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.
Highlights
Renal cell carcinoma (RCC) accounts for 2-3% of all malignancies, with approximately 84,400 new cases and 34,700 cancer-related deaths in Europe in 2013 [1]
Axitinib triggers DNA damage response (DDR) associated with oxidative DNA damage in RCC cells To evaluate whether axitinib treatment could trigger DDR in RCC cells, we initially investigated the presence of γ-H2AX (H2AX), a phosphorylated variant of histone 2A that is associated with DNA double-strand breaks [36]
Since chemotherapeutic agents through the activation of the DDR have been shown to enhance the expression of NKG2D and DNAX accessory molecule-1 (DNAM-1) ligands on human multiple myeloma cells in a reactive oxygen species (ROS)-dependent manner [18], we investigated whether axitinib treatment could modulate the expression of the ligands for NKG2D and DNAM-1 activating natural killer (NK) receptors on A-498 and Caki-2 RCC lines, and the involvement of ROS signaling in this event
Summary
Renal cell carcinoma (RCC) accounts for 2-3% of all malignancies, with approximately 84,400 new cases and 34,700 cancer-related deaths in Europe in 2013 [1]. Tyrosine kinase inhibitors (TKIs) targeting vascular endothelial growth factor receptor (VEGFR), such as sunitinib, sorafenib, pazopanib and axitinib, the anti-VEGF antibody bevacizumab and the mammalian target of rapamycin (mTOR) inhibitors everolimus and temsirolimus, have www.impactjournals.com/oncotarget been sequentially approved by the US Food and Drug Administration (FDA) [5,6,7,8,9,10,11,12]. Axitinib is a potent and selective inhibitor of VEGFR 1, 2, and 3 approved by FDA in 2012 for the treatment of patients with metastatic RCC (mRCC) after failure of one prior systemic therapy. The European Medicines Agency has approved the use of axitinib in 2015 for the treatment of advanced renal carcinoma after failure of prior treatment with sunitinib or interleukin 2 (IL-2) [13]. Its use as first-line therapy for advanced or mRCC was reported [14, 15]
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