Methotrexate (MTX) is mainly used as antimetabolite agents in cancer therapy. It causes serious side effects such as nephrotoxicity,
hepatotoxicity, and anemia. Nigella sativa (NS) is a medicinal herb that has protective effects against the MTX-related side
effects. The study aimed to determine the optimal concentration of the combination of NS-MTX mixture to improve the antitumor
effects of MTX and reduce its related adverse effects. In this study, in vitro evaluation of anti-tumor and anti-angiogenic
activities ofNS,MTX, or their mixture in different concentrations using liver hepatocellular carcinoma and endothelial cells were
performed. The protective effects of NS on normal hepatic and kidney cells against the risks of MTX treatment was also tested
in vitro. Moreover, for in vivo evaluation, male Wistar rats were treated with MTX or a combination of MTX and NS.
Hematological and serological, biochemical, functional, and histopathological studies were performed. The results showed that
the low concentration of NS enhanced the anti-tumor and anti-angiogenic effects ofMTX.Moreover, it had protective effects on
normal hepatic and kidney cells against toxicity induced by MTX treatments. The in vivo results showed significant improvements
in the blood condition and liver functional parameters in MTX-intoxicated rats after treating with NS. NS reduced the
oxidative stress indices in both kidney and liver tissues. The histopathological examinations showed that NS reduced degenerative
changes and necrosis in both kidney and liver. These results suggested that NS in low concentration could improve the
efficacy and the safety of MTX treatment.