Extracellular Vesicle-Related Non-Coding RNAs in Hepatocellular Carcinoma: An Overview.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer. It is a major public health problem worldwide, and it is often diagnosed at advanced stages, when no effective treatment options are available. Extracellular vesicles (EVs) are nanosized double-layer lipid vesicles containing various biomolecule cargoes, such as lipids, proteins, and nucleic acids. EVs are released from nearly all types of cells and have been shown to play an important role in cell-to-cell communication. In recent years, many studies have investigated the role of EVs in cancer, including HCC. Emerging studies have shown that EVs play primary roles in the development and progression of cancer, modulating tumor growth and metastasis formation. Moreover, it has been observed that non-coding RNAs (ncRNAs) carried by tumor cell-derived EVs promote tumorigenesis, regulating the tumor microenvironment (TME) and playing critical roles in the progression, angiogenesis, metastasis, immune escape, and drug resistance of HCC. EV-related ncRNAs can provide information regarding disease status, thus encompassing a role as biomarkers. In this review, we discuss the main roles of ncRNAs present in HCC-derived EVs, including micro(mi) RNAs, long non-coding (lnc) RNAs, and circular (circ) RNAs, and their potential clinical value as biomarkers and therapeutic targets.

The clinical impact of an extra virgin olive oil enriched mediterranean diet on metabolic syndrome: Lights and shadows of a nutraceutical approach.

For years it has been established that the only truly effective treatment of metabolic syndrome (MS) is lifestyle modification to prevent its cardiovascular (e.g., coronary artery disease and atherosclerosis), metabolic (e.g., diabetes mellitus), and hepatic (e.g., steatosis and non-alcoholic steatohepatitis) complications. The focal points of this approach are to increase physical activity and intake of a diet characterized by high quantities of fruits, vegetables, grains, fish, and low-fat dairy products, the so called mediterranean diet (MD); however, the added value of MD is the presence of extra virgin olive oil (EVOO), a healthy food with a high content of monounsaturated fatty acids, especially oleic acid, and variable concentrations (range 50-800 mg/kg) of phenols (oleuropein, ligstroside, and oleocanthal, and their derivatives, phenolic alcohols, such as hydroxytyrosol and tyrosol). Phenolic compounds not only determine EVOO's main organoleptic qualities (oxidative stability, specific flavor, and taste features) but, theoretically, make it a source of antioxidant, anti-inflammatory, insulin-sensitizing, cardioprotective, antiatherogenic, neuroprotective, immunomodulatory, and anticancer activity. Although many studies have been carried out on EVOO's clinical effects and attention toward this dietary approach (healthy and palatable food with strong nutraceutical activity) has become increasingly pressing, there are still many dark sides to be clarified, both in terms of actual clinical efficacy and biochemical and molecular activity. Thus, we reviewed the international literature, trying to show the state of the art about EVOO's clinical properties to treat MS (along with correlated complications) and the future prospective of its nutraceutical use.

Hepatocellular Carcinoma: A Difficult Cancer to Treat.

Hepatocellular carcinoma (HCC) is a very peculiar cancer because it presents several molecular alterations linked to the activation of survival and antiapoptotic signal pathways that are protein in form and not easily targetable by even the newest targeted therapies. In addition, it is almost always a consequence of liver cirrhosis, a serious disease condition in which several drugs are often not tolerated. This is why the study of HCC was such a challenge for Professor Natale D'Alessandro, to whom this work is dedicated, during the latter years of his career. The aim of this review is to summarize studies on different molecules involved in the development, progression, and chemoresistance of HCC, topics on which we have focused our research over the last decade. In particular, we have analyzed the role of inflammatory mediators, such as the cyclooxygenase (COX) enzymes, nuclear factor κB (NF-κB), interleukin 6 (IL-6), as well as other important factors, such as Yin Yang 1 (YY1), in HCC. Moreover, we have reviewed some more recent literature on research aimed at identifying druggable targets in HCC as well as candidate agents for its prevention and treatment.

