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Background Despite the introduction of several novel therapeutic approaches that improved survival, metastatic castration-resistant prostate cancer (mCRPC) remains an incurable disease. Herein we report the synergistic antitumor interaction between two well-known drugs used for years in clinical practice, the antiepileptic agent with histone deacetylase inhibitory activity valproic acid and the cholesterol lowering agent simvastatin, in mCRPC models. Methods Synergistic anti-tumor effect was assessed on PC3, 22Rv1, DU145, DU145R80, LNCaP prostate cancer cell lines and EPN normal prostate epithelial cells, by calculating combination index (CI), caspase 3/7 activation and colony formation assays as well as on tumor spheroids and microtissues scored with luminescence 3D-cell viability assay. Cancer stem cells (CSC) compartment was studied evaluating specific markers by RT-PCR, western blotting and flow cytometry as well as by limiting dilution assay. Cholesterol content was evaluated by 1H-NMR. Overexpression of wild-type YAP and constitutively active YAP5SA were obtained by lipofectamine-based transfection and evaluated by immunofluorescence, western blotting and RT-PCR. 22Rv1 R_39 docetaxel resistant cells were selected by stepwise exposure to increasing drug concentrations. In vivo experiments were performed on xenograft models of DU145R80, 22Rv1 parental and docetaxel resistant cells, in athymic mice. Results We demonstrated the capacity of the combined approach to target CSC compartment by a novel molecular mechanism based on the inhibition of YAP oncogene via concurrent modulation of mevalonate pathway and AMPK. Because both CSCs and YAP activation have been associated with chemo-resistance, we tested if the combined approach can potentiate docetaxel, a standard of care in mCRCP treatment. Indeed, we demonstrated, both in vitro and in vivo models, the ability of valproic acid/simvastatin combination to sensitize mCRPC cells to docetaxel and to revert docetaxel-resistance, by mevalonate pathway/YAP axis modulation. Conclusion Overall, mCRPC progression and therapeutic resistance driven by CSCs via YAP, can be tackled by the combined repurposing of two generic and safe drugs, an approach that warrants further clinical development in this disease.

Breast cancer is one of the most frequently diagnosed forms of cancer and different treatments are used to block its progression. However, it still represents a very common cause of death in women. Doxorubicin (Dox) is reported as an effective agent in breast cancer treatment nonetheless it induces many side‑effects. For this reason, many laboratories are engaged in understanding how it is possible to decrease the drug concentration, considering that one of the possible solutions is to use drug synergy, combining it with natural substances. Recently we showed that a phenolic extract from flaxseed (FS) oil, named PEFSO, induced on MCF‑7 cell line an increase of apoptosis with related modification of G0/G1 phase cell cycle, and the activation of signaling and pro‑oxidant pathways. In this study we present data on the combined effect of Dox and PEFSO on two different breast cancer cell lines to define the conditions to use lower doses of this chemotherapeutic agent. We report the data relating to the ability of this mixture to induce cytotoxicity and apoptosis, cell cycle modification, mitochondrial membrane depolarization and activation of extrinsic and/or intrinsic apoptotic pathway.

Selenoproteins are proteins that contain selenium within selenocysteine residues. To date, twenty-five mammalian selenoproteins have been identified, however, the functions of nearly half of these selenoproteins are unknown. Although alterations in selenoprotein expression and function have been suggested to play a role in cancer development and progression, few detailed studies have been carried out in this field. Network analyses and data mining of publicly available datasets on gene expression levels in different cancers, and the correlations with patient outcome, represent important tools to study the correlation between selenoproteins and other proteins present in the human interactome, and to determine whether altered selenoprotein expression is cancer type-specific, and/or correlated with cancer patient prognosis. Therefore, in the present study, we used bioinformatics approaches to (i) build up the network of interactions between twenty-five selenoproteins and identify the most inter-correlated proteins/genes, which are named HUB nodes, and (ii) analyze the correlation between selenoprotein gene expression and patient outcome in ten solid tumors. Then, considering the need to confirm by experimental approaches the correlations suggested by the bioinformatics analyses, we decided to evaluate the gene expression levels of the twenty-five selenoproteins and six HUB nodes in androgen receptor-positive (22RV1 and LNCaP) and androgen receptor&ndash, negative (DU145 and PC3) cell lines, compared to human nontransformed, and differentiated, prostate epithelial cells (EPN) by RT-qPCR analysis. This analysis confirmed that the combined evaluation of some selenoproteins and HUB nodes could have prognostic value and may improve patient outcome predictions.

