Role of organic cation/carnitine transporter 1 in uptake of phenformin and inhibitory effect on complex I respiration in mitochondria.

PubMed

Shitara, Yoshihisa; Nakamichi, Noritaka; Norioka, Misaki; Shima, Hiroyo; Kato, Yukio; Horie, Toshiharu

2013-03-01

Phenformin causes lactic acidosis in clinical situations due to inhibition of mitochondrial respiratory chain complex I. It is reportedly taken up by hepatocytes and exhibits mitochondrial toxicity in the liver. In this study, uptake of phenformin and [(14)C]tetraethylammonium (TEA) and complex I inhibition by phenformin were examined in isolated liver and heart mitochondria. Uptake of phenformin into isolated rat liver mitochondria was higher than that into heart mitochondria. It was inhibited by several cat ionic compounds, which suggests the involvement of multispecific transport system(s). Similar characteristics were also observed for uptake of TEA; however, uptake of phenformin into mitochondria of organic cation/carnitine transporter 1 (OCTN1) knockout mice was lower than that in wild-type mice, whereas uptake of TEA was comparable between the two strains, suggesting the involvement of distinct transport mechanisms for these two cations in mitochondria. Inhibition by phenformin of oxygen consumption via complex I respiration in isolated rat liver mitochondria was greater than that in heart mitochondria, whereas inhibitory effect of phenformin on complex I respiration was similar in inside-out structured submitochondrial particles prepared from rat livers and hearts. Lactic acidosis provoked by iv infusion of phenformin was weaker in octn1(-/-) mice than that in wild-type mice. These observations suggest that uptake of phenformin into liver mitochondria is at least partly mediated by OCTN1 and functionally relevant to its inhibition potential of complex I respiration. This study was, thus, the first to demonstrate OCTN1-mediated mitochondrial transport and toxicity of biguanide in vivo in rodents.

The effect of phenformin on insulin-stimulated isoaminobutyric acid (AIB) transport by isolated hepatocytes.

PubMed Central

Woods, R. J.; Dandona, P.

1984-01-01

Hepatocytes isolated from adult rat livers by collagenase perfusion were used to investigate the effect of phenformin on amino acid transport by measuring the uptake of aminoisobutyric acid (AIB). At concentrations between 0.01 and 100 micrograms/ml, phenformin hydrochloride did not affect AIB uptake, but concentrations of 500 and 1000 micrograms/ml were inhibitory. Incubating hepatocytes with insulin increased their accumulation of AIB. Inclusion of up to 100 micrograms/ml, phenformin hydrochloride during maximal and submaximal stimulation by insulin did not affect AIB uptake, but inhibition occurred with 500 and 1000 micrograms/ml. The continued presence of phenformin after addition of AIB was not required in order to produce inhibition. Furthermore, increasing the duration of exposure of hepatocytes to phenformin increased the degree of inhibition, suggesting that it is a non-competitive inhibitor. We conclude that phenformin does not enhance the sensitivity of hepatocytes to insulin and that at higher concentrations it may exert an inhibitory effect on basal and insulin stimulated aminoacid transport. PMID:6370292

Lactic acidosis occurring during phenformin therapy

PubMed Central

Tomkins, A. M.; Jones, R.; Bloom, Arnold

1972-01-01

A case of severe lactic acidosis is described in a diabetic taking phenformin who was otherwise healthy. Substitution of metformin for phenformin did not lead to a recurrence of the lactic acidosis. PMID:5049258

Phenformin Induces Cell Cycle Change, Apoptosis, and Mesenchymal-Epithelial Transition and Regulates the AMPK/mTOR/p70s6k and MAPK/ERK Pathways in Breast Cancer Cells.

PubMed

Liu, Zhao; Ren, Lidong; Liu, Chenghao; Xia, Tiansong; Zha, Xiaoming; Wang, Shui

2015-01-01

Breast cancer remains a world-wide challenge, and additional anti-cancer therapies are still urgently needed. Emerging evidence has demonstrated the potent anti-tumor effect of biguanides, among which phenformin was reported to potentially be a more active anti-cancer agent than metformin. However, little attention has been given to the role of phenformin in breast cancer. In this study, we reveal the role of phenformin in cell death of the MCF7, ZR-75-1, MDA-MB-231 and SUM1315 breast cancer cell lines. The respective IC50 values of phenformin in MCF7, ZR-75-1, MDA-MB-231 and SUM1315 cells were 1.184±0.045 mM, 0.665±0.007 mM, 2.347±0.010 mM and 1.885±0.015 mM (mean± standard error). Phenformin induced cell cycle change and apoptosis in breast cancer cells via the AMPK/mTOR/p70s6k and MAPK/ERK pathways. Interestingly, phenformin induced MET (mesenchymal-epithelial transition) and decreased the migration rate in breast cancer cell lines. Furthermore, our results suggest that phenformin inhibits breast cancer cell metastasis after intracardiac injection into nude mice. Taken together, our study further confirms the potential benefit of phenformin in breast cancer treatment and provides novel mechanistic insight into its anti-cancer activity in breast cancer.

Phenformin Induces Cell Cycle Change, Apoptosis, and Mesenchymal-Epithelial Transition and Regulates the AMPK/mTOR/p70s6k and MAPK/ERK Pathways in Breast Cancer Cells

PubMed Central

Liu, Zhao; Ren, Lidong; Liu, Chenghao; Xia, Tiansong; Zha, Xiaoming; Wang, Shui

2015-01-01

Breast cancer remains a world-wide challenge, and additional anti-cancer therapies are still urgently needed. Emerging evidence has demonstrated the potent anti-tumor effect of biguanides, among which phenformin was reported to potentially be a more active anti-cancer agent than metformin. However, little attention has been given to the role of phenformin in breast cancer. In this study, we reveal the role of phenformin in cell death of the MCF7, ZR-75-1, MDA-MB-231 and SUM1315 breast cancer cell lines. The respective IC50 values of phenformin in MCF7, ZR-75-1, MDA-MB-231 and SUM1315 cells were 1.184±0.045 mM, 0.665±0.007 mM, 2.347±0.010 mM and 1.885±0.015 mM (mean± standard error). Phenformin induced cell cycle change and apoptosis in breast cancer cells via the AMPK/mTOR/p70s6k and MAPK/ERK pathways. Interestingly, phenformin induced MET (mesenchymal-epithelial transition) and decreased the migration rate in breast cancer cell lines. Furthermore, our results suggest that phenformin inhibits breast cancer cell metastasis after intracardiac injection into nude mice. Taken together, our study further confirms the potential benefit of phenformin in breast cancer treatment and provides novel mechanistic insight into its anti-cancer activity in breast cancer. PMID:26114294

Repurposing phenformin for the targeting of glioma stem cells and the treatment of glioblastoma

PubMed Central

Jiang, Wei; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Brodie, Ziv; Mikkelsen, Tom; Poisson, Laila; Shackelford, David B.; Brodie, Chaya

2016-01-01

Glioblastoma (GBM) is the most aggressive primary brain tumor with poor prognosis. Here, we studied the effects of phenformin, a mitochondrial complex I inhibitor and more potent chemical analog of the diabetes drug metformin on the inhibition of cell growth and induction of apoptosis of glioma stem cells (GSCs) using both in vitro and in vivo models. Phenformin inhibited the self-renewal of GSCs, decreased the expression of stemness and mesenchymal markers and increased the expression of miR-124, 137 and let-7. Silencing of let-7 abrogated phenformin effects on the self-renewal of GSCs via a pathway associated with inhibition of H19 and HMGA2 expression. Moreover, we demonstrate that phenformin inhibited tumor growth and prolonged the overall survival of mice orthotopically transplanted with GSCs. Combined treatments of phenformin and temozolomide exerted an increased antitumor effect on GSCs in vitro and in vivo. In addition, dichloroacetate, an inhibitor of the glycolysis enzyme pyruvate dehydrogenase kinase, that decreases lactic acidosis induced by biguanides, enhanced phenformin effects on the induction of cell death in GSCs and prolonged the survival of xenograft-bearing mice. Our results demonstrate for the first time that phenformin targets GSCs and can be efficiently combined with current therapies for GBM treatment and GSC eradication. PMID:27486821

Repurposing phenformin for the targeting of glioma stem cells and the treatment of glioblastoma.

PubMed

Jiang, Wei; Finniss, Susan; Cazacu, Simona; Xiang, Cunli; Brodie, Ziv; Mikkelsen, Tom; Poisson, Laila; Shackelford, David B; Brodie, Chaya

2016-08-30

Glioblastoma (GBM) is the most aggressive primary brain tumor with poor prognosis. Here, we studied the effects of phenformin, a mitochondrial complex I inhibitor and more potent chemical analog of the diabetes drug metformin on the inhibition of cell growth and induction of apoptosis of glioma stem cells (GSCs) using both in vitro and in vivo models. Phenformin inhibited the self-renewal of GSCs, decreased the expression of stemness and mesenchymal markers and increased the expression of miR-124, 137 and let-7. Silencing of let-7 abrogated phenformin effects on the self-renewal of GSCs via a pathway associated with inhibition of H19 and HMGA2 expression. Moreover, we demonstrate that phenformin inhibited tumor growth and prolonged the overall survival of mice orthotopically transplanted with GSCs. Combined treatments of phenformin and temozolomide exerted an increased antitumor effect on GSCs in vitro and in vivo. In addition, dichloroacetate, an inhibitor of the glycolysis enzyme pyruvate dehydrogenase kinase, that decreases lactic acidosis induced by biguanides, enhanced phenformin effects on the induction of cell death in GSCs and prolonged the survival of xenograft-bearing mice. Our results demonstrate for the first time that phenformin targets GSCs and can be efficiently combined with current therapies for GBM treatment and GSC eradication.

Phenformin enhances the therapeutic benefit of BRAFV600E inhibition in melanoma

PubMed Central

Yuan, Ping; Ito, Koichi; Perez-Lorenzo, Rolando; Del Guzzo, Christina; Lee, Jung Hyun; Shen, Che-Hung; Bosenberg, Marcus W.; McMahon, Martin; Cantley, Lewis C.; Zheng, Bin

2013-01-01

Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated antitumor activity both in vitro and in vivo. The energy-sensing AMP-activated protein kinase (AMPK) is known to be a major cellular target of biguanides. Based on our discovery of cross-talk between the AMPK and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) signaling pathways, we investigated the antitumor effects of combining phenformin with a BRAF inhibitor PLX4720 on the proliferation of BRAF-mutated melanoma cells in vitro and on BRAF-driven tumor growth in vivo. Cotreatment of BRAF-mutated melanoma cell lines with phenformin and PLX4720 resulted in synergistic inhibition of cell viability, compared with the effects of the single agent alone. Moreover, treatment with phenformin significantly delayed the development of resistance to PLX4720 in cultured melanoma cells. Biochemical analyses showed that phenformin and PLX4720 exerted cooperative effects on inhibiting mTOR signaling and inducing apoptosis. Noticeably, phenformin selectively targeted subpopulations of cells expressing JARID1B, a marker for slow cycling melanoma cells, whereas PLX4720 selectively targeted JARID1B-negative cells. Finally, in contrast to their use as single agents, the combination of phenformin and PLX4720 induced tumor regression in both nude mice bearing melanoma xenografts and in a genetically engineered BRAFV600E/PTENnull-driven mouse model of melanoma. These results strongly suggest that significant therapeutic advantage may be achieved by combining AMPK activators such as phenformin with BRAF inhbitors for the treatment of melanoma. PMID:24145418

Phenformin enhances the therapeutic benefit of BRAF(V600E) inhibition in melanoma.

PubMed

Yuan, Ping; Ito, Koichi; Perez-Lorenzo, Rolando; Del Guzzo, Christina; Lee, Jung Hyun; Shen, Che-Hung; Bosenberg, Marcus W; McMahon, Martin; Cantley, Lewis C; Zheng, Bin

2013-11-05

Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated antitumor activity both in vitro and in vivo. The energy-sensing AMP-activated protein kinase (AMPK) is known to be a major cellular target of biguanides. Based on our discovery of cross-talk between the AMPK and v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) signaling pathways, we investigated the antitumor effects of combining phenformin with a BRAF inhibitor PLX4720 on the proliferation of BRAF-mutated melanoma cells in vitro and on BRAF-driven tumor growth in vivo. Cotreatment of BRAF-mutated melanoma cell lines with phenformin and PLX4720 resulted in synergistic inhibition of cell viability, compared with the effects of the single agent alone. Moreover, treatment with phenformin significantly delayed the development of resistance to PLX4720 in cultured melanoma cells. Biochemical analyses showed that phenformin and PLX4720 exerted cooperative effects on inhibiting mTOR signaling and inducing apoptosis. Noticeably, phenformin selectively targeted subpopulations of cells expressing JARID1B, a marker for slow cycling melanoma cells, whereas PLX4720 selectively targeted JARID1B-negative cells. Finally, in contrast to their use as single agents, the combination of phenformin and PLX4720 induced tumor regression in both nude mice bearing melanoma xenografts and in a genetically engineered BRAF(V600E)/PTEN(null)-driven mouse model of melanoma. These results strongly suggest that significant therapeutic advantage may be achieved by combining AMPK activators such as phenformin with BRAF inhbitors for the treatment of melanoma.

Therapeutic potential of the metabolic modulator phenformin in targeting the stem cell compartment in melanoma.

PubMed

Petrachi, Tiziana; Romagnani, Alessandra; Albini, Adriana; Longo, Caterina; Argenziano, Giuseppe; Grisendi, Giulia; Dominici, Massimo; Ciarrocchi, Alessia; Dallaglio, Katiuscia

2017-01-24

Melanoma is the most dangerous and treatment-resistant skin cancer. Tumor resistance and recurrence are due to the persistence in the patient of aggressive cells with stem cell features, the cancer stem cells (CSC). Recent evidences have shown that CSC display a distinct metabolic profile as compared to tumor bulk population: a promising anti-tumor strategy is therefore to target specific metabolic pathways driving CSC behavior. Biguanides (metformin and phenformin) are anti-diabetic drugs able to perturb cellular metabolism and displaying anti-cancer activity. However, their ability to target the CSC compartment in melanoma is not known. Here we show that phenformin, but not metformin, strongly reduces melanoma cell viability, growth and invasion in both 2D and 3D (spheroids) models. While phenformin decreases melanoma CSC markers expression and the levels of the pro-survival factor MITF, MITF overexpression fails to prevent phenformin effects. Phenformin significantly reduces cell viability in melanoma by targeting both CSC (ALDHhigh) and non-CSC cells and by significantly reducing the number of viable cells in ALDHhigh and ALDHlow-derived spheroids. Consistently, phenformin reduces melanoma cell viability and growth independently from SOX2 levels. Our results show that phenformin is able to affect both CSC and non-CSC melanoma cell viability and growth and suggests its potential use as anti-cancer therapy in melanoma.

Therapeutic potential of the metabolic modulator phenformin in targeting the stem cell compartment in melanoma

PubMed Central

Albini, Adriana; Longo, Caterina; Argenziano, Giuseppe; Grisendi, Giulia; Dominici, Massimo; Ciarrocchi, Alessia; Dallaglio, Katiuscia

2017-01-01

Melanoma is the most dangerous and treatment-resistant skin cancer. Tumor resistance and recurrence are due to the persistence in the patient of aggressive cells with stem cell features, the cancer stem cells (CSC). Recent evidences have shown that CSC display a distinct metabolic profile as compared to tumor bulk population: a promising anti-tumor strategy is therefore to target specific metabolic pathways driving CSC behavior. Biguanides (metformin and phenformin) are anti-diabetic drugs able to perturb cellular metabolism and displaying anti-cancer activity. However, their ability to target the CSC compartment in melanoma is not known. Here we show that phenformin, but not metformin, strongly reduces melanoma cell viability, growth and invasion in both 2D and 3D (spheroids) models. While phenformin decreases melanoma CSC markers expression and the levels of the pro-survival factor MITF, MITF overexpression fails to prevent phenformin effects. Phenformin significantly reduces cell viability in melanoma by targeting both CSC (ALDHhigh) and non-CSC cells and by significantly reducing the number of viable cells in ALDHhigh and ALDHlow-derived spheroids. Consistently, phenformin reduces melanoma cell viability and growth independently from SOX2 levels. Our results show that phenformin is able to affect both CSC and non-CSC melanoma cell viability and growth and suggests its potential use as anti-cancer therapy in melanoma. PMID:28036292

Treatment of Pancreatic Cancer Patient-Derived Xenograft Panel with Metabolic Inhibitors Reveals Efficacy of Phenformin.

PubMed

Rajeshkumar, N V; Yabuuchi, Shinichi; Pai, Shweta G; De Oliveira, Elizabeth; Kamphorst, Jurre J; Rabinowitz, Joshua D; Tejero, Héctor; Al-Shahrour, Fátima; Hidalgo, Manuel; Maitra, Anirban; Dang, Chi V

2017-09-15

Purpose: To identify effective metabolic inhibitors to suppress the aggressive growth of pancreatic ductal adenocarcinoma (PDAC), we explored the in vivo antitumor efficacy of metabolic inhibitors, as single agents, in a panel of patient-derived PDAC xenograft models (PDX) and investigated whether genomic alterations of tumors correlate with the sensitivity to metabolic inhibitors. Experimental Design: Mice with established PDAC tumors from 6 to 13 individual PDXs were randomized and treated, once daily for 4 weeks, with either sterile PBS (vehicle) or the glutaminase inhibitor bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES), transaminase inhibitor aminooxyacetate (AOA), pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA), autophagy inhibitor chloroquine (CQ), and mitochondrial complex I inhibitor phenformin/metformin. Results: Among the agents tested, phenformin showed significant tumor growth inhibition (>30% compared with vehicle) in 5 of 12 individual PDXs. Metformin, at a fivefold higher dose, displayed significant tumor growth inhibition in 3 of 12 PDXs similar to BPTES (2/8 PDXs) and DCA (2/6 PDXs). AOA and CQ had the lowest response rates. Gene set enrichment analysis conducted using the baseline gene expression profile of pancreatic tumors identified a gene expression signature that inversely correlated with phenformin sensitivity, which is in agreement with the phenformin gene expression signature of NIH Library of Integrated Network-based Cellular Signatures (LINCS). The PDXs that were more sensitive to phenformin showed a baseline reduction in amino acids and elevation in oxidized glutathione. There was no correlation between phenformin response and genetic alterations in KRAS, TP53, SMAD4 , or PTEN Conclusions: Phenformin treatment showed relatively higher antitumor efficacy against established PDAC tumors, compared with the efficacy of other metabolic inhibitors and metformin. Phenformin treatment significantly diminished PDAC tumor progression and prolonged tumor doubling time. Overall, our results serve as a foundation for further evaluation of phenformin as a therapeutic agent in pancreatic cancer. Clin Cancer Res; 23(18); 5639-47. ©2017 AACR . ©2017 American Association for Cancer Research.

Phenformin-induced Hypoglycaemia in Normal Subjects*

PubMed Central

Lyngsøe, J.; Trap-Jensen, J.

1969-01-01

Study of the effect of phenformin on the blood glucose level in normal subjects before and during 70 hours of starvation showed a statistically significant hypoglycaemic effect after 40 hours of starvation. This effect was not due to increased glucose utilization. Another finding in this study was a statistically significant decrease in total urinary nitrogen excretion during starvation in subjects given phenformin. These findings show that the hypoglycaemic effect of phenformin in starved normal subjects is due to inhibition of gluconeogenesis. PMID:5780431

Phenformin-loaded polymeric micelles for targeting both cancer cells and cancer stem cells in vitro and in vivo.

PubMed

Krishnamurthy, Sangeetha; Ng, Victor W L; Gao, Shujun; Tan, Min-Han; Yang, Yi Yan

2014-11-01

Conventional cancer chemotherapy often fails as most anti-cancer drugs are not effective against drug-resistant cancer stem cells. These surviving cancer stem cells lead to relapse and metastasis. In this study, an anti-diabetic drug, phenformin, capable of eliminating cancer stem cells was loaded into micelles via self-assembly using a mixture of a diblock copolymer of poly(ethylene glycol) (PEG) and urea-functionalized polycarbonate and a diblock copolymer of PEG and acid-functionalized polycarbonate through hydrogen bonding. The phenformin-loaded micelles, having an average diameter of 102 nm with narrow size distribution, were stable in serum-containing solution over 48 h and non-cytotoxic towards non-cancerous cells. More than 90% of phenformin was released from the micelles over 96 h. Lung cancer stem cells (side population cells, i.e. SP cells) and non-SP cells were sorted from H460 human lung cancer cell line, and treated with free phenformin and phenformin-loaded micelles. The results showed that the drug-loaded micelles were more effective in inhibiting the growth of both SP and non-SP cells. In vivo studies conducted in an H460 human lung cancer mouse model demonstrated that the drug-loaded micelles had greater anti-tumor efficacy, and reduced the population of SP cells in the tumor tissues more effectively than free phenformin. Liver function analysis was performed following drug treatments, and the results indicated that the drug-loaded micelles did not cause liver damage, a harmful side-effect of phenformin when used clinically. These phenformin-loaded micelles may be used to target both cancer cells and cancer stem cells in chemotherapy for the prevention of relapse and metastasis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Direct effects of phenformin on metabolism/bioenergetics and viability of SH-SY5Y neuroblastoma cells.

PubMed

Geoghegan, Fintan; Chadderton, Naomi; Farrar, G Jane; Zisterer, Daniela M; Porter, Richard K

2017-11-01

Phenformin, a member of the biguanides class of drugs, has been reported to be efficacious in cancer treatment. The focus of the current study was to establish whether there were direct effects of phenformin on the metabolism and bioenergetics of neuroblastoma SH-SY5Y cancer cells. Cell viability was assessed using the alamar blue assay, flow cytometry analysis using propidium iodide and annexin V stain and poly (ADP-ribose) polymerase analysis. Cellular and mitochondrial oxygen consumption was determined using a Seahorse Bioscience Flux analyser and an Oroboros Oxygraph respirometer. Cells were transfected using electroporation and permeabilized for in situ mitochondrial functional analysis using digitonin. Standard protocols were used for immunoblotting and proteins were separated on denaturing gels. Phenformin was effective in reducing the viability of SH-SY5Y cells, causing G 1 cell cycle arrest and inducing apoptosis. Bioenergetic analysis demonstrated that phenformin significantly decreased oxygen consumption in a dose- and time-dependent manner. The sensitivity of oxygen consumption in SH-SY5Y cells to phenformin was circumvented by the expression of NADH-quinone oxidoreductase 1, a ubiquinone oxidoreductase, suggesting that complex I may be a target of phenformin. As a result of this inhibition, adenosine monophosphate protein kinase is activated and acetyl-coenzyme A carboxylase is inhibited. To the best of our knowledge, the current study is the first to demonstrate the efficacy and underlying mechanism by which phenformin directly effects the survival of neuroblastoma cancer cells.

Direct effects of phenformin on metabolism/bioenergetics and viability of SH-SY5Y neuroblastoma cells

PubMed Central

Geoghegan, Fintan; Chadderton, Naomi; Farrar, G. Jane; Zisterer, Daniela M.; Porter, Richard K.

2017-01-01

Phenformin, a member of the biguanides class of drugs, has been reported to be efficacious in cancer treatment. The focus of the current study was to establish whether there were direct effects of phenformin on the metabolism and bioenergetics of neuroblastoma SH-SY5Y cancer cells. Cell viability was assessed using the alamar blue assay, flow cytometry analysis using propidium iodide and annexin V stain and poly (ADP-ribose) polymerase analysis. Cellular and mitochondrial oxygen consumption was determined using a Seahorse Bioscience Flux analyser and an Oroboros Oxygraph respirometer. Cells were transfected using electroporation and permeabilized for in situ mitochondrial functional analysis using digitonin. Standard protocols were used for immunoblotting and proteins were separated on denaturing gels. Phenformin was effective in reducing the viability of SH-SY5Y cells, causing G1 cell cycle arrest and inducing apoptosis. Bioenergetic analysis demonstrated that phenformin significantly decreased oxygen consumption in a dose- and time-dependent manner. The sensitivity of oxygen consumption in SH-SY5Y cells to phenformin was circumvented by the expression of NADH-quinone oxidoreductase 1, a ubiquinone oxidoreductase, suggesting that complex I may be a target of phenformin. As a result of this inhibition, adenosine monophosphate protein kinase is activated and acetyl-coenzyme A carboxylase is inhibited. To the best of our knowledge, the current study is the first to demonstrate the efficacy and underlying mechanism by which phenformin directly effects the survival of neuroblastoma cancer cells. PMID:29113281

Phenformin has anti-tumorigenic effects in human ovarian cancer cells and in an orthotopic mouse model of serous ovarian cancer.

PubMed

Jackson, Amanda L; Sun, Wenchuan; Kilgore, Joshua; Guo, Hui; Fang, Ziwei; Yin, Yajie; Jones, Hannah M; Gilliam, Timothy P; Zhou, Chunxiao; Bae-Jump, Victoria L

2017-11-21

Obesity and diabetes have been associated with increased risk and worse outcomes in ovarian cancer (OC). The biguanide metformin is used in the treatment of type 2 diabetes and is also believed to have anti-tumorigenic benefits. Metformin is highly hydrophilic and requires organic cation transporters (OCTs) for entry into human cells. Phenformin, another biguanide, was taken off the market due to an increased risk of lactic acidosis over metformin. However, phenformin is not reliant on transporters for cell entry; and thus, may have increased potency as both an anti-diabetic and anti-tumorigenic agent than metformin. Thus, our goal was to evaluate the effect of phenformin on established OC cell lines, primary cultures of human OC cells and in an orthotopic mouse model of high grade serous OC. In three OC cell lines, phenformin significantly inhibited cellular proliferation, induced cell cycle G1 arrest and apoptosis, caused cellular stress, inhibited adhesion and invasion, and activation of AMPK and inhibition of the mTOR pathway. Phenformin also exerted anti-proliferative effects in seven primary cell cultures of human OC. Lastly, phenformin inhibited tumor growth in an orthotopic mouse model of serous OC, coincident with decreased Ki-67 staining and phosphorylated-S6 expression and increased expression of caspase 3 and phosphorylated-AMPK. Our findings demonstrate that phenformin has anti-tumorigenic effects in OC as previously demonstrated by metformin but it is yet to be determined if it is superior to metformin for the potential treatment of this disease.

Phenformin has anti-tumorigenic effects in human ovarian cancer cells and in an orthotopic mouse model of serous ovarian cancer

PubMed Central

Jackson, Amanda L.; Sun, Wenchuan; Kilgore, Joshua; Guo, Hui; Fang, Ziwei; Yin, Yajie; Jones, Hannah M.; Gilliam, Timothy P.; Zhou, Chunxiao; Bae-Jump, Victoria L.

