Cell Death Induction by the Indirubin Derivative 7BIO and the BH3 Mimetic Drugs ABT-737 and GX15-070 in Medullary Thyroid Carcinoma Cells.

PURPOSE: Patients with advanced and metastasized medullary thyroid carcinoma are difficult to treat since tumor cells do not respond to chemotherapeutic treatment and external radiation. Direct induction of cell death is a new therapeutic approach to therapy-resistant tumor cells. In this study we analyzed the effect of the indirubin-derivative 7BIO and the BH3 mimetic drugs ABT-737 and GX15-070 on cell death induction of TT medullary thyroid carcinoma cells. METHODS: TT medullary thyroid carcinoma cell line was treated with 7BIO, ABT-737 and GX15-070. Cell viability was analyzed by MTT assay, while cell death was determined by caspase 3/7 activity, measurement of caspase cleavage products and lactate dehydrogenase liberation assay. LC3B cleavage was analyzed by western blot. RESULTS: Incubation with all 3 drugs efficiently decreased the number of viable TT cells with IC50 values of 4.1 µM (7BIO), 0.19 µM (ABT-737) and 0.23 µM (GX15-070). The BH3 mimetic ABT-737 caused an apoptotic cell death with caspase activation as expected, while 7BIO- and GX15-070-treatment led to a mixed kind of cell death, where caspase activation was detected but had no effect on viability of TT cells. LC3 conversion as a biochemical marker of autophagic cell death was observed after GX15-070 treatment while LDH release pointed to involvement of necrosis after treatment with all 3 drugs. CONCLUSION: The BH3 mimetic drugs ABT-737 and GX15-070 efficiently killed TT medullary thyroid carcinoma cells with low IC50 values, while the indirubin-derivative 7BIO was also effective but with a higher IC50 value. Although the exact kind of cell death and target molecules of 7BIO and GX15-070 are not yet defined, direct induction of cell death may be a new therapeutic option in medullary thyroid carcinoma cells.

Expression of the cAMP binding protein EPAC1 in thyroid tumors and growth regulation of thyroid cells and thyroid carcinoma cells by EPAC proteins.

The thyrotropin receptor-cAMP pathway is central in growth regulation of thyroid cells and thyroid tumorigenesis, and it regulates expression of thyroid specific genes. Recently, 2 new protein kinase A-independent cAMP effectors named EPAC1 and 2 were described that activate additional intracellular pathways. The aim of our study was to investigate the role of EPAC proteins in growth regulation of thyroid cells and thyroid carcinomas. EPAC1 expression was investigated immunohistochemically in tissues of various thyroid tumors. Utilizing MTT assay, the effect of EPAC stimulation on proliferation in thyroid carcinoma cells and in non-transformed rat FRTL5 cells was investigated. The activation of intracellular signaling pathways was examined by RAP pull-down assay and Western blots. EPAC1 expression was strong in non-oxyphilic follicular thyroid adenomas and carcinomas and in follicular papillary thyroid carcinomas. It was moderate in oxyphilic follicular tumors and classical and tall cell papillary carcinomas. In contrast, EPAC1 expression was low in poorly differentiated carcinomas and very low in anaplastic carcinomas. Thyroid carcinoma cell lines showed no or very weak EPAC1 expression and exhibited no growth-promoting effect after EPAC stimulation. Non-transformed rat FRTL5 cells were growth-stimulated by an EPAC-specific cAMP-analogue and showed EPAC-dependent activation of RAP, ERK, and p70S6 kinase. EPAC1 expression and cellular response to EPAC activation in rat FRTL5 cells reflect cellular responses to cAMP and TSH stimulation in non-transformed thyroid cells. In undifferentiated thyroid carcinomas, loss of EPAC1 expression may be in accordance with the loss of thyroid-specific functions and the loss of responsiveness of the TSHR-cAMP pathway.

B-type natriuretic peptide predicts stroke of presumable cardioembolic origin in addition to coronary artery calcification.

