Nicola Kouttab

SIRT1 confers protection against UVB- and H2O2-induced cell death via modulation of p53 and JNK in cultured skin keratinocytes

SIRT1 is a member of a highly conserved gene family (sirtuins) encoding nicotinamide adenine dinucleotide (NAD)+‐dependent deacetylases, originally found to deacetylate histones leading to increased DNA stability and prolonged survival in yeast and higher organisms, including mammals. SIRT1 has been found to function as a deacetylase for numerous protein targets involved in various cellular pathways, including stress responses, apoptosis and axonal degeneration. However, the role of SIRT1 in ultraviolet (UV) signalling pathways remains unknown. Using cell culture and Western blot analysis in this study we found that SIRT1 is expressed in cultured human skin keratinocytes. Both UV radiation and H2O2, two major inducers of skin cell damage, down‐regulate SIRT1 in a time‐ and dose‐dependent manner. We observed that reactive oxygen species‐mediated JNK activation is involved in this SIRT1 down‐regulation. SIRT1 activator, resveratrol, which has been considered as an important antioxidant, protects against UV‐ and H2O2‐induced cell death, whereas SIRT inhibitors such as sirtinol and nicotinamide enhance cell death. Activation of SIRT1 negatively regulates UV‐ and H2O2‐induced p53 acetylation, because nicotinamide and sirtinol as well as SIRT1 siRNA enhance UV‐ and H2O2‐induced p53 acetylation, whereas SIRT1 activator resveratrol inhibits it. We also found that SIRT1 is involved in UV‐induced AMP‐activated protein kinase (AMPK) and downstream acetyl‐CoA carboxylase (ACC), phosphofructose kinase‐2 (PFK‐2) phosphorylation. Collectively, our data provide new insights into understanding of the molecular mechanisms of UV‐induced skin aging, suggesting that SIRT1 activators such as resveratrol could serve as new anti‐skin aging agents.

EGFR-mediated expression of aquaporin-3 is involved in human skin fibroblast migration

AQP3 (aquaporin-3), known as an integral membrane channel in epidermal keratinocytes, facilitates water and glycerol movement into and out of the skin. Here, we demonstrate that AQP3 is also expressed in cultured human skin fibroblasts, which under normal wound healing processes migrate from surrounding tissues to close the wound. EGF (epidermal growth factor), which induced fibroblast migration, also induced AQP3 expression in a time- and dose-dependent manner. CuSO4 and NiCl2, previously known as AQP3 water transport inhibitors, as well as two other bivalent heavy metals Mn2+ and Co2+, inhibited EGF-induced cell migration in human skin fibroblasts. AQP3 knockdown by small interfering RNA inhibited EGF-induced AQP3 expression and cell migration. Furthermore, an EGFR (EGF receptor) kinase inhibitor, PD153035, blocked EGF-induced AQP3 expression and cell migration. MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase]/ERK inhibitor U0126 and PI3K (phosphoinositide 3-kinase) inhibitor LY294002 also inhibited EGF-induced AQP3 expression and cell migration. Collectively, our findings show for the first time that AQP3 is expressed in human skin fibroblasts and that EGF induces AQP3 expression via EGFR, PI3K and ERK signal transduction pathways. We have provided evidence for a novel role of AQP3 in human skin fibroblast cell migration, which occurs during normal wound healing.

Prediction of well-conserved HIV-1 ligands using a matrix-based algorithm, EpiMatrix

This preliminary study was undertaken to identify new human leucocyte antigens (HLA) ligands from human immunodeficiency virus type 1 (HIV-1) which are highly conserved across HIV-1 clades and which may serve to induce cross-reactive cytotoxic T lymphocytes (CTLs). EpiMatrix was used to predict putative ligands from HIV-1 for HLA-A2 and HLA-B27. Twenty-six peptides that were both likely to bind and also highly conserved across HIV-1 strains in the Los Alamos HIV sequence database were selected for binding assays using the T2 stabilization assay. Two peptides that were also highly likely to bind (for A2 and B27, as determined by EpiMatrix) and well conserved across HIV-1 strains, and had previously been described to bind in the published literature, were also selected to serve as positive controls for the assays. Ten new major histocompatibility complex (MHC) ligands were identified among the 26 study peptides. The control peptides bound, as expected. These data confirm that EpiMatrix can be used to screen HIV-1 protein sequences for highly conserved regions that are likely to bind to MHC and may prove to be highly conserved HIV-1 CTL epitopes.