2017-01-01

Obesity and diabetes have been associated with increased risk and worse outcomes in ovarian cancer (OC). The biguanide metformin is used in the treatment of type 2 diabetes and is also believed to have anti-tumorigenic benefits. Metformin is highly hydrophilic and requires organic cation transporters (OCTs) for entry into human cells. Phenformin, another biguanide, was taken off the market due to an increased risk of lactic acidosis over metformin. However, phenformin is not reliant on transporters for cell entry; and thus, may have increased potency as both an anti-diabetic and anti-tumorigenic agent than metformin. Thus, our goal was to evaluate the effect of phenformin on established OC cell lines, primary cultures of human OC cells and in an orthotopic mouse model of high grade serous OC. In three OC cell lines, phenformin significantly inhibited cellular proliferation, induced cell cycle G1 arrest and apoptosis, caused cellular stress, inhibited adhesion and invasion, and activation of AMPK and inhibition of the mTOR pathway. Phenformin also exerted anti-proliferative effects in seven primary cell cultures of human OC. Lastly, phenformin inhibited tumor growth in an orthotopic mouse model of serous OC, coincident with decreased Ki-67 staining and phosphorylated-S6 expression and increased expression of caspase 3 and phosphorylated-AMPK. Our findings demonstrate that phenformin has anti-tumorigenic effects in OC as previously demonstrated by metformin but it is yet to be determined if it is superior to metformin for the potential treatment of this disease. PMID:29245964

Solid phase extraction--non-aqueous capillary electrophoresis for determination of metformin, phenformin and glyburide in human plasma.

PubMed

Lai, Edward P C; Feng, Sherry Y

2006-10-20

Solid phase extraction (SPE) was coupled at line to capillary electrophoresis (CE) for the determination of three basic and neutral diabetic drugs (metformin, phenformin and glyburide) in human plasma. The SPE procedure employed a C(18) cartridge to remove most of the water and proteins from the plasma sample. Analyte detectability was increased due to trace enrichment during the SPE process. Elution of metformin, phenformin and glyburide was achieved with methanol+3% acetic acid. CE analysis was performed using a non-aqueous buffer, acetonitrile+5mM ammonium acetate+5% acetic acid, which afforded rapid separation of metformin from phenformin within 3 min. Glyburide, with a migration time longer than 6 min, did not cause any interference. The present SPE-CE method, with an electrokinetic injection time of 6s and UV detection at 240 nm, was useful for monitoring down to 1 microg/mL of metformin and phenformin in human plasma. When the electrokinetic injection time was increased to 36s, the detection limits were improved to 12 ng/mL for metformin and 6 ng/mL for phenformin.

Renal Response to Acid Load after Phenformin

PubMed Central

Rooth, Gösta; Bandman, Ulf

1973-01-01

Normally the kidneys respond to an acid load by increased ammonia production. In six patients with adult-type diabetes this response was reduced by a mean 50% after a therapeutic dose of phenformin. The reduced ability to compensate for acid loads may be one factor leading to metabolic acidosis and lactoacidosis sometimes associated with phenformin therapy. PMID:4753235

Pharmacological activators of AMP-activated protein kinase have different effects on Na+ transport processes across human lung epithelial cells.

PubMed

Woollhead, A M; Sivagnanasundaram, J; Kalsi, K K; Pucovsky, V; Pellatt, L J; Scott, J W; Mustard, K J; Hardie, D G; Baines, D L

2007-08-01

AMP-activated protein kinase (AMPK) is activated by metformin, phenformin, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). We have completed an extensive study of the pharmacological effects of these drugs on AMPK activation, adenine nucleotide concentration, transepithelial amiloride-sensitive (I(amiloride)) and ouabain-sensitive basolateral (I(ouabain)) short circuit current in H441 lung epithelial cells. H441 cells were grown on permeable filters at air interface. I(amiloride), I(ouabain) and transepithelial resistance were measured in Ussing chambers. AMPK activity was measured as the amount of radiolabelled phosphate transferred to the SAMS peptide. Adenine nucleotide concentration was analysed by reverse phase HPLC and NAD(P)H autofluorescence was measured using confocal microscopy. Phenformin, AICAR and metformin increased AMPK (alpha1) activity and decreased I(amiloride). The AMPK inhibitor Compound C prevented the action of metformin and AICAR but not phenformin. Phenformin and AICAR decreased I(ouabain) across H441 monolayers and decreased monolayer resistance. The decrease in I(amiloride) was closely related to I(ouabain) with phenformin, but not in AICAR treated monolayers. Metformin and phenformin increased the cellular AMP:ATP ratio but only phenformin and AICAR decreased cellular ATP. Activation of alpha1-AMPK is associated with inhibition of apical amiloride-sensitive Na(+) channels (ENaC), which has important implications for the clinical use of metformin. Additional pharmacological effects evoked by AICAR and phenformin on I(ouabain), with potential secondary effects on apical Na+ conductance, ENaC activity and monolayer resistance, have important consequences for their use as pharmacological activators of AMPK in cell systems where Na+K+ATPase is an important component.

Pharmacological activators of AMP-activated protein kinase have different effects on Na+ transport processes across human lung epithelial cells

PubMed Central

Woollhead, A M; Sivagnanasundaram, J; Kalsi, K K; Pucovsky, V; Pellatt, L J; Scott, J W; Mustard, K J; Hardie, D G; Baines, D L

2007-01-01

Background and purpose: AMP-activated protein kinase (AMPK) is activated by metformin, phenformin, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). We have completed an extensive study of the pharmacological effects of these drugs on AMPK activation, adenine nucleotide concentration, transepithelial amiloride-sensitive (Iamiloride) and ouabain-sensitive basolateral (Iouabain) short circuit current in H441 lung epithelial cells. Experimental approach: H441 cells were grown on permeable filters at air interface. Iamiloride, Iouabain and transepithelial resistance were measured in Ussing chambers. AMPK activity was measured as the amount of radiolabelled phosphate transferred to the SAMS peptide. Adenine nucleotide concentration was analysed by reverse phase HPLC and NAD(P)H autofluorescence was measured using confocal microscopy. Key results: Phenformin, AICAR and metformin increased AMPK (α1) activity and decreased Iamiloride. The AMPK inhibitor Compound C prevented the action of metformin and AICAR but not phenformin. Phenformin and AICAR decreased Iouabain across H441 monolayers and decreased monolayer resistance. The decrease in Iamiloride was closely related to Iouabain with phenformin, but not in AICAR treated monolayers. Metformin and phenformin increased the cellular AMP:ATP ratio but only phenformin and AICAR decreased cellular ATP. Conclusions and implications: Activation of α1-AMPK is associated with inhibition of apical amiloride-sensitive Na+ channels (ENaC), which has important implications for the clinical use of metformin. Additional pharmacological effects evoked by AICAR and phenformin on Iouabain, with potential secondary effects on apical Na+ conductance, ENaC activity and monolayer resistance, have important consequences for their use as pharmacological activators of AMPK in cell systems where Na+K+ATPase is an important component. PMID:17603555

Phenformin in Insulin-Dependent Diabetics

PubMed Central

Bloom, Arnold; Kolbe, R. J.

1970-01-01

Forty-one diabetic patients on insulin were given 100mg. of phenformin daily for six weeks, either before or after a period of six weeks of inert capsules, in a double-blind cross-over trial. Eleven patients while on phenformin noticed hypoglycaemic effects and reduced their insulin on average by almost 20% without resultant rise in blood sugar levels. Twenty-eight patients felt no untoward effects and maintained their usual insulin dose. Phenformin led to improved control of the diabetes, with a significant decrease in blood sugar levels and a significant reduction in the variability of the weekly blood sugar readings. There was no increased ketosis, no change in cholesterol, and no significant loss of weight. PMID:4914612

Comparison of gene expression changes induced by biguanides in db/db mice liver.

PubMed

Heishi, Masayuki; Hayashi, Koji; Ichihara, Junji; Ishikawa, Hironori; Kawamura, Takao; Kanaoka, Masaharu; Taiji, Mutsuo; Kimura, Toru

2008-08-01

Large-scale clinical studies have shown that the biguanide drug metformin, widely used for type 2 diabetes, to be very safe. By contrast, another biguanide, phenformin, has been withdrawn from major markets because of a high incidence of serious adverse effects. The difference in mode of action between the two biguanides remains unclear. To gain insight into the different modes of action of the two drugs, we performed global gene expression profiling using the livers of obese diabetic db/db mice after a single administration of phenformin or metformin at levels sufficient to cause a significant reduction in blood glucose level. Metformin induced modest expression changes, including G6pc in the liver as previously reported. By contrast, phenformin caused changes in expression level of many additional genes. We used a knowledge-based bioinformatic analysis to study the effects of phenformin. Differentially expressed genes identified in this study constitute a large gene network, which may be related to cell death, inflammation or wound response. Our results suggest that the two biguanides show a similar hypoglycemic effect in db/db mice, but phenformin induces a greater stress on the liver even a short time after a single administration. These findings provide a novel insight into the cause of the relatively high occurrence of serious adverse effect after phenformin treatment.

Phenformin-Induced Mitochondrial Dysfunction Sensitizes Hepatocellular Carcinoma for Dual Inhibition of mTOR.

PubMed

Veiga, Sonia Rosa; Ge, Xuemei; Mercer, Carol A; Hernández-Alvarez, María Isabel; Thomas, Hala Elnakat; Hernández-Losa, Javier; Ramón Y Cajal, Santiago; Zorzano, Antonio; Thomas, George; Kozma, Sara C

2018-04-24

Hepatocellular carcinoma (HCC) ranks second in cancer mortality and has limited therapeutic options. We recently described the synergistic effect of allosteric and ATP-site competitive inhibitors against the mammalian target of rapamycin (mTOR) for the treatment of HCC. However, such inhibitors induce glycemia and increase mitochondrial efficiency. Here we determined whether the mitochondrial complex I inhibitor Phenformin could reverse both side effects, impose an energetic-stress on cancer cells and suppress the growth of HCC. Human HCC cell lines were used in vitro to access the signaling and energetic impact of mTOR inhibitors and Phenformin, either alone or in combination. Next, the therapeutic utility of these drugs alone or in combination was investigated pre-clinically in human orthotopic tumors implanted in mice, by analyzing their impact on the tumor burden and overall survival. We found Phenformin caused mitochondrial dysfunction and fragmentation, inducing a compensatory shift to glycolysis. In contrast, dual inhibition of mTOR impaired cell growth and glycolysis, while increasing mitochondrial fusion and efficiency. In a mouse model of human HCC, dual inhibition of mTOR, together with Phenformin, was highly efficacious in controlling tumor burden. However, more striking, pretreatment with Phenformin sensitized tumors to dual inhibition of mTOR, leading to a dramatic improvement in survival. Treatment of HCC cells in vitro with the biguanide Phenformin causes a metabolic shift to glycolysis, mitochondrial dysfunction and fragmentation, and dramatically sensitizes orthotopic liver tumors to dual inhibition of mTOR. We therefore propose this therapeutic approach should be tested clinically in HCC. Copyright ©2018, American Association for Cancer Research.

Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR.

PubMed

Sakamoto, Kei; Göransson, Olga; Hardie, D Grahame; Alessi, Dario R

2004-08-01

Activation of AMP-activated protein kinase (AMPK) by exercise and metformin is beneficial for the treatment of type 2 diabetes. We recently found that, in cultured cells, the LKB1 tumor suppressor protein kinase activates AMPK in response to the metformin analog phenformin and the AMP mimetic drug 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). We have also reported that LKB1 activates 11 other AMPK-related kinases. The activity of LKB1 or the AMPK-related kinases has not previously been studied in a tissue with physiological relevance to diabetes. In this study, we have investigated whether contraction, phenformin, and AICAR influence LKB1 and AMPK-related kinase activity in rat skeletal muscle. Contraction in situ, induced via sciatic nerve stimulation, significantly increased AMPKalpha2 activity and phosphorylation in multiple muscle fiber types without affecting LKB1 activity. Treatment of isolated skeletal muscle with phenformin or AICAR stimulated the phosphorylation and activation of AMPKalpha1 and AMPKalpha2 without altering LKB1 activity. Contraction, phenformin, or AICAR did not significantly increase activities or expression of the AMPK-related kinases QSK, QIK, MARK2/3, and MARK4 in skeletal muscle. The results of this study suggest that muscle contraction, phenformin, or AICAR activates AMPK by a mechanism that does not involve direct activation of LKB1. They also suggest that the effects of excercise, phenformin, and AICAR on metabolic processes in muscle may be mediated through activation of AMPK rather than activation of LKB1 or the AMPK-related kinases.

Treatment of Obese Non-Diabetic Patients with Phenformin. A Double-Blind Cross-Over Trial

PubMed Central

Hart, A.; Cohen, H.

1970-01-01

The effect of 50 mg. of sustained release phenformin capsules administered twice daily was compared with a placebo as an aid to weight reduction in obese non-diabetic patients. Phenformin and placebo were administered alternately in two three-month periods. Weight loss on a 1,200-calorie diet was greater in the first three months than in the second three months regardless of treatment. There was no difference in weight loss between the phenformin and placebo groups in either the first or second three-month period. Serum cholesterol levels tended to fall in association with weight loss, but not apparently in relation to the size of the weight loss or to the treatment. PMID:4904162

Phenformin enhances the therapeutic effect of selumetinib in KRAS-mutant non-small cell lung cancer irrespective of LKB1 status.

PubMed

Zhang, Jun; Nannapaneni, Sreenivas; Wang, Dongsheng; Liu, Fakeng; Wang, Xu; Jin, Rui; Liu, Xiuju; Rahman, Mohammad Aminur; Peng, Xianghong; Qian, Guoqing; Chen, Zhuo G; Wong, Kwok-Kin; Khuri, Fadlo R; Zhou, Wei; Shin, Dong M

2017-08-29

MEK inhibition is potentially valuable in targeting KRAS-mutant non-small cell lung cancer (NSCLC). Here, we analyzed whether concomitant LKB1 mutation alters sensitivity to the MEK inhibitor selumetinib, and whether the metabolism drug phenformin can enhance the therapeutic effect of selumetinib in isogenic cell lines with different LKB1 status. Isogenic pairs of KRAS-mutant NSCLC cell lines A549, H460 and H157, each with wild-type and null LKB1, as well as genetically engineered mouse-derived cell lines 634 ( kras G12D/wt /p53 -/- /lkb1 wt/wt ) and t2 ( kras G12D/wt /p53 -/- / lkb1 -/- ) were used in vitro to analyze the activities of selumetinib, phenformin and their combination. Synergy was measured and potential mechanisms investigated. The in vitro findings were then confirmed in vivo using xenograft models. The re-expression of wild type LKB1 increased phospho-ERK level, suggesting that restored dependency on MEK->ERK->MAPK signaling might have contributed to the enhanced sensitivity to selumetinib. In contrast, the loss of LKB1 sensitized cells to phenformin. At certain combination ratios, phenformin and selumetinib showed synergistic activity regardless of LKB1 status. Their combination reduced phospho-ERK and S6 levels and induced potent apoptosis, but was likely through different mechanisms in cells with different LKB1 status. Finally, in xenograft models bearing isogenic A549 cells, we confirmed that loss of LKB1 confers resistance to selumetinib, and phenformin significantly enhances the therapeutic effect of selumetinib. Irrespective of LKB1 status, phenformin may enhance the anti-tumor effect of selumetinib in KRAS-mutant NSCLC. The dual targeting of MEK and cancer metabolism may provide a useful strategy to treat this subset of lung cancer.

Quasi-equilibrium analysis of the ion-pair mediated membrane transport of low-permeability drugs.

PubMed

Miller, Jonathan M; Dahan, Arik; Gupta, Deepak; Varghese, Sheeba; Amidon, Gordon L

2009-07-01

The aim of this research was to gain a mechanistic understanding of ion-pair mediated membrane transport of low-permeability drugs. Quasi-equilibrium mass transport analyses were developed to describe the ion-pair mediated octanol-buffer partitioning and hydrophobic membrane permeation of the model basic drug phenformin. Three lipophilic counterions were employed: p-toluenesulfonic acid, 2-naphthalenesulfonic acid, and 1-hydroxy-2-naphthoic acid (HNAP). Association constants and intrinsic octanol-buffer partition coefficients (Log P(AB)) of the ion-pairs were obtained by fitting a transport model to double reciprocal plots of apparent octanol-buffer distribution coefficients versus counterion concentration. All three counterions enhanced the lipophilicity of phenformin, with HNAP providing the greatest increase in Log P(AB), 3.7 units over phenformin alone. HNAP also enhanced the apparent membrane permeability of phenformin, 27-fold in the PAMPA model, and 4.9-fold across Caco-2 cell monolayers. As predicted from a quasi-equilibrium analysis of ion-pair mediated membrane transport, an order of magnitude increase in phenformin flux was observed per log increase in counterion concentration, such that log-log plots of phenformin flux versus HNAP concentration gave linear relationships. These results provide increased understanding of the underlying mechanisms of ion-pair mediated membrane transport, emphasizing the potential of this approach to enable oral delivery of low-permeability drugs.

Liver uptake of biguanides in rats.

PubMed

Sogame, Yoshihisa; Kitamura, Atsushi; Yabuki, Masashi; Komuro, Setsuko

2011-09-01

Metformin is an oral antihyperglycaemic agent widely used in the management of non-insulin-dependent diabetes mellitus. The liver is the primary target, metformin being taken up into human and rat hepatocytes via an active transport mechanism. The present study was designed to compare hepatic uptake of two biguanides, metformin and phenformin, in vitro and in vivo. In in vitro experiments, performed using rat cryopreserved hepatocytes, phenformin exhibited a much higher affinity and transport than metformin, with marked differences in kinetics. The K(m) values for metformin and phenformin were 404 and 5.17μM, respectively, with CLint (V(max)/K(m)) values 1.58μl/min per 10(6) cells and 34.7μl/min per 10(6) cells. In in vivo experiments, when (14)C-metformin and (14)C-phenformin were given orally to male rats at a dose of 50mg/kg, the liver concentrations of radioactivity at 0.5 hour after dosing were 21.5μg eq./g with metformin but 147.1μg eq./g for phenformin, ratios of liver to plasma concentrations being 4.2 and 61.3, respectively. In conclusion, the results suggest that uptake of biguanides by rat hepatocytes is in line with the liver distribution found in vivo, phenformin being more efficiently taken up by liver than metformin after oral administration. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Phenformin activates the unfolded protein response in an AMP-activated protein kinase (AMPK)-dependent manner.

PubMed

Yang, Liu; Sha, Haibo; Davisson, Robin L; Qi, Ling

2013-05-10

The cross-talk between UPR activation and metabolic stress remains largely unclear. Phenformin treatment activates the IRE1α and PERK pathways in an AMPK-dependent manner. AMPK is required for phenformin-mediated IRE1α and PERK activation. Our findings demonstrate the cross-talk between UPR and metabolic signals. Activation of the unfolded protein response (UPR) is associated with the disruption of endoplasmic reticulum (ER) homeostasis and has been implicated in the pathogenesis of many human metabolic diseases, including obesity and type 2 diabetes. However, the nature of the signals activating UPR under these conditions remains largely unknown. Using a method that we recently optimized to directly measure UPR sensor activation, we screened the effect of various metabolic drugs on UPR activation and show that the anti-diabetic drug phenformin activates UPR sensors IRE1α and pancreatic endoplasmic reticulum kinase (PERK) in both an ER-dependent and ER-independent manner. Mechanistically, AMP-activated protein kinase (AMPK) activation is required but not sufficient to initiate phenformin-mediated IRE1α and PERK activation, suggesting the involvement of additional factor(s). Interestingly, activation of the IRE1α (but not PERK) pathway is partially responsible for the cytotoxic effect of phenformin. Together, our data show the existence of a non-canonical UPR whose activation requires the cytosolic kinase AMPK, adding another layer of complexity to UPR activation upon metabolic stress.

Phenformin and lactic acidosis: a case report and review.

PubMed

Kwong, S C; Brubacher, J

1998-01-01

Phenformin was removed from the U.S. market 20 years ago because of a high incidence of lactic acidosis. Unfortunately, this medication is still available from foreign sources. Another biguanide, metformin, was reintroduced to the United States market for the treatment of diabetes. Biguanide-induced lactic acidosis should be included in the differential diagnosis of elevated anion gap metabolic acidosis. We present a case of phenformin-induced lactic acidosis in which we were consulted at the local poison control center. We also review its pathophysiology, presentation, and treatment. A review of the actions of phenformin illustrates the mechanism of pathology that may also occur with metformin. Risk factors for the development of lactic acidosis include renal deficiency, hepatic disease, cardiac disease, and drug interaction such as cimetidine.

Graphene and CdS nanocomposite: a facile interface for construction of DNA-based electrochemical biosensor and its application to the determination of phenformin.

PubMed

Zeng, Lijiao; Wang, Rui; Zhu, Lihua; Zhang, Jingdong

2013-10-01

Graphene/cadmium sulphide (GR-CdS) nanocomposite was synthesized via a low temperature process in aqueous solution. The as-prepared nanocomposite was characterized by scanning electron microscopy, UV-visible spectroscopy, Fourier transform infrared spectroscopy, and X-ray diffraction. The impedance analysis indicated that GR-CdS nanocomposite possessed outstanding electrochemical performance for facile electron transfer. When DNA was immobilized on GR-CdS (DNA/GR-CdS) modified electrode, the electrochemical oxidation of guanine and adenine in DNA residue bases was significantly promoted. Due to the interaction of DNA with phenformin, the voltammetric current of guanine or adenine on the DNA/GR-CdS electrode was decreased when phenformin was present in the electrolytic solution. Under optimized conditions, the signal of guanine on DNA/GR-CdS electrode decreased linearly with increasing the concentration of phenformin in the range of 1.0×10(-6)molL(-1) to 1.0×10(-3)molL(-1). The proposed DNA-based electrochemical biosensor was successfully applied to the determination of phenformin in real samples. Copyright © 2013 Elsevier B.V. All rights reserved.

Phenformin enhances the therapeutic effect of selumetinib in KRAS-mutant non-small cell lung cancer irrespective of LKB1 status

PubMed Central

Zhang, Jun; Nannapaneni, Sreenivas; Wang, Dongsheng; Liu, Fakeng; Wang, Xu; Jin, Rui; Liu, Xiuju; Rahman, Mohammad Aminur; Peng, Xianghong; Qian, Guoqing; Chen, Zhuo G.; Wong, Kwok-Kin; Khuri, Fadlo R.; Zhou, Wei; Shin, Dong M.

2017-01-01

MEK inhibition is potentially valuable in targeting KRAS-mutant non-small cell lung cancer (NSCLC). Here, we analyzed whether concomitant LKB1 mutation alters sensitivity to the MEK inhibitor selumetinib, and whether the metabolism drug phenformin can enhance the therapeutic effect of selumetinib in isogenic cell lines with different LKB1 status. Isogenic pairs of KRAS-mutant NSCLC cell lines A549, H460 and H157, each with wild-type and null LKB1, as well as genetically engineered mouse-derived cell lines 634 (krasG12D/wt/p53-/-/lkb1wt/wt) and t2 (krasG12D/wt/p53-/-/lkb1-/-) were used in vitro to analyze the activities of selumetinib, phenformin and their combination. Synergy was measured and potential mechanisms investigated. The in vitro findings were then confirmed in vivo using xenograft models. The re-expression of wild type LKB1 increased phospho-ERK level, suggesting that restored dependency on MEK->ERK->MAPK signaling might have contributed to the enhanced sensitivity to selumetinib. In contrast, the loss of LKB1 sensitized cells to phenformin. At certain combination ratios, phenformin and selumetinib showed synergistic activity regardless of LKB1 status. Their combination reduced phospho-ERK and S6 levels and induced potent apoptosis, but was likely through different mechanisms in cells with different LKB1 status. Finally, in xenograft models bearing isogenic A549 cells, we confirmed that loss of LKB1 confers resistance to selumetinib, and phenformin significantly enhances the therapeutic effect of selumetinib. Irrespective of LKB1 status, phenformin may enhance the anti-tumor effect of selumetinib in KRAS-mutant NSCLC. The dual targeting of MEK and cancer metabolism may provide a useful strategy to treat this subset of lung cancer. PMID:28938614

Biguanides Metformin and Phenformin Generate Therapeutic Effects via AMP-Activated Protein Kinase/Extracellular-Regulated Kinase Pathways in an In Vitro Model of Graves' Orbitopathy.

PubMed

Han, Ye Eon; Hwang, Sena; Kim, Jin Hee; Byun, Jung Woo; Yoon, Jin Sook; Lee, Eun Jig

2018-04-01

It was hypothesized that the biguanides metformin and phenformin, which are anti-hyperglycemic drugs used for diabetes mellitus, would have therapeutic effects in an in vitro model of Graves' orbitopathy (GO). Because adipogenesis, hyaluronan production, and inflammation are considered important in the pathogenesis of GO, this study aimed to determine the therapeutic effects and underlying mechanisms of biguanides on these parameters. In vitro experiments were performed using primary cultured orbital fibroblasts from patients with GO. Orbital preadipocyte fibroblasts were allowed to differentiate into adipocytes and were treated with various concentrations of metformin or phenformin. Oil Red O staining was performed to evaluate lipid accumulation within the cells. Western blot analysis was used to measure the expression of adipogenic transcription factors and the phosphorylation of AMP-activated protein kinase and mitogen-activated protein kinase signaling proteins. Hyaluronan production was measured using enzyme-linked immunosorbent assay, and mRNA levels of proinflammatory molecules were determined using real-time polymerase chain reaction after interleukin (IL)-1β stimulation with or without biguanide treatment. Lipid accumulation during adipogenesis in GO orbital fibroblasts was dose-dependently suppressed by both metformin and phenformin. Adipocyte differentiation was attenuated, and the adipogenic transcription factors peroxisome proliferator-activated receptor γ and CCAAT-enhancer-binding proteins-α/β were downregulated. Furthermore, metformin and phenformin increased the phosphorylation of AMP-activated protein kinase and suppressed extracellular-regulated kinase activation. The IL-1β-induced hyaluronan production and mRNA expression of IL-6, cyclooxygenase-2, and intercellular adhesion molecule-1 were also significantly suppressed after metformin or phenformin co-treatment. The present study indicates that the biguanides metformin and phenformin exert an anti-adipogenic and inhibitory effect on hyaluronan production and expression of pro-inflammatory molecules in GO orbital fibroblasts, suggesting that they could potentially be used for the treatment of GO.

Study of phenformin metabolism in rat liver microsomes by HPLC, CE and on-line HPLC-electrospray ionization mass spectrometry.

PubMed

Llambias, E B; Luo, J

1996-01-01

Methods for the analysis of phenformin and its metabolite by high-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESIMS) are developed. The effects of pH, buffer concentration and proportion of organic modifier on the retention of the compounds in HPLC have been studied. The optimum condition was used for the separation and identification of phenformin and its metabolite in microsomal metabolism by HPLC-ESIMS. A simple CE method is also described for the separation of these compounds. Optimum incubation conditions and cofactor requirements for the formation of 4-hydroxyphenformin by microsomal preparations of rat liver were determined. A linear response in the formation of product was found with increasing concentrations of protein and up to 15 min incubation. High concentrations of phenformin inhibited its metabolite formation, and K(m) was 4 microM.

