MHY1485 ameliorates UV-induced skin cell damages via activating mTOR-Nrf2 signaling.

Ultra Violet (UV)-caused skin cell damage is a main cause of skin cancer. Here, we studied the activity of MHY1485, a mTOR activator, in UV-treated skin cells. In primary human skin keratinocytes, HaCaT keratinocytes and human skin fibroblasts, MHY1485 ameliorated UV-induced cell death and apoptosis. mTOR activation is required for MHY1485-induced above cytoprotective actions. mTOR kinase inhibitors (OSI-027, AZD-8055 and AZD-2014) or mTOR shRNA knockdown almost abolished MHY1485-induced cytoprotection. Further, MHY1485 treatment in skin cells activated mTOR downstream NF-E2-related factor 2 (Nrf2) signaling, causing Nrf2 Ser-40 phosphorylation, stabilization/upregulation and nuclear translocation, as well as mRNA expression of Nrf2-dictated genes. Contrarily, Nrf2 knockdown or S40T mutation almost nullified MHY1485-induced cytoprotection. MHY1485 suppressed UV-induced reactive oxygen species production and DNA single strand breaks in skin keratinocytes and fibroblasts. Together, we conclude that MHY1485 inhibits UV-induced skin cell damages via activating mTOR-Nrf2 signaling.

Analysis of debrided and non-debrided invasive squamous cell carcinoma skin lesions by in vivo reflectance confocal microscopy before and after therapy.

Hyperkeratosis hinders the application of reflectance confocal microscopy (RCM) to image squamous cell carcinoma (SCC). Not all lesions with SCC show hyperkeratosis, and these lesions can be directly imaged. However, lesions with hyperkeratosis can be treated by debriding the hyperkeratotic surface for further imaging. RCM was used to investigate patients with suspected SCC. Lesions without obvious keratosis underwent direct RCM examinations. Lesions with obvious keratosis were treated by debriding the hyperkeratotic surface. The following main RCM criteria were used to diagnose invasive SCC: atypical keratinocytes arranged in nests, islands, and disarrangement patterns; an atypical honeycomb pattern; the absence of a cobblestone pattern; and non-edged dermal papillae. Other characteristics of invasive SCC observed by confocal microscopy included keratin pearl structures, hyperkeratosis, and inflammatory cell infiltration. During the follow-up period after treatment, both the cobblestone pattern and edged dermal papillae were as important as the typical honeycomb pattern in suggesting a normal skin structure. Our findings indicate RCM is a valuable tool to noninvasively examine the histology of invasive SCC before and after therapy.

Gremlin inhibits UV-induced skin cell damages via activating VEGFR2-Nrf2 signaling.

Ultra Violet (UV) radiation induces reactive oxygen species (ROS) production, DNA oxidation and single strand breaks (SSBs), which will eventually lead to skin cell damages or even skin cancer. Here, we tested the potential activity of gremlin, a novel vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) agonist, against UV-induced skin cell damages. We show that gremlin activated VEGFR2 and significantly inhibited UV-induced death and apoptosis of skin keratinocytes and fibroblasts. Pharmacological inhibition or shRNA-mediated knockdown of VEGFR2 almost abolished gremlin-mediated cytoprotection against UV in the skin cells. Further studies showed that gremlin activated VEGFR2 downstream NF-E2-related factor 2 (Nrf2) signaling, which appeared required for subsequent skin cell protection. Nrf2 shRNA knockdown or S40T dominant negative mutation largely inhibited gremlin-mediated skin cell protection against UV. At last, we show that gremlin dramatically inhibited UV-induced ROS production and DNA SSB formation in skin keratinocytes and fibroblasts. We conclude that gremlin protects skin cells from UV damages via activating VEGFR2-Nrf2 signaling. Gremlin could be further tested as a novel anti-UV skin protectant.

An investigation of crosstalk between Wnt/ß-catenin and transforming growth factor-ß signaling in androgenetic alopecia.

