Lignosulfonic acid attenuates NF-κB activation and intestinal epithelial barrier dysfunction induced by TNF-α/IFN-γ in Caco-2 cells.

Lentinula edodes mycelia solid culture extract (MSCE) is used as a medical food ingredient and provides beneficial effects to patients with cancer and chronic type C hepatitis. Low molecular weight lignin (LM-lignin), which is an active component of MSCE, exhibits hepatoprotective, antitumor, antiviral, and immunomodulatory effects. In this study, we investigated the effect of LM-lignin/lignosulfonic acid on intestinal barrier function. Lignosulfonic acid enhanced transepithelial membrane electrical resistance in human intestinal Caco-2 cell monolayers. In Caco-2 cells treated with lignosulfonic acid, expression of claudin-2, which forms high conductive cation pores in tight junctions (TJs), was decreased. Lignosulfonic acid also attenuated the barrier dysfunction that is caused by tumor necrosis factor (TNF)-α and interferon (IFN)-γ in Caco-2 cells. TNF-α- and IFN-γ-induced activation of NF-κB, such as translocation of NF-κB p65 into the nucleus and induction of gene expression, was inhibited by lignosulfonic acid treatment. Furthermore, lignosulfonic acid decreased the TNF-α- and IFN-γ-induced increase in interleukin (IL)-1ß and IL-6 expression in Caco-2 cells. These results suggest that lignosulfonic acid not only enhances TJ barrier function but also restores TJ barrier integrity impaired by inflammatory cytokines. Therefore, lignosulfonic acid may be beneficial for the treatment of inflammation-induced intestinal barrier dysfunction observed in inflammatory bowel disease.

Identification of claudin-4 binder that attenuates tight junction barrier function by TR-FRET-based screening assay.

Claudins are key functional and structural components of tight junctions (TJs) in epithelial cell sheets. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds to claudin-4 and reversibly modulates intestinal TJ seals, thereby enhancing paracellular transport of solutes. However, the use of C-CPE as an absorption enhancer is limited by the molecule's immunogenicity and manufacturing cost. Here, we developed a high-throughput screening system based on the Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) method to identify claudin-4 binders in a library collection of 32,560 compounds. Thiostrepton, identified from the screen, decreased transepithelial electrical resistance and increased flux of 4-kDa fluorescein isothiocyanate-labelled dextran (FD-4) in Caco-2 cell monolayers, a model of intestinal epithelium. Thiostrepton changed the expression, but not the localisation, of TJ components. Treatment of rat jejunum with thiostrepton increased the absorption of FD-4 without tissue toxicity, indicating that thiostrepton is a novel claudin-4 binder that enhances intestinal permeability. The screening system may therefore be a useful tool for identifying claudin-4 binders to enhance drug absorption in mucosa.

Use of cell-based screening to identify small-molecule compounds that modulate claudin-4 expression.

Claudins constitute a family of at least 27 proteins with four transmembrane domains, and play a pivotal role in maintaining tight-junctions seals in diverse epithelial tissues. The expression of claudin-4 often changes in intestinal tissues of inflammatory bowel disease and various human cancers. Therefore, claudin-4 is a promising target for treatment of these diseases. In our previous study, we established a reporter cell line to monitor claudin-4 expression on the basis of a functional claudin-4 promoter. Using this cell line, we have performed a cell-based screen of a library containing 2642 biologically active small-molecule compounds to identify modulators of claudin-4 expression. The screen identified 24 potential modulators of the claudin-4 promoter activity. Fourteen of these compounds (12 of them novel) induced endogenous claudin-4 expression. The identified compounds might serve as lead compounds targeting aberrant gene expression in inflammatory bowel disease.

A simple reporter assay for screening claudin-4 modulators.

