Effects of the Mutant TP53 Reactivator APR-246 on Therapeutic Sensitivity of Pancreatic Cancer Cells in the Presence and Absence of WT-TP53.

The TP53 tumor suppressor is mutated in ~75% of pancreatic cancers. The mutant TP53 protein in pancreatic ductal adenocarcinomas (PDAC) promotes tumor growth and metastasis. Attempts have been made to develop molecules that restore at least some of the properties of wild-type (WT) TP53. APR-246 is one such molecule, and it is referred to as a mutant TP53 reactivator. To understand the potential of APR-246 to sensitize PDAC cells to chemotherapy, we introduced a vector encoding WT-TP53 into two PDAC cell lines, one lacking the expression of TP53 (PANC-28) and one with a gain-of-function (GOF) mutant TP53 (MIA-PaCa-2). APR-246 increased drug sensitivity in the cells containing either a WT or mutant TP53 protein with GOF activity, but not in cells that lacked TP53. The introduction of WT-T53 into PANC-28 cells increased their sensitivity to the TP53 reactivator, chemotherapeutic drugs, and signal transduction inhibitors. The addition of WT-TP53 to PDAC cells with GOF TP53 also increased their sensitivity to the drugs and therapeutics, indicating that APR-246 could function in cells with WT-TP53 and GOF TP53. These results highlight the importance of knowledge of the type of TP53 mutation that is present in cancer patients before the administration of drugs which function through the reactivation of TP53.

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.

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.

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.

Cationic Solid Lipid Nanoparticles as Non Viral Vectors for the Inhibition of Hepatocellular Carcinoma Growth by RNA Interference.

Hepatocellular carcinoma (HCC) is one of the most important causes of cancer deaths worldwide. Gene therapy is a novel approach for treating HCC. A safe and efficient gene delivery method, using viral or non-viral vectors, is a crucial factor for developing a successful HCC gene therapy. Among non-viral vectors, cationic solid lipid nanoparticles (cSLN) have advantages such as biocompatibility and transfection efficiency. In this study, novel cSLN were prepared, characterized and complexed with a plasmid (shNUPR1) capable of inhibiting the expression of the NUPR1 gene, which is involved in HCC growth and chemoresistance. The particles resulted biocompatible, as confirmed by haemolysis and cytotoxicity assays, and was able to protect the shNUPR1 plasmid from degradation by DNase I. We also demonstrated, by carrying out transfection and immunofluorescence studies, that the particles efficiently delivered the shNUPR1 plasmid into HCC cells, causing the downregulation of NUPR1-regulated genes and NUPR1 protein expression. These results suggest that the cSLN obtained could be proposed for further in vivo studies as novel transfection vectors for HCC gene therapy, having shown excellent in vitro transfection efficiency and biocompatibility.

Association Between MICA Gene Variants and the Risk of Hepatitis C Virus-Induced Hepatocellular Cancer in a Sicilian Population Sample.

There are currently no biomarkers that predict hepatocellular carcinoma (HCC) risk in patients with hepatitis C virus (HCV)-related cirrhosis. We investigated the relationships among major histocompatibility complex (MHC) class I chain-related gene A (MICA) polymorphisms, plasma levels of soluble MICA (sMICA), and HCC risk in patients with HCV-related HCC. One hundred fifty-four HCV-related HCC patients, 93 HCV-related liver cirrhosis (LC) cases, and 244 healthy controls, all sampled from the native Sicilian population, were genotyped using the KASP™ single-nucleotide polymorphism genotyping method. The MICA rs2596542 polymorphism showed that the G/G genotype was significantly more frequent in HCC than control subjects and LC patients (p < 0.005). For MICA rs2596538 polymorphism, the C allele and C/C genotype were significantly more frequent in HCC than in controls and LC cases (p < 0.005), after controlling for potential confounders. These results demonstrate that MICA rs2596542G/G, and particularly the rs2596538C/C polymorphism, are associated with the risk of developing HCV-related HCC in a Sicilian population sample. Importantly, using a machine learning classifier, we found that "age" and either rs2596542 or rs2596538 were important discriminating factors for patients with LC and HCC. Finally, sMICA levels significantly increased during HCV-related liver disease progression, while a significant relationship between both rs2596542 and rs2596538 genotypes and sMICA plasma levels was identified in patients with LC and HCC. In summary, the MICA rs2596538 and rs2596542 variants warrant further research for their clinical validity and utility in relationship to the risk of developing HCV-related HCC in independent populations.

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.

Nanoparticles of a polyaspartamide-based brush copolymer for modified release of sorafenib: In vitro and in vivo evaluation.

In this paper, we describe the preparation of polymeric nanoparticles (NPs) loaded with sorafenib for the treatment of hepatocellular carcinoma (HCC). A synthetic brush copolymer, named PHEA-BIB-ButMA (PBB), was synthesized by Atom Trasnfer Radical Polymerization (ATRP) starting from the α-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) and poly butyl methacrylate (ButMA). Empty and sorafenib loaded PBB NPs were, then, produced by using a dialysis method and showed spherical morphology, colloidal size, negative ζ potential and the ability to allow a sustained sorafenib release in physiological environment. Sorafenib loaded PBB NPs were tested in vitro on HCC cells in order to evaluate their cytocompatibility and anticancer efficacy if compared to free drug. Furthermore, the enhanced anticancer effect of sorafenib loaded PBB NPs was demonstrated in vivo by using a xenograft model, by first allowing Hep3B cells to grow subcutaneously into nude mice and then administering sorafenib as free drug or incorporated into NPs via intraperitoneal injection. Finally, in vivo biodistribution studies were performed, showing the ability of the produced drug delivery system to accumulate in a significant manner in the solid tumor by passive targeting, thanks to the enhanced permeability and retention effect.