LKB1 inactivation dictates therapeutic response of non-small cell lung cancer to the metabolism drug phenformin

PubMed Central

Shackelford, David B.; Abt, Evan; Gerken, Laurie; Vasquez, Debbie S.; Seki, Atsuko; Leblanc, Mathias; Wei, Liu; Fishbein, Michael C.; Czernin, Johannes; Mischel, Paul S.; Shaw, Reuben J.

2013-01-01

SUMMARY The LKB1 (also called STK11) tumor suppressor is mutationally inactivated in ~20% of non-small cell lung cancers (NSCLC). LKB1 is the major upstream kinase activating the energy-sensing kinase AMPK, making LKB1-deficient cells unable to appropriately sense metabolic stress. We tested the therapeutic potential of metabolic drugs in NSCLC and identified phenformin, a mitochondrial inhibitor and analog of the diabetes therapeutic metformin, as selectively inducing apoptosis in LKB1-deficient NSCLC cells. Therapeutic trials in Kras-dependent mouse models of NSCLC revealed that tumors with Kras and Lkb1 mutations, but not those with Kras and p53 mutations showed selective response to phenformin as a single agent, resulting in prolonged survival. This study suggests phenformin as a cancer metabolism-based therapeutic to selectively target LKB1-deficient tumors. PMID:23352126

LKB1 inactivation dictates therapeutic response of non-small cell lung cancer to the metabolism drug phenformin.

PubMed

Shackelford, David B; Abt, Evan; Gerken, Laurie; Vasquez, Debbie S; Seki, Atsuko; Leblanc, Mathias; Wei, Liu; Fishbein, Michael C; Czernin, Johannes; Mischel, Paul S; Shaw, Reuben J

2013-02-11

The LKB1 (also called STK11) tumor suppressor is mutationally inactivated in ∼20% of non-small cell lung cancers (NSCLC). LKB1 is the major upstream kinase activating the energy-sensing kinase AMPK, making LKB1-deficient cells unable to appropriately sense metabolic stress. We tested the therapeutic potential of metabolic drugs in NSCLC and identified phenformin, a mitochondrial inhibitor and analog of the diabetes therapeutic metformin, as selectively inducing apoptosis in LKB1-deficient NSCLC cells. Therapeutic trials in Kras-dependent mouse models of NSCLC revealed that tumors with Kras and Lkb1 mutations, but not those with Kras and p53 mutations, showed selective response to phenformin as a single agent, resulting in prolonged survival. This study suggests phenformin as a cancer metabolism-based therapeutic to selectively target LKB1-deficient tumors. Copyright © 2013 Elsevier Inc. All rights reserved.

Metformin and phenformin block the peripheral antinociception induced by diclofenac and indomethacin on the formalin test.

PubMed

Ortiz, Mario I

2012-01-02

Recent evidence has shown that systemic administration of sulfonylureas and biguanides block the diclofenac-induced antinociception, but not the effect produced by indomethacin. However, there are no reports about the peripheral interaction between analgesics and the biguanides metformin and phenformin. Therefore, this work was undertaken to determine whether glibenclamide and glipizide and the biguanides metformin and phenformin have any effect on the peripheral antinociception induced by diclofenac and indomethacin. Diclofenac and indomethacin were administered locally in the formalin-injured rat paw, and the antinociceptive effect was evaluated using the 1% formalin test. To determine whether peripheral antinociception induced by diclofenac or indomethacin was mediated by either the ATP-sensitive K(+) channels or biguanides-induced mechanisms, the effect of pretreatment with the appropriates vehicles or glibenclamide, glipizide, metformin and phenformin on the antinociceptive effect induced by local peripheral diclofenac and indomethacin was assessed. Local peripheral injections of diclofenac (50-200 μg/paw) and indomethacin (200-800 μg/paw) produced a dose-dependent antinociception during the second phase of the test. Local pretreatment with glibenclamide, glipizide, metformin and phenformin blocked the diclofenac-induced antinociception. On the other hand, the pretreatment with glibenclamide and glipizide did not prevent the local antinociception produced by indomethacin. Nonetheless, metformin and phenformin reversed the local antinociception induced by indomethacin. Data suggest that diclofenac could activate the K(+) channels and biguanides-dependent mechanisms to produce its peripheral antinociceptive effects in the formalin test. Likewise, a biguanides-dependent mechanism could be activated by indomethacin consecutively to generate its peripheral antinociceptive effect. Copyright © 2011 Elsevier Inc. All rights reserved.

Lactic acidosis and hyperamylasaemia associated with phenformin therapy

PubMed Central

Williams, D. N.; Knight, A. H.; Goldberg, D. M.

1974-01-01

A case is described of lactic acidosis and hyperamylasaemia in a diabetic with impaired renal function treated with phenformin. Despite normal blood pressure and adequate tissue perfusion, the patient succumbed. No evidence of pancreatitis could be found at autopsy. PMID:4219857

Sustaining pattern of phenformin hydrochloride using various polymers and waxes.

PubMed

Pandey, V P; Kannappan, N; Manavalan, R; Subburaj, T

2002-01-01

The present study was carried out to formulate matrix tablets of phenformin hydrochloride. Granules of phenformin HCl were prepared by using ethyl cellulose, eudragit RS 100, gum acacia, carnauba wax, stearyl alcohol, glyceryl monostearate and triethanol amine. Thus the granules were compressed and fourteen tablets formulations were prepared. All the physical parameters of granules and matrix tablets were studied including compatibility study. One commercial timed disintegration capsule was also included for study and comparison. The results of in vitro studies showed that sustained release matrix tablet might be prepared using carnauba wax, stearyl alcohol, triethanol amine and magnesium stearate.

Targeting oncogenic KRAS in non-small cell lung cancer cells by phenformin inhibits growth and angiogenesis.

PubMed

Wang, Zhi Dong; Wei, Sheng Quan; Wang, Qin Yi

2015-01-01

Tumors require a vascular supply to grow and can achieve this via the expression of pro-angiogenic growth factors. Many potential oncogenic mutations have been identified in tumor angiogenesis. Somatic mutations in the small GTPase KRAS are the most common activating lesions found in human cancer, and are generally associated with poor response to standard therapies. Biguanides, such as the diabetes therapeutics metformin and phenformin, have demonstrated anti-tumor activity both in vitro and in vivo. The extracellular regulated protein kinases (ERK) signaling is known to be a major cellular target of biguanides. Based on KRAS activates several down-stream effectors leading to the stimulation of the RAF/mitogen-activated protein kinase/extracellular signal-regulated kinase (RAF/MEK/ERK) and phosphatidylinositol-3-kinase (PI3K) pathways, we investigated the anti-tumor effects of biguanides on the proliferation of KRAS-mutated tumor cells in vitro and on KRAS-driven tumor growth in vivo. In cancer cells harboring oncogenic KRAS, phenformin switches off the ERK pathway and inhibit the expression of pro-angiogenic molecules. In tumor xenografts harboring the KRAS mutation, phenformin extensively modifies the tumor growth causing abrogation of angiogenesis. These results strongly suggest that significant therapeutic advantage may be achieved by phenformin anti-angiogenesis for the treatment of tumor.

Comparison of potential risks of lactic acidosis induction by biguanides in rats.

PubMed

Bando, Kiyoko; Ochiai, Shoko; Kunimatsu, Takeshi; Deguchi, Jiro; Kimura, Juki; Funabashi, Hitoshi; Seki, Takaki

2010-10-01

Lactic acidosis has been considered to be a side effect of some biguanides, after phenformin was withdrawn from the market because of its association with lactic acidosis. The potential of lactic acidosis induced by biguanides at human therapeutic exposure levels, however, has not been examined. Then, we compared the risk of lactic acid at doses providing exposure levels comparable to human therapeutic doses. Metformin and phenformin were orally administered to rats for up to 28 days, and plasma drug concentrations and blood lactic acid levels were examined. Metformin did not elevate lactic acid levels at the dose corresponding to higher systemic drug exposure than human therapeutic level, even for repeated doses. In contrast, phenformin elevated lactic acid levels at the dose corresponding to lower exposure than human therapeutic level, and sustained high levels were observed up to 24h post-dose; furthermore, these changes were enhanced by repeated doses. Direct comparison at each rat equivalent dose clearly indicated that lactic acid levels of phenformin were higher than those of metformin. These non-clinical findings suggest that metformin dose not increase lactic acid levels like phenformin does, and therefore may not increase the risk for lactic acidosis at human therapeutic exposure level. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Phenformin has a direct inhibitory effect on the ATP-sensitive potassium channel.

PubMed

Aziz, Qadeer; Thomas, Alison; Khambra, Tapsi; Tinker, Andrew

2010-05-25

The biguanides, phenformin and metformin, are used in the treatment of type II diabetes mellitus, as well as being routinely used in studies investigating AMPK activity. We used the patch-clamp technique and rubidium flux assays to determine the role of these drugs in ATP-sensitive K+ channel (K(ATP)) regulation in cell lines expressing the cloned components of K(ATP) and the current natively expressed in vascular smooth muscle cells (VSMCs). Phenformin but not metformin inhibits a number of variants of K(ATP) including the cloned equivalents of currents present in vascular and non-vascular smooth muscle (Kir6.1/SUR2B and Kir6.2/SUR2B) and pancreatic beta-cells (Kir6.2/SUR1). However it does not inhibit the current potentially present in cardiac myocytes (Kir6.2/SUR2A). The highest affinity interaction is seen with Kir6.1/SUR2B (IC50=0.55 mM) and it also inhibits the current in native vascular smooth muscle cells. The extent and rate of inhibition are similar to that seen with the known K(ATP) blocker PNU 37883A. Additionally, phenformin inhibited the current elicited through the Kir6.2DeltaC26 (functional without SUR) channel with an IC50 of 1.78 mM. Phenformin reduced the open probability of Kir6.1/SUR2B channels by approximately 90% in inside-out patches. These findings suggest that phenformin interacts directly with the pore-forming Kir6.0 subunit however the sulphonylurea receptor is able to significantly modulate the affinity. It is likely to block from the intracellular side of the channel in a manner analogous to that of PNU 37883A. Copyright 2010 Elsevier B.V. All rights reserved.

The inhibition of monoamine oxidase by phenformin and pentamidine.

PubMed

Barkhuizen, M; Petzer, A; Petzer, J P

2014-09-01

A computational study has suggested that phenformin, an oral hypoglycaemic drug, may bind to the active sites of the monoamine oxidase (MAO) A and B enzymes. The present study therefore investigates the MAO inhibitory properties of phenformin. Pentamidine, a structurally related diamidine compound, has previously been reported to be a MAO inhibitor and was included in this study as a reference compound. Using recombinant human MAO-A and MAO-B, this study finds that phenformin acts as a moderately potent MAO-A selective inhibitor with an IC50 value of 41 µM. Pentamidine, on the other hand, potently inhibits both MAO-A and MAO-B with IC50 values of 0.61 μM and 0.22 μM, respectively. An examination of the recoveries of the enzymatic activities after dilution and dialysis of the enzyme-inhibitor complexes shows that both compounds interact reversibly with the MAO enzymes. A kinetic analysis suggests that pentamidine acts as a competitive inhibitor with estimated Ki values of 0.41 μM and 0.22 μM for the inhibition of MAO-A and MAO-B, respectively. Phenformin also exhibited a competitive mode of MAO-A inhibition with an estimated Ki value of 65 µM. This study concludes that biguanide and amidine functional groups are most likely important structural features for the inhibition of the MAOs by phenformin and pentamidine, and compounds containing these and closely related functional groups should be considered as potential MAO inhibitors. Furthermore, the biguanide and amidine functional groups may act as useful moieties in the future design of MAO inhibitors. © Georg Thieme Verlag KG Stuttgart · New York.

Malabsorption of Vitamin B12 in Diabetic Patients Treated with Phenformin: A Comparison with Metformin

PubMed Central

Tomkin, Gerald H.

1973-01-01

An investigation into B12 absorption in diabetic patients on long-term phenformin therapy was undertaken and the results were compared to a similar investigation previously reported in diabetics on long-term metformin. Forty-six per cent of patients were found to have B12 malabsorption as shown by abnormal results of Schilling tests. The mechanism of B12 malabsorption is unknown but it is suggested that all patients on long-term phenformin therapy should, like the patients on metformin, have annual serum B12 estimations until the results of a longer follow-up series are known. PMID:4742454

A comparison of uptake of metformin and phenformin mediated by hOCT1 in human hepatocytes.

PubMed

Sogame, Yoshihisa; Kitamura, Atsushi; Yabuki, Masashi; Komuro, Setsuko

2009-11-01

Metformin, a biguanide that has been used to treat type 2 diabetes mellitus, is reportedly transported into human hepatocytes by human organic cation transporter 1 (hOCT1). The objective of this study was to investigate differences in the hepatic uptake of metformin and phenformin, a biguanide derivative similar to metformin. Special focus was on the role of active transport into cells. Experiments were therefore performed using human cryopreserved hepatocytes and hOCT1 expressing oocytes. Both biguanides proved to be good substrates for hOCT1. However, phenformin exhibited a much higher affinity and transport activity, with a marked difference in uptake kinetics compared with metformin. Both biguanides were transported actively by hOCT1, with the active transport components much greater than passive transport components in both cases, suggesting that functional changes in hOCT1 might affect the transport of both compounds to the same degree. This study for the first time produced detailed comparative findings for uptake profiles of metformin and phenformin in human hepatocytes and hOCT1 expressing oocytes. It is considered that hOCT1 may not be the only key factor that determines the frequency of metformin and phenformin toxicity, considering the major contribution of this transporter to the total hepatic uptake and comparable width of their therapeutic concentrations.

[Effect of phenformin hydrochloride on pharmacokinetics of puerarin in rats].

PubMed

Deng, Ying; Li, Ning; Cui, Mei; Xiong, Zhi-li; Li, Fa-mei

2012-10-01

To study the effect of phenformin hydrochloride that may be illegally added in traditional Chinese medicine preparations on the pharmacokinetics of puerarin in rats. Rats were randomly divided into the single pueraria group and the phenformin hydrochloride combined with pueraria group. After oral administration in the two groups, their bloods were sampled at different time points to determine the drug concentration of puerarin in rat blood and calculate pharmacokinetic parameters. After oral administration with pueraria extracts and phenformin hydrochloride combined with pueraria extracts, the two groups showed main pharmacokinetic parameters as follows: Cmax were (2.39 +/- 1.01), (1.03 +/- 0.35) mg x L(-1), respectively; Tmax were (0.50 +/- 0.09), (1.5 +/- 0.5) h, respectively; Ke were (0.153 +/- 0.028), (0.172 +/- 0.042) h(-1), respectively; t(1/2) were (4.65 +/- 0.86), (4.20 +/- 0.81) h, respectively; AUC(0-t), were (5.73 +/- 2.60), (5.45 +/- 1.81) mg x h x L(-1), respectively; AUC(0-infinity) were (6.72 +/- 2.89), (6.26 +/- 1.88) mg x h x L(-1), respectively. Compared with the single puerarin group, the Cmax was significantly decreased (P < 0.05) and the Tmax was markedly longer (P < 0.01) than the hydrochloride combined with pueraria group. Phenformin hydrochloride can slow down the absorption process of puerarin and change the pharmacokinetic process of puerarin to some extent.

Phenformin Activates the Unfolded Protein Response in an AMP-activated Protein Kinase (AMPK)-dependent Manner*

PubMed Central

Yang, Liu; Sha, Haibo; Davisson, Robin L.; Qi, Ling

2013-01-01

Activation of the unfolded protein response (UPR) is associated with the disruption of endoplasmic reticulum (ER) homeostasis and has been implicated in the pathogenesis of many human metabolic diseases, including obesity and type 2 diabetes. However, the nature of the signals activating UPR under these conditions remains largely unknown. Using a method that we recently optimized to directly measure UPR sensor activation, we screened the effect of various metabolic drugs on UPR activation and show that the anti-diabetic drug phenformin activates UPR sensors IRE1α and pancreatic endoplasmic reticulum kinase (PERK) in both an ER-dependent and ER-independent manner. Mechanistically, AMP-activated protein kinase (AMPK) activation is required but not sufficient to initiate phenformin-mediated IRE1α and PERK activation, suggesting the involvement of additional factor(s). Interestingly, activation of the IRE1α (but not PERK) pathway is partially responsible for the cytotoxic effect of phenformin. Together, our data show the existence of a non-canonical UPR whose activation requires the cytosolic kinase AMPK, adding another layer of complexity to UPR activation upon metabolic stress. PMID:23548904

Transport of biguanides by human organic cation transporter OCT2.

PubMed

Sogame, Yoshihisa; Kitamura, Atsushi; Yabuki, Masashi; Komuro, Setsuko; Takano, Mikihisa

2013-06-01

Biguanides have the severe side effect of lactic acidosis. Although both metformin and phenformin are biguanide derivatives, there is a difference in the frequency at which they induce lactic acidosis. However, the reasons for the difference are not clear. Metformin has been reported to be mainly excreted into urine by human organic cation transporter 2 (hOCT2). The present study was designed to investigate the renal transport of metformin and phenformin, focusing on hOCT2, using hOCT2-expressing oocytes. Both biguanides were found to be good substrates for hOCT2. However, phenformin exhibited a higher affinity and transport activity than metformin. The Km values for metformin and phenformin were 235 and 37.4 μM, with CL(int) (V(max)/K(m)) values of 71.9×10⁻³ μL/min per oocyte and 209×10⁻³ μL/min per oocyte, respectively. This is the first report that has compared the transport profiles of these biguanides in hOCT2-expressing oocytes. The results suggest that plasma concentration of phenformin in subjects carrying hOCT2 variant may be higher compared to reference subjects, as reported in metformin. In addition, the relationship between plasma concentration of these biguanides and blood lactate level as well as the possible reasons for the difference in the associated frequency of occurrence of lactic acidosis are discussed. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Phenformin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) activation of AMP-activated protein kinase inhibits transepithelial Na+ transport across H441 lung cells.

PubMed

Woollhead, Alison M; Scott, John W; Hardie, D Grahame; Baines, Deborah L

2005-08-01

Active re-absorption of Na+ across the alveolar epithelium is essential to maintain lung fluid balance. Na+ entry at the luminal membrane is predominantly via the amiloride-sensitive Na+ channel (ENaC) down its electrochemical gradient. This gradient is generated and maintained by basolateral Na+ extrusion via Na+,K+-ATPase an energy-dependent process. Several kinases and factors that activate them are known to regulate these processes; however, the role of AMP-activated protein kinase (AMPK) in the lung is unknown. AMPK is an ultra-sensitive cellular energy sensor that monitors energy consumption and down-regulates ATP-consuming processes when activated. The biguanide phenformin has been shown to independently decrease ion transport processes, influence cellular metabolism and activate AMPK. The AMP mimetic drug 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) also activates AMPK in intact cells. Western blotting revealed that both the alpha1 and alpha2 catalytic subunits of AMPK are present in Na+ transporting H441 human lung epithelial cells. Phenformin and AICAR increased AMPK activity in H441 cells in a dose-dependent fashion, stimulating the kinase maximally at 5-10 mm (P = 0.001, n = 3) and 2 mm (P < 0.005, n = 3), respectively. Both agents significantly decreased basal ion transport (measured as short circuit current) across H441 monolayers by approximately 50% compared with that of controls (P < 0.05, n = 4). Neither treatment altered the resistance of the monolayers. Phenformin and AICAR significantly reduced amiloride-sensitive transepithelial Na+ transport compared with controls (P < 0.05, n = 4). This was a result of both decreased Na+,K+-ATPase activity and amiloride-sensitive apical Na+ conductance. Transepithelial Na+ transport decreased with increasing concentrations of phenformin (0.1-10 mm) and showed a significant correlation with AMPK activity. Taken together, these results show that phenformin and AICAR suppress amiloride-sensitive Na+ transport across H441 cells via a pathway that includes activation of AMPK and inhibition of both apical Na+ entry through ENaC and basolateral Na+ extrusion via the Na+,K+-ATPase. These are the first studies to provide a cellular signalling mechanism for the action of phenformin on ion transport processes, and also the first studies showing AMPK as a regulator of Na+ absorption in the lung.

Phenformin and 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) activation of AMP-activated protein kinase inhibits transepithelial Na+ transport across H441 lung cells

PubMed Central

Woollhead, Alison M; Scott, John W; Hardie, D Grahame; Baines, Deborah L

2005-01-01

Active re-absorption of Na+ across the alveolar epithelium is essential to maintain lung fluid balance. Na+ entry at the luminal membrane is predominantly via the amiloride-sensitive Na+ channel (ENaC) down its electrochemical gradient. This gradient is generated and maintained by basolateral Na+ extrusion via Na+,K+-ATPase an energy-dependent process. Several kinases and factors that activate them are known to regulate these processes; however, the role of AMP-activated protein kinase (AMPK) in the lung is unknown. AMPK is an ultra-sensitive cellular energy sensor that monitors energy consumption and down-regulates ATP-consuming processes when activated. The biguanide phenformin has been shown to independently decrease ion transport processes, influence cellular metabolism and activate AMPK. The AMP mimetic drug 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) also activates AMPK in intact cells. Western blotting revealed that both the α1 and α2 catalytic subunits of AMPK are present in Na+ transporting H441 human lung epithelial cells. Phenformin and AICAR increased AMPK activity in H441 cells in a dose-dependent fashion, stimulating the kinase maximally at 5–10 mm (P = 0.001, n = 3) and 2 mm (P < 0.005, n = 3), respectively. Both agents significantly decreased basal ion transport (measured as short circuit current) across H441 monolayers by approximately 50% compared with that of controls (P < 0.05, n = 4). Neither treatment altered the resistance of the monolayers. Phenformin and AICAR significantly reduced amiloride-sensitive transepithelial Na+ transport compared with controls (P < 0.05, n = 4). This was a result of both decreased Na+,K+-ATPase activity and amiloride-sensitive apical Na+ conductance. Transepithelial Na+ transport decreased with increasing concentrations of phenformin (0.1–10 mm) and showed a significant correlation with AMPK activity. Taken together, these results show that phenformin and AICAR suppress amiloride-sensitive Na+ transport across H441 cells via a pathway that includes activation of AMPK and inhibition of both apical Na+ entry through ENaC and basolateral Na+ extrusion via the Na+,K+-ATPase. These are the first studies to provide a cellular signalling mechanism for the action of phenformin on ion transport processes, and also the first studies showing AMPK as a regulator of Na+ absorption in the lung. PMID:15919715

Inhibition of Growth by Combined Treatment with Inhibitors of Lactate Dehydrogenase and either Phenformin or Inhibitors of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase 3.

PubMed

Lea, Michael A; Guzman, Yolanda; Desbordes, Charles

2016-04-01

Enhanced glycolysis in cancer cells presents a target for chemotherapy. Previous studies have indicated that proliferation of cancer cells can be inhibited by treatment with phenformin and with an inhibitor of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB) namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). In the present work, the action of two inhibitors that are effective at lower concentrations than 3PO, namely 1-(3-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PQP) and 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15) were investigated. The inhibitors of lactate dehydrogenase (LDHA) studied in order of half-maximal inhibitory concentrations were methyl 1-hydroxy-6-phenyl-4-(trifluoromethyl)-1H-indole-2-carboxylate (NHI-2) < isosafrole < oxamate. In colonic and bladder cancer cells, additive growth inhibitory effects were seen with the LDHA inhibitors, of which NHI-2 was effective at the lowest concentrations. Growth inhibition was generally greater with PFK15 than with PQP. The increased acidification of the culture medium and glucose uptake caused by phenformin was blocked by combined treatment with PFKFB3 or LDHA inhibitors. The results suggest that combined treatment with phenformin and inhibitors of glycolysis can cause additive inhibition of cell proliferation and may mitigate lactic acidosis caused by phenformin when used as a single agent. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Genetic disruption of lactate/H+ symporters (MCTs) and their subunit CD147/BASIGIN sensitizes glycolytic tumor cells to phenformin.

PubMed

Marchiq, Ibtissam; Le Floch, Renaud; Roux, Danièle; Simon, Marie-Pierre; Pouyssegur, Jacques

2015-01-01

Rapidly growing glycolytic tumors require energy and intracellular pH (pHi) homeostasis through the activity of two major monocarboxylate transporters, MCT1 and the hypoxia-inducible MCT4, in intimate association with the glycoprotein CD147/BASIGIN (BSG). To further explore and validate the blockade of lactic acid export as an anticancer strategy, we disrupted, via zinc finger nucleases, MCT4 and BASIGIN genes in colon adenocarcinoma (LS174T) and glioblastoma (U87) human cell lines. First, we showed that homozygous loss of MCT4 dramatically sensitized cells to the MCT1 inhibitor AZD3965. Second, we demonstrated that knockout of BSG leads to a decrease in lactate transport activity of MCT1 and MCT4 by 10- and 6-fold, respectively. Consequently, cells accumulated an intracellular pool of lactic and pyruvic acids, magnified by the MCT1 inhibitor decreasing further pHi and glycolysis. As a result, we found that these glycolytic/MCT-deficient cells resumed growth by redirecting their metabolism toward OXPHOS. Third, we showed that in contrast with parental cells, BSG-null cells became highly sensitive to phenformin, an inhibitor of mitochondrial complex I. Phenformin addition to these MCT-disrupted cells in normoxic and hypoxic conditions induced a rapid drop in cellular ATP-inducing cell death by "metabolic catastrophe." Finally, xenograft analysis confirmed the deleterious tumor growth effect of MCT1/MCT4 ablation, an action enhanced by phenformin treatment. Collectively, these findings highlight that inhibition of the MCT/BSG complexes alone or in combination with phenformin provides an acute anticancer strategy to target highly glycolytic tumors. This genetic approach validates the anticancer potential of the MCT1 and MCT4 inhibitors in current development. ©2014 American Association for Cancer Research.

Metformin and phenformin activate AMP-activated protein kinase in the heart by increasing cytosolic AMP concentration.

PubMed

Zhang, Li; He, Huamei; Balschi, James A

2007-07-01

AMP-activated protein kinase (AMPK) acts as a cellular energy sensor: it responds to an increase in AMP concentration ([AMP]) or the AMP-to-ATP ratio (AMP/ATP). Metformin and phenformin, which are biguanides, have been reported to increase AMPK activity without increasing AMP/ATP. This study tests the hypothesis that these biguanides increase AMPK activity in the heart by increasing cytosolic [AMP]. Groups of isolated rat hearts (n = 5-7 each) were perfused with Krebs-Henseleit buffer with or without 0.2 mM phenformin or 10 mM metformin, and (31)P-NMR-measured phosphocreatine, ATP, and intracellular pH were used to calculate cytosolic [AMP]. At various times, hearts were freeze-clamped and assayed for AMPK activity, phosphorylation of Thr(172) on AMPK-alpha, and phosphorylation of Ser(79) on acetyl-CoA carboxylase, an AMPK target. In hearts treated with phenformin for 18 min and then perfused for 20 min with Krebs-Henseleit buffer, [AMP] began to increase at 26 min and AMPK activity was elevated at 36 min. In hearts treated with metformin, [AMP] was increased at 50 min and AMPK activity, phosphorylated AMPK, and phosphorylated acetyl-CoA carboxylase were elevated at 61 min. In metformin-treated hearts, HPLC-measured total AMP content and total AMP/ATP did not increase. In summary, phenformin and metformin increase AMPK activity and phosphorylation in the isolated heart. The increase in AMPK activity was always preceded by and correlated with increased cytosolic [AMP]. Total AMP content and total AMP/ATP did not change. Cytosolic [AMP] reported metabolically active AMP, which triggered increased AMPK activity, but measures of total AMP did not.

