Abstract
Molecular mechanisms underlying the nephrotoxicity associated with bevacizumab are unclear. Endothelin-1 (ET-1) is involved in podocyte injury and proteinuria, and its level increases in most cases of kidney disorders. Forkhead box protein O1 (FoxO1), a transcription factor, is a major determinant of ET-1 promoter activation and is regulated by protein kinase B (Akt) phosphorylation-dependent nuclear exclusion. We evaluated the effect of bevacizumab on ET-1 production in human glomerular microvascular endothelial cells (hGECs). We analyzed the changes in the mRNA and protein levels of ET-1 in hGECs treated with bevacizumab using real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Changes in the protein levels and phosphorylation status of Akt and FoxO1 in hGECs treated with bevacizumab were analyzed by western blotting. After cell lysis, FoxO1 protein was isolated from the cytoplasmic and nuclear fractions. We also investigated the effects of AS1842856 (a FoxO1 inhibitor) on bevacizumab-induced ET-1 production. Bevacizumab significantly and dose-dependently increased the mRNA and protein levels of ET-1 in hGECs (p < 0.05). Bevacizumab treatment also led to a decrease in phosphorylated Akt protein levels. Inhibition of Akt activity by LY294002 promoted ET-1 production. Bevacizumab also induced an increase in FoxO1 protein levels in the nucleus. Inhibition of FoxO1 activity by AS1842856 resulted in decreased ET-1 levels in bevacizumab-treated hGECs. ET-1 axis activation, Akt inactivation, and FoxO1 nuclear localization are the molecular mechanisms underlying bevacizumab-induced nephrotoxicity. Therefore, inhibition of renal ET-1 production could be a promising approach to protect against or treat bevacizumab-induced nephrotoxicity.
Highlights
Bevacizumab is a humanized monoclonal antibody targeting human vascular endothelial growth factor A (VEGFA) and is currently the most commonly used angiogenesis inhibitor for cancer treatment [1,2,3]. e effects of bevacizumab, such as antiangiogenesis and tumor suppression, are associated with the inhibition of VEGFA signaling [4, 5]
We aimed to evaluate the effect of bevacizumab on ET-1 production in human glomerular microvascular endothelial cells. e changes in mRNA and protein levels of ET-1 and the protein levels and phosphorylation status of Akt and forkhead box protein O1 (FoxO1) in hGECs treated with bevacizumab were analyzed
Monolayers of cells that were 90% confluent were serum-starved for 24 h before the experiments were performed. e cells were treated with 0.1 or 1 μM bevacizumab. e rationale for the dose setting of bevacizumab in this study reflects the use of the drug in clinical practice [21]. e cells were incubated with LY294002, a phosphatidylinositol-3 kinase (PI3K)/Akt pathway inhibitor, and AS1842856, a FoxO1 inhibitor, to determine the effect of Akt and FoxO1 inhibition on ET-1 production. e rationale for the dose setting of LY294002 and AS1842856 in this study reflects the minimum concentration needed to inhibit Akt and FoxO1
Summary
Bevacizumab is a humanized monoclonal antibody targeting human vascular endothelial growth factor A (VEGFA) and is currently the most commonly used angiogenesis inhibitor for cancer treatment [1,2,3]. e effects of bevacizumab, such as antiangiogenesis and tumor suppression, are associated with the inhibition of VEGFA signaling [4, 5]. Bevacizumab is a humanized monoclonal antibody targeting human vascular endothelial growth factor A (VEGFA) and is currently the most commonly used angiogenesis inhibitor for cancer treatment [1,2,3]. Bevacizumab treatment is associated with nephrotoxicity, including hypertension, proteinuria, nephrotic syndrome, and renal-limited thrombotic microangiopathy [6]. Bevacizumab-induced nephrotoxicity, characterized by major histological changes observed in glomerular disorders, results in severe defects in the glomerular filtration barrier that prevents the leakage of serum proteins into the urine [7,8,9,10,11]. Whether bevacizumab increases ET-1 production in the vascular endothelium and the molecular mechanisms underlying bevacizumab-induced nephrotoxicity remains unclear. The FoxO1 transcription factor is regulated by protein kinase B ( called Akt) phosphorylation-dependent nuclear exclusion [20]
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