Increased Occurrence of Cutaneous Leiomyomas and Dermatofibromas in Patients with Uterine Leiomyomas without Fumarate Hydratase Gene Mutations.

Leiomyomas are smooth muscle-derived benign neoplasms that can affect all organs, most frequently in the uterus. Fumarate hydratase gene (FH) mutation is characterised by an autosomal dominant disease with increased occurrence of renal tumours, but also by cutaneous (CLs) and uterine leiomyomas (ULs). So far, an increased occurrence of skin tumours in non-mutated patients with ULs has not been verified. To this aim, a case-group of women who were FH non-mutated patients surgically treated for ULs (n = 34) was compared with a control-group (n = 37) of consecutive age-matched healthy women. The occurrence of skin neoplasms, including CLs and dermatofibromas (DFs), was evaluated. Moreover, the microscopic features of FH non-mutated skin tumours were compared with those of an age-matched population group (n = 70) who presented, in their clinical history, only one type of skin tumour and no ULs. Immunohistochemical and in vitro studies analysed TGFß and vitamin D receptor expression. FH non-mutated patients with ULs displayed a higher occurrence of CLs and DFs (p < 0.03 and p < 0.001), but not of other types of skin tumours. Immunohistochemistry revealed a lower vitamin D receptor (VDR) expression in CLs and DFs from the ULs group compared with those from the population group (p < 0.01), but a similar distribution of TGFß-receptors and SMAD3. In vitro studies documented that TGFß-1 treatment and vitamin D3 have opposite effects on α-SMA, TGFßR2 and VDR expression on dermal fibroblast and leiomyoma cell cultures. This unreported increased occurrence of CLs and DFs in FH non-mutated patients with symptomatic ULs with vitamin D deficiency suggests a potential pathogenetic role of vitamin D bioavailability also for CLs and DFs.

Cellular retinol binding protein 1 transfection reduces proliferation and AKT-related gene expression in H460 non-small lung cancer cells.

In recent years, new treatments with novel action mechanisms have been explored for advanced non-small cell lung cancer (NSCLC). Retinoids promote cancer cell differentiation and death and their trafficking and action is mediated from specific cytoplasmic and nuclear receptors, respectively. The purpose of this study was to investigate the effect of Cellular retinol binding protein-1 (CRBP-1) transfection in H460 human NSCLC cell line, normally not expressing CRBP-1. H460 cells were transfected by using a vector pTargeT Mammalian expression system carrying the whole sequence of CRBP-1 gene. For proliferation and apoptosis studies, cells were treated with different concentrations of all-trans Retinoic Acid (atRA) and retinol. AKT-related gene expression was analyzed by using western blot and Signosis array and results analysed by one-way analysis of variance (ANOVA) or by t-student test. CRBP-1+ showed reduced proliferation and viability in basal condition and after atRA treatment when compared to empty-transfected H460 cells. Reduced proliferation in CRBP-1+ H460 cells associated to the down-regulation of pAKT/pERK/pEGFR-related genes. In particular, gene array documented the down-regulation of AKT and Stat-3-related genes, including M-Tor, Akt1, Akt2, Akt3, Foxo1, p27, Jun. Restoration of CRBP-1 expression in H460 cells reduced proliferation and viability in both basal condition and after atRA treatment, likely by down-regulating AKT-related gene level. Further studies are needed to better clarify how those CRBP-1-related intracellular pathways contribute to counteract NSCLC progression in order to suggest a potential tool to improve efficacy of retinoid anti lung cancer adjuvant therapy.

Clusterin exerts a cytoprotective and antioxidant effect in human osteoarthritic cartilage.