Aldehyde dehydrogenase inhibition combined with phenformin treatment reversed NSCLC through ATP depletion

PubMed Central

Lee, Jae-Seon; Nam, Boas; Seong, Tae Wha; Son, Jaekyoung; Jang, Hyonchol; Hong, Kyeong Man; Lee, Cheolju; Kim, Soo-Youl

2016-01-01

Among ALDH isoforms, ALDH1L1 in the folate pathway showed highly increased expression in non-small-cell lung cancer cells (NSCLC). Based on the basic mechanism of ALDH converting aldehyde to carboxylic acid with by-product NADH, we suggested that ALDH1L1 may contribute to ATP production using NADH through oxidative phosphorylation. ALDH1L1 knockdown reduced ATP production by up to 60% concomitantly with decrease of NADH in NSCLC. ALDH inhibitor, gossypol, also reduced ATP production in a dose dependent manner together with decrease of NADH level in NSCLC. A combination treatment of gossypol with phenformin, mitochondrial complex I inhibitor, synergized ATP depletion, which efficiently induced cell death. Pre-clinical xenograft model using human NSCLC demonstrated a remarkable therapeutic response to the combined treatment of gossypol and phenformin. PMID:27384481

Aldehyde dehydrogenase inhibition combined with phenformin treatment reversed NSCLC through ATP depletion.

PubMed

Kang, Joon Hee; Lee, Seon-Hyeong; Lee, Jae-Seon; Nam, Boas; Seong, Tae Wha; Son, Jaekyoung; Jang, Hyonchol; Hong, Kyeong Man; Lee, Cheolju; Kim, Soo-Youl

2016-08-02

Among ALDH isoforms, ALDH1L1 in the folate pathway showed highly increased expression in non-small-cell lung cancer cells (NSCLC). Based on the basic mechanism of ALDH converting aldehyde to carboxylic acid with by-product NADH, we suggested that ALDH1L1 may contribute to ATP production using NADH through oxidative phosphorylation. ALDH1L1 knockdown reduced ATP production by up to 60% concomitantly with decrease of NADH in NSCLC. ALDH inhibitor, gossypol, also reduced ATP production in a dose dependent manner together with decrease of NADH level in NSCLC. A combination treatment of gossypol with phenformin, mitochondrial complex I inhibitor, synergized ATP depletion, which efficiently induced cell death. Pre-clinical xenograft model using human NSCLC demonstrated a remarkable therapeutic response to the combined treatment of gossypol and phenformin.

The genetic control of phenformin 4-hydroxylation.

PubMed Central

Shah, R R; Evans, D A; Oates, N S; Idle, J R; Smith, R L

1985-01-01

Previously published results of phenformin 4-hydroxylation in 195 unrelated white British volunteers and 87 family members of 27 randomly selected probands have been subjected to genetic analysis. The results clearly show that about 9% of this population has a genetically determined defect in carrying out this oxidation reaction. The character for the defect is inherited in a Mendelian autosomal recessive fashion. The polymorphism shows a substantial degree of dominance. PMID:4078865

Metformin and phenformin deplete tricarboxylic acid cycle and glycolytic intermediates during cell transformation and NTPs in cancer stem cells

PubMed Central

Janzer, Andreas; German, Natalie J.; Gonzalez-Herrera, Karina N.; Asara, John M.; Haigis, Marcia C.; Struhl, Kevin

2014-01-01

Metformin, a first-line diabetes drug linked to cancer prevention in retrospective clinical analyses, inhibits cellular transformation and selectively kills breast cancer stem cells (CSCs). Although a few metabolic effects of metformin and the related biguanide phenformin have been investigated in established cancer cell lines, the global metabolic impact of biguanides during the process of neoplastic transformation and in CSCs is unknown. Here, we use LC/MS/MS metabolomics (>200 metabolites) to assess metabolic changes induced by metformin and phenformin in an Src-inducible model of cellular transformation and in mammosphere-derived breast CSCs. Although phenformin is the more potent biguanide in both systems, the metabolic profiles of these drugs are remarkably similar, although not identical. During the process of cellular transformation, biguanide treatment prevents the boost in glycolytic intermediates at a specific stage of the pathway and coordinately decreases tricarboxylic acid (TCA) cycle intermediates. In contrast, in breast CSCs, biguanides have a modest effect on glycolytic and TCA cycle intermediates, but they strongly deplete nucleotide triphosphates and may impede nucleotide synthesis. These metabolic profiles are consistent with the idea that biguanides inhibit mitochondrial complex 1, but they indicate that their metabolic effects differ depending on the stage of cellular transformation. PMID:25002509

Biguanides inhibit complex I, II and IV of rat liver mitochondria and modify their functional properties.

PubMed

Drahota, Z; Palenickova, E; Endlicher, R; Milerova, M; Brejchova, J; Vosahlikova, M; Svoboda, P; Kazdova, L; Kalous, M; Cervinkova, Z; Cahova, M

2014-01-01

In this study, we focused on an analysis of biguanides effects on mitochondrial enzyme activities, mitochondrial membrane potential and membrane permeability transition pore function. We used phenformin, which is more efficient than metformin, and evaluated its effect on rat liver mitochondria and isolated hepatocytes. In contrast to previously published data, we found that phenformin, after a 5 min pre-incubation, dose-dependently inhibits not only mitochondrial complex I but also complex II and IV activity in isolated mitochondria. The enzymes complexes inhibition is paralleled by the decreased respiratory control index and mitochondrial membrane potential. Direct measurements of mitochondrial swelling revealed that phenformin increases the resistance of the permeability transition pore to Ca(2+) ions. Our data might be in agreement with the hypothesis of Schäfer (1976) that binding of biguanides to membrane phospholipids alters membrane properties in a non-specific manner and, subsequently, different enzyme activities are modified via lipid phase. However, our measurements of anisotropy of fluorescence of hydrophobic membrane probe diphenylhexatriene have not shown a measurable effect of membrane fluidity with the 1 mM concentration of phenformin that strongly inhibited complex I activity. Our data therefore suggest that biguanides could be considered as agents with high efficacy but low specifity.

Metformin and phenformin deplete tricarboxylic acid cycle and glycolytic intermediates during cell transformation and NTPs in cancer stem cells.

PubMed

Janzer, Andreas; German, Natalie J; Gonzalez-Herrera, Karina N; Asara, John M; Haigis, Marcia C; Struhl, Kevin

2014-07-22

Metformin, a first-line diabetes drug linked to cancer prevention in retrospective clinical analyses, inhibits cellular transformation and selectively kills breast cancer stem cells (CSCs). Although a few metabolic effects of metformin and the related biguanide phenformin have been investigated in established cancer cell lines, the global metabolic impact of biguanides during the process of neoplastic transformation and in CSCs is unknown. Here, we use LC/MS/MS metabolomics (>200 metabolites) to assess metabolic changes induced by metformin and phenformin in an Src-inducible model of cellular transformation and in mammosphere-derived breast CSCs. Although phenformin is the more potent biguanide in both systems, the metabolic profiles of these drugs are remarkably similar, although not identical. During the process of cellular transformation, biguanide treatment prevents the boost in glycolytic intermediates at a specific stage of the pathway and coordinately decreases tricarboxylic acid (TCA) cycle intermediates. In contrast, in breast CSCs, biguanides have a modest effect on glycolytic and TCA cycle intermediates, but they strongly deplete nucleotide triphosphates and may impede nucleotide synthesis. These metabolic profiles are consistent with the idea that biguanides inhibit mitochondrial complex 1, but they indicate that their metabolic effects differ depending on the stage of cellular transformation.

The metabolic perturbators metformin, phenformin and AICAR interfere with the growth and survival of murine PTEN-deficient T cell lymphomas and human T-ALL/T-LL cancer cells.

PubMed

Rosilio, Célia; Lounnas, Nadia; Nebout, Marielle; Imbert, Véronique; Hagenbeek, Thijs; Spits, Hergen; Asnafi, Vahid; Pontier-Bres, Rodolphe; Reverso, Julie; Michiels, Jean-François; Sahra, Issam Ben; Bost, Fréderic; Peyron, Jean-François

2013-08-09

We show here that the antidiabetic agents metformin and phenformin and the AMPK activator AICAR exert strong anti-tumoural effects on tPTEN-/- lymphoma cells and on human T-ALL cell lines and primary samples. The compounds act by inhibiting tumour metabolism and proliferation and by inducing apoptosis in parallel with an activation of AMPK and an inhibition of constitutive mTOR. In tPTEN-/- cells, the drugs potentiated the anti-leukaemic effects of dexamethasone, and metformin and phenformin synergised with 2-deoxyglucose (2DG) to impair tumour cell survival. In vivo, metformin and AICAR strongly decreased the growth of luciferase-expressing tPTEN-/- cells xenografted in Nude mice, demonstrating that metabolism targeting could be a potent adjuvant strategy for lymphoma/leukaemia treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Immunohistochemical localization of OCT2 in the cochlea of various species

PubMed Central

Gahm, Caroline; Liu, Wei; Ehrsson, Hans; Rask‐Andersen, Helge; Laurell, Göran

2015-01-01

Objective To locate the organic cation transporter 2 (OCT2) in the cochlea of three different species and to modulate the ototoxicity of cisplatin in the guinea pig by pretreatment with phenformin, having a known affinity for OCT2. Study Design Immunohistochemical and in vivo study. Methods Sections from the auditory end organs were subjected to immunohistochemical staining in order to identify OCT2 in cochlea from untreated rats, guinea pigs, and a pig. In the in vivo study, guinea pigs were given phenformin intravenously 30 minutes before cisplatin administration. Electrophysiological hearing thresholds were determined, and hair cells loss was assessed 96 hours later. The total amount of platinum in cochlear tissue was determined using mass spectrometry. Results Organic cation transporter 2 was found in the supporting cells and in type I spiral ganglion cells in the cochlea of all species studied. Pretreatment with phenformin did not reduce the ototoxic side effect of cisplatin. Furthermore, the concentration of platinum in the cochlea was not affected by phenformin. Conclusions The localization of OCT2 in the supporting cells and type I spiral ganglion cells suggests that this transport protein is not primarily involved in cisplatin uptake from the systemic circulation. We hypothesize that OCT2 transport intensifies cisplatin ototoxicity via transport mechanisms in alternate compartments of the cochlea. Level of Evidence N/A. Laryngoscope, 125:E320–E325, 2015 PMID:25892279

Relevance of the OCT1 transporter to the antineoplastic effect of biguanides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Segal, Eric D.; Yasmeen, Amber; Beauchamp, Marie-Claude

2011-11-04

Highlights: Black-Right-Pointing-Pointer siRNA knockdown of OCT1 reduced sensitivity of EOC cells to metformin, but not to another biguanide, phenformin. Black-Right-Pointing-Pointer Suppression of OCT1 also affects the activation of AMP kinase in response to metformin, but not to phenformin. Black-Right-Pointing-Pointer Direct actions of metformin may be limited by low OCT1 expression in EOC tumors. Black-Right-Pointing-Pointer Phenformin could be used as an alternative biguanide. -- Abstract: Epidemiologic and laboratory data suggesting that metformin has antineoplastic activity have led to ongoing clinical trials. However, pharmacokinetic issues that may influence metformin activity have not been studied in detail. The organic cation transporter 1 (OCT1)more » is known to play an important role in cellular uptake of metformin in the liver. We show that siRNA knockdown of OCT1 reduced sensitivity of epithelial ovarian cancer cells to metformin, but interestingly not to another biguanide, phenformin, with respect to both activation of AMP kinase and inhibition of proliferation. We observed that there is heterogeneity between primary human tumors with respect to OCT1 expression. These results suggest that there may be settings where drug uptake limits direct action of metformin on neoplastic cells, raising the possibility that metformin may not be the optimal biguanide for clinical investigation.« less

Phenformin enhances the efficacy of ERK inhibition in NF1-mutant melanoma

PubMed Central

Shaw, Fiona M.; Yao, Zhan; Ran, Yuping; Shakuntala, Tiwari; Merghoub, Taha; Manstein, Dieter; Rosen, Neal; Cantley, Lewis C.; Zippin, Jonathan H.; Zheng, Bin

2017-01-01

Inactivation of the tumor suppressor neurofibromin 1 (NF1) presents a newly characterized melanoma subtype, for which currently no targeted therapies are clinically available. Pre-clinical studies suggest that ERK inhibitors are likely to provide benefit, albeit with limited efficacy as single agent; therefore, there is a need for rationally designed combination therapies. Here, we evaluate the combination of the ERK inhibitor SCH772984 and the biguanide phenformin. Combination of both compounds showed potent synergy in cell viability assays and cooperatively induced apoptosis. Treatment with both drugs was required to fully suppress mTOR signaling, a known effector of NF1 loss. Mechanistically, SCH772984 increased the oxygen consumption rate (OCR), indicating that these cells relied more on oxidative phosphorylation upon treatment. Consistently, SCH772984 increased expression of the mitochondrial transcriptional co-activator PGC1α. In contrast, co-treatment with phenformin, an inhibitor of complex I of the respiratory chain, decreased the OCR. SCH772984 also promoted the expansion of the H3K4 demethylase KDM5B (also known as JARID1B)-positive subpopulation of melanoma cells, which are slow-cycling and treatment-resistant. Importantly, phenformin suppressed this KDM5B-positive population, which reduced the emergence of SCH772984-resistant clones in long-term cultures. Our results warrant the clinical investigation of this combination therapy in patients with NF1 mutant melanoma. PMID:28143781

Destabilization of MYC/MYCN by the mitochondrial inhibitors, metaiodobenzylguanidine, metformin and phenformin

PubMed Central

WANG, STEPHANIE S.; HSIAO, RUTH; LIMPAR, MARIKO M.; LOMAHAN, SARAH; TRAN, TUAN-ANH; MALONEY, NOLAN J.; IKEGAKI, NAOHIKO; TANG, XAO X.

2014-01-01

In the present study, we investigated the anticancer effects of the mitochondrial inhibitors, metaiodobenzylguanidine (MIBG), metformin and phenformin. 131I-MIBG has been used for scintigraphic detection and the targeted radiotherapy of neuroblastoma (NB), a pediatric malignancy. Non-radiolabeled MIBG has been reported to be cytotoxic to NB cells in vitro and in vivo. However, the mechanisms behind its growth suppressive effects have not yet been fully elucidated. Metformin and phenformin are diabetes medications that are being considered in anticancer therapeutics. We investigated the anticancer mechanisms of action of MIBG and metformin in NB. Our data revealed that both drugs suppressed NB cell growth and that the combination drug treatment was more potent. MIBG reduced MYCN and MYC expression in MYCN-amplified and non-MYCN-amplified NB cells in a dose- and time-dependent manner. Metformin was less effective than MIBG in destabilizing MYC/MYCN. The treatment of NB cells with metformin or MIBG resulted in an increased expression of genes encoding biomarkers for favorable outcome in NB [(ephrin (EFN)B2, EFNB3, EPH receptor B6 (EPHB6), neurotrophic tyrosine kinase, receptor, type 1 (NTRK1), CD44 and Myc-interacting zinc finger protein (MIZ-1)] and tumor suppressor genes [(early growth response 1 (EGR1), EPH receptor A2 (EPHA2), growth arrest and DNA-damage-inducible, beta (GADD45B), neuregulin 1 (NRG1), TP53 apoptosis effector (PERP) and sel-1 suppressor of lin-12-like (C. elegans) (SEL1L)]. Accordingly, metformin and MIBG augmented histone H3 acetylation in these cells. Phenformin also exhibited histone modification and was more effective than metformin in destabilizing MYC/MYCN in NB cells. Our data suggest that the destabilization of MYC/MYCN by MIBG, metformin and phenformin and their effects on histone modification are important mechanisms underlying their anticancer effects. PMID:24190252

AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs

PubMed Central

El Hadri, Khadija; Denoyelle, Chantal; Ravaux, Lucas; Viollet, Benoit; Foretz, Marc; Friguet, Bertrand; Rouis, Mustapha; Raymondjean, Michel

2015-01-01

Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs. PMID:26162096

AMPK Signaling Involvement for the Repression of the IL-1β-Induced Group IIA Secretory Phospholipase A2 Expression in VSMCs.

PubMed

El Hadri, Khadija; Denoyelle, Chantal; Ravaux, Lucas; Viollet, Benoit; Foretz, Marc; Friguet, Bertrand; Rouis, Mustapha; Raymondjean, Michel

2015-01-01

Secretory Phospholipase A2 of type IIA (sPLA2 IIA) plays a crucial role in the production of lipid mediators by amplifying the neointimal inflammatory context of the vascular smooth muscle cells (VSMCs), especially during atherogenesis. Phenformin, a biguanide family member, by its anti-inflammatory properties presents potential for promoting beneficial effects upon vascular cells, however its impact upon the IL-1β-induced sPLA2 gene expression has not been deeply investigated so far. The present study was designed to determine the relationship between phenformin coupling AMP-activated protein kinase (AMPK) function and the molecular mechanism by which the sPLA2 IIA expression was modulated in VSMCs. Here we find that 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleotide (AICAR) treatment strongly repressed IL-1β-induced sPLA2 expression at least at the transcriptional level. Our study reveals that phenformin elicited a dose-dependent inhibition of the sPLA2 IIA expression and transient overexpression experiments of constitutively active AMPK demonstrate clearly that AMPK signaling is involved in the transcriptional inhibition of sPLA2-IIA gene expression. Furthermore, although the expression of the transcriptional repressor B-cell lymphoma-6 protein (BCL-6) was markedly enhanced by phenformin and AICAR, the repression of sPLA2 gene occurs through a mechanism independent of BCL-6 DNA binding site. In addition we show that activation of AMPK limits IL-1β-induced NF-κB pathway activation. Our results indicate that BCL-6, once activated by AMPK, functions as a competitor of the IL-1β induced NF-κB transcription complex. Our findings provide insights on a new anti-inflammatory pathway linking phenformin, AMPK and molecular control of sPLA2 IIA gene expression in VSMCs.

Destabilization of MYC/MYCN by the mitochondrial inhibitors, metaiodobenzylguanidine, metformin and phenformin.

PubMed

Wang, Stephanie S; Hsiao, Ruth; Limpar, Mariko M; Lomahan, Sarah; Tran, Tuan-Anh; Maloney, Nolan J; Ikegaki, Naohiko; Tang, Xao X

2014-01-01

In the present study, we investigated the anticancer effects of the mitochondrial inhibitors, metaiodobenzylguanidine (MIBG), metformin and phenformin. 131I-MIBG has been used for scintigraphic detection and the targeted radiotherapy of neuroblastoma (NB), a pediatric malignancy. Non-radiolabeled MIBG has been reported to be cytotoxic to NB cells in vitro and in vivo. However, the mechanisms behind its growth suppressive effects have not yet been fully elucidated. Metformin and phenformin are diabetes medications that are being considered in anticancer therapeutics. We investigated the anticancer mechanisms of action of MIBG and metformin in NB. Our data revealed that both drugs suppressed NB cell growth and that the combination drug treatment was more potent. MIBG reduced MYCN and MYC expression in MYCN-amplified and non-MYCN-amplified NB cells in a dose- and time-dependent manner. Metformin was less effective than MIBG in destabilizing MYC/MYCN. The treatment of NB cells with metformin or MIBG resulted in an increased expression of genes encoding biomarkers for favorable outcome in NB [(ephrin (EFN)B2, EFNB3, EPH receptor B6 (EPHB6), neurotrophic tyrosine kinase, receptor, type 1 (NTRK1), CD44 and Myc-interacting zinc finger protein (MIZ-1)] and tumor suppressor genes [(early growth response 1 (EGR1), EPH receptor A2 (EPHA2), growth arrest and DNA-damage-inducible, beta (GADD45B), neuregulin 1 (NRG1), TP53 apoptosis effector (PERP) and sel-1 suppressor of lin-12-like (C. elegans) (SEL1L)]. Accordingly, metformin and MIBG augmented histone H3 acetylation in these cells. Phenformin also exhibited histone modification and was more effective than metformin in destabilizing MYC/MYCN in NB cells. Our data suggest that the destabilization of MYC/MYCN by MIBG, metformin and phenformin and their effects on histone modification are important mechanisms underlying their anticancer effects.

N-bromosuccinimide-fluorescein based sensitive flow-injection chemiluminescence determination of phenformin.

PubMed

Wang, Zhouping; Zhang, Zhujun; Fu, Zhifeng; Fang, Luqiu; Zhang, Xiao

2004-02-01

A novel and highly sensitive method for the determination of phenformin over the range of 6 x 10(-9) - 1 x 10(-5) g ml(-1) in pharmaceutical formulations with flow-injection chemiluminescence (CL) detection is proposed. The method is based on the CL produced during the oxidation of N-bromosuccinimide (NBS) in an alkaline medium in the presence of fluorescein as an effective energy transfer agent. The use of cetyltrimethylammonium bromide (CTAB) as a sensitizer enhances the signal magnitude by about 100 times. The detection limit is 2 x 10(-9) g ml(-1) (3sigma) with a relative standard deviation of 2.3% (n = 11) at 1 x 10(-7) g ml(-1) phenformin. Ninety samples can be determined per hour. The method was evaluated by carrying out a recovery study and by the analysis of commercial formulations. The obtained results compared well with those by an official method, and demonstrated good accuracy and precision. The possible CL mechanism of the proposed system was also briefly analyzed.

Regulation of Cl(-) secretion by AMPK in vivo.

PubMed

Kongsuphol, Patthara; Hieke, Bernhard; Ousingsawat, Jiraporn; Almaca, Joana; Viollet, Benoit; Schreiber, Rainer; Kunzelmann, Karl

2009-03-01

Previous in vitro studies suggested that Cl(-) currents produced by the cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) are inhibited by the alpha1 isoform of the adenosine monophosphate (AMP)-stimulated kinase (AMPK). AMPK is a serine/threonine kinase that is activated during metabolic stress. It has been proposed as a potential mediator for transport-metabolism coupling in epithelial tissues. All previous studies have been performed in vitro and thus little is known about the regulation of Cl(-) secretion by AMPK in vivo. Using AMPKalpha1(-/-) mice and wild-type littermates, we demonstrate that phenformin, an activator of AMPK, strongly inhibits cAMP-activated Cl(-) secretion in mouse airways and colon, when examined in ex vivo in Ussing chamber recordings. However, phenformin was equally effective in AMPKalpha1(-/-) and wild-type animals, suggesting additional AMPK-independent action of phenformin. Phenformin inhibited CFTR Cl(-) conductance in basolaterally permeabilized colonic epithelium from AMPKalpha1(+/+) but not AMPKalpha1(-/-) mice. The inhibitor of AMPK compound C enhanced CFTR-mediated Cl(-) secretion in epithelial tissues of AMPKalpha1(-/-) mice, but not in wild-type littermates. There was no effect on Ca(2+)-mediated Cl(-) secretion, activated by adenosine triphosphate or carbachol. Moreover CFTR-dependent Cl(-) secretion was enhanced in the colon of AMPKalpha1(-/-) mice, as indicated in Ussing chamber ex vivo and rectal PD measurements in vivo. Taken together, these data suggest that epithelial Cl(-) secretion mediated by CFTR is controlled by AMPK in vivo.

Phenformin Enhances the Efficacy of ERK Inhibition in NF1-Mutant Melanoma.

PubMed

Trousil, Sebastian; Chen, Shuang; Mu, Chan; Shaw, Fiona M; Yao, Zhan; Ran, Yuping; Shakuntala, Tiwari; Merghoub, Taha; Manstein, Dieter; Rosen, Neal; Cantley, Lewis C; Zippin, Jonathan H; Zheng, Bin

2017-05-01

Inactivation of the tumor suppressor neurofibromin 1 (NF1) presents a newly characterized melanoma subtype, for which currently no targeted therapies are clinically available. Preclinical studies suggest that extracellular signal-regulated kinase (ERK) inhibitors are likely to provide benefit, albeit with limited efficacy as a single agent; therefore, there is a need for rationally designed combination therapies. Here, we evaluate the combination of the ERK inhibitor SCH772984 and the biguanide phenformin. A combination of both compounds showed potent synergy in cell viability assays and cooperatively induced apoptosis. Treatment with both drugs was required to fully suppress mechanistic target of rapamycin signaling, a known effector of NF1 loss. Mechanistically, SCH772984 increased the oxygen consumption rate, indicating that these cells relied more on oxidative phosphorylation upon treatment. Consistently, SCH772984 increased expression of the mitochondrial transcriptional coactivator peroxisome proliferator-activated receptor gamma, coactivator 1-α. In contrast, cotreatment with phenformin, an inhibitor of complex I of the respiratory chain, decreased the oxygen consumption rate. SCH772984 also promoted the expansion of the H3K4 demethylase KDM5B (also known as JARID1B)-positive subpopulation of melanoma cells, which are slow-cycling and treatment-resistant. Importantly, phenformin suppressed this KDM5B-positive population, which reduced the emergence of SCH772984-resistant clones in long-term cultures. Our results warrant the clinical investigation of this combination therapy in patients with NF1 mutant melanoma. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Heightening energetic stress selectively targets LKB1-deficient non-small cell lung cancers

PubMed Central

Momcilovic, Milica; McMickle, Robert; Abt, Evan; Seki, Atsuko; Simko, Sarah A.; Magyar, Clara; Stout, David B.; Fishbein, Michael C.; Walser, Tonya C.; Dubinett, Steven M.; Shackelford, David B.

2015-01-01

Inactivation of the LKB1 tumor suppressor is a frequent event in non-small cell lung carcinoma (NSCLC) leading to the activation of mammalian target of rapamycin complex 1 (mTORC1) and sensitivity to the metabolic stress inducer phenformin. In this study, we explored the combinatorial use of phenformin with the mTOR catalytic kinase inhibitor MLN0128 as a treatment strategy for NSCLC bearing co-mutations in the LKB1 and KRAS genes. NSCLC is a genetically and pathologically heterogeneous disease, giving rise to lung tumors of varying histologies that include adenocarcinomas (ADCs) and squamous cell carcinomas (SCCs). We demonstrate that phenformin in combination with MLN0128 induced a significant therapeutic response in KRAS/LKB1 mutant human cell lines and genetically engineered mouse models of NSCLC that develop both ADCs and SCCs. Specifically, we found that KRAS/LKB1 mutant lung ADCs responded strongly to phenformin + MLN0128 treatment, but the response of SCCs to single or combined treatment with MLN0128 was more attenuated due to acquired resistance to mTOR inhibition through modulation of the AKT-GSK signaling axis. Combinatorial use of the mTOR inhibitor and AKT inhibitor MK2206 robustly inhibited the growth and viability of squamous lung tumors thus providing an effective strategy to overcome resistance. Taken together, our findings define new personalized therapeutic strategies that may be rapidly translated into clinical use for the treatment of KRAS/LKB1 mutant adenocarcinomas and squamous cell tumors. PMID:26574479

Heightening Energetic Stress Selectively Targets LKB1-Deficient Non-Small Cell Lung Cancers.

