Sequence-dependent administration of 5-fluorouracil maintains methotrexate antineoplastic activity in human estrogen-negative breast cancer and protects against methotrexate cytotoxicity in human bone marrow

Background: Breast cancer is a leading cause of morbidity and mortality in women in developed countries and in increasingly developing countries. In general, estrogen receptor (ER)-positive breast cancers have a better prognosis and are often more responsive to anti-estrogen therapy. Unfortunately, ER-negative breast cancers are more aggressive and unresponsive to anti-estrogens. The aim of this investigation was to evaluate the 5-fluorouracil (5-FU) and methotrexate (MTX) combination to determine the most effective regimen considering the mechanism of action in treating ER-negative human breast cancer cells and at the same time mitigating methotrexate cytotoxicity in human bone marrow cells. Materials and Methods: In order to determine the sequence-dependent interaction between MTX and 5-FU on proliferation, cell viability was carried out using the Quick Cell Proliferation Assay by exposing the human estrogen negative (MDA-MB-436 and Hs-578T) and bone marrow (HS-5) cells to: (i) MTX and 5-FU alon; (ii) MTX 2 h prior to 5-FU (MTX/5-FU; (iii) 5-FU 2 h prior to MTX (5-FU/MTX). Results: The growth rate in MDA-MB-436 was 23.5±3.98%, in Hs-578T 30±5.9% and HS-5 32±3.1% of the control for MTX/5-FU. Whereas the growth rate in MDA-MB-436 was 28.5±4.1%, in Hs-578T 34.7±3.5% and HS-5 68.6±6.3% of the control for 5-FU/MTX combinations. The later combination exhibits significant protection against MTX cytotoxicity in bone marrow and at same time maintained maximum cytotoxicity in estrogen negative breast cancer cell lines. The findings were further supported by cell flow cytometry, apoptosis and Western blot analysis data. Conclusion: The combination of 5-FU/MTX effectively maintains the maximum inhibitory effect of MTX in ER-negative breast cancer and protects against MTX cytotoxicity in human bone marrow.