Serum Mass Spectrometry Proteomics and Protein Set Identification in Response to FOLFOX-4 in Drug-Resistant Ovarian Carcinoma.

Ovarian cancer is a highly lethal gynecological malignancy. Drug resistance rapidly occurs, and different therapeutic approaches are needed. So far, no biomarkers have been discovered to predict early response to therapies in the case of multi-treated ovarian cancer patients. The aim of our investigation was to identify a protein panel and the molecular pathways involved in chemotherapy response through a combination of studying proteomics and network enrichment analysis by considering a subset of samples from a clinical setting. Differential mass spectrometry studies were performed on 14 serum samples from patients with heavily pretreated platinum-resistant ovarian cancer who received the FOLFOX-4 regimen as a salvage therapy. The serum was analyzed at baseline time (T0) before FOLFOX-4 treatment, and before the second cycle of treatment (T1), with the aim of understanding if it was possible, after a first treatment cycle, to detect significant proteome changes that could be associated with patients responses to therapy. A total of 291 shared expressed proteins was identified and 12 proteins were finally selected between patients who attained partial response or no-response to chemotherapy when both response to therapy and time dependence (T0, T1) were considered in the statistical analysis. The protein panel included APOL1, GSN, GFI1, LCATL, MNA, LYVE1, ROR1, SHBG, SOD3, TEC, VPS18, and ZNF573. Using a bioinformatics network enrichment approach and metanalysis study, relationships between serum and cellular proteins were identified. An analysis of protein networks was conducted and identified at least three biological processes with functional and therapeutic significance in ovarian cancer, including lipoproteins metabolic process, structural component modulation in relation to cellular apoptosis and autophagy, and cellular oxidative stress response. Five proteins were almost independent from the network (LYVE1, ROR1, TEC, GFI1, and ZNF573). All proteins were associated with response to drug-resistant ovarian cancer resistant and were mechanistically connected to the pathways associated with cancer arrest. These results can be the basis for extending a biomarker discovery process to a clinical trial, as an early predictive tool of chemo-response to FOLFOX-4 of heavily treated ovarian cancer patients and for supporting the oncologist to continue or to interrupt the therapy.

A Peptidic Thymidylate-Synthase Inhibitor Loaded on Pegylated Liposomes Enhances the Antitumour Effect of Chemotherapy Drugs in Human Ovarian Cancer Cells.

There is currently no effective long-term treatment for ovarian cancer (OC) resistant to poly-chemotherapy regimens based on platinum drugs. Preclinical and clinical studies have demonstrated a strong association between development of Pt-drug resistance and increased thymidylate synthase (hTS) expression, and the consequent cross-resistance to the hTS inhibitors 5-fluorouracil (5-FU) and raltitrexed (RTX). In the present work, we propose a new tool to combat drug resistance. We propose to treat OC cell lines, both Pt-sensitive and -resistant, with dual combinations of one of the four chemotherapeutic agents that are widely used in the clinic, and the new peptide, hTS inhibitor, [D-Gln4]LR. This binds hTS allosterically and, unlike classical inhibitors that bind at the catalytic pocket, causes cell growth inhibition without inducing hTS overexpression. The dual drug combinations showed schedule-dependent synergistic antiproliferative and apoptotic effects. We observed that the simultaneous treatment or 24h pre-treatment of OC cells with the peptide followed by either agent produced synergistic effects even in resistant cells. Similar synergistic or antagonistic effects were obtained by delivering the peptide into OC cells either by means of a commercial delivery system (SAINT-PhD) or by pH sensitive PEGylated liposomes. Relative to non-PEGylated liposomes, the latter had been previously characterized and found to allow macrophage escape, thus increasing their chance to reach the tumour tissue. The transition from the SAINT-PhD delivery system to the engineered liposomes represents an advancement towards a more drug-like delivery system and a further step towards the use of peptides for in vivo studies. Overall, the results suggest that the association of standard drugs, such as cDDP and/or 5-FU and/or RTX, with the novel peptidic TS inhibitor encapsulated into PEGylated pH-sensitive liposomes can represent a promising strategy for fighting resistance to cDDP and anti-hTS drugs.

Discovery of a benzothiophene-flavonol halting miltefosine and antimonial drug resistance in Leishmania parasites through the application of medicinal chemistry, screening and genomics.