PubMed

Momcilovic, Milica; McMickle, Robert; Abt, Evan; Seki, Atsuko; Simko, Sarah A; Magyar, Clara; Stout, David B; Fishbein, Michael C; Walser, Tonya C; Dubinett, Steven M; Shackelford, David B

2015-11-15

Inactivation of the LKB1 tumor suppressor is a frequent event in non-small cell lung carcinoma (NSCLC) leading to the activation of mTOR complex 1 (mTORC1) and sensitivity to the metabolic stress inducer phenformin. In this study, we explored the combinatorial use of phenformin with the mTOR catalytic kinase inhibitor MLN0128 as a treatment strategy for NSCLC bearing comutations in the LKB1 and KRAS genes. NSCLC is a genetically and pathologically heterogeneous disease, giving rise to lung tumors of varying histologies that include adenocarcinomas and squamous cell carcinomas (SCC). We demonstrate that phenformin in combination with MLN0128 induced a significant therapeutic response in KRAS/LKB1-mutant human cell lines and genetically engineered mouse models of NSCLC that develop both adenocarcinomas and SCCs. Specifically, we found that KRAS/LKB1-mutant lung adenocarcinomas responded strongly to phenformin + MLN0128 treatment, but the response of SCCs to single or combined treatment with MLN0128 was more attenuated due to acquired resistance to mTOR inhibition through modulation of the AKT-GSK signaling axis. Combinatorial use of the mTOR inhibitor and AKT inhibitor MK2206 robustly inhibited the growth and viability of squamous lung tumors, thus providing an effective strategy to overcome resistance. Taken together, our findings define new personalized therapeutic strategies that may be rapidly translated into clinical use for the treatment of KRAS/LKB1-mutant adenocarcinomas and squamous cell tumors. ©2015 American Association for Cancer Research.

Determination of phenformin hydrochloride using molecular imprinting technology coupled with flow-injection chemiluminescence.

PubMed

Liu, Zhenbo; Jia, Fengyan; Wang, Wenwen; Wang, Cuixia; Liu, Yongming

2012-01-01

A novel method was developed using molecular imprinting technology (MIT) coupled with flow-injection chemiluminescence (FI-CL) for highly sensitive detection of phenformin hydrochloride (PH). The phenformin imprinted polymer was synthesized with methacrylic acid (MAA) as a functional monomer and ethylene glycol dimethacrylate (EGDMA) as a cross-linker. Newly synthesized molecularly imprinted polymer (MIP) particles were packed into a column as a selective recognition element for determination of PH. A CL method for the determination of PH was developed based on the CL reaction of PH with N-bromosuccinimide sensitized by eosin Y in basic media. The optimization of detection conditions was investigated. The CL intensity responded linearly to the concentration of PH in the range 0.09-2.0 µg/mL, with a correlation coefficient of 0.9920. The detection limit was 0.031 µg/mL. The relative standard deviation for the determination of 1.0 µg/mL PH solution was 1.0% (n = 11). The method was applied to the determination of PH in urine samples, with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.

Activated AMPK and prostaglandins are involved in the response to conjugated linoleic acid and are sufficient to cause lipid reductions in adipocytes.

PubMed

Jiang, Shan; Chen, Han; Wang, Zhigang; Riethoven, Jean-Jack; Xia, Yuannan; Miner, Jess; Fromm, Michael

2011-07-01

trans-10, cis-12 Conjugated linoleic acid (t10c12 CLA) reduces triglyceride levels in adipocytes. AMP-activated protein kinase (AMPK) and inflammation were recently demonstrated to be involved in the emerging pathways regulating this response. This study further investigated the role of AMPK and inflammation by testing the following hypotheses: (1) a moderate activation of AMPK and an inflammatory response are sufficient to reduce triglycerides, and (2) strong activation of AMPK is also sufficient. Experiments were performed by adding compounds that affect these pathways and by measuring their effects in 3T3-L1 adipocytes. A comparison of four AMPK activators (metformin, phenformin, TNF-α and t10c12 CLA) found a correlation between AMPK activity and triglyceride reduction. This correlation appeared to be modulated by the level of cyclo-oxygenase (COX)-2 mRNA produced. Inhibitors of the prostaglandin (PG) biosynthetic pathway interfered with t10c12 CLA's ability to reduce triglycerides. A combination of metformin and PGH2, or phenformin alone, efficiently reduced triglyceride levels in adipocytes. Microarray analysis indicated that the transcriptional responses to phenformin or t10c12 CLA were very similar, suggesting similar pathways were activated. 3T3-L1 fibroblasts were found to weakly induce the integrated stress response (ISR) in response to phenformin or t10c12 CLA and to respond robustly as they differentiated into adipocytes. This indicated that both chemicals required adipocytes at the same stage of differentiation to be competent for this response. These results support the above hypotheses and suggest compounds that moderately activate AMPK and increase PG levels or robustly activate AMPK in adipocytes may be beneficial for reducing adiposity. Copyright © 2011 Elsevier Inc. All rights reserved.

Structural homologies between phenformin, lipitor and gleevec aim the same metabolic oncotarget in leukemia and melanoma.

PubMed

Somlyai, Gábor; Collins, T Que; Meuillet, Emmanuelle J; Hitendra, Patel; D'Agostino, Dominic P; Boros, László G

2017-07-25

Phenformin's recently demonstrated efficacy in melanoma and Gleevec's demonstrated anti-proliferative action in chronic myeloid leukemia may lie within these drugs' significant pharmacokinetics, pharmacodynamics and structural homologies, which are reviewed herein. Gleevec's success in turning a fatal leukemia into a manageable chronic disease has been trumpeted in medical, economic, political and social circles because it is considered the first successful targeted therapy. Investments have been immense in omics analyses and while in some cases they greatly helped the management of patients, in others targeted therapies failed to achieve clinically stable recurrence-free disease course or to substantially extend survival. Nevertheless protein kinase controlling approaches have persisted despite early warnings that the targeted genomics narrative is overblown. Experimental and clinical observations with Phenformin suggest an alternative explanation for Gleevec's mode of action. Using 13C-guided precise flux measurements, a comparative multiple cell line study demonstrated the drug's downstream impact on submolecular fatty acid processing metabolic events that occurred independent of Gleevec's molecular target. Clinical observations that hyperlipidemia and diabetes are both reversed in mice and in patients taking Gleevec support the drugs' primary metabolic targets by biguanides and statins. This is evident by structural data demonstrating that Gleevec shows pyridine- and phenyl-guanidine homology with Phenformin and identical phenylcarbamoyl structural and ligand binding homology with Lipitor. The misunderstood mechanism of action of Gleevec is emblematic of the pervasive flawed reasoning that genomic analysis will lead to targeted, personalized diagnosis and therapy. The alternative perspective for Gleevec's mode of action may turn oncotargets towards metabolic channel reaction architectures in leukemia and melanoma, as well as in other cancers.

The enhancement of oxidative DNA damage by anti-diabetic metformin, buformin, and phenformin, via nitrogen-centered radicals.

PubMed

Ohnishi, Shiho; Mizutani, Hideki; Kawanishi, Shosuke

2016-08-01

Metformin (N,N-dimethylbiguanide), buformin (1-butylbiguanide), and phenformin (1-phenethylbiguanide) are anti-diabetic biguanide drugs, expected to having anti-cancer effect. The mechanism of anti-cancer effect by these drugs is not completely understood. In this study, we demonstrated that these drugs dramatically enhanced oxidative DNA damage under oxidative condition. Metformin, buformin, and phenformin enhanced generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in isolated DNA reacted with hydrogen peroxide (H2O2) and Cu(II), although these drugs did not form 8-oxodG in the absence of H2O2 or Cu(II). An electron paramagnetic resonance (EPR) study, utilizing alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide as spin trapping agents, showed that nitrogen-centered radicals were generated from biguanides in the presence of Cu(II) and H2O2, and that these radicals were decreased by the addition of DNA. These results suggest that biguanides enhance Cu(II)/H2O2-mediated 8-oxodG generation via nitrogen-centered radical formation. The enhancing effect on oxidative DNA damage may play a role on anti-cancer activity.

Ppm1E is an in cellulo AMP-activated protein kinase phosphatase.

PubMed

Voss, Martin; Paterson, James; Kelsall, Ian R; Martín-Granados, Cristina; Hastie, C James; Peggie, Mark W; Cohen, Patricia T W

2011-01-01

Activation of 5'-AMP-activated protein kinase (AMPK) is believed to be the mechanism by which the pharmaceuticals, metformin and phenformin, exert their beneficial effects for treatment of type 2 diabetes. These biguanide drugs elevate 5'-AMP, which allosterically activates AMPK and promotes phosphorylation on Thr172 of AMPK catalytic α subunits. Although kinases phosphorylating this site have been identified, phosphatases that dephosphorylate it are unknown. The aim of this study is to identify protein phosphatase(s) that dephosphorylate AMPKα-Thr172 within cells. Our initial data indicated that members of the protein phosphatase Mg/Mn(2+)-dependent [corrected] (PPM) family and not those of the PPP family of protein serine/threonine phosphatases may be directly or indirectly inhibited by phenformin. Using antibodies raised to individual Ppm phosphatases that facilitated the assessment of their activities, phenformin stimulation of cells was found to decrease the Mg(2+)/Mn(2+)-dependent [corrected] protein serine/threonine phosphatase activity of Ppm1E and Ppm1F, but not that attributable to other PPM family members, including Ppm1A/PP2Cα. Depletion of Ppm1E, but not Ppm1A, using lentiviral-mediated stable gene silencing, increased AMPKα-Thr172 phosphorylation approximately three fold in HEK293 cells. In addition, incubation of cells with low concentrations of phenformin and depletion of Ppm1E increased AMPK phosphorylation synergistically. Ppm1E and the closely related Ppm1F interact weakly with AMPK and assays with lysates of cells stably depleted of Ppm1F suggest [corrected] that this phosphatase contributes to dephosphorylation of AMPK. The data indicate that Ppm1E and probably PpM1F are in cellulo AMPK phosphatases and that Ppm1E is a potential anti-diabetic drug target. Copyright © 2010 Elsevier Inc. All rights reserved.

Effects of Metformin, Buformin, and Phenformin on the Post Initiation Stage of Chemically-Induced Mammary Carcinogenesis in the Rat

PubMed Central

Zhu, Zongjian; Jiang, Weiqin; Thompson, Matthew D.; Echeverria, Dimas; McGinley, John N.; Thompson, Henry J.

2015-01-01

Metformin is a widely prescribed drug for the treatment of type-2 diabetes. Although epidemiological data have provided a strong rationale for investigating the potential of this biguanide for use in cancer prevention and control, uncertainty exists whether metformin should be expected to have an impact in non-diabetic patients. Furthermore, little attention has been given to the possibility that other biguanides may have anticancer activity. In this study, the effects of clinically relevant doses of metformin (9.3mmol/kg diet), buformin (7.6 mmol/kg diet), and phenformin (5.0 mmol/kg diet) were compared to rats fed control diet (AIN93-G) during the post initiation stage of 1-methyl-1-nitrosourea-induced (50 mg/kg body weight) mammary carcinogenesis (n = 30/group). Plasma, liver, skeletal muscle, visceral fat, mammary gland, and mammary carcinoma concentrations of the biguanides were determined. In comparison to the control group, buformin decreased cancer incidence, multiplicity, and burden; whereas, metformin and phenformin had no statistically significant effect on the carcinogenic process relative to the control group. Buformin did not alter fasting plasma glucose or insulin. Within mammary carcinomas, evidence was obtained that buformin treatment perturbed signaling pathways related to energy sensing. However, further investigation is needed to determine the relative contributions of host systemic and cell autonomous mechanisms to the anticancer activity of biguanides such as buformin. PMID:25804611

Elucidation of a novel phenformin derivative on glucose-deprived stress responses in HT-29 cells.

PubMed

Oh-Hashi, Kentaro; Irie, Nao; Sakai, Takayuki; Okuda, Kensuke; Nagasawa, Hideko; Hirata, Yoko; Kiuchi, Kazutoshi

2016-08-01

Recently, we developed a variety of phenformin derivatives as selective antitumor agents. Based on previous findings, this study evaluated a promising compound, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on the basis of stress responses in the human colon cancer cell line HT-29 under a serum- and glucose-deprived condition. 2-Cl-Phen triggered morphological changes such as shrinkage and plasma membrane disintegration, as well as a decrease in mitochondrial activity and an increase in LDH leakage. To understand intracellular issues relating to 2-Cl-Phen, this study focused on the expression levels of ER stress-inducible genes and several oncogenic genes. Serum and glucose deprivation significantly induced a variety of ER stress-inducible genes, but a 12-h treatment of 2-Cl-Phen down-regulated expression of several ER stress-related genes, with the exception of GADD153. Interestingly, the expression levels of ATF6α, GRP78, MANF, and CRELD2 mRNA were almost completely decreased by 2-Cl-Phen. This study also observed that a 24-h treatment of 2-Cl-Phen attenuated the expression levels of GRP78, GADD153, and c-Myc protein. The decrease in c-Myc protein occurred before the fluctuation of GRP78 protein, while the expression of c-Myc mRNA showed little change with cotreatment of serum and glucose deprivation with 2-Cl-Phen. To further understand the 2-Cl-Phen-induced down-regulation of ATF6-related genes, this study investigated the stability of ATF6α and GRP78 proteins using NanoLuc-tagged constructs. The expression levels of NanoLuc-tagged ATF6α and GRP78 were significantly down-regulated by 2-Cl-Phen in the presence or absence of the translation inhibitor cycloheximide. Taken together, our novel phenformin derivative 2-Cl-Phen has the unique characteristic of diminishing tumor adaptive responses, especially the expression of ATF6-related genes, as well as that of c-Myc protein, in a transcriptional and posttranscriptional manner under a serum- and glucose-deprived condition. Further characterization of cytotoxic mechanisms related to phenformin derivatives may give new insights into developing additional promising anticancer agents.

Suppression of gain-of-function mutant p53 with metabolic inhibitors reduces tumor growth in vivo

PubMed Central

Jung, Chae Lim; Mun, Hyemin; Jo, Se-Young; Oh, Ju-Hee; Lee, ChuHee; Choi, Eun-Kyung; Jang, Se Jin; Suh, Young-Ah

2016-01-01

Mutation of p53 occasionally results in a gain of function, which promotes tumor growth. We asked whether destabilizing the gain-of-function protein would kill tumor cells. Downregulation of the gene reduced cell proliferation in p53-mutant cells, but not in p53-null cells, indicating that the former depended on the mutant protein for survival. Moreover, phenformin and 2-deoxyglucose suppressed cell growth and simultaneously destabilized mutant p53. The AMPK pathway, MAPK pathway, chaperone proteins and ubiquitination all contributed to this process. Interestingly, phenformin and 2-deoxyglucose also reduced tumor growth in syngeneic mice harboring the p53 mutation. Thus, destabilizing mutant p53 protein in order to kill cells exhibiting “oncogene addiction” could be a promising strategy for combatting p53 mutant tumors. PMID:27765910

Suppression of gain-of-function mutant p53 with metabolic inhibitors reduces tumor growth in vivo.

PubMed

Jung, Chae Lim; Mun, Hyemin; Jo, Se-Young; Oh, Ju-Hee; Lee, ChuHee; Choi, Eun-Kyung; Jang, Se Jin; Suh, Young-Ah

2016-11-22

Mutation of p53 occasionally results in a gain of function, which promotes tumor growth. We asked whether destabilizing the gain-of-function protein would kill tumor cells. Downregulation of the gene reduced cell proliferation in p53-mutant cells, but not in p53-null cells, indicating that the former depended on the mutant protein for survival. Moreover, phenformin and 2-deoxyglucose suppressed cell growth and simultaneously destabilized mutant p53. The AMPK pathway, MAPK pathway, chaperone proteins and ubiquitination all contributed to this process. Interestingly, phenformin and 2-deoxyglucose also reduced tumor growth in syngeneic mice harboring the p53 mutation. Thus, destabilizing mutant p53 protein in order to kill cells exhibiting "oncogene addiction" could be a promising strategy for combatting p53 mutant tumors.

Biguanides sensitize leukemia cells to ABT-737-induced apoptosis by inhibiting mitochondrial electron transport

PubMed Central

Velez, Juliana; Pan, Rongqing; Lee, Jason T.C.; Enciso, Leonardo; Suarez, Marta; Duque, Jorge Eduardo; Jaramillo, Daniel; Lopez, Catalina; Morales, Ludis; Bornmann, William; Konopleva, Marina; Krystal, Gerald; Andreeff, Michael; Samudio, Ismael

2016-01-01

Metformin displays antileukemic effects partly due to activation of AMPK and subsequent inhibition of mTOR signaling. Nevertheless, Metformin also inhibits mitochondrial electron transport at complex I in an AMPK-independent manner, Here we report that Metformin and rotenone inhibit mitochondrial electron transport and increase triglyceride levels in leukemia cell lines, suggesting impairment of fatty acid oxidation (FAO). We also report that, like other FAO inhibitors, both agents and the related biguanide, Phenformin, increase sensitivity to apoptosis induction by the bcl-2 inhibitor ABT-737 supporting the notion that electron transport antagonizes activation of the intrinsic apoptosis pathway in leukemia cells. Both biguanides and rotenone induce superoxide generation in leukemia cells, indicating that oxidative damage may sensitize toABT-737 induced apoptosis. In addition, we demonstrate that Metformin sensitizes leukemia cells to the oligomerization of Bak, suggesting that the observed synergy with ABT-737 is mediated, at least in part, by enhanced outer mitochondrial membrane permeabilization. Notably, Phenformin was at least 10-fold more potent than Metformin in abrogating electron transport and increasing sensitivity to ABT-737, suggesting that this agent may be better suited for targeting hematological malignancies. Taken together, our results suggest that inhibition of mitochondrial metabolism by Metformin or Phenformin is associated with increased leukemia cell susceptibility to induction of intrinsic apoptosis, and provide a rationale for clinical studies exploring the efficacy of combining biguanides with the orally bioavailable derivative of ABT-737, Venetoclax. PMID:27283492

Biguanides sensitize leukemia cells to ABT-737-induced apoptosis by inhibiting mitochondrial electron transport.

PubMed

Velez, Juliana; Pan, Rongqing; Lee, Jason T C; Enciso, Leonardo; Suarez, Marta; Duque, Jorge Eduardo; Jaramillo, Daniel; Lopez, Catalina; Morales, Ludis; Bornmann, William; Konopleva, Marina; Krystal, Gerald; Andreeff, Michael; Samudio, Ismael

2016-08-09

Metformin displays antileukemic effects partly due to activation of AMPK and subsequent inhibition of mTOR signaling. Nevertheless, Metformin also inhibits mitochondrial electron transport at complex I in an AMPK-independent manner, Here we report that Metformin and rotenone inhibit mitochondrial electron transport and increase triglyceride levels in leukemia cell lines, suggesting impairment of fatty acid oxidation (FAO). We also report that, like other FAO inhibitors, both agents and the related biguanide, Phenformin, increase sensitivity to apoptosis induction by the bcl-2 inhibitor ABT-737 supporting the notion that electron transport antagonizes activation of the intrinsic apoptosis pathway in leukemia cells. Both biguanides and rotenone induce superoxide generation in leukemia cells, indicating that oxidative damage may sensitize toABT-737 induced apoptosis. In addition, we demonstrate that Metformin sensitizes leukemia cells to the oligomerization of Bak, suggesting that the observed synergy with ABT-737 is mediated, at least in part, by enhanced outer mitochondrial membrane permeabilization. Notably, Phenformin was at least 10-fold more potent than Metformin in abrogating electron transport and increasing sensitivity to ABT-737, suggesting that this agent may be better suited for targeting hematological malignancies. Taken together, our results suggest that inhibition of mitochondrial metabolism by Metformin or Phenformin is associated with increased leukemia cell susceptibility to induction of intrinsic apoptosis, and provide a rationale for clinical studies exploring the efficacy of combining biguanides with the orally bioavailable derivative of ABT-737, Venetoclax.

Effects of metformin, buformin, and phenformin on the post-initiation stage of chemically induced mammary carcinogenesis in the rat.

PubMed

Zhu, Zongjian; Jiang, Weiqin; Thompson, Matthew D; Echeverria, Dimas; McGinley, John N; Thompson, Henry J

2015-06-01

Metformin is a widely prescribed drug for the treatment of type II diabetes. Although epidemiologic data have provided a strong rationale for investigating the potential of this biguanide for use in cancer prevention and control, uncertainty exists whether metformin should be expected to have an impact in nondiabetic patients. Furthermore, little attention has been given to the possibility that other biguanides may have anticancer activity. In this study, the effects of clinically relevant doses of metformin (9.3 mmol/kg diet), buformin (7.6 mmol/kg diet), and phenformin (5.0 mmol/kg diet) were compared with rats fed control diet (AIN93-G) during the post-initiation stage of 1-methyl-1-nitrosourea-induced (50 mg/kg body weight) mammary carcinogenesis (n = 30/group). Plasma, liver, skeletal muscle, visceral fat, mammary gland, and mammary carcinoma concentrations of the biguanides were determined. In comparison with the control group, buformin decreased cancer incidence, multiplicity, and burden, whereas metformin and phenformin had no statistically significant effect on the carcinogenic process relative to the control group. Buformin did not alter fasting plasma glucose or insulin. Within mammary carcinomas, evidence was obtained that buformin treatment perturbed signaling pathways related to energy sensing. However, further investigation is needed to determine the relative contributions of host systemic and cell autonomous mechanisms to the anticancer activity of biguanides such as buformin. ©2015 American Association for Cancer Research.

Levels of lactic acid, normal level & its relation to food, glucose, cholesterol, raised blood urea and phenformin therapy.

PubMed

Patel, J C; Sawant, M S; Amin, B M

2000-01-01

1. The level of lactic acid was found to be 25 mg percent in 95 percent of 186 normal Indians. There was no difference due to sex and age. 2. Level of lactic acid was estimated in blood of normal persons and diabetics Type II patients to observe the effects of food and glucose. There was no change except the level of lactic acid was in higher but in normal range. 3. Hyperglycemia of over 300 mg raised the blood lactic acid in 25 percent of patients. 4. Lactic acid was not affected by hypercholesteremia but was raised in 60 percent of cases with raised blood urea. 5. Lactic acid was found to remain within normal limits in 48 type II diabetics treated with phenformin dose varying from 50 mg to 225 mg per day. The duration of treatment varied from one year to seven years.

Selective inhibition of deactivated mitochondrial complex I by biguanides.

PubMed

Matsuzaki, Satoshi; Humphries, Kenneth M

2015-03-24

Biguanides are widely used antihyperglycemic agents for diabetes mellitus and prediabetes treatment. Complex I is the rate-limiting step of the mitochondrial electron transport chain (ETC), a major source of mitochondrial free radical production, and a known target of biguanides. Complex I has two reversible conformational states, active and de-active. The deactivated state is promoted in the absence of substrates but is rapidly and fully reversed to the active state in the presence of NADH. The objective of this study was to determine the relative sensitivity of active/de-active complex I to biguanide-mediated inhibition and resulting superoxide radical (O₂(•⁻)) production. Using isolated rat heart mitochondria, we show that deactivation of complex I sensitizes it to metformin and phenformin (4- and 3-fold, respectively), but not to other known complex I inhibitors, such as rotenone. Mitochondrial O₂(•⁻) production by deactivated complex I was measured fluorescently by NADH-dependent 2-hydroxyethidium formation at alkaline pH to impede reactivation. Superoxide production was 260.4% higher than in active complex I at pH 9.4. However, phenformin treatment of de-active complex I decreased O₂(•⁻) production by 14.9%, while rotenone increased production by 42.9%. Mitochondria isolated from rat hearts subjected to cardiac ischemia, a condition known to induce complex I deactivation, were sensitized to phenformin-mediated complex I inhibition. This supports the idea that the effects of biguanides are likely to be influenced by the complex I state in vivo. These results demonstrate that the complex I active and de-active states are a determinant in biguanide-mediated inhibition.

Molecular features of biguanides required for targeting of mitochondrial respiratory complex I and activation of AMP-kinase.

PubMed

Bridges, Hannah R; Sirviö, Ville A; Agip, Ahmed-Noor A; Hirst, Judy

2016-08-09

The biguanides are a family of drugs with diverse clinical applications. Metformin, a widely used anti-hyperglycemic biguanide, suppresses mitochondrial respiration by inhibiting respiratory complex I. Phenformin, a related anti-hyperglycemic biguanide, also inhibits respiration, but proguanil, which is widely used for the prevention of malaria, does not. The molecular structures of phenformin and proguanil are closely related and both inhibit isolated complex I. Proguanil does not inhibit respiration in cells and mitochondria because it is unable to access complex I. The molecular features that determine which biguanides accumulate in mitochondria, enabling them to inhibit complex I in vivo, are not known. Here, a family of seven biguanides are used to reveal the molecular features that determine why phenformin enters mitochondria and inhibits respiration whereas proguanil does not. All seven biguanides inhibit isolated complex I, but only four of them inhibit respiration in cells and mitochondria. Direct conjugation of a phenyl group and bis-substitution of the biguanide moiety prevent uptake into mitochondria, irrespective of the compound hydrophobicity. This high selectivity suggests that biguanide uptake into mitochondria is protein mediated, and is not by passive diffusion. Only those biguanides that enter mitochondria and inhibit complex I activate AMP kinase, strengthening links between complex I and the downstream effects of biguanide treatments. Biguanides inhibit mitochondrial complex I, but specific molecular features control the uptake of substituted biguanides into mitochondria, so only some biguanides inhibit mitochondrial respiration in vivo. Biguanides with restricted intracellular access may be used to determine physiologically relevant targets of biguanide action, and for the rational design of substituted biguanides for diverse clinical applications.

