Identification of Dihydrolipoamide Dehydrogenase as Potential Target of Vemurafenib-Resistant Melanoma Cells.

BACKGROUND: Despite recent improvements in therapy, the five-year survival rate for patients with advanced melanoma is poor, mainly due to the development of drug resistance. The aim of the present study was to investigate the mechanisms underlying this phenomenon, applying proteomics and structural approaches to models of melanoma cells. METHODS: Sublines from two human (A375 and SK-MEL-28) cells with acquired vemurafenib resistance were established, and their proteomic profiles when exposed to denaturation were identified through LC-MS/MS analysis. The pathways derived from bioinformatics analyses were validated by in silico and functional studies. RESULTS: The proteomic profiles of resistant melanoma cells were compared to parental counterparts by taking into account protein folding/unfolding behaviors. Several proteins were found to be involved, with dihydrolipoamide dehydrogenase (DLD) being the only one similarly affected by denaturation in all resistant cell sublines compared to parental ones. DLD expression was observed to be increased in resistant cells by Western blot analysis. Protein modeling analyses of DLD's catalytic site coupled to in vitro assays with CPI-613, a specific DLD inhibitor, highlighted the role of DLD enzymatic functions in the molecular mechanisms of BRAFi resistance. CONCLUSIONS: Our proteomic and structural investigations on resistant sublines indicate that DLD may represent a novel and potent target for overcoming vemurafenib resistance in melanoma cells.

Targeting Melanoma-Initiating Cells by Caffeine: In Silico and In Vitro Approaches.

The beneficial effects of coffee on human diseases are well documented, but the molecular mechanisms of its bioactive compounds on cancer are not completely elucidated. This is likely due to the large heterogeneity of coffee preparations and different coffee-based beverages, but also to the choice of experimental models where proliferation, differentiation and immune responses are differently affected. The aim of the present study was to investigate the effects of one of the most interesting bioactive compounds in coffee, i.e., caffeine, using a cellular model of melanoma at a defined differentiation level. A preliminary in silico analysis carried out on public gene-expression databases identified genes potentially involved in caffeine's effects and suggested some specific molecular targets, including tyrosinase. Proliferation was investigated in vitro on human melanoma initiating cells (MICs) and cytokine expression was measured in conditioned media. Tyrosinase was revealed as a key player in caffeine's mechanisms of action, suggesting a crucial role in immunomodulation through the reduction in IL-1ß, IP-10, MIP-1α, MIP-1ß and RANTES secretion onto MICs conditioned media. The potent antiproliferative effects of caffeine on MICs are likely to occur by promoting melanin production and reducing inflammatory signals' secretion. These data suggest tyrosinase as a key player mediating the effects of caffeine on melanoma.

Melanoma Cell Resistance to Vemurafenib Modifies Inter-Cellular Communication Signals.

The therapeutic success of BRAF inhibitors (BRAFi) and MEK inhibitors (MEKi) in BRAF-mutant melanoma is limited by the emergence of drug resistance, and several lines of evidence suggest that changes in the tumor microenvironment can play a pivotal role in acquired resistance. The present study focused on secretome profiling of melanoma cells sensitive or resistant to the BRAFi vemurafenib. Proteomic and cytokine/chemokine secretion analyses were performed in order to better understand the interplay between vemurafenib-resistant melanoma cells and the tumor microenvironment. We found that vemurafenib-resistant melanoma cells can influence dendritic cell (DC) maturation by modulating their activation and cytokine production. In particular, human DCs exposed to conditioned medium (CM) from vemurafenib-resistant melanoma cells produced higher levels of pro-inflammatory cytokines-that potentially facilitate melanoma growth-than DCs exposed to CM derived from parental drug-sensitive cells. Bioinformatic analysis performed on proteins identified by mass spectrometry in the culture medium from vemurafenib-sensitive and vemurafenib-resistant melanoma cells suggests a possible involvement of the proteasome pathway. Moreover, our data confirm that BRAFi-resistant cells display a more aggressive phenotype compared to parental ones, with a significantly increased production of interferon-γ, interleukin-8, vascular-endothelial growth factor, CD147/basigin, and metalloproteinase 2 (MMP-2). Plasma levels of CD147/basigin and MMP-2 were also measured before the start of therapy and at disease progression in a small group of melanoma patients treated with vemurafenib or vemurafenib plus cobimetinib. A significant increment in CD147/basigin and MMP-2 was observed in all patients at the time of treatment failure, strengthening the hypothesis that CD147/basigin might play a role in BRAFi resistance.

