Noriyuki Miyoshi

A Link between Benzyl Isothiocyanate-Induced Cell Cycle Arrest and Apoptosis: Involvement of Mitogen-Activated Protein Kinases in the Bcl-2 Phosphorylation

In the present study, we clarified the molecular mechanism underlying the relationship between benzyl isothiocyanate (BITC)-induced cell cycle arrest and apoptosis and the involvement of mitogen-activated protein kinases (MAPKs). The exposure of Jurkat human T-cell leukemia cells to BITC resulted in the inhibition of the G2-M progression that coincided with the apoptosis induction. The experiment using the phase-specific synchronized cells demonstrated that the G2-M phase-arrested cells are more sensitive to undergoing apoptotic stimulation by BITC than the cells in other phases. We also confirmed that BITC activated c-Jun N-terminal kinase (JNK) and p38 MAPK, but not extracellular signal-regulated kinase, at the concentration required for apoptosis induction. An experiment using a JNK-specific inhibitor SP600125 or a p38 MAPK inhibitor SB202190 indicated that BITC-induced apoptosis might be regulated by the activation of these two kinases. Conversely, BITC is likely to confine the Jurkat cells in the G2-M phase mainly through the p38 MAPK pathway because only the p38 MAPK inhibitor significantly attenuated the accumulation of inactive phosphorylated Cdc2 protein and the G2-M-arrested cell numbers. We reported here for the first time that the antiapoptotic Bcl-2 protein was phosphorylated by the BITC treatment without significant alteration of the Bcl-2 total protein amount. This was abrogated by a JNK specific inhibitor SP600125 at the concentration required for specific inhibition of the c-Jun phosphorylation. Moreover, the spontaneous phosphorylation of antiapoptotic Bcl-2 in the G2-M synchronized cells was enhanced synergistically by the BITC treatment. Involvement of the MAPK activation in the Bcl-2 phosphorylation and apoptosis induction also was observed in HL-60 and HeLa cells. Thus, we identified the phosphorylated Bcl-2 as a key molecule linking the p38 MAPK-dependent cell cycle arrest with the JNK activation by BITC.

Electrophiles in Foods: The Current Status of Isothiocyanates and Their Chemical Biology

Unlike many classical signals and hormones, exposure of the cells to electrophilic molecules potentially induces a series of characteristic and wide-ranging biological responses by covalently attaching with macromolecules such as proteins as well as small cellular reductants. In addition to chemicals originated from xenobiotics or lipid peroxidation, electrophiles in foods have recently attracted much attention. These compounds have recently been found to induce expression of cytoprotective proteins that are involved in the elimination or inactivation of oxidative stress and carcinogenic electrophiles implicated in several pathogeneses. The redox-sensitive regulating systems such as Keap1/Nrf2/ARE play a key role in this induction and thus are considered to be the most important target of electrophiles in foods. This review highlights the food-derived electrophiles as promising protectors against various diseases, with an emphasis on possible molecular mechanisms. Current knowledge of isothiocyanates (ITCs), representative electrophile compounds from cruciferous vegetables, is discussed also, with consideration of the chemistry, metabolism, absorption, and factors influencing the biological activities of ITCs. In addition, this review attempts to provide a balanced perspective on the relative beneficial and harmful effects of the food electrophiles.

Health-promoting effects of green tea

Green tea is manufactured from the leaves of the plant Camellia sinensis Theaceae and has been regarded to possess anti-cancer, anti-obesity, anti-atherosclerotic, anti-diabetic, anti-bacterial, and anti-viral effects. Many of the beneficial effects of green tea are related to the activities of (−)-epigallocatechin gallate (EGCG), a major component of green tea catechins. For about 20 years, we have engaged in studies to reveal the biological activities and action mechanisms of green tea and EGCG. This review summarizes several lines of evidence to indicate the health-promoting properties of green tea mainly based on our own experimental findings.

