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

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

Advances in transient receptor potential vanilloid-2 channel expression and function in tumor growth and progression.

Aim of this review is to study the role of the TRPV2 channel, a member of the TRPV subfamily of TRP channels, in tumor progression. Physiologically, the triggering of TRPV2 by agonists/activators (e.g., growth factors, hormones and cannabinoids), by inducing TRPV2 translocation from the endosome to the plasmatic membrane, inhibit cell proliferation and induce necrosis and/or apoptosis. Thus, loss or alterations of TRPV2 proliferative and apoptotic signals, results in uncontrolled proliferation and augmented resistance to apoptotic stimuli. For example in prostate cancer cells, the TRPV2 activation following lysophospholipid or adrenomedullin stimulation enhances the invasiveness of cancer cells; furthermore, the increased malignancy of castration-resistant prostate cancer cells was associated with enhanced TRPV2 expression, mainly in metastatic prostate cancer cells. In addition, the TRPV2 cellular functions may also to be related to the presence of TRPV2 variants, able to interfere with the physiological functions of normal TRPV2 channels. In this regard, bladder cancer tumors show loss or reduction of a short TRPV2 variant during cancer progression, with increased malignancy and invasiveness. High expression of TRPV2 was also observed more frequently in esophageal squamous cell carcinoma patients with advanced pT stage, lymph node metastasis and advanced pathological stage

Unveiling the Molecular Mechanisms Driving the Capsaicin-Induced Immunomodulatory Effects on PD-L1 Expression in Bladder and Renal Cancer Cell Lines

The blockade of the PD-L1/PD-1 immune checkpoint has promising efficacy in cancer treatment. However, few patients with bladder cancer (BC) or renal cell carcinoma (RCC) respond to this approach. Thus, it is important to implement a strategy to stimulate the immune anti-tumor response. In this scenario, our study evaluated the effects of a low capsaicin (CPS) dose in BC and RCC cell lines. Western blot, qRT-PCR and confocal microscopy were used to assess PD-L1 mRNA and protein expression. Alterations to the cellular oxidative status and changes to the antioxidant NME4 levels, mRNA modulation of cytokines, growth factors, transcriptional factors and oncogene, and the activation of Stat1/Stat3 pathways were examined using Western blot, cytofluorimetry and qRT-PCR profiling assays. In BC, CPS triggers an altered stress oxidative-mediated DNA double-strand break response and increases the PD-L1 expression. On the contrary, in RCC, CPS, by stimulating an efficient DNA damage repair response, thus triggering protein carbonylation, reduces the PD-L1 expression. Overall, our results show that CPS mediates a multi-faceted approach. In modulating PD-L1 expression, there is a rationale for CPS exploitation as a stimulus that increases BC cells' response to immunotherapy or as an immune adjuvant to improve the efficacy of the conventional therapy in RCC patients

Hypofractionated stereotactic radiotherapy in combination with whole brain radiotherapy for brain metastases

Background The efficacy and toxicity of hypofractionated stereotactic radiotherapy (HSRT) in combination with whole brain radiotherapy (WBRT), for the treatment of 1-4 brain metastases, using a non invasive fixation of the skull, was investigated. Methods Between 04/2001 and 01/2006 30 patients with 44 brain metastases underwent irradiation. Every patient received WBRT (10 x 3 Gy); 41/44 lesions received HSRT boost with a median dose fraction of 6 Gy, the fractionation schemes were 3 x 6 Gy and 4 x 8 Gy; a median total dose of 18 Gy was delivered to the tumor isocenter. Results The median survival period was 9.15 months, the actuarial 1-year overall survival and freedom from new brain metastases were 36.6% and 87.9%, respectively; at univariate analysis Karnofsky Performance Status (KPS) was statistically significant (P = 0.05); the actuarial 1-year local control for the 41/44 lesions was 86.1%. No patient had acute or late complications. Conclusions HSRT as a concomitant boost during WBRT is a safe and well tolerated treatment for selected patients with brain metastases

Analysing the performance of MCECs over a wide range of operating temperatures

Hydrogen production through water electrolysis has gained significant attention in the past years as a means of tackling the problem of the imbalance between the intermittent rate of electricity production from renewable sources and the continuous electricity demand from end users. Recently, much of the effort has been shifted toward the electrolysis of steam rather than water, for example in solid oxide cells, which operate at temperatures around 800°C. In this manner, part of the energy required for the conversion to hydrogen is provided as heat rather than electricity. At the same time, the high temperature levels require the use of highly resistant materials, which increase the overall cost of the process. An interesting alternative is represented by molten carbonate electrolysis cells (MCECs), operating at temperatures well below 700°C. In the present work, a molten carbonate cell was operated in a lower temperature range (490-550°C) by changing the composition of the electrolyte mixture. The data obtained, along with experimental results at higher temperature (570-650°C) available in the literature, was analyzed using a 0D model accounting for Ohmic and activation overpotentials to determine the correlation between current and potential. It was found that, while the dependence of Ohmic losses on temperatures is discontinuous when cell operation is switched from the lower to the higher temperature range, activation losses vary with continuity. This result provides important insight on the performance of MCECs that can serve as a basis for future studies

Mitochondrial proteomics: analysis of a whole mitochondrial extract with two-dimensional electrophoresis

Mitochondria are complex organelles, and their proteomics analysis requires a combination of techniques. The emphasis in this chapter is made first on mitochondria preparation from cultured mammalian cells, then on the separation of the mitochondrial proteins with two-dimensional electrophoresis (2DE), showing some adjustment over the classical techniques to improve resolution of the mitochondrial proteins. This covers both the protein solubilization, the electrophoretic part per se, and the protein detection on the gels, which makes the interface with the protein identification part relying on mass spectrometry

Evaluation of anti-inflammatory and immunoregulatory activities of Stimunex® and Stimunex D3® in human monocytes/macrophages stimulated with LPS or IL-4/IL-13

Macrophages exert an important role in maintaining and/or ameliorating the inflammatory response. They are involved in the activation of an immune response to pathogens, with a balance between the immunomodulatory role and tissue integrity maintenance, however, excessive macrophage activity promotes tissue injury and chronic disease pathogenesis. There is a high interest in evaluating the anti-inflammatory properties of new botanical preparations. Stimunex® and Stimunex D3® are two food supplements formulated as syrups, containing the extract of elderflower (Sambucus nigra, Caprifoliaceae), standardized in polyphenol (6%) and anthocyanins (4%), associated with wellmune WGP® β-glucan, with the addiction of vitamin D3 (in Stimunex D3® formulation). The aim of the work was the evaluation of Stimunex® and Stimunex D3® activity in human polarized-macrophages, in order to support their use as supplement for preventing and reducing the inflammatory processes. In primary human stimulated macrophages, both syrups were able to revert LPS- and IL-4/IL-13-mediated response, reducing the release of several pro-inflammatory cytokines. Results support that these standardized botanical preparations fortified with β-glucan, may have a potential use in the prevention and coadjuvant management of inflammatory process as respiratory recurrent infections and other similar conditions. Moreover, the addition of vitamin D3 revealed to be an advantage in Stimunex D3® for its important role in maintaining and enhancing the innate immune response