Background Intramuscular triglycerides (IMTGs) represent an important energy supply and a dynamic fat-storage depot that can expand during periods of elevated lipid availability and a fatty acid source. Ultrasonography (US) of human skeletal muscles is a practical and reproducible method to assess both IMTG presence and entity. Although a crosstalk between cytokines in skeletal muscle and adipose tissue has been suggested in obesity, condition leading to hepatic steatosis (HS) or better defined as nonalcoholic fatty liver disease and cancer, there are still questions to be answered about the role of interferons (IFNs), alpha as well as gamma, and IMTG in obesity. We aimed at discovering any correlation between IFNs and IMTG. Methods We analysed anthropometric data, metabolic parameters and imaging features of a population of 80 obese subjects with low-prevalence of co-morbidities but HS in relation to IFNs serum levels. A population of 38 healthy subjects (21 males) served as controls. The levels of serum IFNs were detected by a magnetic bead-based multiplex immunoassays. Results Serum concentrations of IFN-alpha 2 were increased, while serum levels of IFN-gamma were decreased confronted with those of controls; the severity of IMTG, revealed at US as Heckmatt scores, was inversely predicted by IFN-alpha 2 serum concentrations; IMTG scores were not predicted by serum levels of IFN-gamma; IMTG scores were predicted by HS severity, ascertained at US; HS severity was predicted by visceral adipose tissue, assessed by US, but the latter was not instrumental to IMTG. Discussion and conclusion This study has added some pieces of observation about the cytokine network regulating the interplay between IMTG and obesity in obese patients with HS.

Hepatoblastoma incidence has been associated with different environmental factors even if no data are reported about a correlation between aflatoxin exposure and hepatoblastoma initiation. Considering that hepatoblastoma develops in infants and children and aflatoxin M1 (AFM1), the aflatoxin B1 (AFB1) hydroxylated metabolite, can be present in mothers&rsquo, milk and in marketed milk products, in this study we decided to test the effects of AFM1 on a hepatoblastoma cell line (HepG2). Firstly, we evaluated the effects of AFM1 on the cell viability, apoptosis, cell cycle, and metabolomic and cytokinomic profile of HepG2 cells after treatment. AFM1 induced: (1) a decrease of HepG2 cell viability, reaching IC50 at 9 &micro, M, (2) the blocking of the cell cycle in the G0/G1 phase, (3) the decrease of formiate levels and incremented level of some amino acids and metabolites in HepG2 cells after treatment, and (4) the increase of the concentration of three pro-inflammatory cytokines, IL-6, IL-8, and TNF-&alpha, and the decrease of the anti-inflammatory interleukin, IL-4. Our results show that AFM1 inhibited the growth of HepG2 cells, inducing both a modulation of the lipidic, glycolytic, and amino acid metabolism and an increase of the inflammatory status of these cells.

Introduction: Zearalenone (ZEN) is one of the most widely distributed toxins that contaminates many crops and foods. Its major metabolites are α-Zearalenol (α-zol) and β-Zearalenol. Previous studies showed that ZEN and α-zol have estrogenic properties and are able to induce growth promoting effect in breast tissues. Objectivies: Considering that tumorigenesis is dependent on the reprogramming of cellular metabolism and that the evaluation of the cellular metabolome is useful to understand the metabolic changes that can occur during the cancer development and progression or after treatments, aim of our work is to study, for the first time, the effects of α-zol on the metabolomic profile of an estrogen positive breast cancer cell line, MCF-7, and of an estrogen negative breast cancer cell lines MDA-MB231. Methods: Firstly, we tested the effects of α-zol on the cell viability after 24, 48 and 72 h of treatments with 10−10, 10−8 and 10−6 M concentrations on breast cancer MCF-7 and MDA-MB231 cell lines in comparison to human non-cancerous breast MCF10A cell line. Then, we evaluated cell cycle progression, levels of reactive oxygen species (ROS) and the metabolomic profiling by 1H-NMR approach on MCF-7 and MDA-MB231 before and after 72 h treatments. Principal component analysis was used to compare the obtained spectra. Results: α-zol is resulted able to induce: (i) an increase of the cell viability on MCF-7 cells mainly after 72 h treatment, (ii) a slight decrease of the cell viability on MDA-MB231 cells, and (iii) an increase of cells in S phase of the cell cycle and of ROS only in MCF-7 cells. Moreover, the evaluation of metabolomics profile evidenced that after treatment with α-zol the levels of some metabolites increased in MCF-7 cells whereas decreased slightly in MDA-MB231 cells. Conclusions: Our results showed that α-zol was able to increase the protein biosynthesis as well as the lipid metabolism in MCF-7 cells, and, hence, to induce an estrogen positive breast cancer progression.

