Curcumin induces a fatal energetic impairment in tumor cells in vitro and in vivo by inhibiting ATP-synthase activity.

Curcumin has been reported to inhibit inflammation, tumor growth, angiogenesis and metastasis by decreasing cell growth and by inducing apoptosis mainly through the inhibition of nuclear factor kappa-B (NFκB), a master regulator of inflammation. Recent reports also indicate potential metabolic effects of the polyphenol, therefore we analyzed whether and how it affects the energy metabolism of tumor cells. We show that curcumin (10 µM) inhibits the activity of ATP synthase in isolated mitochondrial membranes leading to a dramatic drop of ATP and a reduction of oxygen consumption in in vitro and in vivo tumor models. The effects of curcumin on ATP synthase are independent of the inhibition of NFκB since the IκB Kinase inhibitor, SC-514, does not affect ATP synthase. The activities of the glycolytic enzymes hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase are only slightly affected in a cell type-specific manner. The energy impairment translates into decreased tumor cell viability. Moreover, curcumin induces apoptosis by promoting the generation of reactive oxygen species (ROS) and malondialdehyde (MDA), a marker of lipid oxidation, and autophagy, at least in part due to the activation of the AMP-activated protein kinase (AMPK). According to the in vitro anti-tumor effect, curcumin (30 mg/kg body weight) significantly delayed in vivo cancer growth likely due to an energy impairment but also through the reduction of tumor angiogenesis. These results establish the ATP synthase, a central enzyme of the cellular energy metabolism, as a target of the antitumoral polyphenol leading to inhibition of cancer cell growth and a general reprogramming of tumor metabolism.

Targeting of Ubiquitin E3 Ligase RNF5 as a Novel Therapeutic Strategy in Neuroectodermal Tumors.

RNF5, an endoplasmic reticulum (ER) E3 ubiquitin ligase, participates to the ER-associated protein degradation guaranteeing the protein homeostasis. Depending on tumor model tested, RNF5 exerts pro- or anti-tumor activity. The aim of this study was to elucidate the controversial role of RNF5 in neuroblastoma and melanoma, two neuroectodermal tumors of infancy and adulthood, respectively. RNF5 gene levels are evaluated in publicly available datasets reporting the gene expression profile of melanoma and neuroblastoma primary tumors at diagnosis. The therapeutic effect of Analog-1, an RNF5 pharmacological activator, was investigated on in vitro and in vivo neuroblastoma and melanoma models. In both neuroblastoma and melanoma patients the high expression of RNF5 correlated with a better prognostic outcome. Treatment of neuroblastoma and melanoma cell lines with Analog-1 reduced cell viability by impairing the glutamine availability and energy metabolism through inhibition of F1Fo ATP-synthase activity. This latter event led to a marked increase in oxidative stress, which, in turn, caused cell death. Similarly, neuroblastoma- and melanoma-bearing mice treated with Analog-1 showed a significant delay of tumor growth in comparison to those treated with vehicle only. These findings validate RNF5 as an innovative drug target and support the development of Analog-1 in early phase clinical trials for neuroblastoma and melanoma patients.

Post-SARS-CoV-2 Acute Telogen Effluvium: An Expected Complication.

Post-SARS-CoV-2 telogen effluvium has been described in case reports of COVID-19 patients. We evaluated the prevalence of post-SARS-CoV-2 telogen effluvium in patients from a single medical center, exploring any causal links with the infection. Our hospital-based, cross-sectional study was conducted with patient participants discharged with a diagnosis of SARS-CoV-2 pneumonia from 1 March to 4 April 2020. All patients were evaluated by the same senior dermatologist; a clinical/dermatoscopic evaluation was performed. Alopecia was assessed in 31.3% of patients, with a significant difference in sex (females 73%, males 26.7%). The average time detected from the onset of the first symptoms to alopecia was 68.43 days. Overall, there were no significant associations between alopecia and COVID-19-related features (length of hospitalization, virologic positivity, or duration of fever), treatment characteristics, or laboratory findings. In this paper, we report that post-infection acute telogen effluvium occurs in a significant number of COVID-19 patients. The burden of this condition may impair the quality of life, with a significant impact on individuals.

