Elena Porcù

Recent advances in vascular disrupting agents in cancer therapy.

Vascular disrupting agents (VDAs) are an important class of compounds that exhibit selective activity against pre-existing tumor vasculature, causing rapid shutdown of the tumor blood flow and consequent necrosis of the tumor mass. The VDAs can be divided into flavonoid compounds, which are related to flavone acetic acid, and tubulin-binding agents. Tubulin-binding agents represent the largest group of VDAs and are characterized by different chemical structures, although most of them are derivatives of the lead compound combretastatin (CA-4). They demonstrated clinical activity, although recent findings have established that they have insufficient activity as single agents. Several resistance mechanisms occur, such as the resistance of the tumor rim cells, while promising results have been described in combination with other chemotherapeutics.

Modeling and Biological Investigations of an Unusual Behavior of Novel Synthesized Acridine-Based Polyamine Ligands in the Binding of Double Helix and G-Quadruplex DNA

Three novel 2,7‐substituted acridine derivatives were designed and synthesized to investigate the effect of this functionalization on their interaction with double‐stranded and G‐quadruplex DNA. Detailed investigations of their ability to bind both forms of DNA were carried out by using spectrophotometric, electrophoretic, and computational approaches. The ligands in this study are characterized by an open‐chain (L1) or a macrocyclic (L2, L3) framework. The aliphatic amine groups in the macrocycles are joined by ethylene (L2) or propylene chains (L3). L1 behaved similarly to the lead compound m‐AMSA, efficiently intercalating into dsDNA, but stabilizing G‐quadruplex structures poorly, probably due to the modest stabilization effect exerted by its protonated polyamine chains. L2 and L3, containing small polyamine macrocyclic frameworks, are known to adopt a rather bent and rigid conformation; thus they are generally expected to be sterically impeded from recognizing dsDNA according to an intercalative binding mode. This was confirmed to be true for L3. Nevertheless, we show that L2 can give rise to efficient π–π and H‐bonding interactions with dsDNA. Additionally, stacking interactions allowed L2 to stabilize the G‐quadruplex structure: using the human telomeric sequence, we observed the preferential induction of tetrameric G‐quadruplex forms. Thus, the presence of short ethylene spacers seems to be essential for obtaining a correct match between the binding sites of L2 and the nucleobases on both DNA forms investigated. Furthermore, current modeling methodologies, including docking and MD simulations and free energy calculations, provide structural evidence of an interaction mode for L2 that is different from that of L3; this could explain the unusual stabilizing ability of the ligands (L2>L3>L1) toward G‐quadruplex that was observed in this study.

Annexin 2A sustains glioblastoma cell dissemination and proliferation

Glioblastoma (GBM) is the most devastating tumor of the brain, characterized by an almost inevitable tendency to recur after intensive treatments and a fatal prognosis. Indeed, despite recent technical improvements in GBM surgery, the complete eradication of cancer cell disseminated outside the tumor mass still remains a crucial issue for glioma patients management. In this context, Annexin 2A (ANXA2) is a phospholipid-binding protein expressed in a variety of cell types, whose expression has been recently associated with cell dissemination and metastasis in many cancer types, thus making ANXA2 an attractive putative regulator of cell invasion also in GBM. Here we show that ANXA2 is over-expressed in GBM and positively correlates with tumor aggressiveness and patient survival. In particular, we associate the expression of ANXA2 to a mesenchymal and metastatic phenotype of GBM tumors. Moreover, we functionally characterized the effects exerted by ANXA2 inhibition in primary GBM cultures, demonstrating its ability to sustain cell migration, matrix invasion, cytoskeletal remodeling and proliferation. Finally, we were able to generate an ANXA2-dependent gene signature with a significant prognostic potential in different cohorts of solid tumor patients, including GBM. In conclusion, we demonstrate that ANXA2 acts at multiple levels in determining the disseminating and aggressive behaviour of GBM cells, thus proving its potential as a possible target and strong prognostic factor in the future management of GBM patients.

Vascular disrupting activity of combretastatin analogues

Tubulin binding agents (TBAs) are drugs commonly used in cancer therapy as antimitotics. In the last years it has been described that TBAs, like combretastatin A-4 (CA-4), present also vascular disrupting activity and among its derivatives we identified three analogues endowed with potent microtubule depolymerizing activity, higher than that of the lead compound. In this paper we have investigated the anti-vascular activity of these derivatives. We tested the anti-angiogenic effects in human umbilical endothelial cells (HUVEC) and in vivo in chick chorioallantoic membrane assay (CAM), and in a syngeneic tumor mouse model. The three molecules, compound 1: 1-(3,4,5-trimethoxyphenyl)-5-(4-ethoxyphenyl)-1H-1,2,4-triazole; compound 2: (1-(3,4,5-trimethoxyphenyl)-5-(4-ethoxyphenyl)-1H-tetrazole, compound-3 (4-amino-2-p-tolylaminothiazol-5-yl)-(3,4,5-trimethoxyphenyl)-methanone) showed a moderate effect on the growth of HUVEC cells at concentrations below 200nM. At lower concentrations (5-20nM), in particular compound 2, they induced inhibition of capillary tube formation, inhibition of endothelial cell migration and affected endothelial cell morphology as demonstrated by the alteration of the microfilaments network. Moreover, they also increased permeability of HUVEC cells in a time dependent manner. In addition, compounds 1 and 3, as well as the reference compound CA-4, inhibited VEGF-induced phosphorylation of VE-cadherin and in addition compound 3 prevented the VEGF-induced phosphorylation of FAK. In CAM assay, both compounds 2 and 3 efficiently counteracted the strong angiogenic response induced by bFGF, even at the lowest concentration used (1pmol/egg). Moreover in a syngenic mouse model, compounds 1-3 after a single i.p. injection (30mg/kg), showed a stronger reduction of microvascular density. Altogether our results identified these derivatives as potential new vascular disrupting agents candidates.

