Effects of metformin on endometrial cancer: Systematic review and meta-analysis.

BACKGROUND: Endometrial cancer is one of the most common gynecological cancers, which is frequently preceded by atypical endometrial hyperplasia, a premalignant lesion. Metformin, an antidiabetic drug, has emerged as a new adjunctive strategy for different cancer types, including endometrial cancer. This systematic review and meta-analysis aimed to evaluate the effects of metformin in atypical endometrial hyperplasia and endometrial cancer patients. METHODS: The search was conducted on January 2017 and the articles were collected in Cochrane, LILACS, PubMed, Scopus and Web of Science. A grey literature search was undertaken using Google SCHOLAR, ProQuest and Open Grey. Nineteen studies were included, which contained information about the following outcomes: reversal of atypical endometrial hyperplasia, cellular proliferation biomarkers expression and overall survival in metformin-users compared to non-users. RESULTS: Metformin was associated with reversion of atypical endometrial hyperplasia to a normal endometrial, and with decreased cell proliferation biomarkers staining, from 51.94% (CI=36.23% to 67.46%) to 34.47% (CI=18.55% to 52.43%). However, there is a high heterogeneity among studies. Metformin-users endometrial cancer patients had a higher overall survival compared to non-metformin users and non-diabetic patients (HR=0.82; CI: 0.70-0.95; p=0.09, I2=40%). CONCLUSION: Regardless the high heterogeneity of the analyzed studies, the present review suggests that adjunct metformin treatment may assist in the reversal of atypical endometrial hyperplasia to normal endometrial histology, in the reduction of cell proliferation biomarkers implicated in tumor progression, and in the improvement of overall survival in endometrial cancer. Further work on prospective controlled trials designed to address the effects of adjunct metformin on clinical outcomes is necessary for definite conclusions.

Radiation induced a supra-additive cytotoxic effect in head and neck carcinoma cell lines when combined with plant extracts from Brazilian Cerrado biome.

OBJECTIVES: Antineoplastic effects of molecules derived from plants have recently gained increasing attention as an additive to traditional therapies. The aim of this study was to evaluate the cytotoxic activity of plant extracts from the Brazilian Cerrado biome associated with radiotherapy in head and neck carcinoma cells (HNSCC). MATERIALS AND METHODS: Fifteen extracts derived from five Cerrado plants were tested in HNSCC cell lines (SCC-25, SCC-9, FaDu) and keratinocyte cells (HaCat). Cell cytotoxicity of extracts and association extract/radiation (2Gy/min) was assessed by MTT assay. Cisplatin (50 µg/mL) was used as a positive control. Extracts with the major cytotoxic activity were selected and their IC50 concentrations were defined. Apoptosis was assessed using flow cytometric analysis. RESULTS: Ten isolated extracts resulted in moderate cytotoxicity (>20 and ≤ 50 % of viable cells), while three extracts induced severe cytotoxic effects (≤ 20 % of viable cells). Plant extracts treatment improved radiotherapy cytotoxicity in all cell lines. Although plant extracts are not as potent as cisplatin plus radiation, in FaDu cells, seven extracts associated with irradiation showed cytotoxic activity similar or better than the association of cisplatin and radiation. Hexanic extract of Erythroxylum daphinites could induce apoptosis in oral cancer cells; however, necrosis was the prevalent kind of death in FaDu cells treated with hexanic extract of Erythroxylum suberosum. CONCLUSIONS: Pre-treatment of HNSCC cells with the extract derived from Cerrado plants followed by irradiation induced a supra-additive cytotoxic effect. CLINICAL RELEVANCE: This study highlights the potential biological relevance of the Cerrado biome when associated with traditional therapy for cancer.

GQ-16, a novel peroxisome proliferator-activated receptor γ (PPARγ) ligand, promotes insulin sensitization without weight gain.

The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the ß-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the ß-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.

Structural basis of GC-1 selectivity for thyroid hormone receptor isoforms.

