Showing papers by "Michele Caraglia published in 2014"

Mir-34: A New Weapon Against Cancer?

Abstract: The microRNA(miRNA)-34a is a key regulator of tumor suppression. It controls the expression of a plethora of target proteins involved in cell cycle, differentiation and apoptosis, and antagonizes processes that are necessary for basic cancer cell viability as well as cancer stemness, metastasis, and chemoresistance. In this review, we focus on the molecular mechanisms of miR-34a-mediated tumor suppression, giving emphasis on the main miR-34a targets, as well as on the principal regulators involved in the modulation of this miRNA. Moreover, we shed light on the miR-34a role in modulating responsiveness to chemotherapy and on the phytonutrients-mediated regulation of miR-34a expression and activity in cancer cells. Given the broad anti-oncogenic activity of miR-34a, we also discuss the substantial benefits of a new therapeutic concept based on nanotechnology delivery of miRNA mimics. In fact, the replacement of oncosuppressor miRNAs provides an effective strategy against tumor heterogeneity and the selective RNA-based delivery systems seems to be an excellent platform for a safe and effective targeting of the tumor.

Short-term diet and moderate exercise in young overweight men modulate cardiocyte and hepatocarcinoma survival by oxidative stress

Abstract: The present study was designed to evaluate the effects of diet lifestyle on extending lifespan and reducing liver cancer risk. Young overweight men (n = 20), without metabolic syndrome, were placed in a 3-week residential program on a low-fat diet and moderate aerobic exercise. In each subject, pre- and postintervention fasting blood were collected for evaluating levels of serum lipids, and oxidative stress markers. Using subject sera and cardiomyocyte (H9C2) culture systems, we measured heat shock protein 27 and 90 expression, lipid accumulation, and oxidative stress marker levels. After 3-weeks of diet, significant reductions (P < 0.05) in body mass index, serum lipids and lipid ratios, and oxidative markers were recorded. In vitro, we observed that the addition of postintervention sera increased H9C2 cell number and reduced HSP27 and 90 expression, mitochondrial superoxide anion, and lipid accumulation with a parallel increase in nitric oxide (NO) production (all P < 0.01). At the same time, postintervention sera decreased human liver hepatocellular carcinoma cell line (HepG-2) proliferation, lipid accumulation, oxidative stress, and extracellular-signal-regulated kinases (ERK1/2) activity. Lifestyle modification in young overweight men, without metabolic syndrome, could ameliorate cardiocyte survival and reduce hepatocellular carcinoma cell proliferation.

High throughput screening for inhibitors of the HECT ubiquitin E3 ligase ITCH identifies antidepressant drugs as regulators of autophagy.

Abstract: Inhibition of distinct ubiquitin E3 ligases might represent a powerful therapeutic tool. ITCH is a HECT domain-containing E3 ligase that promotes the ubiquitylation and degradation of several proteins, including p73, p63, c-Jun, JunB, Notch and c-FLIP, thus affecting cell fate. Accordingly, ITCH depletion potentiates the effect of chemotherapeutic drugs, revealing ITCH as a potential pharmacological target in cancer therapy. Using high throughput screening of ITCH auto-ubiquitylation, we identified several putative ITCH inhibitors, one of which is clomipramine—a clinically useful antidepressant drug. Previously, we have shown that clomipramine inhibits autophagy by blocking autophagolysosomal fluxes and thus could potentiate chemotherapy in vitro. Here, we found that clomipramine specifically blocks ITCH auto-ubiquitylation, as well as p73 ubiquitylation. By screening structural homologs of clomipramine, we identified several ITCH inhibitors and putative molecular moieties that are essential for ITCH inhibition. Treating a panel of breast, prostate and bladder cancer cell lines with clomipramine, or its homologs, we found that they reduce cancer cell growth, and synergize with gemcitabine or mitomycin in killing cancer cells by blocking autophagy. We also discuss a potential mechanism of inhibition. Together, our study (i) demonstrates the feasibility of using high throughput screening to identify E3 ligase inhibitors and (ii) provides insight into how clomipramine and its structural homologs might interfere with ITCH and other HECT E3 ligase catalytic activity in (iii) potentiating chemotherapy by regulating autophagic fluxes. These results may have direct clinical applications.

In vivo activity of MiR-34a mimics delivered by stable nucleic acid lipid particles (SNALPs) against multiple myeloma

Abstract: Multiple myeloma (MM) is a disease with an adverse outcome and new therapeutic strategies are urgently awaited. A rising body of evidence supports the notion that microRNAs (miRNAs), master regulators of eukaryotic gene expression, may exert anti-MM activity. Here, we evaluated the activity of synthetic miR-34a in MM cells. We found that transfection of miR-34a mimics in MM cells induces a significant change of gene expression with relevant effects on multiple signal transduction pathways. We detected early inactivation of pro-survival and proliferative kinases Erk-2 and Akt followed at later time points by caspase-6 and -3 activation and apoptosis induction. To improve the in vivo delivery, we encapsulated miR-34a mimics in stable nucleic acid lipid particles (SNALPs). We found that SNALPs miR-34a were highly efficient in vitro in inhibiting growth of MM cells. Then, we investigated the activity of the SNALPs miR-34a against MM xenografts in SCID mice. We observed significant tumor growth inhibition (p<0.05) which translated in mice survival benefits (p=0.0047). Analysis of miR-34a and NOTCH1 expression in tumor retrieved from animal demonstrated efficient delivery and gene modulation induced by SNALPs miR-34a in the absence of systemic toxicity. We here therefore provide evidence that SNALPs miR-34a may represent a promising tool for miRNA-therapeutics in MM.

