Pancreatic cancer (PC) is one of the deadliest carcinomas and in most cases, which are diagnosed with locally advanced or metastatic disease, current therapeutic options are highly unsatisfactory. Based on the anti-proliferative effects shown by nitroxoline, an old urinary antibacterial agent, we explored a large library of newly synthesised derivatives to unravel the importance of the OH moiety and pyridine ring of the parent compound. The new derivatives showed a valuable anti-proliferative effect and some displayed a greater effect as compared to nitroxoline against three pancreatic cancer cell lines with different genetic profiles. In particular, in silico pharmacokinetic data, clonogenicity assays and selectivity indexes of the most promising compounds showed several advantages for such derivatives, as compared to nitroxoline. Moreover, some of these novel compounds had stronger effects on cell viability and/or clonogenic capacity in PC cells as compared to erlotinib, a targeted agent approved for PC treatment.

The aetiology of breast and ovarian cancer (BC/OC) is multi-factorial. At present, the involvement of base excision repair (BER) glycosylases (MUTYH and OGG1) in BC/OC predisposition is controversial. The present study investigated whether germline mutation status and mRNA expression of two BER genes, MUTHY and OGG1, were correlated with BRCA1 in 59 patients with BC/OC and 50 matched population controls. In addition, to evaluate the relationship between MUTYH, OGG1 and BRCA1, their possible mutual modulation and correlation among mutational spectrum, gene expression and demographic characteristics were evaluated. The results identified 18 MUTYH and OGG1 variants, of which 4 were novel (2 MUTYH and 2 OGG1) in 44 of the 59 patients. In addition, two pathogenic mutations were identified: OGG1 p.Arg46Gln, detected in a patient with BC and a family history of cancer, and MUTYH p.Val234Gly in a patient with OC, also with a family history of cancer. A significant reduced transcript expression in MUTYH was observed (P=0.033) in cases, and in association with the presence of rare variants in the same gene (P=0.030). A significant correlation in the expression of the two BER genes was observed in cases (P=0.004), whereas OGG1 and BRCA1 was significantly correlated in cases (P=0.001) compared with controls (P=0.010). The results of the present study indicated that the relationship among mutational spectrum, gene expression and demographic characteristics may improve the genetic diagnosis and primary prevention of at-risk individuals belonging to families with reduced mRNA expression, regardless of mutation presence.

The use of anthracycline derivatives was approved for the treatment of a broad spectrum of human tumors (i.e., breast cancer). The need to test these drugs on cancer models has pushed the basic research to apply many types of in vitro assays, and, among them, the study of anthracycline-induced apoptosis was mainly based on the application of flow cytometry protocols. However, the chemical structure of anthracycline derivatives gives them a strong autofluorescence effect that must be considered when flow cytometry is used. Unfortunately, the guidelines on the analysis of anthracycline effects through flow cytometry are lacking. Therefore, in this study, we optimized the flow cytometry detection of doxorubicin and epirubicin-treated breast cancer cells. Their autofluorescence was assessed both by using conventional and imaging flow cytometry; we found that all the channels excited by the 488 nm laser were affected. Anthracycline-induced apoptosis was then measured via flow cytometry using the optimized setting. Consequently, we established a set of recommendations that enable the development of optimized flow cytometry settings when the in vitro assays of anthracycline effects are analyzed, with the final aim to reveal a new perspective on the use of those in vitro tests for the further implementation of precision medicine strategies in cancer.

Pancreatic cancer (PC) is one of the most lethal, chemoresistant malignancies and it is of paramount importance to find more effective therapeutic agents. Repurposing of non-anticancer drugs may expand the repertoire of effective molecules. Studies on repurposing of benzimidazole-based anthelmintics in PC and on their interaction with agents approved for PC therapy are lacking. We analyzed the effects of four Food and Drug Administration (FDA)-approved benzimidazoles on AsPC-1 and Capan-2 pancreatic cancer cell line viability. Notably, parbendazole was the most potent benzimidazole affecting PC cell viability, with half maximal inhibitory concentration (IC50) values in the nanomolar range. The drug markedly inhibited proliferation, clonogenicity and migration of PC cell lines through mechanisms involving alteration of microtubule organization and formation of irregular mitotic spindles. Moreover, parbendazole interfered with cell cycle progression promoting G2/M arrest, followed by the emergence of enlarged, polyploid cells. These abnormalities, suggesting a mitotic catastrophe, culminated in PC cell apoptosis, are also associated with DNA damage in PC cell lines. Remarkably, combinations of parbendazole with gemcitabine, a drug employed as first-line treatment in PC, synergistically decreased PC cell viability. In conclusion, this is the first study providing evidence that parbendazole as a single agent, or in combination with gemcitabine, is a repurposing candidate in the currently dismal PC therapy.