Marine sponges are an excellent source of bioactive secondary metabolites for pharmacological applications. In the present study, we evaluated the chemistry, cytotoxicity and metabolomics of an organic extract from the Mediterranean marine sponge Geodia cydonium, collected in coastal waters of the Gulf of Naples. We identified an active fraction able to block proliferation of breast cancer cell lines MCF-7, MDA-MB231, and MDA-MB468 and to induce cellular apoptosis, whereas it was inactive on normal breast cells (MCF-10A). Metabolomic studies showed that this active fraction was able to interfere with amino acid metabolism, as well as to modulate glycolysis and glycosphingolipid metabolic pathways. In addition, the evaluation of the cytokinome profile on the polar fractions of three treated breast cancer cell lines (compared to untreated cells) demonstrated that this fraction induced a slight anti-inflammatory effect. Finally, the chemical entities present in this fraction were analyzed by liquid chromatography high resolution mass spectrometry combined with molecular networking.

In this study, a hydroalcoholic chestnut shell extract was characterized and tested on six different human cell lines. Gallic, ellagic, and syringic acids were the most abundant non-condensed compounds in the chestnut extract, as determined by high performance liquid chromatography (HPLC). Tannins were mainly represented by condensed monomeric units of epigallocatechin and catechin/epicatechin. After 48 h of treatment, only the human hepatoblastoma HepG2 cells reached an inhibition corresponding to IC50 with an increase of apoptosis and mitochondrial depolarization. The cytokinome evaluation before and after treatment revealed that the vascular endothelial growth factor (VEGF) and the tumor necrosis factor (TNF)-α decreased after the treatment, suggesting a potential anti-angiogenic and anti-inflammatory effect of this extract. Moreover, the metabolome evaluation by ¹H-NMR evidenced that the polyphenols extracted from chestnut shell (PECS) treatment affected the levels of some amino acids and other metabolites. Overall, these data highlight the effects of biomolecules on cell proliferation, apoptosis, cell cycle and mitochondrial depolarization, and on cytokinomics and metabolomics profiles.

In recent years, many researchers are focusing their attention on the link between inflammation and cancer. The inflammation is involved in the tumor development and suppression, by stimulating the immune response. In particular, the transition from chronic inflammation to cancer produces angiogenic and growth factors able to repair the tissue and to promote cancer cell survival, implantation, and growth. In this contest, the cytokines contribute to the development of these processes becoming active before and during the inflammatory process and playing an important function at the various disease levels. Thus, these proteins can represent specific markers of tumor development and progression. Therefore the “cytokinome” term is used to indicate the evaluation of cytokine pattern by using an “omics” approach. Newly, specific protein chips of considerable and improved sensitivity are being developed to determine simultaneously several and different cytokines. This can be achieved by a multiplex technology that, through the use of small amounts of serum or other fluids, is used to determine the presence and the levels of underrepresented cytokines. Since this method is an accurate, sensitive, and reproducible cytokine assay, it is already used in many different studies. Thus, this review focuses on the more latest aspects related to cytokinome profile evaluation in different cancers.

Many studies have evidenced that the phenolic components from flaxseed (FS) oil have potential health benefits. The effect of the phenolic extract from FS oil has been evaluated on two human breast cancer cell lines, MCF7 and MDA-MB231, and on the human non-cancerous breast cell line, MCF10A, by SRB assay, cellular death, cell cycle, cell signaling, lipid peroxidation and expression of some key genes. We have evidenced that the extract shows anti-proliferative activity on MCF7 cells by inducing cellular apoptosis, increase of the percentage of cells in G0/G1 phase and of lipid peroxidation, activation of the H2AX signaling pathway, and upregulation of a six gene signature. On the other hand, on the MDA-MB2131 cells we verified only an anti-proliferative activity, a weak lipid peroxidation, the activation of the PI3K signaling pathway and an up-regulation of four genes. Overall these data suggest that the extract has both cytotoxic and pro-oxidant effects only on MCF7 cells, and can act as a metabolic probe, inducing differences in the gene expression. For this purpose, we have performed an interactomic analysis, highlighting the existing associations. From this approach, we show that the phenotypic difference between the two cell lines can be explained through their differential response to the phenolic extract.

