Hideki Kakutani

A Novel Tumor-Targeted Therapy Using a Claudin-4-Targeting Molecule

Carcinogenesis is often accompanied by dysfunctional tight junction (TJs), resulting in the loss of cellular polarity. Claudin, a tetra-transmembrane protein, plays a pivotal role in the barrier and fence functions of TJs. Claudin-4 is deregulated in various cancers, including breast, prostate, ovarian, and gastric cancer. Claudin-4 may be a promising target molecule for tumor therapy, but the claudin-targeting strategy has never been fully developed. In the present study, we prepared a claudin-4-targeting molecule by fusion of the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) with the protein synthesis inhibitory factor (PSIF) derived from Pseudomonas aeruginosa exotoxin. PSIF was not cytotoxic to claudin-4-expressing cells, whereas C-CPE-PSIF was cytotoxic. Cells that express claudin-1, -2, and -5 were less sensitive to C-CPE-PSIF. Pretreatment of the cells with C-CPE attenuated C-CPE-PSIF-induced cytotoxicity, and mutation of C-CPE in the claudin-4-binding residues attenuated the cytotoxicity of C-CPE-PSIF. TJ-undeveloped cells were more sensitive to C-CPE-PSIF than TJ-developed cells. It is noteworthy that polarized epithelial cells are sensitive to C-CPE-PSIF applied to the basal side, whereas the cells were less sensitive to C-CPE-PSIF applied to the apical side. Intratumoral injection of C-CPE-PSIF reduced tumor growth. This is the first report to indicate that a claudin-4-targeting strategy may be a promising method to overcome the malignant tumors.

Domain mapping of a claudin-4 modulator, the C-terminal region of C-terminal fragment of Clostridium perfringens enterotoxin, by site-directed mutagenesis.

A C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is a modulator of claudin-4. We previously found that upon deletion of the C-terminal 16 amino acids, C-CPE lost its ability to modulate claudin-4. Tyrosine residues in the 16 amino acids were involved in the modulation of claudin-4. In the present study, we performed functional domain mapping of the 16-amino acid region of C-CPE by replacing individual amino acids with alanine. To evaluate the ability of the alanine-substituted mutants to interact with claudin-4, we carried out a competition analysis using claudin-4-targeting protein synthesis inhibitory factor. We found that Tyr306Ala, Tyr310Ala, Tyr312Ala, and Leu315Ala mutants had reduced binding to claudin-4 compared to C-CPE. Next, we investigated effects of each alanine-substituted mutant on the TJ-barrier function in Caco-2 monolayer cells. The TJ-disrupting activity of C-CPE was reduced by the Tyr306Ala and Leu315Ala substitutions. Enhancement of rat jejunal absorption was also decreased by each of these mutations. The double mutant Tyr306Ala/Leu315Ala lost the ability to interact with claudin-4, modulate TJ-barrier function, and enhance jejunal absorption. These data indicate that Tyr306 and Leu315 are key residues in the modulation of claudin-4 by C-CPE. This information should be useful for the development of a novel claudin modulator based on C-CPE.

Targeting tight junction proteins-significance for drug development

The choice of drug target and the ability to deliver drug to those targets are pivotal in drug development. Most druggable targets are membrane proteins, such as G-protein-coupled receptors, channels and transporters. However, little attention has been paid to potential druggable targets in the membrane proteins of tight junctions (TJs), through which adjacent cell membranes contact one another. Recent progress in the cell biology of TJs provides new insights into the barrier and fence functions of TJs, suggesting that TJ components are promising candidates for drug discovery. In this review, we summarize the cell biology of TJs and discuss the TJ-based drug discovery.

A claudin-targeting molecule as an inhibitor of tumor metastasis

Tumor metastasis of epithelium-derived tumors is the major cause of death from malignant tumors. Overexpression of claudin is observed frequently in malignant tumors. However, claudin-targeting antimetastasis therapy has never been investigated. We previously prepared a claudin-4-targeting antitumor molecule that consisted of the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) fused to protein synthesis inhibitory factor (PSIF) derived from Pseudomonas exotoxin. In the present study, we investigated whether claudin CPE receptors can be a target for tumor metastasis by using the C-CPE-fused PSIF as a claudin-targeting agent. One of the most popular murine metastasis models is the lung metastasis of intravenously injected B16 cells. Therefore, we first investigated the effects of the C-CPE-fused PSIF on lung metastasis of claudin-4-expressing B16 (CL4-B16) cells. Intravenous administration of the C-CPE-fused PSIF suppressed lung metastasis of CL4-B16 cells but not B16 cells. Injection of C-CPE-fused PSIF also inhibited tumor growth and spontaneous lung metastasis of murine breast cancer 4T1 cells inoculated into the subcutis. Treatment with C-CPE-fused PSIF did not show apparent side effects in mice. These findings indicate that claudin targeting may be a novel strategy for inhibiting some tumor metastases.