ATP depletion alters the mode of cell death induced by benzyl isothiocyanate

Pro-inflammatory death is presumably an undesirable event in cancer prevention process, thus biochemical comprehension and molecular definition of this process could have important clinical implications. In the present study, we examined the cytophysiological conversion of cell death mode by benzyl isothiocyanate (BITC) in human cervical cancer HeLa cells. The detailed studies using flow cytometric and morphological analyses demonstrated that the cells treated with appropriate concentration (25 microM) of BITC showed apoptotic feature, such as chromatin condensation, DNA fragmentation, and preserved plasma membrane integrity, whereas these features were disappeared by treatment with higher concentration (100 microM). The treatment with 2-deoxyglucose, an inhibitor of ATP synthesis, drastically increased in the ratio of necrotic dead cells, while it influences little that of apoptotic cells. Moreover, an analysis using the mitochondrial DNA-deficient HeLa cells demonstrated that the rho degrees cells were more susceptible to the BITC-induced necrosis-like cell death compared to the wild-type (rho(+)) cells, whereas the ROS production was significantly inhibited in the rho degrees cells. It is likely that the BITC-induced ROS is derived from mitochondrial respiratory chain and ruled out the contribution to the mechanism of cell death mode switching. In addition, the BITC treatment resulted in a more rapid depletion of ATP in the rho degrees cells than in the rho(+) cells. Furthermore, a caspase inhibitor, Z-VAD-fmk counteracted not only apoptosis, but also necrosis-like cell death induced by BITC, suggesting that increment in this cell death pattern might be due to the interruption of events downstream of a caspase-dependent pathway. The obtained data suggested that the decline in the intracellular ATP level plays an important role in tuning the mode of cell death by BITC.

Catechol type polyphenol is a potential modifier of protein sulfhydryls: Development and application of a new probe for understanding the dietary polyphenol actions

The oxidation of dietary polyphenols with a catechol structure leads to the formation of an o-quinone structure, which rapidly reacts with sulfhydryls such as glutathione and protein cysteine residues. This modification may be important for understanding the redox regulation of cell functions by polyphenols. In this study, to investigate the catechol modification of protein sulfhydryls, we used 3,4-dihydroxyphenyl acetic acid (DPA) as a model catechol compound and developed a new probe to directly detect protein modification by catechol type polyphenols using a biotinylated DPA (Bio-DPA). The oxidation-dependent electrophilic reactivity of DPA with peptide sulfhydryls was confirmed by both mass spectrometry and nuclear magnetic resonance spectroscopy. When RL34 cells were treated with Bio-DPA, the significant incorporation of Bio-DPA into a 40 kDa protein was observed by Western blot analysis. The band was identified by mass spectrometry as the cytoskeletal protein, beta-actin. This identification was confirmed by the pull-down assay with anti-beta-actin antibody. To examine the reactivity of the catechol type polyphenols, such as flavonoids, to endogenous beta-actin, RL34 cells were coexposed to Bio-DPA and the flavonoids quercetin, (-)-epicatechin, and (-)-epicatechin gallate. Upon exposure of the cells to Bio-DPA in the presence of the flavonoids, we observed a significant decrease in the DPA-modified beta-actin. These results indicate that beta-actin is one of the major targets of protein modification by catechol type polyphenols and that Bio-DPA is an useful probe for understanding the redox regulation by dietary polyphenols. Furthermore, Keap1, a scaffold protein to the actin cytoskeleton controlling cytoprotective enzyme genes, was also identified as another plausible target of the catechol type polyphenols by oxidative modification of the intracellular sulfhydryls. These results provide an alternative approach to understand that catechol type polyphenol is a potential modifier of redox-dependent cellular events through sulfhydryl modification.

Apoptosis Induction by Lectin Isolated from the Mushroom Boletopsis leucomelas in U937 Cells

A 15-kDa lectin was isolated from the edible mushroom Kurokawa by affinity chromatography using N,N′-diacetylchitobiose-Sepharose 4B. The results of microsequencing analysis indicated that the lectin has a partial amino acid sequence similar to the mushroom lectin, Agaricus bisporus agglutinin (ABA). We found that the Kurokawa lectin inhibited proliferation of human monoblastic leukemia U937 cells dose-dependently. Several lines of evidence indicated that this inhibition was due to its apoptosis induction. We observed that the lectin induced apoptotic bodies formation, chromatin condensation, and DNA ladder formation, features of apoptosis. The DNA ladder formation was inhibited by a general inhibitor of caspases, which are known to play essential roles in apoptosis. In contrast, ABA did not have cell growth-inhibiting or apoptosis-inducing activities. Thus, the Kurokawa lectin is the first mushroom lectin with apoptosis-inducing activity.

