Therapeutic Strategies to Improve the Treatment of Pleural Mesothelioma.

Pleural mesothelioma is a rare neoplastic disease with aggressive features. Patient survival is poor due to the lack of early symptoms and the absence of effective therapeutic strategies. The development of pleural mesothelioma is mainly associated with asbestos exposure and related chronic inflammation. From a molecular-based perspective, this disease is a heterogeneous tumor lacking actionable alterations. The median overall survival of patients affected by this tumor does not exceed 16 months from diagnosis. Molecular and biochemical approaches have shown that this disease is characterized by resistance to drug-induced apoptosis associated with the activation of cell survival pathways and expression of anti-apoptotic proteins. Thus, there is an urgent need to develop efficient and safe therapeutic strategies. Here, we review the pharmacological options available for the treatment of this disease with specific reference to the antitumor agents used in systemic therapies. In addition, novel pharmacological approaches, such as drug delivery tools, to improve pleural mesothelioma treatment are discussed.

Identification of oxysterol synthetic analogs as a novel class of late-stage inhibitors of herpes simplex virus 2 replication.

Genital herpes, most frequently caused by herpes simplex virus 2 (HSV-2) infection, is one of the most prevalent sexually transmitted infections. The current rationale for the treatment of HSV-2 infection involves nucleoside analogs (e.g. acyclovir) to suppress reactivation. Enzymatic oxysterols are endogenous 27-carbon atoms molecules produced by enzymatic cholesterol oxidation, and recently emerged as a broad-spectrum host targeting antivirals. In this study, we screened selected members of an in-house synthesized library of oxysterol analogs for their activity against HSV-2, identifying three compounds, named PFM064, PFM067, and PFM069, endowed with 50% effective concentrations (EC50) in the micromolar range, without exerting any apparent cytotoxicity. Moreover, the results obtained showed the ability of the novel derivatives to inhibit both cell-to-cell fusion induced by HSV-2, and the production of an intracellular viral progeny. Further experiments performed with PFM067 (which was selected for more-in-depth studies as the most effective synthetic analog) showed that these molecules act in a late stage of HSV-2 replicative cycle, by sequestering viral glycoproteins in the Golgi compartment, and likely inhibiting the nuclear egress of neo-synthetized viral capsids. Taken together, these results point to PFM067 as a promising chemical scaffold for the development of novel herpetic antivirals.

The deubiquitinase USP8 regulates ovarian cancer cell response to cisplatin by suppressing apoptosis.

The identification of therapeutic approaches to improve response to platinum-based therapies is an urgent need for ovarian carcinoma. Deubiquitinases are a large family of ubiquitin proteases implicated in a variety of cellular functions and may contribute to tumor aggressive features through regulation of processes such as proliferation and cell death. Among the subfamily of ubiquitin-specific peptidases, USP8 appears to be involved in modulation of cancer cell survival by still poorly understood mechanisms. Thus, we used ovarian carcinoma cells of different histotypes, including cisplatin-resistant variants with increased survival features to evaluate the efficacy of molecular targeting of USP8 as a strategy to overcome drug resistance/modulate cisplatin response. We performed biochemical analysis of USP8 activity in pairs of cisplatin-sensitive and -resistant cells and found increased USP8 activity in resistant cells. Silencing of USP8 resulted in decreased activation of receptor tyrosine kinases and increased sensitivity to cisplatin in IGROV-1/Pt1 resistant cells as shown by colony forming assay. Increased cisplatin sensitivity was associated with enhanced cisplatin-induced caspase 3/7 activation and apoptosis, a phenotype also observed in cisplatin sensitive cells. Increased apoptosis was linked to FLIPL decrease and cisplatin induction of caspase 3 in IGROV-1/Pt1 cells, cisplatin-induced claspin and survivin down-regulation in IGROV-1 cells, thereby showing a decrease of anti-apoptotic proteins. Immunohistochemical staining on 65 clinical specimens from advanced stage ovarian carcinoma indicated that 40% of tumors were USP8 positive suggesting that USP8 is an independent prognostic factor for adverse outcome when considering progression free survival as a clinical end-point. Taken together, our results support that USP8 may be of diagnostic value and may provide a therapeutic target to improve the efficacy of platinum-based therapy in ovarian carcinoma.

Targeting of the Lipid Metabolism Impairs Resistance to BRAF Kinase Inhibitor in Melanoma.

