Probiotic fermentation augments the skin anti-photoaging properties of Agastache rugosa through up-regulating antioxidant components in UV-B-irradiated HaCaT keratinocytes.

BACKGROUND: Agastache rugosa (Fisch. & C.A.Mey.) Kuntze (Korean mint) is used to treat diverse types of human disorders in traditional medicine. In recent years, its non-fermented leaf extract (ARE) has been shown to possess protective properties against ultraviolet-B (UV-B) radiation-induced photooxidative stress. The present work aimed to examine whether probiotic bacterial fermentation would potentiate the skin anti-photoaging activity of ARE or not, by comparing the protective properties of ARE and corresponding fermented extract (ARE-F) against UV-B radiation-induced photooxidative stress in HaCaT keratinocytes. METHODS: ARE-F was produced from ARE by the fermentation with Lactobacillus rhamnosus HK-9, a type of Gram-positive probiotic bacterial strain. Anti-photoaging activities were evaluated by analyzing reactive oxygen species (ROS), promatrix metalloproteinases (proMMPs), total glutathione (GSH) and total superoxide dismutase (SOD) in UV-B-irradiated HaCaT keratinocytes. Antiradical activity was determined using 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assay. RESULTS: ARE-F contained higher attenuating activity on the UV-B-induced ROS generation than ARE. Similarly, ARE-F was able to diminish the UV-B-induced proMMP-9 and -2 more effectively than ARE. ARE-F displayed higher tendencies to augment the UV-B-reduced total GSH content and SOD activity than ARE. However, there were no significant difference between ARE and ARE-F in ABTS radical scavenging activities. CONCLUSIONS: The findings suggest that the UV-B radiation-protective activity of ARE is enhanced by probiotic bacterial fermentation, which might improve the therapeutic and cosmetic values of A. rugosa leaves.

Protective properties of geniposide against UV-B-induced photooxidative stress in human dermal fibroblasts.

CONTEXT: Geniposide (genipin-1-O-ß-d-glucoside) is a major bioactive ingredient in the fruits of gardenia [Gardenia jasminoides J. Ellis (Rubiaceae)], a traditional herbal medicine in Asian countries. OBJECTIVE: This work assesses the skin anti-photoaging potential of geniposide in human dermal fibroblasts under UV-B irradiation. MATERIALS AND METHODS: The anti-photoaging property of geniposide, at varying concentrations (5, 12 and 30 µM) treated for 30 min prior to UV-B irradiation, was evaluated by analysing reactive oxygen species (ROS), promatrix metalloproteinase-2 (proMMP-2), glutathione (GSH), superoxide dismutase (SOD), nuclear factor erythroid 2-related factor 2 (Nrf2) and cellular viability. RESULTS: Geniposide suppressed the ROS elevation under UV-B irradiation, which was revealed using three ROS-sensitive fluorescent dyes. The use of 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), dihydroethidium (DHE) and dihydrorhodamine 123 (DHR-123) elicited the IC50 values of 10.5, 9.8 and 21.0 µM, respectively. Geniposide attenuated proMMP-2 at activity and protein levels that were elevated under UV-B-irradiation. Geniposide at 5, 12 and 30 µM augmented the UV-B-reduced total GSH content to 1.9 ± 0.1-, 2.2 ± 0.2- and 4.1 ± 0.2-fold, respectively. Geniposide at 5, 12 and 30 µM upregulated total SOD activity to 2.3 ± 0.1-, 2.5 ± 0.3- and 3.3 ± 0.3-fold, respectively, under UV-B irradiation. The UV-B-reduced Nrf2 levels were also upregulated by geniposide treatment. Geniposide, at the concentrations used, was unable to interfere with cellular viabilities under UV-B irradiation. DISCUSSION AND CONCLUSIONS: After the skin anti-photoaging potential of geniposide may be further verified, it can be utilized as a safer resource in the manufacture of effective anti-aging cosmetics.

Ginsenoside Re improves skin barrier function in HaCaT keratinocytes under normal growth conditions.

