Axitinib induces DNA damage response leading to senescence, mitotic catastrophe, and increased NK cell recognition in human renal carcinoma cells.

Tyrosine kinase inhibitors (TKIs) including axitinib have been introduced in the treatment of renal cell carcinoma (RCC) because of their anti-angiogenic properties. However, no evidence are presently available on a direct cytotoxic anti-tumor activity of axitinib in RCC.Herein we reported by western blot analysis that axitinib treatment induces a DNA damage response (DDR) initially characterized by γ-H2AX phosphorylation and Chk1 kinase activation and at later time points by p21 overexpression in A-498 and Caki-2 RCC cells although with a different potency. Analysis by immunocytochemistry for the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA and flow cytometry using the redox-sensitive fluorescent dye DCFDA, demonstrated that DDR response is accompanied by the presence of oxidative DNA damage and reactive oxygen species (ROS) generation. This response leads to G2/M cell cycle arrest and induces a senescent-like phenotype accompanied by enlargement of cells and increased senescence-associated β-galactosidase activity, which are abrogated by N-acetyl cysteine (NAC) pre-treatment. In addition, axitinib-treated cells undergo to cell death through mitotic catastrophe characterized by micronucleation and abnormal microtubule assembly as assessed by fluorescence microscopy.On the other hand, axitinib, through the DDR induction, is also able to increase the surface NKG2D ligand expression. Accordingly, drug treatment promotes NK cell recognition and degranulation in A-498 RCC cells in a ROS-dependent manner.Collectively, our results indicate that both cytotoxic and immunomodulatory effects on RCC cells can contribute to axitinib anti-tumor activity

Capsaicin triggers autophagic cell survival which drives epithelial mesenchymal transition and chemoresistance in bladder cancer cells in an Hedgehog-dependent manner

Bladder cancer (BC) is a common urologic tumor characterized by high risk of recurrence and mortality. Capsaicin (CPS), used as an intravesical drug for overactive bladder, was demonstrated to induce cell death in different cancer cells including BC cells.Here we found that treatment of high-grade BC cells with high dose of CPS triggers autophagy. Infact, the CPS treatment alters the redox homeostasis by inducing production of radicals, mitochondrial depolarization, alterations of ADP/ATP ratio and activation of AMPK pathway stimulating the autophagic process in BC cells. The inhibition of autophagy, by using the specific inhibitor bafilomycin A or Beclin 1 knock-down, enhanced the CPS-induced cell death, demonstrating that CPS-induced autophagy acts as a pro-survival process in BC cells. By using PCR arrays and FACS analysis, we found that the CPS-treated BC cells displayed typical mesenchymal features of the epithelial mesenchymal transition (EMT) as elongated shape and over-expression of vimentin, α5 and β1 integrin subunits, integrin-like kinase and the anti-apoptotic Bcl-2 proteins. Moreover, we demonstrated that CPS treatment stimulates upregulation of Dhh/Ptch2/Zeb2 members of the Hedgehog signaling pathway, increases CD24, VEGFA and TIMP1 and decreases CD44 and ALCAM mRNA expression levels. By PTCH2 knock-down we found that the Hedgehog signaling pathway is involved in the CPS-induced autophagy and EMT phenotype.Finally, we also showed that the CPS-resistant EMT-positive BC cells displayed an increased drug-resistance to the cytotoxic effects of mitomycin C, gemcitabine and doxorubicine drugs commonly used in BC therapy

Axitinib induces senescence-associated cell death and necrosis in glioma cell lines: The proteasome inhibitor, bortezomib, potentiates axitinib-induced cytotoxicity in a p21(Waf/Cip1) dependent manner.

Glioblastoma is associated with a poor overall survival despite new treatment advances. Antiangiogenic strategies targeting VEGF based on tyrosine kinase inhibitors (TKIs) are currently undergoing extensive research for the treatment of glioma. Herein we demonstrated that the TKI axitinib induces DNA damage response (DDR) characterized by γ-H2AX phosphorylation and Chk1 kinase activation leading to G2/M cell cycle arrest and mitotic catastrophe in U87, T98 and U251 glioma cell lines. Moreover, we found that p21(Waf1/Cip1) increased levels correlates with induction of ROS and senescence-associated cell death in U87 and T98 cell lines, which are reverted by N-acetyl cysteine pretreatment. Conversely, U251 cell line showed a resistant phenotype in response to axitinib treatment, as evidenced by cell cycle arrest but no sign of cell death. The combinatorial use of axitinib with other therapies, with the aim of inhibiting multiple signaling pathways involved in tumor growth, can increase the efficiency of this TKI. Thus, we addressed the combined effects of axitinib with no toxic doses of the proteasome inhibitor bortezomib on the growth of U87 and T98 axitinib- sensitive and axitinib-resistant U251 cell lines. Compared to single treatments, combined exposure was more effective in inhibiting cell viability of all glioma cell lines, although with different cell death modalities. The regulation of key DDR and cell cycle proteins, including Chk1, γ-H2AX and p21(Waf1/Cip1) was also studied in glioma cell lines. Collectively, these findings provide new perspectives for the use of axitinib in combination with Bortezomib to overcome the therapy resistance in gliomas