BACKGROUND AND PURPOSE: B-type natriuretric peptide (BNP) is a marker of cardiac dysfunction that is released from myocytes in response to ventricular wall stress. Previous studies suggested that BNP predicts stroke events in addition to classical risk factors. It was suggested that the BNP-associated risk results from coronary atherosclerosis or atrial fibrillation. METHODS: Three thousand six hundred and seventy five subjects from the population-based Heinz Nixdorf Recall study (45-75 years; 47.6% men) without previous stroke, coronary heart disease, myocardial infarcts, open cardiac valve surgery, pacemakers and defibrillators were followed up over 110.1 ± 23.1 months. Cox proportional hazards regressions were used to examine BNP as a stroke predictor in addition to vascular risk factors (age, gender, systolic blood pressure, low-density lipoprotein, high-density lipoprotein, diabetes, smoking), renal insufficiency, atrial fibrillation/known heart failure and coronary artery calcification. RESULTS: Eighty-nine incident strokes occurred (80 ischaemic, 9 hemorrhagic). Subjects suffering stroke had significantly higher BNP values at baseline than the remaining subjects [26.3 (Q1; Q3 = 12.9; 51.0) vs. 17.4 (9.4; 31.4); P < 0.001]. In a multivariable regression, log10 BNP was an independent stroke predictor [hazard ratio 1.96, 95% confidence interval (CI) 1.13-3.41; P = 0.017] in addition to age (1.24 per 5 years, CI 1.04-1.49; P = 0.016), systolic blood pressure (1.25 per 10 mmHg, CI 1.14-1.38; P < 0.001), smoking (2.05, CI 1.24-3.39; P = 0.005), atrial fibrillation/heart failure (2.25, CI 1.05-4.83; P = 0.037) and computed-tomography-based log10 (coronary artery calcification + 1) (1.47, CI 1.15-1.88; P = 0.002). Log10 BNP predicted stroke in men but not women, both in subjects ≤65 and >65 years. In subsequent analyses, BNP discriminated the incidence of cardioembolic stroke (P for trend = 0.001), but not stroke of macroangiopathic (P = 0.555), microangiopathic (P = 0.809) or unknown (P = 0.367) origin. CONCLUSIONS: BNP predicts presumable cardioembolic stroke independent of coronary calcification.

Cardiac risk in patients with treatment naïve, first-line medically controlled and first-line surgically cured acromegaly in comparison to matched data from the general population.

INTRODUCTION: Coronary risk factors in patients with acromegaly after first-line transsphenoidal surgery (TSS) or first-line somatostatine analogue (SSA) treatment have rarely been examined. Aim of this study was an evaluation of cardiovascular risk factors and left ventricular hypertrophy (LVH) in 3 different patient groups with treatment naïve, active (ACT), first-line medically controlled (MED) and first-line surgically treated (SUR) acromegaly and a calculation of the Framingham Weibull Risk Score (FS). DESIGN: Retrospective comparative matched case-control study. PATIENTS & METHODS: 40 acromegalic patients (cases aged 45-74 years, 23 men) were matched with respect to age and gender to 200 controls from the general population. 13 patients had treatment-naïve acromegaly (ACT), 12 patients were SSA treated (MED) and 15 patients were operated by TSS (SUR). Coronary risk factors were assessed after 12 months of treatment by interviews and direct laboratory measurements. Only patients normalized for IGF-I in MED and SUR group were included. FS and odds ratios (OR) from multiple conditional logistic regression (matched for age and gender, adjusted for BMI) were calculated. RESULTS: Compared to matched controls ACT patients had higher HbA1c levels (6.9±1.4 vs. 5.5±0.7% (p<0.0001)) and an increased prevalence of left ventricular hypertrophy (LVH) (30.8 vs. 3.2% (p=0.007). MED and SUR groups were similar for gender, age, disease duration and IGF-I levels at diagnosis. Compared to matched controls, MED patients had a significantly increased diastolic blood pressure (89±9 vs. 79±11 mmHg (p=0.001), prevalence of LVH (41.7 vs. 1.7% (p<0.0001), prevalence of diabetes mellitus (33.3 vs. 10.0% (p=0.03)), higher HbA1c levels (6.8±1.3 vs. 5.5±0.7% (p=0.0005)) and a higher FS (21.2±9.7 vs. 12.4±7.7% (p=0.002), OR 1.11 [1.02-1.21] (p=0.01)) while in the SUR group only higher prevalences of LVH (40.0 vs. 4.1% (p<0.0001)) and HbA1c levels (6.4±1.2 vs. 5.5±0.8% (p=0.006)) were found compared to controls. CONCLUSION: When comparing treatment naive, medically treated and surgically cured patients with acromegaly to age- and gender-matched subjects from the general population, we have found an increased cardiovascular risk in patients at 12 months after first-line SSA treatment but not in patients after first-line surgery.