The NUPR1/p73 axis contributes to sorafenib resistance in hepatocellular carcinoma.

The multikinase inhibitor sorafenib was the first drug approved by the FDA for treating patients with advanced hepatocellular carcinoma (HCC). However, sorafenib resistance remains a major challenge for improving the effectiveness of HCC treatment. Previously, we identified several genes modulated after sorafenib treatment of human HCC cells, including the stress-inducible nuclear protein 1 (NUPR1) gene. Multiple studies have shown that NUPR1 regulates autophagy, apoptosis, and chemoresistance. Here, we demonstrate that treatment of HCC cells with sorafenib resulted in the activation of autophagic flux. NUPR1 knock-down (KD) in HCC cells was associated with increased p62 expression, suggesting an impairment of autophagic flux, and with a significant increase of cell sensitivity to sorafenib. In NUPR1 KD cells, reduced levels of NUPR1 were associated with the increased expression of p73 as well as its downstream transcription targets PUMA, NOXA, and p21. Simultaneous silencing of p73 and NUPR1 in HCC cells resulted in increased resistance to sorafenib, as compared to the single KD of either gene. Conversely, pharmacological activation of p73, via the novel p73 small molecule activator NSC59984, determined synergistic anti-tumor effects in sorafenib-treated HCC cells. The combination of NSC59984 and sorafenib, when compared to either treatment alone, synergistically suppressed tumor growth of HCC cells in vivo. Our data suggest that the activation of the p73 pathway achieved by NUPR1 KD potentiates sorafenib-induced anti-tumor effects in HCC cells. Moreover, combined pharmacological therapy with the p73 activator NSC59984 and sorafenib could represent a novel approach for HCC treatment.

Nanoalgosomes: Introducing extracellular vesicles produced by microalgae.

Cellular, inter-organismal and cross kingdom communication via extracellular vesicles (EVs) is intensively studied in basic science with high expectation for a large variety of bio-technological applications. EVs intrinsically possess many attributes of a drug delivery vehicle. Beyond the implications for basic cell biology, academic and industrial interests in EVs have increased in the last few years. Microalgae constitute sustainable and renewable sources of bioactive compounds with a range of sectoral applications, including the formulation of health supplements, cosmetic products and food ingredients. Here we describe a newly discovered subtype of EVs derived from microalgae, which we named nanoalgosomes. We isolated these extracellular nano-objects from cultures of microalgal strains, including the marine photosynthetic chlorophyte Tetraselmis chuii, using differential ultracentrifugation or tangential flow fractionation and focusing on the nanosized small EVs (sEVs). We explore different biochemical and physical properties and we show that nanoalgosomes are efficiently taken up by mammalian cell lines, confirming the cross kingdom communication potential of EVs. This is the first detailed description of such membranous nanovesicles from microalgae. With respect to EVs isolated from other organisms, nanoalgosomes present several advantages in that microalgae are a renewable and sustainable natural source, which could easily be scalable in terms of nanoalgosome production.

Sialylation of host proteins as targetable risk factor for COVID-19 susceptibility and spreading: A hypothesis.

Individuals infected with the severe acute respiratory syndrome (SARS)-related coronavirus 2 (SARS-CoV-2) develop a critical and even fatal disease, called Coronavirus disease-19 (COVID-19), that eventually evolves into acute respiratory distress syndrome. The gravity of the SARS-CoV-2 pandemic, the escalating number of confirmed cases around the world, the many unknowns related to the virus mode of action, and the heterogenous outcome of COVID-19 disease in the population ask for the rapid development of alternative approaches, including repurposing of existing drugs, that may dampen virus infectivity. SARS-CoV-2 infects human cells through interaction with sialylated receptors at the surface of epithelial cells, such as angiotensin-converting enzyme 2 (ACE2). Glycan composition on virus entry receptors has been shown to influence the rate of infection of SARS-CoV-2 and spreading of virions has recently been linked to altered lysosomal exocytosis. These processes could concurrently involve the lysosomal system and its glycosidases. We hypothesize that modulating the activity of one of them, the lysosomal sialidase NEU1, could impinge on both the sialylation status of ACE2 and other host receptors as well as the extent of lysosomal exocytosis. Thus NEU1-controlled pathways may represent therapeutic targets, which could impact on SARS-CoV-2 susceptibility, infectivity, and spread.

