Preparation of the Extracellular Domain of Recombinant Human Toll-like Receptor 6.

Toll-like receptors (TLRs) mediate immune responses upon recognition of a variety of ligands. To further elucidate the function of TLRs, it is important to identify novel ligands and their action mechanisms including polymer assembly. In this study, we propose an efficient method for preparation of the extracellular domain of human Toll-like receptor 6 (TLR6ED) in Escherichia coli using the bubbling cultivation method. Our preparation method improved the level of expression of TLR6ED into a soluble fraction as compared with typical cultivation using a rotary shaker. Circular dichroism (CD) experiments confirmed the structural formation of TLR6ED with secondary structure contents similar to leucine-rich repeat (LRR) modules. In addition, we also provided a procedure for preparing this recombinant protein using Sf9 insect cells, which ensures preservation of some key posttranslational modifications often lacking in bacteria-expressed proteins. These materials would be useful for analyzing novel molecules that bind directly to TLR6, complex formations with other regulators including TLR2 and TLR4, and the functional effects of N-linked glycosylation.

Wasabi-derived 6-(methylsulfinyl)hexyl isothiocyanate induces apoptosis in human breast cancer by possible involvement of the NF-κB pathways.

6-(methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a bioactive ingredient of wasabi (Wasabia japonica), which is a popular spice in Japan. 6-MSITC has been reported to inhibit the proliferation of breast cancer and melanoma cell lines. We inoculated 30 female Balb-nu/nu mice with MDA-MB-231 or -453 cells, and orally administered varying concentrations of 6-MSITC for 12 days following tumor growth. The tumor volumes and tumor weights from mice inoculated with MDA-MB-231 cells, and the tumor volumes of MDA-MB-453 cells were significantly inhibited by 6-MSITC on Days 9 and 11 after drug administration. DNA fragmentation, DNA ladder, and caspase 3/7 activity performed in vitro revealed that 6-MSITC induced apoptosis of MDA-MB-231, MDA-MB-453, and MCF-7 cells. Furthermore, nuclear factor-κB (NF-κB) expression in the nuclei and phosphorylation of inhibitor κBα (IκBα) was downregulated by 6-MSITC in a concentration-dependent manner; however, this activity was not observed in MCF-7 cells. Moreover, this downregulation of phosphorylated IκBα by 6-MSITC in MDA-MB-231 and -453 cells supports its inhibitory effects on NF-κB activity. The expression of phosphorylated AKT (pAKT) reduced by 6-MSITC was confirmed in MDA-MB-231 cells. Thus, we conclude that 6-MITC promotes apoptosis of breast cancer cells by inhibiting NF-kB and therefore releasing its control of the PI3K/AKT pathway.

Intracellular accumulation of structurally varied isothiocyanates correlates with inhibition of nitric oxide production in proinflammatory stimuli-activated tumorigenic macrophage-like cells.

In the present study, we analyzed the intracellular accumulation of 6-(methylsulfinyl)hexyl isothiocyanate (6MITC) and its analogs in proinflammatory stimuli-activated J774.1 cells to predict the biological potencies of the ITCs. Our present analyses exhibited that the intracellular accumulation was in the order of 6MITC>2b>2e≈2c>2g>2d>2f>2h. Investigation of reactivity of the ITCs with glutathione (GSH) in the tumor cells revealed partial inhibition of GSH by the ITCs. Furthermore, the inhibition of nitric oxide (NO) production in the tumor cells was ascribed to the intracellularly accumulated ITCs. The NO suppression was correlated with the inhibition of tumor cell growth. Our present results suggest that the intracellular accumulation of the ITCs can be used to predict their biological potencies, such as inhibition of NO production that was correlated with suppression of tumor cell growth. To the best of our knowledge, this is the first report to predict the biological potency of 6MITC and its analogs with their intracellular accumulation.

Utilization of 6-(methylsulfinyl)hexyl isothiocyanate for sensitization of tumor cells to antitumor agents in combination therapies.