[Influence of the calcitonin assay on the definition of biochemical cure in patients with medullary thyroid carcinoma]. / Assay-Abhängigkeit der Calcitonin-Zielbereiche für "biochemisch geheilte" Patienten mit medullärem Schilddrüsenkarzinom.

AIM: Calcitonin (hCT) is an important diagnostic parameter in medullary thyroid carcinoma (MTC). We determined the variability of the reference ranges of several currently available immunometric assays for "biochemically cured" MTC patients. PATIENTS, METHODS: We compared six assays [Nichols ICMA, Biomerica IEMA, Immulite 2000 (Siemens), Calcitonin-IRMA magnum (Medipan), SELco-IRMA (Medipan) and Calcitonin IRMA (Medgenix)] in subgroups of 198 patients with differentiated thyroid cancer (DTC) after total thyroidectomy as a model for curatively treated MTC patients. In addition, hCT was measured after pentagastrin stimulation in 13 DTC patients and 13 patients with MTC. RESULTS: The basal hCT concentrations were below the detection limit of the respective assay in 100% of all thyroidectomized DTC patients for Nichols ICMA (n = 138) and Immulite 2000 (n = 60), in 97% for Biomerica IEMA (n = 57), and in 85% for IRMA magnum (n = 20). However, basal hCT was mostly within the reference range in Selco-IRMA (n = 20) and Medgenix IRMA (n = 76). In all DTC patients and 9/13 MTC patients the pentagastrin stimulated hCT was below the detection limit for the Nichols ICMA and Immulite 2000, all four MTC patients with elevated stimulated hCT developed a recurrence during follow-up. CONCLUSIONS: For assays with high monomer specificity (Nichols ICMA, Biomerica IEMA, Immulite 2000, to a lesser degree IRMA magnum) biochemical cure is defined by basal and stimulated calcitonin levels below the detection limit. For assays with low monomer specificity (SELco-IRMA, IRMA Medgenix) calcitonin levels in the reference range of patients without thyroid diseases are consistent with "biochemical cure".

Measurement of urinary free cortisol by current immunoassays: need for sex-dependent reference ranges to define hypercortisolism.

Urinary free cortisol (UFC) is used to assess disease activity in hypercortisolemic patients. However, reference ranges are often lacking, especially with respect to potential confounding variables. This study analyzed upper limits of normal (ULN, mean + 2 SD) for 2 newer immunoassays, using gas chromatography-mass spectrometry (GC-MS) as reference method. Each 10 healthy subjects were grouped by age (18-29; 30-49; ≥ 50 years), BMI (< 25; ≥ 25 kg/m2), and sex, resulting in a total of 120 controls (60 males; age: 39.3±1.3 years; BMI: 25.9±0.4 kg/m2). ULN were calculated for a radioimmunoassay (RIA, Immunotech) and an electrochemiluminescence immunoassay (ECLIA, Roche) and applied to 12 hypercortisolemic patients (4 males; age: 53.1±3.1 years; BMI: 29.1±1.8 kg/m2). To determine degradation, samples were stored at 4°C (without light) or 22°C (with and without light) for 0, 24, and 72 h. Cortisol concentrations were significantly correlated: r=0.88 for RIA vs. ECLIA, r=0.75 for RIA vs. GC-MS, and r=0.77 for ECLIA vs. GC-MS (always p<0.0001). For each procedure, multiple stepwise regression analysis identified sex as the only significant predictor, resulting in sex-dependent ULN (males vs. females): 294 vs. 208 nmol/24 h (RIA), and 379 vs. 277 nmol/24 h (ECLIA). These ULN classified samples from patients as hypercortisolemic in 100% (RIA) and 95% (ECLIA). Different storage conditions over 72 h did not alter UFC levels significantly. Results of the 3 procedures were well correlated, and the use of assay- and sex-specific ULN allowed excellent identification of hypercortisolic states. UFC is stable over 72 h irrespective of the storage conditions applied.