CXCR4 and CXCR7 transduce through mTOR in human renal cancer cells.

Abstract: Treatment of metastatic renal cell carcinoma (mRCC) has improved significantly with the advent of agents targeting the mTOR pathway, such as temsirolimus and everolimus. However, their efficacy is thought to be limited by feedback loops and crosstalk with other pathways leading to the development of drug resistance. As CXCR4–CXCL12–CXCR7 axis has been described to have a crucial role in renal cancer; the crosstalk between the mTOR pathway and the CXCR4–CXCL12–CXCR7 chemokine receptor axis has been investigated in human renal cancer cells. In SN12C and A498, the common CXCR4–CXCR7 ligand, CXCL12, and the exclusive CXCR7 ligand, CXCL11, activated mTOR through P70S6K and 4EBP1 targets. The mTOR activation was specifically inhibited by CXCR4 antagonists (AMD3100, anti-CXCR4-12G5 and Peptide R, a newly developed CXCR4 antagonist) and CXCR7 antagonists (anti-CXCR7-12G8 and CCX771, CXCR7 inhibitor). To investigate the functional role of CXCR4, CXCR7 and mTOR in human renal cancer cells, both migration and wound healing were evaluated. SN12C and A498 cells migrated toward CXCL12 and CXCL11; CXCR4 and CXCR7 inhibitors impaired migration and treatment with mTOR inhibitor, RAD001, further inhibited it. Moreover, CXCL12 and CXCL11 induced wound healing while was impaired by AMD3100, the anti CXCR7 and RAD001. In SN12C and A498 cells, CXCL12 and CXCL11 promoted actin reorganization characterized by thin spikes at the cell periphery, whereas AMD3100 and anti-CXCR7 impaired CXCL12/CXCL11-induced actin polymerization, and RAD001 treatment further reduced it. In addition, when cell growth was evaluated in the presence of CXCL12, CXCL11 and mTOR inhibitors, an additive effect was demonstrated with the CXCR4, CXCR7 antagonists and RAD001. RAD001-resistant SN12C and A498 cells recovered RAD001 sensitivity in the presence of CXCR4 and CXCR7 antagonists. In conclusion, the entire axis CXCR4–CXCL12–CXCR7 regulates mTOR signaling in renal cancer cells offering new therapeutic opportunities and targets to overcome resistance to mTOR inhibitors.

Medical treatment of orthotopic glioblastoma with transferrin-conjugated nanoparticles encapsulating zoledronic acid

Abstract: Glioblastomas are highly aggressive adult brain tumors with poor clinical outcome. In the central nervous system (CNS) the blood-brain barrier (BBB) is the most important limiting factor for both development of new drugs and drug delivery. Here, we propose a new strategy to treat glioblastoma based on transferrin (Tf)-targeted self-assembled nanoparticles (NPs) incorporating zoledronic acid (ZOL) (NPs-ZOL-Tf). NPs-ZOL-Tf have been assessed on the glioblastoma cell line U373MG-LUC that showed a refractoriness in vitro to temozolomide (TMZ) and fotemustine (FTM). NPs-ZOL-Tf treatment resulted in higher in vitro cytotoxic activity than free ZOL. However, the potentiation of anti-proliferative activity of NPs-ZOL-Tf was superimposable to that one induced by NPs-ZOL (not armed with Tf). On the other hand, NPs-ZOL-Tf showed a higher antitumor efficacy if compared with that one caused by NPs-ZOL in immunosuppressed mice intramuscularly bearing U373MG-LUC xenografts, inducing a significant tumor weight inhibition (TWI). The experiments performed on mice with intracranial U373MG-LUC xenografts confirmed the efficacy of NPs-ZOL-Tf. These effects were paralleled by a higher intratumour localization of fluorescently-labeled-NPs-Tf both in intramuscular and intracranial xenografts. In conclusion, our results demonstrate that the encapsulation of ZOL increases the antitumor efficacy of this drug in glioblastoma through the acquisition of ability to cross the BBB.

Serum oxidative stress markers and lipidomic profile to detect NASH patients responsive to an antioxidant treatment: a pilot study.

