Mitoxantrone dihydrochloride (NSC-301739) is the hydrochloride salt of an anthracenedione antibiotic with antineoplastic activity. It is a type II topoisomerase inhibitor.

In a murine air pouch model of inflammation, methotrexate demonstrated an inhibitory concentration 50 (IC50) of 0.08 mg/kg per week.

Methotrexate inhibits the de novo synthesis of purines and pyrimidines, the formation of polyamines, and the transmethylation reactions of DNA, RNA, phospholipids, and proteins. It suppresses the enzyme thymidylate synthase, leading to a deficiency of thymidylate inside cells, which ultimately results in antiproliferative cytotoxicity. Methotrexate enters cells either through active transport or facilitated diffusion and, once inside, polyglutamation inhibits dihydrofolate reductase, preventing the conversion of dihydrofolate to tetrahydrofolate.

The human breast carcinoma cell lines MDA-MB-231 and MCF-7 are seeded in standard 96-well plates. One day after seeding, the culture medium is changed and replaced by medium containing different concentration of Mitoxantrone (10-5 to 5 μM) with or without DHA (30 μM) during 7 days. Viability of cells are measured as a whole by the tetrazolium salt assay[3].