Theophylline induces differentiation and modulates cytoskeleton dynamics and cytokines secretion in human melanoma-initiating cells.

AIMS: Cutaneous melanoma is the most aggressive skin cancer, derived from neoplastic transformation of melanocytes. Since several evidences highlighted the importance of a hierarchical model of differentiation among cancer cells, closely related to resistance mechanisms and tumor relapse, we investigated the effects of theophylline (Theo), a methylxanthine commonly used in treatment of respiratory diseases, on melanoma cells with different degree of differentiation, including patient-derived melanoma-initiating cells. MATERIALS AND METHODS: The antiproliferative and antimetastatic effects of Theo was demonstrated by cell counting, adhesion and migration assays on A375 and SK-MEL-30 cells. Further, Theo ability to reduce cell growth was highly significant in A375-derived spheroids and in two patient-derived melanoma-initiating cells (MICs). In order to identify pathways potentially involved in the antineoplastic properties of Theo, a comparative mass spectrometry proteomic analysis was used. Then, melanin content, tyrosinase and tissue transglutaminase activities as differentiation markers and actin re-organization through confocal microscopy were evaluated. Furthermore, a secretome profile of MICs after Theo treatments was performed by multiplex immunoassay. KEY FINDINGS: Obtained results demonstrate inhibitory effects of Theo on melanoma cell proliferation and migration, mainly in MICs, together with the induction of differentiation parameters. Moreover, our data indicate that the known anti-melanoma effect of Theo is due also to its ability to interfere with cytoskeleton dynamics and to induce the secretion of inflammatory molecules involved in recruitment of immunosuppressive cells in tumor microenvironment. SIGNIFICANCE: Data strongly suggest that Theo supplement, either as drug or as dietary supply, may represent a potent additional weapon against melanoma.

TNF-alpha and metalloproteases as key players in melanoma cells aggressiveness.

BACKGROUND: Melanoma aggressiveness determines its growth and metastatic potential. This study aimed at identifying new molecular pathways controlling melanoma cell malignancy. METHODS: Ten metastatic melanoma cell lines were characterized by their proliferation, migration and invasion capabilities. The most representative cells were also characterized by spheroid formation assay, gene- and protein- expression profiling as well as cytokines secretion and the most relevant pathways identified through bioinformatic analysis were tested by in silico transcriptomic validation on datasets generated from biopsies specimens of melanoma patients. Further, matrix metalloproteases (MMPs) activity was tested by zymography assays and TNF-alpha role was validated by anti-TNF cell-treatment. RESULTS: An aggressiveness score (here named Melanoma AGgressiveness Score: MAGS) was calculated by measuring proliferation, migration, invasion and cell-doubling time in10human melanoma cell lines which were clustered in two distinct groups, according to the corresponding MAGS. SK-MEL-28 and A375 cell lines were selected as representative models for the less and the most aggressive phenotype, respectively. Gene-expression and protein expression data were collected for SK-MEL-28 and A375 cells by Illumina-, multiplex x-MAP-and mass-spectrometry technology. The collected data were subjected to an integrated Ingenuity Pathway Analysis, which highlighted that cytokine/chemokine secretion, as well as Cell-To-Cell Signaling and Interaction functions as well as matrix metalloproteases activity were significantly different in these two cell types. The key role of these pathways was then confirmed by functional validation. TNF role was confirmed by exposing cells to the anti-TNF Infliximab antibody. Upon such treatment melanoma cells aggressiveness was strongly reduced. Metalloproteases activity was assayed, and their role was confirmed by comparing transcriptomic data from cutaneous melanoma patients (n = 45) and benign nevi (n = 18). CONCLUSIONS: Inflammatory signals such as TNF and MMP-2 activity are key intrinsic players to determine melanoma cells aggressiveness suggesting new venue sin the identification of novel molecular targets with potential therapeutic relevance.

Eosin treatment for psoriasis reduces skin leukocyte infiltration and secretion of inflammatory chemokines and angiogenic factors.