In the present study, we performed in silico and in vitro analyses to evaluate the chemosensitizing effects of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) on tumor cells. Our in silico analyses of the ligand-receptor interactions between 6-MITC and the glutamate cysteine ligase (GCL) catalytic subunit (GCLC) revealed that 6-MITC possibly inhibited GCL enzyme activity, and that Cys-249 and Gln-251 were important residues for stable binding of ligands to GCLC. It was further found that 6-MITC interfered with the hydrogen bonds of the cysteinyl and glutamyl moieties of GSH with Cys-249 and Gln-251, respectively, and possibly overrode the feedback inhibition of GCL enzyme activity by GSH. To the best of our knowledge, this is the first in silico analysis to suggest an overriding effect of 6-MITC on GSH-induced feedback inhibition of GCL. In our in vitro analyses, combined treatment with 6-MITC and L-buthionine-S,R-sulfoximine (BSO) depleted GSH within 4 h in tumorigenic human c-Ha-ras and mouse c-myc-cotransfected highly metastatic serum-free mouse embryo-1 (r/m HM-SFME-1) cells, but did not deplete GSH in normal SFME cells. Furthermore, exposure to 6-MITC plus BSO for 4h, followed by glycyrrhetinic acid (GA) treatment for 3h, eradicated the tumor cells with minimal damage to the normal cells. The present findings suggest that 6-MITC in combination therapies could be used to sensitize tumor cells to antitumor agents, thereby leading to their eradication.

The IL6 gene polymorphism -634C>G and IL17F gene polymorphism 7488T>C influence bone mineral density in young and elderly Japanese women.

Osteoporosis is an important public health problem because of the significant morbidity and mortality associated with its complications, particularly fractures. An important clinical risk factor in the pathogenesis of osteoporosis is the presence of genetic polymorphisms in susceptibility genes. However, few studies have investigated the relevance of these polymorphisms in premenopausal women. Recent studies have demonstrated interactions between bone and immune cells, and that cytokines produced by immune cells regulate bone turnover. In this study, we examined the associations between bone mineral density (BMD) and polymorphisms in genes encoding interleukin (IL)-6 (-634C>G; rs1800796), tumor necrosis factor (TNF)-α (-308G>A; rs1800629), IL-17F (7488T>C; rs763780), transforming growth factor (TGF)-ß (869T>C; rs1800470), osteoprotegerin (OPG; 163A>G; rs3102735) and methylenetetrahydrofolate reductase (MTHFR; 677C>T; rs1801133) in young and elderly Japanese women. Whole-body, lumbar spine (L(1) or L(2)-L(4)), and femoral neck BMD were measured in 100 young subjects (18-23 years), and 100 elderly subjects (60-83 years). Whole-body, lumbar spine, and femoral neck BMD were 1.13±0.06, 1.14±0.12, and 1.00±0.11 g/cm(2), respectively, in young subjects, and 0.92±0.09, 0.86±0.15, and 0.63±0.10 g/cm(2), respectively, in elderly subjects. The frequencies of the IL-6 CC, CG, and GG genotypes were 48%, 49%, and 3%, respectively. The frequencies of the IL17F TT, TC, and CC genotypes were 79%, 15%, and 6%, respectively, in young subjects. Polymorphisms of the IL-6 and IL17F genes were significantly associated with BMD. To our knowledge, this is the first report to examine these associations in a cohort of 200 Japanese women.

Alkyl isothiocyanates suppress epidermal growth factor receptor kinase activity but augment tyrosine kinase activity.

