Masuo Kondoh

Cadmium induces apoptosis partly via caspase-9 activation in HL-60 cells

Cadmium (Cd), a potent immunotoxic metal, induces apoptosis both in vitro and in vivo. However, the mode of action remains unclear. We previously reported that Cd-induced apoptosis was partly dependent on mitochondria. In the present study, we investigated the involvement of caspase-9, which is the apex caspase in the mitochondoria-dependent apoptosis pathway, in Cd-induced apoptosis in human promyelocytic leukemia HL-60 cells. A specific inhibitor of caspase-9, Z-LEHD-FMK, partly inhibited DNA fragmentation induced by Cd treatment in HL-60 cells. Moreover, treatment of HL-60 cells with Cd resulted in the appearance of Cytochrome c (Cyt c), a potent activator of caspase-9, in the cytosol at 3 h, which closely paralleled the activation of caspase-9. Caspase-9 is an initiator caspase that is a potent activator of downstream effector caspases such as caspase-3. Caspase-3 activation was subsequent to the Cyt c release at 6 h. DNA fragmentation, an index of induction of apoptosis, also appeared 6 h after Cd treatment. The effects were more pronounced at 9 h after Cd addition. A broad-specificity inhibitor of caspases, Z-Asp-CH(2)-DCB, inhibited caspase-3 activation and DNA fragmentation induced by Cd in a dose-dependent fashion. The results suggest that Cd-induced apoptosis is partly caused by caspase-9 activation triggered by Cyt c.

Silica nanoparticles as hepatotoxicants.

Nano-size materials are increasingly used in cosmetics, diagnosis, imaging and drug delivery, but the toxicity of the nano-size materials has never been fully investigated. Here, we investigated the relationship between particle size and toxicity using silica particles with diameters of 70, 300 and 1000 nm (SP70, SP300, and SP1000) as a model material. To evaluate acute toxicity, we first performed histological analysis of liver, spleen, kidney and lung by intravenous administration of silica particles. SP70-induced liver injury at 30 mg/kg body weight, while SP300 or 1000 had no effect even at 100 mg/kg. Administration of SP70 dose-dependently increased serum markers of liver injury, serum aminotransferase and inflammatory cytokines. Repeated administration of SP70 twice a week for 4 weeks, even at 10mg/kg, caused hepatic fibrosis. Taken together, nano-size materials may be hepatotoxic, and these findings will be useful for future development in nanotechnology-based drug delivery system.

Specific induction of metallothionein synthesis by mitochondrial oxidative stress

Metallothionein (MT), a sulfhydryl-rich protein, may be increased by administration of a variety of agents, including metals, cytokines and oxidative stress agents. Mitochondria are a major source of reactive oxygen species, but antioxidant systems against mitochondrial free radicals are not fully understood. In this study, we examined the induction of MT synthesis by administration of mitochondrial-specific reactive oxygen generators such as antimycin A (AA), an electron transfer inhibitor, and 2,4-dinitrophenol (DNP), an uncoupling agent. Subcutaneous administration of AA to mice significantly increased the hepatic MT concentration in a dose- and time-dependent manner. AA slightly elevated glutathione peroxidase (GSHPx) activity, but the rate of increase in GSHPx (1.3-fold) was smaller than that in MT (11.8-fold). Other antioxidants such as catalase, manganese-superoxide dismutase (Mn-SOD), copper/zinc-superoxide dismutase (Cu/Zn-SOD) and GSHPx were not activated by AA treatment. Moreover, administration of DNP induced the synthesis of MT in the liver. Although DNP slightly elevated Mn-SOD activity, the rate of increase in Mn-SOD (1.3-fold) was smaller than that in MT (3.7-fold). Other antioxidants such as catalase, Cu/Zn-SOD and GSHPx were not activated by DNP treatment. These data suggest that MT plays a major role in protection against oxidative stress induced in mitochondria.

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.

Cell cycle arrest and antitumor activity of pironetin and its derivatives

The biological effects of pironetin and its derivatives on cell cycle progression and antitumor activity were studied. At 10-20 ng/ml, both pironetin and its demethyl derivative, NK10958P completely inhibited the cell proliferation of 3Y1 cells, however, epoxypironetin showed only a weak inhibitory activity. The cell cycle analysis revealed that these compounds arrested the cell cycle progression at the M-phase in a dose-dependent manner. These antiproliferative effects of pironetin were also observed in the range 5-25 ng/ml with several tumor cell lines. In CDF1-SLC mice bearing P388 leukemia cells, the intraperitoneal administration of 6.3 mg/kg pironetin over a 5-day period showed a moderate antitumor effect (T/C, 128%). As the chemical structure of pironetin is different from other M-phase inhibitors such as colchicine or vinblastine, pironetin will be the lead compound for a potential new antitumor drug.

Mercuric chloride induces apoptosis via a mitochondrial-dependent pathway in human leukemia cells.

