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Metformin Prevents Tobacco Carcinogen–Induced Lung Tumorigenesis
Regan M. Memmott, Jose R. Mercado, Colleen R. Maier, Shigeru Kawabata, Stephen D. Fox and Phillip A. Dennis
Regan M. Memmott
Jose R. Mercado
Colleen R. Maier
Shigeru Kawabata
Stephen D. Fox
Phillip A. Dennis
DOI: 10.1158/1940-6207.CAPR-10-0055 Published September 2010
Activation of the mammalian target of rapamycin (mTOR) pathway is an important and early event in tobacco carcinogen–induced lung tumorigenesis, and therapies that target mTOR could be effective in the prevention or treatment of lung cancer. The biguanide metformin, which is widely prescribed for the treatment of type II diabetes, might be a good candidate for lung cancer chemoprevention because it activates AMP-activated protein kinase (AMPK), which can inhibit the mTOR pathway. To test this, A/J mice were treated with oral metformin after exposure to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Metformin reduced lung tumor burden by up to 53% at steady-state plasma concentrations that are achievable in humans. mTOR was inhibited in lung tumors but only modestly. To test whether intraperitoneal administration of metformin might improve mTOR inhibition, we injected mice and assessed biomarkers in liver and lung tissues. Plasma levels of metformin were significantly higher after injection than oral administration. In liver tissue, metformin activated AMPK and inhibited mTOR. In lung tissue, metformin did not activate AMPK but inhibited phosphorylation of insulin-like growth factor-I receptor/insulin receptor (IGF-1R/IR), Akt, extracellular signal–regulated kinase (ERK), and mTOR. This suggested that metformin indirectly inhibited mTOR in lung tissue by decreasing activation of insulin-like growth factor-I receptor/insulin receptor and Akt upstream of mTOR. Based on these data, we repeated the NNK–induced lung tumorigenesis study using intraperitoneal administration of metformin. Metformin decreased tumor burden by 72%, which correlated with decreased cellular proliferation and marked inhibition of mTOR in tumors. These studies show that metformin prevents tobacco carcinogen–induced lung tumorigenesis and support clinical testing of metformin as a chemopreventive agent. Cancer Prev Res; 3(9); 1066–76. ©2010 AACR.
Read the Perspective on this article by Engelman and Cantley, p. 1049
Lung cancer is the leading cause of cancer-related death in both men and women in the United States, and new targets are needed for the prevention and treatment of lung cancer. The serine-threonine kinase mammalian target of rapamycin (mTOR) could be a good target because it is an important regulator of processes critical to tumorigenesis, such as cellular metabolism, growth, and proliferation. Many cancers, including lung cancer, are characterized by aberrant activation of mTOR (reviewed in ref. 1). Although activating mutations in mTOR itself have not been identified, deregulation of upstream components that regulate mTOR are prevalent in lung cancer. The prototypic mechanism of mTOR regulation in cells is through activation of the phosphatidylinositol 3-kinase/Akt pathway, but mTOR receives input from multiple signaling pathways, including the Liver Kinase β1 (LKB1)/AMP-activated protein kinase (AMPK) pathway (reviewed in ref. 2).
Exposure of A/J mice to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces K-ras–mediated lung tumorigenesis (3). Lung tumors that develop in these mice are characterized by increased activation of the mTOR pathway, and treatment with the mTOR inhibitor rapamycin prevents lung tumorigenesis in this model by 90% (4). Prevention of NNK-induced lung tumorigenesis by rapamycin is associated with inhibition of the mTOR pathway in tumor cells and with decreased lung- and tumor-associated regulatory T cells (Treg; ref. 5), which can suppress lung immunity and create a permissive environment for tumor development (reviewed in ref. 6). This suggests that rapamycin could be an effective chemopreventive agent in patients at high risk of developing lung cancer. However, the development and application of rapamycin as a chemopreventive agent may be limited because of toxicities. The identification of other drugs inhibiting mTOR that are well tolerated could increase opportunities for lung cancer chemoprevention.
One candidate is the biguanide metformin, which is the most widely prescribed drug for the treatment of type II diabetes. Long-term treatment is associated with few adverse effects (7). Metformin is an effective antidiabetic agent because it activates AMPK, a master regulator of cell metabolism (8–10). AMPK activation may also play a role in preventing the formation and progression of cancer because AMPK is activated by the tumor suppressor LKB1 (11). The LKB1/AMPK pathway is linked to inhibition of tumor growth through inhibition of the mTOR pathway (12). Interestingly, metformin use in humans is associated with a decrease in cancer incidence (13, 14), but whether this is related to activation of AMPK is unclear. Because AMPK inhibits the mTOR pathway, drugs that activate AMPK could be tested in the NNK-induced mouse model of lung tumorigenesis.
We investigated if two different routes of administration of metformin were effective in preventing NNK-induced lung tumorigenesis. Administration of oral metformin significantly prevented lung tumorigenesis by 40% to 50%, but only modestly inhibited the mTOR pathway in tumors. Intraperitoneal administration of metformin produced higher plasma concentrations of metformin, which increased the inhibition of the mTOR pathway in tumors and reduced lung tumorigenesis by ∼75%. Inhibition of mTOR in lung tissue was surprisingly independent of AMPK and correlated with decreased circulating levels of insulin-like growth factor-I (IGF-I) and insulin, suggesting that chemoprevention of lung tumors by metformin was indirect. This mechanism of chemoprevention is similar to that observed in diabetics and provides an underpinning for the decreased cancer incidence associated with metformin use in diabetic patients.
The following antibodies were obtained from Cell Signaling Technologies: phospho-AMPK (T172), pan-AMPKα, phospho-acetyl-CoA carboxylase (ACC; S79), phospho-S6 (S235/236), eukaryotic translation initiation factor 4E–binding protein 1 (4E-BP1), phospho-IGF-I receptor/insulin receptor (IGF-1R/IR; Y1135/1146), phospho-Akt (T308), and phospho-extracellular signal–regulated kinase (ERK; T202/Y204). Antimouse/rat Foxp3 (clone FJK-15s) and CD3 antibodies were obtained from eBioscience and Dako, respectively. Ki-67 antibody was purchased from Vision Biosystems. NNK was obtained from EaglePicher Phamaceuticals. Metformin, radioimmunoprecipitation assay (RIPA) buffer, protease inhibitor cocktail, and phosphatase inhibitors were purchased from Sigma Chemicals. Recombinant mouse IGF-I and HUMULIN R were obtained from R&D Systems and Eli Lilly, respectively.
Tumorigenesis and biomarker studies in A/J mice
A/J mice were obtained from The Jackson Laboratory and housed according to the guidelines of the Animal Care and Use Committee of the NIH. For the tumorigenesis studies, 6-week-old female A/J mice were fed the AIN-93G diet (15, 16) and given three weekly injections of 100 mg/kg NNK, as previously described (4). One week following the last dose of NNK, mice were switched to the AIN-93M diet and randomized into a control group or groups that received 1 or 5 mg/mL metformin dissolved in drinking water (n = 15 mice per group). Mice were treated with this dosing schedule with metformin for 13 weeks, and their water was changed weekly. For the tumorigenesis study in which metformin was administered by intraperitoneal injection, mice were treated daily with saline or 250 mg/kg metformin beginning 1 week after the last dose of NNK (n = 15 mice per group). This dosing schedule with metformin was continued for 13 weeks. At the end of the tumorigenesis studies, mice were anesthetized using isoflurane, and blood was collected by cardiac puncture using BD vacutainer vials containing EDTA. Plasma was isolated by centrifugation of the blood samples and sent to the Laboratory of Proteomics and Analytical Technologies (Frederick, MD) for metformin pharmacokinetic analysis. Lung and liver tissues were also harvested, fixed in formalin overnight, and processed by Histoserv, Inc., for immunohistochemical analysis, or stained with H&E or Oil Red O. To determine tumor multiplicity, peripheral lung adenomas were counted using a dissecting microscope. Tumor volume was calculated by multiplying the three dimensions of each tumor. Two dimensions were measured using a micrometer, and the third dimension was estimated by averaging these dimensions. Tumor burden was calculated as the sum of individual lung tumor volumes in each mouse.
For the biomarker studies, 8-week-old female A/J mice were fed the tumorigenic diet AIN-93M and were injected with saline or 250 mg/kg metformin daily × 3 (n = 3 mice per group). Mice were anesthetized with isoflurane 2 hours after the last injection of metformin, and liver or lung tissues were harvested and flash-frozen in liquid nitrogen. Because AMPK is exquisitely sensitive to the metabolic state of the animal, tissues were harvested early in the morning when the mice were in a "fed" state. Biomarker studies were performed at least three times.