Osteoarthritis (OA) is the most common joint disease characterized by destruction of articular cartilage. OA-induced cartilage degeneration causes inflammation, oxidative stress and the hypertrophic shift of quiescent chondrocytes. Clusterin (CLU) is a ubiquitous glycoprotein implicated in many cellular processes and its upregulation has been recently reported in OA cartilage. However, the specific role of CLU in OA cartilage injury has not been investigated yet. We analyzed CLU expression in human articular cartilage in vivo and in cartilage-derived chondrocytes in vitro. CLU knockdown in OA chondrocytes was also performed and its effect on proliferation, hypertrophic phenotype, apoptosis, inflammation and oxidative stress was investigated. CLU expression was upregulated in human OA cartilage and in cultured OA cartilage-derived chondrocytes compared with control group. CLU knockdown reduced cell proliferation and increased hypertrophic phenotype as well as apoptotic death. CLU-silenced OA chondrocytes showed higher MMP13 and COL10A1 as well as greater TNF-α, Nox4 and ROS levels. Our results indicate a possible cytoprotective role of CLU in OA chondrocytes promoting cell survival by its anti-apoptotic, anti-inflammatory and antioxidant properties and counteracting the hypertrophic phenotypic shift. Further studies are needed to deepen the role of CLU in order to identify a new potential therapeutic target for OA.

Cellular retinoic acid binding protein-II expression and its potential role in skin aging.

Skin aging is an intricate biological process consisting of intrinsic and extrinsic alterations of epidermal and dermal structures. Retinoids play an important role in epidermal cell growth and differentiation and are beneficial to counteract skin aging. Cellular retinoic acid binding protein-II (CRABP-II) selectively binds all trans-retinoic acid, the most active retinoid metabolite, contributing to regulate intracytoplasmic retinoid trafficking and keratinocyte differentiation. Immunohistochemistry revealed a reduced epidermal and dermal CRABP-II expression in aged human and mouse skin. To better clarify the role of CRABP-II, we investigated age-related skin changes in CRABP-II knock-out mice. We documented an early reduction of keratinocyte layers, proliferation and differentiation rate, dermal and hypodermal thickness, pilosebaceous units and dermal vascularity in CRABP-II knock-out compared with wild-type mice. Ultrastructural investigation documented reduced number and secretion of epidermal lamellar bodies in CRABP-II knock-out compared with wild-type mice. Cultured CRABP-II knock-out-derived dermal fibroblasts proliferated less and showed reduced levels of TGF-ß signal-related genes, Col1A1, Col1A2, and increased MMP2 transcripts compared with those from wild-type. Our data strongly support the hypothesis that a reduction of CRABP-II expression accelerates and promotes skin aging, and suggest CRABP-II as a novel target to improve the efficacy of retinoid-mediated anti-aging therapies.

Expression and potential role of cellular retinol binding protein I in psoriasis.

Psoriasis is a diffuse chronic skin disorder characterized from accelerated epidermal turnover and inflammatory cell infiltrate. Retinoids influence keratinocyte proliferation and differentiation as well as inflammatory response. Cellular retinol binding protein (CRBPI) regulates intracellular vitamin A bioavailability and contributes to maintain skin homeostasis. The aim of present study was to investigate the expression of CRBPI and its role in the pathogenesis of skin psoriasis. Immunohistochemistry revealed more diffuse and increased CRBPI expression in all epidermal layers of human psoriatic lesions except in the stratum corneum. An imiquimod-induced psoriatic-like model documented the increase of skin lesional area and severity index score as well as of the severity of microscopic features as parakeratosis, papillomatosis and spongiosis in CRBPI-knockout compared to wild-type mice, associated to the increased keratinocyte CK17 and Ki-67 expression and the reduction of CK1, CRABPII and RXRα. Gene array of imiquimod-induced psoriatic skin documented the greater up-regulation of EGF/PDGF-related genes and down-regulation of EGR1 and pro-inflammatory IL-related genes in CRBPI-knockout compared to wild-type mice. Finally, CRBPI transfection in HaCaT cells increased AKT and NF-κB-related genes and proteins and down-regulated IL-2, IL-6 and IL-8 pro-inflammatory signalling. Although not recognized as a psoriatic susceptibility gene in our cohort of patients, the present data strongly supported the potential role of CRBPI to sustain keratinocyte proliferation and differentiation and to counteract pro-inflammatory genes expression in psoriatic lesions.