Starvation-dependent differential stress resistance protects normal but not cancer cells against high-dose chemotherapy.

Strategies to treat cancer have focused primarily on the killing of tumor cells. Here, we describe a differential stress resistance (DSR) method that focuses instead on protecting the organism but not cancer cells against chemotherapy. Short-term starved S. cerevisiae or cells lacking proto-oncogene homologs were up to 1,000 times better protected against oxidative stress or chemotherapy drugs than cells expressing the oncogene homolog Ras2(val19). Low-glucose or low-serum media also protected primary glial cells but not six different rat and human glioma and neuroblastoma cancer cell lines against hydrogen peroxide or the chemotherapy drug/pro-oxidant cyclophosphamide. Finally, short-term starvation provided complete protection to mice but not to injected neuroblastoma cells against a high dose of the chemotherapy drug/pro-oxidant etoposide. These studies describe a starvation-based DSR strategy to enhance the efficacy of chemotherapy and suggest that specific agents among those that promote oxidative stress and DNA damage have the potential to maximize the differential toxicity to normal and cancer cells.

Immunogenicity of human mesenchymal stem cells in HLA-class I-restricted T-cell responses against viral or tumor-associated antigens.

Human mesenchymal stem cells (MSC) are immunosuppressive and poorly immunogenic but may act as antigen-presenting cells (APC) for CD4(+) T-cell responses; here we have investigated their ability to serve as APC for in vitro CD8(+) T-cell responses. MSC pulsed with peptides from viral antigens evoked interferon (IFN)-gamma and Granzyme B secretion in specific cytotoxic T lymphocytes (CTL) and were lysed, although with low efficiency. MSC transfected with tumor mRNA or infected with a viral vector carrying the Hepatitis C virus NS3Ag gene induced cytokine release but were not killed by specific CTL, even following pretreatment with IFN-gamma. To investigate the mechanisms involved in MSC resistance to CTL-mediated lysis, we analyzed expression of human leukocyte antigen (HLA) class I-related antigen-processing machinery (APM) components and of immunosuppressive HLA-G molecules in MSC. The LMP7, LMP10, and ERp57 components were not expressed and the MB-1 and zeta molecules were downregulated in MSC either unmanipulated or pretreated with IFN-gamma. Surface HLA-G was constitutively expressed on MSC but was not involved in their protection from CTL-mediated lysis. MSC supernatants containing soluble HLA-G (sHLA-G) inhibited CTL-mediated lysis, whereas those lacking sHLA-G did not. The role of sHLA-G in such inhibition was unambiguously demonstrated by partial restoration of lysis following sHLA-G depletion from MSC supernatants. In conclusion, human MSC can process and present HLA class I-restricted viral or tumor antigens to specific CTL with a limited efficiency, likely because of some defects in APM components. However, they are protected from CTL-mediated lysis through a mechanism that is partly sHLA-G-dependent.

Effect of starvation on brain glucose metabolism and 18F-2-fluoro-2-deoxyglucose uptake: an experimental in-vivo and ex-vivo study.

