<p><strong><em>Purpose</em></strong></p> <p>To examine the potential of the anti-diabetic agent Metformin (MET) to enhance responses of NSCLC to ionizing radiation (IR).</p> <p><strong><em>Experimental Design</em></strong></p> <p>Human NSCLC A549, H1299 and SK-MES cells were treated with IR, MET or the mTOR inhibitor rapamycin and subjected to proliferation, clonogenic, immunoblotting, cell cycle and apoptosis assays. A549 and H1299 cells were grafted into flanks of immunosuppressed mice and treated with MET and/or IR. Tumours were analyzed by immunoblotting and immunohistochemistry.</p> <p><strong><em>Results</em></strong></p> <p>MET(2.5uM-5mM) caused dose-dependent inhibition of proliferation (10-70%)in all lines, inibited clonogenic survival and sensitized cells to IR. In A549 cellsMET caused inhibition of proliferation comparable to rapamycin, stimulated expression and activation of the ATM and AMPK-p53-p21^cip1and inhibited the Akt-mTOR-4-EBP1 pathway.MET caused G1 arrest of cell cycle, enhanced apoptosis and induced sustained DNA repair foci of gH2AX. MET and IR alone inhibited xenograft growth and combined treatment enhanced that further. IR and MET induced sustained enhancement of expression and activity of ATM-AMPK-p53-p21^cip1and inhibitionof Akt-mTOR-4-EBP1 pathways in tumours also. MET reduced expression of angiogenesis and enhanced expression of apoptosis markers in both control and radiated tumours.</p> <p><strong><em>Conclusions</em></strong></p> <p>Clinically achievable(uM) doses ofMET inhibit human NSCLC cell and tumour growth and sensitize them to IR.This is accompanied by desirable modulation of molecular signals, inhibition of angiogenesis and induction of apoptosis. Our results suggest that MET could be a clinically useful adjunct to radiotherapy in NSCLC and support clinical investigation of MET in combination with radiotherapy.</p> / Master of Science (MSc)

<p>Mnogi lekovi registrovani za razne druge indikacije mogu da deluju selektivno na tumorske receptore, signalne puteve, metaboličke procese, bioenergetske faktore, enzime, proteine, gene koji reguli&scaron;u proliferaciju, apoptozu i neoangiogenezu tumora ne pogađajući ove procese kod zdravih ćelija. Uvođenje novih lekova je izrazito dug, složen i skup proces istraživanja. Kori&scaron;ćenjem principa otkrivanja antikancerskog efekta kod već registrovanih lekova za druge indikacije, direktno se utiče na skraćivanje vremena i tro&scaron;kova istraživanja. Eksperimentalno je ispitana efikasnost antitumorskog delovanja mebendazola, metformina, itrakonazola, diklofenaka, nitroglicerina i deoksiholne kiseline na fibrosarkom hrčka izazvan BHK21/C13 tumorskom ćelijskom linijom praćenjem veličine i histologije lečenih tumora. Eksperimentalno je ispitana mogućnost primene deoksiholne kiseline, nitroglicerina, kofeina i itrakonazola kao adjuvansa u kombinaciji sa pojedinim ispitivanim lekovima (metformin, itrakonazol, diklofenak) za lečenje fibrosarkoma hrčka. Kako je ispitivanje vr&scaron;eno na mladuncima imladim hrčkovima i kako su sarkomi najče&scaron;ći u dečijem uzrastu, definisanje potencijalne antikancerske uloge ispitivanih lekova se odnosi prvenstveno na njihovu primenu u pedijatriji. Pokazano je da metformin, kombinacije metformina sa kofeinom, metformina sa itrakonazolom i metformina sa nitroglicerinom deluju u pogledu svih ispitivanih parametara tumora antitumorski na fibrosarkom hrčka. Kofein, itrakonazol i nitroglicerin pojačavaju antitumorsko dejstvo metformina na fibrosarkom hrčka. Tokom svih eksperimenata realizovanih u okviru ove disertacije, pokazalo se da nije bilo delotvornog tretmana, koji ne sadrži metformin.</p> / <p>Many drugs registered for various other indications can act selectively to tumor receptors, signaling pathways, metabolic processes, bioenergetic factors, enzymes, proteins, genes that regulate proliferation, apoptosis, and neoangiogenesis of the tumor without affecting these processes in the healthy cells. The introduction of new drugs is a very long, complex and expensive process of research. Using the principle of detecting the anticancer effect in already registered drugs for other indications, directly affects the reduction of time and cost of research. The efficacy of mebendazole, metformin, itraconazole, diclofenac, nitroglycerin and deoxycholic acid antitumor activity on hamster fibrosarcinoma induced experimentally