Activation of peroxisome proliferator-activated receptors (PPARs) not only regulates multiple metabolic pathways, but mediates various biological effects related to inflammation and oxidative stress. We investigated the effects of four new PPAR ligands containing a fibrate scaffold-the PPAR agonists (1a (αEC50 1.0 μM) and 1b (γEC50 0.012 μM)) and antagonists (2a (αIC50 6.5 μM) and 2b (αIC50 0.98 μM, with a weak antagonist activity on γ isoform))-on proinflammatory and oxidative stress biomarkers. The PPAR ligands 1a-b and 2a-b (0.1-10 μM) were tested on isolated liver specimens treated with lipopolysaccharide (LPS), and the levels of lactate dehydrogenase (LDH), prostaglandin (PG) E2, and 8-iso-PGF2α were measured. The effects of these compounds on the gene expression of the adipose tissue markers of browning, PPARα, and PPARγ, in white adipocytes, were evaluated as well. We found a significant reduction in LPS-induced LDH, PGE2, and 8-iso-PGF2α levels after 1a treatment. On the other hand, 1b decreased LPS-induced LDH activity. Compared to the control, 1a stimulated uncoupling protein 1 (UCP1), PR-(PRD1-BF1-RIZ1 homologous) domain containing 16 (PRDM16), deiodinase type II (DIO2), and PPARα and PPARγ gene expression, in 3T3-L1 cells. Similarly, 1b increased UCP1, DIO2, and PPARγ gene expression. 2a-b caused a reduction in the gene expression of UCP1, PRDM16, and DIO2 when tested at 10 μM. In addition, 2a-b significantly decreased PPARα gene expression. A significant reduction in PPARγ gene expression was also found after 2b treatment. The novel PPARα agonist 1a might be a promising lead compound and represents a valuable pharmacological tool for further assessment. The PPARγ agonist 1b could play a minor role in the regulation of inflammatory pathways.

Besides its metabolic and endocrine effects, growth hormone (GH)-releasing hormone (GHRH) is involved in the modulation of inflammation. Recently synthetized GHRH antagonist MIA-690 and MR-409, GHRH agonist, developed by us have shown potent pharmacological effects in various experimental paradigms. However, whether their administration modify resistance to chronic inflammatory stimuli in colon is still unknown. Ex vivo results demonstrated that MIA-690 and MR-409 inhibited production of pro-inflammatory and oxidative markers induced by lipopolysaccharide on isolated mouse colon specimens. In vivo, both MIA-690 and MR-409 have also been able to decrease the responsiveness to nociceptive stimulus, in hot plate test. Additionally, both peptides also induced a decreased sensitivity to acute and persistent inflammatory stimuli in male mice, in formalin test and dextran sodium sulfate (DSS)-induced colitis model, respectively. MIA-690 and MR-409 attenuate DSS-induced colitis with particular regard to clinical manifestations, histopathological damage and release of pro-inflammatory and oxidative markers in colon specimens. Respect to MR-409, MIA-690 showed higher efficacy in inhibiting prostaglandin (PG)E2, 8-iso-PGF2α and serotonin (5-HT) levels, as well as tumor necrosis factor (TNF)-α, interleukin (IL)-6 and nitric oxide synthase gene expression in colon specimens of DSS-induced colitis. Furthermore, MIA-690 decreased serum insulin-like growth factor (IGF)-1 levels in mice DSS-treated, respect to MR-409. Thus, our findings highlight the protective effects of MIA-690 and MR-409 on inflammation stimuli. The higher antinflammatory and antioxidant activities observed with MIA-690 could be related to decreased serum IGF-1 levels.