BACKGROUND: The close connection between neuronal activity and glucose consumption accounts for the clinical value of 18F-fluoro-2-deoxyglucose (FDG) imaging in neurodegenerative disorders. Nevertheless, brain metabolic response to starvation (STS) might hamper the diagnostic accuracy of FDG PET/CT when the cognitive impairment results in a severe food deprivation. METHODS: Thirty six-week-old BALB/c female mice were divided into two groups: "control" group (n = 15) were kept under standard conditions and exposed to fasting for 6 h before the study; the remaining "STS" mice were submitted to 48 h STS (absence of food and free access to water) before imaging. In each group, nine mice were submitted to dynamic micro-PET imaging to estimate brain and skeletal muscle glucose consumption (C- and SM-MRGlu*) by Patlak approach, while six mice were sacrificed for ex vivo determination of the lumped constant, defined as the ratio between CMRGlu* and glucose consumption measured by glucose removal from the incubation medium (n = 3) or biochemical analyses (n = 3), respectively. RESULTS: CMRGlu* was lower in starved than in control mice (46.1 ± 23.3 vs 119.5 ± 40.2 nmol × min-1 × g-1, respectively, p < 0.001). Ex vivo evaluation documented a remarkable stability of lumped constant as documented by the stability of GLUT expression, G6Pase activity, and kinetic features of hexokinase-catalyzed phosphorylation. However, brain SUV in STS mice was even (though not significantly) higher with respect to control mice. Conversely, a marked decrease in both SM-MRGlu* and SM-SUV was documented in STS mice with respect to controls. CONCLUSIONS: STS markedly decreases brain glucose consumption without altering measured FDG SUV in mouse experimental models. This apparent paradox does not reflect any change in lumped constant. Rather, it might be explained by the metabolic response of the whole body: the decrease in FDG sequestration by the skeletal muscle is as profound as to prolong tracer persistence in the bloodstream and thus its availability for brain uptake.

Targeting of PHOX2B expression allows the identification of drugs effective in counteracting neuroblastoma cell growth.

The pathogenic role of the PHOX2B gene in neuroblastoma is indicated by heterozygous mutations in neuroblastoma patients and by gene overexpression in both neuroblastoma cell lines and tumor samples. PHOX2B encodes a transcription factor which is crucial for the correct development and differentiation of sympathetic neurons. PHOX2B overexpression is considered a prognostic marker for neuroblastoma and it is also used by clinicians to monitor minimal residual disease. Furthermore, it has been observed that neuronal differentiation in neuroblastoma is dependent on down-regulation of PHOX2B expression, which confirms that PHOX2B expression may be considered a target in neuroblastoma. Here, PHOX2B promoter or 3' untranslated region were used as molecular targets in an in vitro high-throughput approach that led to the identification of molecules able to decrease PHOX2B expression at transcriptional and likely even at post-transcriptional levels. Further functional investigations carried out on PHOX2B mRNA levels and biological consequences, such as neuroblastoma cell apoptosis and growth, showed that chloroquine and mycophenolate mofetil are most promising agents for neuroblastoma therapy based on down-regulation of PHOX2B expression. Finally, a strong correlation between the effect of drugs in terms of down-regulation of PHOX2B expression and of biological consequences in neuroblastoma cells confirms the role of PHOX2B as a potential molecular target in neuroblastoma.

Pancreatic metastasis from mycosis fungoides mimicking primary pancreatic tumor.

Mycosis fungoides (MF) is a cutaneous T-cell lymphoma that can undergo local progression with possible systemic dissemination. We report a case of a patient affected by MF with a pancreatic mass that was a diagnostic challenge between primitive tumor and pancreatic metastasis from MF. Clinical setting findings and imaging studies raised the suspicion of a pancreatic primary neoplasm. A diagnostic clue was provided by the combined histomorphologic/immunohistochemical study of pancreatic and cutaneous biopsies, which revealed a pancreatic localization of MF. Considering the rarity of metastatic localization of MF to the pancreas, we next investigated whether chemokine-chemokine receptor interactions could be involved in the phenomenon to provide new insight into the possible mechanisms underlying metastatic localization of MF to the pancreas. Histological analyses of archival pancreatic tissue demonstrated that glucagon-secreting cells of the pancreatic islets expressed the CCL27 chemokine, which may have attracted in our case metastatic MF cells expressing the complementary receptor CCR10.

Antitumor effect of combined NAMPT and CD73 inhibition in an ovarian cancer model.