Isolation of extracellular vesicles from microalgae: towards the production of sustainable and natural nanocarriers of bioactive compounds.

Safe, efficient and specific nano-delivery systems are essential for current and emerging therapeutics, precision medicine and other biotechnology sectors. Novel bio-based nanotechnologies have recently arisen, which are based on the exploitation of extracellular vesicles (EVs). In this context, it has become essential to identify suitable organisms or cellular types to act as reliable sources of EVs and to develop their pilot- to large-scale production. The discovery of new biosources and the optimisation of related bioprocesses for the isolation and functionalisation of nano-delivery vehicles are fundamental to further develop therapeutic and biotechnological applications. Microalgae constitute sustainable sources of bioactive compounds with a range of sectorial applications including for example the formulation of health supplements, cosmetic products or food ingredients. In this study, we demonstrate that microalgae are promising producers of EVs. By analysing the nanosized extracellular nano-objects produced by eighteen microalgal species, we identified seven promising EV-producing strains belonging to distinct lineages, suggesting that the production of EVs in microalgae is an evolutionary conserved trait. Here we report the selection process and focus on one of this seven species, the glaucophyte Cyanophora paradoxa, which returned a protein yield in the small EV fraction of 1 µg of EV proteins per mg of dry weight of microalgal biomass (corresponding to 109 particles per mg of dried biomass) and EVs with a diameter of 130 nm (mode), as determined by the micro bicinchoninic acid assay, nanoparticle tracking and dynamic light scattering analyses. Moreover, the extracellular nanostructures isolated from the conditioned media of microalgae species returned positive immunoblot signals for some commonly used EV-biomarkers such as Alix, Enolase, HSP70, and ß-actin. Overall, this work establishes a platform for the efficient production of EVs from a sustainable bioresource and highlights the potential of microalgal EVs as novel biogenic nanovehicles.

NUPR1 protects liver from lipotoxic injury by improving the endoplasmic reticulum stress response.

Non-alcoholic fatty liver (NAFL) and related syndromes affect one-third of the adult population in industrialized and developing countries. Lifestyle and caloric oversupply are the main causes of such array of disorders, but the molecular mechanisms underlying their etiology remain elusive. Nuclear Protein 1 (NUPR1) expression increases upon cell injury in all organs including liver. Recently, we reported NUPR1 actively participates in the activation of the Unfolded Protein Response (UPR). The UPR typically maintains protein homeostasis, but downstream mediators of the pathway regulate metabolic functions including lipid metabolism. As increases in UPR and NUPR1 in obesity and liver disease have been well documented, the goal of this study was to investigate the roles of NUPR1 in this context. To establish whether NUPR1 is involved in these liver conditions we used patient-derived liver biopsies and in vitro and in vivo NUPR1 loss of functions models. First, we analyzed NUPR1 expression in a cohort of morbidly obese patients (MOPs), with simple fatty liver (NAFL) or more severe steatohepatitis (NASH). Next, we explored the metabolic roles of NUPR1 in wild-type (Nupr1+/+ ) or Nupr1 knockout mice (Nupr1-/- ) fed with a high-fat diet (HFD) for 15 weeks. Immunohistochemical and mRNA analysis revealed NUPR1 expression is inversely correlated to hepatic steatosis progression. Mechanistically, we found NUPR1 participates in the activation of PPAR-α signaling via UPR. As PPAR-α signaling is controlled by UPR, collectively, these findings suggest a novel function for NUPR1 in protecting liver from metabolic distress by controlling lipid homeostasis, possibly through the UPR.

