Synthesis and biological evaluation of N-biphenyl-nicotinic based moiety compounds: A new class of antimitotic agents for the treatment of Hodgkin Lymphoma.

We previously demonstrated that some N-biphenylanilides caused cell-cycle arrest at G2/M transition in breast cancer cells. Among them we choose three derivatives, namely PTA34, PTA73 and RS35 for experimentation in solid tumor cell lines, classical Hodgkin Lymphoma (cHL) cell lines and bona fide normal cell lines. Almost all tumor cells were sensitive to compounds in the nanomolar range whereas, they were not cytotoxic to normal ones. Interestingly the compounds caused a strong G2/M phase arrest in cHL cell lines, thus, here we investigated whether they affected the integrity of microtubules in such cells. We found that they induced a long prometaphase arrest, followed by induction of apoptosis which involved mitochondria. PTA73 and RS35 induced the mitotic arrest through the fragmentation of microtubules which prevented the kinethocore-mitotic spindle interaction and the exit from mitosis. PTA34 is instead a tubulin-targeting agent because it inhibited the tubulin polymerization as vinblastine. As such, PTA34 maintained the Cyclin B1-CDK1 regulatory complex activated during the G2/M arrest while inducing the inactivation of Bcl-2 through phosphorylation in Ser70, the degradation of Mcl-1 and a strong activation of BIML and BIMS proapoptotic isoforms. In addition PTA34 exerted an antiangiogenic effect by suppressing microvascular formation.

Expression of base excision repair key factors and miR17 in familial and sporadic breast cancer.

Understanding of BRCA1/2 interaction with the base excision repair (BER) pathway could improve therapy based on 'synthetic lethality', whose effectiveness is based on homologous recombination deficiency in cells lacking functional BRCA genes. However, poly (ADP-ribose) polymerase (PARP) inhibitors failed in some patients and for this reason we explored BER key enzyme expression. In this study, the expression of BER enzymes (redox factor 1/apurinic-apyrimidinic endonuclease 1 (REF1/APEX1), NTH endonuclease III-like 1 (NTHL1), 8-oxoguanine DNA glycosylase (OGG1), PARP1) and of the scaffold protein XRCC1 (X-ray repair complementing defective repair in Chinese hamster cells 1) were investigated in familial (BRCA-related and not) and sporadic breast cancer cases. Furthermore, miR17 expression was measured because of its role in the epigenetic regulation of BRCA1. Gene expression was evaluated in BRCA1-mutated cell lines, SUM149PT and SUM1315MO2, and in a BRCA1-proficient triple-negative MDA-MB-231 cell line. A cohort of 27 familial and 16 sporadic breast cancer patients was then examined to confirm results obtained from the cell line model. APEX1/REF1 was found to be upregulated in familial BRCA-wild-type and sporadic cases, indicating this enzyme as a potential therapeutic target. Furthermore, XRCC1 was overexpressed in BRCAX patients; consequently, we suggest to test the effectiveness of inhibitors targeting two different BER components in preclinical studies. XRCC1, which is also involved in the non-homologous end-joining pathway, was found to be downregulated in BRCA2-related patients concurrently with no change in PARP1 expression. Interestingly, no difference in PARP1 and miR17 expression was found in BRCA-related and sporadic breast cancer cases. PARP1 and miR17 could therefore be further investigated as molecular biomarkers of 'BRCAness' phenotype, indicating patients which could really benefit from PARP inhibitor therapies.

New vascular disrupting agents in upper gastrointestinal malignancies.

Antivascular approaches aim to cause rapid and catastrophic shutdown in the vascular function of the tumour, leading to extensive tumour cell death. Tumour vascular disrupting agents (VDAs) are a new class of cancer therapies that target the existing vasculature of tumours, taking advantage of the relative instability of tumour vasculature and its supporting structures. Treatment with VDAs induces a rapid collapse and regression of tumour vessels, with a consequent deprivation of blood and oxygen which leads to ischemic or hemorrhagic necrosis of the tumour. In this review, an overview of the most recently developed vascular disrupting agents is reported, focusing on the biological effects exerted by these compounds on endothelial cells and tumour vasculature, potentially effective in the treatment of several malignancies including upper gastrointestinal tumours. In particular, we have focused on the antimitotic agent combretastatin and its numerous synthetic analogues such as combretastatin A-4-phosphate, OXI4503, and AVE8062, and on the colchicine analogue ZD6126.

