Questioning the oncogenic role of ΔNp73α in different cell lines expressing p53 or not

The recent finding that the 1p36.3 locus gene encodes an array of different p73 isoforms with apparently distinct and sometimes opposing cellular functions, might explain the difficulty in establishing the protein's role as tumor suppressor. Therefore we need to investigate the roles of each of these splicing variants in cellular functions when expressed alone or in combination with other family members, as well as the genetic background on which the proteins are expressed. We investigated, in two p53 null cell lines, the human SCLC line H1299 and a subline derived from the human colon carcinoma cell line HCT116 (HCT116/379.2), the effects of DeltaNp73alpha overexpression on cell growth and the response to anticancer treatment. We generated three different clones overexpressing DeltaNp73alpha under a tetracycline inducible promoter. Immunofluorescent staining and luciferase reporter assays confirmed that clones HCT116/DeltaNA and H1299/DeltaN7 and H1299/DeltaN11 did express a functional, nuclear localized DeltaNp73alpha protein. The stable overexpression of DeltaNp73alpha protein did not confer any cell growth advantage. Doubling time of clones overexpressing DeltaNp73alpha were comparable to counterparts not expressing it. Clonogenic assays showed that the cytotoxic activity of different DNA damaging agents, such as cDDP, UV light and doxorubicin, were comparable in clones expressing DeltaNp73 or not. The overall data argue against an oncogenic role for this isoform. These findings are independent of the p53 status since they overlap with those previously obtained by our group in HCT116 cell lines, wild type for p53

Longitudinal tracking of triple labeled umbilical cord derived mesenchymal stromal cells in a mouse model of Amyotrophic Lateral Sclerosis

The translational potential of cell therapy to humans requires a deep knowledge of the interaction between transplanted cells and host tissues. In this study, we evaluate the behavior of umbilical cord mesenchymal stromal cells (UC-MSCs), labeled with fluorescent nanoparticles, transplanted in healthy or early symptomatic transgenic SOD1G93A mice (a murine model of Amyotrophic Lateral Sclerosis). The double labeling of cells with nanoparticles and Hoechst-33258 enabled their tracking for a long time in both cells and tissues. Whole-body distribution of UC-MSCs was performed by in-vivo and ex-vivo analyses 1, 7, 21 days after single intravenous or intracerebroventricular administration. By intravenous administration cells were sequestered by the lungs and rapidly cleared by the liver. No difference in biodistribution was found among the two groups. On the other hand, UC-MSCs transplanted in lateral ventricles remained on the choroid plexus for the whole duration of the study even if decreasing in number. Few cells were found in the spinal cord of SOD1G93A mice exclusively. No migration in brain parenchyma was observed. These results suggest that the direct implantation in brain ventricles allows a prolonged permanence of cells close to the damaged areas and makes this method of tracking reliable for future studies of efficacy

Early Hemorrhagic Transformation of Brain Infarction: Rate, Predictive Factors, and Influence on Clinical Outcome

Background and Purpose— Early hemorrhagic transformation (HT) is a complication of ischemic stroke but its effect on patient outcome is unclear. The aims of this study were to assess: (1) the rate of early HT in patients admitted for ischemic stroke, (2) the correlation between early HT and functional outcome at 3 months, and (3) the risk factors for early HT. Methods— Consecutive patients with ischemic stroke were included in this prospective study in 4 study centers. Early HT was assessed by CT examination performed at day 5±2 after stroke onset. Study outcomes were 3-month mortality or disability. Disability was assessed using a modified Rankin score (≥3 indicating disabling stroke) by neurologists unaware of the occurrence of HT in the individual cases. Outcomes in patients with and without early HT were compared by χ 2 test. Multiple logistic regression analysis was used to identify predictors for HT. Results— Among 1125 consecutive patients (median age 76.00 years), 98 (8.7%) had HT, 62 (5.5%) had hemorrhagic infarction, and 36 (3.2%) parenchymal hematoma. At 3 months, 455 patients (40.7%) were disabled or died. Death or disability was seen in 33 patients with parenchymal hematoma (91.7%), in 35 patients with hemorrhagic infarction (57.4%) as compared with 387 of the 1021 patients without HT (37.9%). At logistic regression analysis, parenchymal hematoma, but not hemorrhagic infarction, was independently associated with an increased risk for death or disability (OR 15.29; 95% CI 2.35 to 99.35). At logistic regression analysis, parenchymal hematoma was predicted by large lesions (OR 12.20, 95% CI 5.58 to 26.67), stroke attributable to cardioembolism (OR 5.25; 95% CI 2.27 to 12.14) or to other causes (OR 6.77; 95% CI 1.75 to 26.18), high levels of blood glucose (OR 1.01; 95% CI 1.00 to 1.01), and thrombolytic treatment (OR 3.54, 95% CI 1.04 to 11.95). Conclusions— Early HT occurs in about 9% of patients. Parenchymal hematoma, seen in about 3% of patients, is associated with an adverse outcome. Parenchymal hematoma was predicted by large lesions attributable to cardioembolism or other causes, high blood glucose, and treatment with thrombolysis