AIM: We have reported the in vitro and in vivo anticancer activities of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) derived from a Japanese spice, wasabi. In order to obtain some clues about the mechanism of the anticancer activity, we have studied the effect of alkyl isothiocyanates (MITCs) on protein kinase activities. METHODS: The anti-autophosphorylation activity of MITCs with respect to the epidermal growth factor (EGF)-stimulated receptor kinase of A431 epidermoid carcinoma cells was examined by incorporation of radioactive ATP into an acid-insoluble fraction. Their anti-phosphorylation activity with respect to the non-receptor protein kinase was analyzed by a standard SDS-PAGE method. RESULTS: All the tested MITCs interfered with the EGF-stimulated receptor kinase activity in a dose-dependent manner, although their effects were less than 1/10 of that of erbstatin in microg/ml. On the other hand, the MITCs did not interfere with non-receptor kinases (kinase A, kinase C, tyrosine kinase and calmodulin dependent kinase III), but enhanced non-receptor tyrosine kinase. DISCUSSION: A possible anticancer mechanism of MITCs may involve the suppression of EGF receptor kinase activity and augmentation of non-receptor PTK.

The augmenting activity of 6-(methylsulfinyl)hexyl isothiocyanate on cellular glutathione levels is less sensitive to thiol compounds than its cytotoxic activity.

We analyzed the effects of thiol compounds on the biological activities of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC). Thiol compounds abolished the cytotoxic activity of 6-MITC, but did not abolish its activity augmenting cellular total glutathione levels and gamma-glutamylcysteine ligase gene expression. Thiol compounds might play an important role in the augmentation of several significant biological activities by overcoming the inherent limitations of 6-MITC.

Detection of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) and its conjugate with N-acetyl-L-cysteine (NAC) by high performance liquid chromatograpy-atmospheric pressure chemical ionization mass spectrometry (HPLC-MS/APCI).

A method using high-performance liquid chromatography (HPLC) and atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) was established for the detection of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) and its conjugate with N-acetyl-L-cysteine (NAC). The optimal chromatographic conditions were obtained on an ODS column (150 x 4.6 mm, 3 microm) with the column temperature at 37 degrees C. The mobile phase consisted of a methanol-0.1% trifluoroacetic acid (TFA) mixture (50 : 50, v/v), and the flow rate was 0.3 ml/min. The detection wavelength was set at 220 nm. The identities of the peaks were accomplished by comparing retention times (tR), UV and mass data. All calibration curves showed good linear regression (correlation coefficients for 6-MITC and NAC>0.999) within test ranges. The developed method provided satisfactory precision calculated as percent coefficient of variation with overall intra-day and inter-day variations of less than 5% (4.1 and 4.9% for 6-MITC; 4.2 and 4.9% for NAC). Both 6-MITC and NAC had good responses in the positive APCI and formed strong [M+H]+ ions in the full scan spectra at an m/z of 206 and 164, respectively. The presence of the [M+H]+ ion for the 6-MITC/NAC conjugate was also observed at an m/z of 369. To our best knowledge, this is the first report that describes the formation of the 6-MITC/NAC conjugate and its detection method by HPLC-MS.

Increase of visceral fat area in Indonesians and Japanese with normal BMI.

We compared the visceral fat accumulation between Indonesians and Japanese. Non-obese (25>BMI> or =18.5) men aged between their 20s and 50s were collected in Toban including moderately populated middle-sized cities of Hyogo Prefecture in Japan, and Sangsit town and Pedawa village in Indonesia. Their visceral fat accumulation was assessed by determination of visceral fat area (VFA) that was measured through bio-electrical impedance analysis. VFA as well as VFA per body weight in those aged in their 20s did not vary significantly among Toban, Sangsit and Pedawa. In these three districts, they increased with age. Compared with VFA as well as VFA per body weight among those aged in their 20s, they increased in their 30s and over in the urban area of both Japan and Indonesia, whereas it did in their 40s and over in Pedawa, economically poor district. The inhabitants in their 40s and over in Sangsit and those in their 30s and over in Pedawa had less VFA than those with the corresponding age in Toban, while those in the 30s and 40s in Pedawa and those in their 40s in Sangsit had less VFA per body weight than those with corresponding age in Toban. In conclusion, the visceral fat appears to accumulate progressively with aging and urbanization of lifestyle even in those with normal body mass index. It is recommended that some preventive measures against visceral fat accumulation should be taken in their 20s or under for urban dwellers and in their 30s or under for rural inhabitants.

