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.

Kisspeptin-mediated improvement of sensitivity to BRAF inhibitors in vemurafenib-resistant melanoma cells.

Metastatic dissemination is still one of the major causes of death of melanoma's patients. KiSS1 is a metastasis suppressor originally identified in melanoma cells, known to play an important physiological role in mammals' development and puberty. It has been previously shown that expression of KiSS1 could be increased in lung cancer cells using epigenetic agents, and that KiSS1 could have a pro-apoptotic action in combination with cisplatin. Thus, the aim of the present study was to examine in human melanoma vemurafenib sensitive- and -resistant BRAF mutant cells characterized by different mutational profiles and KiSS1, KiSS1 receptor and KiSS1 drug-induced release, if peptides derived from KiSS1 cleavage, i.e., kisspeptin 54, could increase the sensitivity to vemurafenib of human melanoma, using cellular, molecular and biochemical approaches. We found that kisspeptin 54 increases vemurafenib pro-apoptotic activity in a statistically significant manner, also in drug resistant cellular models. The efficacy of the combination appears to reflect the intrinsic susceptibility of each cell line to PLX4032-induced apoptosis, together with the different mutational profile as well as perturbation of proteins regulating the apoptotic pathway, The results presented here highlight the possibility to exploit KiSS1 to modulate the apoptotic response to therapeutically relevant agents, suggesting a multitasking function of this metastasis suppressor.

Ferroptosis-induced Cardiotoxicity and Antitumor Drugs.

The induction of regulated cell death ferroptosis in tumors is emerging as an intriguing strategy for cancer treatment. Numerous antitumor drugs (e.g., doxorubicin, etoposide, tyrosine kinase inhibitors, trastuzumab, arsenic trioxide, 5-fluorouracil) induce ferroptosis. Although this mechanism of action is interesting for fighting tumors, the clinical use of drugs that induce ferroptosis is hampered by cardiotoxicity. Besides in cancer cells, ferroptosis induced by chemotherapeutics can occur in cardiomyocytes, and this feature represents an important drawback of antitumor therapy. This inconvenience has been tackled by developing less or no cardiotoxic antitumor drugs or by discovering cardioprotective agents (e.g., berberine, propofol, fisetin, salidroside, melatonin, epigallocatechin-3gallate, resveratrol) to use in combination with conventional chemotherapeutics. This review briefly summarizes the molecular mechanisms of Fer and describes the Fer-dependent mechanisms responsible for cardiac toxicity developed by cancer-suffering patients following the administration of some chemotherapeutics. Additionally, the pharmacological strategies very recently proposed for potentially preventing this inconvenience are considered.

Radiotherapy-induced ferroptosis for cancer treatment.

Ferroptosis is a regulated cell death mechanism controlled by iron, amino acid and reactive oxygen species metabolisms, which is very relevant for cancer therapy. Radiotherapy-induced ferroptosis is critical for tumor suppression and several preclinical studies have demonstrated that the combination of ionizing radiation with small molecules or nano-systems is effective in combating cancer growth and overcoming drug or ionizing radiation resistance. Here, we briefly overview the mechanisms of ferroptosis and the cross-talk existing between the cellular pathways activated by ferroptosis and those induced by radiotherapy. Lastly, we discuss the recently reported combinational studies involving radiotherapy, small molecules as well as nano-systems and report the recent findings achieved in this field for the treatment of tumors.

The Therapeutic Potential of Pyroptosis in Melanoma.

Pyroptosis is a programmed cell death characterized by the rupture of the plasma membranes and release of cellular content leading to inflammatory reaction. Four cellular mechanisms inducing pyroptosis have been reported thus far, including the (i) caspase 1-mediated canonical, (ii) caspase 4/5/11-mediated non-canonical, (iii) caspase 3/8-mediated and (iv) caspase-independent pathways. Although discovered as a defense mechanism protecting cells from infections of intracellular pathogens, pyroptosis plays roles in tumor initiation, progression and metastasis of tumors, as well as in treatment response to antitumor drugs and, consequently, patient outcome. Pyroptosis induction following antitumor therapies has been reported in several tumor types, including lung, colorectal and gastric cancer, hepatocellular carcinoma and melanoma. This review provides an overview of the cellular pathways of pyroptosis and discusses the therapeutic potential of pyroptosis induction in cancer, particularly in melanoma.

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.

Resveratrol and Prostate Cancer: The Power of Phytochemicals.