HGF/C-MET in breast-cancer bone metastasis : characterization and imaging analysis to evaluate the efficacy of traget therapies

cancer exhibits a propensity to metastatize to bone, resulting in debilitating skeletal complications associated with significant morbidity and poor prognosis. The interactions between metastatic cells and bone are critical to the development and progression of bone metastases and their unravelling could lead to novel therapeutic approaches. This PhD thesis intended to investigate the contribution of the HGH/c-Met pathway to the pathogenesis of bone metastasis. An experimental breast cancer bone metastatic model were used, in combination with in vivo imaging techniques, to demonstrate the involvement of HGF/c-Met system in tumor-bone interaction contributing to bone metastatisation. Moreover, the efficacy of three therapeutic approaches able to interfere with the HGC/c-Met pathway has been investigated. Firstly, I demonstrated that NK4, an HGF antagonist, induces a delay in the progression of bone metastasis probably via its bifunctional properties to act as an HGF antagonist and an angiogenesis inhibitor. Secondly, the efficacy of strategies directed against c-Met receptor, including both a c-Met inhibitor, ARQ197, and a specific shRNA, has been evaluated. Dual c-Met inhibition induces a pronounced tumor growth suppression with concomitant marked decreases of lytic lesions and prolongation of survival. Finally, the effect of targeting PLCyl, one of the c-Met downstream effectors, has been explored. Surprisingly, PLCyl silencing by shRNA significantly accelerates the formation and progression of bone metastases. Overall, these findings highlighted the efficacy of HGF/c- Met inhibition in delaying the onset and progression of bone metastases and strongly suggested that targeting the HGF ligand or the c-Met receptor may have promising therapeutic value in the treatment of breast cancer bone metastases. Despite that, special attention should be paid to block PLCyl since its inhibition could potentiate the aggressive phenotype of breast cancer cells stimulating bone metastatic disease. Therefore, PLCyl should not be considered as a therapeutic candidate for the management of bone metastasis.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Combination of the c-Met Inhibitor Tivantinib and Zoledronic Acid Prevents Tumor Bone Engraftment and Inhibits Progression of Established Bone Metastases in a Breast Xenograft Model

<div><p>Bone is the most common metastatic site for breast cancer. There is a significant need to understand the molecular mechanisms controlling the engraftment and growth of tumor cells in bone and to discover novel effective therapeutic strategies. The aim of this study was to assess the effects of tivantinib and Zoledronic Acid (ZA) in combination in a breast xenograft model of bone metastases. Cancer cells were intracardially implanted into immunodeficient mice and the effects of drugs alone or in combination on bone metastasis were evaluated by <i>in vivo</i> non-invasive optical and micro-CT imaging technologies. Drugs were administered either before (preventive regimen) or after (therapeutic regimen) bone metastases were detectable. In the preventive regimen, the combination of tivantinib plus ZA was much more effective than single agents in delaying bone metastatic tumor growth. When administered in the therapeutic schedule, the combination delayed metastatic progression and was effective in improving survival. These effects were not ascribed to a direct cytotoxic effect of the combined therapy on breast cancer cells <i>in vitro</i>. The results of this study provide the rationale for the design of new combinatorial strategies with tivantinib and ZA for the treatment of breast cancer bone metastases.</p></div