Pharmacological AMP Kinase Activators Target the Nucleolar Organization and Control Cell Proliferation

PubMed Central

Kodiha, Mohamed; Salimi, Ali; Wang, Yi Meng; Stochaj, Ursula

2014-01-01

Aims Phenformin, resveratrol and AICAR stimulate the energy sensor 5′-AMP activated kinase (AMPK) and inhibit the first step of ribosome biogenesis, de novo RNA synthesis in nucleoli. Nucleolar activities are relevant to human health, because ribosome production is crucial to the development of diabetic complications. Although the function of nucleoli relies on their organization, the impact of AMPK activators on nucleolar structures is not known. Here, we addressed this question by examining four nucleolar proteins that are essential for ribosome biogenesis. Methods Kidney cells were selected as model system, because diabetic nephropathy is one of the complications associated with diabetes mellitus. To determine the impact of pharmacological agents on nucleoli, we focused on the subcellular and subnuclear distribution of B23/nucleophosmin, fibrillarin, nucleolin and RPA194. This was achieved by quantitative confocal microscopy at the single-cell level in combination with cell fractionation and quantitative Western blotting. Results AMPK activators induced the re-organization of nucleoli, which was accompanied by changes in cell proliferation. Among the compounds tested, phenformin and resveratrol had the most pronounced impact on nucleolar organization. For B23, fibrillarin, nucleolin and RPA194, both agents (i) altered the nucleocytoplasmic distribution and nucleolar association and (ii) reduced significantly the retention in the nucleus. (iii) Phenformin and resveratrol also increased significantly the total concentration of B23 and nucleolin. Conclusions AMPK activators have unique effects on the subcellular localization, nuclear retention and abundance of nucleolar proteins. We propose that the combination of these events inhibits de novo ribosomal RNA synthesis and modulates cell proliferation. Our studies identified nucleolin as a target that is especially sensitive to pharmacological AMPK activators. Because of its response to pharmacological agents, nucleolin represents a potential biomarker for the development of drugs that diminish diabetic renal hypertrophy. PMID:24498249

Pharmacological AMP-kinase activators have compartment-specific effects on cell physiology.

PubMed

Kodiha, Mohamed; Ho-Wo-Cheong, Dennis; Stochaj, Ursula

2011-12-01

5'-AMP-activated kinase (AMPK) regulates numerous biological events and is an essential target for the treatment of type 2 diabetes. The objectives of the present study were first to determine the compartment-specific effects of three established AMPK activators on Thr172 phosphorylation of the α-subunit, an indicator of AMPK activation. Second, we examined how cytoplasmic and nuclear processes are modulated by pharmacological AMPK activators. Specifically, the impact of phenformin, resveratrol, and 5-aminoimidazole-4-carboxamide riboside (AICAR) on Thr172 phosphorylation in the cytoplasm and nucleus was quantified by different methods. To analyze how these activators change cell physiology, we measured the inactivation of acetyl-CoA-carboxylase 1, a predominantly cytoplasmic enzyme that is crucial for lipid metabolism. As a criterion for activities associated with the nucleus, de novo RNA synthesis in nucleoli was quantified. Our studies demonstrate that pharmacological activators of AMPK can alter the balance between nuclear and cytoplasmic AMPK pools. Thus, phenformin and resveratrol caused a strong activation of AMPK in the cytoplasm, whereas the effect was less pronounced in nuclei. By contrast, AICAR elicited a comparable rise in Thr172 phosphorylation in both compartments. Notably, these activators differed drastically in their effects on physiological processes that are located in distinct subcellular compartments. All compounds led to a substantial inactivation of acetyl-CoA-carboxylase 1 in the cytoplasm, with only minor changes to the nuclear enzyme. In the nucleolus, transcription was strongly inhibited by resveratrol, while a moderate inhibition was observed with phenformin and AICAR. Taken together, the compartment-specific phosphorylation of AMPK and downstream events are determined by the activator.

Pharmacological AMP kinase activators target the nucleolar organization and control cell proliferation.

PubMed

Kodiha, Mohamed; Salimi, Ali; Wang, Yi Meng; Stochaj, Ursula

2014-01-01

Phenformin, resveratrol and AICAR stimulate the energy sensor 5'-AMP activated kinase (AMPK) and inhibit the first step of ribosome biogenesis, de novo RNA synthesis in nucleoli. Nucleolar activities are relevant to human health, because ribosome production is crucial to the development of diabetic complications. Although the function of nucleoli relies on their organization, the impact of AMPK activators on nucleolar structures is not known. Here, we addressed this question by examining four nucleolar proteins that are essential for ribosome biogenesis. Kidney cells were selected as model system, because diabetic nephropathy is one of the complications associated with diabetes mellitus. To determine the impact of pharmacological agents on nucleoli, we focused on the subcellular and subnuclear distribution of B23/nucleophosmin, fibrillarin, nucleolin and RPA194. This was achieved by quantitative confocal microscopy at the single-cell level in combination with cell fractionation and quantitative Western blotting. AMPK activators induced the re-organization of nucleoli, which was accompanied by changes in cell proliferation. Among the compounds tested, phenformin and resveratrol had the most pronounced impact on nucleolar organization. For B23, fibrillarin, nucleolin and RPA194, both agents (i) altered the nucleocytoplasmic distribution and nucleolar association and (ii) reduced significantly the retention in the nucleus. (iii) Phenformin and resveratrol also increased significantly the total concentration of B23 and nucleolin. AMPK activators have unique effects on the subcellular localization, nuclear retention and abundance of nucleolar proteins. We propose that the combination of these events inhibits de novo ribosomal RNA synthesis and modulates cell proliferation. Our studies identified nucleolin as a target that is especially sensitive to pharmacological AMPK activators. Because of its response to pharmacological agents, nucleolin represents a potential biomarker for the development of drugs that diminish diabetic renal hypertrophy.

Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes.

PubMed

Gormand, Amélie; Henriksson, Emma; Ström, Kristoffer; Jensen, Thomas Elbenhardt; Sakamoto, Kei; Göransson, Olga

2011-05-01

AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes remains elusive. Previous studies have identified LKB1 as a major AMPK kinase in muscle, liver, and other tissues. In certain cell types, Ca(2+) /calmodulin-dependent protein kinase kinase β (CaMKKβ) has been shown to activate AMPK in response to increases of intracellular Ca(2+) levels. Our aim was to investigate if LKB1 and/or CaMKK function as AMPK kinases in adipocytes. We used adipose tissue and isolated adipocytes from mice in which the expression of LKB1 was reduced to 10-20% of that of wild-type (LKB1 hypomorphic mice). We show that adipocytes from LKB1 hypomorphic mice display a 40% decrease in basal AMPK activity and a decrease of AMPK activity in the presence of the AMPK activator phenformin. We also demonstrate that stimulation of 3T3L1 adipocytes with intracellular [Ca(2+) ]-raising agents results in an activation of the AMPK pathway. The inhibition of CaMKK isoforms, particularly CaMKKβ, by the inhibitor STO-609 or by siRNAs, blocked Ca(2+) -, but not phenformin-, AICAR-, or forskolin-induced activation of AMPK, indicating that CaMKK activated AMPK in response to Ca(2+) . Collectively, we show that LKB1 is required to maintain normal AMPK-signaling in non-stimulated adipocytes and in the presence of phenformin. In addition, we demonstrate the existence of a Ca(2+) /CaMKK signaling pathway that can also regulate the activity of AMPK in adipocytes. Copyright © 2011 Wiley-Liss, Inc.

Selective Inhibition of Deactivated Mitochondrial Complex I by Biguanides †

PubMed Central

Matsuzaki, Satoshi; Humphries, Kenneth M.

2015-01-01

Biguanides are widely used antihyperglycemic agents for diabetes mellitus and prediabetes treatment. Complex I is the rate limiting step of the mitochondrial electron transport chain (ETC), a major source of mitochondrial free radical production, and a known target of biguanides. Complex I has two reversible conformational states, active and de-active. The deactivated state is promoted in the absence of substrates, but is rapidly and fully reversed to the active state in the presence of NADH. The objective of this study was to determine the relative sensitivity of active/de-active complex I to biguanide-mediated inhibition and resulting superoxide radical (O2•−) production. Using isolated rat heart mitochondria, we show that deactivation of complex I sensitizes it to metformin and phenformin (4- and 3-fold, respectively), but not to other known complex I inhibitors, such as rotenone. Mitochondrial O2•− production by deactivated complex I was measured fluorescently by the NADH-dependent 2-hydroxyethidium formation at alkaline pH to impede reactivation. Superoxide production was 260.4% higher than in active complex I at pH 9.4. However, phenformin treatment of de-active complex I decreased O2•− production by 14.9% while rotenone increased production by 42.9%. Mitochondria isolated from rat hearts subjected to cardiac ischemia, a condition known to induce complex I deactivation, were sensitized to phenformin:mediated complex I inhibition. This supports that the effects of biguanides are likely to be influenced by the complex I state in vivo. These results demonstrate that the complex I active/de-active states are a determinant in biguanide-mediated inhibition. PMID:25719498

Addition of 2-deoxyglucose enhances growth inhibition but reverses acidification in colon cancer cells treated with phenformin.

PubMed

Lea, Michael A; Chacko, Jerel; Bolikal, Sandhya; Hong, Ji Y; Chung, Ryan; Ortega, Andres; desbordes, Charles

2011-02-01

A report that effects of butyrate on some cells may be mediated by activation of AMP-activated protein kinase (AMPK) prompted this study which examines if other AMPK activators can induce differentiation and inhibit proliferation of colon cancer cells in a manner similar to butyrate. Using induction of alkaline phosphatase as a marker, it was observed that compound C, an AMPK inhibitor, is able to reduce the differentiating effect of butyrate on SW1116 and Caco-2 colon cancer cells. Metformin was observed to be less effective than butyrate in the induction of alkaline phosphatase but was more effective as a growth inhibitor. Phenformin was found to be a more potent growth inhibitor than metformin and both compounds cause acidification of the medium when incubated with colon cancer cells. Combined incubation of 2-deoxyglucose with either of the biguanides prevented the acidification of the medium but enhanced the growth inhibitory effects.

Metformin selectively targets redox control of complex I energy transduction.

PubMed

Cameron, Amy R; Logie, Lisa; Patel, Kashyap; Erhardt, Stefan; Bacon, Sandra; Middleton, Paul; Harthill, Jean; Forteath, Calum; Coats, Josh T; Kerr, Calum; Curry, Heather; Stewart, Derek; Sakamoto, Kei; Repiščák, Peter; Paterson, Martin J; Hassinen, Ilmo; McDougall, Gordon; Rena, Graham

2018-04-01

Many guanide-containing drugs are antihyperglycaemic but most exhibit toxicity, to the extent that only the biguanide metformin has enjoyed sustained clinical use. Here, we have isolated unique mitochondrial redox control properties of metformin that are likely to account for this difference. In primary hepatocytes and H4IIE hepatoma cells we found that antihyperglycaemic diguanides DG5-DG10 and the biguanide phenformin were up to 1000-fold more potent than metformin on cell signalling responses, gluconeogenic promoter expression and hepatocyte glucose production. Each drug inhibited cellular oxygen consumption similarly but there were marked differences in other respects. All diguanides and phenformin but not metformin inhibited NADH oxidation in submitochondrial particles, indicative of complex I inhibition, which also corresponded closely with dehydrogenase activity in living cells measured by WST-1. Consistent with these findings, in isolated mitochondria, DG8 but not metformin caused the NADH/NAD + couple to become more reduced over time and mitochondrial deterioration ensued, suggesting direct inhibition of complex I and mitochondrial toxicity of DG8. In contrast, metformin exerted a selective oxidation of the mitochondrial NADH/NAD + couple, without triggering mitochondrial deterioration. Together, our results suggest that metformin suppresses energy transduction by selectively inducing a state in complex I where redox and proton transfer domains are no longer efficiently coupled. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

5'-AMP-activated protein kinase increases glucose uptake independent of GLUT4 translocation in cardiac myocytes.

PubMed

Lee, Christopher T; Ussher, John R; Mohammad, Askar; Lam, Anna; Lopaschuk, Gary D

2014-04-01

Glucose uptake and glycolysis are increased in the heart during ischemia, and this metabolic alteration constitutes an important contributing factor towards ischemic injury. Therefore, it is important to understand glucose uptake regulation in the ischemic heart. There are primarily 2 glucose transporters controlling glucose uptake into cardiac myocytes: GLUT1 and GLUT4. In the non-ischemic heart, insulin stimulates GLUT4 translocation to the sarcolemmal membrane, while both GLUT1 and GLUT4 translocation can occur following AMPK stimulation. Using a newly developed technique involving [(3)H]2-deoxyglucose, we measured glucose uptake in H9c2 ventricular myoblasts, and demonstrated that while insulin has no detectable effect on glucose uptake, phenformin-induced AMPK activation increases glucose uptake 2.5-fold. Furthermore, insulin treatment produced no discernible effect on either Akt serine 473 phosphorylation or AMPKα threonine 172 phosphorylation, while treatment with phenformin results in an increase in AMPKα threonine 172 phosphorylation, and a decrease in Akt serine 473 phosphorylation. Visualization of a dsRed-GLUT4 fusion construct in H9c2 cells by laser confocal microscopy showed that unlike insulin, AMPK activation did not redistribute GLUT4 to the sarcolemmal membrane, suggesting that AMPK may regulate glucose uptake via another glucose transporter. These studies suggest that AMPK is a major regulator of glucose uptake in cardiac myocytes.

Disruption of BASIGIN decreases lactic acid export and sensitizes non-small cell lung cancer to biguanides independently of the LKB1 status.

PubMed

Granja, Sara; Marchiq, Ibtissam; Le Floch, Renaud; Moura, Conceição Souto; Baltazar, Fátima; Pouysségur, Jacques

2015-03-30

Most cancers rely on aerobic glycolysis to generate energy and metabolic intermediates. To maintain a high glycolytic rate, cells must efficiently export lactic acid through the proton-coupled monocarboxylate transporters (MCT1/4). These transporters require a chaperone, CD147/BASIGIN (BSG) for trafficking to the plasma membrane and function.To validate the key role of these transporters in lung cancer, we first analysed the expression of MCT1/4 and BSG in 50 non-small lung cancer (NSCLC) cases. These proteins were specifically upregulated in tumour tissues. We then disrupted BSG in three NSCLC cell lines (A549, H1975 and H292) via 'Zinc-Finger Nucleases'. The three homozygous BSG-/- cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells. Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG-/- cell lines. Both wild-type and BSG-null cells were extremely sensitive to the mitochondria inhibitor metformin/phenformin in normoxia. However, only BSG-null cells, independently of their LKB1 status, remained sensitive to biguanides in hypoxia in vitro and tumour growth in nude mice. Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin.

The biguanides metformin and phenformin inhibit angiogenesis, local and metastatic growth of breast cancer by targeting both neoplastic and microenvironment cells.

PubMed

Orecchioni, Stefania; Reggiani, Francesca; Talarico, Giovanna; Mancuso, Patrizia; Calleri, Angelica; Gregato, Giuliana; Labanca, Valentina; Noonan, Douglas M; Dallaglio, Katiuscia; Albini, Adriana; Bertolini, Francesco

2015-03-15

The human white adipose tissue (WAT) contains progenitors with cooperative roles in breast cancer (BC) angiogenesis, local and metastatic progression. The biguanide Metformin (Met), commonly used for Type 2 diabetes, might have activity against BC and was found to inhibit angiogenesis in vivo. We studied Met and another biguanide, phenformin (Phe), in vitro and in vivo in BC models. In vitro, biguanides activated AMPK, inhibited Complex 1 of the respiratory chain and induced apoptosis of BC and WAT endothelial cells. In coculture, biguanides inhibited the production of several angiogenic proteins. In vivo, biguanides inhibited local and metastatic growth of triple negative and HER2+ BC in immune-competent and immune-deficient mice orthotopically injected with BC. Biguanides inhibited local and metastatic BC growth in a genetically engineered murine model model of HER2+ BC. In vivo, biguanides increased pimonidazole binding (but not HIF-1 expression) of WAT progenitors, reduced tumor microvessel density and altered the vascular pericyte/endothelial cell ratio, so that cancer vessels displayed a dysplastic phenotype. Phe was significantly more active than Met both in vitro and in vivo. Considering their safety profile, biguanides deserve to be further investigated for BC prevention in high-risk subjects, in combination with chemo and/or targeted therapy and/or as post-therapy consolidation or maintenance therapy for the prevention of BC recurrence. © 2014 UICC.

Disruption of BASIGIN decreases lactic acid export and sensitizes non-small cell lung cancer to biguanides independently of the LKB1 status

PubMed Central

Floch, Renaud Le; Moura, Conceição Souto

2015-01-01

Most cancers rely on aerobic glycolysis to generate energy and metabolic intermediates. To maintain a high glycolytic rate, cells must efficiently export lactic acid through the proton-coupled monocarboxylate transporters (MCT1/4). These transporters require a chaperone, CD147/BASIGIN (BSG) for trafficking to the plasma membrane and function. To validate the key role of these transporters in lung cancer, we first analysed the expression of MCT1/4 and BSG in 50 non-small lung cancer (NSCLC) cases. These proteins were specifically upregulated in tumour tissues. We then disrupted BSG in three NSCLC cell lines (A549, H1975 and H292) via ‘Zinc-Finger Nucleases’. The three homozygous BSG−/− cell lines displayed a low MCT activity (10- to 5-fold reduction, for MCT1 and MCT4, respectively) compared to wild-type cells. Consequently, the rate of glycolysis, compared to the wild-type counterpart, was reduced by 2.0- to 3.5-fold, whereas the rate of respiration was stimulated in BSG−/− cell lines. Both wild-type and BSG-null cells were extremely sensitive to the mitochondria inhibitor metformin/phenformin in normoxia. However, only BSG-null cells, independently of their LKB1 status, remained sensitive to biguanides in hypoxia in vitro and tumour growth in nude mice. Our results demonstrate that inhibiting glycolysis by targeting lactic acid export sensitizes NSCLC to phenformin. PMID:25894929

Oxygen consumption is depressed in patients with lactic acidosis due to biguanide intoxication.

PubMed

Protti, Alessandro; Russo, Riccarda; Tagliabue, Paola; Vecchio, Sarah; Singer, Mervyn; Rudiger, Alain; Foti, Giuseppe; Rossi, Anna; Mistraletti, Giovanni; Gattinoni, Luciano

2010-01-01

Lactic acidosis can develop during biguanide (metformin and phenformin) intoxication, possibly as a consequence of mitochondrial dysfunction. To verify this hypothesis, we investigated whether body oxygen consumption (VO2), that primarily depends on mitochondrial respiration, is depressed in patients with biguanide intoxication. Multicentre retrospective analysis of data collected from 24 patients with lactic acidosis (pH 6.93 +/- 0.20; lactate 18 +/- 6 mM at hospital admission) due to metformin (n = 23) or phenformin (n = 1) intoxication. In 11 patients, VO2 was computed as the product of simultaneously recorded arterio-venous difference in O2 content [C(a-v)O2] and cardiac index (CI). In 13 additional cases, C(a-v)O2, but not CI, was available. On day 1, VO2 was markedly depressed (67 +/- 28 ml/min/m2) despite a normal CI (3.4 +/- 1.2 L/min/m2). C(a-v)O2 was abnormally low in both patients either with (2.0 +/- 1.0 ml O2/100 ml) or without (2.5 +/- 1.1 ml O2/100 ml) CI (and VO2) monitoring. Clearance of the accumulated drug was associated with the resolution of lactic acidosis and a parallel increase in VO2 (P < 0.001) and C(a-v)O2 (P < 0.05). Plasma lactate and VO2 were inversely correlated (R2 0.43; P < 0.001, n = 32). VO2 is abnormally low in patients with lactic acidosis due to biguanide intoxication. This finding is in line with the hypothesis of inhibited mitochondrial respiration and consequent hyperlactatemia.

AMPK activators inhibit the proliferation of human melanomas bearing the activated MAPK pathway.

PubMed

Petti, Carlotta; Vegetti, Claudia; Molla, Alessandra; Bersani, Ilaria; Cleris, Loredana; Mustard, Kirsty J; Formelli, Franca; Hardie, Grahame D; Sensi, Marialuisa; Anichini, Andrea

2012-10-01

Raf/MEK/ERK signaling can inhibit the liver kinase B1-AMP-activated protein kinase (LKB1-AMPK) pathway, thus rendering melanoma cells resistant to energy stress conditions. We evaluated whether pharmacological reactivation of the AMPK function could exert antitumor effects on melanoma cells bearing this pathway constitutively active because of a mutation in NRAS or BRAF genes. Nine melanoma cell lines were treated with the AMPK activators 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and phenformin. The activation of AMPK enzymatic activity, phosphorylation of AMPK and acetyl-CoA carboxylase kinase, in-vitro proliferation, cell cycle, and in-vivo growth of xenografts in nude mice were evaluated. AICAR and phenformin promoted phosphorylation and enzymatic activity of AMPK, as well as phosphorylation of the AMPK downstream target acetyl-CoA carboxylase. Drug treatment of either BRAF-mutant or NRAS-mutant melanomas, at doses not inducing cell death, was accompanied by a dose-dependent decrease in melanoma cell proliferation because of cell cycle arrest in either the G0/G1 or the S phase, associated with an increased expression of the p21 cell cycle inhibitor. Melanomas isolated from subcutaneously implanted mice, 25 days from treatment with AICAR, showed increased staining of the senescence-associated marker β-galactosidase, high p21 expression, and evidence of necrosis. Altogether, these results indicate that pharmacological activators of AMPK-dependent pathways inhibit the cell growth of melanoma cells with active Raf/MEK/ERK signaling and provide a rationale for further investigation on their use in combination therapies.

Determination of phenformin hydrochloride employing a sensitive fluorescent probe

NASA Astrophysics Data System (ADS)

Shi, Lin; Xie, Jian-Hong; Du, Li-Ming; Chang, Yin-xia; Wu, Hao

2016-06-01

A complexation of non-fluorescent phenformin hydrochloride (PFH) with cucurbit [7]uril (CB [7]) in aqueous solution was investigated using the fluorescent probe of palmatine (PAL) coupled with CB [7]. The fluorescent probe of CB [7]-PAL exhibited strong fluorescence in aqueous solution, which was quenched gradually with the increase of PFH. This effect is observed because when PFH was added to the host-guest system of CB [7]-PAL, PFH and PAL competed to occupy the CB [7] cavity. Portions of the PAL molecule were expelled from the CB [7] cavity owing to the introduction of PFH. Based on the significant quenching of the supramolecular complex fluorescence intensity, a fluorescence method of high sensitivity and selectivity was developed to determine PFH with good precision and accuracy for the first time. The linear range of the method was 0.005-1.9 μg mL- 1 with a detection limit of 0.003 μg mL- 1. In this work, association constants (K) of PFH with CB [7] were also determined. KCB [7]-PFH = (2.52 ± 0.05) × 105 L mol- 1. The ability of PFH to bind with CB [7] is stronger than that of PAL. The results of a density functional theory calculation authenticated that the moiety of PFH was embedded in the hydrophobic cavity of CB [7] tightly, and the nitrogen atom is located in the vicinity of a carbonyl-laced portal in the energy-minimized structure. The molecular modelling of the interaction between PFH and CB [7] was also confirmed by 1H NMR spectra (Bruker 600 MHz).

Salting-out assisted liquid-liquid extraction coupled with hydrophilic interaction chromatography for the determination of biguanides in biological and environmental samples.

PubMed

Alshishani, Anas; Salhimi, Salizawati Muhamad; Saad, Bahruddin

2018-01-15

A new salting-out assisted liquid-liquid extraction (SALLE) sample preparation method for the determination of the polar anti-diabetic biguanide drugs (metformin, buformin and phenformin) in blood plasma, urine and lake water samples were developed. The SALLE was performed by mixing samples (plasma (0.2mL), urine or lake water (1.0mL)) with acetonitrile (0.4mL for plasma, 0.5mL for urine or lake water), sodium hydroxide powder was then added for the phase separation. The effects of type of salting-out reagent, type of extraction solvent, volumes of acetonitrile and sample, amount of sodium hydroxide, vortexing and centrifugation times on the extraction efficiency were investigated. The upper layer, containing the biguanides, was directly injected into a HPLC unit using ZIC-HILIC column (150mm×2.1mm×3.5μm) and was detected at 236nm. The method was validated and calibration curves were linear with r 2 >0.99 over the range of 20-2000μgL -1 for plasma and 5-2000μgL -1 for urine and lake water samples. The limits of detection were in the range (3.8-5.6)μgL -1 , (0.8-1.5)μgL -1 and (0.3-0.8)μgL -1 for plasma, urine and lake water, respectively. The accuracies in the three matrices were within 87.3-103%, 87.4-109%, 82.2-109% of the nominal concentration for metformin, buformin and phenformin, respectively. The relative standard deviation for inter- and intra -day precision were in the range of 1.0-17% for all analytes in the three matrices. Copyright © 2017 Elsevier B.V. All rights reserved.

Metformin and Its Sulfenamide Prodrugs Inhibit Human Cholinesterase Activity.

PubMed

Markowicz-Piasecka, Magdalena; Sikora, Joanna; Mateusiak, Łukasz; Mikiciuk-Olasik, Elżbieta; Huttunen, Kristiina M

2017-01-01

The results of epidemiological and pathophysiological studies suggest that type 2 diabetes mellitus (T2DM) may predispose to Alzheimer's disease (AD). The two conditions present similar glucose levels, insulin resistance, and biochemical etiologies such as inflammation and oxidative stress. The diabetic state also contributes to increased acetylcholinesterase (AChE) activity, which is one of the factors leading to neurodegeneration in AD. The aim of this study was to assess in vitro the effects of metformin, phenformin, and metformin sulfenamide prodrugs on the activity of human AChE and butyrylcholinesterase (BuChE) and establish the type of inhibition. Metformin inhibited 50% of the AChE activity at micromolar concentrations (2.35  μ mol/mL, mixed type of inhibition) and seemed to be selective towards AChE since it presented low anti-BuChE activity. The tested metformin prodrugs inhibited cholinesterases (ChE) at nanomolar range and thus were more active than metformin or phenformin. The cyclohexyl sulfenamide prodrug demonstrated the highest activity towards both AChE (IC 50  = 890 nmol/mL, noncompetitive inhibition) and BuChE (IC 50  = 28 nmol/mL, mixed type inhibition), while the octyl sulfenamide prodrug did not present anti-AChE activity, but exhibited mixed inhibition towards BuChE (IC 50  = 184 nmol/mL). Therefore, these two bulkier prodrugs were concluded to be the most selective compounds for BuChE over AChE. In conclusion, it was demonstrated that biguanides present a novel class of inhibitors for AChE and BuChE and encourages further studies of these compounds for developing both selective and nonselective inhibitors of ChEs in the future.

Determination of phenformin hydrochloride employing a sensitive fluorescent probe.

