Carla Rozzo

Enhanced anti-tumor activity of a new curcumin-related compound against melanoma and neuroblastoma cells

BackgroundSharing the common neuroectodermal origin, melanoma and neuroblastoma are tumors widely diffused among adult and children, respectively. Clinical prognosis of aggressive neuroectodermal cancers remains dismal, therefore the search for novel therapies against such tumors is warranted. Curcumin is a phytochemical compound widely studied for its antioxidant, anti-inflammatory and anti-cancer properties. Recently, we have synthesized and tested in vitro various curcumin-related compounds in order to select new anti-tumor agents displaying stronger and selective growth inhibition activity on neuroectodermal tumors.ResultsIn this work, we have demonstrated that the new α,β-unsaturated ketone D6 was more effective in inhibiting tumor cells growth when compared to curcumin. Normal fibroblasts proliferation was not affected by this treatment. Clonogenic assay showed a significant dose-dependent reduction in both melanoma and neuroblastoma colony formation only after D6 treatment. TUNEL assay, Annexin-V staining, caspases activation and PARP cleavage unveiled the ability of D6 to cause tumor cell death by triggering apoptosis, similarly to curcumin, but with a stronger and quicker extent. These apoptotic features appear to be associated with loss of mitochondrial membrane potential and cytochrome c release. In vivo anti-tumor activity of curcumin and D6 was surveyed using sub-cutaneous melanoma and orthotopic neuroblastoma xenograft models. D6 treated mice exhibited significantly reduced tumor growth compared to both control and curcumin treated ones (Melanoma: D6 vs control: P < 0.001 and D6 vs curcumin P < 0.01; Neuroblastoma: D6 vs both control and curcumin: P < 0.001).ConclusionsOur data indicate D6 as a good candidate to develop new therapies against neural crest-derived tumors.

Proteomic profiling of human melanoma metastatic cell line secretomes.

During the last few years, the incidence and mortality of human melanoma have rapidly increased. Metastatic spread of malignant melanoma is often associated with cancer progression with poor prognosis and survival. These processes are controlled by dynamic interactions between tumor melanocytes and neighboring stromal cells, whose deregulation leads to the acquisition of cell proliferation capabilities and invasiveness. It is increasingly clear that a key role in carcinogenesis is played by secreted molecules either by tumor and surrounding stromal cells. To address the issue of the proteins secreted during cancer progression, the proteomic profiling of secretomes of cancer cell lines from different melanoma metastases of the same patient (PE-MEL-41, PE-MEL-47, and PE-MEL-43) was performed by applying a shotgun LC-MS/MS-based approach. The results provide a list of candidate proteins associated with the metastatic potential of PE-MEL melanoma cell lines. Among them, several matricellular proteins previously reported as involved in melanoma aggressiveness were identified (i.e., SPARC, osteopontin). In addition, the extracellular matrix protein 1 that stimulates proliferation and angiogenesis of endothelial cells as well as the fibronectin, involved in cell adhesion and motility, were identified. The present work provides the basis to clarify the complex extracellular protein networks implicated in human melanoma cell invasion, migration, and motility.

A novel spontaneous missense mutation in VMD2 gene is a cause of a Best macular dystrophy sporadic case

PURPOSE To report the molecular characterization of a novel VMD2 mutation causing a Best macular dystrophy sporadic case. METHODS All family members underwent ophthalmologic examination and genetic testing by single strand conformation polymorphism analysis and direct sequencing of the VMD2 gene. RESULTS A single T to G transition at nucleotide 663 was identified in one of the VMD2 gene copies of the patient, which results in a Cys to Trp substitution at position 221 in the corresponding protein (C221W). Sequence analysis of the VMD2 exon 6 of both parents of the patient did not reveal any mutation. CONCLUSION These data confirm the involvement of the VMD2 gene in Best macular dystrophy onset, even in sporadic cases of the disease, pointing out the relevance of molecular analysis in the diagnosis of this degenerative retinal disease.

Molecular changes induced by the curcumin analogue D6 in human melanoma cells

BackgroundIn a previous report, we described the in vitro and in vivo antiproliferative and proapoptotic activity of a hydroxylated biphenyl (D6), a structural analogue of curcumin, on malignant melanoma and neuroblastoma tumours. In this paper, we investigated the molecular changes induced by such a compound, underlying cell growth arrest and apoptosis in melanoma cells.ResultsTo shed light on the mechanisms of action of D6, we firstly demonstrated its quick cellular uptake and subsequent block of cell cycle in G2/M phase transition. A gene expression profile analysis of D6-treated melanoma cells and fibroblasts was then carried out on high density microarrays, to assess gene expression changes induced by this compound. The expression profile study evidenced both an induction of stress response pathways and a modulation of cell growth regulation mechanisms. In particular, our data suggest that the antiproliferative and proapoptotic activities of D6 in melanoma could be partially driven by up-regulation of the p53 signalling pathways as well as by down-regulation of the PI3K/Akt and NF-kB pathways. Modulation of gene expression due to D6 treatment was verified by western blot analysis for single proteins of interest, confirming the results from the gene expression profile analysis.ConclusionsOur findings contribute to the understanding of the mechanisms of action of D6, through a comprehensive description of the molecular changes induced by this compound at the gene expression level, in agreement with the previously reported anti-tumour effects on melanoma cells.

