Doppler ultrasonographic measurements of the lateral digital palmar artery in pregnant mares.

Cardiovascular changes have been reported in late pregnancy in mares. However, there are no data on changes in peripheral blood flow. Doppler ultrasound represents a sensitive method for assessing the blood flow directed to the hoof. The aims of this study were to evaluate the blood flow parameters of the lateral palmar digital artery (LPDA) in pregnant mares and to assess intra- and interrater agreement between two observers with different levels of experience. The LPDAs of pregnant Italian Standardbred mares were examined. The vessels were located with B-mode ultrasound and analyzed with color and pulsed wave Doppler. The following parameters were recorded by the operators: heart rate (HR), peak systolic velocity (PSV), end-diastolic velocity (EDV), and resistive index (RI). Measurements were performed between 2 and 3 months of gestation (T1), in the last month of pregnancy (T2) and a week after delivery (T3). Seventeen mares aged 3-18 years met the inclusion criteria. Ultrasound examinations of the LPDA were subjectively easy to perform and well tolerated by the mares. Interrater and intrarater agreement were good and moderate, respectively. The HR was higher at T2 than at T1 and T3. The PSV and RI changed significantly during pregnancy, with higher values at T2 and T3, whereas the EDV remained unchanged throughout the examination. Doppler examination showed that peripheral flow changes were present in mares in late pregnancy. However, the persistence of higher values after delivery invites further investigation to assess the correlation between metabolic/endocrine changes related to pregnancy and Doppler parameters.

A cross-sectional study on performance evaluation in Italian standardbred horses' real-time PCR-positive for Theileria equi.

BACKGROUND: Inflammatory myopathy and perivasculitis have been recently described in horses with chronic equine piroplasmosis (EP). These alterations may be linked to poor performances. The aims of this study were to evaluate the prevalence for EP in clinically healthy Italian Standardbred (IS) racehorses and to compare laboratory parameters and performance metrics between positive and negative horses. Real-time PCR was applied for the detection of T. equi and B. caballi positivity. Haematology parameters, blood chemistry results, subjective muscle mass scores, and performance metrics were compared between PCR-positive and -negative horses. RESULTS: This cross-sectional study included 120 well-trained IS racehorses and was performed over a two-years period. The prevalence of T. equi was 36.3%, whereas all samples were negative for B. caballi. Red blood cells count, haemoglobin concentration, aspartate aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase activities were significantly higher in PCR-positive horses, whereas blood urea nitrogen, globulin concentration and globulin-to-albumin ratio were significantly lower in PCR-positive horses compared to PCR-negative ones. Nonetheless, all values fell within the physiological range. The best racing time, which was selected as the most representative of the performance metrics at the principal component analysis, was not affected by PCR positivity, the muscle mass score or the training yard. The best racing time was significantly better in horses with a mild or no signs of muscular atrophy, within the PCR-positive group. The muscle mass score was associated with the training yard in PCR-negative horses. CONCLUSIONS: Prevalence of T. equi was high in IS racehorses in southern Italy. The absence of obvious changes in haematological and biochemical parameters, as well as performance metrics in positive horses, highlights the need for specific diagnostic tests to identify chronically infected horses.

Computed Tomographic Assessment of Pituitary Gland Dimensions in Domestic Short-Haired Cats.

The detection of subtle changes in the pituitary dimensions has relevant clinical implications. In cats, a few studies have established the cut-off values of the pituitary gland's dimensions using small and inhomogeneous samples. The aims of this study were: to determine by computed tomography (CT) the pituitary linear dimensions and the pituitary-to-brain (P:B) ratio in a sample of domestic short-haired (DSH) cats; to assess the effects of sex, age, and weight on pituitary dimensions; and to evaluate the inter- and intra-observer agreement for such measurements. All skull CTs of DSH cats performed over four years using a multidetector CT and a standardized protocol were retrospectively reviewed. The exclusion criteria were: clinical, laboratory, or CT alterations of the pituitary gland, brain diseases, fractures of the neurocranium, and diabetes. The pituitary dimensions and brain area were assessed by two different observers using multiplanar reconstructions and automated segmentation tools. Fifty-one cats were included in the final sample. The intraclass correlation coefficients for intra- and inter-observer reliability were good/excellent, and moderate/good, respectively. No differences between sexes were detected, and negligible correlations were found between age and weight. According to this study, a pituitary gland with a height > 4 mm or a P:B ratio > 0.49 mm should be considered enlarged.

