title: Axitinib induces DNA damage response leading to senescence, mitotic catastrophe, and increased NK cell recognition in human renal carcinoma cells.
abstract: 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.
authors.affiliation: School of Pharmacy, Experimental Medicine Section, University of Camerino, Camerino, Italy.|Department of Molecular Medicine, Sapienza University, Rome, Italy.
authors.forename: Maria Beatrice
authors.initials: MB
authors.lastname: Morelli
authors.affiliation: School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, Italy.
authors.forename: Consuelo
authors.initials: C
authors.lastname: Amantini
authors.affiliation: Department of Medical Oncology, AOU Ospedali Riuniti, Polytechnic University of the Marche Region, Ancona, Italy.
authors.forename: Matteo
authors.initials: M
authors.lastname: Santoni
authors.affiliation: Department of Molecular Medicine, Sapienza University, Rome, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Soriani
authors.affiliation: School of Pharmacy, Experimental Medicine Section, University of Camerino, Camerino, Italy.
authors.forename: Massimo
authors.initials: M
authors.lastname: Nabissi
authors.affiliation: School of Pharmacy, Experimental Medicine Section, University of Camerino, Camerino, Italy.|Department of Molecular Medicine, Sapienza University, Rome, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Cardinali
authors.affiliation: Department of Molecular Medicine, Sapienza University, Rome, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Santoni
authors.affiliation: School of Pharmacy, Experimental Medicine Section, University of Camerino, Camerino, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Santoni
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D000077784:Axitinib; D002292:Carcinoma, Renal Cell / Q000188:drug therapy* / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D016922:Cellular Senescence / Q000187:drug effects; D004249:DNA Damage; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D007191:Indazoles / Q000494:pharmacology*; D007680:Kidney Neoplasms / Q000188:drug therapy* / Q000235:genetics / Q000276:immunology; D007694:Killer Cells, Natural / Q000187:drug effects / Q000276:immunology; D008938:Mitosis / Q000187:drug effects / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000008:administration & dosage / Q000627:therapeutic use*
chemical_list: D007093:Imidazoles; D007191:Indazoles; D047428:Protein Kinase Inhibitors; D000077784:Axitinib
keywords: NKG2D ligands; cell senescence; mitotic catastrophe; renal cell carcinoma; tyrosine kinase inhibitors