DIAGNOSIS OF ENDOCRINE DISEASE: Drug-induced endocrinopathies and diabetes: a combo-endocrinology overview.

In the currently overwhelming era of polypharmacy, the balance of the dynamic and delicate endocrine system can easily be disturbed by interfering pharmaceutical agents like medications. Drugs can cause endocrine abnormalities via different mechanisms, including direct alteration of hormone production, changes in the regulation of the feedback axis, on hormonal transport, binding and signaling, as well as similar changes to counter-regulatory hormone systems. Furthermore, drugs can interfere with the hormonal assays, leading to erroneous laboratory results that disorientate clinicians from the right diagnosis. The purpose of this review is to cover a contemporary topic, the drug-induced endocrinopathies, which was presented in the monothematic annual Combo Endo Course 2018. This challenging part of endocrinology is constantly expanding particularly during the last decade, with the new oncological therapeutic agents, targeting novel molecular pathways in the process of malignancies. In this new context of drug-induced endocrine disease, clinicians should be aware that drugs can cause endocrine abnormalities via different mechanisms and mimic a variety of clinical scenarios. Therefore, it is extremely important for clinicians not only to promptly recognize drug-induced hormonal and metabolic abnormalities, but also to address the therapeutic issues for timely intervention.

Glucocorticoid receptor signaling and prostate cancer.

Glucocorticoids (GCs) are provided to hormone-refractory prostate cancer (HRPC) patients partly due to the inhibitory effects on adrenal androgen production acting as a pituitary suppressant. Nowadays, the combination of chemotherapy and dexamethasone is a standard treatment for HRPC patients while increasing evidence suggests that a lot of local tissue factors like growth factors, angiogenic/lymphogenic factors, apoptosis-related factors, cytokines related to the transition of prostate cancer from androgen dependence to hormone-refractory status, are among the targets of GR signaling. However, although glucocorticoids have been recognized to be one of a limited number of treatment options for HRPC, the molecular basis of GC-induced effects in prostate cancer remains poorly defined. In this review, we focus on how GCs induce effects via the GR-mediated transcriptional regulation of specific genes known to play key roles in cellular/tissue functions, including growth, apoptosis, inflammation, metastasis, differentiation, cell survival and angiogenesis. In our effort to unravel the molecular interplay of GR signaling with other signaling cascades prevalent in prostate cancer, we also include a detailed description of GR gene and protein structure/function and provide the knowledge gained recently into the mechanism(s) of the cross talk between GR and other signaling cascades via which GCs exert their multiple effects.

Atypical induction of the unfolded protein response by mifepristone.

Mifepristone is a synthetic progesterone antagonist that is being used widely for the treatment of various conditions such as endometriosis, glaucoma, meningiomas, breast, ovarian and prostate cancer, as well as for research purposes, in the conditional induction of gene expression by using artificial plasmid-based systems. Here, we report that exposure of A549 human lung cancer cells to mifepristone caused an atypical induction of the cellular unfolded protein response, as evidenced by the time-dependent stimulation of RNA levels of the chaperone Grp94 and PDIa, as well as the endoplasmic reticulum stress-associated receptors ATF6, PERK and eIF2 but not of their downstream target, transcription factor ATF4. This profile was very different from that of progesterone, which at the same dose as mifepristone, failed to induce all of the ER-stress-related genes examined, apart from PERK. Furthermore, XBP1, a transcription factor that is regulated predominantly by alternative splicing by the IRE1 receptor, remains unspliced and therefore inactive either by mifepristone or progesterone treatment. Finally, the pro-apoptotic molecules CHOP and BIM are only induced in the presence of tunicamycin in the culture medium. Tunicamycin, the most commonly used pharmacologic inducer of ER stress that triggers the canonical ER stress response, was used for comparison purposes. Our results suggest that mifepristone can elicit an atypical ER stress response when used at different doses and for different time points. The subsequent induction of UPR should be taken into consideration when this agent is being used either for therapeutic or for experimental uses.

Estrogen receptor signaling and its relationship to cytokines in systemic lupus erythematosus.