Prostate cancer is the fifth cause of tumor-related deaths in man worldwide. Due to its long latency period, this pathology represents an ideal type of disease for chemopreventive studies. Among the drugs considered thus far for the treatment of prostate cancer, the natural compound resveratrol emerged as very promising. Resveratrol is widely recognized as a chemopreventive agent and was shown to potentiate the antitumor activity of conventional chemotherapeutics in several tumors, including prostate cancer. Here, we overview the literature of the last five years and summarize the recent achievements of resveratrol and its derivatives as antimetastatic agents in prostate cancer. Moreover, drug combination studies as well as nanomedicine approaches proposed to improve resveratrol activity and to overcome delivery drawbacks are addressed. The last part of the review discusses the clinical trials containing resveratrol ongoing on cancer patients.

Androgen Receptor-Directed Molecular Conjugates for Targeting Prostate Cancer.

Due to its central role in the cellular biology of prostate cancer (PC), androgen receptor (AR) still remains an important therapeutic target for fighting this tumor. Several drugs targeting AR have been reported so far, and many new molecules are expected for the future. In spite of their antitumor efficacy, these drugs are not selective for malignant cells and are subjected to AR-mediated activation of drug resistance mechanisms that are responsible for several drawbacks, including systemic toxicity and disease recurrence and metastasis. Among the several strategies considered to overcome these drawbacks, very appealing appears the design of hybrid small-molecule conjugates targeting AR to drive drug action on receptor-positive PC cells. These compounds are designed around on an AR binder, which selectively engages AR with high potency, coupled with a moiety endowed with different pharmacological properties. In this review we focus on two classes of compounds: a) small-molecules and AR-ligand based conjugates that reduce AR expression, which allow down-regulation of AR levels by activating its proteasome-mediated degradation, and b) AR-ligand-based conjugates for targeting small-molecules, in which the AR binder tethers small-molecules, including conventional antitumor drugs (e.g., cisplatin, doxorubicin, histone deacetylase inhibitors, as well as photo-sensitizers) and selectively directs drug action toward receptor-positive PC cells.

Molecular basis for the DNA damage induction and anticancer activity of asymmetrically substituted anthrapyridazone PDZ-7.

Anthrapyridazones, imino analogues of anthraquinone, constitute a family of compounds with remarkable anti-cancer activity. To date, over 20 derivatives were studied, of which most displayed nanomolar cytotoxicity towards broad spectrum of cancer cells, including breast, prostate and leukemic ones. BS-154, the most potent derivative, had IC50 values close to 1 nM, however, it was toxic in animal studies. Here, we characterize another anthrapyridazone, PDZ-7, which retains high cytotoxicity while being well tolerated in mice. PDZ-7 is also active in vivo against anthracycline-resistant tumor in a mouse xenograft model and induces DNA damage in proliferating cells, preferentially targeting cells in S and G2 phases of the cell cycle. Activation of Mre11-Rad50-Nbs1 (MRN) complex and phosphorylation of H2AX suggest double-stranded DNA breaks as a major consequence of PDZ-7 treatment. Consistent with this, PDZ-7 treatment blocked DNA synthesis and resulted in cell cycle arrest in late S and G2 phases. Analysis of topoisomerase IIα activity and isolation of the stabilized covalent topoisomerase IIα - DNA complex in the presence of PDZ-7 suggests that this compound is a topoisomerase IIα poison. Moreover, PDZ-7 interfered with actin polymerization, thereby implying its action as a dual inhibitor of processes critical for dividing cells. Using nuclear magnetic resonance (NMR) spectroscopy we show that PDZ-7 interacts with DNA double helix and quadruplex DNA structure. Taken together, our results suggest that PDZ-7 is a unique compound targeting actin cytoskeleton and DNA.

Synthesis and Evaluation of New Naphthalene and Naphthoquinone Derivatives as Anticancer Agents.

DNA topoisomerase I inhibitors, both synthetic and of natural origin, are receiving increasing consideration primarily as drugs against refractory tumors. Alkannin and shikonin, two enantiomeric dyes from Alkanna tinctoria and Lithospermum erythrorhizon, have been known over many centuries as dyestuff, wound healing, anti-inflammatory, antibacterial and antitumor substances. Although multiple mechanisms appear to be implicated, their potency is associated with the inhibition of topoisomerase I and with the redox properties of the naphthazarin scaffold. Here, the synthesis of new naphthalene and naphthoquinone derivatives inspired by alkannin and shikonin is described and their structural and biological properties were examined. Different oxidation states of the naphthalene nucleus were examined to observe the effect of this parameter on cytotoxicity. Antiproliferative activities against a panel of human cancer cell lines were evaluated and the implication of topoisomerase I was assessed.