Grape (Vitis vinifera L.) pomace is a residue derived from the winemaking process, which contains bioactive compounds displaying noteworthy health-promoting properties. The aim of the present study was to investigate the phenolic composition and protective effects of a water extract of grape pomace (WEGP) in colorectal cancer cell line SW480 and in isolated mouse colon exposed to Escherichia coli lipopolysaccharide (LPS). The extract decreased SW-480 cell viability, as well as vascular endothelial factor A (VEGFA), hypoxia-induced factor 1α (HIF1α), and transient receptor potential M8 (TRPM8) LPS-induced gene expression. Moreover, the extract inhibited mRNA levels of nuclear factor kB (NFkB), cyclooxygenase (COX)-2, tumor necrosis factor (TNF)α, interleukin (IL)-6, IL-1β, IL-10, inducible nitric oxide synthase (iNOS), and interferon (IFN)γ, in isolated colon. Conversely, WEGP increased the gene expression of antioxidant catalase (CAT) and superoxide dismutase (SOD), in the same model. The modulatory effects exerted by WEGP could be related, at least in part, to the phenolic composition, with particular regards to the catechin level. Docking calculations also predicted the interactions of catechin toward TRPM8 receptor, deeply involved in colon cancer; thus further suggesting the grape pomace as a valuable source of bioactive extracts and phytochemicals with protective effects in the colon.

Pancreatic cancer (PC) is one of the deadliest malignancies worldwide, since patients rarely display symptoms until an advanced and unresectable stage of the disease. Current chemotherapy options are unsatisfactory and there is an urgent need for more effective and less toxic drugs to improve the dismal PC therapy. Repurposing of non-oncology drugs in PC treatment represents a very promising therapeutic option and different compounds are currently being considered as candidates for repurposing in the treatment of this tumor. In this review, we provide an update on some of the most promising FDA-approved, non-oncology, repurposed drug candidates that show prominent clinical and preclinical data in pancreatic cancer. We also focus on proposed mechanisms of action and known molecular targets that they modulate in PC. Furthermore, we provide an explorative bioinformatic analysis, which suggests that some of the PC repurposed drug candidates have additional, unexplored, oncology-relevant targets. Finally, we discuss recent developments regarding the immunomodulatory role displayed by some of these drugs, which may expand their potential application in synergy with approved anticancer immunomodulatory agents that are mostly ineffective as single agents in PC.

The aim of the present study was to investigate the possible protective effects of a garlic hydroalcoholic extract on the burden of oxidative stress and inflammation occurring on mouse heart specimens exposed to E. coli lipopolysaccharide (LPS), which is a well-established inflammatory stimulus. Headspace solid-phase microextraction combined with the gas chromatography-mass spectrometry (HS-SPME/GC-MS) technique was applied to determine the volatile fraction of the garlic powder, and the HS-SPME conditions were optimized for each of the most representative classes of compounds. CIEL*a*b* colorimetric analyses were performed on the powder sample at the time of delivery, after four and after eight months of storage at room temperature in the dark, to evaluate the color changing. Freshly prepared hydroalcoholic extract was also evaluated in its color character. Furthermore, the hydroalcoholic extract was analyzed through GC-MS. The extract was found to be able to significantly inhibit LPS-induced prostaglandin (PG) E2 and 8-iso-PGF2α levels, as well as mRNA levels of cyclooxygenase (COX)-2, interleukin (IL)-6, and nuclear factor-kB (NF-kB), in heart specimens. Concluding, our findings showed that the garlic hydroalcoholic extract exhibited cardioprotective effects on multiple inflammatory and oxidative stress pathways.