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.

Biocompatible Lipid Nanoparticles as Carriers To Improve Curcumin Efficacy in Ovarian Cancer Treatment.

Curcumin is a natural molecule with proved anticancer efficacy on several human cancer cell lines. However, its clinical application has been limited due to its poor bioavailability. Nanocarrier-based drug delivery approaches could make curcumin dispersible in aqueous media, thus overtaking the limits of its low solubility. The aim of this study was to increase the bioavailability and the antitumoral activity of curcumin, by entrapping it into nanostructured lipid carriers (NLCs). For this purpose here we describe the preparation and characterization of three kinds of curcumin-loaded NLCs. The nanosystems allowed the achievement of a controlled release of curcumin, the amounts of curcumin released after 24 h from Compritol-Captex, Compritol-Miglyol, and Compritol NLCs being, respectively, equal to 33, 28, and 18% w/w on the total entrapped curcumin. Considering the slower curcumin release profile, Compritol NLCs were chosen to perform successive in vitro studies on ovarian cancer cell lines. The results show that curcumin-loaded NLCs maintain anticancer activity, and reduce cell colony survival more effectively than free curcumin. As an example, the ability of A2780S cells to form colonies was decreased after treatment with 5 µM free curcumin by 50% ± 6, whereas, at the same concentration, the delivery of curcumin with NLC significantly (p < 0.05) inhibited colony formation to approximately 88% ± 1, therefore potentiating the activity of curcumin to inhibit A2780S cell growth. The obtained results clearly suggest that the entrapment of curcumin into NLCs increases curcumin efficacy in vitro, indicating the potential use of NLCs as curcumin delivery systems.

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.

Lipid nanocarriers containing sorafenib inhibit colonies formation in human hepatocarcinoma cells.

Here, the potential of two nanostructured lipid carriers (NLC) for controlled release of sorafenib was evaluated. The obtained systems showed characteristics suitable as drug delivery systems for the treatment of hepatocellular carcinoma (HCC) through parenteral administration. The use of a mixture between a solid lipid (tripalmitin) with a liquid lipid (Captex 355 EP/NF or Miglyol 812) to prepare NLC systems could give a higher drug loading capacity and a longer term stability during storage than that obtained by using only solid lipids. The obtained nanoparticles showed a nanometer size and high negative zeta potential values. Scansion electron microscopy (SEM) of the sorafenib loaded NLC revealed a spherical shape with a diameter <300 nm. In vitro biological studies demonstrated that sorafenib loaded into NLC had enhanced anti-tumor activity compared to that of free drug. This finding raises hope in terms of future drug delivery strategy of sorafenib loaded NLC, that can be useful for therapeutic application in HCC.

Apoptosis and cell growth arrest in A375 human melanoma cells by diorganotin(IV) and triorganotin(IV) complexes of [meso-Tetra(4-sulfonatophenyl)porphine] manganese(III)chloride.

In previous studies we have demonstrated that two derivatives of meso-Tetra(4-sulfonatophenyl)porphine (TPPS), (Bu2Sn)2TPPS and (Bu3Sn)4TPPS, cause apoptotic death of A375 melanoma cells and, at lower concentrations, arrest of cell proliferation. In the present study, we examined if the manganese metal inside the porphyrin cavity could improve the efficacy of this class of compounds. Thus, [meso-Tetra(4-sulfonatophenyl)porphine]Mn(III)Cl (=MnTPPS) derivatives, namely (Me2Sn)2MnTPPS, (Bu2Sn)2MnTPPS, (Me3Sn)4MnTPPS and (Bu3Sn)4MnTPPS, were tested on the A375 human melanoma cell line. A cytotoxicity assay showed that (Bu2Sn)2MnTPPS and (Bu3Sn)4MnTPPS were highly cytotoxic by inducing apoptosis in melanoma cells, as shown by DNA fragmentation analysis and by apoptotic nuclei fluorescence, and when used at lower concentrations, they affected only cellular proliferation. An arrest of cell proliferation was also observed with (Me3Sn)4MnTPPS, but at the highest concentrations used. Moreover, the lower concentration of (Bu3Sn)4MnTPPS induced a change in cell morphology, from a polygonal to an elongated and spindle-shaped phenotype, likewise to its cognate (Bu3Sn)4TPPS, previously tested. Western blotting analysis showed indeed that both tributyltin compounds, i.e. (Bu3Sn)4MnTPPS and (Bu3Sn)4TPPS, lowered levels of the major proteins involved in tumorigenesis: ß-catenin, c-myc and snail. We also demonstrated that all compounds entered the cells and localized in the nuclei. In conclusion, our results show that, in spite of the Mn(III) metal introduction, the butyl derivatives always have a higher efficacy than methyl derivatives, and the tributyltin compounds in particular have an interesting effect in vitro on A375 cell proliferation.