C.A.M. La Porta

Modulation of pro- and anti-apoptotic factors in human melanoma cells exposed to histone deacetylase inhibitors

Valproic acid (VPA, 2-propylpentanoic acid) is an established drug in the long-term therapy of epilepsy. Recently, VPA was demonstrated to inhibit histone deacetylases (HDACs) class I enzyme at therapeutically relevant concentrations, thereby, mimicking the prototypical histone deacetylase inhibitors, tricostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA). In the present study, we investigated the cellular effects of VPA, TSA and SAHA on four human melanoma cell lines (WM115, WM266, A375, SK-Mel28) with particular reference to the modulation of regulators of apoptosis, including Bcl-2, BclXL, Mcl-1, Apaf-1, BclXs, NOXA, TRAIL-R1, TRAIL-R2, caspase 8, and survivin). Firstly, we found that VPA induced apoptosis in two of the four human melanoma cell lines, while both TSA and SAHA exhibited an antiproliferative and apoptotic effects in all four cell lines, a different expression of Bcl-2 and BclXL/S occurred. On the other hand, SAHA and VPA modulated differently pro- and anti-apoptotic factors. In particular, the treatment with VPA enhanced the level of expression of survivin only in VPA-resistant cell lines, whereas down-regulation of survivin was induced by VPA and SAHA in VPA-sensitive cells. In the latter, since activation of caspase 8 was documented, a receptor-mediated apoptosis was suggested. Taken together, our results suggest that HDAC inhibitors may represent a promising therapeutic strategy to treat melanoma.

Increased plasma levels of soluble CD40, together with the decrease of TGFβ1, as possible differential markers of Alzheimer disease

Alzheimers disease (AD) is a progressive neurodegenerative illness and the most frequent cause of dementia in the elderly. The identification of activated microglia within neuritic plaques, coupled with the presence of numerous inflammatory proteins, suggests that inflammation is an integral part of the pathogenetic process in AD. In the present paper we have investigated the levels of circulating inflammatory mediators as potential AD biomarkers concentrating essentially on (a) soluble CD40 (sCD40), a member of the tumor necrosis factor receptor superfamily lacking the membrane-associated endodomain by alternative splicing, and (b) transforming growth factor (TGF)-beta 1, a cytokine deeply involved in AD and playing a protective role on CNS. Decrease of TGF-beta1 in AD patients could enhance the effects of pro-inflammatory cytokines produced by activated microglia as well as the expression of factors, such as the CD40/CD40 ligand complex, by microglia and astrocytes. Total venous blood samples were obtained from 33 patients with clinical diagnosis of possible late-onset AD, 40 healthy age-matched and 11 healthy young individuals. A significant increase of sCD40 levels plasma of AD patients versus healthy controls was measured, concomitantly with a decrease in TGF-beta1 concentration. These variations, however, showed no correlation with the expression of ApoE epsilon 4 allele, which was determined in order to assess the different frequency of this risk factor between AD and control groups. Since no comparable modifications were detected in patients affected by Parkinsons disease or non-AD-based dementia, we propose that sCD40 and TGF-beta1 plasma levels might represent possible differential biomarkers of AD, and be useful pre-mortem to support the clinical diagnosis of late-onset AD.

Decrease of TGF-β1 plasma levels and increase of nitric oxide synthase activity in leukocytes as potential biomarkers of Alzheimer's disease

Abstract A variety of inflammatory proteins have been identified in brains of Alzheimers disease (AD) patients, including inflammatory cytokines, acute phase proteins and complement components. In the present paper we have investigated the levels of circulating inflammatory mediators as potential biomarkers of this disease, concentrating mostly on transforming growth factor beta (TGF-β1) in plasma and on nitric oxide synthase (NOS) activity in leukocytes. Plasma and leukocytes were isolated from 48 sporadic AD and 23 healthy control subjects of same age and sex. Since α2-Macroglobulin (α2M), an acute phase protein possibly involved in AD, is an important modulator of TGF-β1 activity, binding and targeting this cytokine to its appropriate site of action, we have investigated the possible complex between TGF-β1 and α2M in plasma of the same subjects. The results demonstrate a significant reduction of TGF-β1 levels in plasma of AD patients. A complex between α2M and TGF-β1 occurred in AD as well as healthy elderly control subjects, however, with no significant differences. Moreover, α2M appeared to bind only the inactive form of this cytokine. In contrast, NOS activity increased significantly in leukocytes of AD patients. Therefore, we suggest the combined determination of TGF-β1 in the plasma and of NOS activity in the leukocytes as biomarkers of AD.