Leishmaniasis, a major health problem worldwide, has a limited arsenal of drugs for its control. The appearance of resistance to first- and second-line anti-leishmanial drugs confirms the need to develop new and less toxic drugs that overcome spontaneous resistance. In the present study, we report the design and synthesis of a novel library of 38 flavonol-like compounds and their evaluation in a panel of assays encompassing parasite killing, pharmacokinetics, genomics and ADME-Toxicity resulting in the progression of a compound in the drug discovery value chain. Compound 19, 2-(benzo[b]thiophen-3-yl)-3-hydroxy-6-methoxy-4H-chromen-4-one, exhibited a broad-spectrum activity against Leishmania spp. (EC50 1.9 µM for Leishmania infantum, 3.4 µM for L. donovani, 6.7 µM for L. major), Trypanosoma cruzi (EC50 7.5 µM) and T. brucei (EC50 0.8 µM). Focusing on anti-Leishmania activity, compound 19 challenge in vitro did not select for resistance markers in L. donovani, while a Cos-Seq screening for dominant resistance genes identified a gene locus on chromosome 36 that became ineffective at concentrations beyond EC50. Thus, compound 19 is a promising scaffold to tackle drug resistance in Leishmania infection. In vivo pharmacokinetic studies indicated that compound 19 has a long half-life (intravenous (IV): 63.2 h; per os (PO): 46.9 h) with an acceptable ADME-Toxicity profile. When tested in Leishmania infected hamsters, no toxicity and limited efficacy were observed. Low solubility and degradation were investigated spectroscopically as possible causes for the sub-optimal pharmacokinetic properties. Compound 19 resulted a specific compound based on the screening against a protein set, following the intrinsic fluorescence changes.

pH-Promoted Release of a Novel Anti-Tumour Peptide by "Stealth" Liposomes: Effect of Nanocarriers on the Drug Activity in Cis-Platinum Resistant Cancer Cells.

PURPOSE: To evaluate the potential effects of PEGylated pH-sensitive liposomes on the intracellular activity of a new peptide recently characterized as a novel inhibitor of the human thymidylate synthase (hTS) over-expressed in many drug-resistant human cancer cell lines. METHODS: Peptide-loaded pH-sensitive PEGylated (PpHL) and non-PEGylated liposomes (nPpHL) were carefully characterized and delivered to cis-platinum resistant ovarian cancer C13* cells; the influence of the PpHL on the drug intracellular activity was investigated by the Western Blot analysis of proteins involved in the pathway affected by hTS inhibition. RESULTS: Although PpHL and nPpHL showed different sizes, surface hydrophilicities and serum stabilities, both carriers entrapped the drug efficiently and stably demonstrating a pH dependent release; moreover, the different behavior against J774 macrophage cells confirmed the ability of PEGylation in protecting liposomes from the reticuloendothelial system. Comparable effects were instead observed against C13* cells and biochemical data by immunoblot analysis indicated that PEGylated pH-sensitive liposomes do not modify the proteomic profile of the cells, fully preserving the activity of the biomolecule. CONCLUSION: PpHL can be considered as efficient delivery systems for the new promising anti-cancer peptide.

Conformational Propensity and Biological Studies of Proline Mutated LR Peptides Inhibiting Human Thymidylate Synthase and Ovarian Cancer Cell Growth.

LR and [d-Gln4]LR peptides bind the monomer-monomer interface of human thymidylate synthase and inhibit cancer cell growth. Here, proline-mutated LR peptides were synthesized. Molecular dynamics calculations and circular dichroism spectra have provided a consistent picture of the conformational propensities of the [Pro n]-peptides. [Pro3]LR and [Pro4]LR show improved cell growth inhibition and similar intracellular protein modulation compared with LR. These represent a step forward to the identification of more rigid and metabolically stable peptides.

Proteomic and Bioinformatic Studies for the Characterization of Response to Pemetrexed in Platinum Drug Resistant Ovarian Cancer.

