ecular and t?ellulrr Endocrinology Molecular and Cellular Endocrinology 115 (1995) 221-225 Rapid Paper Effect of serum albumin on estrogen metabolism in human cancer cell lines H. Leon Bradlow”, Felice Arcurib, Livio Blasib, Luigi Castagnettabqc “Strang-Cornell Cancer Research Laboratory, Cornell University Medical College, New York, ,YY, USA bHormone Biochemistry Laboratory, University of Palermo School of Medicine, Via Marchese Ugo, 56 90141 Palermo, Italy ‘Experimental Oncology and Molecular Endocrinology Units, Palermo Branch of IST-Genoa, c/o ‘M. Ascoli’ Cancer Hospital Center, Palermo. Italy Received 9 August 1995; accepted 6 September 1995 Abstract The observation that charcoal-treated fetal bovine serum (ctFBS) was able to modify one of main pathways of estrogens in cancer cells in culture, prompted us to initiate the present study. The active component of serum was isolated using native preparative polyacrylamide gel electrophoresis (PAGE). Under analysis with SDS-PAGE, a Mw of 68 kDa and mobility of authentic bovine serum albumin (BSA) was observed. The addition of BSA to the serum free culture medium of HEC 1A human endometrial cancer cell line, resulted in an alteration of estradiol (EJ metabolism similar to that observed in the presence of ctFBS. BSA in fact, much enhanced 16cr-hydroxylation and significantly reduced 2-hydroxylation of E, in HEC 1A cells. Comparable results were obtained with different endometrial (Ishikawa) and mammary (MCF-7) tumor cell lines having a different metabolic conversion rate of E,. Several albumin preparations from either bovine or human serum had the same effect; besides, BSA activity was unaffected by treatment with dextran-charcoal or heat. In the light of the present results, the inclusion of serum albumin (SA) in the formulation of media for studies evaluating steroid metabolism in cultured cells should be carefully considered. Keywords: Cancer cell (human); Fetal bovine serum; Albumin; Estradiol metabolism 1. Introduction components of steroid-depleted serum might modulate estradiol (EJ metabolism. The aim of this study was to The use of varying amounts of serum has been investigate which factor(s) was responsible for the ob- considered mandatory to promote substrate adhesion served changes focusing in particular, on 16a- and and growth of cells in culture. However, in studies 2-hydroxylation of E,. We found that serum albumin evaluating steroid metabolism, the presence of serum (SA) is the main active component of FBS. has always been considered critical since it represents a Recent observations have demonstrated that SA is well-known source of hormones. Therefore, many able to alter metabolic reactions and cellular responses efforts have been made to develop steroid-depleted sera, in normal as well as in tumor cells. SA has been but little or no attention has been given to the role of reported to modify pregnenolone production by imma- other components of serum that might af+ct metabolic ture rat Leydig cells in vitro (Melsnert et al., 1989). transformation of steroids. In our studies on human Singh et al. (1992) identified human SA (HSA) as a endometrial and breast cancer cell lines, we have ob- component of a bioactive fraction able to stimulate the served that the addition to the culture medium of reductive conversion of estrone (E,) to E, in MCF-7 charcoal-treated fetal bovine serum (ctFBS) modifies a cancer cell line. In this paper, we investigate the role for major metabolic pathway of estrogen, indicating that SA in the regulation of E, metabolism, namely en- hancement of 16cr- and reduction of 2-hydroxylase * Corresponding author, Tel.: 39 91 666 4345; Fax: 39 91 666 4352. activity. 0303-7207/95/$09.50 0 1995 - Elsevier Science Ireland Ltd. All rights reserved SSDI 0303-7207(95)03684-Y 222 H.L. Bradlow et al. / Molecular and Crlhlar Endocrinology 115 (1995) 221-225 2. Material and methods exchange and normalized for the amount of DNA, is expressed as percentage of control. 2.1. Chemical Bovine serum albumin (fraction V, fatty acid free, 2.4. Fetal bovine serum fractionation globulin free), human serum albumin (fatty acid free), CtFBS was fractionated by native preparative acry- thyreoglobulin, y-globulin, fetuin, gelatin, dextran, lamide gel electrophoresis (PAGE) using acrylamide dithiothreitol and ethylenediamine tetraacetic acid were concentrations of 7.5% for resolving and 4.0% for obtained from Sigma Chemicals Co. (St. Louis, MO). stacking gels, respectively. Samples of ctFBS were [C16a-3H]E, (SA, 20 Ci/mmol) was prepared as previ- loaded in a 3-mm thick acrylamide gel and run ously described (Schneider et al., 1982). [C2-3H]E, (SA, overnight at 4°C. Dithiothreitol (DTT) and ethylenedi- 15 Ci/mmol) was purchased