Zahlreiche epidemiologische Studien zeigen für das Antidiabetikum Metformin anti-Tumor-Effekte, die bisher ansatzweise für verschiedene Tumorzelllinien in vitro bestätigt wurden. Ziel der vorliegenden Arbeit war, den antiproliferativen Effekt von Metformin an sechs humanen kolorektalen Karzinomzelllinien (Co-lo678, Colo741, HT29, HCT116, LS174T, RKO) zu untersuchen. Zur Identifizierung eines anti-proliferativen Effektes von Metformin bei kolorektalen Karzinomzellen wurde ein Konzentrationsbereich von 1 bis 100 mmol/L untersucht. Die für die Tumorzelllinien bestimmte halbmaximale inhibitorische Konzentration (IC50) von Metformin reichte von 0,8 mmol/L (HT29) über 16 mmol/L (Colo678) bis >40 mmol/L (RKO). Der IC50 für die beiden nicht-transformierten Kontrollzellen lag ebenfalls über 40 mmol/L. Um die Untersuchungen in vitro bei relevanten tumorphysiologischen Bedingungen durchführen zu können, die der Situation von Tumorzellen in einem soliden Tumor wie dem kolorektalen Karzinom möglichst nahekommt, wurden die Zellen bei unterschiedlichen Sauerstoff- und Glukosekonzentrationen kultiviert. Ein permanent erhöhter Blutzuckerspiegel hat sich als grundlegender Faktor für malignes Zellwachstum erwiesen. Der anti-proliferative Effekt von Metformin in Normoxie war nahezu unbeeinflusst von den untersuchten Glukosekonzentrationen (2,5; 5,0; 11 mmol/L). Dagegen nahm in Hypoxie im Vergleich zu Normoxie der antiproliferative Effekt von Metformin bei 4 von 6 Tumorzelllinien um mehr als das Doppelte ab. Ein zentrales Target der Metformin-Wirkung stellt die AMPK, ein wichtiges Enzym für den Energiestoffwechsel der Zelle, dar. Ihre phosphorylierte Form war in den Tumorzelllinien nachzuweisen, doch der anti-proliferative Effekt von Metformin war nicht durch den AMPK-Inhibitor Compound C zu hemmen. Ein antiproliferativer Effekt von Metformin war bei kolorektalen Karzinomzellen nachzuweisen, doch bleiben die durch Metformin ausgelösten molekularen Mechanismen in der Tumorzelle weiterhin wenig verstanden. / Anti-tumor effects of the antidiabetic drug metformin were demonstrated by numerous epidemiological studies, but only verified in a few in vitro studies. The purpose of this study was to examine the antiproliferative effect of metformin in six human colorectal carcinoma cell lines (Colo678, Colo741, HT29, HCT116, LS174T, RKO). To identify an antiproliferative effect a concentration range between 1 to 100 mmol/L was evaluated. The range of the calculated half maximal inhibitory concentration (IC50) for the tumor cell lines reached from 0,8 mmol/L (HT29) over 16 mmol/L (Colo678) to >40 mmol/L (RKO). For both non-transformated control cell lines the IC50 reached also over 40 mmol/L. The cells were cultivated in different oxygen and glucose concentrations for the in vitro simulation of relevant tumorphysiological conditions. Permanently increased blood sugar levels have proven to be a significant parameter for malignant cell growth. The antiproliferative effect of metformin in normoxia remained uneffected by the tested glucose concentrations (2,5; 5,0; 11 mmol/L). In contradiction hypoxia showed a more than 50% decrease of the antiproliferative effect of metformin in 4 out of 6 tumor cell lines. The AMPK - an important enzyme for energy metabolism - acts as a central target of metfomin. The phosphorylated condition of AMPK was detected in the tumor cell lines, despite that the AMPK inhibitor compound C could not affect the antiproliferative effect of metformin. The antiproliferative effect of metformin on colorectal cell lines was verified, while the molecular mechanism in tumor cells remain insufficiently understood.