Integrin up-regulation as marker of neuroblastoma cell differentiation: correlation with neurite extension.

We have characterized the adhesion properties, integrin expression, and morphological changes due to extracellular matrix (ECM)-integrin interactions in a neuronal model. We showed that a modulation of some integrin heterodimers occurs during interferon-γ (IFN-γ) induced neuroblastoma (NB) cell differentiation. To better elucidate the possible implication and function of integrin receptors during neuronal maturation, we analyzed the changes in integrin expression in two human NB cell lines, LAN-5 and GI-LI-N, which represent different stages of neuronal differentiation. These models show opposite morphological maturation after interferon-γ and tumor necrosis factor-α (IFN-γ+TNF) treatment. While LAN-5 cells acquired the ability to extend long and branched neurites, GI-LI-N cells did not. Both cell lines showed enhanced expression of phenotypical and biochemical markers of neural maturation. Moreover, retinoic acid (RA) had different effects on the two NB cell lines: on LAN-5 cells it acts as a differentiation-promoting agent, while on GI-LI-N cells it has an antiproliferative effect, driving them to apoptosis. RT – PCR experiments and immunoprecipitation assays showed a late but marked increase in the expression of α1, α2, α3, and β1 chains after IFN-γ+TNF treatment of LAN-5 cells, and only α1 and β1 chains upon RA induction. Treatment with IFN-γ+TNF induced GI-LI-N cells to show only a late and remarkable increase of α1/β1 heterodimer; on the contrary, RA treatment caused a decrease in all integrin chains. These changes are accompanied in differentiated cells by substantial increases in cell attachment to all purified ECM components tested and an increase of neurite-bearing cells and of average neurite length. In conclusion, these findings indicate a close correlation between up-regulation of integrins and neuronal morphogenesis.

Induction of arginosuccinate synthetase (ASS) expression affects the antiproliferative activity of arginine deiminase (ADI) in melanoma cells

Arginine deiminase (ADI), an arginine-degrading enzyme, has been used in the treatment of tumours sensitive to arginine deprivation, such as malignant melanoma (MM) and hepatocellular carcinoma (HCC). Endogenous production of arginine is mainly dependent on activity of ornithine transcarbamylase (OTC) and argininosuccinate synthetase (ASS) enzymes. We evaluated the effect of ADI treatment on OTC and ASS expression in a series of melanoma cell lines. Twenty-five primary melanoma cell lines and normal fibroblasts as controls underwent cell proliferation assays and Western blot analyses in the presence or absence of ADI. Tissue sections from primary MMs (N = 20) and HCCs (N = 20) were investigated by immunohistochemistry for ASS expression. Overall, 21/25 (84%) MM cell lines presented a cell growth inhibition by ADI treatment; none of them presented constitutive detectable levels of the ASS protein. However, 7/21 (33%) ADI-sensitive melanoma cell lines presented markedly increased expression levels of the ASS protein following ADI treatment, with a significantly higher IC50 median value. Growth was not inhibited and the IC50 was not reached among the remaining 4/25 (16%) MM cell lines; all of them showed constitutive ASS expression. The OTC protein was found expressed in all melanoma cell lines before and after the ADI treatment. Lack of ASS immunostaining was observed in all analyzed in vivo specimens. Our findings suggest that response to ADI treatment in melanoma is significantly correlated with the ability of cells to express ASS either constitutively at basal level (inducing drug resistance) or after the treatment (reducing sensitivity to ADI).

