Anna Ferretta

Effect of resveratrol on mitochondrial function: implications in parkin-associated familiar Parkinson's disease

Mitochondrial dysfunction and oxidative stress occur in Parkinsons disease (PD), but the molecular mechanisms controlling these events are not completely understood. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) is a transcriptional coactivator known as master regulator of mitochondrial functions and oxidative metabolism. Recent studies, including one from our group, have highlighted altered PGC-1α activity and transcriptional deregulation of its target genes in PD pathogenesis suggesting it as a new potential therapeutic target. Resveratrol, a natural polyphenolic compound proved to improve mitochondrial activity through the activation of several metabolic sensors resulting in PGC-1α activation. Here we have tested in vitro the effect of resveratrol treatment on primary fibroblast cultures from two patients with early-onset PD linked to different Park2 mutations. We show that resveratrol regulates energy homeostasis through activation of AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) and raise of mRNA expression of a number of PGC-1αs target genes resulting in enhanced mitochondrial oxidative function, likely related to a decrease of oxidative stress and to an increase of mitochondrial biogenesis. The functional impact of resveratrol treatment encompassed an increase of complex I and citrate synthase activities, basal oxygen consumption, and mitochondrial ATP production and a decrease in lactate content, thus supporting a switch from glycolytic to oxidative metabolism. Moreover, resveratrol treatment caused an enhanced macro-autophagic flux through activation of an LC3-independent pathway. Our results, obtained in early-onset PD fibroblasts, suggest that resveratrol may have potential clinical application in selected cases of PD-affected patients.

Aurora kinase B inhibition reduces the proliferation of metastatic melanoma cells and enhances the response to chemotherapy

BackgroundThe poor response to chemotherapy and the brief response to vemurafenib in metastatic melanoma patients, make the identification of new therapeutic approaches an urgent need. Interestingly the increased expression and activity of the Aurora kinase B during melanoma progression suggests it as a promising therapeutic target.MethodsThe efficacy of the Aurora B kinase inhibitor barasertib-HQPA was evaluated in BRAF mutated cells, sensitive and made resistant to vemurafenib after chronic exposure to the drug, and in BRAF wild type cells. The drug effectiveness has been evaluated as cell growth inhibition, cell cycle progression and cell migration. In addition, cellular effectors of drug resistance and response were investigated.ResultsThe characterization of the effectors responsible for the resistance to vemurafenib evidenced the increased expression of MITF or the activation of Erk1/2 and p-38 kinases in the newly established cell lines with a phenotype resistant to vemurafenib. The sensitivity of cells to barasertib-HQPA was irrespective of BRAF mutational status. Barasertib-HQPA induced the mitotic catastrophe, ultimately causing apoptosis and necrosis of cells, inhibited cell migration and strongly affected the glycolytic metabolism of cells inducing the release of lactate. In association i) with vemurafenib the gain in effectiveness was found only in BRAF(V600K) cells while ii) with nab-paclitaxel, the combination was more effective than each drug alone in all cells.ConclusionsThese findings suggest barasertib as a new therapeutic agent and as enhancer of chemotherapy in metastatic melanoma treatment.

Cytoskeletal Alterations and Biomechanical Properties of parkin-Mutant Human Primary Fibroblasts

AbstractParkinson’s disease (PD) is one of the most common neurodegenerative diseases. Genes which have been implicated in autosomal-recessive PD include PARK2 which codes for parkin, an E3 ubiquitin ligase that participates in a variety of cellular activities. In this study, we compared parkin-mutant primary fibroblasts, from a patient with parkin compound heterozygous mutations, to healthy control cells. Western blot analysis of proteins obtained from patient’s fibroblasts showed quantitative differences of many proteins involved in the cytoskeleton organization with respect to control cells. These molecular alterations are accompanied by changes in the organization of actin stress fibers and biomechanical properties, as revealed by confocal laser scanning microscopy and atomic force microscopy. In particular, parkin deficiency is associated with a significant increase of Young’s modulus of null-cells in comparison to normal fibroblasts. The current study proposes that parkin influences the spatial organization of actin filaments, the shape of human fibroblasts, and their elastic response to an external applied force.

Altered protein expression pattern in skin fibroblasts from parkin-mutant early-onset Parkinson's disease patients.

Parkinsons disease (PD) is the most common neurodegenerative movement disorder caused primarily by selective degeneration of the dopaminergic neurons in substantia nigra. In this work the proteomes extracted from primary fibroblasts of two unrelated, hereditary cases of PD patients, with different parkin mutations, were compared with the proteomes extracted from commercial adult normal human dermal fibroblasts (NHDF) and primary fibroblasts from the healthy mother of one of the two patients. The results show that the fibroblasts from the two different cases of parkin-mutant patients display analogous alterations in the expression level of proteins involved in different cellular functions, like cytoskeleton structure-dynamics, calcium homeostasis, oxidative stress response, protein and RNA processing.

The mitochondrial master regulator gene PGC1alpha in novel sporadic melanoma cell lines: correlations with BRAF mutational status

Background Metabolic reprogramming is commonly found in cancer but it is poorly understood in melanoma. Recent works [1,2] provided new insights concerning molecular mechanisms involved in mitochondrial biogenesis of melanoma. This work aims to find possible correlations between pathways involved in the onset and progression of the disease in order to provide supporting information in this field. In particular we studied the behaviour of the mitochondrial master regulator gene PGC1alpha in novel sporadic melanoma cell lines and its relations with BRAF mutational status.