Aging is a major risk factor for a variety of diseases, including metabolic disease and cancer. Accumulating evidence suggests that the regulation of developmental traits might have long-term effects on aging and health conditions later in life. The current project focuses on understanding the underlying genetic and molecular mechanisms. Nuclear receptor interacting protein 1 (NRIP1, also named RIP140) is a key regulator that modulates transcriptional activity of variety of transcription factors, including estrogen receptor and NF-κB. We previously identified that NRIP1 plays an important role in development and aging. The global depletion of Nrip1 (Nrip1-/-) significantly reduced pre-weaning survival, delayed female sexual maturation, improved insulin sensitivity at young, middle, and old age, as well as increased the longevity of female mice. In the current project, using adipose tissue- derived stem cells (ASCs), we found that the benefits of suppressing NRIP1 on aging and longevity may be related to reduced inflammation, delayed cell senescence and cell growth, and improved stem cell maintenance. The elucidation of the role of NRIP1 in female reproduction, metabolism, inflammation, and cell senescence, encouraged us to further investigate the role of NRIP1 in breast cancer. Importantly, epidemiological studies of human populations found that an early age at menarche significantly associates with risk of breast cancer (BC). During development, depletion of NRIP1 delays female sexual maturation and suppresses the development of mammary glands. Our previous study showed that NRIP1 expression was elevated in breast cancer, and that the depletion of NRIP1 could suppress the onset and progression of breast cancer. Combining this study with the findings of suppressed inflammation and delayed cell senescence, we hypothesized that Nrip1 knockout may have anti-BC effect by improving the stroma cell function and lowering the inflammation in local microenvironment. In this study, we found that NRIP1 is not only involved in tumorigenesis of breast cancer, but also in the cancer progression and the development of drug resistance. Interestingly, suppressing Nrip1 in a tamoxifen-resistant breast cell line restored drug sensitivity. The mechanism of anti-cancer effect may be related to suppressed NF-κB expression and activity, as well as reduced expression of aromatase.While identifying genes that are involved in development and longevity is important to aging research, searching for geroprotective drugs for clinical use may be a more realistic way to improve metabolic traits and delay aging in humans. Metformin is a widely used drug for treating type 2 diabetes (T2D) that has a good safety profile. Also, metformin treatment has been linked to a reduced risk of cancer and cancer-related mortality in retrospective investigations. In this study, heterogeneous mice (UM-HET3) were treated with metformin between the ages of 15 and 56 days. The results show that body weight and food consumption were increased, and sexual maturation was delayed in females. Tail length and circulating insulin-like growth factor 1 (IGF1) levels were significantly increased. Glucose tolerance was improved, but no significant difference in insulin tolerance was found. Circulating adiponectin and insulin levels were altered by metformin treatment. Analysis of quantitative insulin sensitivity check index (QUICKI) suggests that metformin treatment increased insulin sensitivity. These results revealed that treating mice early in life with metformin alters development and metabolism. Importantly, the effects of metformin treatment at an early age may have long-term impacts on health and aging-related traits.

Humans have never before lived as long and we have never had as many cures and treatments for diseases as now. But we still spend the last years of our lives sick with age-related diseases. Some scientists say it is time to grasp the problem by its roots – which in this case is aging itself. A lot of studies show that life can be prolonged in model organisms with the diabetes medication metformin. In recent years, metformin has proven to both expand lifespan and inhibit age-related diseases such as cancer and stroke. Metformin can do this by five different mechanisms; 1) inhibition of mitochondrial complex I, which is a part of the electron transport chain, 2) inhibition of the accumulation of reactive oxygen species, which in turn protects DNA from damage and mutations, 3) activation of AMPK, a kinase that is activated in the body when energy levels are low, 4) decrease of signaling of IGF-1, a growth factor that has been linked to the development of tumors, and 5) inhibition of mTOR, a regulator of cell metabolism, growth, proliferation, survival and autophagy. Studies are constantly proving metformin to be useful in both treatment of diseases and in inhibiting aging. New studies like TAME (Targeting Aging with Metformin), where older patients will try out metformin fors everal years, will shed more light on whether metformin might have the same effect in humans. Metformin could potentially provide a new future of health care where we not only treat diseases, but also prolong aging itself.