by the BHK21/C13 tumor cell line was tested by monitoring the size and histology of the treated tumors. The possibility of using deoxycholic acid, nitroglycerin, caffeine and itraconazole as an adjuvant in combination with investigated drugs (metformin, itraconazole, diclofenac) for the treatment of hamster fibrosarcoma has been experimentally tested. As the examination was carried out on young cubs and young hamsters and that sarcomas are the most common in childhood, defining the potential anti-cancer role of the investigated drugs relates primarily to their application in pediatrics. Metformin, combinations of metformin with caffeine, metformin with itraconazole and metformin with nitroglycerin have shown antitumor action on the hamster fibrosarcoma in terms of all tested tumor parameters. Caffeine, itraconazole and nitroglycerin increase the antitumor effect of metformin on the hamster fibrosarcoma. During all the experiments carried out within this dissertation, there has been no effective treatment, which does not contain metformin.</p>

<p>Prema podacima iz 2011 godine, u Srbiji je približno 630 000 ljudi (8,6%) obolelo od dijabetes melitus-a, a procenjuje se da će taj broj porasti na 730 000 (10,2%) do 2030 godine. Preko 90% obolelih ima tip 2 dijabetes melitus (T2DM). Prva linija medikamentne terapije predstavljaju bigvanidi čiji je najznačajniji predstavnik metformin. Prema literaturnim podacima, u oko 10-30% sluĉajeva, kontinuirana upotreba metformina ima za posledicu smanjenu intestinalnu apsorpciju vitamina B12. Tačan patofiziolo&scaron;ki mehanizam koji dovodi do metforminom indukovane malapsorpcije vitamina B12 nije u potpunosti ispitan i poznat i postoji nekoliko aktuelnih teorija s ciljem obja&scaron;njenja ovog kompleksnog problema. Cilj rada je bio utvrđivanje nivoa, dinamike, trenda i učestalosti promena vitamina B12, holotranskobalamina (B12 aktiv), homocisteina i folne kiseline tokom kontinuirane primene metformina tokom godinu dana. Studija praćenja je sprovedena u Centru za laboratorijsku medicinu a u saradnji sa Klinikom za endokrinologiju, dijabetes i bolesti metabolizma, Kliničkog centra Vojvodine. Ovom studijom je obuhvaćeno 50 ispitanika obolelih od T2DM a u momentu uvođenja metformina. Svim ispitanicima je određivana koncentracija vitamina B12, B12 aktiva, homocisteina i folne kiseline, u momentu uvođenja terapije kao i nakon 4, 8 i 12 meseci primene metformina. Za dvanaest meseci kontinuirane primene metformina, utvrđen je kontinuirani pad i redukcija vrednosti ukupnog vitamina B12 za 25.29 %, odnosno vrednosti B12 aktiva za 23.26 %. U toku ispitivanja, utvrđen je kontinuirani trend porasta vrednosti homocisteina u krvi, s statistički značajnim porastom vrednosti homocisteina nakon osam meseci primene metformina. Po&scaron;av&scaron;i od predpostavki da metformin istovremeno blokira apsorpciju vitamina B12 u gastrointestinalnom traktu kao i raspoloživost iz postojećih, tkivnih rezervi, zatečene količine ovog vitamina u ciljnim ćelijama se postepeno redukuju i tro&scaron;e, rezultujući krajnjem snižavanju nivoa metabolički aktivnih oblika kobalamina, te posledičnoj akumulaciji homocisteina kako u ćelijskom, tako i u vanćelijskom prostoru. Na osnovu dobijenih rezultata ispitivanja može se predložiti opservacija nivoa ukupnog vitamina B12 i homocisteina u krvi pre uvođenja metformina u terapiju tipa 2 dijabetes melitusa kao i dvanaest meseci nakon toga. Na osnovu nivoa jednogodi&scaron;njeg pada koncentracija ukupnog vitamina B12, porasta koncentracija homocisteina, kao i drugih kliničkih i laboratorijskih parametara, može se razmatrati opcija uvođenja supstitucione terapije vitaminom B12 ili dalja opservacija nivoa vitamin B12 u krvi i ćelijskom prostoru.