PubMed

Shi, Lin; Xie, Jian-Hong; Du, Li-Ming; Chang, Yin-xia; Wu, Hao

2016-06-05

A complexation of non-fluorescent phenformin hydrochloride (PFH) with cucurbit [7]uril (CB [7]) in aqueous solution was investigated using the fluorescent probe of palmatine (PAL) coupled with CB [7]. The fluorescent probe of CB [7]-PAL exhibited strong fluorescence in aqueous solution, which was quenched gradually with the increase of PFH. This effect is observed because when PFH was added to the host-guest system of CB [7]-PAL, PFH and PAL competed to occupy the CB [7] cavity. Portions of the PAL molecule were expelled from the CB [7] cavity owing to the introduction of PFH. Based on the significant quenching of the supramolecular complex fluorescence intensity, a fluorescence method of high sensitivity and selectivity was developed to determine PFH with good precision and accuracy for the first time. The linear range of the method was 0.005-1.9 μg mL(-1) with a detection limit of 0.003 μg mL(-1). In this work, association constants (K) of PFH with CB [7] were also determined. KCB [7]-PFH=(2.52±0.05)×10(5) L mol(-1). The ability of PFH to bind with CB [7] is stronger than that of PAL. The results of a density functional theory calculation authenticated that the moiety of PFH was embedded in the hydrophobic cavity of CB [7] tightly, and the nitrogen atom is located in the vicinity of a carbonyl-laced portal in the energy-minimized structure. The molecular modelling of the interaction between PFH and CB [7] was also confirmed by (1)H NMR spectra (Bruker 600 MHz). Copyright © 2016 Elsevier B.V. All rights reserved.

Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dykens, James A.; Jamieson, Joseph; Marroquin, Lisa

2008-12-01

As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanidemore » toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction.« less

Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro.

PubMed

Dykens, James A; Jamieson, Joseph; Marroquin, Lisa; Nadanaciva, Sashi; Billis, Puja A; Will, Yvonne

2008-12-01

As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanide toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction.

Defining the Contribution of AMP-activated Protein Kinase (AMPK) and Protein Kinase C (PKC) in Regulation of Glucose Uptake by Metformin in Skeletal Muscle Cells*

PubMed Central

Turban, Sophie; Stretton, Clare; Drouin, Olivier; Green, Charlotte J.; Watson, Maria L.; Gray, Alexander; Ross, Fiona; Lantier, Louise; Viollet, Benoit; Hardie, D. Grahame; Marette, Andre; Hundal, Harinder S.

2012-01-01

The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GU in L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulated GU was sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interfering AMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide-stimulated GU by ∼20%. Consistent with this, analysis of GU in muscle cells from α1−/−/α2−/− AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ∼35% compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90%) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs. PMID:22511782

LKB1, an upstream AMPK kinase, regulates glucose and lipid metabolism in cultured liver and muscle cells.

PubMed

Imai, Kenta; Inukai, Kouichi; Ikegami, Yuichi; Awata, Takuya; Katayama, Shigehiro

2006-12-22

LKB1 is a 50 kDa serine/threonine kinase that phosphorylates and activates the catalytic subunit of AMPK at its T-loop residue Thr 172. We prepared adenoviruses expressing the constitutive active (wild-type) form (CA) or dominant negative (kinase inactive, D194A mutant) form (DN) of LKB1 and overexpressed these proteins in cultured myotubes (C2C12 cells) and rat hepatoma cells (FAO cells). When analyzed by immunoblotting with the antibody against Thr172-phosphorylated AMPK, the phosphorylation of AMPK was increased (2.5-fold) and decreased (0.4-fold) in cells expressing CA and DN LKB1, respectively, as compared with Lac-Z expressing control cells. Immunoprecipitation experiments, using isoform-specific antibody, revealed these alterations of AMPK phosphorylation to be attributable to altered phosphorylation of AMPK alpha2, but not alpha1 catalytic subunits, strongly suggesting the alpha2 catalytic subunit to be the major substrate for LKB1 in mammalian cells. In addition, adiponectin or AICAR-stimulated AMPK phosphorylation was inhibited by overexpression of DN LKB1, while phenformin-stimulated phosphorylation was unaffected. These results may explain the difference in AMPK activation mechanisms between AMP and phenformin, and also indicate that AMPK phosphorylation by LKB1 is involved in AMP-stimulated AMPK activation. As a downstream target for AMPK, AICAR-induced glucose uptake and ACCbeta phosphorylation were found to be significantly reduced in DN LKB1 expressing C2C12 cells. The expression of key enzymes for gluconeogenesis, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, was also dependent on LKB1 activities in FAO cells. These results demonstrate that LKB1 is a crucial regulator of AMPK activation in muscle and liver cells and, therefore, that LKB1 activity is potentially of importance to our understanding of glucose and lipid metabolism.

Defining the contribution of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) in regulation of glucose uptake by metformin in skeletal muscle cells.

PubMed

Turban, Sophie; Stretton, Clare; Drouin, Olivier; Green, Charlotte J; Watson, Maria L; Gray, Alexander; Ross, Fiona; Lantier, Louise; Viollet, Benoit; Hardie, D Grahame; Marette, Andre; Hundal, Harinder S

2012-06-08

The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GU in L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulated GU was sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interfering AMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide-stimulated GU by ∼20%. Consistent with this, analysis of GU in muscle cells from α1(-/-)/α2(-/-) AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ∼35% compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90%) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs.

Effects of phentermine and phenformin on biomarkers of aging in rats.

PubMed

Anisimov, Vladimir N; Ukraintseva, Svetlana V; Anikin, Ivan V; Popovich, Irina G; Zabezhinski, Mark A; Bertsein, Lev M; Arutjunyan, Alexander V; Ingram, Donald K; Lane, Mark A; Roth, George S

2005-01-01

Caloric restriction (CR) is the only treatment known to substantially prolong both average and maximal life span in experimental animals. Interventions that mimic certain effects of CR could be potential anti-aging treatments in humans. Drugs which reduce appetite (anorexiants) represent one class of candidate treatments. Agents that reduce the glucose utilization by the organism could also represent another class of candidate CR mimetics. In our study, we addressed the following questions: (1) Does treatment with an anorexiant reduce caloric intake and body weight of experimental animals comparable to that caused by CR? (2) Does treatment with an antidiabetic agent influence caloric intake and body weight? (3) Does treatment with any of these drugs affect metabolic parameters of an organism in the way similar to that observed with CR? One hundred and twenty 6-month-old female Wistar-derived LIO rats were randomly subdivided into four groups and exposed to: (1) ad libitum feeding with placebo (controls); (2) the antidiabetic drug phenformin (2 mg/kg); (3) the anorectic drug phentermine (1 mg/kg), and (4) the same amount of food as the group with the least food intake during the previous week (pair-fed controls). Food and water intake, body weight, and rectal temperature were measured weekly during 16 weeks. At the end of the 16th week of the experiment, serum levels of glucose, total beta-lipoprotein and pre-beta-lipoprotein fractions, cholesterol, triglycerides, insulin, total triiodothyronine, and free thyroxine were estimated. The contents of diene conjugates and Schiff's bases, total antioxidant activity, the activities of Cu/Zn superoxide dismutase, glutathione S-transferase, and glutathione peroxidase, and the generation of reactive oxygen species (ROS) were studied in brain and liver homogenates and in the serum. The controls exposed to pair feeding had a significantly reduced food consumption (about 20%) as compared with the ad libitum fed controls and thus exhibited a moderate CR. Treatment with phentermine reduced the caloric intake by about 12% as compared with the ad libitum fed controls. Body weight and water intake in this group were only slightly decreased (by about 2 and 5%, respectively) as compared with the controls. The mean rectal temperature in the phentermine group (38 degrees C) was significantly higher than in the ad libitum fed (37.8 degrees C) and pair-fed (37.6 degrees C) controls. Treatment with phentermine also resulted in significantly reduced ROS levels in all tissues studied, while the highest ROS production was found in ad libitum (blood serum) and pair-fed (brain) controls. Treatment with phenformin did not significantly influence food and water consumption, body weight, and temperature when compared with the ad libitum fed controls. Rats treated with this antidiabetic drug showed intermediate values of ROS generation. Differences among the groups in total antioxidant activity were not obvious. Treatment with phentermine reduces caloric intake slightly less than is commonly observed in CR studies. CR due to forcibly reduced feeding and CR due to substance-suppressed appetite appear to act through different metabolic mechanisms and thus may affect aging and longevity in different ways. Copyright (c) 2005 S. Karger AG, Basel

Modern approach to metabolic rehabilitation of cancer patients: biguanides (phenformin and metformin) and beyond.

PubMed

Berstein, Lev M

2010-08-01

Comparing the experience accumulated for more than 40 years in the Laboratory of Endocrinology of Petrov Institute of Oncology (St Petersburg, Russia) with similar approaches practiced elsewhere, evidence supports the reasonability of metabolic rehabilitation of patients suffering from breast cancer or other hormone-dependent malignancies. The primary objective of such approaches is to improve treatment results by ameliorating hormonal-metabolic disturbances, including excess body fat, glucose intolerance, insulin resistance and manifestations of endocrine-genotoxic switchings, and modify tissue and cellular targets or mechanisms related or nondirectly related to the aforementioned disturbances. The relevant measures may be categorized as pharmacological (antidiabetic biguanides exemplified with metformin being most popular but not exclusive) and nonpharmacological (rational nutrition, moderate physical activity and so forth) and used separately or in different combinations.

Effect of hypolipidemic drugs on basal and stimulated adenylate cyclase activity in tumor cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bershtein, L.M.; Kovaleva, I.G.; Rozenberg, O.A.

1986-02-01

This paper studies adenylate cyclase acticvity in Ehrlich's ascites carcinoma (EAC) cells during administration of drugs with a hypolipidemic action. Seven to eight days before they were killed, male mice ingested the antidiabetic biguanide phenformin, and the phospholipid-containing preparation Essentiale in drinking water. The cAMP formed was isolated by chromatography on Silufol plates after incubation of the enzyme preparation with tritium-ATP, or was determined by the competitive binding method with protein. It is shown that despite the possible differences in the concrete mechanism of action of the hypolipidemic agents chosen for study on the cyclase system, the use of suchmore » agents, offers definite prospects for oriented modification of the hormone sensitivity of tumor cells.« less

Extreme Basicity of Biguanide Drugs in Aqueous Solutions: Ion Transfer Voltammetry and DFT Calculations.

PubMed

Langmaier, Jan; Pižl, Martin; Samec, Zdeněk; Záliš, Stanislav

2016-09-22

Ion transfer voltammetry is used to estimate the acid dissociation constants Ka1 and Ka2 of the mono- and diprotonated forms of the biguanide drugs metformin (MF), phenformin (PF), and 1-phenylbiguanide (PB) in an aqueous solution. Measurements gave the pKa1 values for MFH(+), PFH(+), and PBH(+) characterizing the basicity of MF, PF, and PB, which are significantly higher than those reported in the literature. As a result, the monoprotonated forms of these biguanides should prevail in a considerably broader range of pH 1-15 (MFH(+), PFH(+)) and 2-13 (PBH(+)). DFT calculations with solvent correction were performed for possible tautomeric forms of neutral, monoprotonated, and diprotonated species. Extreme basicity of all drugs is confirmed by DFT calculations of pKa1 for the most stable tautomers of the neutral and protonated forms with explicit water molecules in the first solvation sphere included.

A novel, de novo mutation in the PRKAG2 gene: infantile-onset phenotype and the signaling pathway involved.

PubMed

Xu, Yanchun; Gray, A; Hardie, D Grahame; Uzun, Alper; Shaw, Sunil; Padbury, James; Phornphutkul, Chanika; Tseng, Yi-Tang

2017-08-01

PRKAG2 encodes the γ 2 -subunit isoform of 5'-AMP-activated protein kinase (AMPK), a heterotrimeric enzyme with major roles in the regulation of energy metabolism in response to cellular stress. Mutations in PRKAG2 have been implicated in a unique hypertrophic cardiomyopathy (HCM) characterized by cardiac glycogen overload, ventricular preexcitation, and hypertrophy. We identified a novel, de novo PRKAG2 mutation (K475E) in a neonate with prenatal onset of HCM. We aimed to investigate the cellular impact, signaling pathways involved, and therapeutic options for K475E mutation using cells stably expressing human wild-type (WT) or the K475E mutant. In human embryonic kidney-293 cells, the K475E mutation induced a marked increase in the basal phosphorylation of T172 and AMPK activity, reduced sensitivity to AMP in allosteric activation, and a loss of response to phenformin. In H9c2 cardiomyocytes, the K475E mutation induced inhibition of AMPK and reduced the response to phenformin and increases in the phosphorylation of p70S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1). Primary fibroblasts from the patient with the K475E mutation also showed marked increases in the phosphorylation of p70S6K and 4E-BP1 compared with those from age-matched, nondiseased controls. Moreover, overexpression of K475E induced hypertrophy in H9c2 cells, which was effectively reversed by treatment with rapamycin. Taken together, we have identified a novel, de novo infantile-onset PRKAG2 mutation causing HCM. Our study suggests the K475E mutation induces alteration in basal AMPK activity and results in a hypertrophy phenotype involving the mechanistic target of rapamycin signaling pathway, which can be reversed with rapamycin. NEW & NOTEWORTHY We identified a novel, de novo PRKAG2 mutation (K475E) in the cystathionine β-synthase 3 repeat, a region critical for AMP binding but with no previous reported mutation. Our data suggest the mutation affects AMP-activated protein kinase activity, activates cell growth pathways, and results in cardiac hypertrophy, which can be reversed with rapamycin. Copyright © 2017 the American Physiological Society.

An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis

PubMed Central

Birsoy, Kıvanç; Wang, Tim; Chen, Walter; Freinkman, Elizaveta; Abu-Remaileh, Monther; Sabatini, David M.

2015-01-01

Summary The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation. PMID:26232224

An Essential Role of the Mitochondrial Electron Transport Chain in Cell Proliferation Is to Enable Aspartate Synthesis.

PubMed

Birsoy, Kıvanç; Wang, Tim; Chen, Walter W; Freinkman, Elizaveta; Abu-Remaileh, Monther; Sabatini, David M

2015-07-30

The mitochondrial electron transport chain (ETC) enables many metabolic processes, but why its inhibition suppresses cell proliferation is unclear. It is also not well understood why pyruvate supplementation allows cells lacking ETC function to proliferate. We used a CRISPR-based genetic screen to identify genes whose loss sensitizes human cells to phenformin, a complex I inhibitor. The screen yielded GOT1, the cytosolic aspartate aminotransferase, loss of which kills cells upon ETC inhibition. GOT1 normally consumes aspartate to transfer electrons into mitochondria, but, upon ETC inhibition, it reverses to generate aspartate in the cytosol, which partially compensates for the loss of mitochondrial aspartate synthesis. Pyruvate stimulates aspartate synthesis in a GOT1-dependent fashion, which is required for pyruvate to rescue proliferation of cells with ETC dysfunction. Aspartate supplementation or overexpression of an aspartate transporter allows cells without ETC activity to proliferate. Thus, enabling aspartate synthesis is an essential role of the ETC in cell proliferation. Copyright © 2015 Elsevier Inc. All rights reserved.

Serine deprivation enhances antineoplastic activity of biguanides.

PubMed

Gravel, Simon-Pierre; Hulea, Laura; Toban, Nader; Birman, Elena; Blouin, Marie-José; Zakikhani, Mahvash; Zhao, Yunhua; Topisirovic, Ivan; St-Pierre, Julie; Pollak, Michael

2014-12-15

Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits antineoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here, we show that serine withdrawal increases the antineoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation modified the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, a serine-deficient diet reduced serine levels in tumors and significantly enhanced the tumor growth-inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as antineoplastic agents that target cancer cell energy metabolism. ©2014 American Association for Cancer Research.

S6 Kinase Inhibits Intrinsic Axon Regeneration Capacity via AMP Kinase in Caenorhabditis elegans

PubMed Central

Hubert, Thomas; Wu, Zilu; Chisholm, Andrew D.

2014-01-01

The ability of axons to regrow after injury is determined by the complex interplay of intrinsic growth programs and external cues. In Caenorhabditis elegans mechanosensory neuron, axons exhibit robust regenerative regrowth following laser axotomy. By surveying conserved metabolic signaling pathways, we have identified the ribosomal S6 kinase RSKS-1 as a new cell-autonomous inhibitor of axon regeneration. RSKS-1 is not required for axonal development but inhibits axon regrowth after injury in multiple neuron types. Loss of function in rsks-1 results in more rapid growth cone formation after injury and accelerates subsequent axon extension. The enhanced regrowth of rsks-1 mutants is partly dependent on the DLK-1 MAPK cascade. An essential output of RSKS-1 in axon regrowth is the metabolic sensor AMP kinase, AAK-2. We further show that the antidiabetic drug phenformin, which activates AMP kinase, can promote axon regrowth. Our data reveal a new function for an S6 kinase acting through an AMP kinase in regenerative growth of injured axons. PMID:24431434

Codelivery of dual drugs from polymeric micelles for simultaneous targeting of both cancer cells and cancer stem cells.

PubMed

Krishnamurthy, Sangeetha; Ng, Victor W L; Gao, Shujun; Tan, Min-Han; Hedrick, James L; Yang, Yi Yan

2015-01-01

Phenformin-loaded micelles (Phen M) were used in combination with gemcitabine-loaded micelles (Gem M) to study their combined effect against H460 human lung cancer cells and cancer stem cells (CSCs) in vitro and in vivo. Gem M and Phen M were prepared via self-assembly of a mixture of a diblock copolymer of PEG and urea-functionalized polycarbonate (PEG-PUC) and a diblock copolymer of PEG and acid-functionalized polycarbonate (PEG-PAC) through hydrogen bonding and ionic interactions. Gem M and Phen M were characterized and tested for efficacy both in vitro and in vivo against cancer cells and CSCs. The combination of Gem M/Phen M exhibited higher cytotoxicity against CSCs and non-CSCs than Gem M and Phen M alone, and showed significant cell cycle growth arrest in vitro. The combination therapy had superior tumor suppression and apoptosis in vivo without inducing toxicity to liver and kidney. The combination of Gem M and Phen M may be potentially used in lung cancer therapy.

Activation of AMPK reduces the co-transporter activity of NKCC1.

PubMed

Fraser, Scott A; Davies, Matthew; Katerelos, Marina; Gleich, Kurt; Choy, Suet-Wan; Steel, Rohan; Galic, Sandra; Mount, Peter F; Kemp, Bruce E; Power, David A

2014-01-01

The co-transporter activity of Na(+)-K(+)-2Cl(-) 1 (NKCC1) is dependent on phosphorylation. In this study we show the energy-sensing kinase AMPK inhibits NKCC1 activity. Three separate AMPK activators (AICAR, Phenformin and A-769662) inhibited NKCC1 flux in a variety of nucleated cells. Treatment with A-769662 resulted in a reduction of NKCC1(T212/T217) phosphorylation, and this was reversed by treatment with the non-selective AMPK inhibitor Compound C. AMPK dependence was confirmed by treatment of AMPK null mouse embryonic fibroblasts, where A-769662 had no effect on NKCC1 mediated transport. AMPK was found to directly phosphorylate a recombinant human-NKCC1 N-terminal fragment (1-293) with the phosphorylated site identified as S77. Mutation of Serine 77 to Alanine partially prevented the inhibitory effect of A-769662 on NKCC1 activity. In conclusion, AMPK can act to reduce NKCC1-mediated transport. While the exact mechanism is still unclear there is evidence for both a direct effect on phosphorylation of S77 and reduced phosphorylation of T212/217.

New approach of delivering cytotoxic drugs towards CAIX expressing cells: A concept of dual-target drugs.

PubMed

van Kuijk, Simon J A; Parvathaneni, Nanda Kumar; Niemans, Raymon; van Gisbergen, Marike W; Carta, Fabrizio; Vullo, Daniela; Pastorekova, Silvia; Yaromina, Ala; Supuran, Claudiu T; Dubois, Ludwig J; Winum, Jean-Yves; Lambin, Philippe

2017-02-15

Carbonic anhydrase IX (CAIX) is a hypoxia-regulated and tumor-specific protein that maintains the pH balance of cells. Targeting CAIX might be a valuable approach for specific delivery of cytotoxic drugs, thereby reducing normal tissue side-effects. A series of dual-target compounds were designed and synthesized incorporating a sulfonamide, sulfamide, or sulfamate moiety combined with several different anti-cancer drugs, including the chemotherapeutic agents chlorambucil, tirapazamine, and temozolomide, two Ataxia Telangiectasia and Rad3-related protein inhibitors (ATRi), and the anti-diabetic biguanide agent phenformin. An ATRi derivative (12) was the only compound to show a preferred efficacy in CAIX overexpressing cells versus cells without CAIX expression when combined with radiation. Its efficacy might however not solely depend on binding to CAIX, since all described compounds generally display low activity as carbonic anhydrase inhibitors. The hypothesis that dual-target compounds specifically target CAIX expressing tumor cells was therefore not confirmed. Even though dual-target compounds remain an interesting approach, alternative options should also be investigated as novel treatment strategies. Copyright © 2016 The Authors. Published by Elsevier Masson SAS.. All rights reserved.

Pleiotropic Effects of Biguanides on Mitochondrial Reactive Oxygen Species Production.

PubMed

Pecinova, Alena; Drahota, Zdenek; Kovalcikova, Jana; Kovarova, Nikola; Pecina, Petr; Alan, Lukas; Zima, Michal; Houstek, Josef; Mracek, Tomas

2017-01-01

Metformin is widely prescribed as a first-choice antihyperglycemic drug for treatment of type 2 diabetes mellitus, and recent epidemiological studies showed its utility also in cancer therapy. Although it is in use since the 1970s, its molecular target, either for antihyperglycemic or antineoplastic action, remains elusive. However, the body of the research on metformin effect oscillates around mitochondrial metabolism, including the function of oxidative phosphorylation (OXPHOS) apparatus. In this study, we focused on direct inhibitory mechanism of biguanides (metformin and phenformin) on OXPHOS complexes and its functional impact, using the model of isolated brown adipose tissue mitochondria. We demonstrate that biguanides nonspecifically target the activities of all respiratory chain dehydrogenases (mitochondrial NADH, succinate, and glycerophosphate dehydrogenases), but only at very high concentrations (10 -2 -10 -1  M) that highly exceed cellular concentrations observed during the treatment. In addition, these concentrations of biguanides also trigger burst of reactive oxygen species production which, in combination with pleiotropic OXPHOS inhibition, can be toxic for the organism. We conclude that the beneficial effect of biguanides should probably be associated with subtler mechanism, different from the generalized inhibition of the respiratory chain.

New prodrugs of metformin do not influence the overall haemostasis potential and integrity of the erythrocyte membrane.

PubMed

Markowicz-Piasecka, Magdalena; Sikora, Joanna; Mateusiak, Łukasz; Mikiciuk-Olasik, Elżbieta; Huttunen, Kristiina M

2017-09-15

Although metformin, an oral anti-diabetic drug, has been found to have multidirectional effects over the past decade, it is characterised by unfavourable pharmacokinetic properties. This study discusses the effects of metformin, phenformin and three prodrugs of metformin on the haemostasis and integrity of Red Blood Cells (RBCs). The influence of examined biguanide derivatives on haemostasis was evaluated spectrophotometrically by clot formation and lysis test (CL-test) at 405nm. The extrinsic and intrinsic coagulation pathway were examined by measuring the PT (Prothrombin Time) and aPTT (Activated Partial Tromboplastin Time). Haemolysis assay, microscopy and flow cytometry studies were used to assess the effect of the tested compounds on RBCs. Although none of the tested biguanide derivatives significantly influenced the overall potential of clot formation and fibrinolysis (CL AUC constants), statistically significant changes were seen in the values of the kinetic parameters of fibrinolysis. Furthermore, only prodrug 2, with an 8-carbon alkyl chain, unfavourably affected RBCs by interaction with the erythrocyte membrane leading to significant haemolysis. Our results provide a further insight into the effects of metformin and its prodrugs on haemostasis and RBCs and underscore the necessity for further research. Copyright © 2017 Elsevier B.V. All rights reserved.

Inhibition of Growth of Bladder Cancer Cells by 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one in Combination with Other Compounds Affecting Glucose Metabolism.

PubMed

Lea, Michael A; Altayyar, Mansour; desBordes, Charles

2015-11-01

In seven out of eight human bladder cell lines that were examined herein, growth was more dependent on the presence in the incubation medium of glucose rather than glutamine. The exception was the slowly growing RT4 cells that were more glutamine-dependent. Growth of all the cell lines was reduced by an inhibitor of 6-phosphofructo-2-kinase/2,6-bisphosphatase 3, namely 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO). Growth was also reduced by three compounds that reduce the conversion of glucose to lactate: namely 2-deoxyglucose, butyrate and dichloroacetate. Additive effects were seen when these molecules were combined with 3PO. Treatment of bladder cancer cells with phenformin resulted in growth inhibition that was frequently accompanied by increased glucose uptake and acidification of the medium that was blocked by co-incubation with 3PO. The actions of 3PO suggest that inhibitors of PFKB3 merit further investigation in the treatment of bladder cancer and they may be useful agents in combination with other drugs that inhibit cancer cell proliferation. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Inhibitory effects of a polypeptide thymic factor on the development of 7,12-dimethylbenz(a)anthragene-induced mammary adenocarcinoma in female rats

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anisimov, V.N.; Danetskaya, E.V.; Morozov, V.G.

1980-01-01

It has come to be recognized that tumor growth is accompanied by inhibition of cellular immunity and the function of the T lymphocytes. Restitution of T lymphocyte function by means of several pharmacologic agents such as levamisole, phenformin, or epithalamin (an epiphyseal factor) has, in a number of cases, been accompanied by growth inhibition of both spontaneous and induced tumors. In addition, the importance of the thymus in the regulation of T lymphocytes and in antitumor immunity has been recognized. Several indicators point to the fact that the thymus contains physiologically active substances which stimulate T cell-dependent immunity and preventmore » the occurrence of neoplasms. These considerations have led to attempts at isolation of active thymic factors and studies on their effects on the appearance and growth of tumors. Previously, a thymic factor - thymarin - had been isolated which imparted immunocompetence to the T lymphocytes. This factor differs from other thymic preparations, including thymosine, in terms of a number of physicochemical characteristics and is a polypeptide with a molecular weight of 5000. This study is concerned with its effects on tumor development - mammary gland adenocarcinoma induced in animals with a chemical carcinogen.« less

Propofol induces a metabolic switch to glycolysis and cell death in a mitochondrial electron transport chain-dependent manner.

PubMed

Sumi, Chisato; Okamoto, Akihisa; Tanaka, Hiromasa; Nishi, Kenichiro; Kusunoki, Munenori; Shoji, Tomohiro; Uba, Takeo; Matsuo, Yoshiyuki; Adachi, Takehiko; Hayashi, Jun-Ichi; Takenaga, Keizo; Hirota, Kiichi

2018-01-01

The intravenous anesthetic propofol (2,6-diisopropylphenol) has been used for the induction and maintenance of anesthesia and sedation in critical patient care. However, the rare but severe complication propofol infusion syndrome (PRIS) can occur, especially in patients receiving high doses of propofol for prolonged periods. In vivo and in vitro evidence suggests that the propofol toxicity is related to the impaired mitochondrial function. However, underlying molecular mechanisms remain unknown. Therefore, we investigated effects of propofol on cell metabolism and death using a series of established cell lines of various origins, including neurons, myocytes, and trans-mitochondrial cybrids, with defined mitochondrial DNA deficits. We demonstrated that supraclinical concentrations of propofol in not less than 50 μM disturbed the mitochondrial function and induced a metabolic switch, from oxidative phosphorylation to glycolysis, by targeting mitochondrial complexes I, II and III. This disturbance in mitochondrial electron transport caused the generation of reactive oxygen species, resulting in apoptosis. We also found that a predisposition to mitochondrial dysfunction, caused by a genetic mutation or pharmacological suppression of the electron transport chain by biguanides such as metformin and phenformin, promoted propofol-induced caspase activation and cell death induced by clinical relevant concentrations of propofol in not more than 25 μM. With further experiments with appropriate in vivo model, it is possible that the processes to constitute the molecular basis of PRIS are identified.