Indiana University-Purdue University Indianapolis (IUPUI) / Objectives: The purpose was to evaluate pain scores (SF-36 BPS) among pre-diabetic patients on metformin or placebo to determine if patients on metformin therapy report less pain (higher SF-36 BPS) than patients on placebo. Study design: A descriptive retrospective review of pain scores was conducted using secondary data analyses of the Diabetes Prevention Program (DPP) and Diabetes Prevention Program Outcomes Study (DPPOS) conducted from 1996 to 2008. Patients were randomly assigned to placebo, low (850 mg/day) or high dose (1700 mg/day) metformin groups. Pain scores using the SF-36 BPS standard version were taken before randomization and annually (year one through four). Results: Out of 3,819 patients that participated in the original study, 1,056 patients met the current study criteria. The metformin group included 506 patients and the placebo group included 550 patients. With an alpha level of 0.05 for all analyses, baseline pain scores between the metformin group and placebo group showed no significant difference. Year two showed significance between placebo and metformin pain scores (75.2 vs 78.6). All other years were not significant. Comparing low and high dose metformin and placebo groups, years one, two and three displayed significant differences in pain scores. In years one and two, the high dose metformin group reported less pain than the placebo group (80.7 vs 77.7; 80.1 vs 75.2) and the low dose metformin group (80.7 vs 71.8; 80.1 vs 68.6). In year three, the high dose metformin group had less pain than the low dose metformin group (78.4 vs 70.5).

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19227号 / 医博第4026号 / 新制||医||1011(附属図書館) / 32226 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 坂井 義治, 教授 松原 和夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Methylmercury (MeHg) is a global pollutant that can elicit a range of adverse health effects in both humans and wildlife populations. Humans are often exposed to MeHg through the consumption of contaminated seafood. Developing fetuses are especially susceptible to the effects of MeHg as it can cross the blood-brain barrier and the placenta. At high doses in utero MeHg causes developmental disorders and congenital disabilities, but long-term low-dose effects are still not fully known. Using a culture model of cerebral cortex development, our lab has shown that low-dose MeHg promotes premature neuronal differentiation. Autism spectrum disorder (ASD) has been associated with prenatal MeHg exposure and is correlated with neuronal overproduction, but a cause-effect relationship has not been shown. In this thesis, I aim to test the hypothesis that prenatal exposure to low-dose MeHg can cause ASD-like symptoms in the offspring following premature neuronal differentiation. My results showed that adult mice prenatally exposed to MeHg exhibited key ASD characteristics including impaired communication, reduced sociability, and increased restrictive repetitive behaviours. Furthermore, I explored the underlying cellular and molecular mechanism that promotes premature neuronal differentiation caused by prenatal MeHg exposure. To reverse the MeHg-induced premature neuronal differentiation, I utilized metformin, an FDA-approved diabetes drug. Overall, these findings provide insights into the toxicology of MeHg and its relationship with ASD etiology, including the underlying mechanism, and a potential therapeutic strategy.

Für das klinische Management des Prostatakarzinoms werden nuklearmedizinische Verfahren zunehmend relevant. Bildgebung und Therapie, welche gegen das Prostataspezifische Membranantigen (PSMA) gerichtet sind, werden bereits im klinischen Alltag angewendet. Weitere potenzielle Biomarker des Prostatakarzinoms, wie beispielsweise der CXC-Motiv-Chemokinrezeptor 4 (CXCR4) und der Somatostatinrezeptor Typ 2 (SSTR2), werden zudem als nuklearmedizinische Zielstrukturen diskutiert. Vorangegangene Arbeiten legten einen Zusammenhang zwischen dem Ausmaß der PSMA-Expression und der Sensitivität gegenüber Docetaxel in Prostatakarzinomzellen nahe. Ein Ziel der vorliegenden Arbeit war, diesen Mechanismus genauer zu untersuchen. Dabei wurden die Aktivität onkogener Signalwege, die Proliferation und die CXCR4- sowie die Androgenrezeptor (AR)- Expression in Prostatakarzinomzelllinien mit unterschiedlicher PSMA-Expression durchflusszytometrisch quantifiziert. Im zweiten Projektteil sollte der Einfluss von Metformin und verschiedener, bereits in der Prostatakarzinomtherapie angewandter Medikamente (Docetaxel, Dexamethason, Abirateron und Enzalutamid), auf die Expression von PSMA, CXCR4 und SSTR2 untersucht werden. Die Quantifizierung der Expression erfolgte mittels Durchflusszytometrie. Ein kausaler Mechanismus für den Zusammenhang zwischen PSMA-Expression und Docetaxel-Sensitivität konnte in dieser Arbeit schließlich nicht hergestellt werden. Es zeigten sich jedoch vor