The identification of germline variants predisposing to hereditary nonpolyposis colorectal cancer (HNPCC) is crucial for clinical management of carriers, but several probands remain negative for such variants or bear variants of uncertain significance (VUS). Here we describe the results of integrative molecular analyses in 132 HNPCC patients providing evidences for improved genetic testing of HNPCC with traditional or next generation methods. Patients were screened for: germline allele-specific expression (ASE), nucleotide variants, rearrangements and promoter methylation of mismatch repair (MMR) genes; germline EPCAM rearrangements; tumor microsatellite instability (MSI) and immunohistochemical (IHC) MMR protein expression. Probands negative for pathogenic variants of MMR genes were screened for germline APC and MUTYH sequence variants. Most germline defects identified were sequence variants and rearrangements of MMR genes. Remarkably, altered germline ASE of MMR genes was detected in 8/22 (36.5%) probands analyzed, including 3 cases negative at other screenings. Moreover, ASE provided evidence for the pathogenic role and guided the characterization of a VUS shared by 2 additional probands. No germline MMR gene promoter methylation was observed and only one EPCAM rearrangement was detected. In several cases, tumor IHC and MSI diverged from germline screening results. Notably, APC or biallelic MUTYH germline defects were identified in 2/19 probands negative for pathogenic variants of MMR genes. Our results show that ASE complements gDNA-based analyses in the identification of MMR defects and in the characterization of VUS affecting gene expression, increasing the number of germline alterations detected. An appreciable fraction of probands negative for MMR gene variants harbors APC or MUTYH variants. These results indicate that germline ASE analysis and screening for APC and MUTYH defects should be included in HNPCC diagnostic algorithms.

The spectrum of BRCA1/2 genetic variation in breast-ovarian cancer patients has been scarcely investigated outside Europe and North America, with few reports for South America, where Amerindian founder effects and recent multiracial immigration are predicted to result in high genetic diversity. We describe here the results of BRCA1/BRCA2 germline analysis in an Argentinean series of breast/ovarian cancer patients selected for young age at diagnosis or breast/ovarian cancer family history.

The somatotropic axis, in addition to its well-known metabolic and endocrine effects, plays a pivotal role in modulation of inflammation. Moreover, growth hormone (GH)-releasing hormone (GHRH) has been involved in the development of various human tumors. In this work we aimed to investigate the consequences of GHRH deficiency on the development of inflammation-associated colon carcinogenesis in a mouse model of isolated GH deficiency due to generalized ablation of the GHRH gene [GHRH knock out (GHRHKO)]. Homozygous GHRHKO (-/-) male mice and wild type (C57/BL6, +/+) male mice as control group, were used. After azoxymetane (AOM)/dextran sodium sulfate (DSS) treatment -/- mice displayed higher Disease Activity Index (DAI) score, and more marked weight loss compared to +/+ animals. Additionally, -/- mice showed a significant increase in total tumors, in particular of large size predominantly localized in distal colon. In colonic tissue of AOM/DSS-treated -/- mice we found the presence of invasive adenocarcinomas, dysplasia and colitis with mucosal ulceration. Conversely, AOM/DSS-treated +/+ mice showed only presence of adenomas, without invasion of sub-mucosa. Treatment with AOM/DSS significantly increased prostaglandin (PG)E2 and 8-iso-PGF2α levels along with cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-α, nuclear factor kappa B (NF-kB) and inducible nitric oxide synthase (iNOS) gene expression, in colon specimens. The degree of increase of all these parameters was more markedly in -/- than +/+ mice. In conclusion, generalized GHRH ablation increases colon carcinogenesis responsiveness in male mice. Whether this results from lack of GH or GHRH remains to be established.

Pancreatic cancer (PC) is the fourth most common cause of cancer death. Combination therapies with classical chemotherapeutic agents improved treatment of advanced PC at the cost of a relevant toxicity, but the 5-year survival rate remains below 5%. Consequently, new therapeutic options for this disease are urgently needed. In this study, we explored the effect of two repurposed drug candidates nelfinavir and nitroxoline, approved for non-anticancer human use, in PC cell lines. Nelfinavir and nitroxoline were tested as single agents, or in combinations with or without erlotinib, a targeted drug approved for PC treatment.