Dysregulation of cytokines is among the main abnormalities in Systemic Lupus Erythematosus (SLE). However, although, estrogens, which are known to be involved in lupus disease, influence cytokine production, the underlying molecular mechanisms remain poorly defined. Recent evidence demonstrates the presence of estrogen receptor in various cell types of the immune system, while divergent effects of estrogens on the cytokine regulation are thought to be implicated. In this paper, we provide an overview of the current knowledge as to how estrogen-induced modulation of cytokine production in SLE is mediated by the estrogen receptor while simultaneously clarifying various aspects of estrogen receptor signaling in this disease. The estrogen receptor subtypes, their structure, and the mode of action of estrogens by gene activation and via extranuclear effects are briefly presented. Results regarding the possible correlation between estrogen receptor gene polymorphisms and quantitative changes in the receptor protein to SLE pathology and cytokine production are reviewed.

Adipocyte factors, high-sensitive C-reactive protein levels and lipoxidative stress products in overweight postmenopausal women with normal and impaired OGTT.

OBJECTIVE: In obese postmenopausal women we assessed leptin and adiponectin, high-sensitive C-reactive protein (hsCRP), serum lipids and lipoxidative stress products: oxidized LDL (oxLDL) and malondialdehyde (MDA), in relation to impaired glucose tolerance (IGT). METHODS: Thirty-eight overweight/obese postmenopausal women were included in the study. Eighteen with normal glucose metabolism (NGT) and twenty with IGT, as it is diagnosed by OGTT. Serum leptin, adiponectin, hsCRP and MDA were measured at time 0 and 120 min of OGTT while total-cholesterol, LDL, HDL, triglycerides, oxLDL and anti-oxLDL autoantibodies at time 0. Insulin resistance (HOMA)/sensitivity (QUICKI) indexes were estimated. RESULTS: In subjects with NGT, hsCRP was positively correlated with fasting leptin and HOMA, while in subjects with IGT negatively with QUICKI. In both groups, hsCRP was positively correlated with fasting insulin, body mass index and waist circumference. Fasting adiponectin was positively associated with HDL in both groups and negatively with triglycerides in subjects with NGT as well as with serum glucose levels at time 120 min of OGTT in subjects with IGT. No association was observed between oxLDL and adipokines. A significant positive association was found between oxLDL and HOMA in subjects with IGT. During OGTT there was a significant increase of leptin and MDA levels in both groups. CONCLUSIONS: A relationship exists between obesity, insulin and sub-clinical inflammation. Leptin and lipid peroxidation are linked to hyperglycaemic state while oxLDL might be considered as a predictor of insulin resistance. Adiponectin could exert its antiatherogenic effect through HDL independently of the presence of IGT.

Ursolic acid triggers apoptosis and Bcl-2 downregulation in MCF-7 breast cancer cells.

In this report we determine the ability of ursolic acid (UA) to induce apoptosis and to modulate glucocorticoid receptor (GR) and Activator Protein-1 (AP-1) in MCF-7 cells. The UA-induced apoptosis (53 microM), the PARP cleavage, and the decrease in Bcl-2 protein (53 microM) support the notion that UA induces apoptosis through the intrinsic mitochondrial pathway. UA binds GR (relative binding affinity: 2.57) and translocates GR into nucleus, suggesting its potential as a GR modulator. UA had no effect on GRE- or TRE-driven gene expression. In summary, UA is a GR modulator and may be considered as a potential anticancer agent in breast cancer.

Circulating oxidized LDL levels, current smoking and obesity in postmenopausal women.

OBJECTIVE: The aim of the present study was to estimate circulating oxidized low-density lipoprotein (oxLDL) levels in postmenopausal women and evaluate their association with obesity and smoking status. DESIGN AND METHODS: The study included 135 postmenopausal women aged 52-75 years. Forty of them were overweight (BMI 32.4+/-6.4) and non-smokers (Group A), 40 non-overweight (BMI 22.6+/-1.8) and smokers (Group B) and 55 non-overweight (BMI 23.5+/-1.4) and non-smokers (Group C). oxLDL and antibodies against them (anti-oxLDL) were measured using ELISA. Serum total cholesterol, LDL, HDL and triglycerides were measured in an automated analyzer. RESULTS: Total cholesterol, LDL, HDL and oxLDL serum levels were significantly elevated in Group A as compared to Group B or C, as well as oxLDL in Group B in comparison to Group C (p<0.001). Triglycerides and anti-oxLDL were increased in Group A in comparison to Group C (p=0.043 and 0.023). Total cholesterol, LDL, triglycerides and anti-oxLDL did not differ between Groups B and C, while HDL was decreased in Group B as compared to Group C (p<0.001). A significant positive correlation was found between oxLDL and LDL in Group A (r=0.53, p<0.001) as well as in Group C (r=0.955, p

Walnut extract (Juglans regia L.) and its component ellagic acid exhibit anti-inflammatory activity in human aorta endothelial cells and osteoblastic activity in the cell line KS483.