</p> / <p>According to data from 2011, in Serbia, approximately 630.000 people (8.6%) were diagnosed with diabetes mellitus, and it is estimated that this number will increase to 730.000 (10.2%) by 2030. Over 90% are type 2 diabetes mellitus (T2DM) patients. The first line of medication therapy is metformin. According to the literature data, in about 10-30% of cases, continuous use of metformin causes impared intestinal absorption of vitamin B12. The exact pathophysiological mechanism leading to metformin induced malabsorption of vitamin B12 has not been fully known, and there are several current theories to explain this complex problem. The aim of this study was to determine the level, dynamics, trend and frequency of changes in blood levels of total vitamin B12, holotranscobalamin (B12 active), homocysteine and folic acid during continuous application of metformin, over a year. The study was carried out at the Center of Laboratory Medicine in cooperation with the Clinic for Endocrinology, Diabetes and Metabolic Diseases, Clinical Center of Vojvodina. This study included 50 T2DM patients at the time of the introduction of metformin therapy. Levels of vitamin B12, holotranscobalamin, homocysteine and folic acid are determined before and after 4, 8 and 12 months of metformin administration, to all subjects. After a year of metformin use, the level of total vitamin B12 has been reduced by 25.29%, as well as holotranskobalamin by 23.26%. During the study, a continuous elevation of homocysteine levels was determined, with statistically significant increase in homocysteine values after eight months of metformin administration. Starting from the assumption that metformin blocks the absorption of vitamin B12 in the gastrointestinal tract as well as the availability of existing tissue reserves, the amount of this vitamin in the target cells is gradually reduced, resulting in an extremely low level of metabolically active forms of this vitamin and the consequent accumulation of homocysteine in intracellular and extracellular space. On the basis of the obtained test results, it may suggest observation of the level of total vitamin B12 and homocysteine prior to the introduction of metformin in T2DM therapy and after one year thereafter. Based on the level of one-year decline of total vitamin B12 and the increase of homocysteine concentrations, as well as other clinical and laboratory parameters, substitution therapy with vitamin B12 or further monitoring of laboratory parameters of vitamin B12 metabolism may be proposed.</p>

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

<p>У овој дисертацији испитивана су антитуморска дејства антихипергликемијског лека метформина, вазодилататорног лека нитроглицерина, и комбинација ових лекова са дијагностичким средством 2-деокси-D-глукозом и/или радио и хемио сензибилизатором кофеином на хуманим културама аденокарцинома плућа (A549), колоректалног карцинома (HT29), аденокарцинома цервикса (HeLa), као и на контролној ћелијској култури нормалних фибробласта плућа (МRC 5). In vitro испитивање утицаја метформина, нитроглицерина, 2-деокси-D-глукозе и кофеина на проли- ферацију ћелија карцинома грлића материце (HeLa), ћелијској култури аденокарциномa плућа (A549) и ћелијској линији карцинома дебелог црева (HT29). Ћелије у експоненцијалној фази раста третиране су растућим концентрацијама метформина, нитроглицерина и 2-деокси-D-глукозе и утврдила се дозна зависност цитотоксичног ефекта. Метформин, кофеин и 2-деокси-D-глукоза су утицали на смањење процента преживљавања туморских ћелија, док је применом нитроглицерина овај ефекат изостао, иако у експериментима код истовремене примене нитроглицерина и кофеина постоји пад процента преживелих ћелија. Најпотентнији ефекат је постигнут код истовремене примене метформина и кофеина, док је разлог за одсуство снажног цитотоксичног ефекта метформина и 2-деокси-D-глукозе код комбиноване примене молекуларни механизам деловања појединачних супстанци. Снажан пролиферативни ефекат је евидентиран применом метформина и кофена на здравим фибробластима плућа.</p> / <p>U ovoj disertaciji ispitivana su antitumorska dejstva antihiperglikemijskog leka metformina, vazodilatatornog leka nitroglicerina, i kombinacija ovih lekova sa dijagnostičkim sredstvom 2-deoksi-D-glukozom i/ili radio i hemio senzibilizatorom kofeinom na humanim kulturama adenokarcinoma pluća (A549), kolorektalnog karcinoma (HT29), adenokarcinoma cerviksa (HeLa), kao i na kontrolnoj ćelijskoj kulturi normalnih fibroblasta pluća (MRC 5). In vitro ispitivanje uticaja metformina, nitroglicerina, 2-deoksi-D-glukoze i kofeina na proli- feraciju ćelija karcinoma grlića materice (HeLa), ćelijskoj kulturi adenokarcinoma pluća (A549) i ćelijskoj liniji karcinoma debelog creva (HT29). Ćelije u eksponencijalnoj fazi rasta tretirane su rastućim koncentracijama metformina, nitroglicerina i 2-deoksi-D-glukoze i utvrdila se dozna zavisnost citotoksičnog efekta. Metformin, kofein i 2-deoksi-D-glukoza su uticali na smanjenje procenta preživljavanja tumorskih ćelija, dok je primenom nitroglicerina ovaj efekat izostao, iako u eksperimentima kod istovremene primene nitroglicerina i kofeina postoji pad procenta preživelih ćelija. Najpotentniji efekat je postignut kod istovremene primene metformina i kofeina, dok je razlog za odsustvo snažnog citotoksičnog efekta metformina i 2-deoksi-D-glukoze kod kombinovane primene molekularni mehanizam delovanja pojedinačnih supstanci. Snažan proliferativni efekat je evidentiran primenom metformina i kofena na zdravim fibroblastima pluća.