Ginsenoside Re (Re), a major ginsenoside of ginseng, enhanced the cornified cell envelope (CE) formation in HaCaT keratinocytes under normal conditions. In HaCaT keratinocytes, Re was also able to upregulate filaggrin protein and caspase-14 activity in a concentration-dependent manner. These findings reasonably imply that Re possesses a desirable property of improving skin barrier function.

Contribution of ginsenoside Re to cellular redox homeostasis via upregulating glutathione and superoxide dismutase in HaCaT keratinocytes under normal conditions.

Ginsenoside Re (Re) is one of the main ginsenosides which are known to be responsible for diverse pharmacological properties of ginseng, widely used as a dietary supplement and a general tonic. The present work was undertaken to evaluate the antioxidative property of Re by analyzing reactive oxygen species (ROS), nitric oxide (NO), pro-matrix metalloproteinase-2 (proMMP-2) and -9 (proMMP-9), total glutathione (GSH) and superoxide dismutase (SOD) in normal, unstressed HaCaT keratinocytes. When HaCaT cells were subjected to Re, Re suppressed the ROS and NO levels in a concentration-dependent manner. Re at concentrations used exhibited no cytotoxicity on the cellular viabilities of HaCaT cells. It was also able to attenuate proMMP-2 and -9 at both activity and protein levels. On the contrary, Re was capable of enhancing the total GSH and SOD activity levels. The findings suggest that Re has an antioxidative property through the upregulation of some antioxidant components, including total GSH and SOD, in HaCaT keratinocytes, which then can play its underlying role in maintaining the cellular redox homeostasis.

Ellagic acid plays a protective role against UV-B-induced oxidative stress by up-regulating antioxidant components in human dermal fibroblasts.

Ellagic acid (EA), an antioxidant polyphenolic constituent of plant origin, has been reported to possess diverse pharmacological properties, including anti-inflammatory, anti-tumor and immunomodulatory activities. This work aimed to clarify the skin anti-photoaging properties of EA in human dermal fibroblasts. The skin anti-photoaging activity was evaluated by analyzing the reactive oxygen species (ROS), matrix metalloproteinase-2 (MMP-2), total glutathione (GSH) and superoxide dismutase (SOD) activity levels as well as cell viability in dermal fibroblasts under UV-B irradiation. When fibroblasts were exposed to EA prior to UV-B irradiation, EA suppressed UV-B-induced ROS and proMMP-2 elevation. However, EA restored total GSH and SOD activity levels diminished in fibroblasts under UV-B irradiation. EA had an up-regulating activity on the UV-B-reduced Nrf2 levels in fibroblasts. EA, at the concentrations used, was unable to interfere with cell viabilities in both non-irradiated and irradiated fibroblasts. In human dermal fibroblasts, EA plays a defensive role against UV-B-induced oxidative stress possibly through an Nrf2-dependent pathway, indicating that this compound has potential skin antiphotoaging properties.

The Anti-Diabetic Pinitol Improves Damaged Fibroblasts.

Pinitol (3-O-Methyl-D-chiro-inositol) has been reported to possess insulin-like effects and is known as one of the anti-diabetic agents to improve muscle, liver, and endothelial cells. However, the beneficial effects of pinitol on the skin are not well known. Here, we investigated whether pinitol had effects on human dermal fibroblasts (HDFs), and human dermal equivalents (HDEs) irradiated with ultraviolet A (UVA), which causes various damages including photodamage in the skin. We observed that pinitol enhanced wound healing in UVA-damaged HDFs. We also found that pinitol significantly antagonized the UVA-induced up-regulation of matrix metalloproteinase 1 (MMP1), and the UVA-induced down-regulation of collagen type I and tissue inhibitor of metalloproteinases 1 (TIMP1) in HDEs. Electron microscopy analysis also revealed that pinitol remarkably increased the number of collagen fibrils with regular banding patterns in the dermis of UVA-irradiated human skin equivalents. Pinitol significantly reversed the UVA-induced phosphorylation levels of ERK and JNK but not p38, suggesting that this regulation may be the mechanism underlying the pinitol-mediated effects on UVA-irradiated HDEs. We also observed that pinitol specifically increased Smad3 phosphorylation, which is representative of the TGF-ß signaling pathway for collagen synthesis. These data suggest that pinitol exerts several beneficial effects on UVA-induced damaged skin and can be used as a therapeutic agent to improve skin-related diseases.