Immunoblotting analysis
Frozen tissues were pulverized on dry ice, and lysates were prepared using RIPA buffer supplemented with protease and phosphatase inhibitors. Immunoblotting analysis of tissue lysates was done as described previously (17). For immunoblotting analysis of p-AMPK or p-IGF-IR/IR, antibodies were used at a dilution of 1:500. All other antibodies were used at a dilution of 1:1,000. Densitometry was done using NIH Image software. To determine differences in the phosphorylation of 4E-BP1 using an antibody that recognizes total 4E-BP1, changes in the ratio of the top band (hyperphosphorylated 4E-BP1) to the sum of the bottom two bands (hypophosphorylated 4E-BP1) were quantified.
Formalin-fixed lung tissues were paraffin-embedded, sectioned, and placed on slides (Histoserv). Immunohistochemical analysis was done on lung tissues from five mice per treatment group. Antigen retrieval was done using target retrieval solution (pH 6.0) and a decloaking chamber (Dako). Tissues were incubated in p-AMPK (1:50), p-S6 (1:100), Ki-67 (1:2,000), Foxp3 (1:25), or CD3 (1:600) antibodies per manufacturer's recommendations. To verify staining specificity, tissues were also incubated in the absence of primary antibody. Detection was done using VECTASTAIN Elite ABC kits (Vector Laboratories).
Immunohistochemical analysis for p-AMPK or p-S6 was quantified by assigning a score of absent (0), minimal (1), moderate (2), or high (3) staining to each tumor cell. All tumors in a single randomly selected section of lung tissue containing all lobes were scored. The staining index was calculated for each tumor by multiplying the staining intensity by its distribution. The staining indices were then averaged for all tumors in mice from each treatment group. Ki-67 staining was quantified by counting the number of Ki-67+ cells per tumor, and these values were averaged for all tumors in mice from each treatment group. Lung-associated percent Foxp3+/CD3+ cells were determined by averaging the number of Foxp3+ and CD3+ cells in ten 40× magnification fields containing normal lung parenchyma. Tumor-associated percent Foxp3+/CD3+ cells were determined by counting the number of Foxp3+ or CD3+ cells per tumor. Tumor-associated percent Foxp3+/CD3+ cells were then averaged for all tumors from mice in each treatment group. The investigator was blinded to the identities of the samples.
Pharmacokinetic analysis of metformin plasma levels
Sample preparation and analysis were done as previously described (18). Analysis was done on plasma collected from nine mice per treatment group at the end of the tumorigenesis study using oral administration of metformin, and from six mice injected with 250 mg/kg metformin intraperitoneal daily × 7, 2 hours after the last injection.
Quantitative reverse transcription-PCR for organic cation transporter 1
Lung and liver tissues were harvested from 10-week-old A/J mice (n = 3) and stored in RNAlater (Ambion). RNA was isolated from tissues using an electric homogenizer and the RNeasy kit (Qiagen). Quantitative reverse transcription-PCR (RT-PCR) for organic cation transporter 1 (OCT1) was done using a two-step process. cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit. Quantitative PCR was subsequently done using SYBR Green PCR Master Mix and run on an ABI PRISM 7500 (Applied Biosystems). Expression of OCT1 mRNA was normalized to GAPDH to control for differences in total mRNA between samples. Primer sequences for mouse OCT1 and GAPDH were used as previously published (19).
Blood glucose and plasma IGF-I and insulin analysis
Blood glucose levels were assessed in mice fed the tumorigenic diet AIN-93G/M or the cereal diet NIH-07 for 16 weeks. Mice were fasted for 4 hours before blood collection from the saphenous vein, and glucose levels were measured using a One Touch Ultra2 glucometer (n = 5 mice per diet). Plasma levels of IGF-I and insulin were measured using the Mouse/Rat IGF-I ELISA (Diagnostic Systems Laboratories) and the Rat/Mouse Insulin ELISA kit (Millipore), respectively. These analyses were done on plasma obtained from five mice per treatment group at the end of the tumorigenesis studies.
All analyses were done using a two-tailed Student's t test.
Oral metformin significantly prevents NNK-induced lung tumorigenesis in A/J mice, but only modestly inhibits the mTOR pathway in lung tumors
To investigate if metformin prevents NNK-induced lung tumorigenesis, A/J mice were treated with 1 or 5 mg/mL metformin dissolved in drinking water for 13 weeks, beginning 1 week after administration of NNK (Fig. 1A). These doses of metformin were used because they have been previously shown to activate AMPK in multiple tissues in mice (20, 21). Metformin was well tolerated and did not significantly affect the weight of mice throughout the course of treatment (data not shown). To determine if oral metformin produced levels that could be achieved in humans, plasma concentrations were assessed at the end of 13 weeks. Mice that received metformin at 1 mg/mL achieved 0.45 μg/mL, and mice at 5 mg/mL achieved 1.70 μg/mL, which is consistent with steady-state values that have been reported in patients (0.5-2 μg/mL; Fig. 1B). Although metformin did not affect tumor incidence or pathology, lung tumor multiplicity was decreased by 28% (1 mg/mL) and 32% (5 mg/mL). Treatment with 5 mg/mL metformin also decreased average tumor volume by 34% (Fig. 1C, left and middle). Overall tumor burden was decreased by 39% (1 mg/mL) and 53% (5 mg/mL; Fig. 1C, right). To determine if oral metformin inhibited the mTOR pathway in lung tumors, immunohistochemical analysis was done using antibodies specific for the phosphorylated form of S6, a component downstream of mTOR. Oral metformin inhibited phospho-S6 by 20% in lung tumors (Fig. 1D). Taken together, these results show that oral metformin significantly prevented tumor growth, but to a lesser extent than rapamycin, which previously decreased lung tumorigenesis by 90% in this model system (4).
Oral administration of metformin prevents NNK-induced lung tumorigenesis in A/J mice and is associated with modest inhibition of the mTOR pathway. A, schema for the prevention study with oral metformin in a tobacco carcinogen–driven mouse model of lung tumorigenesis. B, mean plasma levels of metformin in A/J mice treated with 1 or 5 mg/mL metformin orally were determined using liquid chromatography-tandem mass spectrometry. C, tumor multiplicity (left), tumor volume (middle), and tumor burden (right) were assessed after 13 wk of oral administration of 1 or 5 mg/mL metformin in A/J mice. For the dot plots, each point represents a mouse and the lines represent the median values. For the box and whisker graph, boxes indicate interquartile range, lines indicate median, and whiskers indicate minimal and maximal values. n.s., not significant. D, left, representative images of immunohistochemical analysis for p-S6 in airway epithelium (AE) and lung tumors from control mice and from mice that received oral metformin; right, quantification of immunohistochemical analysis. Columns, mean staining indices; bars, SD. Immunohistochemical analysis was done on five mice per treatment group.
Intraperitoneal administration of metformin produces higher plasma levels of metformin that cause tissue-specific modulation of the AMPK and mTOR pathways
Because oral metformin only modestly affected the mTOR pathway in lung tissue, we investigated if another route of administration with metformin might be more effective in modulating this pathway in mice. A short-term biomarker study was done using intraperitoneal injections of metformin because we hypothesized that peak plasma concentrations would be higher than that achieved with oral administration. Pharmacokinetic analysis showed that plasma levels of metformin 2 hours after the last injection were higher (4.0 μg/mL) than with either oral dosing schedule. Immunoblotting analysis of lysates of liver tissue harvested from mice treated with intraperitoneal metformin showed that it activated hepatic AMPK and increased the phosphorylation of a substrate of AMPK, acetyl-CoA carboxylase (Fig. 2A). Additionally, this dosing schedule with metformin inhibited the mTOR pathway in liver tissue, as shown by a trend toward decreased phosphorylation of both S6 and 4E-BP1, although some variability in these markers between mice was observed. Conversely, short-term oral administration of 5 mg/mL metformin did not activate AMPK or inhibit the mTOR pathway in liver tissue (Fig. 2B). This suggests that higher plasma metformin concentrations are better able to activate AMPK and inhibit the mTOR pathway.
Intraperitoneal administration of metformin causes tissue-specific modulation of the AMPK and mTOR pathways in A/J mice. A to C, immunoblotting analysis of components of the AMPK pathway and pathways downstream of IGF-IR/IR that converge on mTOR in liver (A and B) and lung (C) tissues. ACC, acetyl-CoA carboxylase. Tissues were harvested from mice treated short-term with intraperitoneal injections of 250 mg/kg metformin (daily × 3) in two independent studies; representative blots from these studies are shown. Densitometry was done on immunoblots of liver lysates from both studies, which is presented in A (right). B, liver tissue was also harvested from mice treated with 5 mg/mL metformin orally for 7 d for comparison. D, OCT1 expression in liver and lung tissues from A/J mice was assessed by RT-PCR.
Although intraperitoneal administration of metformin activated hepatic AMPK, it did not activate AMPK in lung tissue (Fig. 2C). Nonetheless, metformin still inhibited the mTOR pathway in lung tissue. Akt and ERK phorylation were also inhibited, suggesting that an upstream receptor tyrosine kinase might be inhibited by metformin. Clinical studies have shown that metformin treatment is associated with decreases in plasma levels of insulin and IGF-I in both diabetic and nondiabetic patients (22, 23). Immunoblotting analysis using an antibody specific for the phosphorylated, or active forms, of the IGF-IR and IR showed that metformin decreased their activation in lung tissue. These results could suggest that intraperitoneal administration of metformin inhibits the mTOR pathway in lung tissue independently of AMPK by decreasing the response to insulin or IGF-I.
