Efficacy and safety of once-monthly efpeglenatide in patients with type 2 diabetes: Results of a phase 2 placebo-controlled, 16-week randomized dose-finding study.

AIMS: To determine the optimal dose(s) of once-monthly administration of efpeglenatide, a long-acting glucagon-like peptide-1 receptor agonist (GLP-1RA), in patients with type 2 diabetes (T2D) inadequately controlled on metformin. MATERIALS AND METHODS: In this phase 2, randomized, placebo-controlled, double-blind trial (NCT02081118), patients were randomized 1:1:1:1 to subcutaneous efpeglenatide (8, 12 or 16 mg once monthly; n = 158) or placebo (n = 51). The 16-week treatment period included a 4-week titration phase with once-weekly efpeglenatide 4 mg, followed by one dose of efpeglenatide 8 mg once monthly and two doses of the assigned once-monthly dose. The primary endpoint was change in glycated haemoglobin (HbA1c) from baseline to week 17. RESULTS: All efpeglenatide doses significantly reduced HbA1c versus placebo (P < 0.0001 for all). Overall, the least squares mean difference in HbA1c reductions between efpeglenatide and placebo was -7.7 mmol/mol (-0.71%; baseline to week 17). At week 17, a significantly greater proportion of efpeglenatide patients had an HbA1c level <53 mmol/mol (<7%) versus placebo (48.7% vs. 30.6%; P = 0.0320). Significant body weight loss occurred across all efpeglenatide doses (placebo-corrected reduction -2.0 kg [efpeglenatide overall]; P = 0.0003). The safety profile was consistent with GLP-1RAs, with gastrointestinal (GI) disorders being the most common treatment-emergent adverse events. Fluctuations in effects on glucose levels and rates of GI events occurred between peak and trough efpeglenatide concentrations. CONCLUSIONS: Efpeglenatide once monthly (following once-weekly titration) has significant benefits with regard to HbA1c and weight reduction versus placebo in patients with T2D. Further studies are needed to evaluate the long-term efficacy and safety of efpeglenatide once monthly.

Effect of IL-6 on proliferation of human thyroid anaplastic cancer stem cells.

OBJECTIVE: To investigate the effect of IL-6 on the proliferation of thyroid cancer stem cells. METHODS: HTh74 and HTh74R thyroid cancer stem cells were cultured. The proliferation of thyroid cancer stem cells after IL-6 treatment was assessed by the MTT method. The effect of IL-6 on colony formation was observed by colony formation experiments. The expression of OCT4, ABCG2, CD133, and EMT markers was detected by real-time quantitative PCR. RESULTS: IL-6 promoted the proliferation of HTh74 and HTh74R thyroid cancer stem cells and enhanced sphere formation. However, anti-IL-6 inhibited the proliferation of cancer stem cells. IL-6 promoted colony formation by HTh74 and HTh74R cells and enhanced the expression of stem cell genes OCT4 and ABCG2. The expression of EMT markers E-cadherin was significantly decreased but the expression of vimentin and Snail was increased by IL-6 treatment. CONCLUSIONS: IL-6 promoted proliferation of thyroid cancer stem cells and colony formation, and increased characteristics of thyroid cancer stem cells and EMT. The proliferative effect of thyroid cancer stem cells depends on activation of the IL6/JAK1/STAT3 pathway. These effects may contribute to the development and metastasis of thyroid cancer.

Metformin and thyroid disease.

An intriguing area of research in thyroidology is the recently discovered association of insulin resistance with thyroid functional and morphological abnormalities. Individuals with hyperinsulinemia have larger thyroid gland and a higher prevalence of thyroid nodules and cancer. Accordingly, patients treated with metformin have a smaller thyroid volume and a lower risk of incident goiter, thyroid nodule and cancer. Multiple studies in vitro and in vivo have demonstrated that metformin can inhibit the growth of thyroid cells and different types of thyroid cancer cells by affecting the insulin/IGF1 and mTOR pathways. Besides, metformin treatment was associated with a decrease in the levels of serum thyroid-stimulating hormone (TSH) in diabetic patients possibly by enhancing the effects of thyroid hormones in the pituitary and activating the adenosine monophosphate-activated protein kinase (AMPK). Based on this evidence, metformin appears to be a promising therapeutic tool in patients with thyroid disease. More clinical studies are necessary to evaluate the clinical significance of metformin for the treatment of thyroid diseases.