Differential effects of AMPK agonists on cell growth and metabolism

PubMed Central

Vincent, Emma E.; Coelho, Paula P.; Blagih, Julianna; Griss, Takla; Viollet, Benoit; Jones, Russell G.

2016-01-01

As a sensor of cellular energy status, the AMP-activated protein kinase (AMPK) is believed to act in opposition to the metabolic phenotypes favored by proliferating tumor cells. Consequently, compounds known to activate AMPK have been proposed as cancer therapeutics. However, the extent to which the anti-neoplastic properties of these agonists are mediated by AMPK is unclear. Here we examined the AMPK-dependence of six commonly used AMPK agonists (metformin, phenformin, AICAR, 2DG, salicylate and A-769662) and their influence on cellular processes often deregulated in tumor cells. We demonstrate that the majority of these agonists display AMPK-independent effects on cell proliferation and metabolism with only the synthetic activator, A-769662, exerting AMPK-dependent effects on these processes. We find that A-769662 promotes an AMPK-dependent increase in mitochondrial spare respiratory capacity (SRC). Finally, contrary to the view of AMPK activity being tumor suppressive, we find A-769662 confers a selective proliferative advantage to tumor cells growing under nutrient deprivation. Our results indicate that many of the anti-growth properties of these agonists cannot be attributed to AMPK activity in cells, and thus any observed effects using these agonists should be confirmed using AMPK-deficient cells. Ultimately, our data urge caution, not only regarding the type of AMPK agonist proposed for cancer treatment, but also the context in which they are used. PMID:25241895

Differential effects of AMPK agonists on cell growth and metabolism.

PubMed

Vincent, E E; Coelho, P P; Blagih, J; Griss, T; Viollet, B; Jones, R G

2015-07-01

As a sensor of cellular energy status, the AMP-activated protein kinase (AMPK) is believed to act in opposition to the metabolic phenotypes favored by proliferating tumor cells. Consequently, compounds known to activate AMPK have been proposed as cancer therapeutics. However, the extent to which the anti-neoplastic properties of these agonists are mediated by AMPK is unclear. Here we examined the AMPK dependence of six commonly used AMPK agonists (metformin, phenformin, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), 2-deoxy-D-glucose (2DG), salicylate and A-769662) and their influence on cellular processes often deregulated in tumor cells. We demonstrate that the majority of these agonists display AMPK-independent effects on cell proliferation and metabolism with only the synthetic activator, A-769662, exerting AMPK-dependent effects on these processes. We find that A-769662 promotes an AMPK-dependent increase in mitochondrial spare respiratory capacity. Finally, contrary to the view of AMPK activity being tumor suppressive, we find that A-769662 confers a selective proliferative advantage to tumor cells growing under nutrient deprivation. Our results indicate that many of the antigrowth properties of these agonists cannot be attributed to AMPK activity in cells, and thus any observed effects using these agonists should be confirmed using AMPK-deficient cells. Ultimately, our data urge caution not only regarding the type of AMPK agonist proposed for cancer treatment but also the context in which they are used.

Activation of AMPK improves inflammation and insulin resistance in adipose tissue and skeletal muscle from pregnant women.

PubMed

Liong, Stella; Lappas, Martha

2015-12-01

Gestational diabetes mellitus (GDM) is characterised by maternal peripheral insulin resistance and inflammation. Sterile inflammation and bacterial infection are key mediators of this enhanced inflammatory response. Adenosine monophosphate (AMP)-activated kinase (AMPK), which is decreased in insulin resistant states, possesses potent pro-inflammatory actions. There are, however, no studies on the role of AMPK in pregnancies complicated by GDM. Thus, the aims of this study were (i) to compare the expression of AMPK in adipose tissue and skeletal muscle from women with GDM and normal glucose-tolerant (NGT) pregnant women; and (ii) to investigate the effect of AMPK activation on inflammation and insulin resistance induced by the bacterial endotoxin lipopolysaccharide (LPS) and the pro-inflammatory cytokine IL-1β. When compared to NGT pregnant women, AMPKα activity was significantly lower in women with GDM as evidenced by a decrease in threonine phosphorylation of AMPKα. Activation of AMPK, using two pharmacologically distinct compounds, AICAR or phenformin, significantly suppressed LPS- or IL-1β-induced gene expression and secretion of pro-inflammatory cytokine IL-6, the chemokines IL-8 and MCP-1, and COX-2 and subsequent prostaglandin release from adipose tissue and skeletal muscle. In addition, activators of AMPK decreased skeletal muscle insulin resistance induced by LPS or IL-1β as evidenced by increased insulin-stimulated phosphorylation of IRS-1, GLUT-4 expression and glucose uptake. These findings suggest that AMPK may play an important role in inflammation and insulin resistance.

Metformin Is a Substrate and Inhibitor of the Human Thiamine Transporter, THTR-2 (SLC19A3).

PubMed

Liang, Xiaomin; Chien, Huan-Chieh; Yee, Sook Wah; Giacomini, Marilyn M; Chen, Eugene C; Piao, Meiling; Hao, Jia; Twelves, Jolyn; Lepist, Eve-Irene; Ray, Adrian S; Giacomini, Kathleen M

2015-12-07

The biguanide metformin is widely used as first-line therapy for the treatment of type 2 diabetes. Predominately a cation at physiological pH's, metformin is transported by membrane transporters, which play major roles in its absorption and disposition. Recently, our laboratory demonstrated that organic cation transporter 1, OCT1, the major hepatic uptake transporter for metformin, was also the primary hepatic uptake transporter for thiamine, vitamin B1. In this study, we tested the reverse, i.e., that metformin is a substrate of thiamine transporters (THTR-1, SLC19A2, and THTR-2, SLC19A3). Our study demonstrated that human THTR-2 (hTHTR-2), SLC19A3, which is highly expressed in the small intestine, but not hTHTR-1, transports metformin (Km = 1.15 ± 0.2 mM) and other cationic compounds (MPP(+) and famotidine). The uptake mechanism for hTHTR-2 was pH and electrochemical gradient sensitive. Furthermore, metformin as well as other drugs including phenformin, chloroquine, verapamil, famotidine, and amprolium inhibited hTHTR-2 mediated uptake of both thiamine and metformin. Species differences in the substrate specificity of THTR-2 between human and mouse orthologues were observed. Taken together, our data suggest that hTHTR-2 may play a role in the intestinal absorption and tissue distribution of metformin and other organic cations and that the transporter may be a target for drug-drug and drug-nutrient interactions.

Highly sensitive on-site detection of drugs adulterated in botanical dietary supplements using thin layer chromatography combined with dynamic surface enhanced Raman spectroscopy.

PubMed

Fang, Fang; Qi, Yunpeng; Lu, Feng; Yang, Liangbao

2016-01-01

The phenomenon of botanical dietary supplements (BDS) doped with illegal adulterants has become a serious problem all over the world, which could cause great threat to human's health. Therefore, it is of great value to identify BDS. Herein, we put forward a highly sensitive method for on-site detection of antitussive and antiasthmatic drugs adulterated in BDS using thin layer chromatography (TLC) combined with dynamic surface enhanced Raman spectroscopy (DSERS). Adulterants in BDS were separated on a TLC plate and located under UV illumination. Then DSERS detection was performed using a portable Raman spectrometer with 50% glycerol silver colloid serving as DSERS active substrate. Here, the effects of different solvents on detection efficacy were evaluated using phenformin hydrochloride (PHE) as a probe. It was shown that 50% glycerol resulted in higher SERS enhancement and relatively higher stability. Moreover, practical application of this novel TLC-DSERS method was demonstrated with rapid analysis of real BDS samples and one sample adulterated with benproperine phosphate (BEN) was found. Furthermore, the obtained result was verified by ultra performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QTOF/MS). The sensitivity of the TLC-DSERS technique is 1-2 orders of magnitude higher than that of TLC-SERS technique. The results turned out that this combined method would have good prospects for on-site and sensitive detection of adulterated BDS. Copyright © 2015 Elsevier B.V. All rights reserved.

Inhibiting prenylation augments chemotherapy efficacy in renal cell carcinoma through dual inhibition on mitochondrial respiration and glycolysis.

PubMed

Huang, Jiangrong; Yang, Xiaoyu; Peng, Xiaochun; Huang, Wei

2017-11-18

Prenylation is a posttranslational lipid modification required for the proper functions of a number of proteins involved in cell regulation. Here, we show that prenylation inhibition is important for renal cell carcinoma (RCC) growth, survival and response to chemotherapy, and its underlying mechanism may be contributed to mitochondrial dysfunction. We first demonstrated that a HMG-CoA reductase inhibitor pitavastatin inhibited mevalonate pathway and thereby prenylation in RCC cells. In addition, pitavastatin is effective in inhibiting growth and inducing apoptosis in a panel of RCC cell lines. Combination of pitavastatin and paclitaxel is significantly more effective than pitavastatin or paclitaxel alone as shown by both in vitro cell culture system and in vivo RCC xenograft model. Importantly, pitavastatin treatment inhibits mitochondrial respiration via suppressing mitochondrial complex I and II enzyme activities. Interestingly, different from mitochondrial inhibitor phenformin that inhibits mitochondrial respiration but activates glycolytic rate in RCC cells, pitavastatin significantly decreases glycolytic rate. The dual inhibitory action of pitavastatin on mitochondrial respiration and glycolysis results in remarkable energy depletion and oxidative stress in RCC cells. In addition, inhibition of prenylation by depleting Isoprenylcysteine carboxylmethyltransferase (Icmt) also mimics the inhibitory effects of pitavastatin in RCC cells. Our work demonstrates the previously unappreciated association between prenylation inhibition and energy metabolism in RCC, which can be therapeutically exploited, likely in tumors that largely rely on energy metabolism. Copyright © 2017 Elsevier Inc. All rights reserved.

Metformin and insulin receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vigneri, R.; Gullo, D.; Pezzino, V.

The authors evaluated the effect of metformin (N,N-dimethylbiguanide), a biguanide known to be less toxic than phenformin, on insulin binding to its receptors, both in vitro and in vivo. Specific /sup 125/I-insulin binding to cultured IM-9 human lymphocytes and MCF-7 human breast cancer cells was determined after preincubation with metformin. Specific /sup 125/I-insulin binding to circulating monocytes was also evaluated in six controls, eight obese subjects, and six obese type II diabetic patients before and after a short-term treatment with metformin. Plasma insulin levels and blood glucose were also measured on both occasions. Metformin significantly increased insulin binding in vitromore » to both IM-9 lymphocytes and MCF-7 cells; the maximum increment was 47.1% and 38.0%, respectively. Metformin treatment significantly increased insulin binding in vivo to monocytes of obese subjects and diabetic patients. Scatchard analysis indicated that the increased binding was mainly due to an increase in receptor capacity. Insulin binding to monocytes of normal controls was unchanged after metformin as were insulin levels in all groups; blood glucose was significantly reduced after metformin only in diabetic patients. These data indicate that metformin increases insulin binding to its receptors in vitro and in vivo. The effect in vivo is observed in obese subjects and in obese type II diabetic patients, paralleling the clinical effectiveness of this antidiabetic agent, and is not due to receptor regulation by circulating insulin, since no variation in insulin levels was recorded.« less

The epigenetic regulation of HIF-1α by SIRT1 in MPP{sup +} treated SH-SY5Y cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Su-Yan; Guo, Yan-Jie; Feng, Ya

Both silent information regulator 1 (SIRT1) and hypoxia inducible factor 1 (HIF-1) have been found to play important roles in the pathophysiology of Parkinson's disease (PD). However, their mechanisms and their relationship still require further study. In the present study, we focused on the change and relationship of SIRT1 and HIF-1α in PD. PD cell models were established by using methyl-4-phenylpyridinium (MPP{sup +}), which induced inhibition of cell proliferation, cell cycle arrest and apoptosis. We found that the expression of HIF-1α and its target genes VEGFA and LDHA increased and that SIRT1 expression was inhibited in MPP{sup +} treated cells.more » With further analysis, we found that the acetylation of H3K14 combined with the HIF-1α promoter was dramatically increased in cells treated with MPP{sup +}, which resulted in the transcriptional activation of HIF-1α. Moreover, the acetylation of H3K14 and the expression of HIF-1α increased when SIRT1 was knocked down, suggesting that SIRT1 was involved in the epigenetic regulation of HIF-1α. At last, phenformin, another mitochondrial complex1 inhibitor, was used to testify that the increased HIF-1a was not due to off target effects of MPP{sup +}. Therefore, our results support a link between PD and SIRT1/HIF-1α signaling, which may serve as a clue for understanding PD.« less

A sensitive NanoString-based assay to score STK11 (LKB1) pathway disruption in lung adenocarcinoma

PubMed Central

Chen, Lu; Engel, Brienne E.; Welsh, Eric A.; Yoder, Sean J.; Brantley, Stephen G.; Chen, Dung-Tsa; Beg, Amer A.; Cao, Chunxia; Kaye, Frederic J.; Haura, Eric B.; Schabath, Matthew B.; Cress, W. Douglas

2016-01-01

Introduction Serine/threonine kinase 11 (STK11), better known as LKB1, is a tumor-suppressor commonly mutated in lung adenocarcinoma (LUAD). Previous work has shown that mutational inactivation of the STK11 pathway may serve as a predictive biomarker for cancer treatments including phenformin and COX-2 inhibition. Although immunohistochemistry and diagnostic sequencing are employed to measure STK11 pathway disruption, there are serious limitations to these methods emphasizing the importance to validate a clinically useful assay. Methods An initial STK11 mutation mRNA signature was generated using cell line data and refined using three large, independent patient databases. The signature was validated as a classifier using The Cancer Genome Anatomy Project (TCGA) LUAD cohort as well as a 442-patient LUAD cohort developed at Moffitt. Finally, the signature was adapted into a NanoString -based format and validated using RNA samples isolated from FFPE tissue blocks corresponding to a cohort of 150 LUAD patients. For comparison, STK11 immunochemistry was also performed. Results The STK11 signature was found to correlate with null mutations identified by exon sequencing in multiple cohorts using both microarray and NanoString formats. While there was a statistically significant correlation between reduced STK11 protein expression by IHC and mutation status, the NanoString-based assay showed superior overall performance with a −0.1588 improvement in area under the curve in receiver-operator characteristic curve analysis (p<0.012). Conclusion The described NanoString-based STK11 assay is a sensitive biomarker to study emerging therapeutic modalities in clinical trials. PMID:26917230

Energy metabolism determines the sensitivity of human hepatocellular carcinoma cells to mitochondrial inhibitors and biguanide drugs.

PubMed

Hsu, Chia-Chi; Wu, Ling-Chia; Hsia, Cheng-Yuan; Yin, Pen-Hui; Chi, Chin-Wen; Yeh, Tien-Shun; Lee, Hsin-Chen

2015-09-01

Human hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide particularly in Asia. Deregulation of cellular energetics was recently included as one of the cancer hallmarks. Compounds that target the mitochondria in cancer cells were proposed to have therapeutic potential. Biguanide drugs which inhibit mitochondrial complex I and repress mTOR signaling are clinically used to treat type 2 diabetes mellitus patients (T2DM) and were recently found to reduce the risk of HCC in T2DM patients. However, whether alteration of energy metabolism is involved in regulating the sensitivity of HCC to biguanide drugs is still unclear. In the present study, we treated four HCC cell lines with mitochondrial inhibitors (rotenone and oligomycin) and biguanide drugs (metformin and phenformin), and found that the HCC cells which had a higher mitochondrial respiration rate were more sensitive to these treatments; whereas the HCC cells which exhibited higher glycolysis were more resistant. When glucose was replaced by galactose in the medium, the altered energy metabolism from glycolysis to mitochondrial respiration in the HCC cells enhanced the cellular sensitivity to mitochondrial inhibitors and biguanides. The energy metabolism change enhanced AMP-activated protein kinase (AMPK) activation, mTOR repression and downregulation of cyclin D1 and Mcl-1 in response to the mitochondrial inhibitors and biguanides. In conclusion, our results suggest that increased mitochondrial oxidative metabolism upregulates the sensitivity of HCC to biguanide drugs. Enhancing the mitochondrial oxidative metabolism in combination with biguanide drugs may be a therapeutic strategy for HCC.

Activation of AMPK in human fetal membranes alleviates infection-induced expression of pro-inflammatory and pro-labour mediators.

PubMed

Lim, R; Barker, G; Lappas, M

2015-04-01

In non-gestational tissues, the activation of adenosine monophosphate (AMP)-activated kinase (AMPK) is associated with potent anti-inflammatory actions. Infection and/or inflammation, by stimulating pro-inflammatory cytokines and matrix metalloproteinase (MMP)-9, play a central role in the rupture of fetal membranes. However, no studies have examined the role of AMPK in human labour. Fetal membranes, from term and preterm, were obtained from non-labouring and labouring women, and after preterm pre-labour rupture of membranes (PPROM). AMPK activity was assessed by Western blotting of phosphorylated AMPK expression. To determine the effect of AMPK activators on pro-inflammatory cytokines, fetal membranes were pre-treated with AMPK activators then stimulated with bacterial products LPS and flagellin or viral dsDNA analogue poly(I:C). Primary amnion cells were used to determine the effect of AMPK activators on IL-1β-stimulated MMP-9 expression. AMPK activity was decreased with term labour. There was no effect of preterm labour. AMPK activity was also decreased in preterm fetal membranes, in the absence of labour, with PROM compared to intact membranes. AMPK activators AICAR, phenformin and A769662 significantly decreased IL-6 and IL-8 stimulated by LPS, flagellin and poly(I:C). Primary amnion cells treated with AMPK activators significantly decreased IL-1β-induced MMP-9 expression. The decrease in AMPK activity in fetal membranes after spontaneous term labour and PPROM indicates an anti-inflammatory role for AMPK in human labour and delivery. The use of AMPK activators as possible therapeutics for threatened preterm labour would be an exciting future avenue of research. Copyright © 2015 Elsevier Ltd. All rights reserved.

Determination of drug toxicity using 3D spheroids constructed from an immortal human hepatocyte cell line.

PubMed

Fey, Stephen J; Wrzesinski, Krzysztof

2012-06-01

Numerous publications have documented that the immortal cells grown in three-dimensional (3D) cultures possess physiological behavior, which is more reminiscent of their parental organ than when the same cells are cultivated using classical two-dimensional (2D) culture techniques. The goal of this study was to investigate whether this observation could be extended to the determination of LD(50) values and whether 3D data could be correlated to in vivo observations. We developed a noninvasive means to estimate the amount of protein present in a 3D spheroid from it is planar area (± 21%) so that a precise dose can be provided in a manner similar to in vivo studies. This avoided correction of the actual dose given based on a protein determination after treatment (when some cells may have lysed). Conversion of published in vitro LC(50) data (mM) for six common drugs (acetaminophen, amiodarone, diclofenac, metformin, phenformin, and valproic acid) to LD(50) data (mg compound/mg cellular protein) showed that the variation in LD(50) values was generally less than that suggested by the original LC(50) data. Toxicological analysis of these six compounds in 3D spheroid culture (either published or presented here) demonstrated similar LD(50) values. Although in vitro 2D HepG2 data showed a poor correlation, the primary hepatocyte and 3D spheroid data resulted in a much higher degree of correlation with in vivo lethal blood plasma levels. These results corroborate that 3D hepatocyte cultures are significantly different from 2D cultures and are more representative of the liver in vivo.

Determination of Drug Toxicity Using 3D Spheroids Constructed From an Immortal Human Hepatocyte Cell Line

PubMed Central

Fey, Stephen J.; Wrzesinski, Krzysztof

2012-01-01

Numerous publications have documented that the immortal cells grown in three-dimensional (3D) cultures possess physiological behavior, which is more reminiscent of their parental organ than when the same cells are cultivated using classical two-dimensional (2D) culture techniques. The goal of this study was to investigate whether this observation could be extended to the determination of LD50 values and whether 3D data could be correlated to in vivo observations. We developed a noninvasive means to estimate the amount of protein present in a 3D spheroid from it is planar area (± 21%) so that a precise dose can be provided in a manner similar to in vivo studies. This avoided correction of the actual dose given based on a protein determination after treatment (when some cells may have lysed). Conversion of published in vitro LC50 data (mM) for six common drugs (acetaminophen, amiodarone, diclofenac, metformin, phenformin, and valproic acid) to LD50 data (mg compound/mg cellular protein) showed that the variation in LD50 values was generally less than that suggested by the original LC50 data. Toxicological analysis of these six compounds in 3D spheroid culture (either published or presented here) demonstrated similar LD50 values. Although in vitro 2D HepG2 data showed a poor correlation, the primary hepatocyte and 3D spheroid data resulted in a much higher degree of correlation with in vivo lethal blood plasma levels. These results corroborate that 3D hepatocyte cultures are significantly different from 2D cultures and are more representative of the liver in vivo. PMID:22454432

Silencing Nrf2 impairs glioma cell proliferation via AMPK-activated mTOR inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jia, Yue; Wang, Handong, E-mail: njhdwang@hotmail.com; Wang, Qiang

Gliomas are the leading cause of death among adults with primary brain malignancies. Treatment for malignant gliomas remains limited, and targeted therapies have been incompletely explored. Nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription regulator for antioxidant and detoxification enzymes, is abundantly expressed in cancer cells. In this study, the role and mechanism of Nrf2 in cancer cell proliferation was investigated in multiple glioma cell lines. We first evaluated the expression patterns of Nrf2 in four glioma cell lines and found all four cell lines expressed Nrf2, but the highest level was observed in U251 cells. We further evaluatedmore » the biological functions of Nrf2 in U251 glioma cell proliferation by specific inhibition of Nrf2 using short hairpin RNA (shRNA). We found that Nrf2 depletion inhibited glioma cell proliferation. Nrf2 depletion also decreased colony formation in U251 cells stably expressing Nrf2 shRNA compared to scrambled control shRNA. Moreover, suppression of Nrf2 expression could lead to ATP depletion (with concomitant rise in AMP/ATP ratio) and consequently to AMPK-activated mTOR inhibition. Finally, activation of adenosine monophosphate–activated protein kinase (AMPK) by treated with phenformin, an AMPK agonist, can mimic the inhibitory effect of Nrf2 knockdown in U251 cells. In conclusion, our findings will shed light to the role and mechanism of Nrf2 in regulating glioma proliferation via ATP-depletion-induced AMPK activation and consequent mTOR inhibition, a novel insight into our understanding the role and mechanism of Nrf2 in glioma pathoetiology. To our knowledge, this is also the first report to provide a rationale for the implication of cross-linking between Nrf2 and mTOR signaling.« less

Oncometabolic mutation IDH1 R132H confers a metformin-hypersensitive phenotype.

PubMed

Cuyàs, Elisabet; Fernández-Arroyo, Salvador; Corominas-Faja, Bruna; Rodríguez-Gallego, Esther; Bosch-Barrera, Joaquim; Martin-Castillo, Begoña; De Llorens, Rafael; Joven, Jorge; Menendez, Javier A

2015-05-20

Metabolic flexibility might be particularly constrained in tumors bearing mutations in isocitrate dehydrogenase 1 (IDH1) leading to the production of the oncometabolite 2-hydroxygluratate (2HG). To test the hypothesis that IDH1 mutations could generate metabolic vulnerabilities for therapeutic intervention, we utilized an MCF10A cell line engineered with an arginine-to-histidine conversion at position 132 (R132H) in the catalytic site of IDH1, which equips the enzyme with a neomorphic α-ketoglutarate to 2HG reducing activity in an otherwise isogenic background. IDH1 R132H/+ and isogenic IDH1 +/+ parental cells were screened for their ability to generate energy-rich NADH when cultured in a standardized high-throughput Phenotype MicroArrayplatform comprising >300 nutrients. A radical remodeling of the metabotype occurred in cells carrying the R132H mutation since they presented a markedly altered ability to utilize numerous carbon catabolic fuels. A mitochondria toxicity-screening modality confirmed a severe inability of IDH1-mutated cells to use various carbon substrates that are fed into the electron transport chain at different points. The mitochondrial biguanide poisons, metformin and phenformin, further impaired the intrinsic weakness of IDH1-mutant cells to use certain carbon-energy sources. Additionally, metabolic reprogramming of IDH1-mutant cells increased their sensitivity to metformin in assays of cell proliferation, clonogenic potential, and mammosphere formation. Targeted metabolomics studies revealed that the ability of metformin to interfere with the anaplerotic entry of glutamine into the tricarboxylic acid cycle could explain the hypersensitivity of IDH1-mutant cells to biguanides. Moreover, synergistic interactions occurred when metformin treatment was combined with the selective R132H-IDH1 inhibitor AGI-5198. Together, these results suggest that therapy involving the simultaneous targeting of metabolic vulnerabilities with metformin, and 2HG overproduction with mutant-selective inhibitors (AGI-5198-related AG-120 [Agios]), might represent a worthwhile avenue of exploration in the treatment of IDH1-mutated tumors.

Oncometabolic mutation IDH1 R132H confers a metformin-hypersensitive phenotype

PubMed Central

Cuyàs, Elisabet; Fernández-Arroyo, Salvador; Corominas-Faja, Bruna; Rodríguez-Gallego, Esther; Bosch-Barrera, Joaquim; Martin-Castillo, Begoña; De Llorens, Rafael; Joven, Jorge; Menendez, Javier A.

2015-01-01

Metabolic flexibility might be particularly constrained in tumors bearing mutations in isocitrate dehydrogenase 1 (IDH1) leading to the production of the oncometabolite 2-hydroxygluratate (2HG). To test the hypothesis that IDH1 mutations could generate metabolic vulnerabilities for therapeutic intervention, we utilized an MCF10A cell line engineered with an arginine-to-histidine conversion at position 132 (R132H) in the catalytic site of IDH1, which equips the enzyme with a neomorphic α-ketoglutarate to 2HG reducing activity in an otherwise isogenic background. IDH1 R132H/+ and isogenic IDH1 +/+ parental cells were screened for their ability to generate energy-rich NADH when cultured in a standardized high-throughput Phenotype MicroArrayplatform comprising >300 nutrients. A radical remodeling of the metabotype occurred in cells carrying the R132H mutation since they presented a markedly altered ability to utilize numerous carbon catabolic fuels. A mitochondria toxicity-screening modality confirmed a severe inability of IDH1-mutated cells to use various carbon substrates that are fed into the electron transport chain at different points. The mitochondrial biguanide poisons, metformin and phenformin, further impaired the intrinsic weakness of IDH1-mutant cells to use certain carbon-energy sources. