Human HaCaT Cells Research Articles

Inhibition of TNF-α/IFN-γ induced RANTES expression in HaCaT cell by naringin

Context: Naringin is a bioflavonoid derivative and is predominantly found in Citrus paradisi Macf., Citrus sinensis (Linn.) Osbeck, Citrus unshiu Marc., Citrus reticulata Blanco cv. Nobilis, Citrus tachibana (Makino) Tanaka, Citrus junos Sieb. ex Tanaka (Rutaceae), and related citrus species. It has anti-inflammatory effects that have been well-documented, but the mechanism is poorly characterized.Objective: The effect of naringin on production of RANTES (regulated upon activation normal T-cell expressed and secreted) in human HaCaT cells was investigated here for the first time.Materials and methods: The HaCaT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and the proliferation of cell was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The cells were divided into three groups including control group, tumor necrosis factor alpha (TNF-α)/interferon gamma (IFN-γ)-stimulated group, and naringin pretreatment group (first incubated in the presence of naringin and then exposed to TNF-α/IFN-γ). The concentration of RANTES in the supernatants was determined by enzyme-linked immunosorbent assay (ELISA). The expression of RANTES mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The expression of nuclear factor kappa B (NF-κB) P65 protein was detected with immunocytochemical method and western blot method.Results: Naringin hardly inhibits HaCaT cells growth at concentrations rising from 0.25 to 1 mmol/L. However, RANTES expression detected in supernatant stimulated with TNF-α/IFN-γ reduced 15 and 16%, respectively, when cultured with 0.25, 0.5 mmol/L naringin. Furthermore, 1 mmol/L naringin significantly decreased RANTES mRNA level. Finally, naringin decreased the expression of NF-κB P65 protein in nuclei.Discussion and conclusion: Naringin can inhibit the increased production of RANTES, which is partially via NF-κB-dependent signal pathway.

Involvement of P2Y6 Receptor in p38 MAPK-Mediated COX-2 Expression in Response to UVB Irradiation of Human Keratinocytes

Ultraviolet B (UVB) radiation induces inflammation in human skin. Extracellular nucleotides are released from cells in response to various stimuli and act as intercellular signaling molecules through activation of P2 receptors. In this study, we investigated the involvement of extracellular nucleotides and P2 receptors in UVB-radiation-induced inflammation using human keratinocyte-derived HaCaT cells. UVB radiation induced rapid ATP release from HaCaT cells; this was inhibited by pretreatment with anion transporter blockers or maxi-anion channel blockers. In addition, the radiation-induced activation of p38 MAPK was significantly blocked by pretreatment with ecto-nucleotidase (apyrase) or P2Y6 receptor antagonist (MRS2578). Expression of COX-2, mediated by activation of p38 MAPK, was also induced by UVB radiation. Both pretreatment with MRS2578 and knockdown of the P2Y6 receptor by siRNA transfection attenuated the induction of COX-2 in HaCaT cells exposed to UVB radiation. Our results indicate that UVB radiation evokes ATP release from human keratinocytes and also that activation of P2Y6 receptor mediates the UVB-radiation-induced activation of p38 MAPK and expression of COX-2. Thus P2Y6 receptor is a mediator of UVB-radiation-induced inflammatory responses in keratinocytes.

The peroxisome proliferator-activated receptor β/δ (PPARβ/δ) agonist GW501516 prevents TNF-α-induced NF-κB activation in human HaCaT cells by reducing p65 acetylation through AMPK and SIRT1

