Raloxifene attenuation of 5-FU/methotrexate cytotoxicity in human breast cancer cells: the importance of sequence in combination chemotherapy.

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 microM MTX, 1 microM 5-FU, 10 microM RAL, 10 microM RAL 24 hours prior to 1 microM 5-FU followed 2 hours later by 10 microM MTX, and 1 microM 5-FU 2 hours prior to 10 microM MTX followed 24 hours later by 10 microM 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.

Raloxifene attenuation of methotrexate cytotoxicity in human bone marrow by sequence-dependent administration of raloxifene, 5-FU/methotrexate.

BACKGROUND: Increasing evidence suggests that adjuvant systemic chemotherapy is necessary for the survival of breast cancer patients. Antitumor agents are more effective when used in combination with drugs exhibiting different mechanisms of action than when used alone. Previous studies from this laboratory have shown that raloxifene (RAL) attenuation of 5-fluorouracil/methotrexate (5-FU/MTX) cytotoxicity to breast cancer cells was sequence-dependent. The aim was to evaluate the same combination of RAL, 5-FU and MTX to determine the most effective regimes and cellular mechanisms of action to mitigate MTX cytotoxicity in human bone marrow cells. MATERIALS AND METHODS: The sequence-dependent interaction among MTX, 5-FU and RAL on the proliferation and viability of human bone marrow HS-5 cells was determined by the MTT assay and the Trypan blue dye exclusion assay by exposing the cells to MTX, 5-FU and RAL alone, RAL 24 h prior to 5-FU followed 2 h later by MTX, and 5-FU 2 h prior to MTX followed 24 h later by RAL. The control cells were untreated. RESULTS: The growth rate in MCF-7 in early RAL was 68 +/- 3.07% and late RAL 37 +/- 2.05% of the control rate, whereas in bone marrow the same drug combinations exhibit a significant protection against MTX cytotoxity, with the early RAL combination yielding 81 +/- 3.77% and late 54 +/- 2.74% of the control. The finding was further supported by cell flow cytometry and Western blot analysis. CONCLUSION: Sequence-dependent administration of RAL in combination with 5-FU/MTX may have maximum antineoplastic activity in breast cancer while at the same time provide protection to human bone marrow.

The protection against trimetrexate cytotoxicity in human bone marrow by sequence-dependent administration of raloxifene, 5-fluorouracil/trimetrexate.

BACKGROUND: Currently, one of the most effective strategies for the treatment and prevention of breast cancer is the use of drugs that block estrogen action in the breast. The success of the first clinically relevant selective estrogen receptor modulator (SERM), tamoxifen, provided the foundation for further testing of this drug to reduce breast cancer incidence in high-risk women. However, the negative effects associated with the long-term use of tanrhoxifen have initiated the search for compounds that are more effective but less toxic. The discovery of raloxifene (RAL), which functions as a potent antiestrogen in the breast but an estrogen receptor (ER) agonist in the bone and cardiovascular system with very little uterotropic activity, provided an alternative strategy to the targeted use of tamoxifen. The aim of this study was to evaluate RAL in combination with 5-fluorouracil (5-FU)/trimetrexate (TMX) to determine the most effective regimes and cellular mechanism of action to mitigate trimetrexate cytotoxicity in human bone marrow cells. MATERIALS AND METHODS: The cell viability was performed using the Quick Cell Proliferation Assay by exposing the cells to TMX, 5-FU and RAL alone; RAL 24 h prior to 5-FU followed 2 h by TMX, and 5-FU 2 h prior to TMX followed 24 h by RAL determined the sequence-dependent interaction between TMX, 5-FU and RAL on the proliferation. RESULTS: The growth rate in MCF-7 in late RAL was 34.75 +/- 4.79% of the control, whereas in bone marrow the same drug combination exhibits a significant protection against TMX cytotoxicity with late RAL yielding 51.25 +/- 4.43% of the control. The findings were also supported by Cell flow cytometry and Western blot analysis. CONCLUSION: Sequence-dependent administration of RAL in combination with 5-FU/TMX can act against TMX toxicity in human bone marrow, while not affecting the maximum inhibitory effect of TMX in breast cancer.