Abstract: Liver steatosis can evolve to steatohepatitis (NASH) through a series of biochemical steps related to oxidative stress in hepatocytes. Antioxidants, such as silybin, have been proposed as a treatment of patients with nonalcoholic fatty liver disease (NAFLD) and NASH. In this study, we evaluated, in patients with histologically documented NASH, the oxidant/antioxidant status and lipid “fingerprint” in the serum of NASH patients, both in basal conditions and after 12 months of treatment with silybin-based food integrator Realsil (RA). The oxidant/antioxidant status analysis showed the presence of a group of patients with higher basal severity of disease (NAS scores 4.67 ± 2.5) and a second group corresponding to borderline NASH (NAS scores = 3.8 ± 1.5). The chronic treatment with RA changed the NAS score in both groups that reached the statistical significance only in group 2, in which there was also a significant decrease of serum lipid peroxidation. The lipidomic profile showed a lipid composition similar to that of healthy subjects with a restoration of the values of free cholesterol, lysoPC, SM, and PC only in group 2 of patients after treatment with RA. Conclusion. These data suggest that lipidomic and/or oxidative status of serum from patients with NASH could be useful as prognostic markers of response to an antioxidant treatment.

Advantages and risks of nanotechnologies in cancer patients and occupationally exposed workers

Abstract: Introduction: In recent years, different nanotechnology platforms for drug delivery in the area of medical biology have gained remarkable attention.Areas covered: Nanoparticles (NPs) used as drug delivery vehicles consist of different materials such as natural or synthetic polymers, lipids or metals. They have an ultra-small size, large surface area-to-mass ratio and high reactivity. Although there are many data on the advantages in terms of both higher efficacy and less adverse effects of nanodrugs, several recent findings have reported unexpected toxicities giving origin to nanotoxicology.Expert opinion: Despite the great promise that NPs show, few studies have examined the human body's reaction due to NP exposure in both patients and workers. To perform this type of evaluation, it is necessary to define an adequate index of exposure, and the measure of this index is representative of what the worker is breathing. The properties of the nanomaterials used for designing NPs, such as in the case of poorly ...

The Role of E-Cadherin Down-Regulation in Oral Cancer: CDH1 Gene Expression and Epigenetic Blockage

Abstract: Background: The prognosis of the oral squamous cell carcinoma (OSCC) patients remains very poor, mainly due to their high propensity to invade and metastasize. E-cadherin reduced expression occurs in the primary step of oral tumour progression and gene methylation is a mode by which the expression of this protein is regulated in cancers. In this perspective, we investigated E-cadherin gene (CDH1) promoter methylation status in OSCC and its correlation with Ecadherin protein expression, clinicopathological characteristics and patient outcome. Methods: Histologically proven OSCC and paired normal mucosa were analyzed for CDH1 promoter methylation status and E-cadherin protein expression by methylation-specific polymerase chain reaction and immunohistochemistry. Colocalization of E-cadherin with epidermal growth factor (EGF) receptor (EGFR) was evidenced by confocal microscopy and by immunoprecipitation analyses. Results: This study indicated E-cadherin protein down-regulation in OSCC associated with protein delocalization from membrane to cytoplasm. Low E-cadherin expression correlated to aggressive, poorly differentiated, high grade carcinomas and low patient survival. Moreover, protein down-regulation appeared to be due to E-cadherin mRNA downregulation and CDH1 promoter hypermethylation. In an in vitro model of OSCC the treatment with EGF caused internalization and co-localization of E-cadherin with EGFR and the addition of demethylating agents increased E-cadherin expression. Conclusion: Low E–Cadherin expression is a negative prognostic factor of OSCC and is likely due to the hypermethylation of CDH1 promoter. The delocalization of E-cadherin from membrane to cytoplasm could be also due to the increased expression of EGFR in OSCC and the consequent increase of E-cadherin co-internalization with EGFR.

Transferrin-Conjugated SNALPs Encapsulating 2′-O-Methylated miR-34a for the Treatment of Multiple Myeloma

Abstract: Stable nucleic acid lipid vesicles (SNALPs) encapsulating miR-34a to treat multiple myeloma (MM) were developed. Wild type or completely 2′-O-methylated (OMet) MiR-34a was used in this study. Moreover, SNALPs were conjugated with transferrin (Tf) in order to target MM cells overexpressing transferrin receptors (TfRs). The type of miR-34a chemical backbone did not significantly affect the characteristics of SNALPs in terms of mean size, polydispersity index, and zeta potential, while the encapsulation of an OMet miR-34a resulted in a significant increase of miRNA encapsulation into the SNALPs. On the other hand, the chemical conjugation of SNALPs with Tf resulted in a significant decrease of the zeta potential, while size characteristics and miR-34a encapsulation into SNALPs were not significantly affected. In an experimental model of MM, all the animals treated with SNALPs encapsulating miR-34a showed a significant inhibition of the tumor growth. However, the use of SNALPs conjugated with Tf and encapsulating OMet miR-34a resulted in the highest increase of mice survival. These results may represent the proof of concept for the use of SNALPs encapsulating miR-34a for the treatment of MM.

Pegylated liposomal doxorubicin in the management of ovarian cancer: a systematic review and metaanalysis of randomized trials.