BACKGROUND: Eosin has been traditionally employed as a topical treatment for psoriasis, but the biological mechanism of its therapeutic action has not been fully elucidated. OBJECTIVES: To analyse eosin effects on psoriatic skin in vivo and keratinocytes and endothelial cells in vitro. MATERIALS & METHODS: Skin biopsies were taken from psoriatic plaques before and after a three-day eosin treatment and processed for histological analysis. Cultured human psoriatic keratinocytes and dermal endothelial cells were treated with eosin, and release of inflammatory chemokines was analysed by multiplexed bead-based immunoassay and ELISA. RESULTS: In patients, the three-day eosin treatment significantly reduced the number of infiltrating T lymphocytes, neutrophilic granulocytes, and dermal dendritic cells. A reduction in VEGF-A expression was also observed. In vitro, eosin treatment significantly decreased the release of CCL2, CCL5, and VEGF-A by keratinocytes and angiopoietin-2 by endothelial cells. CONCLUSIONS: Eosin treatment impacts on psoriatic inflammatory infiltrates and dampens the release of proinflammatory chemokines and angiogenic factors.

Molecular mechanisms of cardioprotective effects mediated by transplanted cardiac ckit+ cells through the activation of an inflammatory hypoxia-dependent reparative response.

The regenerative effects of cardiac ckit+ stem cells (ckit+CSCs) in acute myocardial infarction (MI) have been studied extensively, but how these cells exert a protective effect on cardiomyocytes is not well known. Growing evidences suggest that in adult stem cells injury triggers inflammatory signaling pathways which control tissue repair and regeneration. Aim of the present study was to determine the mechanisms underlying the cardioprotective effects of ckit+CSCs following transplantation in a murine model of MI. Following isolation and in vitro expansion, cardiac ckit+CSCs were subjected to normoxic and hypoxic conditions and assessed at different time points. These cells adapted to hypoxia as showed by the activation of HIF-1α and the expression of a number of genes, such as VEGF, GLUT1, EPO, HKII and, importantly, of alarmin receptors, such as RAGE, P2X7R, TLR2 and TLR4. Activation of these receptors determined an NFkB-dependent inflammatory and reparative gene response (IRR). Importantly, hypoxic ckit+CSCs increased the secretion of the survival growth factors IGF-1 and HGF. To verify whether activation of the IRR in a hypoxic microenvironment could exert a beneficial effect in vivo, autologous ckit+CSCs were transplanted into mouse heart following MI. Interestingly, transplantation of ckit+CSCs lowered apoptotic rates and induced autophagy in the peri-infarct area; further, it reduced hypertrophy and fibrosis and, most importantly, improved cardiac function. ckit+CSCs are able to adapt to a hypoxic environment and activate an inflammatory and reparative response that could account, at least in part, for a protective effect on stressed cardiomyocytes following transplantation in the infarcted heart.

Lipid Storage and Autophagy in Melanoma Cancer Cells.

Cancer stem cells (CSC) represent a key cellular subpopulation controlling biological features such as cancer progression in all cancer types. By using melanospheres established from human melanoma patients, we compared less differentiated melanosphere-derived CSC to differentiating melanosphere-derived cells. Increased lipid uptake was found in melanosphere-derived CSC vs. differentiating melanosphere-derived cells, paralleled by strong expression of lipogenic factors Sterol Regulatory Element-Binding Protein-1 (SREBP-1) and Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ). An inverse relation between lipid-storing phenotype and autophagy was also found, since microtubule-associated protein 1A/1B-Light Chain 3 (LC3) lipidation is reduced in melanosphere-derived CSC. To investigate upstream autophagy regulators, Phospho-AMP activated Protein Kinase (P-AMPK) and Phospho-mammalian Target of Rapamycin (P-mTOR) were analyzed; lower P-AMPK and higher P-mTOR expression in melanosphere-derived CSC were found, thus explaining, at least in part, their lower autophagic activity. In addition, co-localization of LC3-stained autophagosome spots and perilipin-stained lipid droplets was demonstrated mainly in differentiating melanosphere-derived cells, further supporting the role of autophagy in lipid droplets clearance. The present manuscript demonstrates an inverse relationship between lipid-storing phenotype and melanoma stem cells differentiation, providing novel indications involving autophagy in melanoma stem cells biology.

Reduction by coffee consumption of prostate cancer risk: Evidence from the Moli-sani cohort and cellular models.