Potential Uses of Olive Oil Secoiridoids for the Prevention and Treatment of Cancer: A Narrative Review of Preclinical Studies.

The Mediterranean diet (MD) is a combination of foods mainly rich in antioxidants and anti-inflammatory nutrients that have been shown to have many health-enhancing effects. Extra-virgin olive oil (EVOO) is an important component of the MD. The importance of EVOO can be attributed to phenolic compounds, represented by phenolic alcohols, hydroxytyrosol, and tyrosol, and to secoiridoids, which include oleocanthal, oleacein, oleuropein, and ligstroside (along with the aglycone and glycosidic derivatives of the latter two). Each secoiridoid has been studied and characterized, and their effects on human health have been documented by several studies. Secoiridoids have antioxidant, anti-inflammatory, and anti-proliferative properties and, therefore, exhibit anti-cancer activity. This review summarizes the most recent findings regarding the pharmacological properties, molecular targets, and action mechanisms of secoiridoids, focusing attention on their preventive and anti-cancer activities. It provides a critical analysis of preclinical, in vitro and in vivo, studies of these natural bioactive compounds used as agents against various human cancers. The prospects for their possible use in human cancer prevention and treatment is also discussed.

Antioxidant Bioprospecting in Microalgae: Characterisation of the Potential of Two Marine Heterokonts from Irish Waters.

Microalgae constitute a heterogeneous and diverse range of organisms capable of accumulating bioactive metabolites, making them promising feedstock for applications in the nutraceutical, functional food, animal feed, biofertilisation or biofuel sectors. There has been renewed interest in recent times in natural sources of antioxidants, particularly as health products and preserving agents. Microalgae strains isolated from aquatic habitats in Ireland were successfully brought into culture. The 91 strains were grown phototrophically in nutrient-enriched media to generate biomass, which was harvested and assessed for antioxidant potential. Extracts were screened for antioxidant activity using a modified volumetric Trolox-ABTS assay and the Folin-Ciocalteu method. Two heterokont marine strains of interest were further studied to ascertain variations in antioxidant capacity across different stages of batch culture growth. The antioxidant activity of extracts of bacillariophyte cf. Stauroneis sp. LACW24 and ocrophyte cf. Phaeothamnion sp. LACW34 increased during growth with a maximum being observed during the late stationary or early death phase (2.5- to 8-fold increases between days 20 and 27). Strains LACW24 and LACW34 contained 5.9 and 3.0 mg g-1 (DW) of the xanthophyll fucoxanthin, respectively. Extracts of strains also showed no cytotoxicity towards mouse cell lines. These results highlight the potential of these strains for biomass valorisation and cultivation upscaling and to be further considered as part of ongoing bioprospecting efforts towards identifying novel species to join the relatively narrow range of commercially exploited marine microalgae species.

The Role of GSK-3 in Cancer Immunotherapy: GSK-3 Inhibitors as a New Frontier in Cancer Treatment.

The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) was initially identified because of its key role in the regulation of glycogen synthesis. However, it is now well-established that GSK-3 performs critical functions in many cellular processes, such as apoptosis, tumor growth, cell invasion, and metastasis. Aberrant GSK-3 activity has been associated with many human diseases, including cancer, highlighting its potential therapeutic relevance as a target for anticancer therapy. Recently, newly emerging data have demonstrated the pivotal role of GSK-3 in the anticancer immune response. In the last few years, many GSK-3 inhibitors have been developed, and some are currently being tested in clinical trials. This review will discuss preclinical and initial clinical results with GSK-3ß inhibitors, highlighting the potential importance of this target in cancer immunotherapy. As described in this review, GSK-3 inhibitors have been shown to have antitumor activity in a wide range of human cancer cells, and they may also contribute to promoting a more efficacious immune response against tumor target cells, thus showing a double therapeutic advantage.

GSK-3 in liver diseases: Friend or foe?