BACKGROUND: Wnt and transforming growth factor-ß (TGF-ß) signaling pathways are known to be involved in the pathogenesis of androgenetic alopecia (AGA). However, the way that Wnt and TGF-ß signaling is altered in patients with AGA and whether there exists a crosstalk between them in pathogenetic process of AGA remain unclear. OBJECTIVES: To investigate the expression of Wnt and TGF-ß signaling and the crosstalk between these 2 signaling pathways in AGA. METHODS: Fifteen male patients with AGA were recruited for our research. Fifteen scalp specimens of the balding were collected from frontal areas, and 9 nonbalding were collected from occipital areas. We analyzed the expression and activation of downstream Wnt and TGF-ß signaling molecules in both balding and nonbalding hair follicles isolated from scalp specimens. Furthermore, we evaluated the activation of Wnt and TGF-ß signaling after either of them was blocked with the inhibitor in balding and nonbalding dermal papilla (DP) cells. RESULTS: Compared with the nonbalding counterparts, the mRNA level of Wnt10a and LEF1 was decreased. But TßRI and TßRII, and the protein expression of TGF-ß1 was elevated in balding hair follicles. To investigate the crosstalk between Wnt and TGF-ß signaling, we used SB431542 to inhibit the TGF-ß signaling in balding DP cells and found that SB431542 significantly attenuated the phosphorylation of Smad2 and Akt. However, the mRNA level of Wnt10a, LEF1, and the nuclear translocation of ß-catenin was increased. On the other hand, we suppressed the Wnt signaling by XAV939 in nonbalding DP cells, which displayed that the level of ß-catenin and LEF1 was significantly inhibited; however, the level of active TGF-ß1 and the phosphorylation of Smad2 and Akt were up-regulated. CONCLUSIONS: These data indicate that crosstalk between Wnt/ß-catenin and TGF-ß signaling pathways may exist as one of the important mechanisms contributing to AGA.

Real-time in vivo confocal laser scanning microscopy of melanin-containing cells: A promising diagnostic intervention.

The use of noninvasive imaging techniques to evaluate different types of skin lesions is increasing popular. In vivo confocal laser scanning microscopy (CLSM) is a new method for high resolution non-invasive imaging of intact skin in situ and in vivo. Although many studies have investigated melanin-containing cells in lesions by in vivo CLSM, few studies have systematically characterized melanin-containing cells based on their morphology, size, arrangement, density, borders, and brightness. In this study, the characteristics of melanin-containing cells were further investigated by in vivo CLSM. A total of 130 lesions, including common nevi, giant congenital pigmented nevi, vitiligo, melasma, melanoma, and chronic eczema, were imaged by in vivo CLSM. This research helps dermatologists understand the characteristics of melanin-containing cells and facilitate the clinical application of melanin-containing cells in the investigation of dermatological disease. In summary, melanin-containing cells include keratinocytes, melanocytes, macrophages, and melanocytic skin tumor cells. Our study presents the CLSM characteristics of melanin-containing cells to potentially facilitate in vivo diagnosis based on shape, size, arrangement, density, borders, and brightness.

HMGB1-mediated autophagy modulates sensitivity of colorectal cancer cells to oxaliplatin via MEK/ERK signaling pathway.

In the present study, we examined the mechanisms of oxaliplatin-induced drug resistance in human colorectal cancer cell lines HT29 and HCT116. Our results demonstrate a significant autophagy expression in CRC cells after an oxaliplatin treatment. Administration of oxaliplatin to human CRC cells significantly enhanced the expression of HMGB1, which regulated the autophagy response and negatively regulate the cell apoptosis. Moreover, a decreased oxaliplatin -induced autophagy response and an increased apoptosis level were detected in stable CRC cells harboring HMGB1 shRNA. Then we noted that HMGB1 significantly induced extracellular signal-regulated kinase (ERK)/Extracellular signal-regulated kinase kinase (MEK) phosphorylation. Taken together, these data suggest that HMGB1-mediated autophagy modulates sensitivity of colorectal cancer cells to oxaliplatin via MEK/ERK signaling pathway.