Epidemiological studies suggest that the incidence of CVD and postmenopausal osteoporosis is low in the Mediterranean area, where herbs and nuts, among others, play an important role in nutrition. In the present study, we sought a role of walnuts (Juglans regia L.) in endothelial and bone-cell function. As the endothelial cell expression of adhesion molecules has been recognised as an early step in inflammation and atherogenesis, we examined the effect of walnut methanolic extract and ellagic acid, one of its major polyphenolic components (as shown by HPLC analysis), on the expression of vascular cell adhesion molecule (VCAM)-1 and intracellular adhesion molecule (ICAM)-1 in human aortic endothelial cells. After incubating the cells with TNF-alpha (1 ng/ml) in the absence and in the presence of walnut extract (10-200 microg/ml) or ellagic acid (10- 7-10- 5 m), the VCAM-1 and ICAM-1 expression was quantified by cell-ELISA. We further evaluated the effect of walnut extract (10-50 microg/ml), in comparison with ellagic acid (10- 9-10- 6m), on nodule formation in the osteoblastic cell line KS483. Walnut extract and ellagic acid decreased significantly the TNF-alpha-induced endothelial expression of both VCAM-1 and ICAM-1 (P < 0.01; P < 0.001). Both walnut extract (at 10-25 microg/ml) and ellagic acid (at 10- 9-10- 8 m) induced nodule formation in KS483 osteoblasts. The present results suggest that the walnut extract has a high anti-atherogenic potential and a remarkable osteoblastic activity, an effect mediated, at least in part, by its major component ellagic acid. Such findings implicate the beneficial effect of a walnut-enriched diet on cardioprotection and bone loss.

The effects of insulin sensitizers on the cardiovascular risk factors in women with polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in pre-menopausal women characterized by menstrual cycle disturbances, chronic anovulation, and clinical and/or biochemical hyperandrogenism. Although, the primary etiology of PCOS remains unknown, insulin resistance/hyperinsulinemia plays a pivotal role in the pathogenesis of the syndrome. A growing body of recent data support that women with PCOS have displayed an increased prevelance of cardiovascular disease (CVD) risk factors putting potentially at a hight risk for heart disease. Most of these CVD risk factors are etiologically correlated with insulin resistance/hyperinsulinemia, highlighting the role of insulin sensitizers in the therapeutic quiver for the chronic treatment of PCOS. In this review, we discuss the current literature on the CVD risk factors in PCOS and the influence of insulin sensitizers upon these risk factors.

Ursolic acid, a naturally occurring triterpenoid, demonstrates anticancer activity on human prostate cancer cells.

PURPOSE: Glucocorticoids are widely used as adjuvant therapy in hormonal refractory prostate cancer; their therapeutic role, however, remains unclear. Ursolic acid, a natural triterpene, structurally similar to dexamethasone, exhibits antitumor effects in various cell types. Our main objective was to investigate the effects of ursolic acid on cell viability, apoptosis and bcl-2 protein, in human hormone refractory and androgen-sensitive prostate cancer cells. METHODS: The ursolic acid-induced changes in cell viability, apoptosis and bcl-2 protein were examined in human hormone refractory prostate cancer PC-3 cells and androgen-sensitive LNCaP cells, by MTT assay, flow cytometry and western blot analysis, respectively. RESULTS: Ursolic acid inhibited significantly the cell viability and induced apoptosis in PC-3 cells at 55 microM and in LNCaP cells at 45 microM associated with a downregulation of bcl-2 protein. CONCLUSIONS: The antiproliferative and apoptotic effects of ursolic acid in PC-3 and LNCaP cells implicate its potential therapeutic use for the treatment of hormone refractory and androgen-sensitive prostate cancer. The downregulation of bcl-2 may be one of the molecular mechanisms via which it induces apoptosis in PC-3 and LNCaP cells.

Evaluation of estrogenic/antiestrogenic activity of Onobrychis ebenoides extract - interaction with estrogen receptor subtypes ERalpha and ERbeta.

