Glycosylation of tyrosinase is a determinant of melanin production in cultured melanoma cells.

The majority of malignant melanoma cell types are able to produce melanin and the degree of melanin synthesis in various types of cultured cell line differs. In this study, we evaluated three types of cultured cell line, MNT­1, HM3KO and G­361, with differing melanin production levels. The level was greatest in the MNT­1 cells, lower in the HM3KO cells and lowest in the G­361 cells. In addition, a positive correlation between melanin production and tyrosinase activity was observed. The molecular masses of tyrosinases from HM3KO and G­361 cells were marginally lower than those from MNT­1 cells. Glycosylation inhibitor treatment on MNT­1 cells caused decreases in the molecular mass of tyrosinase, its activity and melanin production. An immunoprecipitation assay using anti­tyrosinase indicated that the immature glycosylated tyrosinases were associated with a type of chaperone, Hsp70. The interaction between tyrosinase and Hsp70 was also detected in HM3KO and G­361 cells. The results indicated that the immature glycosylation of tyrosinase has a critical effect on the melanin-producing ability of melanoma cells.

Kuromoji (Lindera umbellata) essential oil inhibits LPS-induced inflammation in RAW 264.7 cells.

Kuromoji (Lindera umbellata) essential oil (KEO) has long been used in Japan as a traditional medicine. It contains linalool (C10H18O), a naturally occurring small terpenoid. For this study, we investigated the anti-inflammatory effect of KEO in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Mouse macrophage-like RAW 264.7 cells were stimulated with LPS. Then they were treated with 25 or 50 µg/mL of KEO for 24 h. KEO suppressed LPS-induced pro-inflammatory cytokine production such as that of nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in a dose-dependent manner. In addition, inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) mRNA expression and protein levels were suppressed by treatment with KEO cells. In addition, by treatment with 25 or 50 µg/mL of linalool showed the same anti-inflammatory effect. The results suggest that KEO and linalool can be regarded as a natural resource for use in anti-inflammatory therapeutic products.

Kuromoji (Lindera umbellata) essential oil-induced apoptosis and differentiation in human leukemia HL-60 cells.

Essential oils diluted from certain plants have been shown to have antitumor activity against several human tumor cell lines. Kuromoji (Lindera umbellata) essential oil (KEO) has long been used in Japan as a traditional medicine. KEO and its major chemical constituent, linalool, were investigated in this study for their ability to induce apoptosis and differentiation in human leukemia HL-60 cells. HL-60 cells were treated with 5 or 50 µg/ml KEO or linalool for 24 or 48 h. Then, cell proliferation and apoptosis induction were estimated. In addition, HL-60 cells are known to differentiate into granulocyte or monocytes by a variety of compounds. Therefore, the effect of KEO or linalool on differentiation of HL-60 cells was assessed by Giemsa stain and a nitroblue tetrazolium reduction assay. Cells treated with KEO or linalool for 48 h showed significantly suppressed cell proliferation, with induced apoptosis. Moreover, KEO and linalool promoted cell differentiation. Treatment with KEO cells at the same dose as linalool showed an almost identical effect on HL-60 cells. These results suggest that KEO and linalool have efficacy as anticancer therapeutic products.

Anti-aging effects of high molecular weight proteoglycan from salmon nasal cartilage in hairless mice.

Proteoglycans comprise a family of complex macromolecules consisting of a core protein with covalently attached glycosaminoglycan (GAG) chains. The skin anti-aging effects of oral administration of proteoglycan fractions with different molecular weights from salmon nasal cartilage were investigated in a hairless mouse model of skin aging; aging was caused by repeated ultraviolet B (UVB) irradiation. Three proteoglycan fractions of different molecular weights were prepared from salmon nasal cartilage water extract by ion-exchange column chromatography and gel filtration column chromatography. Physiological and histological analysis of the skin indicated that oral administration of high molecular weight proteoglycan inhibited UVB-induced skin aging, defined as increased erythema, increased transepidermal water loss (TEWL), decreased hydration, and epidermal and dermal hypertrophies. The serum and dorsal skin inflammatory cytokine levels indicated that high molecular weight proteoglycan acts on gut immunity and improves skin by inhibiting surplus inflammatory cytokines produced by UVB irradiation. These results suggest that high molecular weight proteoglycan from salmon nasal cartilage is effective in preventing skin aging.