MicroRNA-222 regulates muscle alternative splicing through Rbm24 during differentiation of skeletal muscle cells

A number of microRNAs have been shown to regulate skeletal muscle development and differentiation. MicroRNA-222 is downregulated during myogenic differentiation and its overexpression leads to alteration of muscle differentiation process and specialized structures. By using RNA-induced silencing complex (RISC) pulldown followed by RNA sequencing, combined with in silico microRNA target prediction, we have identified two new targets of microRNA-222 involved in the regulation of myogenic differentiation, Ahnak and Rbm24. Specifically, the RNA-binding protein Rbm24 is a major regulator of muscle-specific alternative splicing and its downregulation by microRNA-222 results in defective exon inclusion impairing the production of muscle-specific isoforms of Coro6, Fxr1 and NACA transcripts. Reconstitution of normal levels of Rbm24 in cells overexpressing microRNA-222 rescues muscle-specific splicing. In conclusion, we have identified a new function of microRNA-222 leading to alteration of myogenic differentiation at the level of alternative splicing, and we provide evidence that this effect is mediated by Rbm24 protei

Remote landslide mapping using a laser rangefinder binocular and GPS

We tested a high-quality laser rangefinder binocular coupled with a GPS receiver connected to a Tablet PC running dedicated software to help recognize and map in the field recent rainfall-induced landslides. The system was tested in the period between March and April 2010, in the Monte Castello di Vibio area, Umbria, Central Italy. To test the equipment, we measured thirteen slope failures that were mapped previously during a visual reconnaissance field campaign conducted in February and March 2010. For reference, four slope failures were also mapped by walking the GPS receiver along the landslide perimeter. Comparison of the different mappings revealed that the geographical information obtained remotely for each landslide by the rangefinder binocular and GPS was comparable to the information obtained by walking the GPS around the landslide perimeter, and was superior to the information obtained through the visual reconnaissance mapping. Although our tests were not exhaustive, we maintain that the system is effective to map recent rainfall induced landslides in the field, and we foresee the possibility of using the same (or similar) system to map landslides, and other geomorphological features, in other areas

The Use of Premixed Calcium Silicate Bioceramic Sealer with Warm Carrier-Based Technique: A 2-Year Study for Patients Treated in a Master Program

Background: Recently several calcium silicate flowable sealers have been introduced as endodontic materials for the root canal. This clinical study tested the use of a new premixed calcium silicate bioceramic sealer in association with the Thermafil warm carrier-based technique (TF). Epoxy-resin-based sealer with the warm carrier-based technique was the control group. Methodology: Healthy consecutive patients (n = 85) requiring 94 root canal treatments were enrolled in this study and assigned to one filling group (Ceraseal-TF n = 47, AH Plus-TF n = 47) in accordance with operator training and best clinical practice. Periapical X-rays were taken preoperatively, after root canal filling and after 6, 12 and 24 months. Two evaluators blindly assessed the periapical index (PAI) and sealer extrusion in the groups (k = 0.90). Healing rate and survival rate were also evaluated. Chi-square tests was used to analyze significant differences between the groups. Multilevel analysis was performed to evaluate the factors associated with healing status. Results: A total of 89 root canal treatments in 82 patients were analyzed at the end-line (24 months). The total drop-out was 3.6% (3 patients; 5 teeth). A total of 91.1% of healed teeth (PAI 1-2) was observed in Ceraseal-TF, with 88.6% in AH Plus-TF. No significant difference was observed on healing outcome and survival among the two filling groups (p > 0.05). Apical extrusion of the sealers occurred in 17 cases (19.0%). Of these, 6 occurred in Ceraseal-TF (13.3%) and 11 in AH Plus-TF (25.0%). Three Ceraseal extrusions were radiographically undetectable after 24 months. All the AH Plus extrusions did not change during the evaluation time. Conclusions: The combined use of the carrier-based technique and premixed CaSi-based bioceramic sealer showed clinical results comparable with carrier-based technique and epoxy-resin-based sealer. The radiographical disappearance of apically extruded Ceraseal is a possible event in the first 24 months

