Modulatory effect of amifostine (WR-1065) against genotoxicity and oxidative stress induced by methotrexate in human umbilical vein endothelial cells (HUVECs)

Abstract

Amifostine is used in chemotherapy and radiotherapy as a cytoprotective adjuvant alongside DNA-binding chemotherapeutic agents. It functions by reducing free radicals and detoxifying harmful metabolites. Methotrexate, as an antimetabolite drug has been considered for treating various cancers and autoimmune diseases. However, the cytotoxic effects of methotrexate extend beyond tumor cells to crucial organs, including the heart. This study applied the HUVEC cell line as a reference in vitro model for researching the characteristics of vascular endothelium and cardiotoxicity. The current study aimed to assess amifostine’s potential cytoprotective properties against methotrexate-induced cellular damage. Cytotoxicity was measured using the MTT assay. Apoptotic rates were evaluated by Annexin V-FITC/PI staining via flow cytometry. The genoprotective effect of amifostine was determined using the comet assay. Cells were exposed to various amifostine doses (10–200 μg/mL) and methotrexate (2.5 μM) in pretreatment culture condition. Methotrexate at 2.5 μM revealed cytotoxicity, apoptosis, oxidative stress and genotoxicity while highlighting amifostine’s cyto/geno protective properties on HUVECs. Amifostine significantly decreased the levels of ROS and LPO while preserving the status of GSH and SOD activity. Furthermore, it inhibited genotoxicity (tail length, %DNA in tail, and tail moment) in the comet assay. Amifostine markedly attenuated methotrexate-induced apoptotic cell death (early and late apoptotic rates). These findings convey that amifostine can operate as a cytoprotectant agent.

Keywords:

• Amifostine
• methotrexate
• HUVEC
• genoprotective
• apoptosis

Disclosure statement

No potential conflict of interest was reported by the author(s).

Correction Statement

This article has been corrected with minor changes. These changes do not impact the academic content of the article.

Additional information

Funding

The Mazandaran University of Medical Sciences financially supported this study with a grant number of 14412.