Claudin-4, a member of a tetra-transmembrane protein family that comprises 27 members, is a key functional and structural component of the tight junction-seal in mucosal epithelium. Modulation of the claudin-4-barrier for drug absorption is now of research interest. Disruption of the claudin-4-seal occurs during inflammation. Therefore, claudin-4 modulators (repressors and inducers) are promising candidates for drug development. However, claudin-4 modulators have never been fully developed. Here, we attempted to design a screening system for claudin-4 modulators by using a reporter assay. We prepared a plasmid vector coding a claudin-4 promoter-driven luciferase gene and established stable reporter gene-expressing cells. We identified thiabendazole, carotene and curcumin as claudin-4 inducers, and potassium carbonate as a claudin-4 repressor by using the reporter cells. They also increased or decreased, respectively, the integrity of the tight junction-seal in Caco-2 cells. This simple reporter system will be a powerful tool for the development of claudin-4 modulators.

Hepatoprotective effect of syringic acid and vanillic acid on CCl4-induced liver injury.

The mycelia of the edible mushroom Lentinula edodes can be cultured in solid medium containing lignin, and the hot-water extracts (L.E.M.) is commercially available as a nutritional supplement. During the cultivation, phenolic compounds, such as syringic acid and vanillic acid, were produced by lignin-degrading peroxidase secreted from L. edodes mycelia. Since these compounds have radical scavenging activity, we examined their protective effect on oxidative stress in mice with CCl(4)-induced liver injury. We examined the hepatoprotective effect of syringic acid and vanillic acid on CCl(4)-induced chronic liver injury in mice. The injection of CCl(4) into the peritoneal cavity caused an increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. The intravenous administration of syringic acid and vanillic acid significantly decreased the levels of the transaminases. Four weeks of CCl(4) treatment caused a sufficiently excessive deposition of collagen fibrils. An examination of Azan-stained liver sections revealed that syringic acid and vanillic acid obviously suppressed collagen accumulation and significantly decreased the hepatic hydroxyproline content, which is the quantitative marker of fibrosis. Both of these compounds inhibited the activation of cultured hepatic stellate cells, which play a central role in liver fibrogenesis, and maintained hepatocyte viability. These data suggest that the administration of syringic acid and vanillic acid could suppress hepatic fibrosis in chronic liver injury.

Hepatoprotective effect of syringic acid and vanillic acid on concanavalin a-induced liver injury.

The edible mushroom Lentinula edodes (shiitake) contains many bioactive compounds. In the present study, we cultivated L. edodes mycelia in solid medium and examined the hot-water extract (L.E.M.) for its suppressive effect on concanavalin A (ConA)-induced liver injury in mice. ConA injection into the tail vein caused a great increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. The intraperitoneal administration of L.E.M. significantly decreased the levels of the transaminases. L.E.M. contains many bioactive substances, including polysaccharides and glucan, which could be immunomodulators. Since ConA-induced liver injury is caused by the activation of T cells, immunomodulating substances might be responsible for the suppressive effect of L.E.M. L.E.M. also contains phenolic compounds that are produced from lignocellulose by mycelia-derived enzymes. The major phenolics in L.E.M., syringic acid and vanillic acid, were intraperitoneally injected into mice shortly before the ConA treatment. Similar to L.E.M., the administration of syringic acid or vanillic acid significantly decreased the transaminase activity and suppressed the disorganization of the hepatic sinusoids. In addition, the inflammatory cytokines tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and interleukin (IL)-6 in the serum increased rapidly, within 3 h of the ConA administration, but the administration of syringic acid or vanillic acid significantly suppressed the cytokine levels. Together, these findings indicate that the phenolic compounds in L.E.M. are hepatoprotective through their suppression of immune-mediated liver inflammation.

Novel chronotherapeutic rectal aminophylline delivery system for therapy of asthma.