Prevalence of anterior pituitary dysfunction in patients following traumatic brain injury in a German multi-centre screening program.

INTRODUCTION: We determined the prevalence of anterior pituitary dysfunction in a multi-centre screening program across five German endocrine centres in patients rehabilitating from TBI (GCS<13). PATIENTS & METHODS: 246 patients (39+/-14 yrs; 133 males, 12+/-8 months after TBI) underwent a series of baseline endocrine tests with central assessment of TSH, free T4, prolactin, LH, FSH, testosterone (m), estradiol (f), cortisol, GH, and IGF-I. If IGF-I was <-2 SDS dynamic testing was performed. GHD was defined according to BMI-dependent cut-off values for GH response to GHRH+arginine of <4.2, <8.0 and <11.5 ng/ml in obese, overweight and lean subjects, respectively, or <3 micro g/l in ITT. Hypocortisolism was suggested when basal cortisol was <200 nmol/l and confirmed by ITT (peak<500 nmol/l). RESULTS: In TBI patients some degree of impaired pituitary function was shown in 21% (n=52/246). Total, multiple and isolated deficits were present in 1%, 2% and 18%, respectively. 19% had an IGF-I of <-1 SDS, 9% of <-2 SDS. In 5% GHD was confirmed. 9% had hypogonadism. 4% had hypocortisolism and 1% of patients had confirmed ACTH-deficiency. 12% had TSH-deficiency. SUMMARY: In summary, in this large series carried out on an unselected group of TBI survivors we have found hypopituitarism in every fifth patient with predominantly secondary hypogonadism and hypothyreosis. Regarding somatotrope insufficiency IGF-I is decreased in 50% of GHD patients. CONCLUSION: These findings strongly suggest that patients who suffer head trauma should routinely undergo endocrine evaluation.

Differential miRNA expression profiles in variants of papillary thyroid carcinoma and encapsulated follicular thyroid tumours.

BACKGROUND: Recent studies showed a significant upregulation of distinct microRNAs (miRNAs) in papillary thyroid carcinoma (PTC). The objective of this study was to explore whether this upregulation could also be assigned to distinct histomorphological variants of PTC, especially the follicular variant and other encapsulated follicular thyroid tumours. METHODS: We used total RNA of 113 formalin-fixed paraffin-embedded tissues of 50 PTCs ((10 conventional type (PTC-CT), 10 tall cell variants (PTC-TCVs), 30 follicular variants (PTC-FVs)), 10 follicular adenomas (FAs), 10 multinodular goitres (MNGs), 21 follicular thyroid carcinomas and 22 well-differentiated tumours of unknown malignant potential (WDT-UMP) to analyse the miRNA expression pattern of five selected miRNAs (146b, 181b, 21, 221 and 222) using RT-PCR TaqMan miRNA assay to explore the diagnostic utility of this method. RESULTS: The mean values of the expression pattern of all miRNAS in PTCs show a statistically significant difference from those in MNG and FA with fold changes up to 90 for miRNA 146b, P<0.001. No differences in expression pattern could be showed between MNG and FA. The PTC-FVs differ significantly from FA in all five miRNAS, from MNG in three and from WDT-UMP in one miRNA with fold changes between 1.7 and 21.2, but failed to be of diagnostic value regarding individual cases with substantial overlaps. CONCLUSION: We conclude that analysis of a set of five selected miRNAS distinguish common variants of PTC from FA/MNG but failed to be a useful diagnostic method in individual and doubtful cases, especially in the differential diagnosis of encapsulated follicular thyroid tumours.