Predictive factors of lymph node metastasis in papillary thyroid cancer.

This study aimed to evaluate factors that predict lymph node metastasis (LNM) in papillary thyroid cancer (PTC). This retrospective cross-sectional study compared the demographic, clinical, and ultrasonographic findings of patients with PTC with and without LNM. Subgroup analysis was conducted for micro-PTCs (<1 cm). Among total (n = 512; mean age, 47.3 ± 12.7 years) and micro-PTC patients (n = 312), 35.7% and 19.6% had LNM, respectively. Younger age, male sex, tumor size, bilaterality, and suspicious ultrasound features of the tumor were associated with LNM. In multiple logistic regression analysis, among all patients, age, tumor size, and extrathyroidal extension were independent risk factors for LNM (all p<0.05). In the micro-PTC subgroup, age, extrathyroidal extension, bilaterality of tumor, and presence of autoimmune thyroid disease were independent risk and protective factors for LNM (all p<0.05). In the receiver operating characteristic analysis, the accuracy of the multivariable logistic regression model for predicting LNM among all patients and micro-PTC was acceptable (area under the curve = 0.729 and 0.733, respectively). Age, sex, tumor size, and extrathyroidal extension can assist in predicting LNM in PTC patients. Additionally, the bilaterality of tumors and presence of autoimmune thyroid disease can assist in predicting LNM in micro-PTCs.

Reliability and validity of the Korean version of the Neurological Disorders Depression Inventory for Epilepsy (K-NDDI-E).

The Neurological Disorders Depression Inventory for Epilepsy (NDDI-E) was developed as a screening instrument for rapid detection of major depression in people with epilepsy (PWE). We evaluated the reliability and validity of the Korean version of the NDDI-E (K-NDDI-E) in Korean PWE. This study applied to 121 outpatients who underwent psychometric tests including the Mini International Neuropsychiatric Interview-Plus Version 5.0.0, Beck Depression Inventory-II (BDI-II), and K-NDDI-E. The K-NDDI-E was easily comprehended and quickly completed by the patients. Cronbach's α coefficient was 0.898. At a cut off score of 11, the K-NDDI-E had a sensitivity of 84.6%, a specificity of 85.3%, a positive predictive value of 61.1%, and a negative predictive value of 95.3%. The scores of the K-NDDI-E had a positive correlation with those of the BDI-II (p<0.001). In conclusion, the K-NDDI-E is a reliable and valid screening tool to detect major depression in Korean PWE.

Pap1p-dependent upregulation of thioredoxin 3 and thioredoxin reductase genes from the fission yeast under nitrosative stress.

The thioredoxin system, consisting of thioredoxin, thioredoxin reductase, and NADPH, is involved in the response against a variety of stresses. The TRX3(+) and TrxR(+) genes encode thioredoxin 3 and thioredoxin reductase, respectively, in the fission yeast Schizosaccharomyces pombe . Their transcriptional regulations were studied using the lacZ fusion genes. Synthesis of ß-galactosidase from the TRX3(+)-lacZ fusion gene was markedly enhanced by nitric-oxide-generating sodium nitroprusside in the Pap1p-positive cells but not in the Pap1p-negative cells. Similarly, synthesis of ß-galactosidase from the TrxR(+)-lacZ fusion gene was upregulated by sodium nitroprusside in a Pap1p-dependent manner. Synthesis of ß-galactosidase from the TRX3(+)-lacZ and TrxR(+)-lacZ fusion genes was also enhanced by S-nitrosoglutathione in the Pap1p-positive cells but not in the Pap1p-negative cells. In brief, the S. pombe genes encoding thioredoxin 3 and thioredoxin reductase are upregulated under nitrosative stress in a Pap1p-dependent manner.

Comparative Insights into the Skin Beneficial Properties of Probiotic Lactobacillus Isolates of Skin Origin.

