Bonifacio N. Díaz-Chico

Transition to androgen-independence in prostate cancer

Prostate carcinoma is the most frequently diagnosed malignancy and the second leading cause of death as a result of cancer in men in the western countries. Withdrawal of androgens or the peripheral blockage of androgen action remain the critical therapeutic options for the treatment of advanced prostate cancer. However, after initial regression, most of the prostate cancers become androgen-independent and progress further, with eventual fatal outcome. Understanding the mechanisms of transition to androgen independence and tumor progression in prostate cancer is critical to finding new ways to treat aged patients that are ineligible for conventional chemotherapy. A large number of different molecular mechanisms might be responsible for the transition to androgen-independence. Many of these involve the androgen receptor (AR) and its signalling pathways, but they might also include genetic changes that affect several genes, which results in the activation of oncogenes or the inactivation of tumor suppressor genes. Here, we discuss the most recent and relevant findings on androgen resistance in prostate cancer in order provide a comprehensive interpretation of the clinical behaviour of tumors at molecular levels.

Evaluation of acute and chronic hepatotoxic effects exerted by anabolic-androgenic steroid stanozolol in adult male rats.

Abstract Stanozolol (ST) is a 17α-alkyl anabolic-androgenic steroid (17α-AAS) often misused by athletes and bodybuilders. The use of anabolic-steroids by sportsmen and teenagers has increased dramatically, thus raising the question about their hepatotoxicity, specially those such as ST which are orally administered. Previously, we have reported diverse in vivo effects exerted by this steroid and published the existence of a highly specific ST-binding site in male rat liver microsomes. The existence of this binding site, the reported hepatic effects exerted in humans, and the very limited information about its potential hepatotoxicity led us to treat adult male rats acutely and chronically with ST and study different parameters that could indicate liver damage: serum levels of transaminases, concentration of monooxygenase enzymes in liver, liver membrane lipid peroxidation products, liver histopathology, and cell cycle/ploidy status of liver cells. In our study, no changes in serum transaminases or lipid peroxidation levels were obtained. However, acute stanozolol treatment significantly decreased the levels of cytochrome P450 (Cyt. P450) and cytochrome b5 (Cyt. b5) during the first 48 h of treatment, while subsequently, at 72 and 96 h, these microsomal enzymes underwent a significant increase in their levels. In sharp contrast with this response to acute treatment, the content of these two enzymes during chronic treatment showed an important decrease. Interestingly, acutely and chronically ST-treated livers showed slight to moderate inflammatory or degenerative lesions in centrilobular hepatocytes. Flow cytometric analysis demonstrated that both acute and chronic ST treatment were capable of increasing the percentage of S-phase fraction (%SPF) of liver cells. These findings taken together clearly show that this steroid is capable of altering the liver capacity for metabolizing xenobiotics and indicate that high doses of ST could exert a proliferative effect on liver cells. Such data should be considered in risk evaluations for this compound.

Endometrial stromal sarcoma expression of estrogen receptors, progesterone receptors and estrogen-induced srp27 (24K) suggests hormone responsiveness.

The endometrial stroma plays a decisive role in sustaining the gland epithelium along the menstrual cycle, and in preparing the microenvironment that allows embryo implantation. The stroma undergoes important changes during the menstrual cycle that affects both the cell number and differentiation. These changes are regulated by both estrogen and progesterone. Stromal sarcomas are extremely rare, occurring much less than any other uterine tumor. Their origin and biology are poorly understood. The purpose of this work was to try to learn more about the stromal physiology, and also to ascertain whether the stromal sarcoma has characteristics of hormone dependence. We studied the presence of estrogen receptors (ER), progesterone receptors (PR) and the stress-responsive protein of 27K (srp27, a protein first described as an estrogen-induced 24K protein in MCF-7 cells) in both normal stroma and stromal sarcoma. The ER and PR were measured by exchange assays. The srp 27 was studied both by Western-blot and by IHC by means of specific monoclonal antibodies. The stromal sarcomas studied showed a high concentration of both ER (96 to 116 fmol/mg prot.) and PR (565 to 995 fmol/mg prot.). These amounts of ER and PR were higher than the mean found in normal endometrium during the proliferative phase (43 and 637 fmol/mg prot., respectively), and much higher than that of the secretory phase (17 and 229 fmol/mg prot., respectively). The srp27 characterized by Western-blot in both the normal stroma and stromal sarcoma was found to be similar to the srp27 of breast cancer. The IHC results showed a very low expression of srp27 in the stroma during the proliferative phase that increases when the endometrium enters the secretory phase. The low-malignancy grade stromal sarcomas showed abundant expression of srp27, but the high-malignancy grade sarcomas showed no expression of srp27. The obtained results prove the stroma capability to express the srp27. A negative correlation between malignancy of stromal tumors and srp27 expression was found. The presence of ER and PR in some stromal sarcomas proves that they have characteristics of hormone responsiveness. These findings suggest that ER and PR assays should be routinely performed in stromal sarcomas as well as in endometrial adenocarcinomas, and also that antiestrogenic drugs might be considered for the treatment of ER and PR positive stromal sarcomas.