Association of impaired fasting glucose and coronary artery calcification as a marker of subclinical atherosclerosis in a population-based cohort--results of the Heinz Nixdorf Recall Study.

AIMS/HYPOTHESIS: Atherosclerosis and cardiovascular diseases are often present at the time of diagnosis of type 2 diabetes mellitus. Whether subclinical atherosclerosis can be detected in the pre-diabetic (borderline fasting hyperglycemia) state is not clear. This study investigated the association of impaired fasting glucose (IFG) and coronary artery calcification (CAC), a marker of subclinical atherosclerosis, among participants without a history of coronary heart disease or manifest diabetes mellitus. METHODS: Study participants (aged 45-75 years) of the population-based Heinz Nixdorf Recall Study were categorised into those with normal fasting glucose (glucose <6.1 mmol/l) and those with IFG (glucose >or=6.1 to <7.0 mmol/l), excluding participants with a history of CHD or diabetes mellitus. CAC was assessed by electron-beam computed tomography, and risk factors were assessed by extended interviews, anthropometric measurements and laboratory tests. Various CAC cut-off points were used in multiple logistic and ordinal logistic regression models to estimate ORs and 95% CIs. RESULTS: Of the 2,184 participants, more men had IFG than did women (37% vs 22%). Participants with IFG showed a higher prevalence of CAC > 0 (men OR 1.90, 95% CI 1.33-2.70; women 1.63, 1.23-2.15). Risk factor adjustment weakened this association in both sexes (men 1.63, 1.12-1.36; women 1.26, 0.93-1.70). When the age- and sex-specific 75th percentile was used as the cut-off point for CAC, the association further decreased in men (1.10, 0.81-1.50), but became stronger in women (1.41, 1.02-1.94). CONCLUSIONS/INTERPRETATION: These data support the hypothesis that CAC is already present in the pre-diabetic state and that IFG has a modest and independent impact on the atherosclerotic process. Biological sex appears to modify the association between IFG and CAC.

Are symptoms of depression more common in diabetes? Results from the Heinz Nixdorf Recall study.

AIMS: To estimate the association between depressive symptoms and Type 2 diabetes, as well as previously undetected diabetes, in a large population-based sample in Germany and to determine associated variables. METHODS: We used baseline data on 4595 participants (age 45-75 years, 50.2% women) from the German Heinz Nixdorf Recall study, a population-based, prospective cohort study which started in 2000. Diabetes mellitus was assessed by self report (physician diagnosis or medication), undiagnosed diabetes based on blood glucose levels. Depressive symptoms were assessed using the Center for Epidemiologic Studies Depression (CES-D) scale short form (cut-off >or= 15 points). We fitted multiple logistic regression models. RESULTS: The prevalence of diagnosed and previously undetected diabetes was 9.3% (95% confidence interval 8.2-11.6) and 7.6% (6.6-8.8) in men and 6.0% (5.1-7.1) and 3.2% (2.5-4.0) in women, respectively. Compared with non-diabetic women, the prevalence of depressive symptoms was not significantly different in diabetic women (age-adjusted odds ratio, 95% confidence interval 1.48; 0.98-2.24) and women with undiagnosed diabetes (0.67; 0.33-1.36). In men, the prevalence of depressive symptoms tended to be lower in diabetic than in non-diabetic subjects (0.62; 0.35-1.09), but the depressive symptoms were significantly less frequent in men with undiagnosed diabetes (0.30; 0.13-0.70). The pattern remained after further adjustment. Significant associations with depressive symptoms were found for co-morbidities and living without a partner in both women and in men, and for body mass index and activity level in women only. CONCLUSIONS: After adjustment for relevant covariates, the association between depressive symptoms and Type 2 diabetes was heterogenous in our population-based study. In subjects with undiagnosed diabetes, however, depressive symptoms were less frequent in men. Co-morbidities and psychosocial conditions are strongly associated with depressive symptoms.

Expression and mutation analysis of the tyrosine kinase c-kit in poorly differentiated and anaplastic thyroid carcinoma.