1,25 dihydroxyvitamin D3 inhibits the proliferation of thyroid cancer stem-like cells via cell cycle arrest.

BACKGROUND: An anti-proliferative effect of vitamin D has been reported in different carcinomas, including thyroid cancer. Cancer stem cells (CSCs), a very small fraction of cancer cells, are widely believed to be responsible for cancer initiation, relapse and metastasis. OBJECTIVES: We addressed the question as to whether CSCs derived from the anaplastic thyroid carcinoma cell lines SW1736, C643, HTh74 and its doxorubicin- resistant subline HTh74R are also a target of vitamin D action. METHODS: The effect of calcitriol on growth of HTh74, HTh74R, SW1736 and C643 cell lines was investigated by cell viability assays. In stem-enriched cells derived from thyro-spheres cell cycle analysis and apoptotic assays were performed. Furthermore, the role of calcitriol in the formation of cancer thyro-spheres and its putative differentiation-inducing effect were analysed. RESULTS: CSCs isolated as thyro-spheres from all the four anaplastic thyroid carcinoma cells expressed vitamin D receptors as did their parental cells. Calcitriol inhibited proliferation of anaplastic thyroid carcinoma cells with a more pronounced effect on doxorubicin-resistant HTh74R cells, and it significantly reduced the capacity to form stem cell-derived spheres and decreased the size of these spheres that consist of CSCs and their progenitor cells. As revealed by cell cycle analysis, calcitriol induced G2/M phase arrest in thyro-sphere cells derived cells from HTh74, HTh74R and C643 but did not affect apoptosis. Finally, calcitriol altered morphology of CSCs. CONCLUSION: Calcitriol inhibited the growth of CSCs derived from anaplastic thyroid cancer cells. It may also exert a pro-differentiation effect in thyroid CSCs.

Synergistic anti-proliferative effect of metformin and sorafenib on growth of anaplastic thyroid cancer cells and their stem cells.

Sorafenib, a multikinase inhibitor has recently been approved for the treatment of radio-iodine refractory thyroid carcinoma. However, toxic side effects may lead to dose reduction. In the present study, we analyzed whether a combined therapy with metformin may allow a dose reduction of sorafenib without loss of effectiveness at the same time. In HTh74 anaplastic thyroid carcinoma (ATC) cells and its derived doxorubicin-resistant HTh74Rdox cell line, the growth inhibitory effect of sorafenib with or without metformin was investigated. Furthermore, an analysis of cell cycle arrest in response to sorafenib was performed and the ability of a combined treatment to induce apoptosis was analyzed. In addition, the effects on clonal growth and formation of stem cell-derived spheres were assayed. The influence of sorafenib and metformin on MAP kinase pathway was investigated by analysis of ERK phosphorylation. Sorafenib and metformin synergistically inhibited growth of the two thyroid cancer cell lines, with a more pronounced effect on the doxorubicin-resistant HTh74Rdox cell line. The two drugs also synergistically decreased sphere formation, which suggested a specific effect on thyroid cancer stem cells. The addition of metformin enabled a 25% dose reduction of sorafenib without loss of its growth inhibitory efficacy. Sorafenib and metformin synergistically decreased the proliferation of ATC cell lines and the outgrowth of their derived cancer stem cells. A combined treatment enabled a significant dose reduction of sorafenib. In respect to frequent toxic side effects, clinical studies in future should demonstrate whether the addition of metformin may be an advantage in the chemotherapy of patients with radio-iodine­resistant thyroid cancer.

Estrogen and its role in thyroid cancer.

