Small molecules targeted to the microtubule-Hec1 interaction inhibit cancer cell growth through microtubule stabilization.

Highly expressed in cancer protein 1 (Hec1) is a subunit of the kinetochore (KT)-associated Ndc80 complex, which ensures proper segregation of sister chromatids at mitosis by mediating the interaction between KTs and microtubules (MTs). HEC1 mRNA and protein are highly expressed in many malignancies as part of a signature of chromosome instability. These properties render Hec1 a promising molecular target for developing therapeutic drugs that exert their anticancer activities by producing massive chromosome aneuploidy. A virtual screening study aimed at identifying small molecules able to bind at the Hec1-MT interaction domain identified one positive hit compound and two analogs of the hit with high cytotoxic, pro-apoptotic and anti-mitotic activities. The most cytotoxic analog (SM15) was shown to produce chromosome segregation defects in cancer cells by inhibiting the correction of erroneous KT-MT interactions. Live cell imaging of treated cells demonstrated that mitotic arrest and segregation abnormalities lead to cell death through mitotic catastrophe and that cell death occurred also from interphase. Importantly, SM15 was shown to be more effective in inducing apoptotic cell death in cancer cells as compared to normal ones and effectively reduced tumor growth in a mouse xenograft model. Mechanistically, cold-induced MT depolymerization experiments demonstrated a hyper-stabilization of both mitotic and interphase MTs. Molecular dynamics simulations corroborate this finding by showing that SM15 can bind the MT surface independently from Hec1 and acts as a stabilizer of both MTs and KT-MT interactions. Overall, our studies represent a clear proof of principle that MT-Hec1-interacting compounds may represent novel powerful anticancer agents.

Affinity purification-mass spectrometry analysis of bcl-2 interactome identified SLIRP as a novel interacting protein.

Members of the bcl-2 protein family share regions of sequence similarity, the bcl-2 homology (BH) domains. Bcl-2, the most studied member of this family, has four BH domains, BH1-4, and has a critical role in resistance to antineoplastic drugs by regulating the mitochondrial apoptotic pathway. Moreover, it is also involved in other relevant cellular processes such as tumor progression, angiogenesis and autophagy. Deciphering the network of bcl-2-interacting factors should provide a critical advance in understanding the different functions of bcl-2. Here, we characterized bcl-2 interactome by mass spectrometry in human lung adenocarcinoma cells. In silico functional analysis associated most part of the identified proteins to mitochondrial functions. Among them we identified SRA stem-loop interacting RNA-binding protein, SLIRP, a mitochondrial protein with a relevant role in regulating mitochondrial messenger RNA (mRNA) homeostasis. We validated bcl-2/SLIRP interaction by immunoprecipitation and immunofluorescence experiments in cancer cell lines from different histotypes. We showed that, although SLIRP is not involved in mediating bcl-2 ability to protect from apoptosis and oxidative damage, bcl-2 binds and stabilizes SLIRP protein and regulates mitochondrial mRNA levels. Moreover, we demonstrated that the BH4 domain of bcl-2 has a role in maintaining this binding.

Venous Thromboembolism in Cancer: Frequently Asked Questions When Guidelines are Inconclusive.

Management of Venous thromboembolism (VTE) in cancer patients is difficult when guidelines are inconclusive. To share a reasonable and homogeneous behavior in such circumstances, four issues, which are felt as problematic by oncologists and surgeons, have been selected; all were uncovered or only partially covered by current guidelines. Results from the literature and author's specific experience in the field were utilized to suggest reasonable solutions to the raised questions. The reported experience is the first to provide real-world management guidance for VTE in cancer patients. The effort of putting together literature review and author's experience brought to the adoption of a common behavior.

N-terminus-modified Hec1 suppresses tumour growth by interfering with kinetochore-microtubule dynamics.