Nuclear factor (NF)-κB is a ubiquitously expressed transcription factor controlling the expression of numerous genes involved in inflammation. The aim of this study was to evaluate whether activation of the peroxisome proliferator-activated receptor (PPAR) β/δ prevented TNF-α-induced NF-κB activation in human HaCaT keratinocytes and, if so, to determine the mechanism involved. The PPARβ/δ agonist GW501516 inhibited the increase caused by TNF-α in the mRNA levels of the NF-κB target genes interleukin 8 (IL-8), TNF-α and thymic stromal lymphopoietin (TSLP). Likewise, GW501516 prevented the increase in NF-κB DNA-binding activity observed in cells exposed to TNF-α. The reduction in NF-κB activity following GW501516 treatment in cells stimulated with TNF-α did not involve either increased IκBα protein levels or a reduction in the translocation of the p65 subunit of NF-κB. In contrast, GW501516 treatment decreased TNF-α-induced p65 acetylation. Acetylation of p65 is mainly regulated by p300, a transcriptional co-activator that binds to and acetylates p65. Of note, AMP kinase (AMPK) activation phosphorylates p300 and reduces its binding to p65. GW501516 increased AMPK phosphorylation and the subsequent p300 phosphorylation, leading to a marked reduction in the association between p65 and this transcriptional co-activator. In addition, treatment with the PPARβ/δ agonist increased SIRT1 protein levels. Finally, the reduction in IL-8 mRNA levels following GW501516 treatment in TNF-α-stimulated cells was abolished in the presence of the PPARβ/δ antagonist GSK0660, the AMPK inhibitor compound C and the SIRT1 inhibitor sirtinol, indicating that the effects of GW501516 on NF-κB activity were dependent on PPARβ/δ, AMPK and SIRT1, respectively.

CCR10 and CCL27 are overexpressed in cutaneous squamous cell carcinoma

C–C chemokine receptor (CCR)10 is a specific receptor for chemokine ligand (CCL)27, a selective chemoattractant for skin-associated memory T cells to cutaneous sites. In melanoma, CCR10 increases the ability of neoplastic cells to grow, invade tissues, disseminate to lymph nodes, and escape the host immune responses. In this study, we investigated the expression of CCR10 and its ligand CCL27 in squamous cell carcinoma (SCC). CCR10 and CCL27 were expressed in SCC, actinic keratosis (AK), Bowen's disease, and seborrheic keratosis (predominantly prickle cell type), but not in seborrheic keratosis (predominantly basal cell type) and basal cell carcinoma. Furthermore, CCR10 and CCL27 were overexpressed in SCC relative to Bowen's disease, an early stage of SCC. Consistently, a human SCC cell line, A253 cells, and HaCaT cells exhibited CCL27 production that was strongly induced by tumor necrosis factor-α and interleukin-1β. Finally, A253 cells expressed stronger intracellular CCR10 compared to HaCaT cells by flow cytometry. These results suggest that CCR10 and CCL27 overexpression in SCC is related to the progression of SCC and is useful for the diagnosis of SCC.

IGF-IR Internalizes with Caveolin-1 and PTRF/Cavin in Hacat Cells

BackgroundInsulin-like growth factor-I receptor (IGF-IR) is a tyrosine kinase receptor (RTK) associated with caveolae, invaginations of the plasma membrane that regulate vesicular transport, endocytosis and intracellular signaling. IGF-IR internalization represents a key mechanism of down-modulation of receptors number on plasma membrane. IGF-IR interacts directly with Caveolin-1 (Cav-1), the most relevant protein of caveolae. Recently it has been demonstrated that the Polymerase I and Transcript Release Factor I (PTRF/Cavin) is required for caveolae biogenesis and function. The role of Cav-1 and PTRF/Cavin in IGF-IR internalization is still to be clarified.Methodology/Principal FindingsWe have investigated the interaction of IGF-IR with Cav-1 and PTRF/Cavin in the presence of IGF1in human Hacat cells. We show that IGF-IR internalization triggers Cav-1 and PTRF/Cavin translocation from plasma membrane to cytosol and increases IGF-IR interaction with these proteins. In fact, Cav-1 and PTRF/Cavin co-immunoprecipitate with IGF-IR during receptor internalization. We found a different time course of co-immunoprecipitation between IGF-IR and Cav-1 compared to IGF-IR and PTRF/Cavin. Cav-1 and PTRF/Cavin silencing by siRNA differently affect surface IGF-IR levels following IGF1 treatment: Cav-1 and PTRF/Cavin silencing significantly affect IGF-IR rate of internalization, while PTRF/Cavin silencing also decreases IGF-IR plasma membrane recovery. Since Cav-1 phosphorylation could have a role in IGF-IR internalization, the mutant Cav-1Y14F lacking Tyr14 was transfected. Cav-1Y14F transfected cells showed a reduced internalization of IGF-IR compared with cells expressing wild type Cav-1. Receptor internalization was not impaired by Clathrin silencing. These findings support a critical role of caveolae in IGF-IR intracellular traveling.Conclusions/SignificanceThese data indicate that Caveolae play a role in IGF-IR internalization. Based on these findings, Cav-1 and PTRF/Cavin could represent two relevant and distinct targets to modulate IGF-IR function.