Proliferative thyroid diseases are more prevalent in females than in males. Upon the onset of puberty, the incidence of thyroid cancer increases in females only and declines again after menopause. Estrogen is a potent growth factor both for benign and malignant thyroid cells that may explain the sex difference in the prevalence of thyroid nodules and thyroid cancer. It exerts its growth-promoting effect through a classical genomic and a non-genomic pathway, mediated via a membrane-bound estrogen receptor. This receptor is linked to the tyrosine kinase signaling pathways MAPK and PI3K. In papillary thyroid carcinomas, these pathways may be activated either by a chromosomal rearrangement of the tyrosine receptor kinase TRKA, by RET/PTC genes, or by a BRAF mutation and, in addition, in females they may be stimulated by high levels of estrogen. Furthermore, estrogen is involved in the regulation of angiogenesis and metastasis that are critical for the outcome of thyroid cancer. In contrast to other carcinomas, however, detailed knowledge on this regulation is still missing for thyroid cancer.

Effect of 17β-estradiol on proliferation of human thyroid stem cells / 中华内分泌代谢杂志

Objective To investigate the effect of 17β-estradiol on the proliferation of thyroid stem/ progenitor cells.Methods In thyroid stem/progenitor cells derived from nodular goiters,the effects of 17 β-estradiol on thyrosphere formation,estrogen receptor (ER) expression,cyclin D1 expression,and mitogen activated protein kinase (MPAK) pathway were analysed by BrdU ELISA,conventional and realtime PCR,immunofluorensence staining,and Western blot.Results 17β-estradiol induced thyrosphere formation and proliferation of thyroid stem/ progenitor cells.ER-α and ER-β were expressed in thyroid stem and progenitor cells with higher mRNA expression level of ER-α compared to differentiated thyrocytes (8.85-±0.81 vs 1.10 ±0.35,P＜0.01).Stimulation by 1 mmol/L 17β-estradiol increased cyclin D1 mRNA expression and ERK phosphorylation levels,which was blocked by an ER antagonist,ICI 182780.Conclusion Estrogen stimulated the growth of stem cells derived from thyroid nodules via estrogen receptor,suggesting the relevance of increased thyroid stem cell proliferation with higher prevalence of thyroid nodules in women.

Oestrogen action on thyroid progenitor cells: relevant for the pathogenesis of thyroid nodules?

Benign and malignant thyroid nodules are more prevalent in females than in males. Experimental data suggest that the proliferative effect of oestrogen rather than polymorphisms is responsible for this gender difference. This study analysed whether both differentiated thyroid cells and thyroid stem and progenitor cells are targets of oestrogen action. In thyroid stem/progenitor cells derived from nodular goitres, the ability of 17ß-oestradiol (E2) to induce the formation of thyrospheres and the expression of oestrogen receptors (ERs) and the effect of E2 on the growth and expression of markers of stem cells and thyroid differentiation (TSH receptor, thyroperoxidase, thyroglobulin and sodium iodide symporter (NIS)) were analysed. E2 induced thyrosphere formation, albeit to a lower extent than other growth factors. Thyroid stem and progenitor cells expressed ERα (ESR1) and ERß (ESR2) with eight times higher expression levels of ERα mRNA compared with the differentiated thyrocytes. E2 was a potent stimulator of the growth of thyroid stem/progenitor cells. In contrast, TSH-induced differentiation of progenitor cells, in particular, the expression of NIS, was significantly inhibited by E2. In conclusion, oestrogen stimulated the growth and simultaneously inhibited the differentiation of thyroid nodule-derived stem/progenitor cells. From these data and based on the concept of cellular heterogeneity, we hypothesize a supportive role of oestrogen in the propagation of thyroid stem/progenitor cells leading to the selection of a progeny of growth-prone cells with a decreased differentiation. These cells may be the origin of hypofunctioning or non-functioning thyroid nodules in females.

Metformin inhibits goitrogenous effects of type 2 diabetes.