Liver diseases, including hepatitis due to hepatitis B or C virus infection, non-alcoholic fatty liver disease, and hepatocellular carcinoma pose major challenges for overall health due to limited curative treatment options. Thus, there is an urgent need to develop new therapeutic strategies for the treatment of these diseases. A better understanding of the signaling pathways involved in the pathogenesis of liver diseases can help to improve the efficacy of emerging therapies, mainly based on pharmacological approaches, which influence one or more specific molecules involved in key signal transduction pathways. These emerging therapies are very promising for the prevention and treatment of liver diseases. One promising druggable molecular target is the multifunctional serine/threonine kinase, glycogen synthase kinase 3 (GSK-3). In this review, we discuss conditions in which GSK-3 is implicated in liver diseases. In addition, we explore newly emerging drugs that target GSK-3ß, as well as their potential use in and impact on the management of liver diseases.

New landscapes and horizons in hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC), is the sixth most frequent form of cancer and leads to the fourth highest number of deaths each year. HCC results from a combination of environmental factors and aging as there are driver mutations at oncogenes which occur during aging. Most of HCCs are diagnosed at advanced stage preventing curative therapies. Treatment in advanced stage is a challenging and pressing problem, and novel and well-tolerated therapies are urgently needed. We will discuss further advances beyond sorafenib that target additional signaling pathways and immune checkpoint proteins. The scenario of possible systemic therapies for patients with advanced HCC has changed dramatically in recent years. Personalized genomics and various other omics approaches may identify actionable biochemical targets, which are activated in individual patients, which may enhance therapeutic outcomes. Further studies are needed to identify predictive biomarkers and aberrantly activated signaling pathways capable of guiding the clinician in choosing the most appropriate therapy for the individual patient.

RAS/RAF/MEK/ERK, PI3K/PTEN/AKT/mTORC1 and TP53 pathways and regulatory miRs as therapeutic targets in hepatocellular carcinoma.

Introduction: Hepatocellular carcinoma (HCC) is a significant problem globally because of viral infections and the increasing incidence of obesity and fatty liver disease. However, it is difficult to treat because its inherent genetic heterogeneity results in activation of numerous signaling pathways. Kinases have been targeted for decades with varying results, but the development of therapeutic resistance is a major challenge.Areas covered: The key roles of the RAS/RAF/MEK/ERK, PI3K/PTEN/AKT/mTORC1, TP53 microRNAs (miRs) as therapeutic targets are discussed and we suggests novel approaches for targeting miRs or their downstream targets to combat HCC. We performed literature searches using the Medline Database from 2000 to the present.Expert opinion: The involvement of RAS/RAF/MEK/ERK, PI3K/PTEN/AKT/mTORC and TP53 pathways as drivers of the disease and drug resistance is a challenge. Moreover, miRs regulate the expression of key genes in these pathways. What we and others are proposing is the prospect of targeting miRs and their downstream targets to improve conventional approaches to treat HCC. Combination approaches are often promising because multiple signaling pathways are deregulated due to diverse mutations and events.

Synthetic peptide-labelled micelles for active targeting of cells overexpressing EGF receptors.

The goal of nanomedicine is to transport drugs to pathological tissues, reducing side effects while increasing targeting and efficacy. Aggregates grafted by bioactive molecules act as the active targeting agents. Among bioactive molecules, peptides, which are able to recognize overexpressed receptors on cancer cell membranes, appear to be very promising. The aim of this study was to formulate analog peptide-labeled micelles enabled to potentially deliver highly hydrophobic drugs to cancer cells overexpressing epidermal growth factor (EGF) receptor (EGFR). The selected synthetic peptide sequences were anchored to a hydrophobic moiety, aiming to obtain amphiphilic peptide molecules. Mixed micelles were formulated with Pluronic® F127. These micelles were fully characterized by physico-chemical methods, estimating the critical micellar concentration (CMC) by fluorescence. Their sizes were established by dynamic light scattering (DLS) analysis. Then, micelles were also tested in vitro for their binding capacity to human hepatocellular carcinoma (HCC) cell lines overexpressing EGFR.