The aim of this study was to develop a new chronotherapeutic pharmaceutical preparation as a sustained-release suppository for prevention and therapeutic use against bronchial asthma in the early morning. Sustained-release hollow-type (SR-HT) suppositories using sodium alginate (Alg-Na), sodium polyacrylate (PANa) or polyacrylate-PANa co-polymer (PA-PANa) as gelling polymers (gel agent) were prepared and pharmaceutical characteristics of these suppositories were investigated. Type A SR-HT suppositories comprised a suppository shell prepared with oleaginous base and containing aminophylline only or aminophylline with Alg-Na or PANa in the cavity (hollow space). Type B SR-HT suppositories comprised a suppository shell prepared with oleaginous base and gel agent (30%), with aminophylline in the hollow space. In drug-release studies, the acrylate polymer-containing suppositories showed linearity of delayed release rate, providing significantly decreased the highest concentration of theophylline in plasma (C(max)) and delayed the time required to reach C(max) (t(max)) and the mean residence time (MRT) after rectal administrated in rabbits. In particular, suppositories containing PA-PANa maintained significantly higher theophylline concentrations than control suppositories at 12h after rectal administration. Furthermore, histopathological examination indicated that these suppositories using acrylate polymers did not result in rectal lesions. The SR-HT suppository, particularly using PA-PANa as a gel agent, may thus be useful against nocturnal symptoms of asthma. In this study, we confirmed new formulation of sustained-release suppository for chronotherapy of theophylline using oily base material in combination with polymer such as PA-PANa. The hollow-type suppository containing oleaginous base and hydrophilic polymer in the shell could be useful device for rectal administration of various drugs with prolongation of plasma concentration.

Induction of apoptosis by new ent-kaurene-type diterpenoids isolated from the New Zealand liverwort Jungermannia species.

Some diterpenoids show various biological activities, including anti-inflammatory, anti-HIV and anti-tumor activity. Previously, we have focused our research on the apoptosis-inducing properties of diterpenoids and found that some ent-kaurene-type diterpenoids induced apoptosis in human leukemia HL-60 cells. In this study, we have investigated the induction of apoptosis in HL-60 cells by the novel ent-kaurene-type diterpenoids, jungermannenones A (JA), B (JB), C (JC) and D (JD), isolated from the New Zealand liverwort Jungermannia species. Treatment of the cells with each compound for 12 h resulted in cytotoxicity (IC (50) values: A, 1.3; B, 5.3; C, 7.8; D, 2.7 microM) and caused DNA fragmentation and nuclear condensation, both biochemical markers of the induction of apoptosis. Treatment with the compounds resulted in activation of caspases, including caspase-3 and caspase-8. A broad-spectrum inhibitor of caspases, Z-Asp-CH (2)-DCB, attenuated the cytotoxicity induced by these compounds, suggesting that JA, JB, JC and JD induced apoptosis through a caspase-dependent pathway. JA and JD inhibited the activity of nuclear factor-kappaB, which is a transcriptional factor of anti-apoptotic factors. Thus, some of these new ent-kaurene-type diterpenoids may be promising candidates for anti-tumor agents.

Activation of p38 mitogen-activated protein kinase during ent-11alpha-hydroxy-16-kauren-15-one-induced apoptosis in human leukemia HL-60 cells.

Kaurene-type diterpenes possess various biological activities including antitumor and anti-inflammatory effects. Indeed, we have found that an ent-kaurene diterpene, ent-11alpha-hydroxy-16-kauren-15-one (KD), induced apoptosis via caspase-8 activation in human promyelocytic leukemia HL-60 cells. However, the mechanism of caspase-8 activation by KD is not clear. In this study, we investigated the involvement of p38 mitogen-activated protein kinase (p38 (MAPK)) in KD-induced apoptosis. p38 (MAPK) was activated by treatment with KD parallel to DNA ladder formation. Pretreatment with SB203580, a specific inhibitor of p38 (MAPK), attenuated induction of apoptosis by KD and inhibited activation of caspase-8. Cleavage of Bid, a typical substrate of caspase-8, was also inhibited by treatment with SB203580, suggesting that activation of p38 (MAPK) occurs upstream of caspase-8 during KD-induced apoptosis.

An ent-kaurene diterpene enhances apoptosis induced by tumor necrosis factor in human leukemia cells.

Some antitumor agents, including tumor necrosis factor-alpha (TNF-alpha) and camptothecin (CPT), often cause resistance of tumor cells to antitumor agents through activation of the nuclear factor-kappa B (NF-kappa B) pathway that leads to up-regulation of anti-apoptotic proteins. Therefore, co-treatment of an inhibitor of the NF-kappa B pathway with antitumor agents is a useful strategy for chemotherapy. Here we report that ent-11 alpha-hydroxy-16-kauren-15-one (KD) selectively inhibits NF-kappa B-dependent gene expression due to treatment with TNF-alpha. KD in combination with TNF-alpha caused a dramatic increase in apoptosis in human leukemia cells accompanied by activation of caspases. A broad-spectrum inhibitor of caspases decreased the apoptosis induced by treatment with KD and TNF-alpha. KD in combination with CPT also caused an increase in apoptosis. These results suggest that the apoptotic potency of co-treatment of KD with TNF-alpha or CPT is elicited through selective inhibition of NF-kappa B-dependent anti-apoptotic proteins and thus may provide a basis for the development of useful approaches to the treatment of leukemia.