The fact that metformin activates AMPK in liver but not in lung tissue indicates a tissue-specific response to metformin, which could be related to differences in the level of expression of OCT1. This receptor is required for tissue uptake of metformin in mice, and polymorphisms in the gene that encodes OCT1 are associated with decreased response to metformin in diabetic patients (19). Quantitative RT-PCR analysis of liver and lung tissues harvested from A/J mice showed that OCT1 expression was 17-fold higher in liver tissue (Fig. 2D), suggesting that the inability of metformin to activate AMPK in lung tissue was due to diminished uptake.
Intraperitoneal administration of metformin is more effective than oral administration in preventing NNK-induced lung tumorigenesis in A/J mice
Because intraperitoneal administration of metformin significantly inhibited the mTOR pathway in lung tissue, we investigated if this dosing schedule with metformin was more effective in preventing NNK-induced lung tumorigenesis in A/J mice. Mice were fed the tumorigenic AIN-93G/M diets and treated with daily intraperitoneal injections of saline or metformin, beginning 1 week after NNK administration (Fig. 3A). This dosing schedule with metformin was surprisingly well tolerated and did not significantly affect the weight of mice throughout the course of the study (data not shown). Metformin was highly effective. Tumor multiplicity decreased by 66%, average tumor volume decreased by 50%, and overall tumor burden decreased by 72% (Fig. 3B-D). Phosphorylation of S6 in tumors decreased by 40% (Fig. 4A). Thus, intraperitoneal metformin was more effective in preventing NNK-induced lung tumorigenesis and inhibiting mTOR than oral metformin. However, consistent with the results of our short-term biomarker studies, this occurred in the absence of AMPK activation in lung tumors (Supplementary Fig. S1).
Administration of metformin by intraperitoneal injection prevents NNK-induced lung tumorigenesis. A, schema for the prevention study with metformin administered by intraperitoneal injection in a tobacco carcinogen–driven mouse model of lung tumorigenesis. Tumor multiplicity (B), tumor volume (C), and tumor burden (D) were assessed after 13 wk of administration of 250 mg/kg metformin daily by intraperitoneal injection in A/J mice. For the dot plots in B and D, each point represents a mouse and the lines represent the median values. For the box and whisker graph shown in C, boxes indicate interquartile range, lines indicate median, and whiskers indicate minimal and maximal values.
Intraperitoneal administration of metformin inhibits the mTOR pathway in lung tissue, decreases tumor cell proliferation, and decreases lung- and tumor-associated Foxp3+ Treg. A, left, representative images of immunohistochemical analysis for p-S6 in airway epithelium (AE) and lung tumors from vehicle-treated mice and mice that received intraperitoneal metformin; right, quantification of immunohistochemical analysis. Columns, mean staining indices; bars, SD. B, top, representative images of immunohistochemical analysis for Ki-67, a marker of cellular proliferation, in lung tumors from vehicle- and metformin-treated mice; bottom, quantification of immunohistochemical analysis. Columns, mean number of Ki-67+ cells per tumor; bars, SD. C, left, representative images of immunohistochemical analysis for tumor-associated Foxp3+ and CD3+ cells in A/J mice treated with vehicle or intraperitoneal metformin; right, quantification of immunohistochemical analysis for lung- and tumor-associated percent Foxp3+/CD3+ cells (top and bottom, respectively). For A to C, immunohistochemical analysis was done on five mice per treatment group.
Previous studies have shown that metformin inhibits cancer cell proliferation in vitro by a mechanism that is dependent on inhibition of mTOR-induced protein translation (24). To investigate if metformin inhibits tumor cell proliferation in vivo, immunohistochemical analysis was done on lung tissues using an antibody specific for Ki-67, a marker of cellular proliferation (Fig. 4B). We observed that inhibition of NNK-induced lung tumorigenesis by metformin was associated with a 67% decrease in tumor cell proliferation. Terminal deoxyribonucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) analysis showed that metformin did not induce apoptosis in tumor cells (data not shown). Collectively, these data show that prevention of NNK-induced lung tumorigenesis by metformin is associated with decreased tumor cell proliferation, but not increased apoptosis.
The ability of metformin to prevent NNK-induced lung tumorigenesis could also be due to the effects on the tumor microenvironment. We previously showed that NNK increases lung- and tumor-associated Foxp3+ Treg in A/J mice, which are required for K-ras–mediated lung tumorigenesis, and that rapamycin prevented the induction of lung-associated Foxp3+ cells by NNK (5). Because metformin also inhibits the mTOR pathway in NNK-induced lung tumors in A/J mice, we investigated if treatment with metformin was associated with decreases in lung- and tumor-associated Treg. Immunohistochemical analysis using antibodies specific for Foxp3, a marker of Treg, and CD3, a pan-T cell marker, showed that metformin treatment decreased lung-associated Treg by 65% and decreased tumor-associated Treg by 50% (Fig. 4C). Because we have previously shown that Foxp3+ Treg are required for K-ras–mediated lung tumorigenesis (5), decreases in lung- and tumor-associated Treg might contribute to the ability of metformin to prevent NNK-induced lung tumorigenesis in A/J mice.
Metformin is a well-tolerated drug that prevents fatty liver disease in A/J mice fed the tumorigenic diet AIN-93G/M
Because there were no published studies that showed the safety of daily intraperitoneal injections of metformin over many weeks, we harvested livers from mice at the end of the tumorigenesis study to assess hepatotoxicity. Examination of H&E-stained sections showed that livers from the control group contained a large number of vacuoles that stained with Oil Red O (Fig. 5A). These lipid-laden vacuoles were substantially decreased both in size and number in livers from mice treated with metformin (Fig. 5B). The presence of vacuoles was most likely due to diet and not NNK because we did not observe liver vacuolization in mice that were exposed to NNK but were fed the cereal diet NIH-07 (Supplementary Fig. S2). Interestingly, the AIN-93G and AIN-93M diets are reformulations of the AIN-76A diet, which has been previously reported to cause fatty liver disease in rats (25). These results suggest that the tumorigenic AIN-93G and AIN-93M diets induce fatty liver disease in A/J mice, which is prevented by treatment with metformin. To confirm that diabetes was not induced by the AIN-93G/M diet (26), glucose and insulin levels were compared in mice fed either AIN-93G/M or NIH-07 diets. No differences in blood glucose (Fig. 5C) or insulin levels (data not shown) were observed. This shows that metformin prevented tobacco carcinogen–induced tumor growth in a nondiabetic mouse model of lung tumorigenesis.
The tumorigenic diet AIN-93G/M causes fatty liver disease in A/J mice, which is prevented by metformin, but does not cause hyperglycemia. A, H&E (left) or Oil Red O (right) staining of liver sections from A/J mice fed AIN-93G/M and treated with vehicle. B, H&E staining of liver sections from A/J mice fed AIN-93G/M and treated with 250 mg/kg metformin intraperitoneal daily for 13 wk. For A and B, images were taken at ×10 and ×40 magnifications. C, blood glucose levels in A/J mice that were fed the cereal diet NIH-07 or the tumorigenic diet AIN-93G/M (n = 5 mice per group). Columns, mean; bars, SD.
Inhibition of the mTOR pathway in tumors is associated with decreases in levels of circulating IGF-I and insulin
Because we showed that metformin decreased phosphorylation of IGF-IR and/or IR in lung tissue, and metformin is known to decrease circulating levels of these hormones in patients, plasma levels of insulin and IGF-I were assessed at the end of the tumorigenesis studies. Oral and intraperitoneal administration of metformin decreased insulin by ∼20% and 35%, respectively (Fig. 6A). Additionally, oral metformin at either 1 or 5 mg/mL decreased the levels of circulating IGF-I by ∼20% (Fig. 6B, left). Intraperitoneal injection of metformin did not decrease IGF-I levels, but the levels of IGF-I in mice injected with saline were significantly lower than those in mice that were not injected (Fig. 6B, middle and right), suggesting that stress associated with daily injections could have obscured an inhibitory effect of intraperitoneal metformin on the circulating levels of IGF-I. These results support the hypothesis that metformin inhibits the mTOR pathway in normal lung epithelium and in lung tumors by decreasing circulating levels of IGF-I or insulin, which contributes to its ability to prevent NNK-induced lung tumorigenesis.
Metformin-induced inhibition of the mTOR pathway in lung tissue is associated with decreases in circulating levels of IGF-I and insulin. A and B, plasma levels of insulin (A) or IGF-I (B) were assessed by ELISA in A/J mice treated with oral or i.p. administration of metformin at the end of the tumorigenesis studies. Columns, mean; bars, SD. This analysis was done on five mice per treatment group. Plasma IGF-I levels were also assessed in mice that were not injected or given intraperitoneal injections of saline or 250 mg/kg metformin daily × 3 (B, right). C, immunoblotting analysis of the IGF-IR/IR/Akt/mTOR pathway in liver and lung tissues harvested from A/J mice 0.5 h after administration of 0.5 mg/kg IGF-I or 0.75 units of insulin.