OBJECTIVE: Data on the association between type 2 diabetes mellitus (T2DM) and thyroid volume are sparse. An experimental study demonstrated an inhibitory effect of metformin on the growth of human thyroid cells. So far no study on humans has investigated potentially modulating effects of metformin on the association between T2DM and thyroid volume. Therefore, we investigated these effects in a population-based cohort study. DESIGN AND METHODS: We used data from the Study of Health in Pomerania and included 2570 individuals for cross-sectional and 1088 individuals for longitudinal analyses. T2DM was defined by physician-diagnosed self-report or intake of antidiabetic medication. RESULTS: In the cross-sectional data, females with T2DM treated with antidiabetic medication other than metformin had a larger thyroid volume (ß=4.69; 95% CI 1.87 to 7.50) and a higher odds ratio (OR) for goiter (OR=1.71; 95% CI 1.05 to 2.79) than females without T2DM, whereas in males, no such association was detected. In females or males treated with metformin, T2DM was not associated with thyroid volume or goiter. In longitudinal analyses, incident T2DM not treated with metformin was significantly associated with a higher risk for incident goiter in the total population (incidence rate ratio (IRR)=1.70; 95% CI 1.10 to 2.91). Individuals with T2DM having changed from metformin to other antidiabetic agents during follow-up also had a higher risk for incident goiter than individuals without T2DM (IRR=2.71; 95% CI 1.74 to 4.20). CONCLUSIONS: We demonstrate an inhibitory effect of metformin on prevalent and incident goiter. Anti-goitrogenous effects of metformin add to the general benefits of metformin treatment of T2DM.

Metformin inhibits growth of thyroid carcinoma cells, suppresses self-renewal of derived cancer stem cells, and potentiates the effect of chemotherapeutic agents.

CONTEXT: Hyperinsulinemia and insulin resistance are the major reasons for a higher prevalence of several cancer entities in type 2 diabetes mellitus and obesity. Metformin exerts a growth-inhibitory effect by reducing hyperinsulinemia and by a direct cellular action. OBJECTIVE: We investigated the effect of metformin on growth of differentiated human thyroid cells, anaplastic thyroid carcinoma cells, a doxorubicin-resistant thyroid carcinoma cell line, and thyroid cancer stem cells. DESIGN: The antimitogenic effect of metformin was studied in thyroid cells derived from goiters and in thyroid carcinoma cell lines by analysis of cell growth, cell cycle progression, and apoptosis. Furthermore, the influence of pretreatment with insulin or with chemotherapeutic agents on metformin-induced growth inhibition was investigated in thyroid carcinoma cells, in a doxorubicin-resistant thyroid carcinoma cell line, and in derived carcinoma stem cells. RESULTS: Metformin showed an antimitogenic effect by inhibition of cell cycle progression and induction of apoptosis. In addition, metformin antagonized the growth-stimulatory effect of insulin, inhibited clonal cell growth, reduced thyroid cancer sphere formation, and potentiated the antimitogenic effect of chemotherapeutic agents such as doxorubicin and cisplatin on undifferentiated thyroid carcinoma cells. Remarkably, the antiproliferative effect of metformin was even found in a doxorubicin-resistant thyroid carcinoma cell line. The growth-inhibitory effect of metformin was, however, not restricted to differentiated thyroid cells and undifferentiated thyroid carcinoma cells but was also demonstrated in thyroid carcinoma cancer stem cells. CONCLUSIONS: Metformin markedly diminished growth stimulation by insulin and showed an additive antimitogenic effect to chemotherapeutics agents. Therefore, our results suggest this drug as adjuvant treatment for thyroid cancer in type 2 diabetic patients.

Doxorubicin fails to eradicate cancer stem cells derived from anaplastic thyroid carcinoma cells: characterization of resistant cells.