A comparison of apoptosis and necrosis induced by ent-kaurene-type diterpenoids in HL-60 cells.

We previously reported that ent-11alpha-hydroxy-16-kauren-15-one (KD) induces apoptosis through a caspase-dependent pathway and the induction of apoptosis is dependent on its enone group in human leukemia cells. Here we investigated the abilities of some KD-related compounds with enone group (Fig. 1) to induce apoptosis and to activate some caspases. The IC50 values of ent-kaurene-related compounds possessing the enone group, ent-1beta-hydroxy-9(11),16-kauradien-15-one (1), ent-9(11),16-kauradiene-12,15-dione (2) and the rearranged ent-kaurane-type diterpene (3), against HL-60 cells after 12 h of treatment were 40 microM, 1.8 microM and 5.5 microM, respectively. Although treatment with 3 induced apoptosis, DNA ladder formation was not observed after treatment with 1 or 2. Induction of necrosis, as assayed by trypan blue staining, was observed after treatment with 1 or 2. Treatment with compound 1, 2 or 3 induced proteolysis of poly(ADP-ribose) polymerase (PARP), a substrate of caspase-3, and processing of caspase-3. Activation of caspase-8 and processing of Bid, a typical substrate of caspase-8, were also observed on treatment with these compounds. Pretreatment with a broad-spectrum inhibitor of caspases attenuated apoptosis induced by 3 but not necrosis induced by 1 and 2. In summary, KD-related compounds are a unique family of diterpenes that cause either caspase-dependent apoptotic or necrotic cell death.

New ent-kaurene-type diterpenoids possessing cytotoxicity from the New Zealand liverwort Jungermannia species.

Two new ent-kaurene-type and a new rearranged ent-kaurene-type diterpenoids possessing cytotoxicity against a human leukemia cell line have been isolated from the New Zealand liverwort Jungermannia species, together with previously known ent-kaurene-type diterpenoids. Their structures were established based on extensive NMR techniques.

[Apoptosis-inducing properties of ent-kaurene-type diterpenoids from the liverwort Jungermannia truncata].

Ent-11alpha-hydroxy-16-kauren-15-one (1) induced apoptosis in a human leukemia cell line (HL-60 cells), however, the apoptosis-inducing properties of 1 and its related compounds remain to be proved. We examined the involvement of caspases, a family of cysteine aspartic proteases, which play a central role in induction of apoptosis, in apoptosis induced by the compounds in HL-60 cells. Treatment of the cells with compounds 1, 2 and 3 with the enone group at C-15/C-16 caused DNA fragmentation, a sign of induction of apoptosis, and proteolysis of poly(ADP-ribose) polymerase (PARP), a hallmark of caspase activation. Z-Asp-CH2-DCB, abroad spectrum inhibitor of caspases, abolished the appearance of DNA fragmentation and also significantly attenuated the cytotoxic effects. These data suggest that induction of apoptosis by 1 and some of its related compounds are dependent on caspases activation and might be partly involved in the cytotoxicity in HL-60 cells.

Cytotoxic and apoptosis-inducing ent-kaurane-type diterpenoids from the Japanese liverwort Jungermannia truncata NEES.

Five new ent-kaurane-type diterpenoids and a new gymnomitrane (=barbatane)-type sesquiterpenoid have been isolated from the Japanese liverwort Jungermannia truncata NEES, together with twelve previously known ent-kaurane-type diterpenoids. The structures of the new compounds were elucidated by two-dimensional (2D) NMR experiments and chemical reaction. Some of the isolated compounds showed cytotoxicity against human leukemia cell lines and induced apoptosis.