</p> / <p>In this dissertation, the anti-cancer effects of an antihyperglycaemic agent of metformin, a vasodilator drug nitroglycerin, and a combination of these drugs with a 2-deoxy-D-glucose diagnostic agent and / or radio and hemio sensitizer with caffeine on human cultures of adenocarcinoma of the lungs (A549), colorectal carcinoma (HT29), cervix adenocarcinoma (HeLa), as well as on the control cell culture of normal fibroblasts of the lungs (MRC 5). An in vitro study of the effects of metformin, nitroglycerin, 2-deoxy-D-glucose and caffeine on the proliferation of cervical cancer cells (HeLa), cell culture of the lung adenocarcinoma (A549), and colon cancer of the colon (HT29). The cells at the exponential growth stage were treated with rising concentrations of metformin, nitroglycerin and 2-deoxy-D-glucose, and the cytotoxic effect was determined. Metformin, caffeine, and 2-deoxy-D-glucose reduced the number of tumor cells, while nitroglycerin did not it could be concluded. Although there is a decrease in survival in experiments with the simultaneous administration of nitroglycerin and caffeine, the most effective effect is achieved in the simultaneous use of metformin and caffeine, while the reason for the absence of a potent cytotoxic effect of metformin and -deoxy-D-glucose is the molecular mechanism of the action of individual substances. The most significant effect was achieved with the simultaneous administration of metformin and caffeine to the cell culture of lung adenocarcinoma. A potent proliferative effect was recorded using metformin and 2-deoxy-Dglucose on healthy lung fibroblasts.</p>

Submitted in partial compliance with the requirements of the Master’s Degree in Technology: Homoeopathy, Durban University of Technology, Durban, South Africa, 2016, / Background Diabetes mellitus is a metabolic disorder with various aetiologies, characterised by hyperglycaemia, resulting from defects of carbohydrate, fat and protein metabolism due to the deficient action of insulin on target tissues caused by insensitivity to or lack of insulin or both. The long term effects of diabetes mellitus frequently include retinopathy, nephropathy and neuropathy and an increased risk of other diseases such as cardiac, peripheral arterial and cerebrovascular disease. According to the International Diabetes Federation (IDF) 387 million people have diabetes mellitus and this number is predicted to rise to 592 million worldwide by 2035. In 2014 diabetes mellitus caused 4.9 million deaths worldwide and every 7 seconds a person dies from diabetes mellitus. The growing incidence of diabetes mellitus is a worldwide concern because of the increase of economic costs and burden of disease that is due to the cardiovascular complications and the co-morbidities. Objective The aim of this double-blind, randomised clinical trial was to determine the efficacy of Syzygium jambolanum (Java plum) 6CH and Syzygium jambolanum (Java plum) homoeopathic mother tincture on daily fasting blood glucose and glycosylated haemoglobin levels in type 2 diabetes mellitus patients on Metformin® in the treatment of type 2 diabetes mellitus. Material and Method A sample consisted of 24 volunteers selected on the basis of inclusion and exclusion criteria. These participants were then randomly divided into two groups, 13 in the group receiving the homoeopathic potency and 11 in the group receiving the homoeopathic mother tincture. Each participant attended a total of five consultations with the researcher over a period of 14 weeks that included a 2 week baseline period followed by a 12 week treatment period, at the Durban University of Technology (DUT) or Kenneth Gardens Homoeopathic Day Clinic. At each consultation a detailed and comprehensive homoeopathic case history (Appendix D) was taken and a physical examination (Appendix E) was performed by