Formation of cholesterol ozonolysis products in vitro and in vivo through a myeloperoxidase-dependent pathway

3β-Hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol-A) and its aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (secosterol-B) were recently detected in human atherosclerotic tissues and brain specimens, and they may play pivotal roles in the pathogenesis of atherosclerosis and neurodegenerative diseases. However, as their origin remains unidentified, we examined the formation mechanism, the stability, and the fate of secosterols in vitro and in vivo. About 40% of secosterol-A remained unchanged after 3 h incubation in the FBS-free medium, whereas 20% and 40% were converted to its aldehyde-oxidation product, 3β-hydroxy-5-oxo-secocholestan-6-oic acid, and secosterol-B, respectively. In the presence of FBS, almost all secosterol-A was converted immediately to these compounds. Secosterol-B in the medium, with and without FBS, was relatively stable, but ∼30% was converted to its aldehyde-oxidation product, 3β-hydroxy-5β-hydroxy-B-norcholestane-6-oic acid (secoB-COOH). When neutrophil-like differentiated human leukemia HL-60 (nHL-60) cells activated with PMA were cultured in the FBS-free medium containing cholesterol, significantly increased levels of secosterol-A and its aldehyde-oxidation product, but not secosterol-B, were formed. This secosterol-A formation was decreased in the culture of PMA-activated nHL-60 cells containing several reactive oxygen species (ROS) inhibitors and scavengers or in the culture of PMA-activated neutrophils isolated from myeloperoxidase (MPO)-deficient mice. Our results demonstrate that secoterol-A is formed by an ozone-like oxidant generated with PMA-activated neutrophils through the MPO-dependent mechanism.

Chemoprevention of Azoxymethane/Dextran Sodium Sulfate-Induced Mouse Colon Carcinogenesis by Freeze-Dried Yam Sanyaku and Its Constituent Diosgenin

The effects of sanyaku, a traditional Chinese medicine [freeze-dried powder of the yam tuber (Dioscorea)], and its major steroidal saponin constituent, diosgenin, on colon carcinogenesis were investigated. Male ICR mice were subjected to a single intraperitoneal injection of azoxymethane (AOM; 10 mg/kg body weight) followed by administration of 1.5% dextran sodium sulfate (DSS) in drinking water for 7 days to establish carcinogenesis. Commercial diosgenin or sanyaku, which contained diosgenin at 63.8 ± 1.2 mg/kg dry weight, was given in the diet at 20, 100, or 500 mg/kg for 17 weeks. Groups of mice that received diosgenin or sanyaku at all doses yielded significantly less number of colon tumors compared with the AOM/DSS-treated mice. Occurrence of colonic mucosal ulcer and dysplastic crypt induced by AOM/DSS treatment was also significantly decreased by the administration of diosgenin and sanyaku, which was in accordance with the significant reduction of AOM/DSS-mediated increases in expression of inflammatory cytokines such as IL-1β by diosgenin and sanyaku. Furthermore, elevated levels of serum triglyceride in the AOM/DSS-treated mice tended to be reduced in mice given diosgenin and sanyaku. Microarray and real-time reverse transcriptase PCR analyses revealed that diosgenin administration increased 12-fold the expression of lipoprotein lipase, which may contribute to reduced serum triglyceride levels. Other genes altered by diosgenin included those associated with antioxidative stress responses and apoptosis, such as heme oxygenase-1, superoxide dismutase-3, and caspase-6. Our results imply that the Chinese medicine sanyaku and the tubers of various yams containing diosgenin as food could be ingested to prevent colon carcinogenesis in humans. Cancer Prev Res; 4(6); 924–34. ©2011 AACR.