Nicotinamide phosphoribosyltransferase (NAMPT) is a crucial enzyme in the biosynthesis of intracellular NAD+. NAMPT inhibitors have potent anticancer activity in several preclinical models by depleting NAD+ and ATP levels. Recently, we demonstrated that CD73 enables the utilization of extracellular NAD+/nicotinamide mononucleotide (NMN) by converting them to Nicotinamide riboside (NR), which can cross the plasmamembrane and fuel intracellular NAD+ biosynthesis in human cells. These processes are herein confirmed to also occur in a human ovarian carcinoma cell line (OVCAR-3), by means of CD73 or NRK1 specific silencing. Next, we investigated the anti-tumor activity of the simultaneous inhibition of NAMPT (with FK866) and CD73 (with α, ß-methylene adenosine 5'-diphosphate, APCP), in an in vivo human ovarian carcinoma model. Interestingly, the combined therapy was found to significantly decrease intratumor NAD+, NMN and ATP levels, compared with single treatments. In addition, the concentration of these nucleotides in ascitic exudates was more remarkably reduced in animals treated with both FK866 and APCP compared with single treatments. Importantly, tumors treated with FK866 in combination with APCP contained a statistically significant lower proportion of Ki67 positive proliferating cells and a higher percentage of necrotic area. Finally, a slight but significant increase in animal survival in response to the combined therapy, compared to the single agents, could be demonstrated. Our results indicate that the pharmacological inhibition of CD73 enzymatic activity could be considered as a means to potentiate the anti-cancer effects of NAMPT inhibitors.

Divergent targets of glycolysis and oxidative phosphorylation result in additive effects of metformin and starvation in colon and breast cancer.

Emerging evidence demonstrates that targeting energy metabolism is a promising strategy to fight cancer. Here we show that combining metformin and short-term starvation markedly impairs metabolism and growth of colon and breast cancer. The impairment in glycolytic flux caused by starvation is enhanced by metformin through its interference with hexokinase II activity, as documented by measurement of 18F-fluorodeoxyglycose uptake. Oxidative phosphorylation is additively compromised by combined treatment: metformin virtually abolishes Complex I function; starvation determines an uncoupled status of OXPHOS and amplifies the activity of respiratory Complexes II and IV thus combining a massive ATP depletion with a significant increase in reactive oxygen species. More importantly, the combined treatment profoundly impairs cancer glucose metabolism and virtually abolishes lesion growth in experimental models of breast and colon carcinoma. Our results strongly suggest that energy metabolism is a promising target to reduce cancer progression.

Discovery of a novel glucose metabolism in cancer: The role of endoplasmic reticulum beyond glycolysis and pentose phosphate shunt.

Cancer metabolism is characterized by an accelerated glycolytic rate facing reduced activity of oxidative phosphorylation. This "Warburg effect" represents a standard to diagnose and monitor tumor aggressiveness with (18)F-fluorodeoxyglucose whose uptake is currently regarded as an accurate index of total glucose consumption. Studying cancer metabolic response to respiratory chain inhibition by metformin, we repeatedly observed a reduction of tracer uptake facing a marked increase in glucose consumption. This puzzling discordance brought us to discover that (18)F-fluorodeoxyglucose preferentially accumulates within endoplasmic reticulum by exploiting the catalytic function of hexose-6-phosphate-dehydrogenase. Silencing enzyme expression and activity decreased both tracer uptake and glucose consumption, caused severe energy depletion and decreased NADPH content without altering mitochondrial function. These data document the existence of an unknown glucose metabolism triggered by hexose-6-phosphate-dehydrogenase within endoplasmic reticulum of cancer cells. Besides its basic relevance, this finding can improve clinical cancer diagnosis and might represent potential target for therapy.

Fasting induces anti-Warburg effect that increases respiration but reduces ATP-synthesis to promote apoptosis in colon cancer models.