Retrobulbar Filling for Enophthalmos Treatment in Dogs: Technique, Description and Computed-Tomographic Evaluation. Preliminary Cadaveric Study.

A new therapeutic approach for enophthalmos may be retrobulbar lipofilling. This study aims to standardize the intraconal filling technique and to evaluate the degree of eyeball displacement by computed tomography (CT). Skull CT was performed on six dog cadavers before and after intraconal injection of two 5% iodinated, viscoelastic solutions, one per eye, using an ultrasound-guided supratemporal approach. The volume to be injected was calculated using formulas for retrobulbar cone anesthesia. After CT, the dogs underwent necropsy and histopathology to evaluate damages that eventually occurred to retrobulbar structures. Eyeball displacement was estimated using two CT-based methods, named M1 and M2. The Wilcoxon signed-rank test revealed no significant difference between the two injected materials in both M1 (p > 0.99), and M2 (lateral p = 0.84 and rostral p = 0.84 displacement). A statistically significant difference was found between the pre- and post-injection group M1 (p = 0.002), M2 (p = 0.004) for the lateral and (p = 0.003) for rostral displacement. Although the slight eyeball displacement, the retrobulbar filling can lead to enophthalmos resolution. Compared to M1, the M2 method has better-defined anatomical landmarks. Further, preclinical in vivo studies are necessary to assess retrobulbar filling efficacy and safety.

The heart to single vertebra ratio: A new objective method for radiographic assessment of cardiac silhouette size in dogs.

Vertebral heart size (VHS) is widely determined in clinical practice as an objective method to assess the cardiac silhouette dimensions. However, a key limitation is that it is difficult to determine VHS in dogs with vertebral alterations. This retrospective, method comparison, observer agreement study sought to overcome this limitation by using the heart-to-single vertebra ratio (HSVR), by evaluating the level of agreement between VHS and HSVR, as well as the intra- and inter-observer agreement for HSVR. Three independent observers retrospectively evaluated thoracic radiographs obtained over a set time period. Exclusion criteria were the presence of alterations of the thoracic spine and the inability to clearly outline the cardiac silhouette. The lengths of the vertebral bodies, from the fourth to eighth thoracic vertebra, and VHS were measured on each radiograph. The HSVR was calculated by dividing the sum of the cardiac long and short axes by the length of each vertebral body. Eighty dogs of different breeds were included in the final analysis. Lin's concordance correlation coefficients revealed strong correlations between VHS and HSVR (0.91-0.96), and the Bland-Altman plots showed low bias (0.01-0.2) between the methods. The mean absolute errors indicated low average magnitudes of error (0.11-0.28). The intraclass correlation coefficients showed good to excellent inter-observer (0.87-0.92; P = 0.000) and intra-observer (0.87-0.99; P < .001) agreement. In the authors' opinion, this new method, which is less time consuming and more objective, could offer a valuable alternative to VHS.

Description of a low-cost picture archiving and communication system based on network-attached storage.

High costs for installing, maintaining, and updating a standard picture archiving and communication system (PACS) can be prohibitive for small/medium-sized veterinary facilities. The aims of this prospective, exploratory study were to describe the design, implementation, and author experiences for 1 year's use of a low-cost PACS based on network-attached storage. The system described here was easily installed and resiliently stored redundant copies of data. It excellently balanced data recovery, system speed, security, and available memory for storage. A virtual private network also allowed off-site data review. This system can also be used for future off-site backup of data in the cloud.

The Use of Platelet-Rich Plasma for Treatment of Tenodesmic Lesions in Horses: A Systematic Review and Meta-Analysis of Clinical and Experimental Data.