Proteomics and bioinformatics are a useful combined technology for the characterization of protein expression level and modulation associated with the response to a drug and with its mechanism of action. The folate pathway represents an important target in the anticancer drugs therapy. In the present study, a discovery proteomics approach was applied to tissue samples collected from ovarian cancer patients who relapsed after the first-line carboplatin-based chemotherapy and were treated with pemetrexed (PMX), a known folate pathway targeting drug. The aim of the work is to identify the proteomic profile that can be associated to the response to the PMX treatment in pre-treatement tissue. Statistical metrics of the experimental Mass Spectrometry (MS) data were combined with a knowledge-based approach that included bioinformatics and a literature review through ProteinQuest™ tool, to design a protein set of reference (PSR). The PSR provides feedback for the consistency of MS proteomic data because it includes known validated proteins. A panel of 24 proteins with levels that were significantly different in pre-treatment samples of patients who responded to the therapy vs. the non-responder ones, was identified. The differences of the identified proteins were explained for the patients with different outcomes and the known PMX targets were further validated. The protein panel herein identified is ready for further validation in retrospective clinical trials using a targeted proteomic approach. This study may have a general relevant impact on biomarker application for cancer patients therapy selection.

Human Thymidylate Synthase Inhibitors Halting Ovarian Cancer Growth.

Human thymidylate synthase (hTS) has an important role in DNA biosynthesis, thus it is essential for cell survival. TS is involved in the folate pathways, specifically in the de novo pyrimidine biosynthesis. Structure and functions are intimately correlated, account for cellular activity and, in a broader view, with in vivo mechanisms. hTS is a target for anticancer agents, some of which are clinical drugs. The understanding of the detailed mechanism of TS inhibition by currently used drugs and of the interaction with the mechanism of action of other anticancer agents can suggest new perspective of TS inhibition able to improve the anticancer effect and to overcome drug resistance. TS-targeting drugs in therapy today are inhibitors that bind at the active site and that mostly resemble the substrates. Nonsubstrate analogs offer an opportunity for allosteric binding and novel mode of inhibition in the cancer cells. This chapter illustrates the relationship among the large number of hTS actions at molecular and clinical levels, its role as a target for ovarian cancer therapy, in particular in cases of overexpression of hTS and other folate proteins such as those induced by platinum drug treatments, and address the potential combination of TS inhibitors with other suitable anticancer agents.

Inside the biochemical pathways of thymidylate synthase perturbed by anticancer drugs: Novel strategies to overcome cancer chemoresistance.

Our current understanding of the mechanisms of action of antitumor agents and the precise mechanisms underlying drug resistance is that these two processes are directly linked. Moreover, it is often possible to delineate chemoresistance mechanisms based on the specific mechanism of action of a given anticancer drug. A more holistic approach to the chemoresistance problem suggests that entire metabolic pathways, rather than single enzyme targets may better explain and educate us about the complexity of the cellular responses upon cytotoxic drug administration. Drugs, which target thymidylate synthase and folate-dependent enzymes, represent an important therapeutic arm in the treatment of various human malignancies. However, prolonged patient treatment often provokes drug resistance phenomena that render the chemotherapeutic treatment highly ineffective. Hence, strategies to overcome drug resistance are primarily designed to achieve either enhanced intracellular drug accumulation, to avoid the upregulation of folate-dependent enzymes, and to circumvent the impairment of DNA repair enzymes which are also responsible for cross-resistance to various anticancer drugs. The current clinical practice based on drug combination therapeutic regimens represents the most effective approach to counteract drug resistance. In the current paper, we review the molecular aspects of the activity of TS-targeting drugs and describe how such mechanisms are related to the emergence of clinical drug resistance. We also discuss the current possibilities to overcome drug resistance by using a molecular mechanistic approach based on medicinal chemistry methods focusing on rational structural modifications of novel antitumor agents. This paper also focuses on the importance of the modulation of metabolic pathways upon drug administration, their analysis and the assessment of their putative roles in the networks involved using a meta-analysis approach. The present review describes the main pathways that are modulated by TS-targeting anticancer drugs starting from the description of the normal functioning of the folate metabolic pathway, through the protein modulation occurring upon drug delivery to cultured tumor cells as well as cancer patients, finally describing how the pathways are modulated by drug resistance development. The data collected are then analyzed using network/netwire connecting methods in order to provide a wider view of the pathways involved and of the importance of such information in identifying additional proteins that could serve as novel druggable targets for efficacious cancer therapy.