Methods for Elucidation of DNA-Anticancer Drug Interactions and their Applications in the Development of New Drugs.

DNA damaging agents including anthracyclines, camptothecins and platinum drugs are among most frequently used drugs in the chemotherapeutic routine. Due to their relatively low selectivity for cancer cells, administration of these drugs is associated with adverse side effects, inherent genotoxicity with risk of developing secondary cancers. Development of new drugs, which could be spared of these drawbacks and characterize by improved antitumor efficacy, remains challenging yet vitally important task. These properties are in large part dictated by the selectivity of interaction between the drug and DNA and in this way the studies aimed at elucidating the complex interactions between ligand and DNA represent a key step in the drug development. Studies of the drug-DNA interactions encompass determination of DNA sequence specificity and mode of DNA binding as well as kinetic, dynamic and structural parameters of binding. Here, we consider the types of interactions between small molecule ligands and polynucleotides, how they are affected by DNA sequence and structure, and what is their significance for the antitumor activity. Based on this knowledge, we discuss the wide array of experimental techniques available to researchers for studying drug-DNA interactions, which include absorption and emission spectroscopies, NMR, magnetic and optical tweezers or atomic force microscopy. We show, using the clinical and experimental anticancer drugs as examples, how these methods provide various types of information and at the same time complement each other to provide full picture of drug- DNA interaction and aid in the development of new drugs.

Targeting of RET oncogene by naphthalene diimide-mediated gene promoter G-quadruplex stabilization exerts anti-tumor activity in oncogene-addicted human medullary thyroid cancer.

Medullary thyroid cancer (MTC) relies on the aberrant activation of RET proto-oncogene. Though targeted approaches (i.e., tyrosine kinase inhibitors) are available, the absence of complete responses and the onset of resistance mechanisms indicate the need for novel therapeutic interventions. Due to their role in regulation of gene expression, G-quadruplexes (G4) represent attractive targets amenable to be recognized or stabilized by small molecules. Here, we report that exposure of MTC cells to a tri-substituted naphthalene diimide (NDI) resulted in a significant antiproliferative activity paralleled by inhibition of RET expression. Biophysical analysis and gene reporter assays showed that impairment of RET expression was consequent to the NDI-mediated stabilization of the G4 forming within the gene promoter. We also showed for the first time that systemic administration of the NDI in mice xenotransplanted with MTC cells resulted in a remarkable inhibition of tumor growth in vivo. Overall, our findings indicate that NDI-dependent RET G4 stabilization represents a suitable approach to control RET transcription and delineate the rationale for the development of G4 stabilizing-based treatments for MTC as well as for other tumors in which RET may have functional and therapeutic implications.

Platinum-Based Drugs and DNA Interactions Studied by Single-Molecule and Bulk Measurements.

Platinum-containing molecules are widely used as anticancer drugs. These molecules exert cytotoxic effects by binding to DNA through various mechanisms. The binding between DNA and platinum-based drugs hinders the opening of DNA, and therefore, DNA duplication and transcription are severely hampered. Overall, impeding the above-mentioned important DNA mechanisms results in irreversible DNA damage and the induction of apoptosis. Several molecules, including multinuclear platinum compounds, belong to the family of platinum drugs, and there is a body of research devoted to developing more efficient and less toxic versions of these compounds. In this study, we combined different biophysical methods, including single-molecule assays (magnetic tweezers) and bulk experiments (ultraviolet absorption for thermal denaturation) to analyze the differential stability of double-stranded DNA in complex with either cisplatin or multinuclear platinum agents. Specifically, we analyzed how the binding of BBR3005 and BBR3464, two representative multinuclear platinum-based compounds, to DNA affects its stability as compared with cisplatin binding. Our results suggest that single-molecule approaches can provide insights into the drug-DNA interactions that underlie drug potency and provide information that is complementary to that generated from bulk analysis; thus, single-molecule approaches have the potential to facilitate the selection and design of optimized drug compounds. In particular, relevant differences in DNA stability at the single-molecule level are demonstrated by analyzing nanomechanically induced DNA denaturation. On the basis of the comparison between the single-molecule and bulk analyses, we suggest that transplatinated drugs are able to locally destabilize small portions of the DNA chain, whereas other regions are stabilized.

Targeting peptidyl-prolyl isomerase pin1 to inhibit tumor cell aggressiveness.