Drug resistance limits the achievement of persistent cures for the treatment of melanoma, in spite of the efficacy of the available drugs. The aim of the present study was to explore the involvement of lipid metabolism in melanoma resistance and assess the effects of its targeting in cellular models of melanoma with acquired resistance to the BRAF-inhibitor PLX4032/Vemurafenib. Since transcriptional profiles pointed to decreased cholesterol and fatty acids synthesis in resistant cells as compared to their parental counterparts, we examined lipid composition profiles of resistant cells, studied cell growth dependence on extracellular lipids, assessed the modulation of enzymes controlling the main nodes in lipid biosynthesis, and evaluated the effects of targeting Acetyl-CoA Acetyltransferase 2 (ACAT2), the first enzyme in the cholesterol synthesis pathway, and Acyl-CoA Cholesterol Acyl Transferase (ACAT/SOAT), which catalyzes the intracellular esterification of cholesterol and the formation of cholesteryl esters. We found a different lipid composition in the resistant cells, which displayed reduced saturated fatty acids (SFA), increased monounsaturated (MUFA) and polyunsaturated (PUFA), and reduced cholesteryl esters (CE) and triglycerides (TG), along with modulated expression of enzymes regulating biosynthetic nodes of the lipid metabolism. The effect of tackling lipid metabolism pathways in resistant cells was evidenced by lipid starvation, which reduced cell growth, increased sensitivity to the BRAF-inhibitor PLX4032, and induced the expression of enzymes involved in fatty acid and cholesterol metabolism. Molecular targeting of ACAT2 or pharmacological inhibition of SOAT by avasimibe showed antiproliferative effects in melanoma cell lines and a synergistic drug interaction with PLX4032, an effect associated to increased ferroptosis. Overall, our findings reveal that lipid metabolism affects melanoma sensitivity to BRAF inhibitors and that extracellular lipid availability may influence tumor cell response to treatment, a relevant finding in the frame of personalized therapy. In addition, our results indicate new candidate targets for drug combination treatments.

Identification of a ßCD-Based Hyper-Branched Negatively Charged Polymer as HSV-2 and RSV Inhibitor.

Cyclodextrins and cyclodextrin derivatives were demonstrated to improve the antiviral potency of numerous drugs, but also to be endowed with intrinsic antiviral action. They are suitable building blocks for the synthesis of functionalized polymer structures with potential antiviral activity. Accordingly, four water-soluble hyper-branched beta cyclodextrin (ßCD)-based anionic polymers were screened against herpes simplex virus (HSV-2), respiratory syncytial virus (RSV), rotavirus (HRoV), and influenza virus (FluVA). They were characterized by FTIR-ATR, TGA, elemental analyses, zeta-potential measurements, and potentiometric titrations, while the antiviral activity was investigated with specific in vitro assays. The polymer with the highest negative charge, pyromellitic dianhydride-linked polymer (P_PMDA), showed significant antiviral action against RSV and HSV-2, by inactivating RSV free particles and by altering HSV-2 binding to the cell. The polymer fraction with the highest molecular weight showed the strongest antiviral activity and both P_PMDA and its active fractions were not toxic for cells. Our results suggest that the polymer virucidal activity against RSV can be exploited to produce new antiviral materials to counteract the virus dissemination through the air or direct contact. Additionally, the strong HSV-2 binding inhibition along with the water solubility of P_PMDA and the acyclovir complexation potential of ßCD are attractive features for developing new therapeutic topical options against genital HSV-2 infection.

Curcumin and Related Compounds in Cancer Cells: New Avenues for Old Molecules.

Curcumin and related compounds are known for the large spectrum of activities. The chemical features of these compounds are important for their biological effects with a key role for the thiol-reactive α-ß unsaturated carbonyl groups. Curcumin derivatives may overcome the limitation of the bioavailability of the parent compound, while maintaining the key chemical features responsible for biological activities. Curcumin and related compounds show anti-viral, anti-fungal, anti-microbial and anti-tumor activities. The therapeutic effects of curcumin, used as a supplement in cancer therapy, have been documented in various cancer types, in which inhibition of cell growth and survival pathways, induction of apoptosis and other cell death pathways have been reported. Curcumin-induced apoptosis has been linked both to the intrinsic and extrinsic apoptotic pathways. Necroptosis has also been involved in curcumin-induced toxicity. Among curcumin-induced effects, ferroptosis has also been described. The mechanism of curcumin toxicity can be triggered by reactive oxygen species-mediated endoplasmic reticulum stress. Curcumin targets have been identified in the context of the ubiquitin-proteasome system with evidence of inhibition of the proteasome proteolytic activities and cellular deubiquitinases. Curcumin has recently been shown to act on the tumor microenvironment with effects on cancer-associated fibroblasts and immune cells. The related product caffeic acid phenethyl ester has shown promising preclinical results with an effect on the inflammatory microenvironment. Here, we review the mechanisms underlying curcumin and derivatives toxicity towards cancer cells with particular emphasis on cell death pathways and the ubiquitin-proteasome system.

27-Hydroxycholesterol inhibits rhinovirus replication in vitro and on human nasal and bronchial histocultures without selecting viral resistant variants.