Astragaloside IV controls collagen reduction in photoaging skin by improving transforming growth factor-ß/Smad signaling suppression and inhibiting matrix metalloproteinase-1.

Exposure to ultraviolet (UV) light reduces levels of type I collagen in the dermis and results in human skin damage and premature skin aging (photoaging). This leads to a wrinkled appearance through the inhibition of transforming growth factor­ß (TGF­ß)/Smad signaling. UV irradiation increases type I collagen degradation through upregulating matrix metalloproteinase (MMP) expression. Astragaloside IV (AST) is one of the major active components extracted from Astragalus membranaceus. However, its multiple anti­photoaging effects remain to be elucidated. In the present study, the effects of AST against collagen reduction in UV­induced skin aging in human skin fibroblasts were investigated. The expression of type I procollagen (COL1), MMP­1, TGF­ßRⅡ and Smad7 were determined using reverse transcription­polymerase chain reaction, western blotting and ELISA, respectively. UV irradiation inhibits type I collagen production by suppressing the TGF­ß/Smad signaling pathway and increasing COL1 degradation by inducing MMP­1 expression. Transforming growth factor­ß type II protein and COL1 mRNA decreased but MMP­1 and Smad7 levels increased in the photoaging model group, which was reversed by topical application of AST. AST prevents collagen reduction from UV irradiation in photoaging skin by improving TGF­ß/Smad signaling suppression and inhibiting MMP­1, thus AST may be a potential agent against skin photoaging.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-SIN1 association mediates ultraviolet B (UVB)-induced Akt Ser-473 phosphorylation and skin cell survival.

BACKGROUND: The exposure of skin keratinocytes to Ultraviolet (UV) irradiation leads to Akt phosphorylation at Ser-473, which is important for the carcinogenic effects of excessive sun exposure. The present study investigated the underlying mechanism of Akt Ser-473 phosphorylation by UVB radiation. RESULTS: We found that DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and mammalian target of rapamycin (mTOR) complex 2 (mTORC2) were both required for UVB-induced Akt Ser-473 phosphorylation in keratinocytes. Inhibition of DNA-PKcs activity via its inhibitor NU7026, a dominant-negative kinase-dead mutation, RNA interference (RNAi) or gene depletion led to the attenuation of UVB-induced Akt Ser-473 phosphorylation. Meanwhile, siRNA silencing or gene depletion of SIN1, a key component of mTORC2, abolished Akt Ser-473 phosphorylation by UVB. Significantly, we discovered that DNA-PKcs was associated with SIN1 in cytosol upon UVB radiation, and this complexation appeared required for Akt Ser-473 phosphorylation. Meanwhile, this DNA-PKcs-SIN1 complexation by UVB was dependent on epidermal growth factor receptor (EGFR) activation, and was disrupted by an EGFR inhibitor (AG1478) or by EGFR depletion. UVB-induced complexation between DNA-PKcs and mTORC2 components was also abolished by NU7026 and DNA-PKcs mutation. Finally, we found that both DNA-PKcs and SIN1 were associated with apoptosis resistance of UVB radiation, and inhibition of them by NU7026 or genetic depletion significantly enhanced UVB-induced cell death and apoptosis. CONCLUSION: Taken together, these results strongly suggest that DNA-PKcs-mTORC2 association is required for UVB-induced Akt Ser-473 phosphorylation and cell survival, and might be important for tumor cell transformation.

Deguelin induces both apoptosis and autophagy in cultured head and neck squamous cell carcinoma cells.

