was observed 72 h after gene transfer and is restricted to claudin-3-and -4-expressing tumor lines. MCF-7 and HCT116 cells with high claudin-4 expression showed dramatic sensitivity toward CPE toxicity. The claudin-negative melanoma line SKMel-5, however, was insensitive toward CPE gene transfer. The non-viral intratumoral in vivo gene transfer of optCPE led to reduced tumor growth in MCF-7 and HCT116 tumor-bearing mice compared with the vector-transfected control groups. This novel approach demonstrates that CPE gene transfer can be employed for a targeted suicide gene therapy of claudin-3- and -4-overexpressing tumors, leading to the rapid and efficient tumor cell killing in vitro and in vivo.

Use ofClostridium perfringensEnterotoxin and the Enterotoxin Receptor-Binding Domain (C-CPE) for Cancer Treatment: Opportunities and Challenges

Clostridium perfringens enterotoxin (CPE) causes the symptoms associated with several common gastrointestinal diseases. CPE is a 35 kDa polypeptide consisting of three structured domains, that is, C-terminal domain I (responsible for receptor binding), domain II (responsible for oligomerization and membrane insertion), and domain III (which may participate in physical changes when the CPE protein inserts into membranes). Native CPE binds to claudin receptors, which are components of the tight junction. The bound toxin then assembles into a hexameric prepore on the membrane surface, prior to the insertion of this oligomer into membranes to form an active pore. The toxin is especially lethal for cells expressing large amounts of claudin-3 or -4, which includes many cancer cells. Initial studies suggest that native CPE has potential usefulness for treating several cancers where claudin CPE receptors are overexpressed. However, some challenges with immunogenicity, toxicity, and (possibly) the development of resistance may need to be overcome. An alternative approach now being explored is to utilize C-CPE, which corresponds approximately to receptor binding domain I, to enhance paracellular permeability and delivery of chemotherapeutic agents against cancer cells. Alternatively, C-CPE fusion proteins may prove superior to use of native CPE for cancer treatment. Finally, C-CPE may have application for other medical treatments, including vaccination or increasing drug absorption. The coming years should witness increasing exploitation of this otherwise formidable toxin.

Eradication of chemotherapy‐resistant CD44+ human ovarian cancer stem cells in mice by intraperitoneal administration of clostridium perfringens enterotoxin

Emerging evidence has suggested that the capability to sustain tumor formation, growth, and chemotherapy resistance in ovarian as well as other human malignancies exclusively resides in a small proportion of tumor cells termed cancer stem cells. During the characterization of CD44(+) ovarian cancer stem cells, we found a high expression of the genes encoding for claudin-4. Because this tight junction protein is the natural high-affinity receptor for Clostridium perfringens enterotoxin (CPE), we have extensively investigated the sensitivity of ovarian cancer stem cells to CPE treatment in vitro and in vivo. Real-time polymerase chain reaction and flow cytometry were used to evaluate claudin-3/-4 expression in ovarian cancer stem cells. Small interfering RNA knockdown experiments and MTS assays were used to evaluate CPE-induced cytotoxicity against ovarian cancer stem cell lines in vitro. C.B-17/SCID mice harboring ovarian cancer stem cell xenografts were used to evaluate CPE therapeutic activity in vivo. CD44(+) ovarian cancer stem cells expressed claudin-4 gene at significantly higher levels than matched autologous CD44(-) ovarian cancer cells, and regardless of their higher resistance to chemotherapeutic agents died within 1 hour after exposure to 1.0 μg/mL of CPE in vitro. Conversely, small-interfering RNA-mediated knockdown of claudin-3/-4 expression in CD44(+) cancer stem cells significantly protected cancer stem cells from CPE-induced cytotoxicity. Importantly, multiple intraperitoneal administrations of sublethal doses of CPE in mice harboring xenografts of chemotherapy-resistant CD44(+) ovarian cancer stem cells had a significant inhibitory effect on tumor progression leading to the cure and/or long-term survival of all treated animals (ie, 100% reduction in tumor burden in 50% of treated mice; P < .0001). CPE may represent an unconventional, potentially highly effective strategy to eradicate chemotherapy-resistant cancer stem cells.