The genetic plasiticity of viruses is one of the main obstacles to the development of antivirals. The aim of this study has been to assess the ability of two physiologic oxysterols and host-targeting antivirals - namely 25- and 27-hydroxycholesterol (25OHC and 27OHC) - to select resistant strains, using human rhinovirus (HRV) as a challenging model of a viral quasispecies. Moreover, we selected 27OHC for further studies aimed at exploring its potential for the development of antiviral drugs. The results obtained with clonal or serial passage approaches show that 25OHC and 27OHC do not select HRV oxysterol-resistant variants. Moreover, we demonstrate the ability of 27OHC to inhibit the yield of HRV in 3D in vitro fully reconstituted human nasal and bronchial epithelia from cystic fibrosis patients and prevent virus-induced cilia damage. The promising antiviral activity of 27OHC and its competitive advantages over direct-acting antivirals, make this molecule a suitable candidate for further studies to explore its clinical potential.

P301L tau mutation leads to alterations of cell cycle, DNA damage response and apoptosis: Evidence for a role of tau in cancer.

Tau is a microtubule-associated protein, coded by the MAPT gene, which regulates microtubule (MT) polymerization and dynamics. Due to its key role in neurons, it is a major player in neurodegenerative diseases known as "tauopathies". Since tau has emerged as a multitasking protein with a role in genome stability, it may act both in neurodegeneration and cancer. After demonstrating that tau can be considered as a risk factor for cancer, here we explored the mechanisms linking mutated tau to dysregulation of cancer-relevant processes, by employing lymphoblastoid cell lines (LCL) from patients affected by genetic tauopathy carrying the MAPT P301L mutation and healthy controls (wild-type, wt). In mutated LCL, we found reduced sensitivity to MT perturbation, along with decreased G2/M accumulation and cyclin B1 levels. Furthermore, mutated LCL displayed lower levels of phospho-Chk1 and phospho-Chk2 following hydrogen peroxide-induced oxidative stress, indicating a poorly effective DNA damage checkpoint, as well as reduced basal levels of p53. Such cells also exhibited lower levels of Bax and cleaved caspase-3, and increased levels of Cdc25A, upon oxidative stress, accounting for diminished apoptosis. Overall, these findings point to tau as a key player in biological pathways relevant for cancer, as evidenced by the differential response of mutated and wt cells to MT and DNA perturbation. The modulation of p53 is intriguing given its function as guardian of the genome.

The mTOR/4E-BP1/eIF4E Signalling Pathway as a Source of Cancer Drug Targets.

The mechanistic/mammalian target of rapamycin (mTOR) is the crucial hub of signalling pathways that regulate essential steps in the cell life cycle. Once incorporated in the mTORC1 complex, mTOR phosphorylates the eukaryotic initiation factor 4E (eIF4E)- binding protein 1 (4E-BP1), which then releases eIF4E. When not bound to 4EBPs, eIF4E recognizes the mRNA 5'-cap structure and, together with eIF4A and eIF4G, it forms the eIF4F complex that recruits the ribosome on the mRNA. Under normal conditions, the cellular concentration of eIF4E is very low, making eIF4E the limiting factor in the initiation of protein synthesis. The vast majority of cancer types are characterized by the simultaneous deregulation of the mTOR/4E-BP1 signalling pathway and upregulation of eIF4E, which lead to an increased expression of cancer-promoting genes and deregulated cellular growth. Over the last decades, a growing number of selective inhibitors of the mTOR/4E-BP1/eIF4E pathway have been discovered or designed. Several inhibitors with encouraging preclinical results have been tested in clinical trials. This review summarizes the most recent research on drug development against mTOR, 4E-BP1, and eIF4E, describing the design rationale and the available structural and functional data on the most promising compounds.

Caffeic acid phenethyl ester targets ubiquitin-specific protease 8 and synergizes with cisplatin in endometrioid ovarian carcinoma cells.

Deubiquitinases (DUBs) mediate the removal of ubiquitin from diverse proteins that participate in the regulation of cell survival, DNA damage repair, apoptosis and drug resistance. Previous studies have shown an association between activation of cell survival pathways and platinum-drug resistance in ovarian carcinoma cell lines. Among the strategies available to inhibit DUBs, curcumin derivatives appear promising, thus we hypothesized their use to enhance the efficacy of cisplatin in ovarian carcinoma preclinical models. The caffeic acid phenethyl ester (CAPE), inhibited ubiquitin-specific protease 8 (USP8), but not proteasomal DUBs in cell-free assays. When CAPE was combined with cisplatin in nine cell lines representative of various histotypes a synergistic effect was observed in TOV112D cells and in the cisplatin-resistant IGROV-1/Pt1 variant, both of endometrioid type and carrying mutant TP53. In the latter cells, persistent G1 accumulation upon combined treatment associated with p27kip1 protein levels was observed. The synergy was not dependent on apoptosis induction, and appeared to occur in cells with higher USP8 levels. In vivo antitumor activity studies supported the advantage of the combination of CAPE and cisplatin in the subcutaneous model of cisplatin-resistant IGROV-1/Pt1 ovarian carcinoma as well as CAPE activity on intraperitoneal disease. This study reveals the therapeutic potential of CAPE in cisplatin-resistant ovarian tumors as well as in tumors expressing USP8.