Synthesis and Biological Evaluation of 2-Methyl-4,5-Disubstituted Oxazoles as a Novel Class of Highly Potent Antitubulin Agents

Antimitotic agents that interfere with microtubule formation are one of the major classes of cytotoxic drugs for cancer treatment. Multiple 2-methyl-4-(3′,4′,5′-trimethoxyphenyl)-5-substituted oxazoles and their related 4-substituted-5-(3′,4′,5′-trimethoxyphenyl) regioisomeric derivatives designed as cis-constrained combretastatin A-4 (CA-4) analogues were synthesized and evaluated for their antiproliferative activity in vitro against a panel of cancer cell lines and, for selected highly active compounds, interaction with tubulin, cell cycle effects and in vivo potency. Both these series of compounds were characterized by the presence of a common 3′,4′,5′-trimethoxyphenyl ring at either the C-4 or C-5 position of the 2-methyloxazole ring. Compounds 4g and 4i, bearing a m-fluoro-p-methoxyphenyl or p-ethoxyphenyl moiety at the 5-position of 2-methyloxazole nucleus, respectively, exhibited the greatest antiproliferative activity, with IC50 values of 0.35-4.6 nM (4g) and 0.5–20.2 nM (4i), which are similar to those obtained with CA-4. These compounds bound to the colchicine site of tubulin and inhibited tubulin polymerization at submicromolar concentrations. Furthermore, 4i strongly induced apoptosis that follows the mitochondrial pathway. In vivo, 4i in a mouse syngeneic model demonstrated high antitumor activity which significantly reduced the tumor mass at doses ten times lower than that required for CA-4P, suggesting that 4i warrants further evaluation as a potential anticancer drug.

The Novel Antitubulin Agent TR-764 Strongly Reduces Tumor Vasculature and Inhibits HIF-1α Activation

Tubulin binding agents (TBAs) are commonly used in cancer therapy as antimitotics. It has been described that TBAs, like combretastatin A-4 (CA-4), present also antivascular activity and among its derivatives we identified TR-764 as a new inhibitor of tubulin polymerization, based on the 2-(alkoxycarbonyl)-3-(3′,4′,5′-trimethoxyanilino)benzo[b]thiophene molecular skeleton. The antiangiogenic activity of TR-764 (1–10 nM) was tested in vitro on human umbilical endothelial cells (HUVECs), and in vivo, on the chick embryo chorioallantoic membrane (CAM) and two murine tumor models. TR-764 binding to tubulin triggers cytoskeleton rearrangement without affecting cell cycle and viability. It leads to capillary tube disruption, increased cell permeability, and cell motility reduction. Moreover it disrupts adherens junctions and focal adhesions, through mechanisms involving VE-cadherin/β-catenin and FAK/Src. Importantly, TR-764 is active in hypoxic conditions significantly reducing HIF-1α. In vivo TR-764 (1–100 pmol/egg) remarkably blocks the bFGF proangiogenic activity on CAM and shows a stronger reduction of tumor mass and microvascular density both in murine syngeneic and xenograft tumor models, compared to the lead compound CA-4P. Altogether, our results indicate that TR-764 is a novel TBA with strong potential as both antivascular and antitumor molecule that could improve the common anticancer therapies, by overcoming hypoxia-induced resistance mechanisms.

Control of the DNA-Binding and Antiproliferative Properties of Hydroxybenzo[b]quinolizinium Derivatives with pH and Light

The interactions of 8-hydroxybenzo[b]quinolizinium and 9-hydroxybenzo[b]quinolizinium with DNA are investigated in detail. Specifically, spectrophotometric and spectrofluorimetric titrations, thermal DNA-denaturation experiments as well as CD- and LD-spectroscopic analysis show that a pH shift by just one or two orders of magnitude has a significant impact on the interactions of the acidic ligands with the nucleic acid. Both ligands bind with high affinity to DNA at pH 6 (Kb ≈105  m-1 ). At pH 7 or 8, however, the binding interactions are much weaker because of the formation of the corresponding charge-neutral conjugate bases, the affinity to DNA of which is reduced because of the resulting lack of a positive charge. Notably, the variation of DNA affinity occurs in a range that corresponds to the fluctuations of pH values under physiological conditions, so that these ligands may be employed to target DNA in tissue with particular pH values, especially, cancer cells. The antiproliferative activity of the title compounds under different conditions is also investigated. In the absence of irradiation, both compounds show only a modest cytotoxicity toward cancer cells. However, upon irradiation, even at low UV-A doses, a significant reduction of cell viability of tumor cell lines is induced by the ligands.