In recent times, probiotics have been emerging as one of valuable cosmetic resources. This work was undertaken to evaluate and compare the skin beneficial properties of three Lactobacillus strains, namely, L. plantarum SB202, L. fermentum SB101, and L. paraplantarum SB401, originally isolated from the healthy skins of Koreans. The Lactobacillus isolates were individually grown in MRS broth, and the corresponding cell-free conditioned mediums (CMs), LP202, LF101 and LPP401, were prepared for analyzing diverse cosmetic potentials at a comparative perspective. The superoxide radical and nitrite ion scavenging activities of the CMs were in the orders of LPP401 ≥ LF101 > LP202 and LPP401 > LF101≒LPP202, respectively. They attenuated the lipopolysaccharide-induced reactive oxygen species (ROS) and nitrite ion levels in RAW264.7 murine macrophages both in the order of LPP401 ≥ LF101 > LP202, implying their anti-inflammatory properties. They exhibited antityrosinase activities in the order of LPP401 > LF101 ≥ LP202 and diminished α-melanocyte-stimulating hormone-induced melanin levels in B16F10 melanoma cells in the order of LPP401≒LF101 > LP202, suggesting their skin whitening activities. They enhanced cornfield envelope formation in HaCaT keratinocytes in the order of LPP401 > LF101 > LP202. They inhibited the in vitro hyaluronidase and elastase activities in the orders of LPP401 > LP202 ≥ LF101 and LPP401 ≥ LP202 > LF101, respectively. Their enhancing properties on the synthesis of procollagen type I in normal human dermal fibroblasts were in the order of LF101≒LPP401 > >LP202. The CMs possess various cosmetic characteristics, such as antioxidant, skin whitening, antiaging, barrier improving, and anti-inflammatory activities. LPP401, the CM prepared from L. paraplantarum SB401, has been evaluated to be more desirable cosmetic resource than LP202 and LF101.

Investigation of Age-Related Changes in the Skin Microbiota of Korean Women.

The microbiota of human skin is influenced by host and environmental factors. To determine if chronological age influences the composition of the skin microbiota on the forehead and hands, 73 Korean women were sorted into one of three age groups: (1) 10-29 years (n = 24), (2) 30-49 years (n = 21), and (3) 50-79 years (n = 28). From the 73 women, 146 skin samples (two skin sites per person) were collected. 16S rRNA gene amplicon sequencing was then conducted to analyze the skin microbiota. The overall microbial distribution varied on the forehead but was similar on the hands across the three age groups. In addition, the composition of the skin microbiota differed between the forehead and hands. Commensal microbiota, such as Streptococcus, Staphylococcus, Cutibacterium, and Corynebacterium, which contribute to maintaining skin health via dominant occupation, were affected by increasing age on forehead and hand skin. Alpha diversity indices increased significantly with age on forehead skin. This study indicates that older people may be more susceptible to pathogenic invasions due to an imbalanced skin microbiota resulting from age-related changes. The results of our study may help develop new strategies to rebalance skin microbiota shifted during aging.

Anti-Inflammatory, Barrier-Protective, and Antiwrinkle Properties of Agastache rugosa Kuntze in Human Epidermal Keratinocytes.

This work aimed to assess the skin-beneficial properties of Agastache rugosa Kuntze, an herbal medication used to treat different types of disorders in traditional folk medicine. The total phenolic compounds and total antiradical, nitrite scavenging, superoxide scavenging, antielastase, and antihyaluronidase activities of a hot water extract of A. rugosa Kuntze leaves (ARE) were spectrophotometrically determined. Intracellular reactive oxygen species (ROS) was fluorometrically quantitated using 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Inducible nitric oxide synthase (iNOS) and filaggrin were evaluated using Western analysis. Real-time quantitative RT-PCR was used to measure filaggrin mRNA. Caspase-14 activity was determined using a fluorogenic substrate. ARE contained the total phenolic content of 38.9 mg gallic acid equivalent/g extract and exhibited 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical, superoxide radical, and nitrite scavenging activities with the SC50 values of 2.9, 1.4, and 1.7 mg/mL, respectively. ARE exerted suppressive activities on nitric oxide (NO) and ROS levels elevated by lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α) in HaCaT keratinocytes. It attenuated the LPS-stimulated expression of iNOS. ARE augmented the UV-B-reduced filaggrin expression on both protein and mRNA levels and was capable of upregulating the UV-B-reduced caspase-14 activity. ARE inhibited in vitro elastase and hyaluronidase activities associated with the wrinkling process. ARE, at the concentrations used, did not interfere with the viability of HaCaT keratinocytes. These findings preliminarily imply that the leaves of A. rugosa possess desirable cosmetic potentials, such as anti-inflammatory, barrier protective, and antiwrinkle activities, which infers their skin healing potentials.