Mitotic proteins are attractive targets to develop molecular cancer therapeutics due to the intimate interdependence between cell proliferation and mitosis. In this work, we have explored the therapeutic potential of the kinetochore (KT) protein Hec1 (Highly Expressed in Cancer protein 1) as a molecular target to produce massive chromosome missegregation and cell death in cancer cells. Hec1 is a constituent of the Ndc80 complex, which mediates KT-microtubule (MT) attachments at mitosis and is upregulated in various cancer types. We expressed Hec1 fused with enhanced green fluorescent protein (EGFP) at its N-terminus MT-interaction domain in HeLa cells and showed that expression of this modified Hec1, which localized at KTs, blocked cell proliferation and promoted apoptosis in tumour cells. EGFP-Hec1 was extremely potent in tumour cell killing and more efficient than siRNA-induced Hec1 depletion. In striking contrast, normal cells showed no apparent cell proliferation defects or cell death following EGFP-Hec1 expression. Live-cell imaging demonstrated that cancer cell death was associated with massive chromosome missegregation within multipolar spindles after a prolonged mitotic arrest. Moreover, EGFP-Hec1 expression was found to increase KT-MT attachment stability, providing a molecular explanation for the abnormal spindle architecture and the cytotoxic activity of this modified protein. Consistent with cell culture data, EGFP-Hec1 expression was found to strongly inhibit tumour growth in a mouse xenograft model by disrupting mitosis and inducing multipolar spindles. Taken together, these findings demonstrate that stimulation of massive chromosome segregation defects can be used as an anti-cancer strategy through the activation of mitotic catastrophe after a multipolar mitosis. Importantly, this study represents a clear proof of concept that targeting KT proteins required for proper KT-MT attachment dynamics constitutes a powerful approach in cancer therapy.

Safety of fondaparinux in the prevention of venous thromboembolism in elderly medical patients: results of a single-center, retrospective study.

AIM: Venous thromboembolism (VTE) is one of the leading causes of morbidity and mortality in acutely ill medical patients. Fondaparinux is recommended for the prevention of VTE in this setting, but little information is available on its safety and effectiveness in unselected, "real world" patients. The aim of this paper was to assess the safety and efficacy of fondaparinux in elderly acutely ill medical patients. METHODS: Single center, retrospective study. All patients >60 years, admitted for acute medical disease, bedridden for at least four days and treated with fondaparinux were evaluated. Occurrence of objectively documented, symptomatic VTE, and of bleeding events during the treatment period and follow-up were reported. RESULTS: Two hundred and ten patients (median age 81 years) were treated with fondaparinux. Seventy patients received fondaparinux 1.5 mg daily, 140 received the 2.5 mg daily dose. However, 29 patients in the first group (with a CrCl≥50 mL/min) and 84 patients in the last group (with a CrCl<50 mL/min) did not receive the correct dose of fondaparinux. During treatment, one episode (0.48%, 95% CI 0.1% to 2.6%) of major bleeding and 6 episodes (2.86%, 95% CI 1.3% to 6.1%) of clinically relevant non major bleeding were recorded. Only one thromboembolic event (0.48%, 95% CI 0.1% to 2.6%) was documented. Thirty-nine patients died; no death was related to VTE, unlike one death was due to major bleeding. Cancer was the only significant predictor of bleeding at statistical analysis. CONCLUSION: In elderly acutely ill hospitalized medical patients, thromboprophylaxis with fondaparinux 2.5 or 1.5mg daily is safe and effective in preventing VTE without increasing bleeding risk.

The thiazole derivative CPTH6 impairs autophagy.

We have previously demonstrated that the thiazole derivative 3-methylcyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH6) induces apoptosis and cell cycle arrest in human leukemia cells. The aim of this study was to evaluate whether CPTH6 is able to affect autophagy. By using several human tumor cell lines with different origins we demonstrated that CPTH6 treatment induced, in a dose-dependent manner, a significant increase in autophagic features, as imaged by electron microscopy, immunoblotting analysis of membrane-bound form of microtubule-associated protein 1 light chain 3 (LC3B-II) levels and by appearance of typical LC3B-II-associated autophagosomal puncta. To gain insights into the molecular mechanisms of elevated markers of autophagy induced by CPTH6 treatment, we silenced the expression of several proteins acting at different steps of autophagy. We found that the effect of CPTH6 on autophagy developed through a noncanonical mechanism that did not require beclin-1-dependent nucleation, but involved Atg-7-mediated elongation of autophagosomal membranes. Strikingly, a combined treatment of CPTH6 with late-stage autophagy inhibitors, such as chloroquine and bafilomycin A1, demonstrates that under basal condition CPTH6 reduces autophagosome turnover through an impairment of their degradation pathway, rather than enhancing autophagosome formation, as confirmed by immunofluorescence experiments. According to these results, CPTH6-induced enhancement of autophagy substrate p62 and NBR1 protein levels confirms a blockage of autophagic cargo degradation. In addition, CPTH6 inhibited autophagosome maturation and compounds having high structural similarities with CPTH6 produced similar effects on the autophagic pathway. Finally, the evidence that CPTH6 treatment decreased α-tubulin acetylation and failed to increase autophagic markers in cells in which acetyltransferase ATAT1 expression was silenced indicates a possible role of α-tubulin acetylation in CPTH6-induced alteration in autophagy. Overall, CPTH6 could be a valuable agent for the treatment of cancer and should be further studied as a possible antineoplastic agent.