BACKGROUND: Thyroid receptors, TRalpha and TRbeta, are involved in important physiological functions such as metabolism, cholesterol level and heart activities. Whereas metabolism increase and cholesterol level lowering could be achieved by TRbeta isoform activation, TRalpha activation affects heart rates. Therefore, beta-selective thyromimetics have been developed as promising drug-candidates for treatment of obesity and elevated cholesterol level. GC-1 [3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)-phenoxy acetic acid] has ability to lower LDL cholesterol with 600- to 1400-fold more potency and approximately two- to threefold more efficacy than atorvastatin (Lipitor(c)) in studies in rats, mice and monkeys. RESULTS: To investigate GC-1 specificity, we solved crystal structures and performed molecular dynamics simulations of both isoforms complexed with GC-1. Crystal structures reveal that, in TRalpha Arg228 is observed in multiple conformations, an effect triggered by the differences in the interactions between GC-1 and Ser277 or the corresponding asparagine (Asn331) of TRbeta. The corresponding Arg282 of TRbeta is observed in only one single stable conformation, interacting effectively with the ligand. Molecular dynamics support this model: our simulations show that the multiple conformations can be observed for the Arg228 in TRalpha, in which the ligand interacts either strongly with the ligand or with the Ser277 residue. In contrast, a single stable Arg282 conformation is observed for TRbeta, in which it strongly interacts with both GC-1 and the Asn331. CONCLUSION: Our analysis suggests that the key factors for GC-1 selectivity are the presence of an oxyacetic acid ester oxygen and the absence of the amino group relative to T3. These results shed light into the beta-selectivity of GC-1 and may assist the development of new compounds with potential as drug candidates to the treatment of hypercholesterolemia and obesity.

Human thyroid receptor forms tetramers in solution, which dissociate into dimers upon ligand binding.

Thyroid hormone nuclear receptors (TRs) bind to DNA and activate transcription as heterodimers with the retinoid X receptor (RXR) or as homodimers or monomers. RXR also binds to DNA and activates transcription as homodimers but can, in addition, self-associate into homotetramers in the absence of ligand and DNA templates. It is thought that homotetramer formation serves to sequester excess RXRs into an inactive pool within the cell. Here, we report systematic studies of the multimeric state of a recombinant human TRbeta1 truncation (hTRbeta1deltaAB) that encompasses the complete DNA binding domain and ligand binding domain in solution. Native gel electrophoresis, chemical crosslinking, gel filtration, and dynamic light scattering experiments reveal that hTRbeta1deltaAB forms a mixture of monomers, dimers, and tetramers. Like RXR, increasing protein concentration shifts the equilibrium between TR multimers toward tetramer formation, whereas binding of cognate thyroid hormone leads to dissociation of tetramers and increased formation of dimers. This work represents the first evidence that apo-hTRbeta1 forms homotetramers. The findings raise the possibility that tetramer formation provides an additional, and previously unsuspected, level of control of TR activity and that the capacity for homotetramer formation may be more widespread in the nuclear receptor family than previously thought.

Cytotoxic effect of Erythroxylum daphnites extract is associated with G1 cell cycle arrest and apoptosis in oral squamous cell carcinoma.

Plant-derived molecules showing antineoplastic effects have recently gained increased attention as potential adjuvants to traditional therapies for various cancers. Cerrado biome in Brazil contains high floral biodiversity, but knowledge about the potential therapeutic effects of compounds derived from that flora is still limited. The present study investigated the antineoplastic activity of Erythroxylum daphnites Mart., a Brazilian native plant from Cerrado biome, in the SCC-9 oral squamous cell carcinoma cell line. Cells were treated with various concentrations of hexane extract of Erythroxylum daphnites leaves (EDH) and assessed for cytotoxicity, proliferation, and apoptosis. Thin layer chromatography was conducted to characterize the substances present in EDH. Our results showed that EDH exerted anti-proliferative effects in SCC-9 cells by stabilizing the cell cycle at G1 phase in association with reduced intracellular levels of cyclins D and E and increased level of p21. EDH also demonstrated pro-apoptotic properties, as shown by an increased expression of caspase-3. Triterpenes were the major constituents of EDH. Our findings demonstrated a cytotoxic effect of EDH against SCC-9 cells in vitro mediated by the restraint of cellular proliferation and induction of apoptosis. Taken together, these findings support EDH constituents as potential therapeutic adjuvants for oral cancer.

Thyroid hormone export varies among primary cells and appears to differ from hormone uptake.