A protective effect of plant extract from Onobrychis ebenoides on ovariectomy-induced bone loss in rats has been shown. To investigate the molecular mechanisms that underly the beneficial effect of O. ebenoides (Onb) on bone loss, we studied its potential to activate ER subtypes (ERalpha and ERbeta) on transiently transfected HeLa cells with HO-hERalpha or pSG5-hERbeta and 3xERE-TATA-Luc expression vectors. Its impact to stimulate differentiation and mineralization of osteoblasts (KS483 cell line) by Alizarin Red-S staining was also examined. Furthermore we sought to induce for its potential the IGFBP3, a known estrogen-dependent marker in MCF7 breast cancer cells. 17beta-Estradiol and the pure antiestrogen ICI182780 were included to serve as control samples of the estrogenic and antiestrogenic activity respectively. Our data revealed: (1) Onb extract displayed a significant estrogenic activity on both ERalpha and ERbeta subtypes. (2) It exhibited direct action on osteoblasts by inducing mineralization. (3) It showed estrogenic activity in MCF7 cells. These findings suggest that the beneficial effect of Onb extract on bone loss is mediated through an estrogen-like action via activation of ERalpha-ERE and ERbeta-ERE pathways and via direct action on the mineralization process of osteoblasts.

Estrogen receptor alpha gene polymorphism and systemic lupus erythematosus: a possible risk?

Estrogens and their receptors may play a role in the pathogenesis of systemic lupus erythematosus. Genetic alterations in the exon 8-coding region of the estrogen receptor alpha alter the intracellular signalling of estrogens, leading in enhanced or diminished activity. We investigated whether genetic alterations in exon 8 of ERalpha gene are associated with the occurrence and clinical features of lupus disease. The coding region of ERalpha exon 8 was subjected to mutation analysis using the polymerase chain reaction, denaturing gradient gel electrophoresis and sequence analysis, using DNA isolated from whole blood of 36 female patients and 38 healthy females. Clinical and laboratory parameters were available from the patients' files. We identified the codon 594 polymorphism either in homozygous for the wild type gene (ACG/ACG) or heterozygous (ACG/ACA), both in patients and healthy females. Statistical analysis of the genotype and allele distribution revealed that there was a significant difference (chi2 test, P = 0.02 and P = 0.04, respectively) between patients and healthy women. Odds ratio estimate revealed that carriers of ACG/ACA genotype have three-fold higher risk of developing lupus disease (OR = 3.129, 95% CI 1.181-8.292). Moreover, in patients the heterozygous genotype was associated with rash, mouth ulcers and serositis (Fisher's exact test, P = 0.055, P = 0.083, P = 0.065, respectively). The heterozygous patients were associated significantly with an early age at disease onset (ANOVA test, P < 0.05). We conclude that estrogen receptor alpha codon 594 genotype may influence the development of systemic lupus erythematosus at a younger age, as well as a certain disease clinical pattern.

Estrogen receptor alpha gene analysis in osteoporosis and familial osteoporosis.

Estrogens are important determinants of bone mineral density (BMD) mediating their effects via estrogen receptor alpha (ERalpha) and beta (ERbeta). The strong genetic predisposition to osteoporosis, and the fact that alterations in the aminoterminal region of ERalpha have been linked to bone disturbances, prompted us to identify genetic alterations in exon 1 and exon 2 of ERalpha in osteoporotic individuals. Sixty-two unrelated normal subjects (age 46.1+/-9.5 years) and 72 unrelated osteoporotic subjects (age 52.3+/-7.9 years) were studied. Their menopausal status was pre- and perimenopausal. We also included 30 related osteoporotic individuals (mother-daughter or sister-sister relationship) (age 46.2+/-12.8 years) belonging to 14 families who where also pre- and perimenopausal. DNA was extracted from peripheral blood, exons 1 and 2 were amplified by polymerase chain reaction (PCR) and were further submitted to denaturing gradient gel electrophoresis (DGGE), single stranded conformational polymorphism (SSCP), restriction fragment length polymorphism (RFLP) and sequence analysis. Bone turnover markers were also determined. Two polymorphisms were identified in exon 1 (codons 10 and 87) in both normal and osteoporotic women. Statistical analysis revealed no difference (P>0.05) in the ERalpha genotype frequencies within osteoporotic families as compared with the same genotypes in the unrelated normal or osteoporotic subjects. Codon 10, codon 87 polymorphisms were not related to BMD or bone turnover markers. No other mutations were found in exons 1 and 2 in all subjects studied. Genetic alterations in exons 1 and 2 of ERalpha are not associated to osteoporosis and familial osteoporosis. Moreover, the codon 10 and codon 87 polymorphisms do not seem to be correlated with BMD and bone turnover markers.