allem Expressionsmodulationen von PSMA und CXCR4. Mittels Docetaxel konnte z.B. bei C4-2 Zellen eine Verdopplung der PSMA-Expression und eine Verdreifachung der CXCR4-Expression erreicht werden. Darüber hinaus zeigte die Behandlung mit Abirateron eine deutliche Heraufregulation der PSMA- Expression bei LNCaP und C4-2 Zellen, sowie eine Zunahme der CXCR4- Expression bei allen untersuchten Zelllinien. Sollte sich der Einfluss der medikamentösen Behandlung auf die Expression von PSMA und CXCR4 bestätigen, kann dies zukünftig zur verbesserten und individualisierten Diagnostik und Therapie von Prostatakarzinompatienten beitragen. / Nuclear medicine methods are becoming increasingly relevant for the clinical management of prostate carcinoma. Imaging and therapy directed against the prostate specific membrane antigen (PSMA) are already used in clinical practice. Other potential biomarkers of prostate cancer, such as CXC motif chemokine receptor 4 (CXCR4) and somatostatin receptor type 2 (SSTR2), are also being discussed as nuclear medicine targets. Previous work suggested a relationship between the level of PSMA expression and sensitivity to docetaxel in prostate cancer cells. One aim of the present work was to further investigate this mechanism. The activity of oncogenic signalling pathways, proliferation and CXCR4 expression as well as androgen receptor (AR) expression in prostate carcinoma cell lines with different PSMA expression were quantified by flow cytometry. In the second part of the study, the influence of metformin and various drugs already used in prostate carcinoma therapy (docetaxel, dexamethasone, abiraterone and enzalutamide) on the expression of PSMA, CXCR4 and SSTR2 was assessed. Expression was quantified by flow cytometry analysis. A causal mechanism for the connection between PSMA expression and docetaxel sensitivity could, eventually, not be established in this work. However, expression modulations were primarily found for PSMA and CXCR4. Using docetaxel, for instance, a doubling of PSMA expression and a tripling of CXCR4 expression could be achieved in C4-2 cells. In addition, treatment with abiraterone showed a significant upregulation of PSMA expression in LNCaP and C4-2 cells, as well as an increase in CXCR4 expression in all cell lines examined. If the influence of drug treatment on the expression of PSMA and CXCR4 is confirmed, this may contribute to improved and individualised diagnostics and therapy of prostate cancer patients.

Metformin is the most common first-line oral therapeutic agent used in the treatment of type-2 diabetes. Because of its widespread use, metformin has been increasingly detected in wastewater effluent. It is partially bio-transformed into guanylurea is subsequently released into aquatic environments. Since the literature concerning the effect of metformin and guanyl urea on early life stage of fish is scant, the aim of this research was to understand the potential influence of metformin and guanylurea on developmental, cardiometabolic and behavioral responses in zebrafish embryos, from the 4 cell stage (3 hours post fertilization, hpf) to first feed (120 hpf). To this end, embryos were exposed to environmentally relevant (0.4, 4, 40 μg·L−1) and supra-environmental (400 and 4000 μg·L−1) concentrations of the two chemicals. Metformin caused an increased mortality and spinal abnormalities in all concentrations compared to controls. and increased pericardial and yolk sac edema at the highest tested concentration. Metformin did not cause alterations in hatch or heart rate over the examined developmental stages. In addition, metformin did not cause alterations in general swimming, light-dark movement, startle response or thigmotaxis, irrespective of exposure concentration. Exposure to guanylurea over the same developmental stages caused a significant difference in mortality at 40 μg·L−1 only. Guanylurea did not cause alterations to any of the other tested endpoints. Our data suggests that metformin and guanylurea caused modest impacts to embryonic development of zebrafish at these concentrations. / Thesis / Master of Science (MSc) / Pharmaceuticals have been detected at the ng to µg L-1 range in aqueous environments for decades. These compounds are designed to be biologically active at low concentrations and can cause elicit adverse effects in non-target species. Among the more recently detected compounds are the antihyperglycemic drug metformin and its biotransformation product (guanylurea), which have been the focus of few studies in fish. This thesis addresses multiple knowledge gaps by examining the potential impacts of metformin and guanylurea during the embryonic and early larval zebrafish period (3-120 hours post-fertilization). Exposure to metformin resulted in increased mortality and abnormalities. Guanylurea exposure increased mortality at one dose. We suggest that metformin and guanylurea cause modest effects in developing larval zebrafish.

