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

// Maria Beatrice Morelli 1,2 , Consuelo Amantini 3 , Matteo Santoni 4 , Alessandra Soriani 2 Massimo Nabissi 1 , Claudio Cardinali 1,2 , Angela Santoni 2 and Giorgio Santoni 1 1 School of Pharmacy, Experimental Medicine Section, University of Camerino, Camerino, Italy 2 Department of Molecular Medicine, Sapienza University, Rome, Italy 3 School of Biosciences and Veterinary Medicine, University of Camerino, Camerino, Italy 4 Department of Medical Oncology, AOU Ospedali Riuniti, Polytechnic University of the Marche Region, Ancona, Italy Correspondence to: Matteo Santoni, email: // Keywords : renal cell carcinoma, tyrosine kinase inhibitors, cell senescence, mitotic catastrophe, NKG2D ligands Received : August 07, 2015 Accepted : September 12, 2015 Published : October 14, 2015 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.