Overexpression of novel protein kinase C delta in BL6 murine melanoma cells inhibits the proliferative capacity in vitro but enhances the metastatic potential in vivo.

&NA; In this study we analysed the effect of overexpressing novel protein kinase C &dgr; isoform (n‐PKC &dgr;) on melanin synthesis and metastatic potential in the highly metastatic BL6 murine melanoma cells. The proliferative capacity in vitro and into matrigel in vivo were also examined. Although murine melanocytes express the n‐PKC &dgr; isoform, BL6 cells do not express this isoform at levels detectable by Western blot analysis. In untransfected and transfected cells we also studied the effect of 12‐O‐tetra‐decanoylphorbol‐13‐acetate (TPA), a modulator of specific isoforms of PKC, and of bryostatin 1, a potent immunomodulator and antineoplastic drug and a partial agonist of PKC. Our results demonstrate a pivotal role for this isoform in melanin synthesis and the close relationship between n‐PKC &dgr; expression and its association with the particulate fraction, melanogenesis and metastatic potential. In fact, heterogeneous BL6 cells overexpressing n‐PKC &dgr; and all the clones isolated showed increased intracellular melanin and metastatic capacity. TPA and bryostatin 1 decreased n‐PKC &dgr; expression, the intracellular melanin level and metastatic capacity in both cell lines. Therefore both treatments were able to abolish the effects of overexpressing n‐PKC &dgr;.

Inhibition of protein kinase C-alpha isoform enhances the P-glycoprotein expression and the survival of LoVo human colon adenocarcinoma cells to doxorubicin exposure.

The aim of the present paper was to analyse the effect of long-term inhibitory treatment, for at least 7 days, of individual protein kinase C (PKC) isoforms on the survival of LoVo human colon adenocarcinoma cells to doxorubicin exposure. The treatment for 2 h, after plating the cells, and after 3 days with 1 microM Gö6976, a specific inhibitor of protein kinase C (PKC)-alpha and -betal isoforms, induced on day 7 in LoVo cell lines (WT) a significant increased survival when these cells were exposed to increasing doxorubicin concentrations. In contrast, resistant LoVo cells (DX) did not show significant changes in the survival to doxorubicin exposure when incubated with the inhibitor of the same specific PKC isoforms. In addition, Gö6976 reduced the PKC-alpha activity (the main calcium-dependent PKC isoforms expressed) in both cell lines with contemporary increased expression. Under such conditions, an increased nuclear activity and an increased P-glycoprotein expression occurred only in WT-treated cells with respect to untreated cells. Taken together, our data indicate a specific relationship between PKC-alpha inhibition, the increased nuclear PKC-alpha activity as well as the increased expression of P-glycoprotein, possibly causing the acquisition of a resistant phenotype in WT LoVo cells.

Higher levels of melanin and inhibition of cdk2 activity in primary human melanoma cells WM115 overexpressing nPKCdelta.

Many studies have attempted to define the state of differentiation of melanoma cells and to correlate it with other critical parameters of malignancy such as the tumorigenic and metastatic nature of the cells. In the present paper we focused on the possible relationships between the novel protein kinase C isoform nPKCδ, melanin synthesis and proliferative capacity in a primary human melanoma cell line WM115. Cells were transfected to produce overexpression of this isoform and the effects on melanin synthesis, cyclin-E dependent kinase (cdk2) activity and cyclin E expression were studied. It was shown that translocation of nPKCδ into the nucleus affects melanin synthesis and inhibits cdk2 activity. As a compensatory effect, the level of cyclin E increases. In view of these results we suggest a model for the role of nPKCδ in melanoma cells that may offer a new therapeutic perspective.