Head and neck squamous cell carcinoma (HNSCC) represents more than 5% of all cancers diagnosed annually in United States and around the world. Despite advances in the management of patients with this disease, the survival has not been significantly improved, and the search for potential alternative therapies is encouraging. Here we demonstrate that deguelin administration causes a significant HNSCC cell death. Deguelin induces both cell apoptosis and autophagy by modulating multiple signaling pathways in cultured HNSCC cells. Deguelin inhibits Akt signaling, and down-regulates survivin and cyclin-dependent kinase 4 (Cdk4) expressions, by disrupting their association with heat shock protein-90 (Hsp-90). Deguelin induces ceramide production through de novo synthase pathway to promote HNSCC cell death. Importantly, increased ceramide level activates AMP-activated protein kinase (AMPK), which then directly phosphorylates Ulk1 and eventually leads to cell autophagy. We found that a low dose of deguelin sensitized HNSCC cells to 5-FU. Finally, using a nude mice Hep-2 xenograft model, we also showed a significant anti-tumor ability of deguelin in vivo. Together, we suggest that deguelin may represent a novel and effective chemo-agent against HNSCC.

Ultra-violet B (UVB)-induced skin cell death occurs through a cyclophilin D intrinsic signaling pathway.

UVB-induced skin cell damage involves the opening of mitochondrial permeability transition pore (mPTP), which leads to both apoptotic and necrotic cell death. Cyclophilin D (Cyp-D) translocation to the inner membrane of mitochondrion acts as a key component to open the mPTP. Our Western-Blot results in primary cultured human skin keratinocytes and in HaCaT cell line demonstrated that UVB radiation and hydrogen peroxide (H(2)O(2)) induced Cyp-D expression, which was inhibited by anti-oxidant N-acetyl cysteine (NAC). We created a stable Cyp-D deficiency skin keratinocytes by expressing Cyp-D-shRNA through lentiviral infection. Cyp-D-deficient cells were significantly less susceptible than their counterparts to UVB- or H(2)O(2)-induced cell death. Further, cyclosporine A (Cs-A), a Cyp-D inhibitor, inhibited UVB- or H(2)O(2)-induced keratinocytes cell death. Reversely, over-expression of Cyp-D in primary keratinocytes caused spontaneous keratinocytes cell death. These results suggest Cyp-D's critical role in UVB/oxidative stress-induced skin cell death.

Sphingosine kinase-1 inhibition sensitizes curcumin-induced growth inhibition and apoptosis in ovarian cancer cells.

Recent published studies suggest that increasing levels of ceramides enhance the chemo-sensitivity of curcumin. Using in vitro approaches, we analyzed the impact of sphingosine kinase-1 (SphK-1) inhibition on ceramide production, and evaluated SphK1 inhibitor II (SKI-II) as a potential curcumin chemo-sensitizer in ovarian cancer cells. We found that SphK1 is overexpressed in ovarian cancer patients' tumor tissues and in cultured ovarian cancer cell lines. Inhibition of SphK1 by SKI-II or by RNA interference (RNAi) knockdown dramatically enhanced curcumin-induced apoptosis and growth inhibition in ovarian cancer cells. SKI-II facilitated curcumin-induced ceramide production, p38 activation and Akt inhibition. Inhibition of p38 by the pharmacological inhibitor (SB 203580), a dominant-negative expression vector, or by RNAi diminished curcumin and SKI-II co-administration-induced ovarian cancer cell apoptosis. In addition, restoring Akt activation introducing a constitutively active Akt, or inhibiting ceramide production by fumonisin B1 also inhibited the curcumin plus SKI-II co-administration-induced in vitro anti-ovarian cancer effect, suggesting that ceramide accumulation, p38 activation and Akt inhibition are downstream effectors. Our findings suggest that low, well-tolerated doses of SKI-II may offer significant improvement to the clinical curcumin treatment of ovarian cancer.

Perifosine sensitizes UVB-induced apoptosis in skin cells: new implication of skin cancer prevention?

We demonstrate here that a relative low dose of perifosine significantly enhanced UVB-induced apoptosis in skin cells (keratinocytes and fibroblasts), associated with a significant increase of reactive oxygen species (ROS) and ceramide production as well as multiple perturbations of diverse cell signaling pathways, shifting to a significant pro-apoptosis outcomes. Perifosine inhibited UVB-induced pro-survival Akt/mammalian target of rapamycin (mTOR) and ERK activation, while facilitating pro-apoptotic AMP-activated protein kinas (AMPK), c-Jun-NH(2)-kinase (JNK), and p53 activation; these signaling changes together promoted a striking increase in skin cell apoptosis and a significantly reduced amount of DNA damages. Our results suggest that perifosine may represent a novel skin cancer prevention strategy.