Comparison of tissue transglutaminase 2 and bone biological markers osteocalcin, osteopontin and sclerostin expression in human osteoporosis and osteoarthritis.

Osteoporosis (OP) and osteoarthritis (OA) are the most common joint diseases, with a high incidence in the elderly population. OP is characterized by trabecular bone remodeling and reabsorption, whereas articular cartilage and subchondral bone remodeling are major features of OA. Although classically considered as independent or even conflicting processes, clinical coexistence of OP and OA was recently described. Transglutaminase 2 (TG2) expression is considered a biomarker of OA, but its role in osteoporotic bone remodeling is still uncertain. We investigated TG2 and bone biological markers (Osteocalcin, Osteopontin, and Sclerostin) in osteoporotic and osteoarthritic osteocartilagineous tissue (n = 54) and human chondrocyte cultures in vitro by immunohistochemistry, immunofluorescence and RT-PCR. Histomorphometric evaluation of bone trabecular remodeling was also performed. In cartilage, TG2 expression was faint in control and OP and significantly less than in OA and OP + OA chondrocytes; the opposite was found for Osteocalcin, whereas Osteopontin and Sclerostin expression was similar. In the subchondral trabecular bone, osteocytes/osteoblasts TG2 expression was slight and similar comparing control, OP, OA, and OP + OA group, whereas Osteocalcin and Osteopontin expression was lower in OP compared to control, OA and OP + OA. Increased TG2 and reduced Osteocalcin expression were maintained in human osteoarthritic chondrocytes in vitro. Histomorphometric analysis confirmed reduced trabecular bone mass in OP and OP + OA compared with OA patients. TG2 represented a suitable biomarker of osteoarthritic chondrocyte activation, whereas osteocalcin and osteopontin characterized osteoporotic osteocyte/osteoblast changes; differences were lost in OP + OA patients, suggesting careful consideration when coexistence of the two diseases occurs.

Loss of CRABP-II Characterizes Human Skin Poorly Differentiated Squamous Cell Carcinomas and Favors DMBA/TPA-Induced Carcinogenesis.

Retinol and its derivatives play an important role in epidermal growth and differentiation and represent chemopreventive agents in nonmelanoma skin cancer. Retinoic acid binding protein II (CRABP-II) is a cytoplasmic receptor that critically regulates all-trans-retinoic acid (ATRA) trafficking. We documented the marked reduced expression of CRABP-II and its promoter methylation in human poorly differentiated squamous cell carcinomas. To investigate the role of CRABP-II in skin carcinogenesis we used skin lesion induction by dimethylbenz[a]anthracene/12-O-tetradecanoyl-phorbol-13-acetate in CRABP-II-knockout C57BL/6 mice. We observed earlier and more diffuse epidermal dysplasia, greater incidence and severity of tumors, reduced expression of cytokeratin 1/cytokeratin 10 and involucrin, increased proliferation, and impaired ATRA inhibition of tumor promotion compared with wild-type animals. CRABP-II-transfected HaCaT, FaDu, and A431 cells showed expression of differentiation markers, retinoic acid receptor-ß/-γ signaling, ATRA sensitivity, and suppression of EGFR/v-akt murine thymoma viral oncogene homolog 1 (AKT) pathways in a fatty acid binding protein 5/peroxisome proliferator-activated receptor-ß/-δ-independent manner. The opposite was true in keratinocytes isolated from CRABP-II-knockout mice. Finally, CRABP-II accumulation induced ubiquitination-associated reduction of EGFR. Our results showed reduced CRABP-II expression in human poorly differentiated squamous cell carcinomas, and its gene deletion favored experimental skin carcinogenesis and impaired ATRA antitumor efficacy, likely modulating EGFR/AKT pathways and retinoic acid receptor-ß/-γ signaling. Therapeutic interventions aimed at restoring CRABP-II-mediated signaling may amplify therapeutic retinoid efficacy in nonmelanoma skin cancer.

Vitamin A, cancer treatment and prevention: the new role of cellular retinol binding proteins.