Multiple studies demonstrated biological activities of aged black garlic, including anti-inflammatory, antioxidant, and cardioprotective effects. We aimed to investigate the protective effects of an aged black garlic water extract (ABGE) alone or in association with multivitamins consisting of combined Vitamins D, C, and B12, on mouse heart specimens exposed to E. coli lipopolysaccharide (LPS). Moreover, we studied the hydrogen sulphide (H2S) releasing properties and the membrane hyperpolarization effect of the Formulation composed by ABGE and multivitamins, using Human Aortic Smooth Muscle Cells (HASMCs). ABGE, vitamins D and C, and the Formulation suppressed LPS-induced gene expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, interleukin (IL)-6, nuclear factor-kB (NF-kB), and inducible nitric oxide synthase (iNOS) on mouse heart specimens. The beneficial effects induced by the extract could be related to the pattern of polyphenolic composition, with particular regard to gallic acid and catechin. The Formulation also increased fluorescence values compared to the vehicle, and it caused a significant membrane hyperpolarization of HASMCs compared to ABGE. To conclude, our present findings showed that ABGE, alone and in association with multivitamins, exhibited protective effects on mouse heart. Moreover, the Formulation increased intracellular H2S formation, further suggesting its potential use on cardiovascular disease.

Paragangliomas (PGLs) are infiltrating autonomic nervous system tumors that cause important morbidity. At present, surgery is the only effective therapeutic option for this rare tumor. Thus, new agents for PGL treatment should be identified. Using unique PGL cell models established in our laboratory, we evaluated the effect of dichloroacetate (DCA) as single agent or in a novel combination with other metabolic drugs, including GW6471 and metformin. DCA and metformin had not been tested before in PGL. DCA reduced PGL cell viability and growth through mechanisms involving reactivation of PDH complex leading to promotion of oxidative metabolism, with lowering of lactate and enhanced ROS production. This resulted in cell cycle inhibition and induction of apoptosis in PGL cells, as shown by flow cytometry and immunoblot analyses. Moreover, DCA drastically impaired clonogenic activity and migration of PGL cells. Also metformin reduced PGL cell viability as single agent and the combinations of DCA, GW6471 and metformin had strong effects on cell viability. Furthermore, combined treatments had drastic and synergistic effects on clonogenic ability. In conclusion, DCA, GW6471 and metformin as single agents and in combination appear to have promising antitumor effects in unique cell models of PGL.

Pulmonary carcinoids, distinct in typical and atypical, represent 2-5% of all primary lung tumors. The aim of this study was to investigate the molecular alterations correlated with the development of this form of neoplasms. A collection of 38 paraffin-embedded apparently sporadic carcinoids was investigated, through a combined study, for protein expression/localization of menin, p53, β-catenin and E-cadherin and for mutational analysis of the MEN1, TP53 and CTNNB1 genes. Menin was expressed in 71% of cases, with a prevalent cytoplasmic (c) localization, β-catenin was expressed in 68.4% of cases, of which 36.8% with a membranous (m) and 31.6% with a cytoplasmic localization. Membranous E-cadherin immunoreactivity was detected in 84.2% cases, nuclear p53 expression in 5.3% of cases. Positive correlation was found between c-menin and c-β-catenin expression (rho=0.439, P=0.008). In addition, m-β-catenin showed a positive correlation with both c-β-catenin and E-cadherin expression (rho=0.380, P=0.022 and rho=0.360, P=0.040, respectively). With regard to the E-cadherin/β-catenin complex, we found also a significant positive correlation between c-menin and 'disarrayed' β-catenin expression (rho=0.481, P=0.007). MEN1 gene variants were characterized in 34% of cases. c-menin was more highly expressed in tumors with MEN1 variants, compared to tumors without MEN1 variants (P=0.023). Three nucleotide variants of TP53 were also detected. This study confirms the involvement of the MEN1 gene in the development of sporadic pulmonary carcinoids, demonstrates the accumulation of menin in the cytoplasm, and indicates that the disarrayed pattern of the complex significantly correlates with c-menin accumulation.

We recently identified nitroxoline as a repurposed drug candidate in pancreatic cancer (PC) showing a dose-dependent antiproliferative activity in different PC cell lines. This antibiotic is effective in several in vitro and animal cancer models. To date, the mechanisms of nitroxoline anticancer action are largely unknown. Using shotgun proteomics we identified 363 proteins affected by nitroxoline treatment in AsPC-1 pancreatic cancer cells, including 81 consistently deregulated at both 24- and 48-hour treatment. These proteins previously unknown to be affected by nitroxoline were mostly downregulated and interconnected in a single highly-enriched network of protein-protein interactions. Integrative proteomic and functional analyses revealed nitroxoline-induced downregulation of Na/K-ATPase pump and β-catenin, which associated with drastic impairment in cell growth, migration, invasion, increased ROS production and induction of DNA damage response. Remarkably, nitroxoline induced a previously unknown deregulation of molecules with a critical role in cell bioenergetics, which resulted in mitochondrial depolarization. Our study also suggests that deregulation of cytosolic iron homeostasis and of co-translational targeting to membrane contribute to nitroxoline anticancer action. This study broadens our understanding of the mechanisms of nitroxoline action, showing that the drug modulates multiple proteins crucial in cancer biology and previously unknown to be affected by nitroxoline.

