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

Termostabilnost topljivih i vezanih peroksidaza iz artičoke te matematički model njihove kinetike inaktivacije

Soluble peroxidases (SP), and ionically (IBP) and covalently (CBP) bound peroxidases were extracted from leaves and edible part of artichoke heads (heart). The peroxidase (POD) forms showed a characteristic electrophoretic pattern; in particular SP and IBP forms showed nearly the same pattern, while CBP was quite different. Several basic and acid POD isoforms were present in SP and CBP forms, whereas in IBP only basic components were found. The thermal stability of different POD forms was tested at different temperatures (70, 80, 90 and 100 °C). The leaf POD forms showed greater heat stability compared to the head ones. The heat sensitivity of three POD forms was different: the bound forms were characterized by a greater heat stability than the soluble form. A series-type mathematical model has been developed to describe the inactivation kinetics of artichoke POD. The activation energies for the three POD forms of artichoke leaves and hearts were estimated by an Arrhenius-type relationship. The second step of the inactivation requires more energy to be carried out in all POD forms, especially in SP and IBP.Iz lišća i jestivog dijela glavice artičoke ekstrahirane su topljive te ionski i kovalentno vezane peroksidaze. Elektroforezom je dobivena karakteristična slika, pri čemu su topljive i ionski vezane peroksidaze imale gotovo isti, a kovalentno vezane sasvim drukčiji raspored. U topljivim i kovalentno vezanim peroksidazama nađeno je nekoliko kiselih i lužnatih izoformi, a u ionski vezanim samo lužnate. Toplinska stabilnost pojedinih peroksidaza ispitana je pri različitim temperaturama (70, 80, 90 i 100 °C). Izoforme iz lišća pokazale su veću termostabilnost od onih iz glavice artičoke. Vezani oblici peroksidaza bili su stabilniji od topljivih. Razvijen je matematički model (series-type mathematical model) za opisivanje kinetike inaktivacije peroksidaza artičoke. Energija aktivacije triju peroksidaza iz lišća i glavice artičoke procijenjena je pomoću Arrheniusova modela, pa je zaključeno da je potrebno utrošiti više energije za provođenje drugog koraka inaktivacije u svim peroksidazama, a osobito u topljivim i ionski vezanim

Cereal foods fortified with by-products from the olive oil industry

Abstract The oil industry produces large volume of waste, olive mill waste water (OMWW) and olive paste (OP), which represents a disposal and a potential environmental pollution problem. They are also promising sources of valuable compounds that can be recovered and used. The effects of OMWW and OP addition to bread and pasta, separately and combined, were studied. Both sensory and chemical properties as related to phenols content and antioxidant activity of raw materials, and fortified bread and pasta were evaluated. Results suggested that the enrichment of bread and pasta with OMWW slightly improved the chemical quality without compromising the sensory properties. While, foods enrichment with OP had considerably improved chemical quality, the sensory acceptability was worse due to the bitter and spicy taste of OP. To choose the best cereal food between bread and pasta to be enriched, a mathematical model, the whole quality index (WQI), was used. Bread was better than pasta for re-using olive oil by-products. Between OMWW and OP, the latter was more suitable for food fortification, despite the sensory problems of the enriched product. Also, when the two by-products were combined, the best product continued to be the bread

Green bean biofortification for Si through soilless cultivation: Plant response and Si bioaccessibility in pods

Food plants biofortification for micronutrients is a tool for the nutritional value improvement of food. Soilless cultivation systems, with the optimal control of plant nutrition, represent a potential effective technique to increase the beneficial element content in plant tissues. Silicon (Si), which proper intake is recently recommended for its beneficial effects on bone health, presents good absorption in intestinal tract from green bean, a high-value vegetable crop. In this study we aimed to obtain Si biofortified green bean pods by using a Si-enriched nutrient solution in soilless system conditions, and to assess the influence of boiling and steaming cooking methods on Si content, color parameters and Si bioaccessibility (by using an in vitro digestion process) of pods. The Si concentration of pods was almost tripled as a result of the biofortification process, while the overall crop performance was not negatively influenced. The Si content of biofortified pods was higher than unbiofortified also after cooking, despite the cooking method used. Silicon bioaccessibility in cooked pods was more than tripled as a result of biofortification, while the process did not affect the visual quality of the product. Our results demonstrated that soilless cultivation can be successfully used for green bean Si biofortification

Biokemijska svojstva topljivih i vezanih peroksidaza ekstrahiranih iz glavica i lišća artičoke