PURPOSE: Because the peptidyl-prolyl isomerase PIN1 interacts with multiple protein kinases and phosphoproteins into a network orchestrating the cellular response to various stimuli, there is an increasing interest in exploiting its potential as therapeutic target. In the present study, the effect of targeting PIN1 was investigated in 2 human cancer cell lines characterized by increased aggressive potential, high expression of erbB receptor family members, and defective p53. METHODS: PIN1 silencing was carried out in skin squamous cell carcinoma A431 cells displaying elevated EGFR/HER1 levels and in ovarian adenocarcinoma SKOV-3 cells displaying high levels of erbB2 (HER2). Nonoverlapping siRNA duplexes targeting different regions of PIN1 mRNA were transfected in tumor cells, which were analyzed using Western blotting for the expression of selected proteins. In vivo tumorigenicity studies were carried out in athymic nude mice. RESULTS: A431 and SKOV-3 cell systems were found to be a source of cells with increased aggressive potential, i.e., cancer stem cell-like cells, as defined by the capability to grow as spheres. A marked decrease of PIN1 levels and of sphere-forming capability was observed in PIN1-silenced cells. The expression of phospho-p38 decreased following PIN1 silencing in A431 and SKOV-3 cells, as well as phospho-EGFR levels in A431 - silenced cells. PIN1 inhibition prolonged latency and reduced tumor take and growth of SKOV-3 cells in nude mice. CONCLUSIONS: Our results support that PIN1 may be a valuable target to hit in cancer cells characterized by increased aggressive potential, overexpression of erbB receptor family members, and defective p53.

Engineering Nanomedicines to Overcome Multidrug Resistance in Cancer Therapy.

Drug resistance to conventional antitumor drugs represents one of the major causes of treatment failure in patients affected by tumors. Two main types of drug resistance to anticancer drugs are found in tumors, namely intrinsic resistance, in which tumor cells are inherently resistant to chemotherapy, and acquired resistance, which results from previous drug exposure. Tumor cells resistant to a chemotherapeutic agent become cross-resistant to both similar and structurally unrelated classes of antitumor drugs, a biological mechanism known as multi drug resistance (MDR). Among the strategies considered to overcome MDR, nanovector-mediated drug administration represents an innovative and promising alternative. In this review, we report a number of nanovectors including polymer-drug conjugates, polymeric micelles, nanotubes, LbL nanocapsules, and silica and gold nanoparticles. These systems are designed for the efficient delivery of anthracyclines, vinca alkaloids, taxanes, and others drugs. The development of these nanovectos to specifically overcome MDR and their mechanisms of action are covered and discussed. Finally, we discuss challenges and opportunities for further development of nanodevices-based chemotherapies to circumvent MDR through the design of nanovectors for the delivery of multiple cargoes.

Redox-Sensitive PEG-Polypeptide Nanoporous Particles for Survivin Silencing in Prostate Cancer Cells.

We report the engineering of intracellular redox-responsive nanoporous poly(ethylene glycol)-poly(l-lysine) particles (NPEG-PLLs). The obtained particles exhibit no toxicity while maintaining the capability to deliver a small interfering RNA sequence (siRNA) targeting the anti-apoptotic factor, survivin, in prostate cancer cells. The redox-mediated cleavage of the disulfide bonds stabilizing the NPEG-PLL-siRNA complex results in the release of bioactive siRNA into the cytosol of prostate cancer PC-3 cells, which, in turn, leads to the effective silencing (∼59 ± 8%) of the target gene. These findings, obtained under optimal conditions, indicate that NPEG-PLLs may protect the therapeutic nucleic acid in the extracellular and intracellular environments, thus preventing the occurrence of competitive interactions with serum and cytosolic proteins as well as degradation by RNase. The intracellular trafficking and final fate of the NPEG-PLLs were investigated by a combination of deconvolution microscopy, fluorescence lifetime imaging microscopy, and super-resolution structured illumination microscopy. A significant impairment of cell survival was observed in cells concomitantly exposed to paclitaxel and siRNA-loaded NPEG-PLLs. Overall, our findings indicate that NPEG-PLLs represent a highly loaded depot for the delivery of therapeutic nucleic acids to cancer cells.

Unravelling "off-target" effects of redox-active polymers and polymer multilayered capsules in prostate cancer cells.