Effects of tamoxifen on serum lipid and apolipoprotein levels in postmenopausal patients with breast cancer

Serum lipids and apolipoprotein levels were measured in twenty postmenopausal women with primary breast cancer, before and three months after tamoxifen therapy (10 mg twice a day). Tamoxifen caused a significant reduction in total serum cholesterol (10%;P < 0.02), and in low-density lipoprotein cholesterol (17%;P < 0.01), and a significant 47 % increase in the subclass 2 of the high density lipoprotein cholesterol (P< 0.01). In addition, tamoxifen caused a 16% increase in apolipoprotein A-I, a 12% decrease in apolipoprotein B (P < 0.05), and a 37% reduction in the serum concentration of lipoprotein(a) (P< 0.01). These results show that tamoxifen brings about an important improvement in serum lipid profile.

Microsatellite Instability Predicts Clinical Outcome in Radiation-Treated Endometrioid Endometrial Cancer

PURPOSE To elucidate whether microsatellite instability (MSI) predicts clinical outcome in radiation-treated endometrioid endometrial cancer (EEC). METHODS AND MATERIALS A consecutive series of 93 patients with EEC treated with extrafascial hysterectomy and postoperative radiotherapy was studied. The median clinical follow-up of patients was 138 months, with a maximum of 232 months. Five quasimonomorphic mononucleotide markers (BAT-25, BAT-26, NR21, NR24, and NR27) were used for MSI classification. RESULTS Twenty-five patients (22%) were classified as MSI. Both in the whole series and in early stages (I and II), univariate analysis showed a significant association between MSI and poorer 10-year local disease-free survival, disease-free survival, and cancer-specific survival. In multivariate analysis, MSI was excluded from the final regression model in the whole series, but in early stages MSI provided additional significant predictive information independent of traditional prognostic and predictive factors (age, stage, grade, and vascular invasion) for disease-free survival (hazard ratio [HR] 3.25, 95% confidence interval [CI] 1.01-10.49; p = 0.048) and cancer-specific survival (HR 4.20, 95% CI 1.23-14.35; p = 0.022) and was marginally significant for local disease-free survival (HR 3.54, 95% CI 0.93-13.46; p = 0.064). CONCLUSIONS These results suggest that MSI may predict radiotherapy response in early-stage EEC.

Photoaffinity Labeling Identification of a Specific Binding Protein for the Anabolic Steroids Stanozolol and Danazol: An Oligomeric Protein Regulated by Age, Pituitary Hormones, and Ethinyl Estradiol

We have demonstrated previously that both rat and human liver microsomes contain a highly specific binding protein for the anabolic steroids stanozolol (ST) and danazol (DA). In this study we solubilized the male rat liver ST-binding protein (STBP) and investigated the following parameters: 1) pharmacological properties, 2) hydrodynamic properties, 3) peptidic composition, 4) the effects of age and hypophysectomy, and 5) inducibility by 17a-ethinyl estradiol. We found that STBP is an integral protein bound to the endoplasmic reticulum. 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) provided its optimal solubilization without changes in its pharmacological properties, i.e. high specificity for ST and danazol, between natural steroids and ligands of low affinity glucocorticoid-binding sites or of progesterone-binding sites. Hydrodynamic properties of the STBP showed that it has a molecular mass of at least 118 kDa. SDS-PAGE of covalently labeled STBP under nonreducing conditions showed that [ 3 H]ST binds to a 110-kDa protein. The STBP was resolved under reducing conditions into three peptides of 55, 31, and 22 kDa, respectively. STBP increased from immature to adult rats, and it dramatically decreased after hypophysectomy. Unlike the 22-kDa peptide, both the 55- and 31-kDa peptides drastically decreased in both immature and hypophysectomized rats. 17a-Ethinyl estradiol administration to immature or hypophysectomized rats induced the 55- and 31-kDa [ 3 H]STBP to a greater extent than the 22-kDa peptide. Thus, STBP appears as an oligomeric protein composed of hormone-regulated peptides. The availability of solubilized STBP and the fact that it can be induced in vivo represent major steps toward the purification and functional significance of this unique steroid-binding protein. (Endocrinology 141: 3377‐3387, 2000)

Validation of a differential PCR and an ELISA procedure in studying HER-2/neu status in breast cancer

HER‐2/neu oncogene status and total cellular p185HER‐2 content were simultaneously analyzed in 415 invasive breast‐cancer specimens by differential PCR and ELISA respectively. Mathematical analysis of the data led us to establish a cut‐off value of 1.7 for the ratio between the intensity of the HER‐2/neu gene band and the reference gene band, to consider the HER‐2/neu gene amplified, and of 260 fmol/mg protein, to consider p185HER‐2 over‐expressed. Of the 415 tumors studied, 15% showed a diverse degree of HER‐2/neu gene amplification. Of these tumors, 87% showed over‐expression of the p185HER‐2. Of the remaining 352 specimens that did not display HER‐2/neu gene amplification, 97% showed no p185HER‐2 over‐expression (p < 0.0001). In 40 selected samples with a p185HER‐2 level lower than 260 fmol/mg protein, the degree of p185HER‐2 phosphorylation was very low or undetectable. Conversely, 38 of 46 selected tumors with a p185HER‐2 level higher than 260 fmol/mg protein exhibited a considerable degree of p185HER‐2 phosphorylation (p < 0.0001). Our data suggest that: (i) differential PCR and ELISA, which are relatively simple procedures, give similar information on HER‐2/neu status in breast cancer; and (ii) given the large series analyzed, the cutoff values established can be considered as safe values for determining whether, in a given tumor, the HER‐2/neu oncogene is amplified or p185HER‐2 is over‐expressed.