We characterized T3 efflux in primary cultures of cells derived from human placenta, neonatal rat cardiac myocytes, and rat inner medullary collecting ducts (IMCD). The T3 efflux rate was highest in placenta cells, followed by ventriculocytes, atriocytes, and IMCD cells. Verapamil reversibly blocked [125I]T3 efflux in these cells in a manner that correlated with their T3 efflux rate. Thus, verapamil inhibition of [125I]T3 efflux in placenta cells led to a 432% increase in the [125I]T3 content compared with 33% increase in IMCD cells. Several unlabeled iodothyronines, but not TRIAC, differentially blocked [125I]T3 efflux such as (T4 > T3 > rT3 = D-T3 > D-T4) in placenta cells and (T4 > rT3 = D-T4 = T3 > D-T3) in ventriculocytes, suggesting tissue-specific differences in the carriers/transporters responsible for T3 efflux. This hypothesis draws further support from the fact that D-T3 inhibited [125I]T3 efflux in placenta cells, but not in ventriculocytes. TRIAC did not affect T3 efflux in ventriculocytes or placenta cells, but it greatly inhibited [125I]T3 uptake in these cells, suggesting that [125I]T3 uptake and efflux mechanisms are distinct and appear to be mediated by distinct carrier/transporter proteins. Collectively, these data suggest that differences in thyroid hormone transport in target cells may provide an important mechanism for regulating hormone action in a tissue-specific fashion.

Cytotoxic effect of Pouteria torta leaf extracts on human oral and breast carcinomas cell lines.

AIMS: This study aimed at investigating the cytotoxic activity and the type of cell death induced by Pouteria torta (P. torta) leaf extracts on human oral squamous cell carcinoma and breast carcinoma cell lines. MATERIAL AND METHODS: The effects of P. torta leaf hexanic (PTH), ethanolic (PTE) and aqueous (PTA) extracts at the concentration of 500 mg/mL were evaluated on OSCC-3 and MCF-7 cell lines, using crystal violet staining after 24 and 48 h of treatment. To obtain the dose-response curve, cells were treated with decreasing concentrations of the extracts (1000, 750, 500, 250, 125 mg/mL) for 24 h. To investigate the mechanism of cell death (apoptosis vs. necrosis), DNA fragmentation assay was performed. RESULTS: All extracts were cytotoxic to both OSCC-3 and MCF-7, albeit at differing levels. PTH and PTE were effective at the concentration of 500 µg/mL, resulting in nearly 50% of cell death in both cancer cell lines. PTA was more effective at lower concentrations, with more significant cell death at 125 g/mL. Treatment with PTA and PTE caused apoptosis in MCF-7, whereas in OSCC-3 cells, the same effect could only be caused by PTH. On the other hand, PTA was able to induce necrosis in OSCC-3. CONCLUSIONS: These findings demonstrated that P. torta leaf extracts may contain useful compounds to combat oral and breast cancer, and this study highlights the potential biological relevance of the Brazilian Cerrado Biome in cancer therapy.

Helix 12 dynamics and thyroid hormone receptor activity: experimental and molecular dynamics studies of Ile280 mutants.

Nuclear hormone receptors (NRs) form a family of transcription factors that mediate cellular responses initiated by hormone binding. It is generally recognized that the structure and dynamics of the C-terminal helix 12 (H12) of NRs' ligand binding domain (LBD) are fundamental to the recognition of coactivators and corepressors that modulate receptor function. Here we study the role of three mutations in the I280 residue of H12 of thyroid hormone receptors using site-directed mutagenesis, functional assays, and molecular dynamics simulations. Although residues at position 280 do not interact with coactivators or with the ligand, we show that its mutations can selectively block coactivator and corepressor binding, and affect hormone binding affinity differently. Molecular dynamics simulations suggest that ligand affinity is reduced by indirectly displacing the ligand in the binding pocket, facilitating water penetration and ligand destabilization. Mutations I280R and I280K link H12 to the LBD by forming salt bridges with E457 in H12, stabilizing H12 in a conformation that blocks both corepressor and coactivator recruitment. The I280M mutation, in turn, blocks corepressor binding, but appears to enhance coactivator affinity, suggesting stabilization of H12 in agonist conformation.

Cytotoxic effect of tobacco extracts on human oral squamous cell carcinoma cell-line.

