Raloxifene Attenuation of 5-FU/Methotrexate Cytotoxicity in Human Breast Cancer Cells: The Importance of Sequence in Combination Chemotherapy

Abstract

Background: The selective estrogen receptor modulator raloxifene (RAL), used to treat and prevent osteoporosis, is under investigation for its use in the treatment and prevention of breast cancer. RAL in combination with the antimetabolites methotrexate (MTX) and 5-fluorouracil (5-FU) has not been extensively studied. Because RAL and the antimetabolites target different phases of the cell cycle and exhibit different mechanisms of action and clinical toxicity, the effects of sequence of administration on the growth inhibition of MCF-7 human breast cancer cells were investigated. Materials and Methods: MCF-7 human breast cancer cells were exposed to vehicle alone, 10 μM MTX, 1 μM 5-FU, 10 μM RAL, 10 μM RAL 24 hours prior to 1 μM 5-FU followed 2 hours later by 10 μM MTX, and 1 μM 5-FU 2 hours prior to10 μM MTX followed 24 hours later by 10 μM RAL. The cells were evaluated for viability and proliferation. The retinoblastoma (Rb) protein, a cell cycle regulator which when phosphorylated allows the progression of cells from G₁- to S-phase, was used as a marker to determine the effects of early RAL and late RAL on cellular progression at the molecular level. Results: Early RAL administration exhibited a cell viability of 66.83±6.17% of the control. However, late RAL administration exhibited cell viability 39.40±17.03% of the control. Late RAL was a more cytotoxic combination than RAL alone or early RAL. These findings from manual cell counting were also supported by cell flow cytometric analysis and Western blot data. Conclusion: Late RAL in combination with 5-FU and MTX, due to greater cytotoxicity, is a more desirable combination to treat breast cancer than RAL alone.