Recombinant FNIII9-10-derived extracellular signaling effects on the physiology of dermal fibroblasts during in vitro culture.

Previous reports showed that fibronectin (FN) was effective in stimulating the recovery of damaged dermis. However, native FN has multifunctional domains transmitting beneficial as well as unbeneficial signals to dermal tissue cells through the mediation of integrin heterodimers. The use of a functional domain [FN type III9-10 fragments (FNIII9-10)] providing beneficial effects on the physiology of dermal tissue cells would enhance an in vitro culture system for dermal fibroblasts (DFs). We therefore investigated the FNIII9-10-derived extracellular signaling effect on the physiology of DFs during in vitro culture. Recombinant FNIII9-10 proteins were constructed and their functionality was determined by observing the adhesion of adult human DFs (aHDFs) to recombinant FNIII9-10 and of low adhesion integrin α5ß1- and αvß3-blocked aHDFs to recombinant FNIII9-10. Cellular proliferation, morphology, and senescence were measured and compared in the aHDFs cultured on native FN and recombinant FNIII9-10 for short or long periods. The results show that recombinant FNIII9-10-derived extracellular signaling stimulated increased proliferation of aHDF (both in short- and long-term cultures) and inhibited the generation of morphological abnormalities (in short- and long-term cultures) and cellular senescence (long-term culture) when compared with native FN-derived extracellular signaling. Our results suggest that, instead of native FN, recombinant FNIII9-10 better enhanced the in vitro culture of aHDFs while diminishing the adverse effects associated with the use of human-derived materials.

Screening of integrins localized on the surface of human epidermal melanocytes.

In vivo, melanocytes occupy three-dimensional (3D) space. Nevertheless, most experiments involving melanocytes are performed in a two-dimensional microenvironment, resulting in difficulty obtaining accurate results. Therefore, it is necessary to construct an artificial in vivo-like 3D microenvironment. Here, as a step towards engineering a precisely defined acellular 3D microenvironment supporting the maintenance of human epidermal melanocytes (HEMs), we examined the types of integrin heterodimers that are expressed transcriptionally, translationally, and functionally in HEMs. Real-time PCR and fluorescent immunoassay analyses were used to elucidate the expression of integrin α and ß subunit genes at the transcriptional and translational levels, respectively. The functionality of the presumed integrin heterodimers was confirmed using attachment and antibody-inhibition assays. Among the genes encoding 12 integrin subunits (α1, α2, α3, α4, α5, α6, α7, αV, ß1, ß3, ß5, and ß8) showing significantly higher transcription levels, proteins translated from the integrin α2, α4, α5, ß1, ß3, and ß5 subunit genes were detected on the surface of HEMs. These HEMs showed significantly increased adhesion to collagen I, fibronectin, laminin, and vitronectin, and functional blockade of the integrin α2 subunits significantly inhibited adhesion to collagen I, fibronectin, and laminin. In addition, there was no significant inhibition of the adhesion to fibronectin or vitronectin in HEMs with functional blockade of the integrin α4, α5, or αV subunits. These results indicate that the active integrin α2ß1 heterodimer and the inactive integrin α4, α5, αV, ß3, and ß5 subunits are all localized on the surface of HEMs.

Ginsenoside Rc protects against UVB­induced photooxidative damage in epidermal keratinocytes.