Gefitinib-induced epidermal growth factor receptor-independent keratinocyte apoptosis is mediated by the JNK activation pathway

Gefitinib (ZD1839) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor with a significant antitumour effect on various cancers. Skin toxicity induced by gefitinib is common, and has been shown to be related to the inhibition of EGFR signalling pathways. However, other mechanisms may be involved in gefitinib-induced skin toxicity. To study the possible EGFR-independent mechanisms of gefitinib-induced skin toxicity. The human immortalized keratinocyte cell line HaCaT and human lung adenocarcinoma cell lines (A549 and PC9) were treated with different concentrations of gefitinib for 24, 48 and 72 h. Cell viability was measured by MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] after EGFR gene silencing. The signalling pathways were investigated by immunoblot analysis. Keratinocyte apoptosis was evaluated by nuclear condensation and flow cytometric analysis. Gefitinib maintained its cytotoxicity to HaCaT cells after EGFR gene silencing, indicating that an EGFR-independent mechanism exists. Increased phosphorylation of p38 mitogen-activated protein kinase and JNK by gefitinib was observed in a dose-dependent manner in HaCaT cells. The JNK inhibitor, SP600125, attenuated the gefitinib-induced cytotoxicity and apoptosis of HaCaT cells. Immunohistochemical examination of patient specimens showed an increased expression of phosphorylated JNK in lesional epidermis compared with nonlesional epidermis. Gefitinib can induce keratinocyte apoptosis through an EGFR-independent JNK activation pathway.

Sulforaphane suppresses TARC/CCL17 and MDC/CCL22 expression through heme oxygenase-1 and NF-κB in human keratinocytes

Sulforaphane (4-methylsulfinylbutyl isothiocyanate, SFN) from broccoli has been used a chemopreventive photochemical as detoxification of xenobiotics and anti-inflammatory, however, there is no studies for Th2 chemokine expression through heme oxygenase-1 and NF-κB in keratinocytes. Atopic dermatitis is a chronically relapsing pruritic inflammatory skin disease. SFN is demonstrated to have anti-inflammatory and anti-oxidant effects. This study aimed to define whether and how SFN regulates Th2-related chemokine production in human HaCaT keratinocytes. The level of chemokine expression was measured by reverse transcription polymerase chain reaction (RT-PCR) and signaling study was performed by Western blot analysis. Chemokine production was determined by enzyme-linked immunosorbent assay. Pretreatment with SFN suppressed interferon-γ (IFN-γ) and tumor necrosis factor (TNF)-α- induced thymus- and activation-regulated chemokine (TARC/CCL17) and macrophage-derived chemokine (MDC/CCL22) production in HaCaT keratinocytes. SFN inhibited IFN-γ and TNF-α-induced NF-κB activation as well as STAT1 activation. Interestingly, pretreatment with SFN result in significantly suppressed IFN-γ and TNF-α-induced TARC/CCL17 and MDC/CCL22 production through the induction of HO-1. This suppression was completely abolished by HO-1 siRNA. Furthermore, Carbon monoxide, but not other end products of HO-1 activity, also suppressed IFN-γ and TNF-α-induced TARC/CCL17 and MDC/CCL22 production. These results demonstrate that SFN has an inhibitory role in IFN-γ and TNF-α-induced production of TARC/CCL17 and MDC/CCL22 in human HaCaT cells by inhibition of NF-κB activation and induction of HO-1.