The Hsp90 inhibitor 17-AAG represses calcium-induced cytokeratin 1 and 10 expression in HaCaT keratinocytes.

Hsp90 is essential for maintaining the activity of numerous signaling factors, and plays a key role in cellular signal transduction networks. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is an ansamycin antibiotic that binds to Hsp90 and inhibits its function. HaCaT human keratinocytes were used to investigate the cellular and molecular functions of Hsp90 in keratinocyte differentiation. Inhibition of Hsp90 by 17-AAG leads to downregulation of the differentiation markers cytokeratin 1 and cytokeratin 10 at the protein and mRNA levels.

Hsp40 regulates the amount of keratin proteins via ubiquitin-proteasome pathway in cultured human cells.

Keratins represent important structural components of intermediate filament proteins. Their expression profiles are remarkably tissue-specific. Recent data have shown that keratins associate with many proteins including heat shock proteins (HSP). We recently identified cell-specific keratin and HSP expression. We aimed to gain further insight into the regulation of keratins by specific inhibition through knockdown of Hsp40 in human keratinocyte cells. Keratin-HSP interaction in HaCaT cell lysate was evaluated by immunoprecipitation followed by Western blotting. Immunofluorescence, was used to examine the co-localization of keratins and Hsp40. Hsp40 depletion led to an increase in the levels of keratin proteins (K5, K14, K10) and a decrease in keratin ubiquitination without influencing keratin gene expression. Our results demonstrate direct or indirectly association of Hsp40 and imply that expressed keratin proteins were regulated by Hsp40 depending on the ubiquitin-proteasome pathway in HaCaT. Furthermore, the K10 differentiation marker was increased by knockdown of Hsp40. The results presented in this study indicate that Hsp40 is related to the differentiation exchange of keratin pairs.

Anti-aging effects of extracts prepared from salmon nasal cartilage in hairless mice.

The skin anti-aging effects of orally administered salmon nasal cartilage extract (SNCE), which includes abundant proteoglycan, were investigated using a hairless mouse skin-aging model, in which aging was caused by repetitive ultraviolet B (UV-B) irradiation. Physiological analysis of the skin surface following repetitive UV-B irradiation of 8 weeks revealed inhibition of erythema levels and reduction of transepidermal water loss (TEWL) due to oral administration of SNCE. Similarly, inhibitory actions of epidermal and dermal hypertrophy were revealed by hematoxylin and eosin staining. Furthermore, effects on the hydration level of the skin surface by SNCE administration were indicated at 4 weeks of UV-B irradiation, but greater effects were not apparent. These results indicate that SNCE may serve as an anti-aging agent for healthy skin.

Extract of passion fruit (Passiflora edulis) seed containing high amounts of piceatannol inhibits melanogenesis and promotes collagen synthesis.

The effect of passion fruit, the fruit of Passiflora edulis , on melanin inhibition and collagen synthesis was studied using cultured human melanoma and fibroblast cells. Passion fruit was divided into three parts, rind (PF-R), pulp (PF-P), and seed (PF-S), and each part was extracted using 80% ethanol. The concentration of polyphenols was higher in PF-S than in PF-R or PF-P. Treatment of melanoma cells with PF-S led to inhibition of melanogenesis. In addition, the production of total soluble collagen was elevated in dermal fibroblast cells cultured in the presence of PF-S. PF-R and PF-P did not yield these effects. Furthermore, the removal of polyphenols from PF-S led to the abolishment of the effects described above. We discovered that piceatannol (3,4,3',5'-tetrahydroxy-trans-stilbene) is present in passion fruit seeds in large amounts and that this compound is the major component responsible for the PF-S effects observed on melanogenesis and collagen synthesis.

