Resveratrol Treatment in Human Parkin-Mutant Fibroblasts Modulates cAMP and Calcium Homeostasis Regulating the Expression of Mitochondria-Associated Membranes Resident Proteins.

Parkin plays an important role in ensuring efficient mitochondrial function and calcium homeostasis. Parkin-mutant human fibroblasts, with defective oxidative phosphorylation activity, showed high basal cAMP level likely ascribed to increased activity/expression of soluble adenylyl cyclase and/or low expression/activity of the phosphodiesterase isoform 4 and to a higher Ca2+ level. Overall, these findings support the existence, in parkin-mutant fibroblasts, of an abnormal Ca2+ and cAMP homeostasis in mitochondria. In our previous studies resveratrol treatment of parkin-mutant fibroblasts induced a partial rescue of mitochondrial functions associated with stimulation of the AMPK/SIRT1/PGC-1α pathway. In this study we provide additional evidence of the potential beneficial effects of resveratrol inducing an increase in the pre-existing high Ca2+ level and remodulation of the cAMP homeostasis in parkin-mutant fibroblasts. Consistently, we report in these fibroblasts higher expression of proteins implicated in the tethering of ER and mitochondrial contact sites along with their renormalization after resveratrol treatment. On this basis we hypothesize that resveratrol-mediated enhancement of the Ca2+ level, fine-tuned by the ER-mitochondria Ca2+ crosstalk, might modulate the pAMPK/AMPK pathway in parkin-mutant fibroblasts.

Behind the Scene: Exploiting MC1R in Skin Cancer Risk and Prevention.

Melanoma and non-melanoma skin cancers (NMSCs) are the most frequent cancers of the skin in white populations. An increased risk in the development of skin cancers has been associated with the combination of several environmental factors (i.e., ultraviolet exposure) and genetic background, including melanocortin-1 receptor (MC1R) status. In the last few years, advances in the diagnosis of skin cancers provided a great impact on clinical practice. Despite these advances, NMSCs are still the most common malignancy in humans and melanoma still shows a rising incidence and a poor prognosis when diagnosed at an advanced stage. Efforts are required to underlie the genetic and clinical heterogeneity of melanoma and NMSCs, leading to an optimization of the management of affected patients. The clinical implications of the impact of germline MC1R variants in melanoma and NMSCs' risk, together with the additional risk conferred by somatic mutations in other peculiar genes, as well as the role of MC1R screening in skin cancers' prevention will be addressed in the current review.

Clinical course of choroidal neovascular membrane in West Nile virus chorioretinitis: a case report.

BACKGROUND: This report describes the clinical course of choroidal neovascular membrane (CNV) in West Nile virus-associated chorioretinitis. CASE PRESENTATION: A 28-year-old Italian woman was referred to our institution because of reduced visual acuity in the left eye dating back 4 months. A diagnosis of retinal vasculitis in the right eye and chorioretinitis with CNV in the left eye was made. A complete workup for uveitis revealed positivity only for anti-West Nile virus immunoglobulin M (IgM), while immunoglobulin G (IgG) was negative. Whole-body computed tomography and nuclear magnetic resonance imaging of the brain were also negative. Therefore, the patient was treated with a combination of oral prednisone (starting dose 1 mg/kg per day) and three intravitreal injections of bevacizumab 1.25 mg/0.05 ml, 1 month apart. Fourteen days from starting corticosteroid therapy and after the first intravitreal injection, the patient experienced increased visual acuity to 0.4. Response to therapy was monitored by clinical examination, ocular coherence tomography (OCT), OCT angiography and retinal fluorescein angiography. Three months later, resolution of CNV in the left eye was achieved and no signs of retinal vasculitis were detected in the right eye, while serum IgM for West Nile virus turned negative and IgG positive. CONCLUSION: CNV may be a complication of West Nile virus-associated chorioretinitis, and only subclinical retinal vasculitis may also be found even in non-endemic regions.

Effect of Cladribine on Neuronal Apoptosis: New Insight of In Vitro Study in Multiple Sclerosis Therapy.