Activating transcription factor 3 (ATF3) expression is increased in erythema multiforme and is regulated by IFN‐γ in human keratinocytes

Activating transcription factor 3 (ATF3) expression is increased in erythema multiforme and is regulated by IFN-gamma in human keratinocytes. Experimental Dermatology 2010; 19: e310-e313. Abstract: Activating transcription factor 3 (ATF3) is a member of the ATF/cyclic AMP responsive element-binding protein (CREB) family of transcription factors and is involved in the regulation of immune responses, apoptosis, DNA repair and oncogenesis. The epidermal expression of ATF3 in the setting of cutaneous inflammation has not been well characterized. To examine the expression of ATF3 in the setting of inflammatory skin disease, ATF3 protein expression was analysed by immunohistochemistry (IHC). We found diffuse epidermal ATF3 protein expression in skin biopsies of erythema multiforme (EM). Given the role of interferon (IFN)-gamma in erythema multiforme, we sought to examine the impact of IFN-gamma on ATF3 expression in human keratinocytes. IFN-gamma induced ATF3 mRNA and protein in primary human keratinocytes and HaCaT cells. Thus, epidermal ATF3 expression can be increased in the setting of inflammatory skin diseases and is regulated by IFN-gamma in human keratinocytes.

Enhancement of P53-Mutant Human Colorectal Cancer Cells Radiosensitivity by Flavonoid Fisetin

The aim of this study was to investigate whether fisetin is a potential radiosensitizer for human colorectal cancer cells, which are relatively resistant to radiotherapy. Cell survival was examined by clonogenic survival assay, and DNA fragmentation was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The effects of treatments on cell cycle distribution and apoptosis were examined by flow cytometry. Western blot analysis was performed to ascertain the protein levels of gamma-H2AX, phospho-Chk2, active caspase-3, PARP cleavage, phospho-p38, phospho-AKT, and phospho-ERK1/2. Fisetin pretreatment enhanced the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells but not human keratocyte HaCaT cells; it also prolonged radiation-induced G(2)/M arrest, enhanced radiation-induced cell growth arrest in HT-29 cells, and suppressed radiation-induced phospho-H2AX (Ser-139) and phospho-Chk2 (Thr-68) in p53-mutant HT-29 cells. Pretreatment with fisetin enhanced radiation-induced caspase-dependent apoptosis in HT-29 cells. Fisetin pretreatment augmented radiation-induced phosphorylation of p38 mitogen-activated protein kinase, which is involved in caspase-mediated apoptosis, and SB202190 significantly reduced apoptosis and radiosensitivity in fisetin-pretreated HT-29 cells. By contrast, both phospho-AKT and phospho-ERK1/2, which are involved in cell proliferation and antiapoptotic pathways, were suppressed after irradiation combined with fisetin pretreatment. To our knowledge, this study is the first to provide evidence that fisetin exerts a radiosensitizing effect in p53-mutant HT-29 cells. Fisetin could potentially be developed as a novel radiosensitizer against radioresistant human cancer cells.

Amniotic membrane induces epithelialization in massive posttraumatic wounds

Large-surface or deep wounds often become senescent in the inflammatory or proliferation stages and cannot progress to reepithelialization. This failure makes intervention necessary to provide the final sealing epithelial layer. The best current treatment is autologous skin graft, although there are other choices such as allogenic or autologous skin substitutes and synthetic dressings. Amniotic membrane (AM) is a tissue of interest as a biological dressing due to its biological properties and immunologic characteristics. It has low immunogenicity and beneficial reepithelialization effects, with antiinflammatory, antifibrotic, antimicrobial, and nontumorigenic properties. These properties are related to its capacity to synthesize and release cytokines and growth factors. We report the use of AM as a wound dressing in two patients with large and deep traumatic wounds. Negative pressure wound therapy followed by AM application was capable of restoring skin integrity avoiding the need for skin graft reconstruction. AM induced the formation of a well-structured epidermis. To understand this effect, we designed some assays on human keratinocyte-derived HaCaT cells. AM treatment of HaCaT induced ERK1/2 and SAP/JNK kinases phosphorylation and c-jun expression, a gene critical for keratinocytes migration; however, it did not affect cell cycle distribution. These data suggest that AM substantially modifies the behavior of keratinocytes in chronic wounds, thereby allowing effective reepithelialization.