Abstract: Ovarian cancer is the leading cause of death among gynecological tumors. Carboplatin/paclitaxel represents the cornerstone of front-line treatment. Instead, there is no consensus for management of recurrent/progressive disease, in which pegylated liposomal doxorubicin (PLD) ± carboplatin is widely used. We performed a systematic review and metaanalysis to evaluate impact of PLD-based compared with no-PLD-based regimens in the ovarian cancer treatment. Data were extracted from randomized trials comparing PLD-based treatment to any other regimens in the January 2000-January 2013 time-frame. Study end-points were overall survival (OS), progression free survival (PFS), response rate (RR), CA125 response, and toxicity. Hazard ratios (HRs) of OS and PFS, with 95% CI, odds ratios (ORs) of RR and risk ratios of CA125 response and grade 3-4 toxicity, were extracted. Data were pooled using fixed and random effect models for selected endpoints. Fourteen randomized trials for a total of 5760 patients were selected and included for the final analysis, which showed no OS differences for PLD-based compared with other regimens (pooled HR: 0.94; 95% CI: 0.88-1.02; P = 0.132) and a significant PFS benefit of PLD-based schedule (HR: 0.91; 95% CI: 0.86-0.96; P = 0.001), particularly in second-line (HR: 0.85; 95% CI: 0.75-0.91) and in platinum-sensitive (HR: 0.83; 95% CI: 0.74-0.94) subgroups. This work confirmed the peculiar tolerability profile of this drug, moreover no difference was observed for common hematological toxicities and for RR, CA125 response. PLD-containing regimens do not improve OS when compared with any other schedule in all phases of disease. A marginal PFS advantage is observed only in platinum-sensitive setting and second-line treatment.

Gemcitabine, oxaliplatin, levofolinate, 5-fluorouracil, granulocyte-macrophage colony-stimulating factor, and interleukin-2 (GOLFIG) versus FOLFOX chemotherapy in metastatic colorectal cancer patients: the GOLFIG-2 multicentric open-label randomized phase III trial.

Abstract: The GOLFIG-2 phase III trial was designed to compare the immunobiological activity and antitumor efficacy of GOLFIG chemoimmunotherapy regimen with standard FOLFOX-4 chemotherapy in frontline treatment of metastatic colorectal cancer (mCRC) patients. This trial was conceived on the basis of previous evidence of antitumor and immunomodulating activity of the GOLFIG regimen in mCRC. GOLFIG-2 is a multicentric open/label phase III trial (EUDRACT: 2005-003458-81). Chemo-naive mCRC patients were randomized in a 1:1 ratio to receive biweekly standard FOLFOX-4 or GOLFIG [gemcitabine (1000 mg/m(2), day 1); oxaliplatin (85 mg/m(2), day 2); levofolinate (100 mg/m(2), days 1-2), 5-fluorouracil (5-FU) (400 mg/m(2) in bolus followed by 24 h infusion at 800 mg/m(2),days 1-2), sc. GM-CSF (100 μg, days 3-7); sc. aldesleukin (0·5 MIU bi-daily, days 8-14 and 17-30)] treatments. The study underwent early termination because of poor recruitment in the control arm. After a median follow-up of 43.83 months, GOLFIG regimen showed superiority over FOLFOX in terms of progression-free survival [median 9·23 (95% confidence interval (CI), 6·9-11.5) vs. median 5.70 (95% CI, 3.38-8.02) months; hazard ratio (HR): 0.52 (95% CI, 0.35-0.77), P=0·002] and response rate [66.1% (95% CI, 0.41-0.73) vs. 37·0% (95% CI, 0.28-0.59), P=0.002], with a trend to longer survival [median 21.63 (95% CI, 18.09-25.18) vs. 14.57 mo (95% CI, 9.07-20.07); HR: 0·79 (95% CI, 0.52-1.21); P=0.28]. Patients in the experimental arm showed higher incidence of non-neutropenic fever (18.5%), autoimmunity signs (18.5%), an increase in the number of monocytes, eosinophils, CD4(+) T lymphocytes, natural killer cells, and a decrease in immunoregulatory (CD3(+)CD4(+)CD25(+)FoxP3(+)) T cells. Taken together, these findings provide proof-of-principle that GOLFIG chemoimmunotherapy may represent a novel reliable option for first-line treatment of mCRC.

Sphingosine analog fingolimod (FTY720) increases radiation sensitivity of human breast cancer cells in vitro

Abstract: Radiotherapy is one of the most effective therapeutic strategies for breast cancer patients, although its efficacy may be reduced by intrinsic radiation resistance of cancer cells. Recent investigations demonstrate a link between cancer cell radio-resistance and activation of sphingosine kinase (SphK1), which plays a key role in the balance of lipid signaling molecules. Sphingosine kinase (SphK1) activity can alter the sphingosine-1-phosphate (S1P)/ceramide ratio leading to an imbalance in the sphingolipid rheostat. Fingolimod (FTY720) is a novel sphingosine analog and a potent immunosuppressive drug that acts as a SphK1 antagonist, inhibits the growth, and induces apoptosis in different human cancer cell lines. We sought to investigate the in vitro radiosensitizing effects of FTY720 on the MDA-MB-361 breast cancer cell line and to assess the effects elicited by radiation and FTY720 combined treatments. We found that FTY720 significantly increased anti-proliferative and pro-apoptotic effects induced by a single dose of ionizing radiation while causing autophagosome accumulation. At the molecular level, FTY720 significantly potentiated radiation effects on perturbation of signaling pathways involved in regulation of cell cycle and apoptosis, such as PI3K/AKT and MAPK. In conclusion, our data highlight a potent radiosensitizing effect of FTY720 on breast cancer cells and provide the basis of novel therapeutic strategies for breast cancer treatment.

