OBJECTIVE:Pharmacologic disruption of microtubule function may provide effective therapy for advanced epithelial ovarian cancer, as has been observed in clinical trials using taxol. However, the limited availability of taxol and taxol's side effects emphasize a need to develop alternative antimicrotubule agents. Estramustine (EM) inhibits binding of microtubule-associated proteins (MAPs) to microtubules, promotes microtubule disassembly, disrupts spindle formation, and in duces metaphase arrest in human prostate carcinoma and glioma cells in culture. We studied the effect of EM on DNA synthesis and on the cell cycle in four human ovarian carcinoma cell lines and examined the cell lines for evidence of MAP-like immunoreactivity.
METHODS:The effect of EM on DNA synthesis and on the cell cycle was determined using [3H]thymidine incorporation assays and flow cytometry, respectively. Microtubule-associated pro tein-like immunoreactivity was determined using monoclonal antibodies directed against MAP 1A, MAP 1B, and MAP 2(2A + 2B) for Western analysis after sodium dodecyl sulfate- polyacrylamide gel electrophoresis.
RESULTS:We demonstrated a dose-dependent inhibitory response to EM in BIXLER, DK2NMA, and SKOV3. BIX3 showed a dose-dependent inhibitory response to EM concen trations from 25 μ g/mL to 100 μg/mL, but a stimulatory response at 10 μ g/mL. Estramustine inhibited exponentially growing cells by causing mitotic arrest with subsequent accumulation of cells in G2/M phase of the cell cycle in all four cell lines. We found MAP 1A, MAP 1B, and MAP 2-like immunoreactivity in all four cells lines studied.
CONCLUSIONS: These results are consistent with a MAP-microtubule mechanism of action for EM in ovarian carcinoma cells and provide reason to conduct further study of EM for potential use in the treatment of human epithelial ovarian cancer. (J Soc Gynecol Invest 1994;1:97-103)
McGuire WP, Rowinsky EK, Rosenshein NB, et al. Taxol: A unique antineoplastic agent with significant activity in advanced ovarian epithelial neoplasms. Ann Intern Med1989;111:273-9.
2.
Rowinsky EK, McGuire WP, Guarnieri T., Fisherman JS, Christian MC, Donehower RCCardiac disturbances during the administration of taxol. J Clin Oncol1991;9:1704-12.
3.
Jönsson G., Högberg B., Nilsson T.Treatment of advanced prostatic carcinoma with estramustine phosphate (Estracyt®) . Scand J Urol Nephrol1977;11:231-8.
4.
Stearns ME, Tew KDAntimicrotubule effects of estramustine, an antiprostatic tumor drug. Cancer Res1985;45: 3891-7.
5.
Stearns ME, Wang M., Tew KD, Binder LIEstramustine binds a MAP-1-like protein to inhibit microtubule assembly in vitro and disrupt microtubule organization in DU 145 cells. J Cell Biol1988;107:2647-56.
6.
Eklov S., Nilsson S., Larson A., Björk P., Hartley-Asp B.Evidence for a non-estrogenic cytostatic effect of estramustine on human prostatic carcinoma cells in vivo. Prostate1992;20:43-50.
7.
Fridén B., Wallin M., Deinum J., Prasad V., Luduena R.Effect of estramustine phosphate on the assembly of trypsin-treated microtubules reconstituted from purified tubulin with either tau, MAP-2, or the tubulin-binding fragment of MAP-2. Arch Biochem Biophys1987 ;257:123-30.
8.
Stearns ME, Tew KDEstramustine binds MAP-2 to inhibit microtubule assembly in vitro. J Cell Sci1988;89:331-42.
9.
Mareel MM, Storme Ga, Dragonetti CH, et al. Antiinvasive activity of estramustine on malignant MO4 mouse cells and on DU-145 human prostate carcinoma cells in vitro . Cancer Res1988;48:1842-9.
10.