the researcher. Participants were required to fill in a log sheet (Appendix C1 and C2) with their fasting blood glucose readings daily for 14 weeks, which included a 2 week baseline period followed by a 12 week treatment period. Participants were also required to have their glycosylated haemoglobin measurements tested pre- and post-treatment. Results Both groups reflected a statistically significant reduction in fasting blood glucose levels as compared to the baseline. The mean fasting blood glucose level in week 1 was 11.8802 whereas in week 13 the mean blood glucose level was 8.6590 with a p value ≤ 0.05 for the Syzygium jambolanum 6CH group and the mean blood glucose level in week 1 was 9.0338 with a standard whereas in week 13 the mean blood glucose level was 6.8591 with a p value ≤ 0.05 in the Syzygium jambolanum homoeopathic mother tincture. However there was no significant differences between the two groups (Syzygium jambolanum 6CH and Syzygium jambolanum homoeopathic mother tincture), the significance score was 0.623 when comparing reduction in fasting blood glucose levels. Both groups reflected a statistically non-significant reduction in the glycosylated haemoglobin (HbA1C) and there were no significant differences between the two groups when comparing reduction in HbA1C levels. Conclusion Both homoeopathic preparations of Syzygium jambolanum (mother tincture and 6CH) significantly reduce fasting blood glucose levels in patients with type 2 diabetes mellitus. This result suggests that Syzygium jambolanum has beneficial anti-diabetic effects and warrants further investigation. / M

The analysis of pharmacotherapy by patients suffering with DM in Greece I Author: Georgios Kalaitzidis Tutor: Professor RNDr.Jiri Vlcek,CSc. Department of Social and Clinical Pharmacy, Charles University in Prague, Faculty of Pharmacy in Hradec Kralove. Introduction: The diabetes in developed countries concerns 11% of people over 70 years and is the cause of 3% of total deaths in general population. Aim: The aim of the study was to assess the Pharmacotherapy of Diabetes mellitus type II in a pharmacy of a small town of Greece, Veria. Methods: It is retrospective cross-sectional study, which was conducted in a pharmacy in a small town of Greece, Veria. The study population consists of 60 patients with known Type II diabetes Melitus. The data collection was performed by a self-reported questionnaire, which was created and developed by the researcher and filled by the respondents. Results: The mean age of the sample was 56.5±17.5 years. Most of them were females (n=40). Most of the patients knew their fasting glucose level (93.3%,n=56).Of the patients who knew their fasting glucose level, 36 (64.3%) patients had high fasting glucose level and 20 (35.7%) had physiological fasting glucose level. From all the patients(n=60), some of them visited their physician every 6 months (n=24), and every 3 months...

Objetivo: Avaliar o controle glicêmico em pacientes que utilizaram metformina ou insulina para tratamento do diabetes gestacional, identificando fatores preditores da necessidade de insulina complementar nas pacientes que tiveram, como terapêutica inicial, a metformina. Método: pacientes com DG que não obtiveram controle glicêmico com dieta e exercícios físicos foram randomizadas para receber metformina (n=46) ou insulina (n=46). Os critérios de inclusão foram: gestação única, realização de dieta e exercícios físicos por período mínimo de uma semana, sem controle glicêmico satisfatório, ausência de fatores de risco para acidose láctica, ausência de anormalidades anatômicas e/ou cromossômicas do produto conceptual. Foram excluídas as gestantes que apresentaram perda de seguimento pré-natal. Resultado: A comparação das médias glicêmicas pré-tratamento medicamentoso não mostrou diferença estatisticamente significativa entre os grupos (p=0,790). Porém, após a introdução dos medicamentos, foram observados médias glicêmicas menores no grupo metformina, ao longo do dia (p=0,020), principalmente, após o jantar (p=0,042). Pacientes que utilizaram metformina tiveram menor ganho de peso (p=0,002) e, também, menor frequência de hipoglicemia neonatal (p=0,032). Doze pacientes do grupo metformina (26,08%) necessitaram de insulina complementar para controle glicêmico. A idade gestacional precoce (odds ratio 0,71, CI95% 0,52-0,97; p=0,032) e a média glicêmica