Exploitation of viral properties for intracellular delivery.

Abstract: Nanotechnology is an expanding area of study with potentially pivotal applications in a discipline as medicine where new biomedical active molecules or strategies are continuously developing. One of the principal drawbacks for the application of new therapies is the difficulty to cross membranes that represent the main physiological barrier in our body and in all living cells. Membranes are selectively permeable and allow the selective internalization of substances; generally, they form a highly impermeable barrier to most polar and charged molecules, and represent an obstacle for drug delivery, limiting absorption to specific routes and mechanisms. Viruses provide attracting suggestions for the development of targeted drug carriers as they have evolved naturally to deliver their genomes to host cells with high fidelity. A detailed understanding of virus structure and their mechanisms of entry into mammalian cells will facilitate the development and analysis of virus-based materials for medical applications. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Urotensin II receptor determines prognosis of bladder cancer regulating cell motility/invasion.

Abstract: Background: Non Muscle Invasive Bladder Transitional Cancer (NMIBC) and Muscle Invasive Bladder Transitional Cancer (MIBC)/invasive have different gene profile and clinical course. NMIBC prognosis is not completely predictable, since the relapse rate is higher than 20%, even in the form of MIBC. The aim of this study is to evaluate if UTR expression can discriminate between NMIBC and MIBC and predict the risk of relapses in NMIBCs. Methods: We have investigated upon urotensin-II (UII) receptor (UTR) expression in vivo in 159 patients affected by NMIBC. The biological role of UTR was also investigated in vitro. UTR expression was evaluated in a tissue-micro-array, consisting of normal, NMIBC and invasive bTCC samples. Results: UTR discriminated between NMIBC and MIBC and showed a significant correlation between low UTR expression and shorter disease free survival in NMIBC. The superagonist UPG84 induced growth suppression at nM concentrations on 3/4 cell lines. Bladder cancer cell treatment with the antagonist urantide or the knock-down of UTR with a specific shRNA significantly blocked both the motility and invasion of bladder cancer cells. Conclusions: The evaluation of UTR expression can discriminate between NMIBC at high and low risk of relapse. Moreover, our data suggest that UTR is involved in the regulation of motility, invasion and proliferation of bladder cancer cells. High UTR expression is an independent prognostic factor of good prognosis for NMIBC regulating motility and invasion of bladder cancer cells.

Myrtucommulone production by a strain of Neofusicoccum australe endophytic in myrtle (Myrtus communis).

Abstract: Myrtucommulones are acylphloroglucinol compounds reported from myrtle (Myrtus communis) and a few more plant species belonging in the Myrtaceae that have recently attracted the attention of pharmacologists for their anti-oxidant, anti-inflammatory and anti-tumor properties. An endophytic strain of Neofusicoccum australe recovered from a myrtle branch was selected based on the bioactivity of its culture extracts, and found to produce myrtucommulones A and D. A mixture of these compounds induced anti-proliferative effects on the human prostatic cancer cell lines DU145 and PC3, with a IC₅₀ of respectively 4.64 and 3.11 mg/l. Along the lines of recent evidences of the ability by endophytic fungi to produce bioactive compounds originally extracted from their host plants, this is the first report of myrtucommulones as secondary metabolites of an endophytic fungal strain. The availability of a microbial strain to be cultured in vitro may provide access to more substantial amounts of these products for further investigations in view of their possible pharmaceutical use.

Urotensin-II receptor is over-expressed in colon cancer cell lines and in colon carcinoma in humans

Abstract: Background Urotensin (U)-II receptor (UTR) has been previously reported to be over-expressed in a number of tumours. Whether UTR-related pathway plays a role in colon carcinogenesis is unknown. Methods We evaluated UTR protein and mRNA expression in human epithelial colon cancer cell lines and in normal colon tissue, adenomatous polyps and colon cancer. U-II protein expression was assessed in cancer cell lines. Moreover, we evaluated the effects of U-II(4-11) (an UTR agonist), antagonists and knockdown of UTR protein expression through a specific shRNA, on proliferation, invasion and motility of human colon cancer cells. Results Cancer cell lines expressed U-II protein and UTR protein and mRNA. By immunohistochemistry, UTR was expressed in 5–30% of epithelial cells in 45 normal controls, in 30–48% in 21 adenomatous polyps and in 65–90% in 48 colon adenocarcinomas. UTR mRNA expression was increased by threefold in adenomatous polyps and eightfold in colon cancer, compared with normal colon. U-II(4-11) induced a 20–40% increase in cell growth while the blockade of the receptor with specific antagonists caused growth inhibition of 20–40%. Moreover, the knock down of UTR with a shRNA or the inhibition of UTR with the antagonist urantide induced an approximately 50% inhibition of both motility and invasion. Conclusions UTR appears to be involved in the regulation of colon cancer cell invasion and motility. These data suggest that UTR-related pathway may play a role in colon carcinogenesis and that UTR may function as a target for therapeutic intervention in colon cancer.