Increasing ceramides sensitizes genistein-induced melanoma cell apoptosis and growth inhibition.

The aim of the current study is to investigate the effect of ceramides on genistein-induced anti-melanoma effects in vitro. We found that exogenously added cell-permeable short-chain ceramides (C6) dramatically enhanced genistein-induced growth inhibition and apoptosis in cultured melanoma cells. Genistein treatment only induced a moderate intracellular ceramides accumulation in B16 melanoma cells. Two different agents including 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a ceramide glucosylation inhibitor, and the sphingosine kinase-1 (SphK1) inhibitor II (SKI-II), a sphingosine (ceramides precursor) phosphorylation inhibitor, both facilitated genistein-induced ceramides accumulation and melanoma cell apoptosis. Co-administration of ceramide (C6) and genistein induced a significant Akt inhibition and c-jun-NH(2)-kinase (JNK) activation, caspase-3 cleavage and cytochrome c release. Caspase-3 inhibitor z-DVED-fmk, JNK inhibitor SP 600125, or to restore Akt activation by introducing a constitutively active form of Akt (CA-Akt) diminished ceramide (C6) and genistein co-administration-induced in vitro anti-melanoma effect. Our study suggests that increasing cellular level of ceramides may sensitize genistein-induced anti-melanoma effects.

Protective effect of astragaloside IV against matrix metalloproteinase-1 expression in ultraviolet-irradiated human dermal fibroblasts.

Ultraviolet (UV) irradiation induces skin photoaging associated with up-regulated matrix metalloproteinase (MMP) expression. Inhibition of MMP expression is suggested to alleviate photoaging induced by UV irradiation. Astragaloside IV (As-IV), one of the main active ingredients of Astragalus membranaceus (Fisch) Bge, has been reported to have various biological activities. However, its anti-photoaging effect has not been examined to date. In the present study, we observed the effect of As-IV on matrix metalloproteinase-1 (MMP-1) expression in UV-irradiated human dermal fibroblasts (HDFs). We found that treatment with As-IV significantly decreased UV-induced MMP-1 expression at the messenger RNA and protein levels. In addition, western blotting analysis revealed that As-IV concentration-dependently suppressed UV-induced phosphorylation of extracellular-regulated protein kinase, Jun-N-terminal kinase and p38 mitogen-activated protein kinase (MAPK). Furthermore, treatment with As-IV markedly inhibited UV-induced nuclear factor kappaB (NF-κB) activity. These results suggest that As-IV down-regulates UV-induced MMP-1 expression, perhaps through suppression of MAPK and NF-κB activation in HDFs. As-IV is thus a potential agent for the management of skin photoaging.

Regiodivergent annulation of alkynyl indoles to construct spiro-pseudoindoxyl and tetrahydro-ß-carbolines.

Regiodivergent annulations of 3-phenoxy alkynyl indoles have been developed and tuned by protective groups through gold catalysis. With electron-donating protective groups, the substrate followed a C3-selective annulation and gave structurally interesting tetrahydro-ß-carboline derivatives possessing potential bioactivity. Using electron-withdrawing protective groups, the substrate underwent a C2-selective annulation and afforded the structurally useful spiro-pseudoindoxyl found in natural indole alkaloids. Notably, an interesting and unusual 1, 2-migration of the phenoxy group was found in the C3-selective process.

Increased MAPK and NF-κB expression of Langerhans cells is dependent on TLR2 and TLR4, and increased IRF-3 expression is partially dependent on TLR4 following UV exposure.