pré-tratamento medicamentoso (odds ratio 1,061, CI95% 1,001-1,124; p=0,046) foram identificadas como preditoras da necessidade de insulina complementar. Conclusão: A metformina foi eficaz em propiciar controle glicêmico adequado, com menor ganho de peso e menor frequência de hipoglicemia neonatal. Foi identificado grupo de pacientes com maior probabilidade de necessitar de complementação com insulina para atingir controle glicêmico / Objective: To evaluate glycemic control in women receiving metformin or insulin for the treatment of gestational diabetes, and to identify factors predicting the need for supplemental insulin in women initially treated with metformin. Methods: Women with gestational diabetes who did not achieve glycemic control with diet and exercise were randomized to receive either metformin (n=46) or insulin (n=46). Criteria for inclusion were singleton pregnancy, diet and exercise for a minimum period of one week without satisfactory glycemic control, absence of risk factors for lactic acidosis, and absence of anatomic and/or chromosome anomalies of the conceptus. Patients who were lost to prenatal follow-up were excluded. Results: Comparison of mean pretreatment glucose levels showed no significant difference between groups (P=.790). However, lower mean glucose levels across the day were observed in the metformin group after introduction of the drug (P=.020), especially after dinner (P=.042). Women using metformin presented less weight gain (P=.002) and a lower frequency of neonatal hypoglycemia (P=.032). Twelve women in the metformin group (26.08%) required supplemental insulin for glycemic control. Early gestational age (OR=0.71, 95%CI: 0.52-0.97; P=.032) and mean pretreatment glucose level (OR=1.061, 95%CI: 1.001-1.124; P=.046) were identified as predictors of insulin need. Conclusion: Metformin was found to provide adequate glycemic controlwith lower mean glucose levels across the day, less weight gain and a lower frequency of neonatal hypoglycemia. Logistic regression analysis showed that gestational age at diagnosis and mean pretreatment glucose level were predictors of the need for supplemental insulin therapy in women initially treated with metformin

Os tumores de musculatura liso do útero se desenvolvem a partir do miométrio e podem apresentar carcterísticas clínicas malignas e benignas. Dentre eles, o leiomiossarcoma (LMS) é o tumor maligno mais comum, com altas taxas de metástase e recidiva, mesmo sendo diagnosticado em estágios iniciais. Já os leiomiomas (LM) são os tumores benignos mais frequentes em mulheres em idade reprodutiva. Ambos possuem mesma diferenciação celular, porém com comportamentos clínico e biológico bastante distintos, e até o momento não se dispõe de tratamento específico ou curativo. Nesse contexto, a busca por novos alvos moleculares pode contribuir não só para um melhor entendimento dessas neoplasias, como também para a descoberta de novas terapias. Em estudo prévio foi observada a expressão aumentada de FOXO3a nos sarcomas uterinos, em comparação aos LMs e ao miométrio adjacente (MM). Além disso, sua expressão foi crescente de acordo com o potencial de malignidade do tumor. Assim, o objetivo do presente estudo foi avaliar in vitro o efeito de terapia alvo específica para FOXO3a em células de LM e LMS. Para isto, linhagens celulares de MM (ATCC PCS-460-011), LM (THESCs - CRL-4003) e LMS (SK-UT-1 - HTB-114) foram caracterizadas quanto à expressão basal de FOXO3a (gene e proteína) e submetidas a tratamento com Genisteína e Metformina ou inativação do gene por siRNA. Os efeitos dos tratamentos foram avaliados por PCR em tempo real, Western Blot, imunocitoquímica, ensaios de proliferação, migração e apoptose. Nossos resultados mostraram que todos os tratamentos realizados interferiram na capacidade de proliferação e migração das células, com maior inibição após as 48 horas nos LMS e 72h nos LM. O efeito obtido na transfecção com siRNA apresentou maior eficiência após 48 h da transfecção nos LMS e 72h nos LM. Os efeitos da inibição de FOXO3a foram maiores na proliferação e migração dos LM, porém os resultados não foram estatisticamente significativos. Dentre as substâncias testadas, a Metformina apresentou maior efeito sobre a proliferação, migração e viabilidade das linhagens celulares. A Genisteína também apresentou efeito inibitório nas células, porém o controle com