Introduction: docetaxel (DTX) represents the standard of care first line treatment of castration-resistant prostate cancer (PCa). However, the onset of systemic side effects hampers patient's compliance and DTX resistance invariably emerges, leading to disease relapse, suggesting the need of novel combination strategies. Cancer stem cells (CSC) drive PCa survival and metastasis and are thought to be responsible for the development of resistance to DTX. The mevalonate pathway (MVP) plays a critical role in PCa progression and is implicated in cell stemness, proliferation, and organ size regulation through the YAP-TAZ signaling axis. Here, we evaluate the combination treatment of valproic acid (VPA), an histone deacetylase (HDAC) inhibitor and the cholesterol-lowering drug simvastatin (SIM), an inhibitor of HMGCoA reductase (HMGCR), the rate limiting enzyme in MVP, alone and plus DTX in PCa models. Method: synergistic antiproliferative effects were assessed on LNCAP, 22Rv1, DU145, PC3 and DU14580 SIM-resistant cell lines and normal epithelial prostate EPN cells, by calculating combination index (CI) according to Chou and Talalay method. Apoptosis was measured by FACS analysis and caspase assay. Tumor spheroids were obtained by low attach systems and scored with luminescence 3D-cell viability assay. Proteins and genes expression was assessed by western blot and real time PCR analysis. In vivo experiments were performed on xenograft models in athymic mice. Cholesterol content was evaluated by nuclear magnetic resonance (1H-NMR). Results: synergistic antiproliferative and proapoptotic effect of VPA-SIM was observed in all PCa cell lines tested, except in EPN cells and was confirmed in PCa spheroids. SIM-dependent cholesterol content downmodulation was potentiated by VPA and mevalonic acid, the product of HMGCR activity, reverts all the antitumor combined effect, suggesting the involvement of MVP in the synergistic interaction. Mechanistically, the combination is able to induce a reduction of HMGCR mRNA expression and the inhibitory phosphorylation of HMGCR and YAP, followed by a reduction of CTGF, BRCA5 and Cyr61 YAP-target genes, as well as a reduction of a CSC-marker gene such as NANOg. Notably, the VPA-SIM combination, sensitizes PCa cells to DTX treatment in sensitive and DTX-resistant cells, as shown by CI calculation, apoptosis, and CSC enriched-spheroid experiments. The synergistic interaction of the triple combination was also confirmed in vivo in both DU145R80 and 22Rv1 xenograft models. Conclusions: Overall, this study suggests that the combination of two safe generic drugs such as VPA and SIM, may improve the therapeutic index of DTX and revert DTX-resistance, representing an innovative and feasible antitumor strategy for the treatment of prostate cancer that warrants further clinical evaluation. Citation Format: Federica Iannelli, Maria Serena Roca, Chiara Ciardiello, Simona De Rienzo, Rita Lombardi, Angela Sorice, Susan Costantini, Tania Moccia, Maria Rita Milone, Biagio Pucci, Alfredo Budillon, Francesca Bruzzese. Synergistic antitumor interaction of valproic acid and simvastatin sensitizes prostate cancer to docetaxel by targeting cancer stem cells compartment via YAP-pathway modulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2877.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is still one of the most fatal cancers. Hence, it needs to identify always new putative markers to improve its diagnosis and prognosis. The selenium is an essential trace mineral implicated as a key factor in the early stage of cancer and exerts its biological function through the selenoproteins. In the last years our group has been studying the involvement of some selenoproteins in HCC. However, no many data are reported in literature about the correlation between HCC and the glutathione peroxidases (GPXs), both selenium and non selenium-containing GPXs. In this paper we have evaluated the GPX4 and GPX7 expression in some paraffin-embedded tissues from liver biopsy of patients with hepatitis C virus (HCV)-related cirrhosis and HCC by immunohistochemistry and RT-qPCR analysis. Our results evidenced that i) GPX4 and GPX7 had a statistically significant over-expression in HCC tissues compared to cirrhotic counterparts used as non tumor tissues, and ii) their expression was higher in grade III HCC tissues with respect to grade I-II samples. Therefore, we propose to use GPX4 and GPX7 as possible markers for improving HCC diagnosis/prognosis.

Correction for ‘Metabolomic profiling and biochemical evaluation of the follicular fluid of endometriosis patients’ by Marianna Santonastaso et al., Mol. BioSyst., 2017, DOI: 10.1039/c7mb00181a.