Implications of cholesterol autoxidation products in the pathogenesis of inflammatory diseases.

There is rising interest in non-enzymatic cholesterol oxidation because the resulting oxysterols have biological activity and can be used as non-invasive markers of oxidative stress in vivo. The preferential site of oxidation of cholesterol by highly reactive species is at C7 having a relatively weak carbon-hydrogen bond. Cholesterol autoxidation is known to proceed via two distinct pathways, a free radical pathway driven by a chain reaction mechanism (type I autoxidation) and a non-free radical pathway (type II autoxidation). Oxysterols arising from type II autoxidation of cholesterol have no enzymatic correlates, and singlet oxygen ((1)ΔgO2) and ozone (O3) are the non-radical molecules involved in the mechanism. Four primary derivatives are possible in the reaction of cholesterol with singlet oxygen via ene addition and the formation of 5α-, 5β-, 6α- and 6β-hydroxycholesterol preceded by their respective hydroperoxyde intermediates. The reaction of ozone with cholesterol is very fast and gives rise to a complex array of oxysterols. The site of the initial ozone reaction is at the Δ5,6 -double bond and yields 1,2,3-trioxolane, a compound that rapidly decomposes into a series of unstable intermediates and end products. The downstream product 3β-hydroxy-5-oxo-5,6-secocholestan-6-al (sec-A, also called 5,6-secosterol), resulting from cleavage of the B ring, and its aldolization product (sec-B) have been proposed as a specific marker of ozone-associated tissue damage and ozone production in vivo. The relevance of specific ozone-modified cholesterol products is, however, hampered by the fact sec-A and sec-B can also arise from singlet oxygen via Hock cleavage of 5α-hydroperoxycholesterol or via a dioxietane intermediate. Whatever the mechanism may be, sec-A and sec-B have no enzymatic route of production in vivo and are reportedly bioactive, rendering them attractive biomarkers to elucidate oxidative stress-associated pathophysiological pathways and to develop pharmacological agents.

Quercetin-3-O-glucuronide inhibits noradrenaline-promoted invasion of MDA-MB-231 human breast cancer cells by blocking β2-adrenergic signaling

Endogenous catecholamines such as adrenaline (A) and noradrenaline (NA) are released from the adrenal gland and sympathetic nervous system during exposure to stress. The adrenergic system plays a central role in stress signaling, and excessive stress was found to be associated with increased production of reactive oxygen species (ROS). Overproduction of ROS induces oxidative damage in tissues and causes the development of diseases such as cancer. In this study, we investigated the effects of quercetin-3-O-glucuronide (Q3G), a circulating metabolite of quercetin, which is a type of natural flavonoid, on the catecholamine-induced β2-adrenergic receptor (β2-AR)-mediated response in MDA-MB-231 human breast cancer cells expressing β2-AR. Treatment with A or NA at concentrations above 1μM generated significant levels of ROS, and NA treatment induced the gene expression of heme oxygenase-1 (HMOX1), and matrix metalloproteinase-2 (MMP-2) and -9 (MMP9). Inhibitors of p38 MAP kinase (SB203580), cAMP-dependent protein kinase (PKA) (H-89), activator protein-1 (AP-1) transcription factor (SR11302), and NF-κB and AP-1 (Tanshinone IIA) decreased MMP2 and MMP9 gene expression. NA also enhanced cAMP induction, RAS activation and phosphorylation of ERK1/2. These results suggested that the cAMP-PKA, MAPK, and ROS-NF-κB pathways are involved in β2-AR signaling. Treatment with 0.1μM Q3G suppressed ROS generation, cAMP and RAS activation, phosphorylation of ERK1/2 and the expression of HMOX1, MMP2, and MMP9 genes. Furthermore, Q3G (0.1μM) suppressed invasion of MDA-MB-231 breast cancer cells and MMP-9 induction, and inhibited the binding of [(3)H]-NA to β2-AR. These results suggest that Q3G may function to suppress invasion of breast cancer cells by controlling β2-adrenergic signaling, and may be a dietary chemopreventive factor for stress-related breast cancer.