Vasoactive intestinal peptide induces vascular endothelial growth factor production in human HaCaT keratinocytes via MAPK pathway

Psoriasis is a chronic skin disease characterized by abnormal keratinocyte proliferation and differentiation, inflammation, and angiogenesis. Although dysfunction of the immune system is known to be an important factor in the pathogenesis of psoriasis, there is also strong evidence that psychological stresses are involved. Neuropeptides are thought to be main mediators of neurogenic inflammation, presumably involved in the pathogenesis of psoriasis. Vasoactive intestinal peptide (VIP) is one of the major neuropeptides in human and rodent skin. In the present study, we examined the effect and mechanism of VIP on vascular endothelial growth factor (VEGF) production by HaCaT cells which is a spontaneous, immortalized, human keratinocyte cell line. Our data indicate the mRNA and protein levels of VEGF by VIP were increased in a concentration-dependent manner. However, this increase was abrogated by pretreatment with an extracellular signal-regulated kinase (ERK) inhibitor PD98059 or p38MAPK inhibitor SB203580; pretreatment with c-Jun N-terminal kinase (JNK) inhibitor SP600125 did not attenuate the effects of VIP on the expression of VEGF. In addition, VIP treatment induced rapid phosphorylation of ERK1/2 and p38MAPK, and PD98059 and SB203580 were able to inhibit VIP-induced phosphorylation of ERK1/2 and p38MAPK, respectively. These results suggest that VIP increases the expression of VEGF through the ERK1/2 and p38MAPK signaling pathway in human HaCaT cells.

326 Targets of “angioprevention”: from inflammatory angiogenesis to hypoxia

The increasing amount of nanotechnological products, found in our environment and those applicable in engineering, material sciences and medicine has stimulated a growing interest in examining their long-term impact on genetic and epigenetic processes. We examined here the epigenomic response to nm-SiO2 particles in human HaCaT cells and methyltransferases (DNMTs) and DNA-binding domain proteins (MBDs) induced by nano-SiO2 particles. Nm-SiO2 treatment induced global hypoacetylation implying a global epigenomic response. The levels of DNMT1, DNMT3a and methyl-CpG binding protein 2 (MBD2) were also decreased in a dose dependent manner at mRNA and protein level. Epigenetic changes may have long-term effects on gene expression programming long after the initial signal has been removed, and if these changes remain undetected, it could lead to long-term untoward effects in biological systems. These studies suggest that nanoparticles could cause more subtle epigenetic changes which merit thorough examination of environmental nanoparticles and novel candidate nanomaterials for medical applications.

SiO 2 nanoparticles induce global genomic hypomethylation in HaCaT cells

The increasing amount of nanotechnological products, found in our environment and those applicable in engineering, material sciences and medicine has stimulated a growing interest in examining their long-term impact on genetic and epigenetic processes. We examined here the epigenomic response to nm-SiO 2 particles in human HaCaT cells and methyltransferases (DNMTs) and DNA-binding domain proteins (MBDs) induced by nano-SiO 2 particles. Nm-SiO 2 treatment induced global hypoacetylation implying a global epigenomic response. The levels of DNMT1, DNMT3a and methyl-CpG binding protein 2 (MBD2) were also decreased in a dose dependent manner at mRNA and protein level. Epigenetic changes may have long-term effects on gene expression programming long after the initial signal has been removed, and if these changes remain undetected, it could lead to long-term untoward effects in biological systems. These studies suggest that nanoparticles could cause more subtle epigenetic changes which merit thorough examination of environmental nanoparticles and novel candidate nanomaterials for medical applications.

Prenylated chalcone from Sophora flavescens suppresses Th2 chemokine expression induced by cytokines via heme oxygenase-1 in human keratinocytes

We recently reported the inhibitory effect of an oral administration of a Sophora flavescens Aiton methanol extract on the development of atopic dermatitis in the NC/Nga model mice. Heme oxygenase (HO)-1 has recently emerged as an important cytoprotective enzyme against oxidative stress and inflammatory responses in many cell types. The aim of this study was to investigate the possible mechanism by which prenylated chalcone (PC, 7,9,2',4'-tetrahydroxy-8-isopentenyl-5-methoxychalcone), a natural product isolated from S. flavescens, inhibited cytokine-induced Th2 chemokine expression in human keratinocytes, HaCaT cells. The level of chemokine expression was measured by reverse transcription-polymerase chain reaction and HO-1 study was performed by Western blot analysis. Interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha)-induced thymus- and activation-regulated chemokine (TARC/CCL17), macrophage-derived chemokine (MDC/CCL22), cutaneous T-cell attracting chemokine (CTACK/CCL27) expression in a dose-dependent manner. Interestingly, PC significantly suppressed IFN-gamma and TNF-alpha-induced TARC/CCL17, MDC/CCL22 and CTACK/CCL27 expression via the induction of heme oxygenase (HO)-1. This suppression was completely restored by HO-1 siRNA, suggesting a direct role of HO-1 for the suppressive effect. Furthermore, exogenous CO, but not other end products of HO-1 activity, also suppressed IFN-gamma and TNF-alpha-induced TARC/CCL17, MDC/CCL22 and CTACK/CCL27 expression. These results demonstrate that prenylated chalcone induces HO-1 expression, which in turn HO-1 and/or CO suppresses Th2 chemokine expressions induced by cytokines in human HaCaT cells.