Antiproliferative activity of extracts prepared from three species of Reishi on cultured human normal and tumor cell lines.

The present study investigated the growth of human fibrosarcoma (HT-1080) and fibroblast (SF-TY) cells in combination with water-soluble (WS) and high molecular component (HMC) fractions prepared from Reishi (R), Rokkaku-Reishi (2R) and Apple Rokkaku-Reishi (A2R). Each WS fraction exhibited dose-and time-dependent inhibition of the growth of the HT-1080 and SF-TY cells. The extracts exhibited marked antiproliferative activity against the HT-1080 cells. The HMC fractions inhibited cell growth dose-and time-dependently in the HT-1080 cells only, and not in the SF-TY cells, suggesting that HMC fractions selectively inhibit HT-1080 cells. Among the HMC fractions, A2R is a strong candidate for anti-tumor targeting since its fraction exhibited better inhibition than the R and 2R fractions. Furthermore, the volume of the A2R fraction was approximately five times greater than that of the others, and included four proteins (molecular mass 9, 13, 22 and 40 kDa) detected by SDS-PAGE. Three of these (13, 22 and 40 kDa) were confirmed to be glycosylated with the Periodic Acid-Schiff Stain kit. These results suggest that A2R may possess anti-tumor activity and, in particular, that the protein components of A2R may act to selectively inhibit the growth of HT-1080 cells.

Impaired activation of caspase cascade during cell death induced by newly synthesized singlet oxygen generator, 1-buthylnaphthalene-4-propionate endoperoxide.

Endoperoxides of naphthalene derivatives generate singlet oxygen under physiological conditions. Here we have synthesized a new endoperoxide of a naphthalene derivative, 1-buthylnaphthalene-4-propionate endoperoxide (BNPE), and studied its cytotoxic properties on HepG2 and HaCaT cells. BNPE induced cell death at much lower concentration than 1-methylnaphthalene-4-propionate endoperoxide (MNPE) and naphthalene dipropionate endoperoxide (NDPE). A positive correlation exists between the amount of endoperoxide incorporated into cells and its cytotoxic ability. The cytotoxic effect of BNPE was attenuated by alpha-tocopherol but not by sodium azide. In contrast, the effects of MNPE and NDPE were attenuated by both alpha-tocopherol and sodium azide. The caspase cascade in cells treated with endoperoxide was impaired. Caspase activity in a soluble protein fraction were inhibited similarly by the above three endoperoxides. These results suggest an abortive apoptotic pathway due to the suppression of caspase activation is a general feature of cell death induced by singlet oxygen.

Unexpected expression of Hsp47, a replacement of one amino acid (Val 7 Leu) in the amino terminal region, in cultured human tumorigenic cell lines.

BACKGROUND: In general, it has been stated that keratin (K) molecules are glycosylated. During biochemical studies of K subunits, we encountered a glycoprotein that does not judge K subunits. OBJECTIVE: This study was intended to elucidate how the above glycoprotein co-exists in the K fraction prepared from ISO-HAS (cultured angiosarcoma cell line). METHODS: We analyzed and sequenced a remarkable spot, which was shown as a glycoprotein by periodic acid Sciff's (PAS) staining, in the K fraction prepared from ISO-HAS. RESULTS: The glycoprotein was identified as an N-terminal amino acid sequence covering 10 residues of the spot. A homology search showed that it was identical to that of Hsp47 (matured type), except for one amino acid (seventh amino acid: Val 7 Leu). Similar results were confirmed for four other tumorigenic cell line types. Subsequent PAS staining using the same samples after 2D-PAGE revealed no glycosylated Ks. CONCLUSION: No glycosylated Ks were found by PAS staining in the K fraction prepared from four tumorigenic cell line types. During K preparation from cultured human tumor cell lines, Hsps might be associated with K expression in tumor cells.