Current chemotherapy with doxorubicin fails to eradicate anaplastic thyroid cancer or even to stop tumor progress which may be due to the failure of these drugs to effectively target putative cancer stem cells. To test this hypothesis, anaplastic thyroid cell lines were characterized by FACS for their content of cancer stem cells, their in vitro sphere-forming capacity and their expression of multidrug resistance transporters of the ABC gene family which may confer drug resistance to the cells. Cells were treated with doxorubicin in short-term and long-term culture up to 6 months to establish a resistant cell line. The survival of cancer and cancer stem cells and the differential expression of transporters were analyzed. Anaplastic thyroid cancer cell lines that consisted of 0.4-0.8% side population cells, expressed ABCG2 and multi-drug-resistant 1 (MDR1) transporters. Treatment with doxorubicin gradually killed the non-side population of cancer cells derived from anaplastic thyroid carcinoma cells. This conferred a growth advantage to cancer stem cells which in turn overgrew the culture. Resistant cell line consisted of a 70% side population fraction enriched with Oct4-positive cancer stem cells. Inhibition of ABCG2 and/or MDR1 revealed that resistance of cancer stem cells to doxorubicin may be mainly due to the expression of these ABC transporters that were highly up-regulated in the resistant subline. The poor outcome of chemotherapy with doxorubicin in anaplastic thyroid carcinoma may be partly explained by up-regulation of ABCG2 and MDR1 transporters that confers resistance to cancer stem cells. Thus an effective treatment of anaplastic thyroid cancer has not only to destroy cancer cells that represent the bulk of tumor cell population but also cancer stem cells that may drive tumor progression.

Limitations of the HOMA-B score for assessment of beta-cell functionality in interventional trials-results from the PIOglim study.

BACKGROUND: Drugs with unspecific stimulating effects on beta-cell secretion increase the homeostasis model assessment (HOMA)-B score, indicating improved beta-cell "function." We investigated whether the beta-cell protection provided by adding pioglitazone (PIO) to glimepiride (GLIM) in comparison to up-titrating the GLIM dose alone is reflected by appropriate changes in several measures of beta-cell function, including HOMA-B score. METHODS: This double-blind, parallel prospective 6-month study was performed with 82 patients (47 men, 35 women; age, 61 +/- 9 years; duration of disease, 5.3 +/- 4.4 years; body mass index, 32.6 +/- 6.0 kg/m(2); hemoglobin A1c [HbA1c], 7.3 +/- 0.7%) with GLIM monotherapy (1-3 mg). They were randomized to receive a GLIM + PIO combination with up-titration (2 mg + 30 mg/4 mg + 30 mg/4 mg + 4 mg) or to remain on GLIM (up-titration 4/5/6 mg). Observation parameters determined at baseline and end point included HOMA-B, HOMA-IR, HbA1c, glucose, insulin, and intact proinsulin. RESULTS: There was a slight increase in the HOMA-B score in the GLIM group but not in the GLIM + PIO arm (baseline/end point: for GLIM, 71 +/- 48/88 +/- 64; for PIO + GLIM, 74 +/- 56/69 +/- 52). Improvements in the other observation parameters were predominantly detected in the PIO + GLIM group (HbA1c, 7.20 +/- 0.61%/6.36 +/- 0.90%; HOMA-IR, 7.0 +/- 4.5/4.1 +/- 2.1; intact proinsulin, 12.4 +/- 10.3/7.6 +/- 4.8 pmol/L [all P < 0.05 vs. baseline]) compared with the GLIM group (HbA1c, 7.45 +/- 0.69%/7.15 +/- 0.97% [P < 0.05]; HOMA-IR, 7.4 +/- 4.5/7.5 +/- 4.3 [not significant]; intact proinsulin, 17.3 +/- 21.6/16.3 +/- 15.5 pmol/L [not significant]). CONCLUSIONS: The PIO + GLIM combination led to overall improvement of laboratory biomarkers for beta-cell function, except for HOMA-B. Glimepiride up-titration had no such effects but increased the HOMA-B score. HOMA-B seems to provide misleading results when used as a diagnostic tool in patients treated with sulfonylurea drugs. A corrective term for consideration of proinsulin in the HOMA-B equation may address this limitation.

Stem cells derived from goiters in adults form spheres in response to intense growth stimulation and require thyrotropin for differentiation into thyrocytes.