Tumor chemoresistance is associated with high aerobic glycolysis rates and reduced oxidative phosphorylation, a phenomenon called "Warburg effect" whose reversal could impair the ability of a wide range of cancer cells to survive in the presence or absence of chemotherapy. In previous studies, Short-term-starvation (STS) was shown to protect normal cells and organs but to sensitize different cancer cell types to chemotherapy but the mechanisms responsible for these effects are poorly understood. We tested the cytotoxicity of Oxaliplatin (OXP) combined with a 48hour STS on the progression of CT26 colorectal tumors. STS potentiated the effects of OXP on the suppression of colon carcinoma growth and glucose uptake in both in vitro and in vivo models. In CT26 cells, STS down-regulated aerobic glycolysis, and glutaminolysis, while increasing oxidative phosphorylation. The STS-dependent increase in both Complex I and Complex II-dependent O(2) consumption was associated with increased oxidative stress and reduced ATP synthesis. Chemotherapy caused additional toxicity, which was associated with increased succinate/Complex II-dependent O(2) consumption, elevated oxidative stress and apoptosis .These findings indicate that the glucose and amino acid deficiency conditions imposed by STS promote an anti-Warburg effect characterized by increased oxygen consumption but failure to generate ATP, resulting in oxidative damage and apoptosis.

Immunosuppressive microenvironment in neuroblastoma.

According to the cancer immunoediting model, the interplay between tumor cells and the host immune system is crucial for the control of tumor growth. NB is a pediatric tumor that presents with metastatic disease at diagnosis in about 50% of the cases, the majority of which have poor prognosis. In this Review article, immune escape pathways adopted by human neuroblastoma (NB) cells are reviewed. These include intrinsic defects of tumor cells such impaired expression of the HLA class I related antigen processing machinery and functional alterations of the tumor microenvironment (TM) induced by NB cell-derived immunosuppressive molecules as MICA and HLA-G. Finally, examples of therapeutic interventions targeting the TM are discussed to emphasize the concept that successful cancer treatment may be achieved using this strategy.

Role of BAFF in Opsoclonus-Myoclonus syndrome, a bridge between cancer and autoimmunity.

OMS is a rare paraneoplastic disorder that affects adults and children. Pediatric OMS is often associated with NB, a common, solid tumor of childhood, derived from the sympathetic nervous system. The detection of autoantibodies and lymphocytic infiltration in NB patients led to advance an autoimmune hypothesis for the pathogenesis of OMS-related NB. BAFF is a potent modulator of B cell growth and survival upon interaction with its receptors BAFF-R and BCMA. The aim of this study was to investigate mechanism(s) involved in ectopic lymphoid neogenesis in OMS-associated NB. We investigated BAFF, BAFF-R, and BCMA expression in NB tumors associated or not with OMS. Furthermore, we evaluated BAFF expression and secretion in NB cell lines, treated or untreated with differentiating agents. Immunohistochemically, lymphocytes infiltrating NB tumors from patients, with or without OMS, expressed BAFF, BAFF-R, and BCMA, whereas neuroblasts expressed BAFF and BCMA but not BAFF-R. By flow cytometry, BAFF was found to be consistently expressed in NB cell lines. Similarly to the results obtained in tissue lesions, BCMA but not BAFF-R was detected on the surface of all NB cell lines under basal conditions. De novo synthesis of BAFF-R and up-regulation of BCMA were observed in NB cell lines upon treatment with IFN-γ or 13-cis retinoic acid. This study provides new insights in the mechanisms driving the neogenesis of lymphoid follicles and in the functional interactions between tumor and immune cells in OMS-associated NB.

Immunosuppressive treatments in acute myocardial infarction and stroke.