To investigate the responsiveness of lung and liver tissues to exogenously administered IGF-I or insulin, IGF-I or insulin was injected into the peritoneal cavity and tissues were processed for immunoblotting analysis. IGF-I rapidly activated the IGF-IR/Akt/mTOR pathway in lung tissue (Fig. 6C). It also increased phosphorylation of IGF-IR and Akt in liver tissue but did not affect the phosphorylation of S6. The tissue-specific response to insulin was opposite that of IGF-I, in that insulin had no effect on the mTOR pathway in lung tissue, but increased the phosphorylation of the IR, Akt, and S6 in liver tissue. These data suggest that lung tissue is more responsive to IGF-I and that liver tissue is more responsive to insulin. Given that metformin decreased the levels of IGF-I and insulin, prevention of lung tumorigenesis by metformin might be related to multiple mechanisms in different organs.
A majority of lung cancer cases are associated with tobacco use (85-90%), and more than 90 million current or former smokers in the United States are at permanent increased risk of developing lung cancer (27). Thus, there is a great need for the development of new chemotherapeutic strategies for the prevention of this disease. Our studies show that metformin is effective in preventing tumor growth in a mouse model of tobacco carcinogen–induced lung tumorigenesis. Oral administration of 1 or 5 mg/mL metformin decreased lung tumor burden in mice by 38% and 53%, respectively. The steady-state levels of metformin in mice given 5 mg/mL are similar to those in diabetic patients using metformin, suggesting the possibility that clinical prevention of lung cancer could be achieved with standard oral dosing. Despite this efficacy of oral metformin, intraperitoneal administration of metformin was more effective than oral administration and decreased tumor burden by 72%. Because plasma levels of metformin were higher using this route of administration, these results could support the development of more potent analogues of metformin such as phenformin. Phenformin is more effective at decreasing hyperglycemia in diabetic patients, and preclinical studies show that phenformin is a more potent activator of AMPK and activates AMPK in a broader range of tissues than metformin (21). However, phenformin treatment is associated with lactic acidosis in patients, which could limit its clinical potential as a chemopreventive agent (28).
Inhibition of tumorigenesis by metformin could be related to mTOR inhibition within tumors that is an indirect effect of diminished circulating levels of growth factors such as insulin and IGF-I. Inhibition of mTOR correlated with decreased tumor cell proliferation in A/J mice, which is consistent with in vitro studies that show that metformin is a cytostatic agent that decreases cancer cell proliferation by inhibiting mTOR-dependent protein translation (24). Metformin inhibited mTOR in lung tissue independently of AMPK activation, which suggests that lung tissue does not respond directly to metformin due to insufficient uptake. Consistent with this, lung tissues from A/J mice express 17-fold less OCT1 than liver tissues, which did show AMPK activation after metformin administration. Inhibition of the mTOR pathway by metformin was associated with decreased phosphorylation of IGF-IR/IR and decreased levels of circulating IGF-I and insulin, suggesting that they might contribute to tumorigenesis in this model. In agreement with this, a recent study using mice genetically engineered to overexpress IGF-I in lung tissues showed that IGF-I enhances NNK-induced lung tumorigenesis in FVB mice (29). These data suggest that the prevention of lung tumorigenesis by metformin could be due to effects on other tissues that decrease the circulating levels of growth factors.
This is the first study to show the safety and efficacy of metformin in a tobacco carcinogen–driven mouse model of lung tumorigenesis, but other studies have investigated the ability of metformin to prevent tumor growth in vivo. For example, studies performed using multiple xenograft models have shown that treatment with metformin modestly inhibited tumor growth (30–32). The modest effect of metformin observed in some of these xenograft models could be due to the use of immunocompromised mice or the fact that the subcutaneous compartment where tumors are placed is not analogous to the microenvironment within organs. A role for the microenvironment in the response to metformin is supported by the fact that metformin decreased lung- and tumor-associated Treg, which is a requirement for K-ras–induced lung tumorigenesis originally described in studies using rapamycin. In a syngeneic orthotopic model of lung cancer, LLC1, investigators showed that metformin effectively inhibited tumor growth, but only in mice that were fed a high-calorie diet (33). However, the role of the mTOR pathway in promoting tumor growth in this model is not clear, and inhibition of the mTOR pathway in tumors was not assessed. Metformin was also tested in a Pten+/−Lkb1fl/+ mouse model (21). Pten and LKB1 are tumor suppressors that negatively regulate mTOR, and tissues from these mice were characterized by activation of the mTOR pathway. However, administration of metformin to Pten+/−Lkb1fl/+ mice only increased tumor latency by 1 month and did not affect tumor incidence or morphology. Although this study used a similar oral dosing schedule, they did not observe decreases in serum levels of insulin. This could be related to loss of LKB1 and/or Pten in different tissues such as liver. Collectively, these studies suggest that indirect effects of metformin on the tumor microenvironment and on circulating levels of growth factors could be important in the prevention of tumorigenesis.
Metformin is a promising candidate for lung cancer chemoprevention. Clinical studies have shown that long-term treatment with metformin is associated with few adverse effects in diabetic, as well as nondiabetic, patient populations (7, 34). Similarly, the dosing schedules with metformin used in our studies seemed to be well tolerated and did not cause weight loss or hepatotoxicity. Metformin also prevented diet-induced fatty liver disease in A/J mice. This effect of metformin has been previously shown in mouse models of hepatosteatosis (35), as well as in patients with nonalcoholic fatty liver disease (reviewed in ref. 36). Although the tumorigenic diets used in our studies caused lipid accumulation in the liver, they did not induce hyperglycemia or hyperinsulinemia. This shows that metformin was effective in preventing tobacco carcinogen–induced tumor growth in a nondiabetic mouse model of lung tumorigenesis. An additional advantage of metformin as a chemopreventive agent for lung cancer is that it inhibits tumor formation independently of AMPK activation in lung tissue. Metformin activates AMPK by a mechanism that is dependent on its upstream kinase, the tumor suppressor LKB1 (37). Somatic mutations in LKB1 are an early and frequent event in lung cancer and are associated with smoking (38–40). Because the effects of metformin are independent of AMPK in lung tissue, the presence of LKB1 mutations would not preclude response to metformin. Taken together, the safety and efficacy of metformin provide strong rationale for a clinical prevention trial with metformin in heavy smokers who are at high risk of developing lung cancer.
Note: Supplementary data for this article are available at Cancer Prevention Research Online (http://cancerprevres.aacrjournals.org/).
Revision received April 13, 2010.
Accepted May 17, 2010.
Chiang GG,
Abraham RT
. Targeting the mTOR signaling network in cancer. Trends Mol Med 2007;13:433–42.
Memmott RM,
Dennis PA
. Akt-dependent and -independent mechanisms of mTOR regulation in cancer. Cell Signal 2009;21:656–64.
Belinsky SA,
Devereux TR,
Maronpot RR,
Stoner GD,
Anderson MW
. Relationship between the formation of promutagenic adducts and the activation of the K-ras protooncogene in lung tumors from A/J mice treated with nitrosamines. Cancer Res 1989;49:5305–11.
Granville CA,
Warfel N,
Tsurutani J,
. Identification of a highly effective rapamycin schedule that markedly reduces the size, multiplicity, and phenotypic progression of tobacco carcinogen-induced murine lung tumors. Clin Cancer Res 2007;13:2281–9.
Balogh A,
. A central role for Foxp3+ regulatory T cells in K-Ras-driven lung tumorigenesis. PLoS One 2009;4:e5061.
Zou W
. Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol 2006;6:295–307.
Bolen S,
Feldman L,
Vassy J,
. Systematic review: comparative effectiveness and safety of oral medications for type 2 diabetes mellitus. Ann Intern Med 2007;147:386–99.
Hundal HS,
Ramlal T,
Reyes R,
Leiter LA,
Klip A
. Cellular mechanism of metformin action involves glucose transporter translocation from an intracellular pool to the plasma membrane in L6 muscle cells. Endocrinology 1992;131:1165–73.
Stumvoll M,
Nurjhan N,
Perriello G,
Dailey G,
Gerich JE
. Metabolic effects of metformin in non-insulin-dependent diabetes mellitus. N Engl J Med 1995;333:550–4.
Zhou G,
Myers R,
. Role of AMP-activated protein kinase in mechanism of metformin action. J Clin Invest 2001;108:1167–74.
Shaw RJ,
Kosmatka M,
Bardeesy N,
. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc Natl Acad Sci U S A 2004;101:3329–35.
Manning BD,
. The LKB1 tumor suppressor negatively regulates mTOR signaling. Cancer Cell 2004;6:91–9.