Curcumin attenuates EGF-induced AQP3 up-regulation and cell migration in human ovarian cancer cells

ObjectiveAquaporin (AQP) water channels are expressed in high-grade tumor cells of different tissue origins. Based on the involvement of AQPs in angiogenesis and cell migration as well as our previous studies which show that AQP3 is involved in human skin fibroblasts cell migration, in this study, we investigated whether AQP3 is expressed in cultured human ovarian cancer cell line CaOV3 cells, and whether AQP3 expression in these cells enhances cell migration and metastatic potential.MethodsCultured CaOV3 cells were treated with EGF and/or various reagents and subjected to cell migration assay by phagokinetic track mobility assay or biochemical analysis for expression or activation of proteins by SDS-PAGE/Western blot analysis.ResultsIn this study, we demonstrate that AQP3 is expressed in CaOV3 cells. EGF induces CaOV3 migration and up-regulates AQP3 expression. EGF-induced cell migration is inhibited by specific AQP3 siRNA knockdown or AQP3 water transport inhibitor CuSO4 and NiCl2. We also find that curcumin, a well known anti-ovarian cancer drug, down-regulates AQP3 expression and reduces cell migration in CaOV3, and the effects of curcumin are mediated, at least in part, by its inhibitory effects on EGFR and downstream AKT/ERK activation.ConclusionsCollectively, our results provide evidence for AQP3-facilitated ovarian cancer cell migration, suggesting a novel function for AQP3 expression in high-grade tumors. The results that curcumin inhibits EGF-induced up-regulation of AQP3 and cell migration, provide a new explanation for the anticancer potential of curcumin.

Rearrangement in the Breakpoint Cluster Region and the Clinical Course in Philadelphia-Negative Chronic Myelogenous Leukemia

We have followed one patient with Philadelphia (Ph)-negative chronic myelogenous leukemia and identified an additional four patients from the literature who showed the rearrangement in the breakpoint cluster region (bcr) on chromosome 22 characteristic of Ph-positive chronic myelogenous leukemia. The clinical course of these five patients was similar to that of Ph-positive patients, with easily controlled leukocyte counts, a prolonged benign phase, and prolonged survival. Furthermore, we have shown, for the first time, that bcr rearrangement in Ph-negative chronic myelogenous leukemia can result in expression of the aberrant 210-kilodalton bcr-abl fusion protein, which has been strongly implicated in Ph-positive leukemogenesis. Research data pertaining to possible cytogenetic mechanisms leading to production of p210bcr-abl in the absence of the Ph chromosome are reviewed. Molecular analysis provides an important tool for classifying and predicting prognosis of some patients with Ph-negative chronic myelogenous leukemia.

Combined cytotoxic action of paclitaxel and ceramide against the human Tu138 head and neck squamous carcinoma cell line

Purpose: Paclitaxel, a chemotherapeutic agent used in the treatment of recalcitrant ovarian and breast as well as other neoplasms, is being investigated for the treatment of squamous cell carcinoma of the head and neck. Our previous studies have demonstrated that exogenously added ceramide enhances apoptosis in paclitaxel-exposed human leukemic cells. In this study, we showed that exogenous ceramide augmented paclitaxel-induced apoptosis in Tu138 cells in vitro when added simultaneously in combination with the paclitaxel. Methods: The combined cytotoxic effects of paclitaxel and ceramide exposure against Tu138 cells were assessed by an MTT dye assay, cell cycle analysis, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay, and isobologram analysis for synergistic activity. Results: The MTT dye assay results indicated augmentation of time- and concentration-dependent paclitaxel-mediated cell cytotoxicity by simultaneous ceramide treatment. Paclitaxel treatment of Tu138 cells also resulted in an accumulation of cells in the G2-M phase of the cell cycle. This paclitaxel-mediated G2-M phase accumulation decreased significantly with the addition of ceramide, indicating that combined paclitaxel/ceramide treatment resulted in the elimination of Tu138 cells from the S and/or G2-M phases of the cell cycle. Furthermore, ceramide enhancement of paclitaxel-mediated apoptosis was also detected by the TUNEL assay. Conclusion: Our results suggest that paclitaxel/ceramide combination therapy may be an attractive alternative to conventional methods of chemotherapy for head and neck cancer, and should be further explored.