Retinol and vitamin A derivatives influence cell differentiation, proliferation, and apoptosis and play an important physiologic role in a wide range of biological processes. Retinol is obtained from foods of animal origin. Retinol derivatives are fundamental for vision, while retinoic acid is essential for skin and bone growth. Intracellular retinoid bioavailability is regulated by the presence of specific cytoplasmic retinol and retinoic acid binding proteins (CRBPs and CRABPs). CRBP-1, the most diffuse CRBP isoform, is a small 15 KDa cytosolic protein widely expressed and evolutionarily conserved in many tissues. CRBP-1 acts as chaperone and regulates the uptake, subsequent esterification, and bioavailability of retinol. CRBP-1 plays a major role in wound healing and arterial tissue remodelling processes. In the last years, the role of CRBP-1-related retinoid signalling during cancer progression became object of several studies. CRBP-1 downregulation associates with a more malignant phenotype in breast, ovarian, and nasopharyngeal cancers. Reexpression of CRBP-1 increased retinol sensitivity and reduced viability of ovarian cancer cells in vitro. Further studies are needed to explore new therapeutic strategies aimed at restoring CRBP-1-mediated intracellular retinol trafficking and the meaning of CRBP-1 expression in cancer patients' screening for a more personalized and efficacy retinoid therapy.

High expression of cellular retinol binding protein-1 in lung adenocarcinoma is associated with poor prognosis.

PURPOSE: Adenocarcinoma, the most common non-small cell lung cancer is a leading cause of death worldwide, with a low overall survival (OS) despite increasing attempts to achieve an early diagnosis and accomplish surgical and multimodality treatment strategies. Cellular retinol binding protein-1 (CRBP-1) regulates retinol bioavailability and cell differentiation, but its role in lung cancerogenesis remains uncertain. EXPERIMENTAL DESIGN: CRBP-1 expression, clinical outcome and other prognostic factors were investigated in 167 lung adenocarcinoma patients. CRBP-1 expression was evaluated by immunohistochemistry of tissue microarray sections, gene copy number analysis and tumor methylation specific PCR. Effects of CRBP-1 expression on proliferation/apoptosis gene array, protein and transcripts were investigated in transfected A549 lung adenocarcinoma cells. RESULTS: CRBP-1(High) expression was observed in 62.3% of adenocarcinomas and correlated with increased tumor grade and reduced OS as an independent prognostic factor. CRBP-1 gene copy gain also associated with tumor CRBP-1(High) status and dedifferentiation. CRBP-1-transfected (CRBP-1(+)) A549 grew more than CRBP-1(-) A549 cells. At >1µM concentrations, all trans-retinoic acid and retinol reduced viability more in CRBP-1(+) than in CRBP-1(-) A549 cells. CRBP-1(+) A549 cells showed up-regulated RARα/ RXRα and proliferative and transcriptional genes including pAkt, pEGFR, pErk1/2, creb1 and c-jun, whereas RARß and p53 were strongly down-regulated; pAkt/pErk/ pEGFR inhibitors counteracted proliferative advantage and increased RARα/RXRα, c-jun and CD44 expression in CRBP-1(+) A549 cells. CONCLUSION: CRBP-1(High) expression in lung adenocarcinoma correlated with increased tumor grade and reduced OS, likely through increased Akt/Erk/EGFR-mediated cell proliferation and differentiation. CRBP-1(High) expression can be considered an additional marker of poor prognosis in lung adenocarcinoma patients.

Antioxidant treatment prevents serum deprivation- and TNF-α-induced endothelial dysfunction through the inhibition of NADPH oxidase 4 and the restoration of ß-oxidation.