from New England Nu- aminetetraacetic acid (EDTA), 1 mM each, were in- clear (Boston, MA). cluded in both stacking and resolving gel as protective agents. The following day the gel was cut horizontally 2.2. Cell culture in fractions of 8.0 mm each, and proteins were electroe- HEC 1A and Ishikawa human endometrial cancer luted in dialysis bags (Mw cut-off, 3.5 kDa) in 0.1 M cell lines were kindly supplied by Dr. E. Gurpide sodium-phosphate buffer, pH 7.4, DTT and 1 mM (Mount Sinai School of Medicine, New York, USA). EDTA, at 4°C. The content of the dialysis bags was MCF-7 human breast cancer cell line was obtained concentrated using Microsep 10 K centrifugal concen- from Michigan Cancer Foundation (Detroit, MI). Cells trators (Filtron Technology Co., Northborough, MA), were routinely maintained at 37°C in a humidified assayed for protein following the method of Bradford atmosphere of 95% air, 5% CO, in MEM containing (1976) and tested for bioactivity. Gel electrophoresis of 8.63 mg/l phenol red as pH indicator and supplemented proteins was carried out in 10.5% polyacrylamide in the with 10% FBS, 10 mM L-glutamine, 100 III/ml peni- presence of sodium dodecyl sulfate (SDS) as described cillin, 100 pg/ml streptomycin, 0.25 pug/ml fungizone in by Laemmli (1970). Proteins were stained with silver loo-mm tissue culture plates. according to the procedure of Wray et al. (1981). 2.3. Bioactivity assay 2.5. Statistical analysis The assay is based on the capability of protein prepa- Statistical comparisons were performed using one rations to modify 16cw-and 2-hydroxylation of E, in sample two tailed Student’s t-test. Probability values of cultured cells. The extent of C16a- and C2-hydroxylase less than 0.05 were considered significant. activity was measured by determining 3H,0 formation in cells incubated in the presence either of [C16a-3H]E, 3. Results or [C2-3H]E2. The cells were grown to confluence, trypsinized and seeded in 24-well culture dishes (1 x Fig. 1 shows the effect of ctFBS on the metabolic 10’ cells per well, four wells for each experimental conversion of E, by HEC 1A human endometrial can- condition) in maintenance medium. The next day the cer cell line. The presence of ctFBS in the culture medium was removed and, after washing the cultures medium resulted in a dramatic increase in 16a-hydroxy- three times with HBSS, changed to serum-free, phenol red free, D-MEM medium containing either vehicle 350 1 control or Jest proteins in aqueous solution. Unless ..I I T otherwise indicated, proteins were tested at a concen- tration of 2.0 mg/ml. After 24 h, the medium was i replaced with fresh medium that contained ‘H-E, at a 6; 250 concentration of 1 x 10 - 9 M in ethanol (final alco- b 200 holic concentration = 0.1%). After 48 h of incubation at & 37°C in a humidified atmosphere of 95% air, 5% CO,, 7c1 150 aliquots of 700 ,ul of medium were diluted to 1.5 ml t with distilled water, lyophilized and the sublimate was k 100 Untreated Control a counted for radioactivity in duplicate in a liquid scintil- 50 lation counter. The 3H released from specifically labeled E, provided an indirect measurement of the regiospe- 0 cific hydroxylation of the steroid (Schneider et al., 16a-hydroxylation 2-hydroxylation 1982). The cell layer was lysed in 0.1% SDS and DNA Fig. I. Elect of ctFBS on 16a- and 2-hydroxylation of EZ in HEC 1A content was determined by a spectrofluorimetric cells. The results are means f SD (16a-hydroxylation, n = 5; 2-hy- method (Hinegardner, 1971). The extent of 16c(- and droxylation, n = 3). ***P = 0.0025; **P = 0.019 (two tailed Student’s 2-hydroxylation of E,, corrected for the non-specific 3H t-test). H.L. Bradlow et al. / Molecular and Cellular Endocrinology 115 (1995) 221-225 223 12.5 225 E 10.0 200 95,000 .F t;al 7.5 175 a’ 68,000 I 5.0 150 2.5 125 43,000 0.0 100 1 2 3 4 5 6 7 6 9 1011 Fraction Number 36,000 Fig. 2. Effect of ctFBS fractions obtained by native polyacrylamide gel electrophoresis on 16a-hydroxylase activity in HEC IA cells. Proteins were tested at the concentration of 0.5 m&ml. 29,000 lase activity (271% &- 56.5 with respect to control; P = 0.0025) and in a significant decrease of 2-hydroxy- lase (78.6% & 5.2 of control; P = 0.019) as reflected by the 16a-/2_hydroxylation ratio (3.1 -fold of control). Fig. 3. SDS-PAGE of fraction # 10 from native polyacrylamide gel To identify the component of serum responsible for this electrophoresis of ctFBS. The protein standards used (with their M,) effect, ctFBS was fractionated by native preparative were phosphorylase B (95 000 Da), bovine serum albumin (68 000 PAGE electrophoresis and the fractions tested for Da), ovalbumin (43 000 Da), lactate dehydrogenase (36 000 Da) and carbonic anhydrase (29 000 Da).