Meta-analytic data on the effect of coffee in prostate cancer risk are controversial. Caffeine as a bioactive compound of coffee has not yet been studied in deep in vitro. Our study aimed at evaluating in a population cohort the effect of Italian-style coffee consumption on prostate cancer risk and at investigating in vitro the potential antiproliferative and antimetastatic activity of caffeine on prostate cancer cell lines. 6,989 men of the Moli-sani cohort aged ≥50 years were followed for a mean of 4.24 ± 1.35 years and 100 new prostate cancer cases were identified. The European Prospective Investigation into Cancer and Nutrition-Food Frequency Questionnaire was used for the dietary assessment and the evaluation of Italian-style coffee consumption. Two human prostate cancer cell lines, PC-3 and DU145, were tested with increasing concentrations of caffeine, and their proliferative/metastatic features were evaluated. The newly diagnosed prostate cancer participants presented lower coffee consumption (60.1 ± 51.3 g/day) compared to the disease-free population (74.0 ± 51.7 g/day) (p < 0.05). Multiadjusted analysis showed that the subjects at highest consumption (>3 cups/day) had 53% lower prostate cancer risk as compared to participants at the lowest consumption (0-2 cups/day) (p = 0.02). Both human prostate cancer cell lines treated with caffeine showed a significant reduction in their proliferative and metastatic behaviors (p < 0.05). In conclusion, reduction by Italian-style coffee consumption of prostate cancer risk (>3 cups/day) was observed in epidemiological level. Caffeine appeared to exert both antiproliferative and antimetastatic activity on two prostate cancer cell lines, thus providing a cellular confirmation for the cohort study results.

Transglutaminase type 2 affects cell migration through post-translational modification of platelet-derived growth factor-BB.

Migration is a key cellular function with important implications in cell physiology. Impairment of such function is observed in angiogenesis, cancer, central nervous system development, and many other physiological and pathological events. Serum is considered among the most potent physiological chemotactic stimuli. Transglutaminase 2 (TG2) is involved in most of the mentioned processes, suggesting the hypothesis that TG2 may modulate cell movement and chemotaxis by acting on serum factors. Cell biology and biochemistry studies confirmed this hypothesis, showing that human serum contains potent chemotactic signals significantly impaired by activated TG2. Bioinformatics studies indicated that one potent serum factor potential substrate of TG2-dependent transamidation is platelet-derived growth factor-BB (PDGF-BB). Cell biology and immunometric experiments carried out with U87MG human glioma cell line showed that human recombinant PDGF-BB pre-incubated with calcium-activated TG2 lost about 70 % of its chemotactic activity and antigenicity. These data indicate that PDGF-BB is a substrate of TG2-transamidating activity, and such modification may play a key role in the modulation of PDGF's chemotactic features. Further, these findings suggest a novel point of view to study the extracellular functions of TG2 and to understand how protein signals, such as growth factors and cytokines, act in the extracellular space to reach their specific targets.

PDGFR-alpha inhibits melanoma growth via CXCL10/IP-10: a multi-omics approach.

Melanoma is the most aggressive skin-cancer, showing high mortality at advanced stages. Platelet Derived Growth Factor Receptor-alpha (PDGFR-alpha) potently inhibits melanoma- and endothelium-proliferation and its expression is significantly reduced in melanoma-biopsies, suggesting that melanoma progression eliminates cells expressing PDGFR-alpha. In the present study transient overexpression of PDGFR-alpha in endothelial (HUVEC) and melanoma (SKMel-28, A375, Preyer) human-cells shows strong anti-proliferative effects, with profound transcriptome and miRNome deregulation. PDGFR-alpha overexpression strongly affects expression of 82 genes in HUVEC (41 up-, 41 down-regulated), and 52 genes in SKMel-28 (43 up-, 9 down-regulated). CXCL10/IP-10 transcript showed up to 20 fold-increase, with similar changes detectable at the protein level. miRNA expression profiling in cells overexpressing PDGFR-alpha identified 14 miRNAs up- and 40 down-regulated, with miR-503 being the most down-regulated (6.4 fold-reduction). miR-503, miR-630 and miR-424 deregulation was confirmed by qRT-PCR. Interestingly, the most upregulated transcript (i.e., CXCL10/IP-10) was a validated miR-503 target and CXCL10/IP-10 neutralization significantly reverted the anti-proliferative action of PDGFR-alpha, and PDGFR-alpha inhibition by Dasatinb totally reverted the CXCL10/IP10 induction, further supporting a functional interplay of these factors. Finally, integration of transcriptomics and miRNomics data highlighted several pathways affected by PDGFR-alpha.This study demonstrates for the first time that PDGFR-alpha strongly inhibits endothelial and melanoma cells proliferation in a CXCL10/IP-10 dependent way, via miR-503 down-regulation.