Libby G,
Donnelly LA,
Donnan PT,
Alessi DR,
Morris AD,
Evans JM
. New users of metformin are at low risk of incident cancer: a cohort study among people with type 2 diabetes. Diabetes Care 2009;32:1620–5.
Evans JM,
Emslie-Smith AM,
Morris AD
. Metformin and reduced risk of cancer in diabetic patients. BMJ 2005;330:1304–5.
Hecht SS,
Morse MA,
Amin S,
. Rapid single-dose model for lung tumor induction in A/J mice by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and the effect of diet. Carcinogenesis 1989;10:1901–4.
Reeves PG,
Nielsen FH,
Fahey GC Jr.
AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 1993;123:1939–51.
Gills JJ,
Hollingshead M,
. Phosphatidylinositol ether lipid analogues induce AMP-activated protein kinase-dependent death in LKB1-mutant non small cell lung cancer cells. Cancer Res 2008;68:580–8.
Wang Y,
Tang Y,
Gu J,
Fawcett JP,
Bai X
. Rapid and sensitive liquid chromatography-tandem mass spectrometric method for the quantitation of metformin in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci 2004;808:215–9.
Shu Y,
Sheardown SA,
Brown C,
. Effect of genetic variation in the organic cation transporter 1 (OCT1) on metformin action. J Clin Invest 2007;117:1422–31.
Ma TC,
Buescher JL,
Oatis B,
. Metformin therapy in a transgenic mouse model of Huntington's disease. Neurosci Lett 2007;411:98–103.
Huang X,
Wullschleger S,
Shpiro N,
. Important role of the LKB1-AMPK pathway in suppressing tumorigenesis in PTEN-deficient mice. Biochem J 2008;412:211–21.
Berker B,
Emral R,
Demirel C,
Corapcioglu D,
Unlu C,
Kose K
. Increased insulin-like growth factor-I levels in women with polycystic ovary syndrome, and beneficial effects of metformin therapy. Gynecol Endocrinol 2004;19:125–33.
Goodwin PJ,
Pritchard KI,
Ennis M,
Clemons M,
Graham M,
Fantus IG
. Insulin-lowering effects of metformin in women with early breast cancer. Clin Breast Cancer 2008;8:501–5.
Dowling RJ,
Zakikhani M,
Fantus IG,
Pollak M,
Sonenberg N
. Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells. Cancer Res 2007;67:10804–12.
Medinsky MA,
Popp JA,
Hamm TE,
Dent JG
. Development of hepatic lesions in male Fischer-344 rats fed AIN-76A purified diet. Toxicol Appl Pharmacol 1982;62:111–20.
Surwit RS,
Feinglos MN,
Rodin J,
. Differential effects of fat and sucrose on the development of obesity and diabetes in C57BL/6J and A/J mice. Metabolism 1995;44:645–51.
IARC. Tobacco smoking and tobacco smoke. IARC monographs on the evaluation of the carcinogenic risks of chemicals to humans. Lyon (France): IARC; 2004.
McGuinness ME,
Talbert RL
. Phenformin-induced lactic acidosis: a forgotten adverse drug reaction. Ann Pharmacother 1993;27:1183–7.
Kim WY,
Jin Q,
Oh SH,
. Elevated epithelial insulin-like growth factor expression is a risk factor for lung cancer development. Cancer Res 2009;69:7439–48.
Buzzai M,
Jones RG,
Amaravadi RK,
. Systemic treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell growth. Cancer Res 2007;67:6745–52.
Ben Sahra I,
Laurent K,
Loubat A,
. The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level. Oncogene 2008;27:3576–86.
Phoenix KN,
Vumbaca F,
Claffey KP
. Therapeutic metformin/AMPK activation promotes the angiogenic phenotype in the ERα negative MDA-MB-435 breast cancer model. Breast Cancer Res Treat 2009;113:101–11.
Algire C,
Blouin MJ,
Shuai JH,
Pollak M
. Metformin attenuates the stimulatory effect of a high-energy diet on in vivo LLC1 carcinoma growth. Endocr Relat Cancer 2008;15:833–9.
Tang T,
Lord JM,
Norman RJ,
Yasmin E,
Balen AH
. Insulin-sensitising drugs (metformin, rosiglitazone, pioglitazone, d-chiro-inositol) for women with polycystic ovary syndrome, oligo amenorrhoea and subfertility. Cochrane Database Syst Rev 2009;CD003053.
Lin HZ,
Yang SQ,
Chuckaree C,
Kuhajda F,
Ronnet G,
Diehl AM
. Metformin reverses fatty liver disease in obese, leptin-deficient mice. Nat Med 2000;6:998–1003.
Stein LL,
Dong MH,
Loomba R
. Insulin sensitizers in nonalcoholic fatty liver disease and steatohepatitis: current status. Adv Ther 2009;26:893–907.
Lamia KA,
Vasquez D,
. The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin. Science 2005;310:1642–6.
Ghaffar H,
Sahin F,
Sanchez-Cepedes M,
. LKB1 protein expression in the evolution of glandular neoplasia of the lung. Clin Cancer Res 2003;9:2998–3003.
Matsumoto S,
Iwakawa R,
Takahashi K,
. Prevalence and specificity of LKB1 genetic alterations in lung cancers. Oncogene 2007;26:5911–8.
Ji H,
Ramsey MR,
Hayes DN,
. LKB1 modulates lung cancer differentiation and metastasis. Nature 2007;448:807–10.
You are going to email the following Metformin Prevents Tobacco Carcinogen–Induced Lung Tumorigenesis
Cancer Prev Res September 1 2010 (3) (9) 1066-1076; DOI: 10.1158/1940-6207.CAPR-10-0055


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Cricket Probe
Welcome to our blog. As enthusiasts of cricket we present you the pre-match build up's, post match analysis and of course the famous on-going cricketing controversies. We value your feedback, please leave a comment on our stories and also tell us what content will intrigue you to comeback and read again.
New Zealand vs England
Contemporary Cricket, interesting or not?
By CricketProbe April 22, 2018
Gone are those days when the test matches used to last for all the 5 days, gone are those days when cricket was considered to be an even contest between the bat and the ball, gone are those days when the pitch was just a platform/turf which is used for the sport.
With the new generation bats, the short boundaries, and the lack of quality in the bowling, the gentleman's sport is tending towards batsmen.
Sachin Tendulkar is considered as one of the greatest batsmen, we all are aware of his achievements and records, Ricky Pointing is one the Australian greats, Jacques Kallis from South Africa, Inzamam from Pakistan, and many more. One common factor among all these players and what made them great was their resilience and playing in the same era facing quality bowlers.
Talking about the bowlers do we remember Courtney Walsh and Curtly Ambrose from West Indies? Glenn Mcgrath and Brett Lee from Australia, Shaun Pollock and Ntini from South Africa, Wasim and Waqar from Pakistan and many more. These are some of the prominent bowlers that cricket has ever witnessed.
The common factor between these batsmen and bowlers was that they have played in the same era and most of the batsmen have faced these bowlers and showed why they are remembered as great players, and most of these bowlers have picked up these batsmen and showed why they are remembered as great players.
Cricket as a sport was enjoyed and embraced when there was an even competition between the bat and the ball.
That was the time when the average score of an ODI was 250, and test matches were played all the five days.
With the contemporary cricket what we witness is completely contrasting, the average score in an ODI is close to 300, and it is very rare to witness a test match get into the fifth day.
The runs are flowing like water gushing through the opened dam gates, the wickets are getting dried as a drought-stricken place. The reason is still a poking enigma, there sure are some class bowlers to push the batsmen on to the back foot.
However, we cannot compare the Cricket of both the eras, the conditions are different, the players are different, the politics are different, the mindset is different and many more.
With all this said is this Cricket interesting anymore? or will that Cricket remain interesting forever?
Stalwart of England Cricket call it quits!
By CricketProbe September 14, 2018
As we all thought the master blaster's test record is in jeopardy by this English Stalwart, with the run of time this stalwart call it quits from the international Cricket at the age of 33- Still young enough to call it quits. We are now aware of which English player has announced his retirement lately, the reason is simple as we expect it to be so from his nature- which is always down to earth.
Carrying the English opening duties on his shoulder for 12 years and delivering in most of the cases talks about his grit and potential. England's test team without Cook's name is unseen, a record of representing a national team continuously without missing a match is held by our Cook- talks about his strength and fitness.
The legacy was incepted in 2006 when Cook has been handed his debut cap against India at Nagpur by Flintoff, he announced his arrival by scoring a brilliant century in England's second innings, from then he has cooked many innings and has become an unprecede…
En-route English Soil
By CricketProbe May 07, 2018
The only country where he hasn't sparked up to his potential, the only nation where he likes to quench his thirst for runs, the only place where Indian fans would love to see him score big is England and our captain Kohli is all set to begin his county stint with Surrey.
With this news rolling out in the social media many of his fans are gearing up to witness their hero in the county colors. Surrey not only managed to bag the big brand Virat for a month they managed to get him for a nominal value.