TP-0903 inhibits neuroblastoma cell growth and enhances the sensitivity to conventional chemotherapy

Abstract Neuroblastoma (NB) is an embryonal tumor with low cure rate for patients classified as high‐risk. This class of NB tumors shows a very complex genomic background and requires aggressive treatment strategies. In this work we evaluated the efficacy of the novel multi‐kinase inhibitor TP‐0903 in impairing NB cells’ growth, proliferation and motility. In vitro studies were performed using cell lines with different molecular background, and in vivo studies were done using the zebrafish experimental model. Our results confirmed a strong cytotoxicity of TP‐0903 already at the sub‐micro molar concentrations. The observed cytotoxicity of TP‐0903 was irreversible and the resulting apoptosis was caspase dependent. In addition, TP‐0903 impaired colony formation and neurosphere creation. Depending on the molecular background of the selected NB cell lines, TP‐0903 influenced either their capacity to migrate, to complete their cell cycle or both. Likewise, TP‐0903 reduced NB cells intravasation in vitro and in vivo. Importantly, TP‐0903 showed remarkable pharmacological efficacy not only as a mono‐treatment, but also in combination with conventional chemotherapy drugs (ATRA, cisplatin, and VP16) in different types of NB cells. In conclusion, the multi‐kinase activity of TP‐0903 allowed the impairment of several biological processes required for expansion of NB cells, making them more vulnerable to the conventional chemotherapeutics. Altogether, our results support the eligibility of TP‐0903 for further (pre)clinical assessments in NB.

Glucocorticoid resistance is reverted by LCK inhibition in pediatric T-cell acute lymphoblastic leukemia

Pediatric T-acute lymphoblastic leukemia (T-ALL) patients often display resistance to glucocorticoid (GC) treatment. These patients, classified as prednisone poor responders (PPR), have poorer outcome than do the other pediatric T-ALL patients receiving a high-risk adapted therapy. Because glucocorticoids are administered to ALL patients during all the different phases of therapy, GC resistance represents an important challenge to improving the outcome for these patients. Mechanisms underlying resistance are not yet fully unraveled; thus our research focused on the identification of deregulated signaling pathways to point out new targeted approaches. We first identified, by reverse-phase protein arrays, the lymphocyte cell-specific protein-tyrosine kinase (LCK) as aberrantly activated in PPR patients. We showed that LCK inhibitors, such as dasatinib, bosutinib, nintedanib, and WH-4-023, are able to induce cell death in GC-resistant T-ALL cells, and remarkably, cotreatment with dexamethasone is able to reverse GC resistance, even at therapeutic drug concentrations. This was confirmed by specific LCK gene silencing and ex vivo combined treatment of cells from PPR patient-derived xenografts. Moreover, we observed that LCK hyperactivation in PPR patients upregulates the calcineurin/nuclear factor of activated T cells signaling triggering to interleukin-4 (IL-4) overexpression. GC-sensitive cells cultured with IL-4 display an increased resistance to dexamethasone, whereas the inhibition of IL-4 signaling could increase GC-induced apoptosis in resistant cells. Treatment with dexamethasone and dasatinib also impaired engraftment of leukemia cells in vivo. Our results suggest a quickly actionable approach to supporting conventional therapies and overcoming GC resistance in pediatric T-ALL patients.

BMP9 counteracts the tumorigenic and pro-angiogenic potential of glioblastoma

Glioblastoma multiforme (GBM) is a highly vascularized and aggressive brain tumor, with a strong ability to disseminate and invade the surrounding parenchyma. In addition, a subpopulation of GBM stem cells has been reported to possess the ability to transdifferentiate into tumor-derived endothelial cells (TDECs), supporting the resistance to anti-angiogenic treatments of newly formed blood vessels. Bone Morphogenetic Protein 9 (BMP9) is critically involved in the processes of cancer cell differentiation, invasion and metastasis, representing a potential tool in order to impair the intrinsic GBM aggressiveness. Here we demonstrate that BMP9 is able to trigger the activation of SMADs in patient-derived GBM cells, and to strongly inhibit proliferation and invasion by reducing the activation of PI3K/AKT/MAPK and RhoA/Cofilin pathways, respectively. Intriguingly, BMP9 treatment is sufficient to induce a strong differentiation of GBM stem-like cells and to significantly counteract the already reported process of GBM cell transdifferentiation into TDECs not only in in vitro mimicked TDEC models, but also in vivo in orthotopic xenografts in mice. Additionally, we describe a strong BMP9-mediated inhibition of the whole angiogenic process engaged during GBM tumor formation. Based on these results, we believe that BMP9, by acting at multiple levels against GBM cell aggressiveness, can be considered a promising candidate, to be further developed, for the future therapeutic management of GBM.