Induction of differentiation and apoptosis by interferon-γ in human neuroblastoma cells in vitro as a dual and alternative early biological response

Interferon-γ (IFN-γ) has a well known differentiation-promoting activity on several neuroblastoma (NB) cell lines and has also been reported to induce apoptosis in different cellular models. We have investigated the potential of IFN-γ to trigger, besides differentiation, programmed cell death in NB cells and the relationship between these processes. Nine NB cell lines, characterized by different phenotypic and maturational features, were cultured in the presence of IFN-γ (1000 IU/ml) for up to 5 days with either only one treatment at the start of the culture or renewing the culture medium (with or without IFN-γ) every other day. Neuronal differentiation was assessed by evaluation of morphological changes and expression of mature cytoskeletal proteins, while apoptosis was evaluated at the desired times by fluorescent and electronic microscopy, DNA content analysis and DNA fragmentation assay. Our findings show that apoptosis is an early (mainly non post-differentiative) event and is much more evident following a single IFN-γ administration. Moreover, IFN-γ-triggered apoptosis is independent of the cellular phenotype (schwannian or neuronal) and appears to be mutually exclusive with respect to differentiation at the single cell level. Our results strengthen the potential of IFN-γ as a promising therapeutic agent for NB.

Antitumoral effect of vanadium compounds in malignant melanoma cell lines

In this study we evaluated the anticancer activity against malignant melanoma (MM) of four different vanadium species: the inorganic anion vanadate(V) (indicated with VN), and three oxidovanadium(IV) complexes, [VIVO(dhp)2] where dhp- is the anion 1,2-dimethyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS2), [VIVO(mpp)2] where mpp- is 1-methyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS3), and [VIVO(ppp)2] where ppp- is 1-phenyl-2-methyl-3-hydroxy-4(1H)-pyridinonate (indicated with VS4). The antitumor effects of these compounds were studied against two different MM cell lines (A375 and CN-mel) and a fibroblast cell line (BJ) as normal control. All tested V compounds exert antiproliferative activity on MM cells in a dose dependent manner (IC50 ranges from 2.4μM up to 14μM) being A375 the most sensitive cell line. VN and VS2 were the two most active compounds against A375 (IC50 of 4.7 and 2.6μM, respectively), causing apoptosis and cell cycle block. The experimental data indicate that the cell cycle arrest occurs at different phases for the two V species analyzed (G2 checkpoint for VN and G0/G1 for VS2), showing the importance of the chemical form in determining their mechanism of action. These results add more insights into the landscape of vanadium versatility in biological systems and into its role as a potential cancer therapeutic agent.

Vitamin D status and risk for malignant cutaneous melanoma: recent advances.

Cutaneous malignant melanoma, whose incidence is increasing steadily worldwide, is the result of complex interactions between individual genetic factors and environmental risk factors. Ultraviolet radiation represents the most important environmental risk factor for the development of skin cancers, including melanoma. Sun exposure and early sunburn during childhood are the principal causes of cutaneous melanoma insurgence in adults, with double the risk relative to a nonexposed population. Consequently, ultraviolet protection has long been recognized as an important measure to prevent such a malignancy. Biological and epidemiological data suggest that vitamin D status could affect the risk of cancer and play a role in cancer prevention by exerting antiproliferative effects. Solar radiations are critical for vitamin D synthesis in humans; however, uncontrolled and intensive sun exposure is dangerous to skin health and may contribute toward the development of cutaneous malignant melanoma. An optimum balance between sun protection and exposure is thus advocated. Additional research is required to confirm the preventive role of vitamin D in melanoma incidence or a positive influence on patient outcome.

Protein expression changes induced in a malignant melanoma cell line by the curcumin analogue compound D6

BackgroundWe have previously demonstrated that the hydroxylated biphenyl compound D6 (3E,3′E)-4,4′-(5,5′,6,6′-tetramethoxy-[1,1′-biphenyl]-3,3′-diyl)bis(but-3-en-2-one), a structural analogue of curcumin, exerts a strong antitumor activity on melanoma cells both in vitro and in vivo. Although the mechanism of action of D6 is yet to be clarified, this compound is thought to inhibit cancer cell growth by arresting the cell cycle in G2/M phase, and to induce apoptosis through the mitochondrial intrinsic pathway. To investigate the changes in protein expression induced by exposure of melanoma cells to D6, a differential proteomic study was carried out on D6-treated and untreated primary melanoma LB24Dagi cells.MethodsProteins were fractionated by SDS-PAGE and subjected to in gel digestion. The peptide mixtures were analyzed by liquid chromatography coupled with tandem mass spectrometry. Proteins were identified and quantified using database search and spectral counting. Proteomic data were finally uploaded into the Ingenuity Pathway Analysis software to find significantly modulated networks and pathways.ResultsAnalysis of the differentially expressed protein profiles revealed the activation of a strong cellular stress response, with overexpression of several HSPs and stimulation of ubiquitin-proteasome pathways. These were accompanied by a decrease of protein synthesis, evidenced by downregulation of proteins involved in mRNA processing and translation. These findings are consistent with our previous results on gene expression profiling in melanoma cells treated with D6.ConclusionsOur findings confirm that the curcumin analogue D6 triggers a strong stress response in melanoma cells, turning down majority of cell functions and finally driving cells to apoptosis.