BACKGROUND: Cladribine (2-CdA) can cross the blood-brain barrier, resulting in inhibition of DNA synthesis and repair and disruption of cellular proliferation in actively dividing lymphocytes. No data on effect on neurons are available. AIM: To study "in vitro" 2-CdA apoptotic effects on neurons in healthy donor and multiple sclerosis patient lymphocytes. METHODS: Neuroblastoma cells were co-cultured with lymphocytes, with and without 2-CdA. RESULTS: Apoptosis increased in lymphocytes with 2-CdA; increase was also observed when lymphocytes were cultured with neuronal cells. However, neurons were not affected by 2-CdA for apoptosis. CONCLUSIONS: 2-CdA causes peripheral and central lymphocyte death preserving neurons, with a reasonable impact on inflammation and neuroprotection.

Tomatine Displays Antitumor Potential in In Vitro Models of Metastatic Melanoma.

There is a growing interest in the cytotoxic effects of bioactive glycoalkaloids, such as α-tomatine on tumor cells. Here, for the first time, we determine the antitumor potential of tomatine, a mixture of α-tomatine and dehydrotomatine, in metastatic melanoma (MM) cell lines harboring different BRAF and MC1R variants. We performed cytotoxicity experiments and annexin-V/propidium iodide staining to assess the apoptotic/necrotic status of the cells. ER stress and autophagy markers were revealed by Western Blot, whereas antiangiogenic and vascular-disrupting effects were evaluated through a capillary tube formation assay on matrigel and by ELISA kit for VEGF release determination. Cell invasion was determined by a Boyden chamber matrigel assay. Tomatine reduced 50% of cell viability and induced a concentration-dependent increase of apoptotic cells in the range of 0.5-1 µM in terms of α-tomatine. The extent of apoptosis was more than two-fold higher in V600BRAF-D184H/D184H MC1R cells than in BRAF wild-type cells and V600BRAF-MC1R wild-type cell lines. Additionally, tomatine increased the LC3I/II autophagy marker, p-eIF2α, and p-Erk1/2 levels in BRAF wild-type cells. Notably, tomatine strongly reduced cell invasion and melanoma-dependent angiogenesis by reducing VEGF release and tumor-stimulating effects on capillary tube formation. Collectively, our findings support tomatine as a potential antitumor agent in MM.

Human Ovarian Cancer Tissue Exhibits Increase of Mitochondrial Biogenesis and Cristae Remodeling.

Ovarian cancer (OC) is the most lethal gynecologic cancer characterized by an elevated apoptosis resistance that, potentially, leads to chemo-resistance in the recurrent disease. Mitochondrial oxidative phosphorylation was found altered in OC, and mitochondria were proposed as a target for therapy. Molecular evidence suggests that the deregulation of mitochondrial biogenesis, morphology, dynamics, and apoptosis is involved in carcinogenesis. However, these mitochondrial processes remain to be investigated in OC. Eighteen controls and 16 OC tissues (serous and mucinous) were collected. Enzymatic activities were performed spectrophotometrically, mitochondrial DNA (mtDNA) content was measured by real-time-PCR, protein levels were determined by Western blotting, and mitochondrial number and structure were measured by electron microscopy. Statistical analysis was performed using Student's t-test, Mann-Whitney U test, and principal component analysis (PCA). We found, in OC, that increased mitochondrial number associated with increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and mitochondrial transcription factor A (TFAM) protein levels, as well as mtDNA content. The OC mitochondria presented an increased maximum length, as well as reduced cristae width and junction diameter, associated with increased optic atrophy 1 protein (OPA1) and prohibitin 2 (PHB2) protein levels. In addition, in OC tissues, augmented cAMP and sirtuin 3 (SIRT3) protein levels were observed. PCA of the 25 analyzed biochemical parameters classified OC patients in a distinct group from controls. We highlight a "mitochondrial signature" in OC that could result from cooperation of the cAMP pathway with the SIRT3, OPA1, and PHB2 proteins.

Increased Levels of cAMP by the Calcium-Dependent Activation of Soluble Adenylyl Cyclase in Parkin-Mutant Fibroblasts.