While BCCI is skeptical about Surrey managing Virat's image, they agreed to keep the promotions at minimum and focus more on cricket.
Indian skipper will feature in at least six matches starting from June 1st. Both Virat and Alec Stewart (Surrey's director of cricket) expressed their happiness.
Virat says "It has long been an ambition of mine to play county cricket and I am thankful to Alec Stewart and Surrey for allowing me the opportunity to join them during their 2018 se…
Dhamaka in Delhi
Well well, if you have missed out tonight's encounter between Delhi and Hyderabad, then you have certainly missed out on some serious run feast. Winning the toss and electing to bat first, Delhi has lost couple of early wickets as Sunrisers were again spot on with their line and lengths.
However, their misery wasn't amplified as Pant took up the responsibility and annihilated the Sunrisers bowlers to every nook and corner of the park. Very seldom you find a guy like Maxwell being veiled by his partner's innings; it was the case today when Pant has out shadowed every one until something unprecedented incident took place.
Scoring his maiden century, Pant has smashed all the bowlers of SRH; known for their bowling SRH has turned the tide and chased down 188 like a boss.
At the end of first innings, it certainly looked like an uphill task for the Sunrisers. But what we have witnessed was just scintillating. SRH batsmen were all over the DD bowlers and never let the required r…
Australia vs SouthAfrica
Ball-tampering
England vs India
Kings X1 Punjab
Pakistan vs WestIndies

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LOS ANGELES, April 7, 2016—Researchers from Stanford University and the University of California, Berkeley report in today's edition of Science that volunteer canvassers trained in an approach developed by the Los Angeles LGBT Center reduced voters' prejudice against transgender people.
Last year the study's authors set off a firestorm of debate about the need for transparency in social science research when they raised doubts about a now-retracted article in Science about the voter canvassing methods developed and implemented by the Center to promote support for marriage equality. They discovered the data irregularities in the article, authored by Michael J. LaCour and Donald P. Green, while beginning their own study of the Center's canvassing methods to reduce anti-transgender prejudice.
"We found that a single, approximately 10-minute conversation with a stranger produced large reductions in prejudice that persisted for at least the three months studied to date, were resistant to counterargument, and affected political attitudes," said David Broockman, an assistant professor of political economy at Stanford University's Graduate School of Business.
He and Joshua Kalla, a Ph.D. student in political science at the University of California at Berkeley, co-authored the study.
Kalla said "The decline in prejudice against transgender people achieved by the canvassers is comparable to the decrease in prejudice against gay and lesbian people that took more than a decade to achieve."
The Los Angeles LGBT Center developed and implemented the canvassing model the researchers studied. Dave Fleischer, director of the center's Leadership LAB, welcomed the academics' independent measurement of its canvassing program.
"Our ability to change voters' hearts and minds has been measured, this time for real," Fleischer said, contrasting the intensely reviewed work of Broockman and Kalla to the earlier, retracted paper.
When Broockman and Kalla both were graduate students at UC Berkeley last year, they uncovered data irregularities in a December 2014 Science article studying the center's innovative grassroots approach. That article was retracted after Broockman and Kalla ( along with political science and biostatistics professor Peter Aronow of Yale University ) uncovered the irregularities. Broockman, Kalla and Aronow subsequently won a Leamer-Rosenthal Prize for Open Social Science for going public with their concerns.
"Our study's findings differ from the retracted one's in an important way," Kalla said. "We found that canvassers do not need to be members of an affected group to lastingly reduce prejudice against that group. Both transgender and non-transgender allies were effective at reducing prejudice against transgender people." However, the study does find that experienced canvassers appear especially effective.
The research measured the impact of an approach developed by the Los Angeles LGBT Center that the Center calls "deep canvassing." It differs from a conventional canvass conversation in both length and reciprocity, with canvassers taking 10-15 minutes as they listen to voters' experiences and respond conversationally rather than reciting a script or talking points. These conversations involve voters engaging in deep reflection on their experiences and views, a process involving so-called "active processing" of their ideas.
In the conversations, canvassers asked voters to reflect on experiences voters had when treated differently and with LGBT people. Psychologists call this exercise "analogic perspective-taking," as it involves considering what another's experience is like by likening it to one's own.
From January through June 2015, the Los Angeles LGBT Center partnered with SAVE, South Florida's largest and longest-serving LGBT organization, based in Miami. After the Miami-Dade County Commission voted to include transgender people in the county's human rights ordinance, the two organizations canvassed voters in conservative Miami neighborhoods in expectation of a ballot measure to repeal the ordinance. The study of the canvassing began in June 2015.
The result: with a rigorous randomized trial — just like a clinical drug trial — Broockman and Kalla found that the deep-canvass conversations resulted in a significant reduction in anti-transgender prejudice among approximately one in 10 voters. As in a clinical drug trial, the researchers calculated this impact by comparing those canvassed to a randomly assigned control group of voters who opened their doors but were assigned to receive a conversation about recycling instead.
The researchers also found that among the entire treatment group of voters who were canvassed, there was a reduction of anti-transgender prejudice comparable to the decline of prejudice against gay and lesbian people that took place over more than a decade, between 1998 and 2012.
In repeated re-measurement, the impact of the canvassing remained intact for at least the three months studied to date. This enduring effect stands in contrast to other published measurements of conventional attempts at voter persuasion and prejudice reduction through TV ads or mail, or in standard phone or canvass conversations.
"The immediate practical value is that we know we are reducing anti-transgender prejudice here in Miami," said Justin Klecha, director of campaigns at SAVE. The organization has continued deep canvassing in its ongoing campaign to pass statewide non-discrimination protections and make Miami-Dade County a more accepting place for transgender people to live and work.
"These conversations are a real game-changer for us here in Florida," said Tony Lima, executive director of SAVE. "Because of these conversations and their impact, we're getting closer to being the first state in the South to pass statewide protections for LGBT people."
Commenting on the significance of the findings to the broad LGBT movement for equality, Los Angeles LGBT Center CEO Lorri L. Jean said "When our community faces anti-LGBT ballot measures, it has become abundantly clear we need more than 30-second ads to win. We've long believed that if LGBT people and our allies could meet and engage voters in heartfelt conversations, we could reduce their prejudice. It's exciting to have the data to prove that deep canvassing works, and gratifying to share this powerful new approach with LGBT leaders and progressive allies throughout the country."
Neither of the study's authors is affiliated with the Los Angeles LGBT Center or SAVE, nor did they receive compensation from them for the research.
"These results suggest that campaigns could be more effective by engaging in more one-on-one conversations with voters, rather than merely flooding airwaves and mailboxes," he said. "The fact that minds could be changed across partisan lines and on a controversial social issue is encouraging for the power of face-to-face discourse."
"The bottom line is that we have new insight into how to reduce prejudice against transgender people," said Fleischer. "Considering the recent loss at the ballot box in Houston, new anti-LGBT legislation in North Carolina and Mississippi, and the threat of future anti-LGBT ballot measures and bills, this study has real practical importance. We in the LGBT community, and our allies, can put ourselves in a better position to win if we start having deep-canvass conversations now, well in advance of a flash point."


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About Kathy Etchingham
Home Learn About Hendrix About Kathy Etchingham
The recreation of Jimi Hendrix's flat at 23 Brook Street, London was done using evidence from the photo shoots and films captured here, but also through invaluable discussion with Kathy Etchingham. Her recollections of the time she spent living under the same roof as Jimi Hendrix informed much of what is seen on display today.
Hendrix met Kathy Etchingham on his first day in London in September 1966, and they embarked on what was to be a two and half year relationship. Kathy was the inspiration behind Hendrix's classic songs 'Gypsy Eyes', 'The Wind Cries Mary' & perhaps even 'Foxy Lady.
Much of the information below is taken from Kathy Etchingham's autobiography Through Gypsy Eyes: My Life, The Sixties and Jimi Hendrix, which is available exclusively from our online shop.
Jimi and Kathy pose during a Melody Maker session on 25 February, 1969. © Mirrorpix
About Kathy:
Born: Kathleen Mary Etchingham, 18th June 1946, Derby
Moved to London: 1962, Aged 16
Met Jimi: 24 September 1966
Relationship with Jimi Ended: 9th April 1969
Kathleen (Kathy) Etchingham was born at number 7 Raven Street, Derby on the 18th June 1946. She had an older brother, John, and grew up in a house in the centre of Derby with him, her mother Lil and father Charles. It was a typical city-centre terraced house, with a typical post-war routine (an outside toilet, a tin bath to be placed in front of the fire on bath days), the sort of existence that the BBC makes "genteel" drama series out of, according to Kathy, but not one of poverty. It was the norm for much of the British population following the war's economic toll, and with her father holding down an engineering job at Rolls Royce, it was relatively secure – on paper, at least. What was less secure were the relationships within it, especially that of her parents, who were much older than the average parent, especially at the time – her mother was approaching fifty when Kathy started school – and they were not perhaps cut out for the demands of two young children.