Levels of tetrabromobisphenol A, tribromobisphenol A, dibromobisphenol A, monobromobisphenol A, and bisphenol a in Japanese breast milk.

The levels of bisphenol A (BPA) and tetrabromobisphenol A (TeBBPA) were determined in breast milk samples from 19 Japanese mothers. BPA and TeBBPA levels were 36 ng/g lipid (range: 1.4-380 ng/g lipid) and 1.9 ng/g lipid (range: N.D. - 8.7 ng/g lipid), respectively. Tribromobisphenol A (TriBBPA) was similarly detected in all samples (mean: 5.5 ng/g lipid). We investigated the alteration of BPA-related compounds in breast milk over a period of three months. No trend could be observed for time-dependent changes in TeBBPA levels. High levels of TriBBPA were detected in breast milk samples with a high concentration of TeBBPA. We further examined concentration changes in BPA-related compounds in the breast milk of two donors over a period of 24 h. The results suggested that TriBBPA was a debrominated metabolite of TeBBPA, which had been ingested via food consumption and immediately transferred to the breast milk. On the basis of the present results, we estimated and compared the daily intake of BPA, TriBBPA, and TeBBPA from breast milk for infants. The estimated average intake of TriBBPA was 4 times higher than TeBBPA, at 48 and 12 ng/kg/day, respectively. The level of TeBBPA in breast milk was low, suggesting a low risk of causing adverse health effects. In conclusion, the concentration of both TriBBPA and TeBBPA must be determined in breast milk to accurately clarify the exposure of these compounds to infants.

Mucosal vaccination using claudin-4-targeting.

Mucosa-associated lymphoid tissue (MALT) plays pivotal roles in mucosal immune responses. Efficient delivery of antigens to MALT is a critical issue for the development of mucosal vaccines. Although claudin-4 is preferentially expressed in MALT in the gut, a claudin-4-targeting approach for mucosal vaccination has never been developed. In the present study, we found that claudin-4 is expressed in nasal MALT, and we prepared a fusion protein of ovalbumin (OVA) as a model antigen with a claudin-4-binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) (OVA-C-CPE). Nasal immunization with OVA-C-CPE, but not a mixture of OVA and C-CPE, induced the production of OVA-specific serum IgG and nasal, vaginal and fecal IgA. Deletion of the claudin-4-binding region in OVA-C-CPE attenuated the induction of the immune responses. OVA-C-CPE immunization activated both Th1 and Th2 responses, and nasal immunization with OVA-C-CPE showed anti-tumor activity in mice inoculated with OVA-expressing thymoma cells. These results indicate that the claudin-4-targeting may be a potent strategy for nasal vaccination.

Claudin-4-targeting of diphtheria toxin fragment A using a C-terminal fragment of Clostridium perfringens enterotoxin

Claudin (CL)-4, a tight junction protein, is overexpressed in some human neoplasias, including ovarian, breast, pancreatic and prostate cancers. The targeting of CL-4 is a novel strategy for tumor therapy. We previously found that the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds to CL-4. In the present study, we genetically prepared a novel CL-4-targeting molecule (DTA-C-CPE) by fusion of C-CPE and diphtheria toxin fragment A (DTA). Although DTA is not toxic to CL-4-expressing L cells, even at 20 microg/ml, DTA-C-CPE is toxic to CL-4-expressing L cells at 1 microg/ml. DTA-C-CPE-induced cytotoxicity was attenuated by pretreatment of the cells with C-CPE but not bovine serum albumin, indicating that DTA-C-CPE may bind to CL-4-expressing L cells through its C-CPE domain. To evaluate the specificity of DTA-C-CPE, we examined its cytotoxic effects in L cells that express CL-1, -2, -4 or -5. We found that DTA-C-CPE was toxic to only CL-4-expressing L cells. Thus, C-CPE may be a promising ligand for the development of cancer-targeting systems.