veículo também apresentou o mesmo efeito citotóxico. De modo geral, os efeitos obtidos com os fármacos, foram tempo e concentração dependentes. Em conjunto, nossos resultados sugerem um relevante do FOXO3a nos tumores de musculatura lisa uterinos, além de apresentá-lo como potencial alvo para terapia específica / Smooth muscle tumors of the uterus develop from the myometrium and may present benign and malignant clinical features. Among them, leiomyosarcoma (LMS) is the most frequent malignant tumor, with high rates of metastasis and relapse, even when diagnosed in early stages. On the other hand, leiomyomas (LM) are the most frequent benign tumors in women of reproductive age. Both have the same cellular differentiation, but with very different clinical and biological behaviors, and so far no specific or curative treatment is available. In this context, the search for new molecular targets can contribute not only for a better understanding of these neoplasms, but also for the discovery of new therapies. In a previous study, increased expression of FOXO3a in uterine sarcomas was observed, compared to LMs and adjacent myometrium (MM). In addition, its expression was increasing according to the malignancy potential of the tumor. Thus, the aim of the present study was to evaluate in vitro, the effect of specific targeted therapy for FOXO3a on LM and LMS cells. For this, MM (ATCC PCS-460-011), LM (THESCs-CRL-4003) and LMS (SK-UT-1-HTB-114) cell lines were characterized for basal expression of FOXO3a (gene and protein) and subsequently submitted to treatment with metformin and genistein, or silencing of FOXO3a by siRNA. The effects of the treatments were evaluated by real-time PCR, Western Blot, immunocytochemistry, proliferation, migration and apoptosis assays. Our results showed that all treatments interfered in the proliferation and migration capacity of the cells, with greater inhibition after 48 hours for LMS and 72 hours LM. The effect obtained in the transfection with siRNA showed higher efficiency after 48 hours of transfection in LMS and 72 hours in LM. The effects of inhibition of FOXO3a were greater in the proliferation and migration of the LM, but the results were not statistically significant. Among the substances tested, Metformin had a greater effect on proliferation, migration and viability of the cell lines. Genistein also had an inhibitory effect on the cells, but the control with the vehicle also presented the same cytotoxic effect. In general, the effects obtained with the drugs were time and concentration dependent. Together, our results suggest a relevant role of FOXO3a in uterine smooth muscle tumors, in addition to presenting it as a potential target for specific therapy

OBJETIVO: avaliação da expressão gênicado receptor do fator de crescimento semelhante à insulina de (Insulin-Like Growth Factor- IGF) 1 em células da granulosa luteinizadas do cumulusde mulheres não obesas, com sensibilidade à insulina normal, com síndrome dos ovários policísticos (SOP) tratadas e não tratadas com metformina. MODELO DO ESTUDO: prospectivo, longitudinal, randomizado. PACIENTES E MÉTODOS: avaliamos 12 mulheres com ciclosovulatórios, 9 mulheres com SOP e 8 mulheres com SOP e tratadas com metformina, ao menos 8 semanas na dose de 1.700 mg/dia. Todos os grupos foram similares com relação ao peso, ao índice de massa corporal (IMC), à circunferência da cintura e com sensibilidade à insulina normal. Todas as mulheres foram submetidas à estimulação ovariana controlada com uso de agonista de GnRH em protocolo longo e gonadotrofinas para ciclos de FIV/ICSI. As células da granulosa do cumulusforam obtidas por microdissecção dos cinco maiores folículos pré-ovulatórios. A expressão gênica do receptor de IGF-1 foi determinada com técnica da Reação da Polimerase em Cadeia a partir da Transcrição Reversa (Reverse transcriptase - Polymerase Chain ReactionRT-PCR) emiquantitativa. Foram avaliadas as concentrações séricas e foliculares de estradiol, progesterona, testosterona, hormônio folículo estimulante (Follicle-Stimulating Hormone- FSH), hormônio luteinizante (Luteinizing Hormone - LH), Sex Hormone-Binding Globulin(SHBG), glicose, insulina e IGF-1. Para análise estatística, foram utilizados os testes: ANOVA, Newman-Keuls, coeficiente de Pearsone regressão linear múltipla, sendo considerado nível de significância de 5%. RESULTADOS: não