Abstract 341: Effects of immunosuppressive cyclosporin A on epidermal keratinocytes

Abstract Non-melanoma skin cancer, the most common neoplasia after solid organ transplantation, causes serious morbidity and mortality and is related to sun exposure. Cyclosporin A (CsA) has been used widely to prevent rejection in organ transplantation. The mechanism of CsA action in causing cancer was thought to be well understood via immunosuppression. Here, we show that CsA promotes primary skin tumor growth in immune-deficient mice. CsA treatment at therapeutic plasma concentrations promotes growth and survival from removal of extracellular matrix or UVB radiation. CsA treatment enhances AKT activation following serum treatment and UVB radiation. Furthermore we found that PTEN expression, the negative regulator of AKT activation, is reduced significantly upon CsA treatment in human HaCaT and A431 cells and in mouse skin in vivo. Accordingly, the enhanced AKT activation in CsA-treated cells upon UVB radiation is observed in PTEN knockdown keratinocytes. CsA-induced PTEN suppression is critical for increased AKT activation. Inhibition of AKT activation abolishes CsA-promoted growth and survival, indicating that AKT hyperactivation is essential for the enhanced growth and survival of CsA-treated cells. In addition, mTOR signaling as a known AKT downstream target is required for CsA-enhanced growth and survival. Taken together, we have identified PTEN/AKT pathway as new molecular targets of CsA in epidermal keratinocytes, suggesting a previously unknown mechanism in CsA-enhanced skin carcinogenesis. Our findings challenge assumptions about how CsA-associated tumors arise in skin. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 341.

Calcium hypochlorite induces apoptosis in cultured human skin keratinocytes

Calcium hypochlorite compositions are primarily used for disinfectant and general sanitation purposes in swimming pools. It has not been shown that calcium hypochlorite induces apoptosis in human skin cells. Therefore, we undertook this study to investigate whether and how calcium hypochlorite induces skin cell apoptosis using human skin keratinocytes cell line HaCaT cells. After treatment of HaCaT cells with different doses of calcium hypochlorite (Pulsar® Plus Briquettes), cell proliferation was measured using an MTT Dye assay. Detection of specific proteins from apoptosis pathways were analyzed using Western blot at different time intervals. Increased calcium hypochlorite concentrations lead to increased apoptosis in cultured HaCaT cells. It is also shown that prolonged exposure to calcium hypochlorite result in an increased rate of apoptosis. Although it is known that calcium hypochlorite is an effective disinfectant in swimming pools, little is known about its effects on human skin cells. Our results thus suggest that prolonged exposure at specific concentrations of calcium hypochlorite lead to varying rates of apoptosis in human keratinocytes.

Immunosuppressive Cyclosporin A Activates AKT in Keratinocytes through PTEN Suppression: IMPLICATIONS IN SKIN CARCINOGENESIS