Cancer is a public health problem worldwide. Incidences of oral carcinomas are increasing in the last decades, and the developed countries are the most affected. Current therapeutic options for this type of cancer are aggressive and/or invasive, including surgery, radiotherapy and chemotherapy. In addition, they have not yet translated into an improvement of life quality or expectancy to patients. In this scenario, new therapeutics are urgently needed and actively sought after. The goal of this study was to investigate the cytotoxic effects of tobacco crude extract (TCE) and two fractions thereof in the human lineage of oral squamous cell carcinoma, OSCC-3. Exposure of human oral cancer cells to TCE-induced cell death and decreased cell viability in a dose-dependent manner. Of the fractions tested, one was able to induce significant cell death (over 50%) after 48 h treatment. DNA fragmentation and caspase-3 activation indicated that the type of cell death induced by TCE and its fraction was apoptosis. Our results indicate that tobacco contains compounds that could be useful in inducing apoptosis in cancer cells. More specifically, because of the neutral chemical nature of the fraction capable of inducing apoptosis, we postulate that the putative compound responsible for the cell death is non-polar. Further investigation is needed to uncover its chemical nature and structure.

Thyroid hormone response element organization dictates the composition of active receptor.

Thyroid hormone (triiodothyronine, T(3)) is known to activate transcription by binding heterodimers of thyroid hormone receptors (TRs) and retinoid X receptors (RXRs). RXR-TRs bind to T(3) response elements (TREs) composed of direct repeats of the sequence AGGTCA spaced by four nucleotides (DR-4). In other TREs, however, the half-sites can be arranged as inverted palindromes and palindromes (Pal). Here we show that TR homodimers and monomers activate transcription from representative TREs with alternate half-site placements. TR beta activates transcription more efficiently than TR alpha at an inverted palindrome (F2), and this correlates with preferential TR beta homodimer formation at F2 in vitro. Furthermore, reconstruction of TR transcription complexes in yeast indicates that TR beta homodimers are active at F2, whereas RXR-TRs are active at DR-4 and Pal. Finally, analysis of TR beta mutations that block homodimer and/or heterodimer formation reveal TRE-selective requirements for these surfaces in mammalian cells, which suggest that TR beta homodimers are active at F2, RXR-TRs at DR-4, and TR monomers at Pal. TR beta requires higher levels of hormone for activation at F2 than other TREs, and this differential effect is abolished by a dimer surface mutation suggesting that it is related to composition of the TR.TRE complex. We propose that interactions of particular TR oligomers with different elements play unappreciated roles in TRE-selective actions of liganded TRs in vivo.

Thyroid hormone receptor binding to DNA and T3-dependent transcriptional activation are inhibited by uremic toxins.

BACKGROUND: There is a substantial clinical overlap between chronic renal failure (CRF) and hypothyroidism, suggesting the presence of hypothyroidism in uremic patients. Although CRF patients have low T3 and T4 levels with normal thyroid-stimulating hormone (TSH), they show a higher prevalence of goiter and evidence for blunted tissue responsiveness to T3 action. However, there are no studies examining whether thyroid hormone receptors (TRs) play a role in thyroid hormone dysfunction in CRF patients. To evaluate the effects of an uremic environment on TR function, we investigated the effect of uremic plasma on TRbeta1 binding to DNA as heterodimers with the retinoid X receptor alpha (RXRalpha) and on T3-dependent transcriptional activity. RESULTS: We demonstrated that uremic plasma collected prior to hemodialysis (Pre-HD) significantly reduced TRbeta1-RXRalpha binding to DNA. Such inhibition was also observed with a vitamin D receptor (VDR) but not with a peroxisome proliferator-activated receptor gamma (PPARgamma). A cell-based assay confirmed this effect where uremic pre-HD ultrafiltrate inhibited the transcriptional activation induced by T3 in U937 cells. In both cases, the inhibitory effects were reversed when the uremic plasma and the uremic ultrafiltrate were collected and used after hemodialysis (Post-HD). CONCLUSION: These results suggest that dialyzable toxins in uremic plasma selectively block the binding of TRbeta1-RXRalpha to DNA and impair T3 transcriptional activity. These findings may explain some features of hypothyroidism and thyroid hormone resistance observed in CRF patients.