The effect of intraperitoneal administration of clostridium perfringens enterotoxin on chemotherapy-resistant CD44+ human ovarian cancer stem cells in mice.

e13077 Background: Emerging evidence has suggested that the capability to sustain tumor formation, growth and chemotherapy-resistance in ovarian, as well as other human malignancies, exclusively resides in a small proportion of tumor cells termed cancer stem cells (CSCs). During the characterization of CD44+ ovarian CSCs, we found a high expression of the genes encoding for claudin-4. Because this tight junction protein is the natural high affinity receptor for Clostridium perfringens enterotoxin (CPE), we have extensively investigated the sensitivity of ovarian CSC to CPE treatment in vitro and in vivo. Methods: Real-time polymerase-chain-reaction and flow cytometry were used to evaluate claudin-3/-4 expression in ovarian CSCs. Small-interfering-RNA-knockdown experiments, and MTS-assays were used to evaluate CPE-induced cytotoxicity against ovarian CSC lines in vitro. C.B-17/SCID mice harboring ovarian CSC xenografts were used to evaluate CPE therapeutic activity in vivo. Results: CD44+ ovarian CSCs expressed claudin-4 gene at significantly higher levels than matched autologous CD44- ovarian cancer cells and, regardless of their higher resistance to chemotherapeutic agents, died within 1 hour after exposure to 1.0μg/ml of CPE in vitro. Conversely, small-interfering-RNA–mediated knockdown of claudin-3/-4 expression in CD44+ CSCs significantly protected CSCs from CPE-induced-cytotoxicity. Importantly, multiple intraperitoneal administrations of sublethal doses of CPE in mice harboring xenografts of chemotherapy resistant CD44+ ovarian CSCs had a significant inhibitory effect on tumor progression leading to the cure and/or long-term survival of all treated animals. Conclusions: CPE may represent an unconventional, potentially highly effective strategy to eradicate chemotherapy-resistant CSCs.

Effects of Clostridium perfringens enterotoxin via claudin-4 on normal human pancreatic duct epithelial cells and cancer cells

The tight junction protein claudin-4 is frequently overexpressed in pancreatic cancer, and is also a receptor for Clostridium perfringens enterotoxin (CPE). The cytotoxic effects of CPE are thought to be useful as a novel therapeutic tool for pancreatic cancer. However, the responses to CPE via claudin-4 remain unknown in normal human pancreatic duct epithelial (HPDE) cells. We introduced the human telomerase reverse transcriptase (hTERT) gene into HPDE cells in primary culture as a model of normal HPDE cells in vitro. hTERT-HPDE cells treated with or without 10% FBS and pancreatic cancer cell lines PANC-1, BXPC3, HPAF-II and HPAC were treated with CPE. In Western blotting, the expression of claudin-4 protein in hTERT-HPDE cells treated with 10% FBS was as high as it was in all of the pancreatic cancer cell lines. In hTERT-HPDE cells with or without 10% FBS, cytotoxicity was not observed at any concentration of CPE, whereas in all pancreatic cancer cell lines, CPE had a dose-dependent cytotoxic effect. In hTERT-HPDE cells with 10% FBS, claudin-4 was localized in the apical-most regions, where there are tight junction areas, in which in all pancreatic cancer cell lines claudin-4 was found not only in the apical-most regions but also at basolateral membranes. In hTERT-HPDE cells with 10% FBS after treatment with CPE, downregulation of barrier function and claudin-4 expression at the membranes was observed. In HPAC cells, the sensitivity to CPE was significantly decreased by knockdown of claudin-4 expression using siRNA compared to the control. These findings suggest that, in normal HPDE cells, the lack of toxicity of CPE was probably due to the localization of claudin-4, which is different from that of pancreatic cancer cells. hTERT-HPDE cells in this culture system may be a useful model of normal HPDE cells not only for physiological regulation of claudin-4 expression but also for developing safer and more effective therapeutic methods targeting claudin-4 in pancreatic cancer.

Clostridium perfringens enterotoxin carboxy-terminal fragment is a novel tumor-homing peptide for human ovarian cancer

BackgroundDevelopment 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.MethodsBecause 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.ResultsUsing 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.ConclusionsBased 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.

On the Interaction of Clostridium perfringens Enterotoxin with Claudins

Clostridium perfringens causes one of the most common foodborne illnesses, which is largely mediated by the Clostridium perfringens enterotoxin (CPE). The toxin consists of two functional domains. The N-terminal region mediates the cytotoxic effect through pore formation in the plasma membrane of the mammalian host cell. The C-terminal region (cCPE) binds to the second extracellular loop of a subset of claudins. Claudin-3 and claudin-4 have been shown to be receptors for CPE with very high affinity. The toxin binds with weak affinity to claudin-1 and -2 but contribution of these weak binding claudins to CPE-mediated disease is questionable. cCPE is not cytotoxic, however, it is a potent modulator of tight junctions. This review describes recent progress in the molecular characterization of the cCPE-claudin interaction using mutagenesis, in vitro binding assays and permeation studies. The results promote the development of recombinant cCPE-proteins and CPE-based peptidomimetics to modulate tight junctions for improved drug delivery or to treat tumors overexpressing claudins.