[Show abstract][Hide abstract] 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 M(W) 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 (E2) metabolism similar to that observed in the presence of ctFBS. BSA in fact, much enhanced 16 alpha-hydroxylation and significantly reduced 2-hydroxylation of E2 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 E2. 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.
No preview · Article · Jan 1996 · Molecular and Cellular Endocrinology
[Show abstract][Hide abstract] ABSTRACT: The main goal of the present work was to compare the ability of human prostate cancer (PCa) cells to metabolize testosterone (T) in living conditions. To this end we studied three different human PCa cell lines (LNCaP, DU145 and PC3) having different hormone-sensitive status and capability of response to androgens. We used an original approach which allows the evaluation of conversion metabolic rates in growing cells after administration of labeled steroid precursor (presently T), at physiological concentrations (1-10 nM). Analysis of both precursor degradation and formation of several products was carried out using reverse phase-high performance liquid chromatography (RP-HPLC) and "on line" radioactive detection. Comparison of the three human PCa cells revealed that their metabolic aptitude differed in many respects: (i) rates of precursor degradation, (ii) different products' formation, and (iii) extent of conjugate production. In detail, PC3 cells quickly degraded T and exhibited high formation rates of androstenedione (A-4-ene-Ad); both DU145 and LNCaP cells mostly retained high levels of unconverted T, with a limited production of A-4-ene-Ad and its 17-keto derivatives (if any). Either LNCaP or DU145 cells generated a relatively high amount of dihydrotestosterone (DHT). In contrast, neither DHT nor its main metabolites were detected in PC3 cells at both short and longer incubation times. As expected, T degradation and A-4-ene-Ad production were highly correlated (r = 0.97; P < 0.03); similarly, A-4-ene-Ad and DHT formation showed a negative, significant correlation. Negligible production of conjugates was noted in both PC3 and DU145 cells, whilst it was remarkable in LNCaP cells (ranging from 43 to 57%). Overall, our data indicate that human PCa cells degrade T quite differently, favoring alternatively reductive or oxidative patterns of androgen metabolism.
No preview · Article · Jun 1994 · The Journal of Steroid Biochemistry and Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: The main goal of this study was to ascertain whether routine transurethral resection (TUR) of prostate may provide useful material for the evaluation of androgen receptor (AR) status. At the same time, either intracellular distribution of binding affinity and capacity of receptor molecules were particularly taken into account. Based on our previous findings in breast and endometrial cancer, we suggest that a "functional" receptor status may correspond to the presence of type I (high affinity, low capacity) AR in both soluble and nuclear fractions. However, the precise significance of type II (lower affinity, higher capacity) binding sites remains to be clarified. Ten samples of large prostatic adenomas, obtained by transvesical adenomectomy (TVA), were compared with ten parallel specimens obtained by an in vitro TUR, whereby a pure cutting current was used. The AR assay was carried out with a standard competition method using tritiated mibolerone as the radioligand and Scatchard analysis for data processing. No significant difference between the TUR and TVA groups emerged concerning type I AR content of soluble, nuclear or soluble together with nuclear fractions; this was also true when the results were expressed either as fmol/ml homogenate or as fmol/mg DNA. Similarly, concentrations of type II AR in TVA and TUR samples did not differ significantly in either cell compartment, although they were widely scattered, especially in the soluble fraction. In the light of our findings, it is suggested that TUR specimens represent suitable material for receptor studies, provided that only cutting current is employed and that the use of coagulation current, to control bleeding from the prostatic bed, is confined to the final step of the TUR procedure.
No preview · Article · Feb 1991 · Urological Research
[Show abstract][Hide abstract] ABSTRACT: We briefly review some biochemical aspects of benign breast disease (BBD), mainly focusing on free and conjugate estrogen content of breast cyst fluid (BCF), also in relation to cyst type. Evidence is reported that high K(+)-type I-cysts clearly associate with low Cl- levels and accumulate significantly higher quantities of dehydroepiandrosterone sulfate (DHAS) and estrone-3-sulfate (E1S). In spite of the limited number of cases, both increasing DHAS and E1S levels correlate with the increment of K+ to Na+ ratio. A positive correlation was also found between DHAS and E1S. Using electrochemical detection (ECD) on-line to high performance liquid chromatography (HPLC) in the reverse phase mode, we also studied the free estrogen profile. We observed that in type I BCF there are significantly increased amounts of free estrone (E1). The E1S to E1 ratio was significantly different in the two cyst subpopulations; again, a positive correlation was found between free and sulfated E1 (r = 0.820, p less than 10(-6). This last, together with other experimental observations, allows us to hypothesize that in BCF a main pathway of steroids should be E1S----E1. Besides, high specific activity of sulfatase, as well as beta-glucuronidase enzymes, has been demonstrated for BBD. Preliminary information is also reported concerning the BCF pattern of free estrogens, including the highly polar ones, i.e., catecholestrogens (CCE) and the parent methoxy (MeO) conjugates, which represent, in BCF, a predominant portion of all free estrogens. Both CCE levels and ratios appear unevenly distributed in the two different cyst types. In addition, some BCFs show very high concentrations of 16 alpha-OH-E1. Further studies are needed to answer the main question: whether estrogen patterns could represent additive parameters to further categorize breast cystic disease (BCD) or whether they are of minor interest to determine patients' risk of developing breast cancer.
No preview · Article · Feb 1990 · Annals of the New York Academy of Sciences