Ovulation induction in rabbit does submitted to artificial insemination by adding lecirelin to the semen dose: preliminary results

The aim of this study was to analyze the effect of intravaginal administration of lecirelin on ovulation induction in rabbit does. Eighty pluriparous does (Martini genetic strain) were submitted to AI using a seminal dose of 0.5 ml containing about 10 million sperms. To stimulate ovulation, 4 homogeneous groups were submitted to different treatments: Control Group: 0.2 ml intramuscular administration of lecirelin (Dalmarelin, Fatro\uae); 0.2 Group: 0.2 ml intravaginal administration of lecirelin; 0.6 Group: 0.6 ml intravaginal administration of lecirelin; 2.0 Group: 2 ml intravaginal administration of lecirelin. In groups receiving an intra-vaginal administration, 25 \ub5g/ml Dalmarelin was diluted in the seminal dose using benzilic alcohol (20 mg/ml) as excipient. Blood samples were collected from all females, to determine LH prior (-60, -30 and 0 minutes) and (30, 60, 90, 120 and 180 minutes) after AI, and progesterone once a week for 4 weeks. After 7 days from AI, 10 does per group were euthanized in order to analyze the ovarian status. The does of control group showed a high LH peak after 30 minutes from AI; whereas intra-vaginal administration of 0.2 and 0.6 ml determined a lower increase of LH blood concentration after 2 hours. The highest dose did not produce any LH or progesterone increase. The ovary status showed a higher number of corpora lutea in Control group (P<0.05), followed by 0.2 and 0.6 ones, whereas embryos were recorded only in Control and 0.2 groups. The unsuccessful of the other experimental groups could be ascribed to the negative effect of benzilic alcohol on seminal characteristic. Only 30% of 0.2 group does were pregnant and the prolificacy was 8 kits/doe. Compared to the control group, the progesterone concentration in pregnant does showed lower value in 0.2 group. In conclusion, in spite of the obtained results, it will be necessary to test different Dalmarelin formulations (lower volume, different excipient) to recommend the minimal dose to inject intravaginally for inducing ovulation

Native immunity and oxidative traits of growing rabbits

[EN] The evaluation of animal welfare through innate immunity (Serum Bactericidal Activity - SBA, Hemolytic Complement Assay - HCA, lysozyme) and the antioxidant status of the body (Reactive Oxygen Substances - ROS and Antioxidant Power of plasma, AP) offers a reliable prognostic and diagnostic tool. The aim of the present study was to investigate trends and correlations between some traits of innate immunity and the oxidative status of fattening rabbits at different ages. Blood samples from 120 New Zealand White fattening rabbits at 45, 55, 65, and 75 d of age were collected and analyzed. The results showed that SBA did not have a normal distribution because of numerous 0 values. Data distribution was normal when only SBA > 0 values were considered. Lysozyme (mean value 27.19 microg/mL) and HCA (mean value 50.84 CH50% ) had stable trends at different ages and showed a tendency that was comparable to that obtained in other animal species. On the contrary, SBA (mean value 42.15%) showed an unexpected positive correlation with lysozyme (P<0.001) and a negative correlation with HCA (P<0.001). Oxygen free-radicals are involved in the pathogenesis of several diseases and oxidative stress alters immune competence. In this experiment, ROS and AP showed mean values of 0.60 mmol H2 O2 and 421.67 micromol HClO, respectively. In this context positive correlation coefficients between oxidative status traits and immune traits (P<0.001) were found, although at a very low level; and surprisingly, only ROS and SBA did not show any significant correlation. In this study it emerged that, even in the absence of evident pathologies, the immune and oxidative traits of fattening rabbits could be affected by environmental stress (weaning, cage, neighbors)Funded by Ricerca Corrente 2006 Istituto Zooprofilattico Sperimentale dell’Umbria e MarcheMoscati, L.; Dal Bosco, A.; Battistacci, L.; Cardinali, R.; Mugnai, C.; Castellini, C. (2008). Native immunity and oxidative traits of growing rabbits. World Rabbit Science. 16(4). doi:10.4995/wrs.2008.616SWORD16