The clinical progression of B cell chronic lymphocytic leukemia (CLL) is associated with immune cell dysfunction and a strong decrease of miR-181b-5p (miR-181b), promoting the death of CLL cells. Here we investigated whether the reduction of miR-181b impairs the immune response in CLL. We demonstrate that activated CD4+ T cells increase miR-181b expression in CLL through CD40-CD40L signaling, which enhances the maturation and activity of cytotoxic T cells and, consequently, the apoptotic response of CLL cells. The cytotoxic response is facilitated by a depletion of the anti-inflammatory cytokine interleukin 10, targeted by miR-181b. In vivo experiments in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice confirmed that miR-181b promotes the apoptotic death of CLL cells only when functional T cells are restored. Overall, our findings suggest that the reinstatement of miR-181b in CLL cells could be an exploitable adjuvant therapeutic option for the treatment of CLL.

Bridelia species have been used in traditional African medicine for the management of diverse human ailments. In the current work, the detailed phytochemical profiles of the extracts of the stem bark of B. speciosa were evaluated and the antioxidant and enzyme inhibitory properties of the extracts were assessed. The anti-bacterial and anti-mycotic effects of the extracts were evaluated against selected pathogen strains. Additionally, the anti-proliferative effects were studied on the liver cancer HepG2 cell line. Finally, the putative protective effects were assessed on isolated rat liver that was challenged with lipopolysaccharide (LPS). The results revealed the presence of 36 compounds in the ethyl acetate extract, 44 in the methanol extract, and 38 in the water extract. Overall, the methanol extract showed the highest antioxidant activity, particularly in LPS-stimulated rat liver. Additionally, this extract exerted the highest antimycotic effect on C. albicans, whereas the water extract showed a promising anti-proliferative effect on liver cancer HepG2 cells. The methanol extract was also the most active as enzyme inhibitor, against acetylcholinesterase and butyrylcholinesterase. The current study appraises the antioxidant and enzyme inhibition properties of B. speciosa methanol extract and showed that this specie could be a promising source of biologically active phytochemicals, with potential health uses.

Immune checkpoint inhibitors (ICIs) induce durable clinical responses only in a subset of advanced non-small cell lung cancer (NSCLC) patients. There is a need to identify mechanisms of ICI resistance and immunotherapy biomarkers to improve clinical benefit. In this study, we evaluated the prognostic and predictive value of circulating endothelial and leukocyte-derived extracellular vesicles (EV) in patients with advanced NSCLC treated with anti-PD-1/PD-L1 agents. In addition, the relationship between total blood circulating EV proteome and response to ICIs was investigated. An optimized flow cytometry method was employed for the identification and subtyping of blood circulating EVs in 59 patients with advanced NSCLC. Blood samples were collected from patients receiving anti-PD-1/PD-L1 inhibitors (n = 31) or chemotherapy (n = 28). An exploratory proteomic analysis of sorted blood EVs was conducted in a subset of patients. Our results show that a low blood concentration of circulating endothelial-derived EVs before treatment was strongly associated to longer overall survival (p = 0.0004) and higher disease control rate (p = 0.045) in patients treated with ICIs. Interestingly, shotgun proteomics revealed that EVs of responders to anti-PD-1 therapy had a specific protein cargo before treatment. In addition, EV protein cargo was specifically modulated during immunotherapy. We identified a previously unknown association between circulating endothelial-derived extracellular vesicle concentration and immunotherapy-related clinical outcomes. We also observed differences in circulating extracellular vesicle proteome according to anti-PD-1-based treatment response in NSCLC patients. Overall, these results may contribute to the identification of novel circulating biomarkers for rational immunotherapy approaches in patients affected by NSCLC.