Targeting HSP90 with the small molecule inhibitor AUY922 (luminespib) as a treatment strategy against hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant tumor that responds very poorly to existing therapies, most probably due to its extraordinary inter- and intra-tumor molecular heterogeneity. The modest therapeutic response to molecular targeted agents underlines the need for new therapeutic approaches for HCC. In our study, we took advantage of well-characterized human HCC cell lines, differing in transcriptomic subtypes, DNA mutation and amplification alterations, reflecting the heterogeneity of primary HCCs, to provide a preclinical evaluation of the specific heat shock protein 90 (HSP90) inhibitor AUY922 (luminespib). Indeed, HSP90 is highly expressed in different tumor types, but its role in hepatocarcinogenesis remains unclear. Here, we analyzed HSP90 expression in primary human HCC tissues and evaluated the antitumor effects of AUY922 in vitro as well as in vivo. HSP90 expression was significantly higher in HCC tissues than in cirrhotic peritumoral liver tissues. AUY922 treatment reduced the cell proliferation and viability of HCC cells in a dose-dependent manner, but did not do so for normal human primary hepatocytes. AUY922 treatment led to the upregulation of HSP70 and the simultaneous depletion of HSP90 client proteins. In addition, in a cell type-dependent manner, treatment induced either both caspase-dependent ß-catenin cleavage and the upregulation of p53, or Mcl-1 expression, or NUPR1 expression, which contributed to the increased efficacy of, or resistance to, treatment. Finally, in vivo AUY922 inhibited tumor growth in a xenograft model. In conclusion, HSP90 is a promising therapeutic target in HCC, and AUY922 could be a drug candidate for its treatment.

Preclinical evaluation of antitumor activity of the proteasome inhibitor MLN2238 (ixazomib) in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is one of the common malignancies and is an increasingly important cause of cancer death worldwide. Surgery, chemotherapy, and radiation therapy extend the 5-year survival limit in HCC patients by only 6%. Therefore, there is a need to develop new therapeutic approaches for the treatment of this disease. The orally bioavailable proteasome inhibitor MLN2238 (ixazomib) has been demonstrated to have anticancer activity. In the present study, we investigated the preclinical therapeutic efficacy of MLN2238 in HCC cells through in vitro and in vivo models, and examined its molecular mechanisms of action. MLN2238 inhibited cell viability in human HCC cells HepG2, Hep3B, and SNU475 in a time- and dose-dependent manner. Flow cytometry analysis demonstrated that MLN2238 induced G2/M cell cycle arrest and cellular apoptosis in HCC cells. Cell cycle arrest was associated with increased expression levels of p21 and p27. MLN2238-induced apoptosis was confirmed by caspase-3/7 activation, PARP cleavage and caspase-dependent ß-catenin degradation. In addition, MLN2238 activated ER stress genes in HCC cells and increased the expression of the stress-inducible gene nuclear protein-1. Furthermore, MLN2238 treatment induced upregulation of myeloid cell leukemia-1 (Mcl-1) protein, and Mcl-1 knockdown sensitized HCC cells to MLN2238 treatment, suggesting the contribution of Mcl-1 expression to MLN2238 resistance. This result was also confirmed using the novel Mcl-1 small molecule inhibitor A1210477. Association of A1210477 and MLN2238 determined synergistic antitumor effects in HCC cells. Finally, in vivo orally administered MLN2238 suppressed tumor growth of Hep3B cells in xenograft models in nude mice. In conclusion, our results offer hope for a new therapeutic opportunity in the treatment of HCC patients.