Molecular analysis of estrogen receptor alpha and beta in lupus patients.

BACKGROUND: In female patients with systemic lupus erythematosus (SLE), we identified estrogen receptor ERa, ERb and ERa variant transcripts in peripheral blood mononuclear cells (PBMC). Exon 1 and 2 of ERa gene was subjected to mutation analysis to assess whether possible nucleotide alterations are linked to the disease. METHODS: The whole coding sequence of ERa was analysed by reverse transcription polymerase chain reaction (RT-PCR) and cDNA sequencing in PBMC prepared from 19 SLE patients and 12 healthy females. ERa exon 1 and exon 2 were subjected to mutation analysis using DNA isolated from whole blood of 21 SLE patients and 29 healthy females. The aminoterminal coding sequence of ERb was also analysed by RT-PCR. RESULTS: Wild type ERa and ERa splicing variants with deletions in exons 2, 5 and 7 were detected both in healthy individuals and in SLE patients, with no qualitative difference in their expression among the two populations. In ERa exon 1, the polymorphisms identified codon 10 and codon 87, both in patients and in healthy individuals who were not associated with the disease. No other mutations were present in ERa exon 1 or ERa exon 2 in all subjects studied. ERb was expressed in both populations. CONCLUSION: PBMC of SLE patients express wild type ERa, ERb and the same ERa variants as do healthy individuals. Genetic alterations in exon 1 and exon 2 of the ERa gene are not linked with SLE disease.

The oestrogen receptor codon 10 polymorphism detected in breast cancer is also present in non-malignant cells.

The effect of oestrogens on oestrogen-receptive organs and cells is mediated via intracellular receptors (ERalpha and ERbeta). Oestrogen receptor gene polymorphisms in the region encoding the N-terminal portion of the protein are reportedly associated with pathological conditions including breast cancer, hypertension, spontaneous abortion and coronary heart disease. A silent mutation in codon 10 of exon 1, detected in ER-negative and ER-positive human breast cancer cell lines, in breast tumors and blood DNA from breast cancer patients, has been recognized as a polymorphic site. In this study we examined, by denaturing gradient-gel electrophoresis and DNA sequence analysis, the possible presence of a codon 10 polymorphic site in normal oestrogen target organs and cells such as the uterus (myometrium and endometrium), in the placenta and peripheral blood mononuclear cells and in a benign uterus tumour (leiomyoma). We have detected ER codon 10 polymorphism in these samples and have compared them to those observed in breast cancer samples. All tissues and cells studied were homozygous for the wild-type gene, and were heterozygous as well as homozygous for the codon-10-variant type. These results indicate that the presence of the codon-10-variant type is not a characteristic of breast cancer. Out current findings suggest that further investigations are warranted to elucidate the possible linkage of ER codon 10 polymorphism to physiological and pathological conditions.

Detection of oestrogen receptor variants in endometrium, myometrium, leiomyoma and peripheral blood mononuclear cells: comparison to variants present in breast cancer.

Oestradiol has mitogenic and regulatory effects on various organs and cells, mediated mainly by its nuclear receptor (ER). The presence of aberrant ER forms in Oestrogen-dependent tumours has been discussed in correlation with tumour progression. ER variants, generated by alternative splicing, have been detected in human breast cancer, but also in normal mammary glands, therefore their role in tumorigenesis has been questioned. We have investigated, by the use of the reverse transcription polymerase chain reaction amplification technique, the possible existence of ER variants in other normal oestrogen target organs and cells, such as uterus (myometrium and endometrium), in peripheral blood mononuclear cells and in a benign uterus tumour (leiomyoma). We have detected variant ER in these samples and have compared the variant profile to that observed in breast cancer. All tissues and cells studied expressed both wild-type ER and variant species. Variant forms encompassed ER with deletions of exons 2, 5 and 7. Variants with exon 5 deleted were detected only in peripheral blood mononuclear cells and in breast cancer. Variants with exons 2 and 7 deleted were present in all specimens tested. These results corroborate previous findings that the presence of ER variants is not a characteristic of breast cancer. The physiological significance and possible clinical relevance of the variant ER forms remain to be elucidated.