Soluble (SP), ionically bound (IBP) and covalently bound (CBP) peroxidases (POD) from artichoke leaves and heads have been characterized for the main biochemical parameters. The three PODs, in both leaves and heads, showed the major apparent catalytic efficiency (vmax,app/Km,app) towards ferulic acid, even though, in some cases, they showed higher affinity (Km,app) for other substrates. In leaves, SP and IBP showed higher Km,app for ferulic and chlorogenic acids, and CBP for ferulic and caffeic acids. In heads, SP showed higher Km,app for chlorogenic acid, IBP for caffeic and ferulic acids, and CBP for ferulic acid. It was shown that pH optimum for PODs ranged between 5.0 and 6.0 in leaves. In heads, pH optimum for SP and IBP was 5.5, while CBP presented a very low activity in a wide pH range. All PODs showed high thermal stability but different ability to regenerate: the bound forms were more able to regenerate than the soluble one. The results obtained show that (i) CBP from heads is able to work under very different cellular conditions, (ii) all PODs, in both tissues, have a high apparent catalytic efficiency for ferulic acid, which could explain the effective involvement of POD in lignin biosynthesis, (iii) in heads, high Km,app of SP for chlorogenic acid, particularly abundant in artichoke, could justify the possible involvement of PODs in browning mechanism, and (iv) in heat-processed artichoke, the ability of PODs to regenerate could contribute to oxidation and loss of product quality.Okarakterizirana su mnoga biokemijska svojstva topljivih, ionski i kovalentno vezanih peroksidaza ekstrahiranih iz lišća i glavica artičoke. Sva tri oblika peroksidaza, iz lišća i glavica, pokazala su veliku prividnu katalitičku djelotvornost (vmax,app/Km,app) prema feruličnoj kiselini, iako su u nekoliko slučajeva imala veći afinitet (Km,app) prema drugim supstratima. Topljive i ionski vezane peroksidaze iz lišća artičoke imale su veći afinitet za feruličnu i klorogenu kiselinu, a kovalentno vezane za feruličnu i kafeinsku kiselinu. Topljive peroksidaze iz glavica artičoke pokazale su veći afinitet prema klorogenoj kiselini, ionski vezane prema kafeinskoj i feruličnoj, a kovalentno vezane peroksidaze samo prema feruličnoj kiselini. Optimalna pH-vrijednost za aktivnost peroksidaza iz lišća bila je od 5 do 6, a za aktivnost topljivih i ionski vezanih peroksidaza iz glavica 5,5. Kovalentno vezane peroksidaze ekstrahirane iz glavica artičoke imale su malu aktivnost pri svim pH-vrijednostima. Sve su peroksidaze imale veliku termostabilnost, ali različitu sposobnost regeneracije: vezani su se oblici bolje regenerirali od topljivih oblika. Iz dobivenih rezultata izvedeni su sljedeći zaključci: (i) kovalentno vezane peroksidaze iz glavica djeluju u različitim staničnim uvjetima, (ii) sve ekstrahirane peroksidaze imaju veliku prividnu katalitičku djelotvornost prema feruličnoj kiselini, što objašnjava ulogu peroksidaza u biosintezi lignina, (iii) veliki afinitet topljive peroksidaze iz glavica prema klorogenoj kiselini, osobito zastupljenoj u artičokama, mogao bi objasniti ulogu peroksidaza u posmeđivanju glavica i (iv) sposobnost regeneracije peroksidaza uzrokuje oksidaciju i gubitak kakvoće artičoke tijekom kuhanja

An Integrated Caco-2TC7cells/biosensors Device for the Real Time Monitoring of Intestinal Glucose and Polyphenols Absorption and Hypoglycemic Effect of Phytochemicals

Abstract An integrated device, for real-time monitoring of glucose and phenols absorption, that consists of a sensors/biosensors system (SB) and a Caco-2TC7 human intestinal cell culture, is shown here. The SB was made of a glucose oxidase-based biosensor, a sentinel platinum sensor, a laccase/tyrosinase-based biosensor and a sentinel carbon sensor located in the basolateral compartment (BC) of a cell culture plate. This system was able to monitor the glucose absorption and the hypoglycemic effect induced by different polyphenols and could be proposed to provide an effective strategy to manage postprandial hyperglycemia with natural compounds

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

Post-transcriptional regulation of 5'-untranslated regions of human Transient Receptor Potential Vanilloid type-1 (TRPV-1) channels: role in the survival of glioma patients

The Transient Receptor Potential Vanilloid type-1 (TRPV1) channel is a non-selective cation channel belonging to the Transient Receptor Potential family; variation of its expression has been correlated to glioma progression. In human, TRPV1 transcripts display a remarkable homogeneity differing only for the 5'-untranslated region (5'UTR) sequence that generates four variants encoding the same protein. Herein, we investigated the role of the 5'UTR sequences in TRPV1 transcripts stability, regulation of translation, expression in glioma cells and tissues. In addition, the expression of 5'UTR TRPV1 variants as prognostic factor in the survival of glioblastoma patients was evaluated. The expression level for each 5'UTR and their stability was evaluated by RT-PCR analysis. The effect of rapamycin and interferon-gamma in 5'UTR-regulating TRPV1 translation was determined by western blot analysis in glioma cell lines. We demonstrated that the 5'UTR influences the stability and translation efficacy of TRPV1 transcripts, and that TRPV1 variant three (TRPV1v3) was the most stable and the only variant expressed in GBM samples and in glioma stem-like cells. Furthermore, we found that TRPV1v3 expression levels correlate with patient's survival, suggesting that it may represent a potential prognostic marker for patients with glioma