Pivotal roles of glycogen synthase-3 in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers in the world, and represents the second most frequently cancer and third most common cause of death from cancer worldwide. At advanced stage, HCC is a highly aggressive tumor with a poor prognosis and with very limited response to common therapies. Therefore, there is still the need for new effective and well-tolerated therapeutic strategies. Molecular-targeted therapies hold promise for HCC treatment. One promising molecular target is the multifunctional serine/threonine kinase glycogen synthase kinase 3 (GSK-3). The roles of GSK-3ß in HCC remain controversial, several studies suggested a possible role of GSK-3ß as a tumor suppressor gene in HCC, whereas, other studies indicate that GSK-3ß is a potential therapeutic target for this neoplasia. In this review, we will focus on the different roles that GSK-3 plays in HCC and its interaction with signaling pathways implicated in the pathogenesis of HCC, such as Insulin-like Growth Factor (IGF), Notch, Wnt/ß-catenin, Hedgehog (HH), and TGF-ß pathways. In addition, the pivotal roles of GSK3 in epithelial-mesenchymal transition (EMT), invasion and metastasis will be also discussed.

Oleocanthal exerts antitumor effects on human liver and colon cancer cells through ROS generation.

The beneficial health properties of the Mediter-ranean diet are well recognized. The principle source of fat in Mediterranean diet is extra-virgin olive oil (EVOO). Oleocanthal (OC) is a naturally occurring minor phenolic compound isolated from EVOO, which has shown a potent anti-inflammatory activity, by means of its ability to inhibit the cyclooxygenase (COX) enzymes COX-1 and COX-2. A large body of evidence indicates that phenols exhibit anticancer activities. The aim of the present study was to evaluate the potential anticancer effects of OC in hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) models. A panel of human HCC (HepG2, Huh7, Hep3B and PLC/PRF/5) and CRC (HT29, SW480) cell lines was used. Cells were treated with OC, and cell viability and apoptosis were evaluated. Compared with classical commercially available COX inhibitors (ibuprofen, indomethacin, nimesulide), OC was more effective in inducing cell growth inhibition in HCC and CRC cells. Moreover, OC inhibited colony formation and induced apoptosis, as confirmed by PARP cleavage, activation of caspases 3/7 and chromatin condensation. OC treatment in a dose dependent-manner induced expression of γH2AX, a marker of DNA damage, increased intracellular ROS production and caused mitochondrial depolarization. Moreover, the effects of OC were suppressed by the ROS scavenger N-acetyl-L-cysteine. Finally, OC was not toxic in primary normal human hepatocytes. In conclusion, OC treatment was found to exert a potent anticancer activity against HCC and CRC cells. Taken together, our findings provide preclinical support of the chemotherapeutic potential of EVOO against cancer.

A PTEN inhibitor displays preclinical activity against hepatocarcinoma cells.

Phosphatase and tensin homolog (PTEN) gene is considered a tumor suppressor gene. However, PTEN mutations rarely occur in hepatocellular carcinoma (HCC), whereas heterozygosity of PTEN, resulting in reduced PTEN expression, has been observed in 32-44% of HCC patients. In the present study, we investigated the effects of the small molecule PTEN inhibitor VO-OHpic in HCC cells. VO-OHpic inhibited cell viability, cell proliferation and colony formation, and induced senescence-associated ß-galactosidase activity in Hep3B (low PTEN expression) and to a lesser extent in PLC/PRF/5 (high PTEN expression) cells, but not in PTEN-negative SNU475 cells. VO-OHpic synergistically inhibited cell viability when combined with PI3K/mTOR and RAF/MEK/ERK pathway inhibitors, but only in Hep3B cells, and significantly inhibited tumor growth in nude mice bearing xenografts of Hep3B cells. Therefore, we demonstrated for the first time that VO-OHpic inhibited cell growth and induced senescence in HCC cells with low PTEN expression, and that the combination of VO-OHpic with PI3K/mTOR and RAF/MEK/ERK inhibitors resulted in a more effective tumor cell kill. Our findings, hence, provide proof-of-principle evidence that pharmacological inhibition of PTEN may represent a promising approach for HCC therapy in a subclass of patients with a low PTEN expression.