P-glycoprotein binding and modulation of the multidrug-resistant phenotype by estramustine
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L A Speicher1, L R Barone, A E Chapman
1Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, PA 19111.
Background:
Previous preclinical studies of combinations of estramustine and vinblastine or paclitaxel (Taxol) have shown that it is possible to achieve a greater than additive cytotoxicity with these antimicrotubule drug combinations. Phase II studies in hormone-refractory prostate cancer have demonstrated clinical antitumor activity of sufficient magnitude to stimulate further laboratory and clinical studies of these drugs combinations.
Purpose:
Our purpose was to characterize the interactions of estramustine with P-glycoprotein and to determine its effects.
Methods:
Standard laboratory techniques were used to study the effects of estramustine on intracellular drug concentrations, cytotoxicity, and induction of messenger RNA (mRNA) for the MDR1 (also known as PGY1) gene. Using a photoaffinity analogue of estramustine 17-0-[[2-[3-(4-azido-3-[125I]-iodophenyl) propionamido]ethyl]-carbamyl]estradiol-3-N-bis(2-chloroethyl)ca rba mate ([125I]AIPP-estramustine), binding to the membrane proteins of human ovarian (SKOV3) and their multidrug-resistant counterpart SKVLB1 cells was studied. Southern-blot analysis was performed on DNA extracted from human prostate carcinoma wild-type DU145, estramustine-resistant cell line (E4), and SKVLB1 cells.
Results:
Membrane fractions from SKOV3 and SKVLB1 cells were analyzed for proteins that could be photoaffinity labeled with [125I]AIPP-estramustine. Competitive inhibition of this binding was achieved with excess concentrations of (in order of efficacy) estramustine, vinblastine, verapamil, progesterone, and to a lesser degree, by paclitaxel but not with estramustine phosphate, estradiol, and estriol. SKVLB1 cells accumulated much less [3H]vinblastine and [3H]paclitaxel than did SKOV3 cells. Estramustine caused a concentration-dependent enhancement of drug accumulation in the SKVLB1 cells to a maximum of approximately 12-fold. No effect of estramustine was apparent for the wild-type SKOV3 cells. In comparison with verapamil, estramustine was less effective as a modulator; however estramustine demonstrated good chemosensitizing activity in combination with actinomycin D and vinblastine. Neither short-term, low-dose no longer-term, higher concentration were found to produce measurable transcript (mRNA for the MDR1 gene levels. Such data suggest that, at least levels. Such data suggest that, at least for two distinct human cell line (SKOV3 and DU145), estramustine does not induce the overexpression of the MDR1 gene.
Conclusion:
It is apparent from the P-glycoprotein data that estramustine interacts with this efflux pump, altering intracellular drug accumulation. Overall, the nonempiric basis for including estramustine in clinical protocols that contain other multidrug-resistant drugs is strengthened by the present data.
Insights
Estramustine interacts with P-glycoprotein, enhancing drug accumulation in multidrug-resistant cells. This study supports estramustine
Area of Science:
- Pharmacology
- Oncology
- Molecular Biology
Background:
- Preclinical studies suggest synergistic cytotoxicity with estramustine and antimicrotubule agents.
- Phase II trials show clinical antitumor activity in hormone-refractory prostate cancer.
Purpose of the Study:
- Characterize estramustine's interaction with P-glycoprotein.
- Determine estramustine's effects on drug accumulation and multidrug resistance.
Main Methods:
- Photoaffinity labeling with [125I]AIPP-estramustine to study binding to P-glycoprotein.
- Assessed intracellular drug concentrations and cytotoxicity in drug-resistant cell lines.
- Analyzed MDR1 gene expression using Southern-blot analysis.
Main Results:
- Estramustine binds to P-glycoprotein, with competitive inhibition by vinblastine and paclitaxel.
- Estramustine enhanced intracellular accumulation of vinblastine and paclitaxel in resistant cells.
- Estramustine did not induce overexpression of the MDR1 gene.
Conclusions:
- Estramustine interacts with P-glycoprotein, modulating intracellular drug accumulation.
- Findings support the use of estramustine in combination therapies for multidrug-resistant cancers.