gender differences in patients with acute ischemic stroke

2test. Multiple logistic regression analysis was used to identify any independent predictors of outcome. A total of 1136 patients were included in this study; of these, 494 (46%) were female. Women were statistically older compared with men: 76.02 (± 12.93) and 72.68 (± 13.27) median years of age, respectively. At admission, females had higher NIH Stroke Scale scores compared with males (9.4 [± 6.94] vs 7.6 [± 6.28] for men; p = 0.0018). Furthermore, females tended to have more cardioembolic strokes (153 [30%] vs 147 [23%] for men; p = 0.004). Males had lacunar and atherosclerotic strokes more often (146 [29%] vs 249 [39%] for men; p = 0.002, and 68 [13%] vs 123 [19%] for men; p = 0.01, respectively). The mean modified Rankin Scale score at 3 months was also significantly different between genders, at 2.5 (± 2.05) for women and 2.1 (± 2.02) for men (p = 0.003). However, at multivariate analysis, female gender was not an indicator for negative outcome. It was concluded that female gender was not an independent factor for negative outcome. In addition, both genders demonstrated different stroke pathophysiologies. These findings should be taken into account when diagnostic workup and treatment are being planned

<i>In vivo</i> effects of tivantinib and ZA single agent and in combination in a preventive schedule of bone metastases.

<p>(<b>A–B</b>) <i>In vivo</i> bioluminescence imaging (BLI) of 1833/TGL-i.c. injected athymic nude mice (4 week old) treated with tivantinib (120 mg/Kg; <i>n</i> = 7), ZA (100 mg/Kg; <i>n</i> = 5) and tivantinib+ZA (<i>n</i> = 5). (<b>A</b>) Ventral images from a representative nude mouse for each group 24 days after implant. Pseudocolor scale bars are consistent for all images of ventral views, in order to show relative changes at metastatic sites over time. (<b>B</b>) Average growth of bone metastasis in the hindlimbs of controls and treated mice: photon counts from the hindlimb (right and left) regions of tumor-bearing mice were quantified and displayed over time. The data are presented as mean ± SE. **, p<0.005; ***, p<0.0001. (<b>C</b>) The presence of tumor-induced osteolytic lesions were detected by weekly micro-CT scans. Representative 3D reconstruction of micro-CT images of the hindlimbs of controls and treated mice at day 24 after xenografting are reported. Similar results were obtained in the second experiment conducted independently. Red circles indicate osteolysis, the circle’s size was proportional to the extent of bone lesion. Numbers at the bottom of the images represent the number of mice. (<b>D</b>) Kaplan-Meier survival plot of the survival rate of 1833/TGL xenografted controls and treated-mice.</p

Antitumor activity of tivantinib and ZA alone and in combination against subcutaneous breast cancer xenografts.

<p>A breast cancer xenograft model was established using 1833 cells (7.5×10⁶) implanted s.c. into the flanks of female athymic nude mice (6-week old). When tumor volume reached 70 mm³ animals were randomized into 4 groups: tivantinib (300 mg/Kg), ZA (100 mg/Kg), tivantinib+ZA-treated mice and controls treated with vehicle (PEG 400∶20% vit. E TPGS solution [60∶40]). Tivantinib and vehicle were administered p.o. in a volume of 10 mL/kg of body weight daily until the end of the experiment. ZA was administered i.p. every 2 days starting 13 days after implant till the end of the experiment. (<b>A</b>) Tumor growth inhibition. Tumor size was measured using Vernier caliper twice a week until the animals were sacrificed after 27 days of treatment. Tumor weight was calculated by the formula: Tumor weight (mg) = (length×width2)/2. (<b>B</b>) Relative body weight of mice bearing subcutaneous tumor xenografts and treated with vehicle or drugs. Body weights were measured twice weekly. Results are expressed as mean ± SD; <i>n</i> = 9–10.</p

Effect of combination on <i>in vitro</i> cytotoxicity and cell migration.

<p>(<b>A</b>) In vitro cytotoxic activity of tivantinib alone (▪) or in combination with ZA (□) against 1833/TGL cells. Values represent the percentage of controls. (<b>B</b>) 1833/TGL cells were seeded at the concentration of 1×10⁵ cells/mL on a 12-well culture plate in six-well plates. On day 0, for each well, a wound was made in the center of the monolayer of confluent cells with a sterile plastic pipette tip and vehicle or drugs were added. The plate was placed under a motorized inverted microscope and wounds were photographed at 0 (<i>t</i>₀), 24, and 48 hours after wounding. Representative photographs of a filed of view from the different experimental conditions are shown. (<b>C</b>) Quantitative analysis: the level of cell migration was quantified as the percentage of wound closure at each time point after the wound scratch. Values represent averages ± SE of two independent experiments, each consisting of 2 replicates. Statistical analysis were performed at 24 and 48 hours after wounding.</p

Scheme showing the experimental protocol used.

<p>Scheme showing the experimental protocol used.</p