Synergistic antiproliferative and antiangiogenic effects of EGFR and mTOR inhibitors.

Single-agent therapy with molecularly targeted agents has shown limited success in tumor growth control, mainly because escape or resistance mechanisms are activated once a signalling molecule is inhibited. Rational combinations of target-specific agents could counteract this response providing a useful strategy in cancer treatment. In this regard, the EGFR and mTOR inhibitors have been used together to generate a synergistic effect and maximize the efficacy of each individual agent. Overall, the in vivo and in vitro evidences support the utilization of combinations targeting EGFR and mTOR, for malignancies characterized by deregulated EGFR/PI3K/Akt/ mTOR signalling cascade; whereas the clinical experience points out that the assessment of the therapeutic value of such combination awaits further investigations.

Synthetic lethality to overcome cancer drug resistance.

A large body of evidence point out that the onset of synthetic lethality may provide a useful tool for amplifying the efficacy of drugs in anticancer regimens, to uncover interdependence between genes and to identify predictive factors that would be extremely useful to guide in the selection of more effective targeted drugs and drug combinations for each patient. Here, we provide an overview on the exploitation of synthetic lethality to overcome drug resistance to conventional chemotherapy in several types of solid tumors. We report recent findings on cellular markers and gene mutations which are specifically essential for the viability of cancer cells and for resistance to chemotherapeutics. In addition, new molecularly targeted strategies to overcome drug resistance are suggested.

Synthesis, characterization and biological evaluation of ureidofibrate-like derivatives endowed with peroxisome proliferator-activated receptor activity.

A series of ureidofibrate-like derivatives was prepared and assayed for their PPAR functional activity. A calorimetric approach was used to characterize PPARγ-ligand interactions, and docking experiments and X-ray studies were performed to explain the observed potency and efficacy. R-1 and S-1 were selected to evaluate several aspects of their biological activity. In an adipogenic assay, both enantiomers increased the expression of PPARγ target genes and promoted the differentiation of 3T3-L1 fibroblasts to adipocytes. In vivo administration of these compounds to insulin resistant C57Bl/6J mice fed a high fat diet reduced visceral fat content and body weight. Examination of different metabolic parameters showed that R-1 and S-1 are insulin sensitizers. Notably, they also enhanced the expression of hepatic PPARα target genes indicating that their in vivo effects stemmed from an activation of both PPARα and γ. Finally, the capability of R-1 and S-1 to inhibit cellular proliferation in colon cancer cell lines was also evaluated.

Aurora B kinase inhibitor AZD1152: determinants of action and ability to enhance chemotherapeutics effectiveness in pancreatic and colon cancer.

BACKGROUND: AZD1152, the prodrug for AZD1152-hydroxyquinazoline pyrazol anilide (HQPA), is a selective inhibitor of Aurora B kinase activity. Preclinical evaluation of AZD1152 has been reported in several human cancer models. The potentiality of this compound in combination therapy warrants further investigation in solid tumours. EXPERIMENTAL DESIGN: This study explored the effects of AZD1152-HQPA in colon and pancreatic tumour cells. The antitumour properties of AZD1152, either as single agent or in combination with chemotherapeutics, were evaluated in each study model. The efficacy and the toxicity of AZD1152 alone and in combination with gemcitabine were validated in pancreatic tumour xenograft model. RESULTS: AZD1152-HQPA treatment resulted in a dramatic increase of chromosome number, modification of cell cycle and induction of apoptosis. The most effective combination was that with chemotherapeutics given soon after AZD1152 in both tumour cell types. The effectiveness of the sequential schedule of AZD1152 with gemcitabine was confirmed in nude mice bearing MiaPaCa-2 tumours, showing inhibition of tumour volumes and delaying of tumour growth after the interruption of the treatments. Here we show that AZD1152-HQPA enhances oxaliplatin and gemcitabine effectiveness in colon and pancreatic cancer, respectively. First, we provide advances into administration schedules and dosing regimens for the combination treatment in in vivo pancreatic tumour.

