N-Acetyl-L-Cysteine (NAC) Blunts Axitinib-Related Adverse Effects in Preclinical Models of Glioblastoma

Abstract

Objective: Axitinib is a tyrosine kinase inhibitor characterized by a strong affinity for Vascular Endothelial Growth Factor Receptors (VEGFRs). It was approved in 2012 by Food and Drug Administration and European Medicines Agency as a second line treatment for advanced renal cell carcinoma and is currently under evaluation in clinical trial for the treatment of other cancers. Glioblastoma IDH-wild type (GBM) is a highly malignant brain tumor characterized by diffusely infiltrative growth pattern and by a prominent neo-angiogenesis. In GBM, axitinib has demonstrated a limited effectiveness as a monotherapy, while it was recently shown to significantly improve its efficacy in combination treatments. In preclinical models, axitinib has been reported to trigger cellular senescence both in tumor as well as in normal cells, through a mechanism involving intracellular reactive oxygen species (ROS) accumulation and activation of Ataxia Telangiectasia Mutated kinase (ATM). Limiting axitinib-dependent ROS increase by antioxidants prevents senescence specifically in normal cells, without affecting tumor cells.

Methods: We used brain tumor xenografts obtained by engrafting Glioma Stem Cells (GSCs) into the brain of immunocompromised mice, to investigate the hypothesis that the antioxidant molecule N-Acetyl-L-Cysteine (NAC) might be used to reduce senescence-associated adverse effects of axitinib treatment without altering its anti-tumor activity.

Results: We demonstrate that the use of the antioxidant molecule N-Acetyl-Cysteine (NAC) in combination with axitinib stabilizes tumor microvessels in GBM tumor orthotopic xenografts, eventually resulting in vessel normalization, and protects liver vasculature from axitinib-dependent toxicity.

Conclusion: Overall, we found that NAC co-treatment allows vessel normalization in brain tumor vessels and exerts a protective effect on liver vasculature, therefore minimizing axitinib-dependent toxicity.

Keywords: N‐acetyl‐L‐cysteine; axitinib; brain tumor xenograft; endothelium; glioblastoma IDH‐wild type; glioma stem cells; therapy; toxicity.

FIGURE 1

Axitinib triggers cell senescence and ROS accumulation in GSC. (A) Axitinib triggers senescence in GSC#1 and GSC#61, as addressed by SA‐β‐gal staining 4 and 7 days posttreatment, respectively. The percentage of SA‐β‐gal‐positive cells increases with axitinib and axitinib + NAC cotreatment. Magnification 20× , scale bar 100 μm. (B) Real‐time PCR experiments on GSC#1 and GSC#61 cotreated with axitinib and NAC show that ROS buffering by NAC do not prevent axitinib‐dependent gene expression change in LMNB1, a well‐recognized marker of cellular senescence. Relative quantities were calculated normalizing for TBP and are given relative to control (untreated). n = 3 biological replicates, *p < 0.05; **p < 0.01; ***p < 0.001. (C) GSCs were treated with axitinib or axitinib + NAC, stained with the redox‐sensitive fluorescent dye DCFHDA and analyzed by plate reader 3 days postdrug treatment. A significant increase in ROS‐associated fluorescence was observed in the presence of axitinib. NAC cotreatment protects cells from axitinib‐induced oxidative stress.

FIGURE 2

Axitinib effect on GSC‐derived 3D spheroid cultures. (A) 3D tumor spheroids were established in ultralow attachment multiwell plates (ULA) in a collagen matrix and treated for 7 days with vehicle (CTR), 5 mM NAC, 2.5 μM (GSC#1) and 10 μM (GSC#61) axitinib or with a combination of axitinib plus NAC. Axitinib significantly impairs the ability of tumor spheroids to grow. NAC cotreatment does not impair the antitumor effect of axitinib as addressed in terms of spheroid area. (B) Combined fluorescence images of GSC‐derived 3D tumor spheroids. Tumor spheroids were treated with vehicle (CTR), NAC, axitinib or axitinib‐NAC. After 7 days of treatment, tumor spheroids were stained with calcein, propidium, and Hoechst, for staining metabolically active cells, dead cells, and cell nuclei, respectively, and analyzed by confocal microscope. Overall, axitinib decreases the live/dead cells ratio. NAC cotreatment does not prevent the antitumor effect of axitinib (B). Magnification 10×, scale bar 100 μm. n = 10 biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

Axitinib and axitinib‐NAC treatments reduce expression of Ki67 proliferative marker and of senescence‐associated marker Lamin B1 in GSC#1 brain orthotopic xenografts. (A) Both axitinib and axitinib‐NAC treatments significantly impair the expression of the proliferative marker Ki67 and (B) of the senescence‐associated marker Lamin B1 in GFP‐expressing GSC#1 brain tumor xenografts, as addressed by immunostaining with anti Ki67 and anti‐Lamin B1 antibodies, respectively. Magnification 20×, scale bar 100 μm (A); Magnification 40×, scale bar 100 μm (B). ***p < 0.001.

FIGURE 4

NAC rescues the axitinib‐dependent reduction in the endothelial cell markers CD31 and lectin in GSC#1 brain orthotopic xenografts. Axitinib significantly reduces the density of endothelial cells in the brain tumor tissue as addressed by lectin (A) and CD31 (B) immunostaining, respectively. Axitinib‐NAC cotreatment protects endothelial cells from axitinib‐dependent depletion, as addressed by lectin (A) and CD31 (B) immunostaining and also confirmed by CD31‐lectin costaining (C). Magnification 20×, scale bar 100 μm (A); magnification 40×, scale bar 100 μm (B) and (C). ***p < 0.001.

FIGURE 5

NAC cotreatment protects liver endothelium from axitinib toxicity and oxidative stress. Axitinib significantly reduces the density of endothelial cells in the liver of mice bearing brain tumor xenografts, as addressed by CD31 (A) and lectin (B) immunostaining, respectively. Magnification 40×, scale bar 100 μm. (C) NAC effectively counteracts axitinib‐induced ROS accumulation in liver tissue as addressed by DHE staining. Magnification 10×, scale bar 100 μm. ***p < 0.001.