Lil and Charles held it together whilst Kathy and John were small, holding family lunches on a Sunday where Lil's mother, known by all as 'Black Nana', would come and read tea-leaves. Nana's parents had moved to Britain from Hungary, and were Roma. The spiritual practices of her ancestors were continued by Nana and Lil; both were "forever poring over the tea-leaves together", and regular consulters of mediums and clairvoyants. Kathy was not so convinced, and even as a child found it all "transparently ridiculous" as she was ferried along to yet another mystic with a stick of liquorice to keep her quiet.
Kathy was, however, absorbed by the stories of her Nana's second husband: Jim Clarkin. Jim had been a vaudeville actor in his younger years and claimed to have walked the streets of New York with Charlie Chaplin in search of work; a story he was fond of retelling. Kathy was fascinated by the prospect of such distant places, hopeful that she would one day be able to travel further afield than Ireland – the farthest her elders would ever travel.
As Kathy got older, her parent's relationship began to fragment, culminating in Lil leaving them for a lodger who had been living in the house with them. Her father began to drink heavily and lost his job, and Kathy and her brother were left to effectively fend for themselves at the age of 11. Eventually, after a period in which Kathy was almost certainly malnourished – her hair was falling out and her gums were bleeding – and their house was lost after a family had forcibly taken over the lease from her father. At this point the family stepped in, and Kathy and John were sent to live with an aunt in Dublin – a place from which they promptly escaped and caught a ferry back to England.
After a period of being passed around various family members, Nana and the family scraped together the finances to send Kathy to a convent boarding school in Skerries, just north of Dublin. The constant movement of places and schools meant that Kathy could never make a group of firm friends as a child, and naturally became more introverted and independent as a result. The convent school's regime, unsurprisingly, did little to change this – but as "bleak and depressing" as it was, it was "no worse" than any of the other households she had been forced to live in.
After less than a year, however, Kathy's mother Lil miraculously reappeared and she was taken from the convent back to England, this time to Chester, to live in another house in another city with another set of adults who had never really been there for her.
It was this combination of constant movement juxtaposed with periods of stifling confinement that set the tone for Kathy to make her own way in the world. Escaping a life in which her decisions were made for her was what drove Kathy to make her way to London, aged just 16, and it is here where our story begins (in fact we'll be skipping ahead four years but you get the idea).
Kathy Etchingham, back at the Hendrix Flat almost 50 years after she lived there with Jimi. © REUTERS/Peter Nicholls
Meeting Jimi and the journey to 23 Brook Street:
Jimi Hendrix did not know London without Kathy Etchingham. They met on the first day he arrived, and he sought her out from the crowd with some "corny" line or other, but, coming from Jimi Hendrix, it worked. The world may not have known Jimi at all had Kathy not been with him that night: following their night together Jimi looked the wrong way before stepping out into oncoming traffic, Kathy dragging him back by his jacket in the nick of time.
They became a couple soon after, and lived with Chas Chandler, Jimi's manager, and his girlfriend, first in Montagu Square, then in Berkeley Place. They lived the lives of those in the London music scene at the time – late nights, late mornings – and spent time in the flats and houses of the biggest names of the era. Their first flat in Montagu Square was owned by none other than Ringo Starr, and was rented to them when he heard that they had been living in a cheap hotel filled with dry-rot; the money from Jimi's early work yet to materialise.
It was not long before they were required to move, though, after Ringo had received multiple complaints from the neighbours – Kathy concedes that they often "rolled home in the small hours", and, being in a residential area, were bound to wake up the neighbours. Chas Chandler maintained that they had been forcibly removed as there was a clause in the lease which said "no blacks", and it was actually this that the neighbours complained about. Kathy thinks it likely that if that was the case, it was a pretext used to rid themselves of the noisy neighbours. It is likely that one fed into the other, and the combination of Hendrix's blackness, the successful rock-star lifestyle and his white girlfriend was something the residents of Montague Square were unable to stomach. One only has to look at the discriminatory Commonwealth Immigrant Acts of 1962 and '68 to gauge the general distrust felt toward non-white immigrants during Jimi's time in Britain.
Their next home was a more settled affair, again sharing with Chas Chandler and his girlfriend, this time in Berkeley Square. The reason for moving on from this flat was an internal one: Chas and Jimi had fallen out on tour, and been told that they were no longer welcome. Finding a place for them to live alone proved more difficult than anticipated, given their more than healthy bank balance. It was the general suspicion that this was down to what the "fuddy-duddy" estate agents of London's wealthy districts thought of Jimi, both his fast-living reputation and his ethnicity. Once the couple were not allowed into Claridge's when they tried to get in. Kathy is reticent to give a reason for this in an interview, but it is clearly not that they were unable to afford a meal there.
There was hope, however, in the form of the landlord of 23 Brook Street in Mayfair, who also owned the Mr. Love restaurant on the ground floor. The rent was thirty pounds a week and he had no hang-ups with them as tenants, so long as the rent was paid. It was perfect for them. Its central location made it a short journey to the clubs they socialised in, and it was surrounded only by offices, so there were no neighbours. Useful for Jimi to able to play as loud as he liked to his heart's content, and have musicians over to jam through the night.
A who's who of musicians ascended those creaking stairs and stumbled across those sloping floors – something anybody who's visited the museum will empathise with! One interviewer of Kathy said that "every picture of Jimi is iconic", and that "no-one associates normality with him". 23 Brook Street, however, represented exactly that. It was "a proper home… one where you got up in the morning and had a cup of tea". It was the antithesis to every buzzword of excess associated with the life of Jimi; they spent much of their time playing board games like Twister, watching Coronation Street (he had a particular soft spot for the character of Ena Sharples, being "nothing like anybody he'd seen in America") and living a life as close to that of an average young couple as they could manage.
Jimi was involved in the interior decorating, too, and insisted that the carpets be replaced within a week of his return from touring in late 1968. He and Kathy went to John Lewis to pick out the material, to bemused stares from onlookers.
The flat was very private and didn't even have a doorbell, so many would-be visitors' knocks simply weren't heard over the music. Even when they did have visitors they would usually only have four or five over at a time; if they "wanted to be in a crowd" they would head to one of the nearby clubs. Clearly, it has to be said, there was some excess in terms of certain recreational activities (Jimi's famous vision of Handel in full regalia appearing to him in a mirror has more than a hint of psychedelic origins), but this was definitely not the flat's primary purpose. In essence, it represented calm. A sanctuary from the relentless storm of Hendrix's meteoric rise to fame, and one which they both cherished. Jimi could not be a performer 24/7 and it was here that he was able to shed his showman's "persona" and be himself, which is consistently described as quiet, easy company; nothing like the centre of attention he was when he picked up a guitar.
The musical heritage of the place was not lost on Jimi, and he certainly believed that Handel's musical spirit lived on within the walls; his subsequent purchase of Messiah and Belshazzar helped him tap into this musical heritage. Now that combined heritage lives on through Handel & Hendrix in London, and much thanks must be given to Kathy that it became a reality.
The unveiling of the blue plaque at 23 Brook Street in 1997 with The Jimi Hendrix Experience bass player Noel Redding, Kathy and Pete Townshend of The Who.
In the late 1990s Kathy successfully campaigned to have Jimi recognised alongside Handel as an equal with his own blue plaque, and the Handel House Museum's opening in 2001 was the first step to bringing the musical heritage of the building back to life. As the Hendrix Flat was beginning to take shape, Kathy made sure that their old bedroom was restored to its former glory. The photos taken from the time were a good start, but they were largely black and white; as we all know, Jimi would not have lived in a room without vibrancy and colour, and Kathy was on hand to get it just right. The room is the same as it was; the architecture outside the window, the wooden beams in the room and the décor that hangs. Kathy says it is akin to time travel when you step into their old room, and it really does feel like something frozen in time. It took 15 years for the concept, support and funding to come together, but I think we can all agree that it was well worth the wait.
Handel House Audio Guide
Exhibition Room – Furniture & Artwork
Handel's Bedroom – Furniture & Artwork
Handel's Composition Room – Furniture & Artwork
Handel's Dressing Room – Furniture & Artwork
Handel's Music Room – Furniture & Artwork
Hendrix Flat Audio Guide
Hendrix Flat – More Information
Our Collection in Focus
George Frideric Handel Bust
Handel's Music and Musical Instruments
Hendrix's Fireplace
Jimi Hendrix's Mirror
Jimi's Record Collection
Jimi's Red Velvet Jacket
Joseph Goupy's Caricature of Handel
Mezzotint of Louis-François Roubiliac (sculptor)
Portrait of Johann Christoph Pepusch (Composer)
Portrait of John Gay (Writer of The Beggar's Opera)
Portrait of Thomas Britton
Rich's Glory or his Triumphant Entry into Covent-Garden, Etching
Susanna Cibber Portrait
The Castrati
The First Guitar Hendrix Played in London
The Handel & Hendrix Home School
Handel Activities
18th-century parlour games
Commission a new piece of music
Cook a Georgian recipe
Draw your own caricature
Flautotonic's Family Concert
Georgian Botanical Prints
Learn the Ritornello
Make your own potpourri
Hendrix Activities
1. Name Your Band
2. Release an Album
3. Make Your Own Instruments
4. Dress-Up Time
5. Gig Night!
Send us a postcard
Create Your Own Festival
Recreate the Hendrix Photo!