Thesis (DMed (Obstetrics and Gynaecology))--Stellenbosch University, 2008. / Chapter 1 presents a literature study on the diagnostic debate of PCOS. The literature study includes a discussion of the recent Rotterdam consensus statement regarding the diagnosis of PCOS. This is followed by a discussion on the essential work-up of the patient presenting with PCOS. Finally, chapter 1 presents a discussion on the complexity of the different variations in women presenting with PCOS. Chapter 2 is a literature review on ovulation induction methods in patients who present with PCOS. This literature study puts special emphasis on the different available methods used for ovulation induction in women with PCOS and the profounding effect weight loss will have in managing these patients. This chapter also addresses the use of newer agents, like aromatase inhibitors (Letrozole), and the current role of each of these agents in ovulation induction protocols. Chapter 3 is a literature overview on the effect of Metformin in Clomiphene-resistant PCOS women. The inclusion criteria of this review was all prospective randomized trials where Metformin was added for ovulation in the Clomiphene-resistant PCOS patient. The data is presented as a metaanalysis. Chapter 4 is a prospective randomise control trial to evaluate the benefit of metformin if added to Clomiphene in a primary ovulation induction protocol in comparison to Clomiphene alone. This chapter also evaluates all factors influencing ovulation outcome. Finally in the discussion section all the recent studies published addressing this topic were reviewed. Chapter 5 is a literature review to evaluate the classification systems for semen parameters and the in vivo fertility potential. This data is also used to establish fertility/subfertility thresholds for semen parameters. This chapter also presents the results of a prospective and retrospective study of the semen analysis of the partners of women with PCOS. We believe that this population presents the best reference group to study the semen profile of the general male population. Chapter 6 is a summary of the results of these studies and serves as an evidence based approach for ovulation induction in women with PCOS.

Background: Two-thirds of U.S. adult women are overweight or obese. High body mass index (BMI) and adult weight gain are risk factors for a number of chronic diseases, including postmenopausal breast cancer. The higher postmenopausal breast cancer risk in women with elevated BMI is likely to be attributable to related metabolic disturbances including altered circulating sex steroid hormones and adipokines, elevated pro-inflammatory cytokines, and insulin resistance. Metformin is a widely used antidiabetic drug that has demonstrated favorable effects on metabolic disturbances and as such may lead to lower breast cancer risk in obese women. Further, the anti-proliferative effects of metformin suggest it may decrease breast density, an accepted biomarker of breast cancer risk. Methods/design: This is a Phase II randomized, double-blind, placebo-controlled trial of metformin in overweight/obese premenopausal women who have elements of metabolic syndrome. Eligible participants will be randomized to receive metformin 850 mg BID (n=75) or placebo (n=75) for 12 months. The primary endpoint is change in breast density, based on magnetic resonance imaging (MRI) acquired fat-water features. Secondary outcomes include changes in serum insulin levels, serum insulin-like growth factor (IGF)-1 to insulin-like growth factor binding protein (IGFBP)-3 ratio, serum IGF-2 levels, serum testosterone levels, serum leptin to adiponectin ratio, body weight, and waist circumference. Exploratory outcomes include changes in metabolomic profiles in plasma and nipple aspirate fluid. Changes in tissue architecture as well as cellular and molecular targets in breast tissue collected in a subgroup of participants will also be explored. Discussion: The study will evaluate whether metformin can result in favorable changes in breast density, select proteins and hormones, products of body metabolism, and body weight and composition. The study should help determine the potential breast cancer preventive activity of metformin in a growing population at risk for multiple diseases.