All-trans retinoic acid attenuates ultraviolet radiation-induced down-regulation of aquaporin-3 and water permeability in human keratinocytes

One of the major characteristics of human skin photoaging induced by ultraviolet (UV) radiation is the dehydration of the skin. Water movement across plasma membrane occurs via diffusion through lipid bilayer and via aquaporins (AQPs). We find that UV induces aquaporin‐3 (AQP3) down‐regulation in human skin keratinocytes. MEK/ERK inhibitors PD98059 and U0126 inhibit UV‐induced down‐regulation of AQP3. Antioxidant N‐acetyl‐L‐cysteine or NAC blocks UV‐induced MEK/ERK activation and down‐regulation of AQP3. All‐trans retinoic acid or atRA, while alone inducing AQP3 expression, attenuates UV‐induced down‐regulation of AQP3 and water permeability. Using special inhibitors, we find that activation of EGFR and inhibition on ERK activation are involved in atRAs protective effects against UV‐induced AQP3 down‐regulation. Using specific AQP3s water transport inhibitors and siRNA knockdown, we observe that AQP3 is involved in cell migration and in vitro wound healing. UV‐induced AQP3 down‐regulation results in reduced water permeability, decreased cell migration, and delayed wound healing, which are attenuated by atRA pretreatment. We conclude that atRA protects against UV‐induced down‐regulation AQP3 and decrease in water permeability, reduction in cell migration and delayed in vitro wound healing via trans‐activation of EGFR and inhibition on ROS‐mediated MEK/ERK pathway. This novel finding provides evidence to support possible involvement of AQP3 in UV induced skin dehydration. J. Cell. Physiol. 215: 506–516, 2008.

Stored red blood cell supernatant facilitates thrombin generation.

BACKGROUND: Observational studies have reported that patients transfused with red blood cells (RBCs) have a worse clinical outcome than untransfused patients and that storage age of RBCs at the time of transfusion may be an independent predictor of this adverse clinical outcome.

Paclitaxel-induced apoptosis in Jurkat, a leukemic T cell line, is enhanced by ceramide

We hypothesized that the lipid second messenger, ceramide, and microtubule-directed chemotherapeutic agents might engage converging pathways in inducing apoptosis. Our studies demonstrated that simultaneous treatment of Jurkat cells with paclitaxel and ceramide enhanced paclitaxel-induced cell growth inhibition. Cell cycle analysis indicated a significant increase in the hypodiploid population over that observed with paclitaxel treatment alone. Morphologic evaluation and a TUNEL assay confirmed a dramatic increase in apoptosis in Jurkat cells treated with the combination of these two agents. This is the first demonstration that paclitaxel and ceramide interact in a supra-additive manner to decrease leukemic T-cell growth, suggesting a possible application of paclitaxel and ceramide in combination therapy.

ATP-sensitive potassium channel: A novel target for protection against UV-induced human skin cell damage

Ultraviolet radiation (UV) induces cell damages leading to skin photoaging and skin cancer. ATP‐sensitive potassium (KATP) channel openers (KCOs) have been shown to exert significant myocardial preservation and neuroprotection in vitro and in vivo, and yet the potential role of those KCOs in protection against UV‐induced skin cell damage is unknown. We investigated the effects of pinacidil and diazoxide, two classical KCOs, on UV‐induced cell death using cultured human keratinocytes (HaCat cells). Here, we demonstrated for the first time that Kir 6.1, Kir 6.2 and SUR2 subunits of KATP channels are functionally expressed in HaCaT cells and both non‐selective KATP channel opener pinacidil and mitoKATP (mitochondrial KATP) channel opener diazoxide attenuated UV‐induced keratinocytes cell death. The protective effects were abolished by both non‐selective KATP channel blocker glibenclamide and selective mitoKATP channel blocker 5‐hydroxydecanoate (5‐HD). Also, activation of KATP channel with pinacidil or diazoxide resulted in suppressive effects on UV‐induced MAPK activation and reactive oxygen species (ROS) production. Unexpectedly, we found that the level of intracellular ROS was slightly elevated in HaCaT cells when treated with pinacidil or diazoxide alone. Furthermore, UV‐induced mitochondrial membrane potential loss, cytochrome c release and ultimately apoptotic cell death were also inhibited by preconditioning with pinacidil and diazoxide, and their effects were reversed by glibenclamide and 5‐HD. Taken together, we contend that mitoKATP is likely to contribute the protection against UV‐induced keratinocytes cell damage. Our findings suggest that KATP openers such as pinacidil and diazoxide may be utilized to prevent from UV‐induced skin aging. J. Cell. Physiol. 212: 252–263, 2007.