Erlotinib: early clinical development in brain cancer.

Abstract: Introduction: Glioblastoma (GBM) is the most common brain cancer in adults. It is also, unfortunately, the most aggressive type and the least responsive to therapy. Overexpression of EGFR and/or EGFRvIII is frequently found in GBM and is frequently associated with the more malignant phenotype of the disease and a poor clinical outcome. EGFR-targeted therapy represents a promising anti-GBM therapy. Two EGFR kinase inhibitors, gefitinib and erlotinib have been tested in clinical trials for malignant gliomas. However, the clinical efficacy of EGFR-targeted therapy has been only modest in GBM patients. Areas covered: The authors provide an evaluation of erlotinib as a potential therapy for GBM. The authors highlight experiences drawn from clinical trials and discuss the challenges, which include the insufficient penetration through the blood–brain barrier (BBB) and chemoresistance. Expert opinion: Malignant brain tumours have a very complex signalling network that is not only driven by EGFR. This complexity d...

Keratin 5 expression in squamocellular carcinoma of the head and neck

Abstract: Keratin 5 (K5) is present in the basal layer of a stratified squamous keratinized and non-keratinized epithelium. K5 and K14 have been demonstrated in the mucosa and tumors of the oral cavity, oropharynx, hypopharynx and larynx, and in the mitotic active basal cells of a stratified squamous epithelium. The aim of the present study was to assess K5 expression in squamocellular carcinoma with various localizations in the head and neck. A total of 13 biopsy fragments were included from patients diagnosed with squamocellular carcinoma of the larynx area (n=2), pharynx (n=2), hard palate (n=1), tongue (n=2), submandibular (n=1), lip (n=1), gingival sulcus (n=1), nasal pyramid (n=1), maxilla (n=1) and zygomatic (n=1). The immunohistochemical staining for K5 was evaluated according to the following score criteria: 0 (0% positive cells); 1 ( 30% positive cells). K5 expression was observed in all squamocellular carcinomas included in the present study with scores between 1 and 3. For well- and moderately-differentiated histopathological types, a maximum score of 3 was recorded for all of the cases, not including the laryngeal area, which presented a score of 2. The following scores were identified in the regions of the poorly differentiated carcinomas: Jaw, 3; gingival sulcus, 2; and tongue and submandibular area, 1. These observations may aid with an improved stratification of head and neck squamocellular carcinoma, thus improving the diagnosis and treatment strategies for this type of cancer.

Expression and localization of serine protease Htra1 in neuroblastoma: correlation with cellular differentiation grade.

Abstract: Neuroblastoma (NB) is a paediatric tumor that arises from neural crest and shows heterogeneous clinical and biological features. The serine-protease high temperature requirement A1 (HtrA1) has a pivotal role in both cell proliferation and differentiation. Here we report the expression and localization of HtrA1 in NB tumor samples to assess HtrA1 role as a possible new biomarker of cellular differentiation in NB patients. HtrA1 protein expression by Western Blot assay was performed in 60 tissue samples of 50 children with NB and 10 children with ganglioneuroblastoma (GNB). HtrA1 was expressed in 56/60 (93.3 %) samples with different expression levels: low levels in 36/56 samples (64.3 %) and high levels in 20/56 (35.7 %). Higher levels were found in 1, 2 and 4s stages (80 %), whereas 3 and 4 stages (20 %) showed a low expression, with a statistically significant difference (p = 0.003). Among not amplified N-MYC group, 28 (60 %) had low/absent expression of HtrA1: seven with recurrent disease and negative outcome and 21 in continuous complete remission (CCR), whereas all samples with high expression of HtrA1 (17/44) were in CCR (p = 0.03). The immunohistochemical analysis showed localization of HtrA1 in differentiated areas higher than in undifferentiated areas where the protein was absent. Moreover, HtrA1 was highly expressed in all GNB samples. In conclusion, the over-expression of HtrA1 is correlated to cellular differentiation grade and stage of NB at diagnosis. Moreover, HtrA1 could represent a new marker of undifferentiation and biological aggressiveness of NB.

Nanoparticles for the delivery of zoledronic acid to prostate cancer cells: a comparative analysis through time lapse video-microscopy technique.