AIMS: Oxidative stress plays a pivotal role in the impaired endothelial function occurring in vascular diseases. Antioxidant strategies induce a clinical advantage in patients with endothelial dysfunction and atherosclerosis and protect from oxidative damage, but the underlying molecular mechanisms have been poorly evaluated. The aim of this study was to analyze the effects and mechanisms of action of antioxidant regimens on endothelial function. METHODS AND RESULTS: Antioxidant efficacy of N-acetylcysteine, ascorbic acid and propionyl-L-carnitine was evaluated in serum-deprived and TNF-α-stimulated human umbilical vein endothelial cells in vitro. Cell adhesion molecule (CAM) expression was evaluated by blot and real-time PCR, and inflammatory cytokine secretion was evaluated by ELISA; leukocyte adhesion and reactive oxygen species assays and NADPH oxidase 4 isoform (Nox4) expression analyses by blots were also performed. Antioxidant pretreatment restored serum-deprived and TNF-α-induced impaired mitochondrial ß-oxidation by reducing flavin adenine dinucleotide level and counteracting increased CAM and Nox4 expression, leukocyte adhesion and inflammatory cytokine secretion. Specific inhibition by plumbagin and siNox4 prevented TNF-α- and serum deprivation-induced detrimental effects, confirming that endothelial oxidative stress and inflammation were Nox4 dependent. CONCLUSIONS: Our findings documented Nox4 as a main actor in oxidative stress-induced endothelial dysfunction and further clarify the molecular basis of antioxidant treatment efficacy.

Adult adipose-derived stem cells and breast cancer: a controversial relationship.

Breast cancer is the most common cancer in women and autologous fat grafting is an important clinical application in treatment of post-surgical deformities. The simplicity of fat grafting procedures and the absence of subsequent visible scar prompted an increasing interest for this technique. The plasticity of adipose-derived stem cells (ASCs) obtained from stromal vascular fraction (SVF) of adult adipose tissue provided exciting perspectives for regenerative medicine and surgery. The recent discovery that SVF/ASC enrichment further ameliorates clinical efficacy of grafting ASCs suggest as ASC-mediated new adipogenesis and vasculogenesis. ASC adipogenic differentiation involves Akt activity and EGFRs, FGFRs, ERbB2 receptor-mediated pathways that also play a pivotal role in the regulation of breast cancer growth. Moreover, the finding that platelet-derived growth factors and hormones improved long-term maintenance of fat grafting raises new concerns for their use during breast reconstruction after cancer surgery. However, it remains unclear whether grafted or resident ASCs may increase the risk of de novo cancer development or recurrence. Preliminary follow-up studies seem to support the efficacy and safety of SVF/ASCs enrichment and the additional benefit from the combined use of autologous platelet-derived growth factors and hormones during breast reconstruction procedures. In the present review we highlighted the complex interplay between resident or grafted ASCs, mature adipocytes, dormant or active breast cancer cells and tumor microenvironment. Actually, data concerning the permissive role of ASCs on breast cancer progression are contrasting, although no clear evidence speaking against their use exists.

CRBP-1 expression in ovarian cancer: a potential therapeutic target.

BACKGROUND/AIM: Cellular retinol binding protein-1 regulates retinol bioavailability and contributes to cell differentiation maintenance, but its role in ovarian carcinogenesis remains uncertain. We investigated CRBP-1 expression in ovarian tumors and CRBP-1 signaling-regulated pathways. MATERIALS AND METHODS: We performed immunohistochemistry, methylation-specific PCR, gene copy number analysis in ovarian tumors and proliferation/apoptosis evaluation, gene array, blot and real-time PCR in CRBP-1-transfected A2780 ovarian cancer cells. RESULTS: CRBP-1 expression was reduced or absent in G2 and G3 ovarian carcinomas. CRBP-1 silencing in 60% of G2 and 66.7% of G3 carcinomas was due to CRBP-1 promoter methylation. A2780 CRBP-1-transfected cells showed increased retinol-induced apoptosis, retinoid-induced reduced clonogenicity and down-regulation of proliferation and transcription genes, including AKT1, AKT3, EGFR, FOS, JUN, STAT1 and STAT5A. CONCLUSION: CRBP-1 loss in G2/G3 ovarian carcinomas and increased apoptotic susceptibility to retinoids in CRBP-1-transfected-A2780 cells suggest CRBP-1 screening as a target to ensure efficacy of an adjuvant retinoid therapy.