Involvement of BH4 domain of bcl-2 in the regulation of HIF-1-mediated VEGF expression in hypoxic tumor cells.

In addition to act as an antiapoptotic protein, B-cell lymphoma (bcl)-2 can also promote tumor angiogenesis. In this context, we have previously demonstrated that under hypoxia bcl-2 promotes hypoxia-inducible factor-1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in melanoma and breast carcinoma. Here, we report on the role of the BH4 domain in bcl-2 functions, by showing that removal of or mutations at the BH4 domain abrogate the ability of bcl-2 to induce VEGF protein expression and transcriptional activity under hypoxia in human melanoma cells. We have also extended this observation to other human tumor histotypes, such as colon, ovarian and lung carcinomas. The involvement of BH4 on HIF-1α protein expression, stability, ubiquitination and HIF-1 transcriptional activity was also demonstrated in melanoma experimental model. Moreover, we validated the role of the BH4 domain of bcl-2 in the regulation of HIF-1/VEGF axis, demonstrating that BH4 peptide is sufficient to increase HIF-1α protein half-life impairing HIF-1α protein ubiquitination, and to enhance VEGF secretion in melanoma cells exposed to hypoxia. Finally, we found that the mechanism by which bcl-2 regulates HIF-1-mediated VEGF expression does not require BH1 and BH2 domains, and it is independent of antiapoptotic and prosurvival function of bcl-2.

Smoking reduction in smokers compliant to a smoking cessation trial with nicotine patch.

The aim of this retrospective analysis was to assess the extent of smoking reduction in smokers who were compliant to a smoking cessation trial with nicotine patch, and failed to completely quit smoking. Out of 297 smokers in total, 237 participants received active treatment (60 received placebo). Eighty treated subjects attended all the scheduled visits and were classified as either abstainers (nonsmokers), regular smokers or occasional smokers. Compared to the remaining 157 participants, these 80 subjects had significantly lower mean baseline daily cigarette consumption (24 versus 30; p < 0.001), expired carbon monoxide levels (25 versus 33 ppm; p < 0.001), plasma nicotine and cotinine levels, and Fagerström Tolerance Questionnaire score (5.7 versus 7.0; p < 0.001). All subjects received active treatment for up to 18 weeks (full dose for 12 weeks plus tapering dose for 6 weeks), with follow-up visits scheduled up to 1 yr. A statistically significant reduction in cigarette consumption (versus baseline) was observed among both the occasional (-99%) and regular (-77%) smokers between week 1 and week 52 (p < 0.001). Concomitant smoking and patch use was well tolerated since adverse events were infrequent, mild and transient. Thus, in addition to those subjects who successfully quit smoking, a further group of subjects who attended all the follow-up visits during the smoking cessation trial significantly reduced their mean daily cigarette consumption.

Computerized impedance plethysmography in the diagnosis of delayed deep vein thrombosis after total hip replacement.

Although noninvasive techniques have been extensively evaluated in the diagnosis of deep vein thrombosis (DVT), few data exist about the role of computerized impedance plethysmography (CIP) in the diagnosis of delayed DVT after surgery, when patients are at home and the risk of DVT is still high. The aim of this study was to evaluate the reliability of CIP in the diagnosis of proximal and distal delayed DVT in both symptomatic and asymptomatic patients who had undergone elective total hip replacement (THR). Bilateral CIP of lower limbs was performed in 61 patients on days 5, 9, 15 and 45 after THR; for comparison, ascending phlebography was performed on days 45 after THR in all patients on the operated limb or on the limb with positive results on CIP. The overall rate of DVT diagnosed by phlebography was 16.3% (10/61). The sensitivity and specificity of CIP for all DVT were 20% and 98%, respectively, while the accuracy was 85%. The results were not influenced by the presence of symptoms or signs of the lower limbs. Similar results were obtained when considering proximal DVT only. In conclusion, the low sensitivity of computerized impedance plethysmography means that it cannot be used in screening for delayed deep vein thrombosis after total hip replacement, in both symptomatic and asymptomatic patients. However, because of its high specificity, when the results of computerized impedance plethysmography are positive phlebography should be performed to confirm the diagnosis of deep vein thrombosis.

Nasal ventilation in COPD exacerbations: early and late results of a prospective, controlled study.

Noninvasive positive pressure mechanical ventilation (NIPPV) in exacerbated chronic obstructive pulmonary disease (COPD) has been investigated early and after 1 yr of follow-up. To this end, 30 patients were enrolled in a prospective, controlled trial: 15 had early administration of NIPPV (Group A), 15 had medical therapy only (Group B); assignment was made on the basis of equipment availability only. In-hospital mortality, need for endotracheal intubation and mean length of hospitalization were lower in Group A, though the difference was not statistically significant. Arterial oxygen tension in arterial blood (Pa,O2), carbon dioxide tension in arterial blood (Pa,CO2) and HCO3- improved significantly in both groups from admission to discharge: 45.8+/-8.6 versus 64.9+/-10.0; 59.4+/-11.8 versus 48.6+/-7.3; 34.3+/-4.3 versus 30.1+/-3.4 in group A; 49.2+/-11.4 versus 60.9+/-8.2; 52.6+/-15.9 versus 44.4+/-8.7; 31.7+/-5.9 versus 28.0+/-3.6 in group B, respectively, p<0.05 for all comparisons; pH, percentage forced expiratory volume in one second (FEV1) and tidal volume (VT) improved significantly in patients of group A only: 7.36+/-0.04 versus 7.41+/-0.02; 39.8+/-13.6 versus 49.4+/-11.7; 0.71+/-0.3 versus 0.84+/-0.4, respectively, p<0.05. During follow-up, 3, 6, and 12 months survival rates were significantly higher in Group A than in Group B (p<0.02). Hospital new admissions over 1 yr were more frequent in Group B (n=6, incidence rate: 0.216%) than in Group A (n=4, incidence rate: 0.084%). Therefore, noninvasive positive pressure mechanical ventilation may be added to "conventional" medical therapy in exacerbated chronic obstructive pulmonary disease.

Mortality rates for respiratory disorders in Italy (1979-1990).

Trends in mortality rates for respiratory disorders were investigated in Italy from 1979 to 1990, using data from the Italian Central Statistical Institute (ISTAT). Mortality from lung cancer increased in all age groups, except for those aged 45-64 yrs after 1985. Respiratory diseases showed a consistent reduction; in particular, mortality from emphysema decreased slowly, and mortality from chronic bronchitis showed a significant reduction in all age groups. However, mortality from asthma increased markedly in all age groups up to 1985, and then levelled off and slightly decreased, although remaining at a higher level than in the 1970s. In 1990, data stratified for age group and gender indicated a higher mortality rate in males, that tended to be age-dependent, with the highest rate ratio male/female in those aged 65-74 yrs. Overall, these data indicate a trend to increased mortality from lung cancer and asthma in Italy in the 1980s.

Evaluation of DNA damage in leukocytes of ex-smokers by single cell gel electrophoresis.

Single cell gel electrophoresis (SCGE), or comet assay, appears to be a promising tool to estimate DNA damage at the single cell level and it provides information on the presence of damage among individual cells. A follow-up study of 90 smokers who ceased smoking was undertaken to determine the possible decrease of DNA damage in their leukocytes. Before beginning the trial, volunteers smoked on average 26.1 +/- 8.4 cigarettes/day. Comet length did not correlate with the number of cigarettes/day or with the condensate tar content. At the end of the study, 28 volunteers had abandoned the trial, 40 volunteers relapsed into smoking at different times, but with a reduced number of cigarettes/day, whereas 22 fully succeeded in smoking cessation. Throughout the 5 sampling times, a great variability of comet length at individual level was found. However, after 1 year of follow-up, comet length means were found to be significantly shorter (p < 0.0001) in those volunteers who completely quit smoking compared to those who relapsed into smoking (27.2 +/- 1.6 vs. 31.9 +/- 5.1 microns, respectively), irrespective of the amount of cigarettes previously smoked. No effect of age or sex was found. Six months later, these results were confirmed by a further study carried out on a reduced sample of volunteers. The present data strongly suggest that, in spite of the great variability observed, 1 year of smoking cessation is associated with a significant reduction of DNA damage in circulating leukocytes.