Abstract: Time-lapse live cell imaging is a powerful tool for studying the responses of cells to drugs. Zoledronic acid (ZOL) is the most potent aminobiphosphonate able to induce cell growth inhibition at very low concentrations. The lack of clear evidence of ZOL-induced anti-cancer effects is likely due to its unfavorable pharmacokinetic profile. The use of nanotechnology-based formulations allows overcoming these limitations in ZOL pharmaco-distribution. Recently, stealth liposomes (LIPOs) and new self-assembly PEGylated nanoparticles (NPs) encapsulating ZOL were developed. Both the delivery systems showed promising anticancer activity in vitro and in vivo. In this work, we investigated the cytostatic effect of these novel formulations (LIPOs and NPs) compared with free ZOL on 2 different prostate cancer cell lines, PC 3 and DU 145 and on prostate epithelial primary cells EPN using time lapse video-microscopy (TLVM). In PC3 cells, free ZOL showed a significant anti-proliferative effect but this effect was lower than that induced by LIPOs and NPs encapsulating ZOL; moreover, LIPO-ZOL was more potent in inducing growth inhibition than NP-ZOL. On the other hand, LIPO-ZOL slightly enhanced the free ZOL activity on growth inhibition of DU 145, while the anti-proliferative effect of NP-ZOL was not statistically relevant. These novel formulations did not induce anti-proliferative effects on EPN cells. Finally, we evaluated cytotoxic effects on DU145 where, LIPO-ZOL induced the highest cytotoxicity compared with NP-ZOL and free ZOL. In conclusion, ZOL can be transformed in a powerful anticancer agent, if administered with nanotechnology-based formulations without damaging the healthy tissues.

Treatment of c‑kit positive adenoid cystic carcinoma of the tongue: A case report

Abstract: Adenoid cystic carcinoma (ACC) or 'cylindroma' is a malignant tumor that often occurs in the areas of the head and neck, affecting the secretory glands and the major and minor salivary glands. The present study describes a case of a patient who presented with a posterior tongue lesion. The case is of a 71-year-old female with an asymptomatic volume growth of the posterior left tongue perceived 8 months prior, and neoplastic cells positive for c-kit. A computed tomography of the head and neck showed asymmetry of the base of the tongue, which was enlarged in the left portion. A physical examination revealed a nodule on the posterior left tongue of ~3 cm in diameter, while the cervical lymph node chain had a normal size and consistency. Surgical exeresis of the tongue lesion and cervical lymph node dissection were performed. Subsequent to surgical removal of the cancer cells and adjuvant radiotherapy, the patient showed excellent health, although the follow-up remains in progress. ACC, one of the most biologically destructive tumors of the head and neck, is locally aggressive and gives rise to distant metastases. The tongue is the place of origin in 3.4-17.1% of cases. The treatment for ACC consists of primary surgical resection with adjuvant radiotherapy. To prevent the risk for distant metastasis, it is necessary to remove the first echelon nodes and monitor the patient with a long-term follow-up.