Evaluation of acute hepatotoxic effects exerted by environmental estrogens nonylphenol and 4-octylphenol in immature male rats

Nonylphenol (NP) and 4-Octylphenol (4OP) have shown estrogenic properties both in vivo and in vitro. Researchers have known for years that estrogens induce a wide number of hepatotoxic actions in rodents. In order to study the acute hepatic effects exerted by NP and 4OP on rat liver the following endpoints were evaluated: relative liver weight (RLW), morphology, cell cycle and ploidy status, monooxygenase enzymes content and levels of both, cytosolic estrogen receptor (cER) and microsomal binding sites for estrogens (mEBS). Immature male Sprague-Dawley rats were injected intraperitoneally (i.p.) with 60 mg/kg of NP or 4OP for 1, 5 or 10 days. Despite the fact that RLW of the animals was not modified but any treatment, the histopathological study revealed the presence of an increase in the percentage of both, mitotic activity and Ki-67-labeling index (LI) in the livers from animals treated with alkylphenols in absence of any degenerative lesion. Furthermore, all the livers from alkylphenols-treated groups showed the presence of abnormal mitosis and c-mitosis. Although the levels of both, cER and cytochrome P450 (Cyt. P450) were not affected by any treatment, concentration of the mEBS was decreased after 10 days of treatment with alkylphenols. These findings taken together suggest that the exposition to alkylphenols induce cell proliferation and spindle disturbances and that these compounds are capable of modulating the expression of putative membrane receptors for estrogens.

Quantitative analysis of p185HER‐2/neu protein in breast cancer and its association with other prognostic factors

The total cellular p185HER‐2/neu protein (p185) content was measured by ELISA in 346 invasive primary breast cancers, and the results were compared with those of estrogen (ER) and progesterone (PR) receptors, pS2 and Cathepsin D (Cat D) content. At a cut‐off level of 260 fmol/mg protein, 53 of the 346 tumors (15%) were p185‐positive. A significant positive correlation was observed between p185 levels and those of Cat D, and a weaker, though significant, positive correlation with ER, and pS2 levels, but not with those of PR. However, when only the 293 p185‐negative tumors were considered, the correlation between p185 and ER improved substantially, and statistical significance was reached for PR. p185‐positive tumors exhibited lower ER and PR content and higher Cat D content than p185‐negative tumors. The pS2 content, in contrast, did not undergo significant variation. Tumors considered to be p185‐positive were significantly more frequently positive for Cat D at the cut‐off of 45 pmol/mg protein, and were more frequently negative for ER and/or PR, but only significant at the cut‐off of 15 fmol/mg or higher for both steroid receptors. Finally, p185 status was not associated with menopausal status, tumor size, axillary‐lymph‐node invasiveness or distant metastases. These results suggest that 260 fmol/mg protein as the cut‐off for p185 allows the identification of a tumoral sub‐population with a more aggresive phenotype.Int. J. Cancer 74:175–179, 1997.

Serum Resistin and Polymorphisms of Androgen Receptor CAGn and GGNn and Aromatase TTTAn

There is evidence that androgens are regulators of insulin resistance (IR), and may be involved in the regulation of resistin, a cytokine that has been related with IR. Earlier studies found that androgen receptor length polymorphisms CAGn and GGNn and the aromatase polymorphism TTTAn may influence receptor or enzyme activity and serum concentrations of androgens. This study was designed to determine whether polymorphism length was related to serum resistin concentration and to other variables related with IR. In 1,580 persons chosen randomly from the general population of the Canary Islands (Spain), we measured polymorphism length, waist circumference, waist/hip ratio, BMI, and serum glucose concentration. In smaller subgroups, we also measured C‐peptide (n = 677), resistin (n = 583), and leptin concentration (n = 754) and estimated IR (homeostasis model assessment‐IR (HOMA2‐IR)). In men, polymorphism length correlated with resistin concentration (CAGn, r = 0.13, P = 0.031; TTTAn, r = 0.15, P = 0.005; GGNn, r = −0.15, P = 0.026), and the correlations were confirmed in multivariate regression models. The length of CAGn and TTTAn correlated inversely with C‐peptide (r = −0.13, P = 0.016 and r = −0.21, P < 0.001, respectively) and with estimated IR (r = −0.12, P = 0.032 and r = −0.19, P = 0.001, respectively). In men, length of the CAGn, GGNn, and TTTAn was associated with serum resistin concentration. These results support the hypothesis that androgens may be involved in the regulation of resistin. Resistin may be a link between IR and androgens.