Carbon nanopowder acts as a Trojan-horse for benzo(α)pyrene in Danio rerio embryos

Abstract Carbon-based nanoparticles (CBNs) are largely distributed worldwide due to fossil fuel combustion and their presence in many consumer products. In addition to their proven toxicological effects in several biological models, attention in recent years has focussed on the role played by CBNs as Trojan-horse carriers for adsorbed environmental pollutants. This role has not been conclusively determined to date because CBNs can decrease the bioavailability of contaminants or represent an additional source of intake. Herein, we evaluated the intake, transport and distribution of one of the carbon-based powders, the so-called carbon nanopowder (CNPW), and benzo(α)pyrene, when administered alone and in co-exposure to Danio rerio embryos. Data obtained by means of advanced microscopic techniques illustrated that the “particle-specific” effect induced a modification in the accumulation of benzo(α)pyrene, which is forced to follow the distribution of the physical pollutant instead of its natural bioaccumulation. The combined results from functional proteomics and gene transcription analysis highlighted the different biochemical pathways involved in the action of the two different contaminants administered alone and when bound together. In particular, we observed a clear change in several proteins involved in the homeostatic response to hypoxia only after exposure to the CNPW or co-exposure to the mixture, whereas exposure to benzo(α)pyrene alone mainly modified structural proteins. The entire dataset suggested a Trojan-horse mechanism involved in the biological impacts on Danio rerio embryos especially due to different bioaccumulation pathways and cellular targets.

Human skin-derived fibroblasts used as a ‘Trojan horse’ for drug delivery

Drug toxicity currently represents the main challenge of tumour chemotherapy. Our group recently developed a new method for drug delivery inspired by the ‘Trojan Horse’ concept. Human mesenchymal stem cells (hMSCs) have been shown to play the role of new ‘horses’ in delivering anti‐tumour agents, without involving any genetic manipulation. As human stromal dermal fibroblasts (hSDFs) represent an interesting alternative to hMSCs, being easy to isolate, they could be an ideal candidate for this kind of procedure.

Inhibition of PKCα decreases the gelatinase activity and the angiogenic and metastatic ability of the highly metastatic B16 murine melanoma cells

Protein kinase C (PKC) comprises a family of at least 11 isoforms that play a pivotal role in the angiogenic and metastatic process. In this study we analysed the effect of two PKC isoform-selective inhibitors, Gö6976 an inhibitor of c-PKCα and βI, and bisindolylmaleimide (BIM) that prevents the effect of phorbol ester, on the gelatinolytic, angiogenic and metastatic capacity of the low metastatic B16F1 and the highly metastatic BL6 murine melanoma cells. The treatment with BIM did not modify these processes in either cell line, while Gö6976 induced a significant decrease in the angiogenic, gelatinase and metastatic potential in the BL6 cells. Both inhibitors inversely modulated VEGF and bFGF expression. Nitric oxide synthase (NOS), however, showed no change in activity. Taken together our results demonstrate an involvement of the c-PKCα isoform in the metastatic and angiogenic process, possibly by reducing the gelatinolytic activity. Thus, the c-PKCα isoform may be a novel target for therapy.

Cellular pathways affected by carbon nanopowder-benzo(α)pyrene complex in human skin fibroblasts identified by proteomics

One of the crucial and unsolved problems of the airborne carbon nanoparticles is the role played by the adsorbed environmental pollutants on their toxicological effect. Indeed, in the urban areas, the carbon nanoparticles usually adsorb some atmospheric contaminants, whose one of the leading representatives is the benzo(α)pyrene. Herein, we used the proteomics to investigate the alteration of toxicological pathways due to the carbon nanopowder-benzo(α)pyrene complex in comparison with the two contaminants administered alone on human skin-derived fibroblasts (hSDFs) exposed for 8 days in semi-static conditions. The preliminary confocal microscopy observations highlighted that carbon-nanopowder was able to pass through the cell membranes and accumulate into the cytoplasm both when administered alone and with the adsorbed benzo(α)pyrene. Proteomics revealed that the effect of carbon nanopowder-benzo(α)pyrene complex seems to be related to a new toxicological behavior instead of simple additive or synergistic effects. In detail, the cellular pathways modulated by the complex were mainly related to energy shift (glycolysis and pentose phosphate pathway), apoptosis, stress response and cellular trafficking.