Hartley-Asp B.Estramustine-induced mitotic arrest in two human prostatic carcinoma cell lines DU 145 and PC-3. Prostate1984;5:93-100.
11.
Sheridan VR , Speicher LA, Tew KDThe effects of estramustine on metaphase and anaphase in DU 145 prostatic carcinoma cells. Eur J Cell Biol1991;54:268-76.
12.
von Schoultz E., Lundblad D., Bergh J., Grankvist K., Henriksson R.Estramustine binding protein and antiproliferative effect of estramustine in human glioma cell lines. Br J Cancer1988;58:326-9.
13.
Kacinski BM , Carter D., Mittal K., et al. Ovarian adenocarcinomas express fms-complementary transcripts and fms antigen, often with coexpression of CSF-1. Am J Pathol1990; 137:135-47.
14.
King BL, Carter D., Foellmer HG, Kacinski BMNeu proto-oncogene amplification and expression in ovarian adenocarcinoma cell lines. Am J Pathol1992;140:23-31.
15.
Jett JHMathematical analysis of DNA histograms from asynchronous and synchronous cell populations. In: Lutz D, ed. Pulse cytophotometry. Belgium: European Press, 1978:93-102.
16.
Wallin M., Deinum J., Fridén B.Interaction of estramustine phosphate with microtubule-associated proteins . FEBS Lett1985;179:289-93.
17.
Hartley-Asp B., Gunnarsson POGrowth and cell survival following treatment with estramustine, nor-nitrogen mustard, estradiol and testosterone of a human prostatic cancer cell line (DU 145). J Urol1982;127:818-22.
18.
Tew KDThe mechanism of action of estramustine. Semin Oncol1983;10(suppl 3):21-6.
19.
Matus A., Riederer B.Microtubule-associated proteins in the developing brain. Ann NY Acad Sci1986;466:167-79.
20.
Wiche G., Herrman H., Dalton JM, et al. Molecular aspects of MAP-1 and MAP-2: Microheterogeneity, in vitro localization and distribution in neuronal and non-neuronal cells. Ann NY Acad Sci1986;466:180-98.
21.
Wiche G.High-Mr microtubule-associated proteins: Properties and functions . Biochem J1989;259:1-12.
22.
von Schoultz E., Grankvist K., Gustavsson H., Henriksson R.Effects of estramustine on DNA and cell membrane in malignant glioma cells. Acta Oncol (Stockh) 1991;30:719-23.
23.
Sloboda RD, Rudolph SA, Rosenbaum JL, Greengard P.Cyclic AMP-dependent endogenous phosphorylation of a microtubule-associated protein. Proc Natl Acad Sci USA1975;72:177-81.
24.
Schoenfeld TA, McKerracher L., Obar R., Vallee RBMAP 1A and MAP 1B are structurally related microtubule associated proteins with distinct developmental patterns in the CNS. J Neurosci1989;9:1712-30.
25.
O'Brian CA, Liskamp RM, Solomon DH, Weinstein IBInhibition of protein kinase C by tamoxifen. Cancer Res1985;45:2462-5.
26.
Su H., Mazzei GJ, Vogler WR, Kuo JFEffect of tamoxifen, a nonsteroidal antiestrogen, on phospholipid/calciumdependent protein kinase and phosphorylation of its endogenous substrate proteins from the rat brain and ovary. Biochem Pharmacol1985;34:3649-53.
27.
Pollack IF, Randall MS, Kristofik MP, Kelly RH, Selker RG, Vertosick FT Jr.Effect of tamoxifen on DNA synthesis and proliferation of human malignant glioma lines in vitro. Cancer Res1990;50:7134-8.
28.
Piepmeier JM , Keefe DL, Weinstein MA, et al. Estramustine and estrone analogs rapidly and reversibly inhibit deoxyribonucleic acid synthesis and alter morphology in cultured human glioblastoma cells. Neurosurgery1993;32: 422-30.