Aloe-emodin exerts a potent anticancer and immunomodulatory activity on BRAF-mutated human melanoma cells.

Aim of this study was to extend the knowledge on the antineoplastic effect of aloe-emodin (AE), a natural hydroxyanthraquinone compound, both in metastatic human melanoma cell lines and in primary stem-like cells (melanospheres). Treatment with AE caused reduction of cell proliferation and induction of SK-MEL-28 and A375 cells differentiation, characterized by a marked increase of transamidating activity of transglutaminase whose expression remained unmodified. In vitro antimetastatic property of AE was evaluated by adhesion and Boyden chamber invasion assays. The effect of AE on melanoma cytokines/chemokines production was determined by a multiplex assay: interestingly AE showed an immunomodulatory activity through GM-CSF and IFN-γ production. We report also that AE significantly reduced the proliferation, stemness and invasive potential of melanospheres. Moreover, AE treatment significantly enhanced dabrafenib (a BRAF inhibitor) antiproliferative activity in BRAF mutant cell lines. Our results confirm that AE possesses remarkable antineoplastic properties against melanoma cells, indicating this anthraquinone as a promising agent for differentiation therapy of cancer, or as adjuvant in chemotherapy and targeted therapy. Further, its mechanisms of action support a potential efficacy of AE treatment to counteract resistance of BRAF-mutated melanoma cells to target therapy.

Inhibition of cell proliferation, migration and invasion of B16-F10 melanoma cells by α-mangostin.

In this study, we have evaluated the potential antineoplastic effects of α-mangostin (α-M), the most representative xanthone in Garcinia mangostana pericarp, on melanoma cell lines. This xanthone markedly inhibits the proliferation of high-metastatic B16-F10 melanoma cells. Furthermore, by deeply analyzing which steps in the metastatic process are influenced by xanthone it was observed that α-M strongly interferes with homotypic aggregation, adhesion, plasticity and invasion ability of B16-F10 cells, probably by the observed reduction of metalloproteinase-9 activity. The antiproliferative and antimetastatic properties of α-M have been established in human SK-MEL-28 and A375 melanoma cells. In order to identify pathways potentially involved in the antineoplastic properties of α-M, a comparative mass spectrometry proteomic approach was employed. These findings may improve our understanding of the molecular mechanisms underlying the anti-cancer effects of α-M on melanoma.

Differential denaturation of serum proteome reveals a significant amount of hidden information in complex mixtures of proteins.

UNLABELLED: Recently developed proteomic technologies allow to profile thousands of proteins within a high-throughput approach towards biomarker discovery, although results are not as satisfactory as expected. In the present study we demonstrate that serum proteome denaturation is a key underestimated feature; in fact, a new differential denaturation protocol better discriminates serum proteins according to their electrophoretic mobility as compared to single-denaturation protocols. Sixty nine different denaturation treatments were tested and the 3 most discriminating ones were selected (TRIDENT analysis) and applied to human sera, showing a significant improvement of serum protein discrimination as confirmed by MALDI-TOF/MS and LC-MS/MS identification, depending on the type of denaturation applied. Thereafter sera from mice and patients carrying cutaneous melanoma were analyzed through TRIDENT. Nine and 8 protein bands were found differentially expressed in mice and human melanoma sera, compared to healthy controls (p<0.05); three of them were found, for the first time, significantly modulated: α2macroglobulin (down-regulated in melanoma, p<0.001), Apolipoprotein-E and Apolipoprotein-A1 (both up-regulated in melanoma, p<0.04), both in mice and humans. The modulation was confirmed by immunological methods. Other less abundant proteins (e.g. gelsolin) were found significantly modulated (p<0.05). CONCLUSIONS: i) serum proteome contains a large amount of information, still neglected, related to proteins folding; ii) a careful serum denaturation may significantly improve analytical procedures involving complex protein mixtures; iii) serum differential denaturation protocol highlights interesting proteomic differences between cancer and healthy sera.