From Protein Synthesis to Molecular Biology: The Appealing Tale of eIF-5A

Abstract: Posttranslational hypusine modification of lysine 50 of the eukaryotic initiation factor 5A (eIF-5A) is associated with the function of this factor in cell proliferation and differentiation and is involved in tumor formation, progression and maintenance (for a review see ref. 1). In a Molecular Therapy Issue, Francis et al. report that knock down of hypusine formation in eIF-5A through RNA interference strategies is a promising approach for the treatment of B-cell neoplasia2. The authors had previously employed this strategy in models of multiple myeloma to abrogate hypusination of eIF-5A by codelivery of an siRNA targeted to the eIF-5A mRNA along with a plasmid encoding a mutated form of eIF-5a that can be not hypusinated.3 The new twist in the follow-up study is that the authors have demonstrated synergistic activity of this strategy when coupled with well-established modalities of treatment of patients affected by lymphoproliferative diseases, suggesting that this approach could translate readily to a clinical setting. Hypusine modification of eIF-5A is involved in regulation of its activity in both protein synthesis and eukaryotic cell growth. Several strategies have been used to inhibit hypusine formation. N1-guanyl-1,7-diaminoheptane (GC7), a nonspecific inhibitor of one of the two enzymes that mediate the hypusination, efficiently inhibits tumour cell proliferation. However, it gives rise to in vivo toxicity owing to its effect upon other enzymes.4,5 A way to limit the toxicity of anti-cancer agents is to combine them with other nontoxic drugs that exhibit synergistic inhibition of cell proliferation. In this light, the anticancer cytokine interferon α (IFNα) showed synergistic anticancer activity with GC 7 (ref. 6) and IFNα-mediated cell growth inhibition was associated with decreased hypusine synthesis and altered eIF-5A function.7 However, eIF-5A is expressed as two different isoforms with different functions: eIF5A1, which in its nonhypusinated form induces mytochondria-mediated apoptosis and eIF5A2, which in its nonhypusinated form loses its oncogenic potential.8 Interestingly, GC7 also increases the response of bladder cancer cells to doxorubicin, likely due to its ability to antagonize epithelial-mesenchymal transition through inhibition of eIF-5A2 hypusination and function.9 On these bases, the hypusination of both isoforms is essential to the oncogenic activity of eIF-5A and a benefit of the strategy used by Francis et al. is that it completely abrogates hypusine formation in eIF-5A without inducing nonspecific side effects. Lymphoproliferative diseases represent an attractive target for this approach since there is an urgent need for new therapeutic strategies in chemo- or immuno-resistant patients. However, a previous report by Scuoppo et al. revealed a tumor suppressor function for hypusinated eIF5A in lymphoproliferative diseases.10 These contradictory results were observed in an Eµ-Myc overexpression mouse model of lymphomagenesis that is very similar to Burkitt lymphoma, in which the lack of eIF5A hypusination promotes c-Myc-driven tumorigenesis. This is however the only report of tumor suppressor function of eIF-5A and, therefore merits additional investigation. As noted above, the same research group demonstrated the activity of SNS01 in models of multiple myeloma.3 This work advances the strategy reported in the previous report by demonstrating synergistic activity with bortezomib and an additive effect when combined with lenalidomide in inhibiting the growth of lymphoproliferative diseases. The use of combinatorial strategies in the treatment of cancer is an important challenge that requires determination of the optimal combination of complementary agents and their sequence of administration. The choice of optimal therapeutic strategy also depends upon the detection of predictive markers of response to therapy. In the case of eIF-5A targeted strategies, predictive biomarkers have yet to be determined and relevant animal studies are strongly warranted. Moreover, Francis et al. employed polyethylenimine (PEI)-based nanoparticles to deliver their nucleic acid therapy to the tumor tissues. PEI promotes self-assembly of nanoparticles so as to protect their contents from serum nucleases and is an efficient system for delivery of nucleic acids in vivo and in humans. However, only certain PEIs can be used for in vivo applications: they must exhibit an overall positive charge that allows them to bind to the negatively charged heparan sulphate proteoglycans on the cell surface. This can induce different side effects, such as liver necrosis, adhesion of aggregated platelets, shock after systemic injection at higher doses and stimulation of immune system and lung inflammation.11 Delivery strategies can be optimized through the use of second-generation nucleic acid-nanocarriers such as stabilized nucleic acid lipid particles (SNALPs). It has been demonstrated that SNALPs are able to efficiently deliver miRNAs in the treatment of lymphoproliferative diseases and multiple myeloma without signs of toxic side effects.12,13 SNALPs can also allow the functionalization of their surface with macrophage escaping molecules (such as polyethylene glycol), active targeting moieties (such as transferrin) or cell penetrating peptides. It was recently reported that miRNAs can be suitable tools to inhibit cell growth through the interference with eIF-5A.14 The advantage of miRNAs as a therapeutic derives from their ability to inhibit multiple intracellular targets and to give rise to pleiotropic interfering functions upon multiple pathways involved in tumorigenesis and cancer cell proliferation. In conclusion, the manuscript by Francis et al. opens an appealing scenario in which a factor traditionally involved in protein synthesis machinery regulation becomes a molecular target for in vivo anti-cancer approaches. The latter are based on both the use of RNA interference techniques and nanotechnology-based delivery strategies. Based on the results from Francis et al., exploratory clinical trials are strongly warranted and the optimization based on the search for both the optimal weapon and delivery strategy and on the finding of predictive biomarkers is still required (for a summary, see Figure 1). Figure 1 Historical development of molecular therapy aimed to block hypusine formation in eIF-5A. Initially GC7 was assessed to inhibit hypusine formation by enzyme inhibition (deoxyhypusine synthase inhibition) (from the right to the left). In this article, it ...

Basal Cell Carcinoma: Molecular and Pathological Features

Abstract: Basal cell carcinoma is the most common malignant tumour of humans, recording a progressive increase in incidence during the last decades. Basal cell carcinomas are locally destructive malignancies, mainly involving sun-exposed areas, such as head and neck skin. Basal cell carcinoma derives from basaloid epithelia located in the follicular bulges, and its development seems to be related to the deregulation of Hedgehog signalling pathway, primarily studied in the Gorlin-Goltz syndrome, characterised by a wide range of developmental abnormalities and predisposition to neoplasm, such as basal cell carcinomas. Basal cell carcinoma shows a clinical favourable behaviour, but some pathological features could suggest a more aggressive clinical course. In addition, the high incidence is responsible of a significant increasing workload for the health service, in relation to their treatment. Thus, novel therapy approaches have been developed in order to improve the treatment.

Impact of anemia management with EPO on psychologic distress in cancer patients: results of a multicenter patient survey

Abstract: : Aim: We investigated the role of erythropoietin (EPO) in reducing anemia and preventing the development of psychological distress in patients treated with chemotherapy. Patients & methods: This prospective observational study enrolled 591 adult patients receiving EPO at a dose of 30,000 IU administered once weekly for chemotherapy-induced anemia (mean baseline hemoglobin [Hb] level was 9.55 g/dl) over a 12-month period. Results: The majority of patients (371 [71%] patients) achieved a Hb increase >2 g/dl after 4 weeks of treatment. Interestingly, the nonresponder group had a statistically significant deterioration of their psychological conditions as indicated by psychological distress score (p = 0.01). However, within the group of responders to EPO, the Psychological Distress Inventory score remained unchanged. In the present study, severe side effects associated with EPO were not recorded. Conclusion: Hb increase, induced by EPO, ameliorates the psychological conditions of cancer patients.