foram observadas diferenças com relação à expressão gênica do receptor de IGF-1 nos três grupos analisados (P>0,05). O número de oócitos (20,4 vs. 13,1 vs.11,5, P= 0,009), os níveis séricos de estradiol (1.896,00 pcg/mL vs. 985,20 pcg/mL vs.908,10 pcg/mL,P = 0,03) e testosterona (1,43 ng/mL vs.0,89 ng/mL vs. 0,82 ng/mL pcg/mL,P = 0,02) foram maiores no grupo de mulheres com SOP não tratadas com metformina em comparação com as mulheres com ciclos ovulatórios e tratadas com metformina, respectivamente. As mulheres com ciclos ovulatórios (50.710±42.520 ng/mL) apresentaram maiores concentrações foliculares de progesterona quando comparados com as mulheres com SOP tratadas (13.660±5.212 ng/mL) e não tratadas com metformina (17.680±6.644 ng/mL) (P=0,01). Na avaliação da regressão múltipla, a testosterona sérica não sofreu influência da expressão gênica do receptor de IGF-1 ou do IMC. CONCLUSÕES: as altas concentrações séricas de estradiol e testosterona, maior número de oócitos no grupo de mulheres com SOP não tratadas com metformina nos levam a concluir que mulheres com SOP provavelmente têm uma maior sensibilidade à estimulação da esteroidogênese ovariana quando comparadas com mulheres sem essa doença, embora não tenhasido encontrada diferença na expressão do receptor de IGF-1 nos trêsgrupos analisados. A similaridade dos resultados deste estudo entre mulheres com SOP tratadas com metformina e com ciclos ovulatórios nos levam a \"hipotetizar\" que um dos possíveis mecanismos de ação da metformina no sistema IGF-1 nas células da granulosa do cumulus poderia ser por mecanismos pós-receptores. / OBJECTIVE: evaluation of the gene expression of the IGF-I receptor in luteinized cumulus granulosa cells from non-obese women with normal insulin sensitivity and with polycystic ovarian syndrome (PCOS)treated or nor with metformin. STUDY MODEL: prospective,longitudinal, randomized. PATIENTS AND METHODS: we evaluated 12 women withovulatory cycles and 9 women with PCOS who had been treated for at least 8 weeks with a metformin dose of 1700 mg/day. All groups were similar interms of weight, body mass index (BMI), and waist circumference and all had normal insulin sensitivity. All women were submitted to controlled ovarian stimulation with a GnRH agonist in a long protocol and with gonadotropins for IVF/ICSI cycles. Cumulus granulosa cells were obtained by microdissection of the five largest pre-ovulatory follicles. Gene expression of the IGF-1 receptor was determined by semiquantitative RT-PCR. Serum and follicular concentrations of estradiol, progesterone, testosterone, FSH, LH, insulin, SHBG, and IGF-1 were determined. Data were analyzed statistically by ANOVA and by the Newman-Keuls test and the Pearson coefficient and linear multiple regression were calculated, with the level of significance set at 5%. RESULTS: no difference in geneexpression of the IGF-I receptor were observed between the three groups studied (P>0.05). The number of oocytes (20.4 vs. 13.1 vs. 11.5, P= 0.009) and the serum levels of estradiol (1,896.00 pcg/mL vs. 985.20 pcg/mL vs.908.10 pcg/mL,P = 0.03) and testosterone (1.43 ng/mL vs.0.89 ng/mL vs. 0.82 ng/mL pcg/mL,P = 0.02) were higher in the group of women with PCOS not treated with metformin than in women with ovulatory cycles and in women treated with metformin, respectively. The women with ovulatory cycles (50.710±42.520 ng/mL) presented higher follicular concentrations of progesterone compared with women with PCOS treated (13.660±5.212 ng/mL) or not with metformin (17.680±6.644 ng/mL) (P=0.01). Multiple regression revealed that serum testosterone was not affected by the gene expression of the IGF-1 receptor or by BMI. CONCLUSIONS: the high serum concentrations of estradiol and testosterone and the larger number of oocytes in the group ofwomen with PCOS not treated with metformin lead us to conclude that women with PCOS probably have greater sensitivity to stimulation ofovarian steroidogenesis than women without the disease, although no difference was detected in the expression of the IGF-I receptor between the three groups studied. The similarity ofthe present results for the women with PCOS treated with metformin and for the women with ovulatory cycles leads us to hypothesize that one of the possible mechanisms of action of metformin on the IGF-1 system in cumulus granulosa cells may be of the post-receptor type.