Genus Pouteria: chemistry and biological activity / Gênero Pouteria: química e atividade biológica

The genus Pouteria belongs to the family Sapotaceae and can be widely found around the World. These plants have been used as building material, as food, because the eatable fruits, as well as remedies in folk medicine. Some biological activities have been reported to species of this genus such as antioxidant, anti-inflammatory, antibacterial and antifungal. However, the real potential of this genus as source of new drugs or phytomedicines remains unknown. Therefore, a review of the so far known chemical composition and biological activities of this genus is presented to stimulate new studies about the species already reported moreover that species have no reference about chemistry or biological activities could be found until now.

O gênero Pouteria pertence à família Sapotaceae e pode ser encontrado em muitos continentes. As plantas desse gênero têm sido utilizadas na construção civil, na alimentação e também na medicina popular. Algumas atividades biológicas são reportadas às espécies desse gênero, tais como, antioxidante, anti-inflamatória, antibacteriana e antifúngica, mas seu real potencial como fonte de novos fármacos ainda é pouco conhecido. Assim, uma revisão sobre a composição química e as atividades biológicas de Pouteria é apresentada, com o intuito de estimular a continuação dos estudos das espécies aqui citadas, e a investigação de outras espécies para as quais não foram encontrados relatos.

Analysis of P-glycoprotein-mediated membrane transport in human peripheral blood lymphocytes using the UIC2 shift assay.

BACKGROUND: During transport-associated adenosine triphosphate hydrolysis, P-glycoprotein (Pgp) undergoes conformation transitions detected by UIC2, a functional anti-Pgp monoclonal antibody. A newly developed UIC2 shift assay is based on increased UIC2 reactivity in the presence of Pgp substrates. All peripheral blood leukocytes express low Pgp levels. The existing antibody-based detection methods are limited in their sensitivity and require additional techniques to simultaneously analyze Pgp expression and efflux, making it difficult to ascertain the physiologic role of Pgp-mediated transport. METHODS: We validated the UIC2 shift assay against UIC2 immunostaining and DiOC(2) efflux. The UIC2 shift assay was then used to characterize Pgp functional expression and its physiologic substrates in peripheral blood leukocytes. RESULTS: A strong correlation was observed between the UIC2 shift assay versus immunostaining and dye efflux tests. The UIC2 shift assay showed improved sensitivity (compared with conventional UIC2 staining) and allowed for simultaneous detection of Pgp expression and function. Using this assay, we identified several new Pgp substrates, including monensin and retinol, and confirmed that interleukin-2 and interferon-gamma can be transported by Pgp. CONCLUSIONS: Our findings validate the use of the UIC2 shift assay in MDR1 detection and support the idea that Pgp plays a physiologic role in immunoregulation.

Thyroid hormone transport is disturbed in erythrocytes from patients with chronic renal failure on hemodialysis.

AIMS: To now, there are no studies reporting whether thyroid hormones (THs) transport play a role in thyroid hormone dysfunction observed in chronic renal failure (CRF). Therefore, the aim of this study was to investigate the transport of THs in erythrocytes from patients with CRF on hemodialysis (HD). METHODS: [125I]-L-triiodothyronine ([125I]T3) and [125I]-L-thyroxine ([125I]T4) erythrocytes uptake was measured at 1 min and 5 min. To study L-triiodothyronine (LT3) and L-thyroxine (LT4) efflux from erythrocytes, we preloaded the cells during 180 min with [125I]T3 or [125I]T4 and measured their [125I]T3 or [125I]T4 efflux during 60 min. RESULTS: [125I]T3 uptake in erythrocytes from uremic patients pre-HD was higher than control subjects by 50% at 1 min and by 55% at 5 min. However, [125I]T4 uptake in erythrocytes from uremic patients was significantly lower at 1min (88%) and at 5 min (63%). LT3 efflux rate was lower and LT4 efflux was significantly higher than in control subjects. After 60-min of efflux, LT3 remained in erythrocytes was 80% higher and LT4 was 57% lower than in normal individuals. Neither [125I]T3 and [125I]T4 uptake, nor efflux rates were changed by hemodialysis. CONCLUSION: Despite the fact that uremic patients on hemodialysis show low serum levels of LT3, changes in LT3 influx and efflux could act as a compensatory mechanism that neutralize thyroid hormone dysfunction in order to maintain the euthyroid state.