Mercurial compounds modulate immunologic functions by inducing cytotoxicity. Although mercury chloride (HgCl(2)) is known to induce apoptosis in various immune system cells, the mechanism of the induction of apoptosis is poorly understood. In this study, we examined the activation of caspase-3, an important cysteine aspartic protease, during HgCl(2)-induced apoptosis in a human leukemia cell line (HL-60 cells). Both DNA fragmentation, a characteristic of apoptotic cells, and proteolysis of poly(ADP-ribose) polymerase (PARP), a substrate of caspase-3, occurred at 6 h after HgCl(2) treatment in HL-60 cells. These results suggest that the activation of caspase-3 was involved in HgCl(2)-induced apoptosis. The release of cytochrome c (Cyt c) from mitochondria into the cytosol, which is an initiator of the activation of caspase cascades, was also observed in HgCl(2)-treated HL-60 cells. Moreover, the release of Cyt c from mitochondria was observed in HgCl(2)-treated mitochondria isolated from mice liver, and this was followed by mitochondrial permeability transition (PT). The PT was inhibited by cyclosporin A (CsA), a potent inhibitor of PT. CsA also suppressed the occurrence of DNA fragmentation induced by HgCl(2) treatment in HL-60 cells. Taken together, these findings indicate that HgCl(2) is a potent inducer of apoptosis via Cyt c release from the mitochondria in HL-60 cells.

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.

Histological analysis of 70-nm silica particles-induced chronic toxicity in mice.

Nano-sized silica is a promising material for disease diagnosis, cosmetics and drugs. For the successful application of nano-sized material in bioscience, evaluation of nano-sized material toxicity is important. We previously found that nano-sized silica particles with a diameter of 70 nm showed acute liver failure in mice. Here, we performed histological analysis of major organs such as the liver, spleen, lung, kidney, brain and heart in mice, chronically injected with 70-nm silica particles for 4 weeks. Histological analysis revealed hepatic microgranulation and splenic megakaryocyte accumulation in these 70-nm silica particles treated mice, while the kidney, lung, brain and heart remained unaffected. Thus, liver and spleen appear to be the major target organs for toxicity by the chronic administration of the 70-nm silica particles.

Development of high-fat-diet-induced obesity in female metallothionein-null mice

Oxidative stress accelerates adipocyte differentiation and lipid accumulation, leading to endoplasmic reticulum (ER) stress, which causes insulin resistance. Because metallothionein (MT) has a role in prevention of oxidative and ER stress, we examined the effects of MT on the development of obesity induced by 27 wk of a high‐fat diet (HFD) in female MT‐I‐ and MT‐II‐null (MT−/−) and wild‐type (MT+/+) mice. Body weight, fat mass, and plasma cholesterol increased at a greater rate in MT−/− mice fed an HFD than in MT −/− mice fed a control diet (CD) and MT+/+ mice fed an HFD, indicating that MT−/− mice fed an HFD became obese and hypercholesterolemic and that MT could prevent HFD‐induced obesity. The observed increases in the levels of plasma leptin and leptin mRNA in the white adipose tissue of MT−/− mice fed the HFD suggested a leptin‐resistant state. Enhanced expression of a mesoderm‐specific transcript, which regulates the enlargement of fat cells, was accompanied by enlarged adipocytes in the white adipose tissue of young MT −/− mice before obesity developed after 3 and 8 wk of feeding the HFD. Thus, MT may have a preventive role against HFD‐induced obesity by regulating adipocyte enlargement and leptin signaling.—Sato, M., Kawakami, T., Kondoh, M., Takiguchi, M., Kadota, Y., Himeno, S., Suzuki, S. Development of high‐fat‐diet‐induced obesity in female metallothionein‐null mice. FASEBJ. 24, 2375–2384 (2010). www.fasebj.org

A claudin-4 modulator enhances the mucosal absorption of a biologically active peptide

Biologics, such as peptides, proteins and nucleic acids, are emerging pharmaceuticals. Passage across the epithelium is the first step in the absorption of biologics. Tight junctions (TJ) function as seals between adjacent epithelial cells, preventing free movement of solutes across the epithelium. We previously found that modulation of a key TJ component, claudin-4, is a potent method to enhance jejunal absorption when we used dextran as a model drug and the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) as a claudin-4 modulator. Here, we investigated whether the claudin-4 modulator enhances jejunal, nasal and pulmonary absorption of a biologics human parathyroid hormone derivative, hPTH(1-34). The claudin-4 modulator enhanced nasal but not jejunal and pulmonary absorption of hPTH(1-34). C-CPE is hydrophobic with low solubility of less than 0.3mg/ml, but deletion of 10 amino acids at the N-terminal of C-CPE increased its solubility by 30-fold. Moreover, the N-terminal truncated C-CPE bound to claudin-4, modulated the TJ-barrier and enhanced jejunal absorption of dextran. The N-terminal-truncated C-CPE also enhanced jejunal and pulmonary absorption of hPTH(1-34). This report is the first to indicate that a claudin-4 modulator may be a promising enhancer of the jejunal, pulmonary and nasal absorption of a peptide drug.