Preventive effect of oral administration of 6-(methylsulfinyl)hexyl isothiocyanate derived from wasabi (Wasabia japonica Matsum) against pulmonary metastasis of B16-BL6 mouse melanoma cells.

AIM: Effect of oral administration of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) or a 6-MITC-containing T-wasabi fraction from wasabi root (Wasabia japonica Matsum) to inhibit the macroscopic pulmonary metastasis was studied with a murine B16-BL6 melanoma model. METHOD: Two administration routes, subcutaneous or intravenous, and two administration times, prior to or concomitant with tumor inoculation, of 6-MITC or T-wasabi against the metastatic foci formation in C57BL/6J mouse lungs were compared. RESULTS: The number of metastasized foci per lung in either subcutaneous or intravenous injection was significantly reduced by intake of 6-MITC or a T-wasabi fraction. The maximum reduction by a T-wasabi fraction reached to 82%. Fifty-six percent of foci formation was inhibited by a 2 week-prior administration of 6-MITC (200 microM), whereas only 27% inhibition was obtained by a concomitant administration with tumor inoculation. Neither 6-MITC nor T-wasabi at tested concentrations showed any toxic effects. DISCUSSION: Together with our previous results, a component of the Japanese pungent spice, wasabi appears to inhibit not only tumor cell growth but also tumor metastasis. Therefore, 6-MITC from wasabi is apparently a useful dietary candidate for controlling tumor progression.

Selective sensitivity to wasabi-derived 6-(methylsulfinyl)hexyl isothiocyanate of human breast cancer and melanoma cell lines studied in vitro.

Recently, attention has focused on the anticancer properties of an aromatic component 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) in a typical Japanese spice, wasabi. In this paper, anticancer activity of 6-MITC in vitro was studied by using a human cancer cell (HCC) panel. 6-MITC directly affected the cells in the HCC panel and inhibited their growth in culture. The mean concentration required to inhibit 50% of control cell growth was 3.9 microM, which is a sufficiently low dosage for practical use. The suppression influenced not only the cell growth, but also the survival of these cells. The mean concentration to suppress cells to a 50% survival was 43.7 microM. The reduction activity of 6-MITC was differential, and it suppressed specific cells. These severely suppressed cell lines included breast cancer and melanoma cell lines. For example, one melanoma line was seriously damaged at a concentration of 0.3 microM of 6-MITC. Compared with other MITCs (2-MITC, 4-MITC and 8-MITC), 6-MITC showed the most effective suppression and with the most specific manner of the cells mentioned above. A "COMPARE" analysis using a computerized algorithm, which was based on the HCC database, suggested that the suppression mechanism of 6-MITC is unique and may be different from that of other known chemicals. The actual mechanism may not a simple one but may involve multiple pathways. On account of its sufficiently small size, 6-MITC is a new possible candidate for controlling cancer cells.

Augmented gene expression of quinone reductase by 6-(methylsulfinyl)hexyl isothiocyanate through avoiding its cytotoxicity.

We examined both the induction of quinone reductase (QR) by 6-(methylsulfinyl)hexyl isothiocyanate and its cytotoxicity in Hepa1c1c7 cells, and compared the sensitivity of these two responses to NAC. QR activity was increased by 6-(methylsulfinyl)hexyl isothiocyanate in a dose-dependent manner. At 80 microM, the compound was significantly toxic to cells, but the resulting QR inhibition was dose-dependently overcome by NAC. Augmentation of QR activity by 6-(methylsulfinyl)hexyl isothiocyanate seemed to be due to augmented expression of QR mRNA, which was significantly increased by the compound. Inhibition of QR gene expression was seen at 80 microM and could be overcome by NAC. Optimal induction of QR gene expression by the compound (at 40 microM) was slightly but significantly inhibited by 10 mM NAC but not by 1 mM. The present study suggests that induction of Phase 2 detoxification enzymes by isothiocyanate compounds may be further enhanced by suppression of their inherent cytotoxic activity.