Atherosclerosis is an inflammatory disease that involves formation of atherosclerotic lesions characterized by deposition of lipids and cell debris in the arterial wall, fibrosis and recruitment of various cell types including smooth muscle, endothelial, immune and foam cells. Progressive enlargement of the atherosclerotic plaques together with development of necrosis, intraplaque hemorrhage and ulceration results into rupture of the plaques, with subsequent exposure to thrombotic material and occlusion of the artery. These phenomena culminate in myocardial infarction when they occur in the coronary arteries or stroke when cerebral arteries are affected. Several lines of evidence indicated that innate and adaptive immunity tightly regulate atherogenesis. In particular, activated T cells influence the stability of the atherosclerotic plaque and promote disease progression. Experiments performed on suitable animal models allowed to identify CD4+ lymphocytes as the major T cell subpopulation involved in atherogenesis. Furthermore, immunophenotypic and functional analyses demonstrated that human and murine plaques contain predominantly CD4+ T Helper (TH)1 cells producing proinflammatory cytokines. Taken together, these observations have fostered the evaluation in preclinical models of whether or not immunosuppressive drugs may represent an efficacious therapeutic strategy for atherosclerosis-associated diseases. This review focuses on the role of innate and adaptive immunity in atherogenesis and atherosclerosis progression, and discusses the potential immunosuppressive approaches for the treatment of patients affected by acute myocardial infarction and stroke, with particular emphasis on the use of tacrolimus and stem cell transplantation.

Close interactions between mesenchymal stem cells and neuroblastoma cell lines lead to tumor growth inhibition.

Mesenchymal stem cells (MSCs) have attracted much interest in oncology since they exhibit marked tropism for the tumor microenvironment and support or suppress malignant cell growth depending on the tumor model tested. The aim of this study was to investigate the role of MSCs in the control of the growth of neuroblastoma (NB), which is the second most common solid tumor in children. In vivo experiments showed that systemically administered MSCs, under our experimental conditions, did not home to tumor sites and did not affect tumor growth or survival. However, MSCs injected intratumorally in an established subcutaneous NB model reduced tumor growth through inhibition of proliferation and induction of apoptosis of NB cells and prolonged the survival of hMSC-treated mice. The need for contact between MSCs and NB cells was further supported by in vitro experiments. In particular, MSCs were found to be attracted by NB cells, and to affect NB cell proliferation with different results depending on the cell line tested. Moreover, NB cells, after pre-incubation with hMSCs, acquired a more invasive behavior towards CXCL12 and the bone marrow, i.e., the primary site of NB metastases. In conclusion, this study demonstrates that functional cross-talk between MSCs and NB cell lines used in our experiments can occur only within short range interaction. Thus, this report does not support the clinical use of MSCs as vehicles for selective delivery of antitumor drugs at the NB site unless chemotherapy and/or radiotherapy create suitable local conditions for MSCs recruitment.

Expression of P2X7 receptor increases in vivo tumor growth.

The P2X7 receptor is an ATP-gated ion channel known for its cytotoxic activity. However, recent evidence suggests a role for P2X7 in cell proliferation. Here, we found that P2X7 exhibits significant growth-promoting effects in vivo. Human embryonic kidney cells expressing P2X7 exhibited a more tumorigenic and anaplastic phenotype than control cells in vivo, and the growth rate and size of these tumors were significantly reduced by intratumoral injection of the P2X7 inhibitor-oxidized ATP. The accelerated growth of P2X7-expressing tumors was characterized by increased proliferation, reduced apoptosis, and a high level of activated transcription factor NFATc1. These tumors also showed a more developed vascular network than control tumors and secreted elevated amounts of VEGF. The growth and neoangiogenesis of P2X7-expressing tumors was blocked by intratumoral injection of the VEGF-blocking antibody Avastin (bevacizumab), pharmacologic P2X7 blockade, or P2X7 silencing in vivo. Immunohistochemistry revealed strong P2X7 positivity in several human cancers. Together, our findings provide direct evidence that P2X7 promotes tumor growth in vivo.

Fasting cycles retard growth of tumors and sensitize a range of cancer cell types to chemotherapy.