Toll-like receptors (TLRs) and epidermal Langerhans cells (LCs) play a crucial role in innate and adaptive immunity. To date, the pattern of TLR expression has not been fully analyzed. The effects of ultraviolet (UV) light on TLR expression and the downstream signaling cascades of human LC have not been examined. In this study, we purified human epidermal LCs using a density gradient centrifugation method and an immunomagnetic microbead method. We found that cultured purified LCs from human skin express mRNAs encoding TLR2, TLR4, TLR5 and TLR7-9. The expression of TLR2 and TLR4 protein was confirmed by Western blot analysis. The results showed for the first time that UV exposure up-regulated the mRNA and protein expression of TLR2 and TLR4 in human LCs. We also found that UV exposure-induced up-regulated MAPK and NF-κB/p65 expression was dependent on TLR2 and TLR4, and up-regulated IRF-3 expression was partially dependent on TLR4. In conclusion, UV light up-regulates the expression of TLR2, TLR4 and downstream signaling molecules MAPK, NF-κB/p65 and IRF-3 in human LCs. This suggests that UV light has a significant effect on skin immune responses.

IPL irradiation rejuvenates skin collagen via the bidirectional regulation of MMP-1 and TGF-ß1 mediated by MAPKs in fibroblasts.

The efficacy of intense pulsed light (IPL) in remodeling the extracellular matrix of aged skin had been proven by an increasing number of clinical trials. However, because of the lack of research about the underlying molecular and signaling mechanisms, its efficiency had not been accepted universally. A potential mechanism of IPL rejuvenation effects is due to its different effects on diverse cytokines, the impact of IPL on them may determine the phenotype and prognosis of the aged skin. We designed this study to evaluate the impact of IPL on the secretion of matrix metalloproteinase-1 (MMP-1), transforming growth factor-ß1 (TGF-ß1), and the mitogen-activated protein kinase (MAPK) signaling pathway in human skin fibroblasts, and tried to study the respective functions of MAPKs as mediators of the MMP-1, TGF-ß1 secretion. Results showed that the MMP-1 secretion was only enhanced by IPL at 10 J/cm(2); while the TGF-ß1 secretion was inhibited by IPL when the fluence was below 36 J/cm(2), but enhanced at 72 J/cm(2). Meanwhile, ERK inhibitor PD98059 decreased MMP-1 secretion, but did not show a significant influence on TGF-ß1; JNK inhibitor SP600125 increased the secretion of MMP-1 and decreased the TGF-ß1 secretion; P38 inhibitor SB203580 had no significant influence on MMP-1 but increased the secretion of TGF-ß1. Our findings indicated that the bidirectional influence of IPL on the secretion of MMP-1 and TGF-ß1 is a potential mechanism of its skin rejuvenation effect; and the secretion of these two cytokines can be mediated by MAPKs.

Trans-Zeatin attenuates ultraviolet induced down-regulation of aquaporin-3 in cultured human skin keratinocytes.

Solar ultraviolet (UV) irradiation is one of the most significant extrinsic factors contributing to skin photoaging. One major characteristic of photoaging induced by UV is water loss of the skin. Water movement across the plasma membrane can occur via two pathways: by diffusion through the lipid bilayer and by membrane-inserted water channels (aquaporins). In this study we demonstrate that UV induces aquaporin-3 (AQP3) downregulation in cultured keratinocytes (HaCaT cells). PD98059 and U0126, MEK/ERK inhibitors, inhibit UV-induced AQP3 loss. Trans-Zeatin (tZ), which alone induces AQP3 expression, attenuates UV-induced loss of AQP3. We found that tZ inhibits UV-induced MEK/ERK activation; the latter serves as the key signal pathway mediating UV-induced AQP3 loss. Using specific AQP3 siRNA knockdown, we found AQP3 is involved in wound healing in human skin keratinocytes. Loss-of-AQP3-mediated delayed wound healing in UV-radiated skin keratinocytes is attenuated by tZ pretreatment. tZ pretreatment also attenuates UV-induced decreased water permeability in HaCaT cells. We concluded that UV radiation downregulates AQP3 in HaCaT cells. MEK/ERK activation is involved in this process. tZ treatment attenuates UV-induced AQP3 loss, in vitro wound healing delay and water permeability decrease. This work provides a new explanation for the anti-photoaging potential of tZ.