Resveratrol Modulation of Protein Expression in parkin-Mutant Human Skin Fibroblasts: A Proteomic Approach

In this study, we investigated by two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) analysis the effects of resveratrol treatment on skin primary fibroblasts from a healthy subject and from a parkin-mutant early onset Parkinsons disease patient. Parkin, an E3 ubiquitin ligase, is the most frequently mutated gene in hereditary Parkinsons disease. Functional alteration of parkin leads to impairment of the ubiquitin-proteasome system, resulting in the accumulation of misfolded or aggregated proteins accountable for the neurodegenerative process. The identification of proteins differentially expressed revealed that resveratrol treatment can act on deregulated specific biological process and molecular function such as cellular redox balance and protein homeostasis. In particular, resveratrol was highly effective at restoring the heat-shock protein network and the protein degradation systems. Moreover, resveratrol treatment led to a significant increase in GSH level, reduction of GSSG/GSH ratio, and decrease of reduced free thiol content in patient cells compared to normal fibroblasts. Thus, our findings provide an experimental evidence of the beneficial effects by which resveratrol could contribute to preserve the cellular homeostasis in parkin-mutant fibroblasts.

Detrimental effects of melanocortin‐1 receptor (MC1R) variants on the clinical outcomes of BRAF V600 metastatic melanoma patients treated with BRAF inhibitors

Melanocortin‐1 receptor (MC1R) plays a key role in skin pigmentation, and its variants are linked with a higher melanoma risk. The influence of MC1R variants on the outcomes of patients with metastatic melanoma (MM) treated with BRAF inhibitors (BRAFi) is unknown. We studied the MC1R status in a cohort of 53 consecutive BRAF‐mutated patients with MM treated with BRAFi. We also evaluated the effect of vemurafenib in four V600BRAF melanoma cell lines with/without MC1R variants.

New insight into the role of metabolic reprogramming in melanoma cells harboring BRAF mutations.

This study explores the V600BRAF-MITF-PGC-1α axis and compares metabolic and functional changes occurring in primary and metastatic V600BRAF melanoma cell lines. V600BRAF mutations in homo/heterozygosis were found to be correlated to high levels of pERK, to downregulate PGC-1α/β, MITF and tyrosinase activity, resulting in a reduced melanin synthesis as compared to BRAFwt melanoma cells. In this scenario, V600BRAF switches on a metabolic reprogramming in melanoma, leading to a decreased OXPHOS activity and increased glycolytic ATP, lactate, HIF-1α and MCT4 levels. Furthermore, the induction of autophagy and the presence of ER stress markers in V600BRAF metastatic melanoma cells suggest that metabolic adaptations of these cells occur as compensatory survival mechanisms. For the first time, we underline the role of peIF2α as an important marker of metastatic behaviour in melanoma. Our results suggest the hypothesis that inhibition of the glycolytic pathway, inactivation of peIF2α and a reduction of basal autophagy could be suitable targets for novel combination therapies in a specific subgroup of metastatic melanoma.

Barasertib: a novel approach for the treatment of metastatic melanoma

Background Metastatic melanoma represents the most deadly form of skin cancer. The poor response to chemotherapy and the brief response to the anti-BRAF vemurafenib in selected population of patients, make the identification of new therapeutic approaches an urgent need. Our goal is the evaluation of the efficacy of barasertib, an aurora B kinase inhibitor impairing cytokinesis, in both mutated and non-mutated melanoma cell lines. Materials and methods Panel of melanoma cells: BRAFV600E mutated cells (MBA72 and Hmel1), the same cell in which the resistance to vemurafenib was induced by chronic exposure to it (MBA72R and Hmel1R) and BRAF wt (HBL and LND1). Cells were characterized for vemurafenib and barasertib effectiveness on cell growth by MTT assay after 3 and 6 days of continuous exposure. Cell cycle was determined by flowcytometry and migration was evaluated by wound-healing assay. Results Cells with BRAFV600E mutation are sensitive to vemurafenib conversely, those with BRAF wt and the resistant ones showed an IC50 of at least 10 folds higher. 3 days-barasertib exposure strongly reduced cell growth (30-60% at 30 and 300nM, respectively) in all cell lines; when the drug was given together with vemurafenib, no gain in effectiveness was evident. Prolonged exposure to barasertib (6 days) showed a progressive increase of effectiveness particularly in cells BRAF wt. The analysis of cell death mechanisms involved in determining the effectiveness of barasertib and vemurafenib showed that the first drug induced both apoptosis and necrosis conversely, the latter mainly apoptosis. Cell cycle analysis demonstrated that barasertib induced an increase in cell size and in polyploidia, suggesting also the mitotic catastrophe as a further cell death mechanism. Moreover, the anti-metastatic behaviour of this agent has been evaluated in function of drug concentration and time exposure. Preliminary results showed a strong reduction of cell migration after drug exposure. Conclusions

The relevance of BRAF G469A mutation in determining the response to therapy in metastatic melanoma

Background BRAF G469A is a missense mutation within exon 11 of the BRAF gene resulting in a constitutively active state of the enzyme responsible also for the protein localization to the mitochondria. The BRAF G469A mutation, frequently recovers in lung cancer, is rare in melanoma and uncertain is its association with a more aggressive disease. BRAF G469A mutation is frequently associated to MAP kinase cascade signaling activation, however no evidence currently exists about its role in determining sensitivity/resistance to BRAF inhibitors vemurafenib or dabrafenib. In our Institute, a patient with metastatic melanoma (MM) was treated with fotemustine, however the disease progressed. From patient biopsy, a new metastatic melanoma cell line has been established and named Mo-1. Here, we investigated the sensitivity of Mo-1 cells to vemurafenib and abraxane, both already approved for the treatment of melanoma carrying BRAF V600 mutations, comparing it with that found in MM cells wild type for BRAF or mutated in V600. Furthermore, a biomarker for the response to abraxane is hypothesized.