[CANCER RESEARCH 53, 2071-2075, May I, 1993] Growth Inhibition of Hormone-responsive and -resistant Human Breast Cancer Cells in Culture by /V1,A^-BisiethyOspermine1 Nancy E. Davidson,2 Amy R. Mank, Laura J. Prestigiacomo, Raymond J. Bergeron, and Robert A. Casero, Jr. The Johns Hopkins Oncology Center. Baltimore. Maryland 21231 IN. E. D.. A. R. M., L. J. P., R. A. C.¡.and the Department of Medicinal Chemistry. J. Hillis Miller Health Center. University of Florida, Gainesville. Florida 32610 [R. J. B.J ABSTRACT Previous studies have documented differential sensitivity of human lung cancer and melanoma cell lines to the cytotoxic effects of A", A"2-bis- (ethyl)spermine (BESpm). We show here that BESpm can significantly inhibit the growth of six human breast cancer cell lines with 50% inhib itory concentration in the UM range. The degree of inhibition does not correlate with estrogen receptor status. Detailed studies with estrogen receptor-positive MCF-7 and estrogen receptor- negative Hs578t cells show a similar dose-response curve with concentrations of 1-10 UMresult ing in maximal growth inhibition. Growth inhibition in both lines is as sociated with an 8-12-fold induction of the polyamine catabolic enzyme, spermidine/spermine JV'-acetyltransferase, and a progressive decrease in intracellular polyamine levels over 6 days even though steady-state levels of BESpm are achieved within 24 h. Similar studies on WTMCF7 and AdrRMCF7 cells show that the acquisition of resistance to hormonal or doxorubicin therapy is not associated with resistance to the growth-inhib itory effects of BESpm. These results suggest that BESpm exerts similar growth-inhibitory effects against both hormone-responsive and -unre sponsive human breast cancer cells, a finding which has significance for the potential use of polyamine analogues in treating human breast cancer. INTRODUCTION Intracellular polyamines play an important role in the proliferation of normal and malignant cells. The recognition of their critical role in cell growth and differentiation has led to the development of several inhibitors of polyamine biosynthesis, particularly DFMO,3 which is directed against ODC, the first enzyme in polyamine biosynthesis (1-5). Recently, however, attention has been focused on other steps in the polyamine-metabolic pathway as potential targets for intervention (6). In particular the /V,yv'-bis(ethyl) analogues of spermine were found to down-regulate ODC, deplete intracellular polyamine pools, and inhibit cell growth, suggesting a failure to substitute for the depleted natural polyamines (7). BESpm is a representative compound for this family of agents and its cytostatic and cytotoxic effects have been studied in several tumor models (8, 9-12). In addition to down-regulating the biosynthesis of the polyamines, BESpm induces the activity of the rate-limiting enzyme in polyamine catabolism, SS AT (9, 10). A possible role for SS AT in human breast carcinogenesis has been suggested by the finding that SSAT activity is significantly elevated in primary human breast cancer specimens com pared with surrounding normal breast tissue ( 13). SSAT is induced in a variety of cell types in response to several different stimuli including hormonal stimulation and exposure to polyamines and polyamine analogues (14-19). Although many cell types respond to BESpm treatment with a severalfold induction of SSAT, a few respond with extreme induction of SSAT (9, 10). Recent work using human lung Received 9/18/92; accepted 2/24/93. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by NIH Grants CA37606, CA49634. and CA51085. 2 To whom correspondence should be addressed, at The Johns Hopkins Oncology Center. 422 N. Bond Street, Baltimore, MD 21231. 1The abbreviations used are: DFMO. a-difluoromethylomithine; BESpm, JV'JV'Mns- (ethyl)spermine; SSAT, spermidine/spermine /V'-acetyltransferase; ODC, omithine decar- boxylase. cancer and melanoma cell lines as model systems suggests that a correlation between SSAT superinduction and growth inhibition is observed in that bis(ethyl)polyamines are cytotoxic primarily to SSAT "superinducer" cell types. For example, BESpm-mediated killing of human lung cancer cell lines is typically associated with a > 100-fold induction in SSAT activity to levels of >1000 pmol/mg protein/min within 24 h, whereas cell types which demonstrate cytostatic growth effects manifest a smaller induction of SSAT in response to BESpm treatment (9). Metastatic breast cancer is a common disease. A major clinical problem is that tumors which are initially responsive to both hormonal and chemotherapeutic approaches generally progress to more aggres sive forms which are poorly responsive to either category of agents (20). The need for antineoplastic agents with novel mechanisms of action is therefore great. Previous studies have shown that growth of established human breast cancer cell lines can be inhibited by DFMO in culture (21-24). Evidence that hormone-responsive, but not hor mone-resistant, human breast cancer cells are sensitive to the antipro- liferative effect of DFMO has been presented by some investigators (21, 22) and refuted by others (23). Thus the major goal of this work was to characterize the effects of BESpm on a panel of hormone- responsive and -unresponsive human breast cancer cells. In addition we sought to correlate any change in growth with the effects of BESpm on intracellular polyamine levels and the induction of SSAT at the enzyme and steady-state mRNA levels. MATERIALS AND METHODS Chemicals. BESpm was synthesized as previously described (25, 26). It was maintained as a 10 mM stock in 0.1 M HC1 and diluted in media for cell treatment as described below. Cell Culture and Growth Studies. The acquisition and maintenance of the estrogen-responsive MCF-7, T47D. and ZR-75-1 and estrogen-unresponsive MDA-MB-231, MDA-MB-468, and Hs578t human breast cancer cell lines have been described previously (27). WTMCF7 and AdrRMCF7 cells were the gift of Dr. K. Cowan (National Cancer Institute, Bethesda, MD). The AdrRM- CF7 cell line was selected by serial passage of WTMCF7 cells in the presence of increasing concentrations of doxorubicin and was maintained in 10 UM doxorubicin as described previously (28). AdrRMCF7 cells were passaged in doxorubicin-free medium for at least four passages before use. For cell growth studies cells were plated in quadruplicate 12-mm wells under routine culture conditions. The plating density ranged from 20,000 to 30,000 cells/well, de pending on the particular cell line under study. After 24 h the medium was replaced by medium with 0.01 M HC1 vehicle or varying concentrations of BESpm. The final concentration of HC1 did not exceed 0.01 M. Medium was replaced every 3 days and the number of adherent cells was determined by Coulter Counter after 6 days. Results are expressed as N¡/NHwhere Na is the number of cells on the day of BESpm or vehicle addition and N, is the number of cells after 6 days of BESpm or vehicle (9). Analysis of Polyamine Content and SSAT Activity. The polyamine con tent of treated and untreated cells was determined by the precolumn dansyla- tion, reversed-phase high-performace liquid Chromatographie methods of Kabra et al. (29). 1,7-Diaminoheptane was used as the internal standard. The level of detection of this method is 5 pmol of the individual polyamines. SSAT activity of cellular extracts was measured as reported previously (19). Enzyme activity is expressed as pmol /V'-[l'lC]acetylspermidine formed/ing protein/min. Protein concentrations were determined by the method of Brad ford (30). 2071 Research. on February 19, 2016. © 1993 American Association for Cancer cancerres.aacrjournals.org Downloaded from