Redox-active polymers and carriers are oxidizing nanoagents that can potentially trigger intracellular off-target effects. In the present study, we investigated the occurrence of off-target effects in prostate cancer cells following exposure to redox-active polymer and thin multilayer capsules with different chemical properties. We show that, depending on the intracellular antioxidant capacity, thiol-functionalized poly(methacrylic acid), PMA(SH) triggers cell defense responses/perturbations that result in off-target effects (i.e., induction of autophagy and down-regulation of survivin). Importantly, the conversion of the carboxyl groups of PMA(SH) into the neutral amides of poly(hydroxypropylmetacrylamide) (pHPMA(SH)) nullified the off-target effects and cytotoxicity in tested cell lines. This suggests that the simultaneous action of carboxyl and disulfide groups in PMA(SH) polymer or capsules may play a role in mediating the intracellular off-target effects. Our work provides evidence that the rational design of redox-active carriers for therapeutic-related application should be guided by a careful investigation on potential disturbance of the cellular machineries related to the carrier association.

Fhit Nuclear Import Following EGF Stimulation Sustains Proliferation of Breast Cancer Cells.

The tumor-suppressor protein fragile histidine triad (Fhit) exerts its functions in the cytoplasm, although some reports suggest that it may also act in the nucleus. We previously showed that cytosolic Fhit protein levels in cancer cell lines stimulated to proliferate were reduced by proteasomal degradation. Here, we demonstrate that Fhit is physiologically present in the nucleus of breast cancer cell lines and tissues at a low level and that proliferative stimulation increases nuclear levels. Breast cancer cells expressing the FhitY114F mutant, which do not undergo proteasomal degradation, contained mutated Fhit in the nucleus, while cells treated with a proteasome inhibitor accumulated nuclear Fhit during proliferation. Thus, Fhit nuclear shuttling and proteasome degradation phenomena occur independently. When Fhit was coupled to a nuclear localization sequence, the proliferation rate of the transfected cells increased together with levels of proliferation pathway mediators cyclin D1, phospho-MAPK, and phospho-STAT3. Fhit nuclear translocation upon mitogenic stimulation may represent a new regulatory mechanism that allows rapid restoration of Fhit cytoplasmic levels and promotes the proliferation cascade activated by mitogenic stimulation.

Clinicopathological impact of ABCC1/MRP1 and ABCC4/MRP4 in epithelial ovarian carcinoma.

Ovarian cancer is the main cause of death from gynaecological malignancies. In spite of the efficacy of platinum-paclitaxel treatment in patients with primary epithelial ovarian carcinoma, platinum-based chemotherapy is not curative and resistance remains one of the most important causes of treatment failure. Although ABC transporters have been implicated in cellular resistance to multiple drugs, the clinical relevance of these efflux pumps is still poorly understood. Thus, we examined the prognostic role of transporters of the MRP family (i.e., ABCC1/MRP1, ABCC4/MRP4) to gain insights into their clinical impacts. A case material of 127 patients with ovarian carcinoma at different stages and histotypes was used. The expression of MRP1 and MRP4 was examined by immunohistochemistry using tissue microarrays in tumor specimens collected at the time of initial surgery expression. We found an association between MRP1 expression and grading, and we observed that MRP4 displayed an unfavourable impact on disease relapse in multivariate analysis (HR = 2.05, 95% CI: 1.01-4.11; P = 0.045). These results suggest that in epithelial ovarian cancer, MRP1 may be a marker for aggressiveness because its expression was associated with tumor grade and support that MRP4 may play an unfavourable role in disease outcome.

Proteomic analysis of cellular response to novel proapoptotic agents related to atypical retinoids in human IGROV-1 ovarian carcinoma cells.

Novel agents characterized by the scaffold of the atypical retinoid ST1926, but containing different chemical functions (carboxylic or hydroxamic acid), exhibit potent proapoptotic activity. In the present paper, we show that the treatment of the IGROV-1 ovarian cancer cell line with compounds sharing structural features with ST1926 (ST1898, ST3595, ST3056) determines a strong inhibition of proliferation mainly due to apoptotic cell death. In an effort to understand the mechanism of action of these compounds, we performed a proteomics analysis of IGROV-1 total lysates and nuclear extracts. Using this approach, we found that deregulation of calcium homeostasis, oxidative stress, cytoskeleton reorganization, and deregulation of proteasome function may represent important pathways involved in response of IGROV-1 cells to the studied compounds. The most prominent effect was down-regulation of factors involved in protein degradation, an event more marked in cells treated with ST3595. In addition, we identified proteins specifically modulated by each treatment, including prohibitin and cochaperone P23 (ST1898), pre-mRNA splicing factor SF2p32 and clathrin light chain (ST3595), as well as Far upstream element (FUSE) binding protein 1 and DNA-binding protein B (ST3056). By identifying proteins modulated by novel proapoptotic agents, this study provides insights into critical aspects of their mechanism of action.