Non-melanoma skin cancer, the most common neoplasia after solid organ transplantation, causes serious morbidity and mortality and is related to sun exposure. Cyclosporin A (CsA) has been used widely to prevent rejection in organ transplantation. The mechanism of CsA action in causing cancer was thought to be well understood via immunosuppression. Here, we show that CsA promotes primary skin tumor growth in immune-deficient mice and keratinocyte growth in vitro. In addition, CsA enhances keratinocyte survival from removal of extracellular matrix or UVB radiation. At the molecular level, CsA increases AKT activation after serum treatment and UVB irradiation. Furthermore we found that expression of PTEN, the negative regulator of AKT activation, is significantly reduced post-CsA in human HaCaT and A431 cells and in mouse skin in vivo. CsA-induced PTEN down-regulation occurs at the transcription level and is epidermal growth factor receptor-dependent. Such PTEN suppression is required for increased AKT activation. Inhibition of AKT activation abolishes CsA-promoted growth and survival, indicating that AKT hyperactivation is essential for both growth and survival of CsA-treated cells. In addition, mTOR signaling as a known AKT downstream pathway is required for CsA-enhanced growth and survival. Taken together, we have identified the PTEN/AKT pathway as new molecular targets of CsA in epidermal keratinocytes, suggesting a previously unknown mechanism in CsA-enhanced skin carcinogenesis. Our findings challenge assumptions about how CsA-associated tumors arise in skin.

Cyclobutane pyrimidine dimers are photosensitised by carprofen plus UVA in human HaCaT cells

Every year in the UK about 75,000 cases of non-melanoma skin cancer (NMSC) are registered, and about 9500 people are diagnosed with cutaneous melanoma (CM). The main risk factor for these cancers is exposure to sunlight. The effects of light on skin are wavelength dependent, with wavelengths in the UVB waveband (280–315nm) being the most carcinogenic. UVB is directly absorbed by DNA, producing dimeric pyrimidine photoproducts including cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimodone photoproducts (6-4PP). However UVA (315–400nm) can also produce CPD, induce skin tumours in mice, and has been shown to be mutagenic in cell culture. Although the precise role of UVA in human skin cancer remains to be elucidated, it comprises the major portion of solar UV radiation, transmits through window glass and can be delivered in high doses from tanning lamps. Non-steroidal anti-inflammatory drugs (NSAIDs), in particular the 2-aryl propionic acid derivatives, are a well-documented group of photosensitising chemicals producing clinical phototoxic and photoallergic reactions. We have used carprofen, a model compound from this group to see if it could amplify the effects of UVA and contribute to the formation of CPD by UVA. Preliminary work has shown that carprofen combined with low doses of UVA (λmax: 365nm; 5J/cm2) can produce both strand breaks (SB) and CPD in human skin or blood cells. CPD were detected indirectly by both an immunofluorescence method and as T4 endonuclease V sensitive sites in the comet assay. These findings show that compounds other than fluoroquinolones and psoralen derivatives may contribute to CPD formation in skin cells in combination with UVA.

Epigallocatechin-3-gallate has an Anti-inflammatory Effect by Regulating Macrophage Migration Inhibitory Factor-induced T Helper Related Chemokines and Cytokines in Human HaCaT Cells

Epigallocatechin-3-gallate has an Anti-inflammatory Effect by Regulating Macrophage Migration Inhibitory Factor-induced T Helper Related Chemokines and Cytokines in Human HaCaT Cells

Identification of a Novel Family of Laminin N-terminal Alternate Splice Isoforms

The laminins are a family of heterotrimeric basement membrane proteins that play roles in cellular adhesion, migration, and tissue morphogenesis. Through in silico analysis of the laminin-encoding genes, we identified a novel family of alternate splice isoforms derived from the 5′-end of the LAMA3 and LAMA5 genes. These isoforms resemble the netrins in that they contain a laminin N-terminal domain followed by a short stretch of laminin-type epidermal growth factor-like repeats. We suggest the terms LaNt (laminin N terminus) α3 and LaNt α5, for the predicted protein products of these mRNAs. RT-PCR confirmed the presence of these transcripts at the mRNA level. Moreover, they exhibit differential, tissue-specific, expression profiles. To confirm the existence of LaNt α3 protein, we generated an antibody to a unique domain within the putative polypeptide. This antibody recognizes a protein at the predicted molecular mass of 64 kDa by immunoblotting. Furthermore, immunofluorescence analyses revealed a basement membrane staining in epithelial tissue for LaNt α3 and LaNt α3 localized along the substratum-associated surface of cultured keratinocytes. We have also tested the functionality LaNt α3 through RNAi-mediated knockdown. Keratinocytes exhibiting specific knockdown of LaNt α3 displayed impaired adhesion, stress resistance, and reduced ability to close scratch wounds in vitro.