OBJECTIVE: This study aimed to analyze under which conditions quiescent stem cells derived from human goiters can be propagated to outgrow and whether these cells have retained the capacity to differentiate into thyroid cells. DESIGN: Stem cells were isolated by fluorescence-activated cell sorting as a side population by the Hoechst 33342 efflux technique. Growth pattern of stem cells and cocultures of stem cells with thyrocytes grown as monolayer and in Matrigel was investigated. Expression of stem cell markers, endodermal markers, and thyroid-specific markers was analyzed by RT-PCR. In stem cell-derived thyrocytes, embedded in collagen to form follicles, TSH-dependent (125)iodide uptake was measured. RESULTS: Stem cells were isolated as a side population from a non-side population fraction that consisted of endodermal marker-positive cells and thyroid cells. Intense growth stimulation of stem cells in coculture with thyrocytes resulted in formation of nonadherent, three-dimensional spheres that consisted of highly proliferating stem cells with their characteristic expression profiles. In response to TSH and serum, sphere-derived progenitor cells differentiated into thyrocytes that expressed paired box gene 8, thyroglobulin, sodium iodide symporter, thyroid-stimulating hormone receptor, and thyroperoxidase mRNA and showed TSH-dependent (125)iodide uptake. CONCLUSION: Quiescent stem cells derived from goiters can be propagated to form spheres that consist of highly proliferating stem cells that are able to differentiate TSH dependently into thyroid cells. Compared with thyrocytes, stem cells display a much higher proliferation rate on acute growth stimulation, which may suggest a putative role of the offspring of stem cells in the chronic growth factor-stimulated nodular transformation of the thyroid.

Expression of endoderm stem cell markers: evidence for the presence of adult stem cells in human thyroid glands.

OBJECTIVE: Adult stem cells have been detected in several human tissues. The object of this study was to investigate whether they also occur in the human thyroid gland. DESIGN: The expression of the stem cell marker Oct- 4 and the early endodermal markers GATA-4 and HNF4alpha was analyzed in histologic slides and cultured cells derived from goiters, in the FRTL5 cell line, and the HTh74, HTC, C643, and XTC133 thyroid carcinoma cell lines. MAIN OUTCOME: Stem cell markers were detectable in all primary cultures whereas in the differentiated FRTL5 cell line no expression was observed. Expression of stem cell marker mRNA was not affected by thyrotropin (TSH) stimulation and did not decrease when cells underwent several passages. Immunostaining of cultured cells and of histologic slides of goitrous tissues showed only single cells that were immunoreactive for Oct-4, GATA-4, and HNF4a. Expression of Oct-4 but not of endodermal marker GATA-4 was also detectable in some thyroid carcinoma cell lines. Fluorescence-activated cell sorter (FACS) analysis demonstrated cell populations that were positive for either Oct-4, GATA-4, or HNF4alpha but negative for thyroglobulin. When these putative, FACS-sorted stem cell populations were further analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR), expression of all stem cell markers and of Pax8 but not of thyroglobulin mRNA was detectable. CONCLUSIONS: These data provide evidence for the presence of adult stem and precursor cells of endodermal origin in the human thyroid gland.

Hyperplasia versus adenoma in endocrine tissues: are they different?

The traditional view holds that hyperplasia of endocrine glands is secondary to oversecretion of a trophic hormone. However, in most cases, the mechanism underlying this growth is the spontaneous proliferation of benign neoplasias. Pathologists still depend on subtle morphological criteria to delineate and further classify these tumours. Owing to their variable architecture, a bewildering nomenclature has emerged for these tumours, exemplified by the many names applied to the goitrous thyroid gland: hyperplasia, adenomatous goitre, adenomatoid nodules, benign nodular thyroid disease, adenoma, etc. This article reviews the evidence suggesting that: (1) the varied types of benign neogeneration of endocrine tissue, the spectrum of which ranges from 'simple hyperplasia' to 'true adenoma', involve the same process; (2) even clonality of a growing lesion cannot distinguish hyperplasia from neoplasia; and (3) the basic processes in both cases are not different from those that cause benign tumours in other organs.