Validation of gefitinib effectiveness in a broad panel of head and neck squamous carcinoma cells.

Recently improved understanding of the pathogenesis of human head and neck squamous cell carcinoma (HNSCC) has led to the development of new, molecular-based therapeutic strategies, one of the more promising is the utilisation of tyrosine kinase (TK) inhibitors, targeting epidermal growth factor receptor (EGFR). In this study, we tested for gefitinib effectiveness in a broad panel of 12 newly established HNSCC cell lines, investigating its ability to reduce cell growth, to induce apoptosis and to modulate cell cycle and various EGFR pathway-related targets. Gefitinib IC50 values ranged between 0.064 and 33 microM, its capability to induce apoptosis and cell accumulation in G0/G1 phase was cell line-specific, and the main EGFR-related pathway involved in gefitinib activity was PI3K/Akt/mTor. We characterised our in vitro panel extensively, with the aim to identify predictive factors for gefitinib effectiveness; all cell lines were free of human papillomavirus infection, two were positive for Fhit expression, four expressed wild-type p53, and all of them variously expressed the other two p53 family members, p63 and p73. The comparison between the targets analysed and gefitinib effectiveness evidenced the absence of a clear relationship, excluding them as predictive factors for gefitinib efficacy. Our results confirmed the in vitro efficacy of an anti-EGFR approach, but other targets than those analysed here should be characterised in order to identify valid predictive factors for gefitinib utilisation.

Laminin-5 offsets the efficacy of gefitinib ('Iressa') in hepatocellular carcinoma cells.

Prognosis and survival of patients with hepatocellular carcinoma (HCC) is still very poor, and no therapies are currently available to inhibit tumour growth and metastases. Recently, we reported that the expression of an extracellular matrix component (ECM), namely Laminin-5 (Ln-5), is directly related to poor prognosis in HCC patients. The aim of our study is to investigate the preclinical effect of gefitinib in an in vitro HCC model. We found that the IC(50) of gefitinib in HCC cells ranged from 0.7 to 10.0 muM, whereas Ln-5 inhibited the activity of gefitinib in a dose-dependent manner. Complete inhibition of phosphorylated (p)-EGFR (epidermal growth factor receptor) was obtained within 6 h exposure to gefitinib and complete restoration of the receptor status was obtained after 24 h. A downstream effect yields a decrease in p-Akt and p-Erk 1/2. The addition of exogenous Ln-5 has no effect on p-EGFR, whereas it restores p-Erk 1/2 and p-Akt. Consistently, Ln-5 induces recovery of HCC cells from Gefitinib-induced apoptosis. In conclusion, gefitinib inhibits HCC cell growth and we report for the first time that Ln-5, but not other ECM molecules, reduces the ability of gefitinib to inhibit cell growth via Akt. As patients with HCC have different Ln-5 expression levels, these results may help to better understand which patients might benefit from gefitinib treatment.

Human leucocyte elastase and cathepsin G: structural and functional characteristics.

Two of the major enzymes present in an released from neutrophil granulocytes are the endoproteinases elastase and cathepsin G. While the former is believed to be one of the major causative agents responsible for tissue destruction in emphysema and rheumatoid arthritis, little is known about the function of cathepsin G. We have recently developed simple procedures for isolating the isoenzymes of each type of proteinase as well as for their specific controlling plasma inhibitors. We have also prepared synthetic substrates and inhibitor analogues. Some sequence studies have been initiated and the results indicate homology of these enzymes not only with each other and with the pancreatic proteinases but also between cathepsin G and proteolytic enzymes present in muscle and mast cell tissue. Significantly, both types of enzyme can degrade the structural protein myosin, as well as elastin and proteoglycan. However, their relative importance in muscle protein turnover or muscle disease has not yet been clarified.