Almost half of autosomal recessive early-onset parkinsonism has been associated with mutations in PARK2, coding for parkin, which plays an important role in mitochondria function and calcium homeostasis. Cyclic adenosine monophosphate (cAMP) is a major second messenger regulating mitochondrial metabolism, and it is strictly interlocked with calcium homeostasis. Parkin-mutant (Pt) fibroblasts, exhibiting defective mitochondrial respiratory/OxPhos activity, showed a significant higher value of basal intracellular level of cAMP, as compared with normal fibroblasts (CTRL). Specific pharmacological inhibition/activation of members of the adenylyl cyclase- and of the phosphodiesterase-families, respectively, as well as quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis, indicate that the higher level of cAMP observed in Pt fibroblasts can contribute to a higher level of activity/expression by soluble adenylyl cyclase (sAC) and to low activity/expression of the phosphodiesterase isoform 4 (PDE4). As Ca2+ regulates sAC, we performed quantitative calcium-fluorimetric analysis, showing a higher level of Ca2+ in the both cytosol and mitochondria of Pt fibroblasts as compared with CTRL. Most notably, inhibition of the mitochondrial Ca2+ uniporter decreased, specifically the cAMP level in PD fibroblasts. All together, these findings support the occurrence of an altered mitochondrial Ca2+-mediated cAMP homeostasis in fibroblasts with the parkin mutation.

Translational control mechanisms in cutaneous malignant melanoma: the role of eIF2α.

BACKGROUND: Melanoma cells develop adaptive responses in order to cope with particular conditions of tumor microenvironment, characterized by stress conditions and deregulated proliferation. Recently, the interplay between the stress response and the gene expression programs leading to metastatic spread has been reported. METHODS: We evaluated levels and localization of eIF2α/peIF2α in V600BRAF and wtBRAF metastatic melanoma cell lines by means of western blot and confocal microscopy analyses. Furthermore, we performed a sequence analyses and structure and dynamics studies of eIF2α protein to reveal the role of eIF2α and its correlations in different pathways involved in the invasive phase of melanoma. RESULTS: We found peIF2α both in cytoplasm and nucleus. Nuclear localization was more represented in V600BRAF melanoma cell lines. Our studies on eIF2α protein sequence indicated the presence of a predicted bipartite NLS as well as a nuclear export signal NES and an S1 domain, typical of RNA interacting proteins. Furthermore, we found high levels of transcription factor EB (TFEB), a component of the MiT/TFE family, and low ß-catenin levels in V600BRAF cells. CONCLUSIONS: Based on our results, we suggest that peIF2α nuclear localization can be crucial in ER stress response and in driving the metastatic spread of melanoma, through lysosomal signaling and Wnt/ß-catenin pathway. In conclusion, this is the first evidence of nuclear localization of peIF2α, representing a possible target for future therapeutic approaches for metastatic melanoma.

Mitochondrial Dynamics of Proximal Tubular Epithelial Cells in Nephropathic Cystinosis.

Nephropathic cystinosis is a rare lysosomal storage disorder caused by mutations in CTNS gene leading to Fanconi syndrome. Independent studies reported defective clearance of damaged mitochondria and mitochondrial fragmentation in cystinosis. Proteins involved in the mitochondrial dynamics and the mitochondrial ultrastructure were analyzed in CTNS-/- cells treated with cysteamine, the only drug currently used in the therapy for cystinosis but ineffective to treat Fanconi syndrome. CTNS-/- cells showed an overexpression of parkin associated with deregulation of ubiquitination of mitofusin 2 and fission 1 proteins, an altered proteolytic processing of optic atrophy 1 (OPA1), and a decreased OPA1 oligomerization. According to molecular findings, the analysis of electron microscopy images showed a decrease of mitochondrial cristae number and an increase of cristae lumen and cristae junction width. Cysteamine treatment restored the fission 1 ubiquitination, the mitochondrial size, number and lumen of cristae, but had no effect on cristae junction width, making CTNS-/- tubular cells more susceptible to apoptotic stimuli.

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 V600 BRAF melanoma cell lines with/without MC1R variants. We found a significant correlation between the presence of MC1R variants and worse outcomes in terms of both overall response rate (ORR; 59% versus 95%, P = 0.011 univariate, P = 0.028 multivariate analysis) and progression-free survival (PFS) shorter than 6 months (72% versus 33%, P = 0.012 univariate, P = 0.027 multivariate analysis). No difference in overall survival (OS) was reported, probably due to subsequent treatments. Data in vitro showed a significant different phosphorylation of Erk1/2 and p38 MAPK during treatment, associated with a greater increase in vemurafenib IC50 in MC1R variant cell lines.

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/ß, 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.

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

BACKGROUND: The 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. METHODS: The 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. RESULTS: The 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. CONCLUSIONS: These findings suggest barasertib as a new therapeutic agent and as enhancer of chemotherapy in metastatic melanoma treatment.

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

Mitochondrial dysfunction and oxidative stress occur in Parkinson's 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-1α's 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.