Comic Competition
About Handel
Handel's London
Opera Synopses
Hendrix at Home: A Bluesman in Mayfair
Jimi Hendrix's Record Collection
Recreating Jimi Hendrix's Home Audio Setup
The Eighteenth Century
Handel's Event Music
GCSE Set Works
New Artist Programme
LHF School Opera Project
Baroque central


I spent the majority of elementary school in backwards hats (paired with oversized animal shirts and leggings), so I hadn't exactly given them much thought since then. But this one feels dressed up in the most understated way without having to commit to a sports team. I've also been wearing this colorblock coat nonstop - it's one of the most versatile pieces in my closet and makes even the most androgynous of outfits a bit more pulled together.


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The magical land of Oz coming to Bridgnorth Endowed Leisure Centre... - Why not head down the yellow brick road to Bridgnorth Endowed Leisure Centre in February to watch...
Happy new year, and happy new resolutions - We know that January is the month when health and fitness are at the top of the agenda and internet...
Here's a goal that will get you going... - Why not join the scores of swimmers in Halo, or the thousands across the country, who use January...
Welcoming our new Chair of the Board of Trustees…. - Halo is hugely excited to welcome Reg Curtis as its new Chair of the Board of Trustees. Bringing a...
Introducing Hereford Leisure Centre's NEW fast track entry system… - If you're one of the thousands of people who come into Hereford Leisure Centre each month, we've...
Halo Leisure - Halo Staff Awards celebrates 15 years of employee-led success
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Halo Staff Awards celebrates 15 years of employee-led success
Halo Leisure, the social enterprise and registered health and fitness charity, welcomed employees from across 22 sites in England and Wales to its annual Staff Awards evening on Saturday 25th November. The second event of its kind to take place, the awards celebrated the effort put into the organisation by its hard-working employees.
Halo staff swapped their trainers, trunks, t-shirts and trackies for ties, tux, suits, frocks and stilettos for the evening, which proved to be a roaring success from start to finish.
With so many fantastic employees doing such a great job, each and every one deserved an award. But, of course, it wouldn't be a very interesting night if that was the case. So in no particular order, the winners were:
Volunteer of the Year 2017: Julie O'Connor
Julie is a regular sight in the pool at Pencoed where she is a popular swimming teacher. From everyone at Halo, especially those you have introduced to the world of water, thank you for all your time and effort . We are proud to have you part of the team.
Halo Leisure's Manager of the Year 2017: Karl Paterson
Karl's half-full approach to life and management makes him a standout leader at Halo centres in Maesteg, Ogmore and Garw. A self-confessed disciple of Disney, 'nothing is too much trouble' for Karl when it comes to supporting the local community to get more physically active.
Halo Leisure's Employee of the Year 2017: Stuart James
As a 16-year-old member of the Welsh youth triathlon team, Stuart knew his future lay in leisure. Lifeguarding was the starting point for a varied career in the industry which has led to Stuart recently being appointed as Customer Services Manager, with a specialism in operations, at Bridgend Life Centre. We're lucky to have you, Stuart.
Halo Leisure's Outstanding Achievement 2017: Dana Machackova
"I have the best job I could wish for" says Dana who is a popular member of the fitness team at Halo centre in Ross-On-Wye. Dana combines her love of sport both professionally and personally. As a fitness professional, she encourages people to get the most from their workouts and, on a personal front, pushes herself to amazing achievements whether in the pool or on the road or bike. Finishing 13th in her first Ironman led to Dana qualifying for European middle distance events. More recently she took the Best Women top 10 position in the British Championships. She is now part of Team GB and has her sights set on representing her country in Ibiza next year. Dana - we are honoured and proud to have you part of Team Halo. Good luck…….
Halo Leisure's Health and Safety Award: Bridgnorth Leisure Centre
The centre has recently undergone a major refurbishment. Despite all the building work and disruption, the team worked tirelessly to make sure the facilities were clean, safe and welcoming whilst we had the builders in. With the new gym, exercise studio and changing facilities all in place, the team are just as passionate about helping existing customers maintain their motivation as well as encouraging even more new people to get more active more often.
Halo Leisure's Customer Choice Award 2017: Ian Scrivens
What can we say? The staff and customers at Halo Ross-On-Wye just love Ian. From swimming lessons and gym workouts to group exercise classes, Ian has helped hundreds of local people of all ages and abilities to introduce physical activity into their lives and maintain an exercise routine. Customers call him the 'all-rounder' as there isn't a role he isn't involved with or passionate about. We would like to thank Ian for his role in helping more people get more active more often.
Halo Leisure's Centre of Year 2017: Ross-On-Wye Swimming Pool
Ross has long been recognised as outstanding. Several years ago, the centre was runner-up in UK Active's National Leisure Centre of the Year. Customers are greeted with a smile and their first name - giving a real community feel to the place and placing the centre at the top of Halo's customer service rankings. With every aspect of operations from front of house, swimming lessons, gym workouts, group exercise classes, health and safety and housekeeping to maintenance being carried out to the highest standards - it is no surprise that Ross is the 2017 centre of the year. Keep up the great work.
Halo Leisure's Long Service Award: Sally Davis
From a career starting in Bridgend County Borough Councils leisure department to her current role as manager of Halo centres in Ynysawdre, Pencoed and Pyle - Sally has been committed to helping local people get more active for 25 years. We are so very proud to have such pedigree on our team.
Halo Leisure's Innovation Champion 2017: Cathy Fletcher
As an organisation, innovation is at our core. We are always looking at new ways to help us meet our mission of "Creating Healthier Communities". Cathy, in particular, is passionate on this front and constantly comes up with new ideas, products, campaigns, offers and services to make our customers get the most from their workouts and enjoy their Halo experience. Thanks Cathy!
Don't they scrub up well? We would love to hear your ideas on how we can get more people more active more often - share your thoughts at http://haloleisure.org.uk/contact-form.
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® Halo Leisure is a Social Enterprise and registered charity managing leisure facilities in Wales and England.


Free to play and accessible through web browser (Facebook) Swords of Divinity offers a fantasy based MMORPG that focuses on building up a team of mercenaries and battling through PVE and PVP focused content, arming a team, levelling them up and unlocking new features.
The core of the game focuses on building a team of mercenaries and keeping them trained up to make the player's team stronger and increase their overall "Combat Power", the numerical representation of how powerful your team is based on the characters, their levels, abilities and gear. As the players main hero level increases they will gain access to new features that allow the take on more challenging content as well as elements that help their characters become even stronger.
Each class has a number of different skills which are unlocked, as they are the main character to a combat team the Mercenary companions that a player recruits will revolve primarily around their main character; determining their strategy and combat style.
Mercenaries are hired companions that join a player's group, the player manages their team able to switch around which mercenaries fight in their battle formation from those they have recruited. Mercs are recruited from the Tavern area by acquiring various "Wines" using Wine Tokens, the quality of the drink determines the quality of Merc a player can recruit Rum (green) recruits uncommon (green) Mercs, Gin (blue) recruits common (blue) Mercs, Whiskey (purple) recruits rare (purple) Mercs and finally Legendary Hero Shards (orange) recruit Legendary Heroes. The quality of a merc represents their stats, grade and combat power, as well as typically how expensive they are in Wine Tokens to recruit.
Mercs, and the player's Hero, can be upgraded in a wide variety of ways usually requiring currency or some type of special item to boost the character and increase their stats, level or abilities. By spending EXP potions a Merc can be levelled up as high as the Hero, once reaching certain levels a Merc can then be boosted to grant them extra stats and more skills. Players can equip and upgrade various items and weapons, attaching gem boosts, upgrading mounts and more.
The game offers a wide array of PVE options with a quest campaign, stage based repeatable dungeons for loot, even a World Boss where player teams battle against powerful enemies as many times as possible earning rewards for dealing as much damage as they can. PVP mostly revolves around the PVP Arena where players challenge each other's teams in the hopes of climbing the ranks and earning rewards based off their position.
The game has a number of mini-games that add a different element to common features, such as a prayer system where the gods hurl down benefits and gifts to the player and they move a jug left to right with the arrow keys to catch the gifts. A stamina replenishment system has players clicking a gong as fast as they can which shows their character eating food in a tavern as quickly as possible, building up their strength, and from time to time they must quickly click some ale to wash it down.
Some of the features in the game have restrictions on them only allowing players to attempt them a set number of times, others use up the players Stamina which replenishes slowly over time. Players can spend premium Gold currency purchased with real cash to refresh cooldowns or increase energy, as well as this they can use it to purchase VIP that grants numerous benefits such as increasing character stats, daily Silver, and various other privileges.