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

Effect of LPS-induced inflammatory state on some aspects of reproductive function of rabbit does

Effetto dell\u2019infiammazione indotta con LPS su alcuni aspetti della funzione riproduttiva nelle coniglie. Scopo della ricerca \ue8 stato quello di studiare un modello d\u2019induzione sperimentale di infiammazione con lipopolisaccaridi (LPS) microbici nella coniglia fattrice e l'eventuale effetto sulla risalita degli spermatozoi. Due gruppi di 6 coniglie fattrici sono state inoculate per via intra-peritoneale rispettivamente con LPS di E. coli 0127:B8 (100 \ub5g/kg peso vivo), o con soluzione fisiologica (controllo). Sono stati rilevati per 72 ore temperatura rettale e il numero dei leucociti; dopo inseminazione artificiale \ue8 stata valutata la risalita degli spermatozoi nel tratto riproduttivo femminile e la situazione ovarica. L\u2019infiammazione sperimentale ha indotto un rilevante incremento della temperatura rettale e sostanziali modifiche a livello di leucociti che sono comunque scomparse entro 72 ore. Anche il numero di spermatozoi risaliti \ue8 stato significativamente pi\uf9 basso a livello di corna uterine e addirittura nullo a livello dell\u2019ovidutto. In conclusione si pu\uf2 affermare che \ue8 possibile costruire un modello d\u2019induzione dello stato infiammatorio nella coniglia mediante inoculazione intra-peritoneale di 100 \ub5g LPS/kg di peso vivo.The efficiency of the cycled production system in rabbit farms is greatly conditioned by the fertility rate of does. Nulliparous does generally exhibit high fertility rate (Castellini et al., 1998), whereas the reproductive performances of multiparous does goes down. One reason of this reduction is related to the use of intensive reproductive rhythms which implies an overlapping between lactation and insemination which often produces a severe energy deficit. As in other mammals, lactation shows a strong hormonal antagonism with the reproductive activity. An other cause of hypo-fertility depends on the sanitary condition of does. Genital tract inflammation and/or infection is one of the major causes of infertility (Gram et al., 2002) and often determined by incorrect practices of artificial insemination (AI). It has been demonstrated that uterine infection negatively affects fertility (Facchin et al., 1999) and prolongs the life span of corpora lutea (Boiti et al., 1999) due to uterine leukocytes infiltration, reduced prostaglandins synthesis and increased spermatozoa reabsorption. Lipopolysaccarides (LPS), constituents of the Gram-negative germ wall, are potent stimulators of prostaglandins synthesis and are widely used to simulate inflammation in several district and organs. The aim of the paper was to verify the effect of an LPS-induced inflammatory state on some aspects of reproductive function of non-lactating rabbit does

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

Food restriction during pregnancy in rabbits: effects on hormones and metabolites involved in energy homeostasis and metabolic programming

This study examined the effects of food restriction during rabbit pregnancy on hormones and metabolites involved in energy homeostasis and metabolic programming. Pregnant does were assigned to four groups: the control group was fed a standard ration while the others received a restricted amount of food (30% restriction) during early (0-9 days), mid (9-18 days), and late (19-28 days) pregnancy. The pregnancy induced a coordinated range of adaptations to fulfil energy requirements of both mother and foetus, such as hyperleptinaemia and hyperinsulinaemia, reduced insulin sensitivity, increased cortisol and non-esterified fatty acid. Food restriction altered leptin, insulin, T3, non-esterified fatty acids and glucose concentrations depending on the gestational phase in which it was applied. Collectively, present data confirm that the endocrinology of pregnancy and the adaptive responses to energy deficit make the rabbit an ideal model for studying nutritional-related disorders and foetal programming of metabolic disease. (C) 2014 Elsevier Ltd. All rights reserved