Helicobacter pylori VacA enhances prostaglandin E2 production through induction of cyclooxygenase 2 expression via a p38 mitogen-activated protein kinase/activating transcription factor 2 cascade in AZ-521 cells.

Treatment of AZ-521 cells with Helicobacter pylori VacA increased cyclooxygenase 2 (COX-2) mRNA in a time- and dose-dependent manner. A p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, blocked elevation of COX-2 mRNA levels, whereas PD98059, which blocks the Erk1/2 cascade, partially suppressed the increase. Consistent with involvement of p38 MAPK, VacA-induced accumulation of COX-2 mRNA was reduced in AZ-521 cells overexpressing a dominant-negative p38 MAPK (DN-p38). Phosphatidylinositol-specific phospholipase C, which inhibits VacA-induced p38 MAPK activation, blocked VacA-induced COX-2 expression. In parallel with COX-2 expression, VacA increased prostaglandin E(2) (PGE(2)) production, which was inhibited by SB203580 and NS-398, a COX-2 inhibitor. VacA-induced PGE(2) production was markedly attenuated in AZ-521 cells stably expressing DN-p38. VacA increased transcription of a COX-2 promoter reporter gene and activated a COX-2 promoter containing mutated NF-kappaB or NF-interleukin-6 sites but not a mutated cis-acting replication element (CRE) site, suggesting direct involvement of the activating transcription factor 2 (ATF-2)/CREB-binding region in VacA-induced COX-2 promoter activation. The reduction of ATF-2 expression in AZ-521 cells transformed with ATF-2-small interfering RNA duplexes resulted in suppression of COX-2 expression. Thus, VacA enhances PGE(2) production by AZ-521 cells through induction of COX-2 expression via the p38 MAPK/ATF-2 cascade, leading to activation of the CRE site in the COX-2 promoter.

Effects of angiotensin II receptor signaling during skin wound healing.

The tissue angiotensin (Ang) system, which acts independently of the circulating renin Ang system, is supposed to play an important role in tissue repair in the heart and kidney. In the skin, the role of the system for wound healing has remained to be ascertained. Our study demonstrated that oral administration of selective AngII type-1 receptor (AT(1)) blocker suppressed keratinocyte re-epithelization and angiogenesis during skin wound healing in rats. Immunoprecipitation and Western blot analysis indicated the existence of AT(1) and AngII type-2 receptor (AT(2)) in cultured keratinocytes and myofibroblasts. In a bromodeoxyuridine incorporation study, induction of AT(1) signaling enhanced the incorporation into keratinocytes and myofibroblasts. Wound healing migration assays revealed that induction of AT(1) signaling accelerated keratinocyte re-epithelization and myofibroblasts recovering. In these experiments, induction of AT(2) signaling acted vice versa. Taken together, our study suggests that skin wound healing is regulated by balance of opposing signals between AT(1) and AT(2).

Occurrence and comparison of the expressed keratins in cultured human fibroblasts, endothelial cells and their sarcomas.

We investigated keratin (K) expression in cultured fibroblasts, endothelial cells and their sarcomas by using two-dimensional gel electrophoresis and electron microscopy techniques. Although the fibroblast and endothelial cell lines were derived from mesenchyme, we confirmed Ks in both cell lines. The K in two cultured cell lines consisted of K14 and K16, together with vimentin. In addition to the above Ks, K5 and K8/K17 were comprised in each cell line, respectively. On the other hand, the cultured fibrosarcomas contained K8 and K18 in addition to the Ks present in the cultured fibroblasts, except K17. Moreover, cultured angiosarcomas showed the same Ks expression as those of the cultured fibrosarcomas, except vimentin. However, electron microscopy showed that the extremely thin fiber-like substances existed or at least did not form filamentous structures in four cultured cell types.