A Novel Screening System for Claudin Binder Using Baculoviral Display

Recent progress in cell biology has provided new insight into the claudin (CL) family of integral membrane proteins, which contains more than 20 members, as a target for pharmaceutical therapy. Few ligands for CL have been identified because it is difficult to prepare CL in an intact form. In the present study, we developed a method to screen for CL binders by using the budded baculovirus (BV) display system. CL4-displaying BV interacted with a CL4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE), but it did not interact with C-CPE that was mutated in its CL4-binding region. C-CPE did not interact with BV and CL1-displaying BV. We used CL4-displaying BV to select CL4-binding phage in a mixture of a scFv-phage and C-CPE-phage. The percentage of C-CPE-phage in the phage mixture increased from 16.7% before selection to 92% after selection, indicating that CL-displaying BV may be useful for the selection of CL binders. We prepared a C-CPE phage library by mutating the functional amino acids. We screened the library for CL4 binders by affinity to CL4-displaying BV, and we found that the novel CL4 binders modulated the tight-junction barrier. These findings indicate that the CL-displaying BV system may be a promising method to produce a novel CL binder and modulator.

The application of an alanine-substituted mutant of the C-terminal fragment of Clostridium perfringens enterotoxin as a mucosal vaccine in mice

Efficient delivery of antigen to mucosal immune tissues is an essential part of mucosal vaccination. Claudin-4 is expressed on the epithelial cells that cover the mucosal immune tissues. We previously found that claudin-4-targeting is a promising strategy for mucosal vaccination by using a claudin-4 binder, the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Substitution of Asn and Ser at positions 309 and 313, respectively, with alanine increased the affinity of C-CPE for claudin-4. However, application of the C-CPE mutant as a mucosal vaccine has never been tried. Here, we investigated whether the C-CPE mutant could serve as a mucosal vaccine. We used ovalbumin (OVA) as a model antigen and fused the C-CPE mutant to it. The resultant fusion protein was bound to claudin-4. When mice were immunized with the C-CPE mutant-fused OVA, OVA-specific serum IgG and nasal IgA increased relative to levels in mice immunized with a C-CPE-fused antigen. Immunization with the C-CPE mutant-fused OVA activated Th1- and Th2-type responses and led to increased anti-tumor activity against OVA-expressing thymoma cells relative to that of mice immunized with the C-CPE-fused antigen. These findings suggest that the alanine-substituted C-CPE mutant shows promise as a claudin-targeted mucosal vaccine.

In vitro and in vivo induction of cytochrome P450 by coplanar polychlorinated/brominated biphenyls (Co-PXBs) providing high TEQ in mother's milk in Japan.

Coplanar polychlorinated/brominated biphenyls (Co-PXBs) are environmental pollutants previously identified in market fish samples. In this study, we observed that mothers milk in Japan is contaminated with Co-PXBs. Based on assumption that the toxicity of the same congener of PXDDs/DFs and Co-PXBs is nearly equal to that of the corresponding PCDDs/DFs and Co-PCBs, respectively, the toxic equivalent (TEQ) concentration was 10% of the total TEQ concentration (∑PCDDs/DFs, ∑PXDDs/DFs, ∑Co-PCBs and ∑Co-PXBs) in the milk. This observation suggested that humans, and especially infants, are exposed to high levels of Co-PXBs, which might cause adverse effects. However, the toxicity of Co-PXBs has to date not been reported. We assessed the toxic potency of Co-PXBs by studying their effect on the activity of cytochrome P450. Only the mRNA level and activity of CYP1A increased in a dose-dependent manner upon exposure to Co-PXBs. Substitution of bromine for chlorine into Co-PCBs provided higher CYP1A activity in in vitro and in vivo experiments. The expression level of aryl hydrocarbon receptor (AhR) mRNA was not altered, but luciferase activity, an indicator of AhR transcriptional activity, increased following treatment with Co-PXBs. The results suggest that CYP1A induction by Co-PXBs depended on AhR transcriptional activity and not on AhR expression. Although the TEFs of Co-PXBs are not set, if Co-PXBs are included in these calculations because of their higher toxicity compared to Co-PCBs, exposure to Co-PXBs cannot be neglected when assessing human health risks.