Short-term starvation (or fasting) protects normal cells, mice, and potentially humans from the harmful side effects of a variety of chemotherapy drugs. Here, we show that treatment with starvation conditions sensitized yeast cells (Saccharomyces cerevisiae) expressing the oncogene-like RAS2(val19) to oxidative stress and 15 of 17 mammalian cancer cell lines to chemotherapeutic agents. Cycles of starvation were as effective as chemotherapeutic agents in delaying progression of different tumors and increased the effectiveness of these drugs against melanoma, glioma, and breast cancer cells. In mouse models of neuroblastoma, fasting cycles plus chemotherapy drugs--but not either treatment alone--resulted in long-term cancer-free survival. In 4T1 breast cancer cells, short-term starvation resulted in increased phosphorylation of the stress-sensitizing Akt and S6 kinases, increased oxidative stress, caspase-3 cleavage, DNA damage, and apoptosis. These studies suggest that multiple cycles of fasting promote differential stress sensitization in a wide range of tumors and could potentially replace or augment the efficacy of certain chemotherapy drugs in the treatment of various cancers.

Receptor activator of NF- κB ligand (RANKL) increases the release of neutrophil products associated with coronary vulnerability.

The "blood vulnerability", resulting from the complex balance between serum molecules and inflammatory cell atherosclerotic activities, is a major determinant in the evaluation of the "global patient cardiovascular vulnerability". In the present study, we focused on the role of the soluble receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL, a potential marker of coronary calcification and vulnerability) in the release of neutrophilic proteases. Then, the association between these mediators and the degree of coronary calcification (assessed by coronary calcium score [CCS]) was investigated in 20 subjects (aged ≥65 years) asymptomatic for cardiovascular disease. Results showed that RANKL dose-dependently induced matrix metalloprotease (MMP)-8 and MMP-9 release from human primary neutrophils cultured in Teflon dishes (suspension condition, mimicking cells circulating in the blood stream). Conversely, when adherent to polystyrene, neutrophils became unresponsive to RANKL. RANKL did not influence the release of other neutrophilic products in suspension and adherence cultures as well as neutrophil migration. RANKL-induced release of MMPs was dependent on the activation of defined intracellular signalling pathways (PI3K/Akt and ERK1/2). In asymptomatic subjects, serum levels of RANKL, MMP-8 and MMP-9 positively correlated with CCS, reflecting a potential relationship between circulating RANKL and coronary calcification. In conclusion, RANKL increased the release of neutrophilic products potentially related to the "blood" vulnerability via defined intracellular pathways. Serum levels of RANKL might represent a potential biomarker of coronary calcification and related cardiovascular risk.

Structure-activity relationships of novel substituted naphthalene diimides as anticancer agents.

Novel 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) derivatives were synthesized and evaluated for their antiproliferative activity on a wide number of different tumor cell lines. The prototypes of the present series were derivatives 1 and 2 characterized by interesting biological profiles as anticancer agents. The present investigation expands on the study of structure-activity relationships of prototypes 1 and 2, namely, the influence of the different substituents of the phenyl rings on the biological activity. Derivatives 3-22, characterized by a different substituent on the aromatic rings and/or a different chain length varying from two to three carbon units, were synthesized and evaluated for their cytostatic and cytotoxic activities. The most interesting compound was 20, characterized by a linker of three methylene units and a 2,3,4-trimethoxy substituent on the two aromatic rings. It displayed antiproliferative activity in the submicromolar range, especially against some different cell lines, the ability to inhibit Taq polymerase and telomerase, to trigger caspase activation by a possible oxidative mechanism, to downregulate ERK 2 protein and to inhibit ERKs phosphorylation, without acting directly on microtubules and tubuline. Its theoretical recognition against duplex and quadruplex DNA structures have been compared to experimental thermodynamic measurements and by molecular modeling investigation leading to putative binding modes. Taken together these findings contribute to define this compound as potential Multitarget-Directed Ligands interacting simultaneously with different biological targets.