Ginsenoside Rc (Rc) is a major ginsenoside isolated from Panax ginseng, and has exhibited pharmacological effects on skin cells. The present study aimed to investigate the putative skin­protective properties of Rc, including its anti­photoaging and barrier function­protective effects, in human HaCaT keratinocytes exposed to UVB radiation. The protective properties of Rc were evaluated through the assessment of keratinocyte viability, reactive oxygen species (ROS) production, total glutathione (GSH) and superoxide dismutase (SOD) activity, caspase­14, matrix metalloproteinase (MMP)­2 and ­9 activity, and MMP­2, MMP­9 and filament aggregating protein (filaggrin) expression following UVB irradiation. Treatment with Rc was revealed to prevent the UVB­induced increase in ROS production and pro­MMP­2 and ­9 levels in HaCaT keratinocytes. In addition, treatment with Rc resulted in enriched GSH contents and enhanced SOD activity following exposure to UVB radiation. Furthermore, Rc treatment enhanced caspase­14 activity and counteracted the UVB­induced downregulation in filaggrin expression. However, no significant difference was identified between Rc­treated and normal groups in terms of keratinocyte viability, regardless of exposure to radiation. The present findings suggested that Rc may exert anti­photoaging and barrier function­protective effects in keratinocytes, and thus protect the skin against photooxidative stress induced by exposure to UV radiation.

Attenuating properties of Agastache rugosa leaf extract against ultraviolet-B-induced photoaging via up-regulating glutathione and superoxide dismutase in a human keratinocyte cell line.

Agastache rugosa Kuntze, known as a Korean mint, is an herbal medicine that has been used for the treatment of diverse kinds of symptoms in traditional medicine. This work was undertaken to assess the protective properties of A. rugosa leaves against UV-B-induced photoaging in HaCaT keratinocytes. They were evaluated via analyzing reactive oxygen species (ROS), promatrix metalloproteinase-2 (proMMP-2) and -9 (proMMP-9), total glutathione (GSH), total superoxide dismutase (SOD), cellular viability, flavonoid content and in vitro radical scavenging activity. Total flavonoid content of ARE, a hot water extract of A. rugosa leaves, was 22.8±7.6mg of naringin equivalent/g ARE. ARE exhibited ABTS(+) radical scavenging activity with an SC50 of 836.9µg/mL. ARE attenuated the UV-B-induced ROS generation. It diminished the UV-B-induced elevation of proMMP-2 and -9 at both activity and protein levels. On the contrary, ARE was able to enhance the UV-B-reduced total GSH and total SOD activity levels. ARE, at the used concentrations, was unable to interfere with the cellular viabilities of HaCaT keratinocytes under UV-B irradiation. Taken together, ARE possesses a protective potential against UV-B-induced photoaging in HaCaT keratinocytes, possibly based upon up-regulating antioxidant components, including total GSH and SOD. These findings reasonably suggest the use of A. rugosa leaves as a photoprotective resource in manufacturing functional cosmetics.

Stereospecificity of ginsenoside Rg2 epimers in the protective response against UV-B radiation-induced oxidative stress in human epidermal keratinocytes.

Ginseng, referring to the dried roots of Panax ginseng C.A. Meyer, has been known as a famous traditional folkloric medicine in East Asian countries for a long time. In recent years, it has been gaining a worldwide popularity as a dietary herbal supplement. Ginsenosides are bioactive ingredients that are responsible for most pharmacological efficacies of ginseng. Ginsenoside Rg2 (Rg2), one of minor protopanaxatriol (PPT)-type ginsenosides, exists in two epimeric forms, 20(S)-ginsenoside Rg2 [20(S)-Rg2] and 20(R)-ginsenoside Rg2 [20(R)-Rg2]. This work was undertaken to assess and compare their skin anti-photoaging properties. When they were applied to HaCaT keratinocytes prior to the irradiation, 20(S)-Rg2 only could attenuate the UV-B-induced intracellular reactive oxygen species (ROS) elevation, which were detected using three fluorescent ROS dyes, such as 2',7'-dichlorodihydrofluorescein diacetate, dihydroethidium and dihydrorhodamine 123. 20(S)-Rg2 but not 20(R)-Rg2 significantly attenuated the UV-B-induced promatrix metalloproteinase-2 (proMMP-2) gelatinolytic activity and protein levels. Likewise, 20(S)-Rg2 only augmented the UV-B-reduced total glutathione (GSH) and superoxide dismutase (SOD) activity levels in a concentration-dependent manner. Neither of the two Rg2 epimers was cytotoxic to HaCaT keratinocytes, regardless of UV-B irradiation. Taken together, of the two Rg2 epimers, 20(S)-Rg2 only possesses the stereospecific protective properties against the UV-B-induced skin photoaging in HaCaT keratinocytes.