The use of platelet-rich plasma (PRP) to enhance tenodesmic lesion healing has been questioned over the years. The aim of this study was to evaluate current literature to establish the effectiveness of PRP for treating tenodesmic lesions through a systematic review, in accordance with the PRISMA guidelines, and a meta-analysis. Studies comparing PRP with placebo or other treatments for horses with tenodesmic injuries or evaluated PRP effect on tendon and ligament explants were included. Outcomes were clinical, ultrasound, histologic, molecular evaluation, and adverse effects. Two authors independently extracted data and assessed each study's risk of bias. Treatment effects were evaluated using risk ratios for dichotomous data, together with 95% CI. Data were pooled using the random-effects model. The quality of the evidence for each outcome was assessed using GRADE criteria. Twenty-four trials met inclusion criteria for systematic review, while fifteen studies were included in the meta-analysis. Results showed no significant differences in the outcomes between PRP and control groups. Finally, there is no definitive evidence that PRP enhances tendons and ligaments healing. Therefore, there is a need for more controlled trials to draw a firmer conclusion about the efficacy of PRP as a treatment for tenodesmic lesions in the horse.

Ultrasonographic assessment of normal jugular veins in Standardbred horses.

BACKGROUND: Ultrasonography (US) is the recommended imaging technique to evaluate jugular veins. This prospective randomized clinical study was designed to collect a series of B-mode US measurements of manually distended jugular veins in healthy Italian Standardbreds and to find possible correlations between ultrasound measurements and animal morphometric characteristics. Forty-two horses, eight males and 34 females (range 3-22 years; bodyweight 494.4 ± 41.7 kg), were included in the study. The diameters and wall thicknesses of both jugular veins were measured at three different sites of the neck. The differences in ultrasound measurements based on scans, age, gender, side, and site of the neck were evaluated by ANOVA or by the Kruskal-Wallis test. The effects of the morphometric measures on each ultrasound parameter were evaluated by MANOVA (P < 0.05). RESULTS: The ultrasound measurements did not differ significantly between the three different sites or between genders; hence, they were pooled together in the results. On the transverse scan, the mean dorsoventral and lateromedial diameters were 1.58 ± 0.23 and 2.20 ± 0.25 cm, respectively; the mean superficial and deep wall thicknesses (SWT and DWT) were 0.07 ± 0.01 and 0.08 ± 0.01 cm, respectively. On the longitudinal scan, the mean dorsoventral diameter was 1.59 ± 0.26 cm, and the SWT and DWT were both 0.08 ± 0.01 cm. Neck length, from the caudal edge of the mandible to the thoracic inlet, was related to the dorsoventral diameter in both longitudinal and transverse scan and to the SWT and DWT in transverse scan, whereas height at the withers (measured with tape) and estimated weight were related to the wall thickness. Dividing the subjects into groups by age in years ("young" 3-7, "mature" 8-14, "old" > 14), differences were found for the lateromedial diameter in the transverse scan and the SWT on the longitudinal scan. The main limitation of this study was that only one operator performed the measurements. CONCLUSION: The US measurements of the jugular veins and their relationship with morphometric measures reported in this manuscript might be considered as guidelines both for early diagnosis and monitoring jugular vein abnormalities in healthy Italian Standardbred horses.

Nicotine increases colon cancer cell migration and invasion through epithelial to mesenchymal transition (EMT): COX-2 involvement.

Cigarette smoking is a recognized risk factor for colon cancer and nicotine, the principal active component of tobacco, plays a pivotal role in increasing colon cancer cell growth and survival. The aim of this study was to determine the effect of nicotine on cellular Caco-2 and HCT-8 migration and invasion, focusing on epithelial to mesenchymal transition (EMT) induction, and COX-2 pathway involvement. In both these cell lines, treatment with nicotine increased COX-2 expression and the release of its enzymatic product PGE2 . Moreover, nicotine-stimulated cells showed increased migratory and invasive behavior, mesenchymal markers up-regulation and epithelial markers down-regulation, nuclear translocation of the ß-catenin, increase of MMP-2 and MMP-9 activity, and enhanced NF-κB expression. Noticeably, all these effects are largely mediated by COX-2 activity, as simultaneous treatment of both cell lines with nicotine and NS-398, a selective COX-2 inhibitor, greatly reduced the number of migrating and invading cells and reverted nicotine-induced EMT. These findings emphasize that nicotine triggers EMT, leading hence to increased migration and invasiveness of colon cancer cells. Thereby, the use of COX-2 inhibitor drugs might likely counteract nicotine-mediated EMT effects on colon cancer development and progression.

Inflammatory Myopathy in Horses With Chronic Piroplasmosis.

Horses affected by chronic piroplasmosis may develop poor performance and muscle atrophy. Here we investigate the pathological and immunopathological aspects of myopathy occurring in chronic equine piroplasmosis. The study included 16 horses serologically positive for equine piroplasms presenting with clinical signs and supporting serum biochemical evidence of a myopathy. Skeletal muscle was evaluated by histopathology, immunohistochemistry, indirect immunofluorescence, and molecular detection of piroplasms and inflammatory cytokines in skeletal muscle. Histologic lesions included muscle fiber atrophy (100% of cases), degenerative changes (13/16, 81%), and perivascular perimysial and endomysial lymphocytic infiltrates (81% of cases). In 15 cases (94%), muscle fibers had strong immunostaining for major histocompatibility complex classes I and II. T lymphocyte populations were mainly CD3+, CD8+, and CD4+ in equal proportions, with a lower number of CD79α+ cells. The serum from affected horses was tested by indirect immunofluorescence for binding of IgG, IgM, or IgA to sections of normal equine muscle to detect circulating autoantibodies against muscle antigen(s). In all cases, distinct sarcolemmal staining was detected in sections incubated with serum from affected horses, in contrast to sections incubated with phosphate-buffered saline or equine control sera. Reverse transcription polymerase chain reaction (RT-PCR) testing of muscles from affected animals revealed a significant increase of interferon-γ, interleukin-12, and tumor necrosis factor-α gene expression compared to healthy controls. Theileria equi or Babesia caballi was not detected in samples of affected muscle by RT-PCR. Thus, inflammatory myopathy associated with equine piroplasmosis may involve an autoimmune pathogenesis with upregulation of inflammatory cytokines that may cause myofiber atrophy and degeneration.

Paradoxical E-cadherin increase in 5FU-resistant colon cancer is unaffected during mesenchymal-epithelial reversion induced by γ-secretase inhibition.

AIM: Presenilin-1 (PS1), the main component of γ-secretase activity support a key role during Epithelial-Mesenchymal Transition (EMT) and chemoresistance acquisition by triggering a complex sequence of molecular events, including E-cadherin down-regulation. However, we hypothesize that EMT and chemoresistance should be deemed separate processes in HCT-8 colon cancer cells. MAIN METHODS: HCT-8 and HCT-8FUres invasion was evaluated by trans-well assay. uPA activity was detected by zymography. Prostaglandin E2 levels were quantified using an ELISA kit. E-cadherin FL and CTF2, PS1, Notch1, Cyclin D1, COX2, SNAI1 and α-SMA expression were determined using Western blot technique. ß-Catenin localization was observed by confocal microscopy. Cell apoptosis was evaluated by cytofluorimetric assay, and measurement of caspase-3 and cl-PARP. γ-Secretase activity was inhibited by DAPT, a γ-secretase inhibitor. KEY FINDINGS: Chemoresistant HCT-8 underwent EMT that can be efficiently reversed by inhibiting PS1 activity, leading thus to a normalization of mostly of the pivotal features showed by the invasive cancer phenotype. Indeed, we observed decreased SNAI1 and Notch 1 activation, altogether with reduced E-cadherin cleavage. Concomitantly, resistant HCT-8 invasiveness was almost completely abolished. However, such reversion was not followed by any increase in apoptotic rate, not by changes in E-cadherin levels. Indeed, despite HCT-8FUres underwent an undeniable EMT, full-length E-cadherin levels were found remarkably higher than those observed in wild HCT-8. SIGNIFICANCE: High E-cadherin concentration in presence of enhanced γ-secretase activity is incontestably a paradoxically result, highlighting that E-cadherin loss is not a pre-requisite for EMT. Additionally, EMT and chemoresistance acquisition in HCT-8 should be considered as distinct processes.

Combination of antibodies directed against different ErbB3 surface epitopes prevents the establishment of resistance to BRAF/MEK inhibitors in melanoma.

Patients with metastatic melanoma bearing V600 mutations in BRAF oncogene clinically benefit from the treatment with BRAF inhibitors alone or in combination with MEK inhibitors. However, a limitation to such treatment is the occurrence of resistance. Tackling the adaptive changes helping cells survive from drug treatment may offer new therapeutic opportunities. Very recently the ErbB3 receptor has been shown to act as a central node promoting survival of BRAF mutated melanoma. In this paper we first demonstrate that ErbB3/AKT hyperphosphorylation occurs in BRAF mutated melanoma cell lines following exposure to BRAF and/or MEK inhibitors. This strongly correlates with increased transcriptional activation of its ligand neuregulin. Anti-ErbB3 antibodies impair the establishment of de novo cell resistance to BRAF inhibition in vitro. In order to more potently ablate ErbB3 activity we used a combination of two anti-ErbB3 antibodies directed against distinct epitopes of its extracellular domain. These two antibodies in combo with BRAF/MEK inhibitors potently inhibit in vitro cell growth and tumor regrowth after drug withdrawal in an in vivo xenograft model. Importantly, residual tumor masses from mice treated by the antibodies and BRAF/ERK inhibitors combo are characterized almost exclusively by large necrotic areas with limited residual areas of tumor growth. Taken together, our findings support the concept that triple therapy directed against BRAF/MEK/ErbB3 may be able to provide durable control of BRAF mutated metastatic melanoma.

Multiwalled carbon nanotube buckypaper induces cell cycle arrest and apoptosis in human leukemia cell lines through modulation of AKT and MAPK signaling pathways.

MWCNT buckypaper (BP) shows physico-chemical and mechanical properties that make it potentially useful as a substrate in nano-bio interface research including in tissue engineering. When used as a scaffold material, BP comes into contact with host cells and surrounding tissues; therefore it is critical to determine its biocompatibility and interaction with living systems. The aim of this study was to investigate BP effects on cell growth, apoptosis and reactive oxygen species (ROS) production in three human leukemia cell lines HL-60, U-937 and K-562. BP was able to induce both the reduction of cell proliferation, associated with an arrest in G0/G1 phase of cell cycle and the increase of apoptosis in leukemic cell lines, thus exerting both cytostatic and cytotoxic effects. The growth inhibitory effect was likely mediated by the decrease of cyclins D, E, A, B1 levels and CDK4 expression; meanwhile, the apoptotic effect, not mediated by ROS production, was presumably due to the combined action of the survival and pro-apoptotic AKT and MAPK signal transduction pathways. These results raised the issue of biocompatibility of MWCNT BP for the creation of carbon nanotubes based scaffolds to utilize as prostheses in tissue engineering.

Lung cancer stem cell lose their stemness default state after exposure to microgravity.

Microgravity influences cell differentiation by modifying the morphogenetic field in which stem cells are embedded. Preliminary data showed indeed that stem cells are committed to selective differentiation when exposed to real or simulated microgravity. Our study provides evidence that a similar event occurs when cancer stem cells (CSCs) are cultured in microgravity. In the same time, a significant increase in apoptosis was recorded: those data point out that microgravity rescues CSCs from their relative quiescent state, inducing CSCs to lose their stemness features, as documented by the decrease in ALDH and the downregulation of both Nanog and Oct-4 genes. Those traits were stably acquired and preserved by CSCs when cells were placed again on a 1 g field. Studies conducted in microgravity on CSCs may improve our understanding of the fundamental role exerted by biophysical forces in cancer cell growth and function.

Phenotypic switch induced by simulated microgravity on MDA-MB-231 breast cancer cells.

Microgravity exerts dramatic effects on cell morphology and functions, by disrupting cytoskeleton and adhesion structures, as well as by interfering with biochemical pathways and gene expression. Impairment of cells behavior has both practical and theoretical significance, given that investigations of mechanisms involved in microgravity-mediated effects may shed light on how biophysical constraints cooperate in shaping complex living systems. By exposing breast cancer MDA-MB-231 cells to simulated microgravity (~0.001 g), we observed the emergence of two morphological phenotypes, characterized by distinct membrane fractal values, surface area, and roundness. Moreover, the two phenotypes display different aggregation profiles and adherent behavior on the substrate. These morphological differences are mirrored by the concomitant dramatic functional changes in cell processes (proliferation and apoptosis) and signaling pathways (ERK, AKT, and Survivin). Furthermore, cytoskeleton undergoes a dramatic reorganization, eventually leading to a very different configuration between the two populations. These findings could be considered adaptive and reversible features, given that, by culturing microgravity-exposed cells into a normal gravity field, cells are enabled to recover their original phenotype. Overall these data outline the fundamental role gravity plays in shaping form and function in living systems.

Grape seed extract suppresses MDA-MB231 breast cancer cell migration and invasion.

BACKGROUND AND AIM: Breast cancer remains a leading cause of mortality among women. In metastasis, cascade migration of cancer cells and invasion of extracellular matrix (ECM) represent critical steps. Urokinase-type plasminogen activator (uPA), as well as metalloproteinases MMP-2 and MMP-9, strongly contribute to ECM remodelling, thus becoming associated with tumour migration and invasion. In addition, the high expression of cytoskeletal (CSK) proteins, as fascin, has been correlated with clinically aggressive metastatic tumours, and CSK proteins are thought to affect the migration of cancer cells. Consumption of fruits and vegetables, characterized by high procyanidin content, has been associated to a reduced mortality for breast cancer. Therefore, we investigated the biological effect of grape seed extract (GSE) on the highly metastatic MDA-MB231 breast cancer cell line, focusing on studying GSE ability in inhibiting two main metastatic processes, i.e., cell migration and invasion. METHODS: After MDA-MB231 breast cancer cells stimulated with GSE migration and invasion were evaluated by means of trans-well assays and uPA as well as MMPs activity was detected by gelatin zymography. Fascin, ß-catenin and nuclear factor-κB (NF-κB) expression were determined using western blot technique. ß-Catenin localization was observed by confocal microscopy. RESULTS: We observed that high concentrations of GSE inhibited cell proliferation and apoptosis. Conversely, low GSE concentration decreased cell migration and invasion, likely by hampering ß-catenin expression and localization, fascin and NF-κB expression, as well as by decreasing the activity of uPA, MMP-2 and MMP-9. CONCLUSIONS: These results make GSE a powerful candidate for developing preventive agents against cancer metastasis.

Nicotine increases survival in human colon cancer cells treated with chemotherapeutic drugs.

Cigarette smoking is implicated in the development of colon cancer. Furthermore, nicotine increases cell proliferation and inhibits apoptosis through α7-nicotinic acetylcholine receptor (α7-nAChR) activation in human colon carcinoma cells. An open issue is whether nicotine interfere with colorectal cancer pharmacological treatment, by inhibiting drug-mediated apoptosis. To assess this hypothesis, we evaluated nicotine effect on Caco-2 and HCT-8 colon cancer cells, treated with 5-Fluorouracil (5-FU) and Camptothecin (CPT), chemotherapeutics commonly utilized as adjuvant treatment of colon cancer. Nicotine decreased anti-proliferative and pro-apoptotic effects exerted by chemotherapeutics on both cell lines. These effects partially reverted by exposure to α-bungarotoxin (α-BTX), an inhibitor of α7-nAChR. Nicotine addition to Caco-2 and HCT-8, treated with 5-FU or CPT, decreased the cleavage of substrate of caspase 3 and 7, poly-ADP-ribose polymerase (PARP). Moreover, P-ERK/ERK ratio was modified by nicotine addition to 5-FU and CPT treated cells in an opposite manner. However, when co-administrating PD98059, an ERK phosphorylation inhibitor, an increased apoptosis was observed. In Caco-2 and HCT-8 nicotine reverted 5-FU and CPT apoptotic effects through AKT phosphorylation, as demonstrated by apoptotic increase in presence of LY294002, an AKT phosphorylation inhibitor. Nicotine interfered with colorectal cancer pharmacological treatment in vitro by inhibiting apoptosis induced by chemotherapeutic drugs. Nicotine anti-apoptotic effects were exerted through ERK and AKT pathway activation.

Combination therapy with anti-ErbB3 monoclonal antibodies and EGFR TKIs potently inhibits non-small cell lung cancer.

Personalized therapy of advanced non-small cell lung cancer (NSCLC) has been improved by the introduction of EGFR tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib. EGFR TKIs induce dramatic objective responses and increase survival in patients bearing sensitizing mutations in the EGFR intracytoplasmic tyrosine kinase domain. However, virtually all patients develop resistance, and this is responsible for disease relapse. Hence several efforts are being undertaken to understand the mechanisms of resistance in order to develop combination treatments capable to sensitize resistant cells to EGFR TKIs. Recent studies have suggested that upregulation of another member of the EGFR receptor family, namely ErbB3 is involved in drug resistance, through increased phosphorylation of its intracytoplasmic domain and activation of PI3K/AKT signaling. In this paper we first show, by using a set of malignant pleural effusion derived cell cultures (MPEDCC) from patients with lung adenocarcinoma, that surface ErbB3 expression correlates with increased AKT phosphorylation. Antibodies against ErbB3, namely A3, which we previously demonstrated to induce receptor internalization and degradation, inhibit growth and induce apoptosis only in cells overexpressing surface ErbB3. Furthermore, combination of anti-ErbB3 antibodies with EGFR TKIs synergistically affect cell proliferation in vitro, cause cell cycle arrest, up-regulate p21 expression and inhibit tumor growth in mouse xenografts. Importantly, potentiation of gefitinib by anti-ErbB3 antibodies occurs both in de novo and in ab initio resistant cells. Anti-ErbB3 mAbs strongly synergize also with the dual EGFR and HER2 inhibitor lapatinib. Our results suggest that combination treatment with EGFR TKI and antibodies against ErbB3 should be a promising approach to pursue in the clinic.

Grape seed extract triggers apoptosis in Caco-2 human colon cancer cells through reactive oxygen species and calcium increase: extracellular signal-regulated kinase involvement.

Grape seed extract (GSE) from Italia, Palieri and Red Globe cultivars inhibits cell growth and induces apoptosis in Caco-2 human colon cancer cells in a dose-dependent manner. In order to investigate the mechanism(s) supporting the apoptotic process, we analysed reactive oxygen species (ROS) production, intracellular Ca2+ handling and extracellular signal-regulated kinase (ERK) activation. Upon exposure to GSE, ROS and intracellular Ca2+ levels increased in Caco-2 cells, concomitantly with ERK inactivation. As ERK activity is thought to be essential for promoting survival pathways, inhibition of this kinase is likely to play a relevant role in GSE-mediated anticancer effects. Indeed, pretreatment with N-acetyl cysteine, a ROS scavenger, reversed GSE-induced apoptosis, and promoted ERK phosphorylation. This effect was strengthened by ethylene glycol tetraacetic acid-mediated inhibition of extracellular Ca2+ influx. ROS and Ca2+ influx inhibition, in turn, increased ERK phosphorylation, and hence almost entirely suppressed GSE-mediated apoptosis. These data suggested that GSE triggers a previously unrecognised ERK-based mechanism, involving both ROS production and intracellular Ca2+ increase, eventually leading to apoptosis in cancer cells.

Nicotine stimulates proliferation and inhibits apoptosis in colon cancer cell lines through activation of survival pathways.

BACKGROUND: Colorectal cancer is one of the leading causes of cancer-related death throughout the world, and the risk to develop this malignant disease seems to be associated with long-term cigarette smoking. Nicotine, one of the major components of cigarette smoking, can stimulate cell proliferation and suppress apoptosis both in normal cells and in several human cancer cell lines derived from various organs. However, although nicotine appears to have a role in stimulating cell proliferation of colon cancer cells, there is no information on its role in inhibiting apoptosis in these cells. MATERIALS AND METHODS: Human colorectal cancer cell lines Caco-2 and HCT-8 were treated with 1 µM nicotine alone or in combination with 1 µM α-BTX in complete or in serum free medium. Cell proliferation and apoptosis were determined by cell count performed with a cell counter and by cytofluorimetric assay respectively. PI3K/Akt and PKC/ERK1/2 pathways, survivin, and P-Bcl2 (Ser70) were investigated by Western blot analysis. RESULTS: Nicotine induced an increase in cell proliferation and a decrease of apoptosis in Caco-2 and HCT-8 cells. Both cell growth and apoptosis appear to be mediated by α7-nicotinic acetylcholine receptors, since treatment with α-Bungarotoxin inhibited these processes. Nicotine induced a statistically significant increase in the expression of PI3K and in P-Akt/Akt ratio as well as in the expression of PKC, ERK1/2, survivin, and P-Bcl2 (Ser70) in both cell lines. CONCLUSIONS: Nicotine, contained in cigarette smoking, could participate in colon cancer development and progression by stimulating cell proliferation and suppressing physiological apoptosis.