Poorly differentiated and anaplastic thyroid carcinoma are aggressive tumors failing to res-pond to conventional therapy. Imatinib mesylate offers an effective therapeutic option in patients with various types of malignancies by inhibiting tyrosine kinases such as c-kit. In this study we investigated c-kit expression in anaplastic and poorly differentiated thyroid carcinoma compared to differentiated carcinoma and adenoma and the presence of c-kit mutations. In total, 224 thyroid tissues were analyzed by immunohistochemistry. Mutation analysis of exon 9, 11, 13, and 17 of the c-kit gene was performed in anaplastic and poorly differentiated carcinoma. c-Kit expression was negative in all anaplastic thyroid carcinoma, while c-kit expression of poorly differentiated carcinoma showed a high variability with a more intense staining in tumors showing obvious differentiated malignant follicular tumor areas. Differentiated carcinoma showed a slight, but not significantly stronger c-kit expression than poorly differentiated carcinoma. All tumors revealed wild type sequences of c-kit gene in exons 9, 11, 13, and 17. The low or lacking c-kit expression in undifferentiated thyroid carcinoma together with the lack of mutations argue against a crucial role of c-kit in thyroid carcinoma cell proliferation. Further molecular targets of imatinib mesylate have to be analyzed to estimate a potential benefit of this drug for patients with dedifferentiated thyroid carcinoma.

Effects of estrogen replacement therapy on bone and glucose metabolism in a male with congenital aromatase deficiency.

Little is known about the impact of estrogen replacement therapy for bone formation, glucose metabolism and hormonal parameters on males with aromatase deficiency. Transdermal estrogen (TE) replacement was initiated at 100 microg/week in months 0-3, 50 microg/week in months 3-6, 25 microg/week in months 6-12, 75 microg/week in months 12-24, and 25 microg/week in months 24-36 to substitute for the deficiency in a 27-year-old homozygous male with a mutation on the CYP19 gene. Estradiol levels increased from<10 at baseline to 45, 12, 27 and 17 pg/ml (normal range 10-50) after 6, 12, 24 and 36 months, and inversely correlated to LH and FSH levels. Testosterone levels changed from 31.2 nmol/l at baseline to 3.8, 22.1, 7.1 and 22.0 nmol/l (9.5-30) after 6, 12, 24 and 36 months, respectively, and correlated closely to basal and stimulated LH and FSH levels at 100 microg GnRH. Bone maturation progressed, and metacarpal and phalangeal epiphysis closed after 12 months. Spongiosa-hydroxyapatite of the radius assessed by quantitative computed tomography changed from 52 to 83, 51, 69 and 71 mg/cm3 (120-160); bone mineral density of the lumbar spine assessed by dual energy X-ray-absorptiometry (normal value>1.150) increased from 0.971 (T-Score -2.24) to 1.043 (-1.64), 1.065 (-1.46), 1.128 (-0.93) g/cm2 and 1.021 (-1.82) after 6, 12, 24 and 36 months of TE, respectively. Osteocalcin as a bone formation parameter and aminoterminal collagen type I telopeptide as a bone resorption parameter increased during high-dose estrogen supplementation, and then decreased during the lower doses. Lipoprotein (a) increased from 20 mg/dl at baseline to 60 and 62 mg/dl after 6 and 12 months, and then decreased to 24 and 25 mg/dl after 24 and 36 months, respectively, while total cholesterol, HDL, LDL and triglycerides did not change. AUC glucose decreased continuously after oral glucose load, and HOMA IR reached its lowest value the 75 microg weekly estradiol dose. This study confirms the role of estrogens in achieving bone mineralization and maturation in human males. Additionally, estradiol has dual negative feedback sites that on the hypothalamus to decrease GnRH pulse frequency, and on the pituitary to decrease responsiveness to GnRH. The improvement in glucose metabolism after estrogen replacement therapy suggests a probable role of sex steroids in insulin sensitivity. The optimal weekly dose of transdermal estrogen replacement for normalizing estrogen levels and maintain bone mass in adult males with aromatase deficiency may be 50-75 microg spread over two doses.