Up-regulation of defense enzymes is responsible for low reactive oxygen species in malignant prostate cancer cells.

Reactive oxygen species (ROS) are involved in a diversity of important phenomena in the process of tumor development. To investigate the alterations of oxidative stress and their related systems in tumor progression, a variety of components in the antioxidative stress defense system were examined in prostate cancer cell lines, PC3 and LNCaP. Cell surface molecules involved in metastasis were expressed highly in PC3 cells compared with LNCaP cells, and strong invasion ability was shown in PC3 cells only. ROS level in LNCaP cells was twice higher than that in PC3 cells, although nitric oxide (NO) level was similar between the two cell lines. The content of GSH increased up to about 2-fold in PC3 compared with LNCaP. Activities of glutathione reductase, thioredoxin reductase, and glutathione S-transferase except catalase are significantly higher in PC3 cells than in LNCaP cells. Furthermore, oxidative stress-inducing agents caused down-regulation of GSH and glutathione S-transferase much more significantly in LNCaP cells than in PC3 cells. These results imply that malignant tumor cells may maintain low ROS content by preserving relatively high anti-oxidative capacity, even in the presence of stressful agents.

Characterization and regulation of the gene encoding monothiol glutaredoxin 3 in the fission yeast Schizosaccharomyces pombe.

Glutaredoxins (Grxs) are thioloxidoreductases which are required for maintaining thiol/disulfide equilibrium in living cells. The Grx3 gene, which encodes one of the three monothiol Grxs in the fission yeast Schizosaccharomyces pombe, was characterized, and its transcriptional regulation studied. Genomic DNA encoding Grx3 was isolated by PCR, and a plasmid pTT3 carrying this DNA was produced. The DNA sequence has 1,267 bp, which would encode a monothiol Grx of 166 amino acids with a molecular mass of 18.3 kDa. The putative protein has 27% homology with Grx5, and contains many hydrophobic amino acid residues in its N-terminal region. S. pombe cells harboring pTT3 had increased Grx activity and enhanced survival on minimal medium plates containing aluminum (5 mM), BSO (0.05 mM), menadione (0.01 mM) or cadmium (0.2 mM). The 568 bp upstream region of Grx3 was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp367R to generate fusion plasmid pMJS10. Potassium chloride (KCl) and metals including aluminum and cadmium enhanced the synthesis of beta-galactosidase from the fusion gene. The synthesis of beta-galactosidase was also enhanced, in a Pap1-dependent manner, by fermentable carbon sources such as glucose (at low concentrations) and sucrose, but not by non-fermentable carbon sources such as ethanol and acetate. Grx3 mRNA increased in response to treatment with BSO. These observations indicate that S. pombe Grx3 is involved in the response to stress, and is regulated by stress.

Up-regulation of glutathione biosynthesis in NIH3T3 cells transformed with the ETV6-NTRK3 gene fusion.

The ETV6-NTRK3 gene fusion, first identified in the chromosomal translocation in congenital fibrosarcoma, encodes a chimeric protein tyrosine kinase with potent transforming activity. ETV6-NTRK3-dependent transformation involves the joint action of NTRK3 signaling pathways, and aberrant cell cycle progression resulting from activation of Mek1 and Akt. The level of glutathione (GSH) was found to be markedly increased in ETV6-NTRK3-transformed NIH3T3 cells. The activities of the two GSH biosynthetic enzymes as well as of glutathione peroxidase, together with their mRNAs, were also higher in the transformed cells. The transformed cells were able to grow in the presence of GSH-depleting agents, whereas the control cells were not. L-Buthionine-(S,R)-sulfoximine (BSO) inhibited activation of Mek1 and Akt in the transformed NIH3T3 cells. These observations imply that up-regulation of GSH biosynthesis plays a central role in ETV6-NTRK3-induced transformation.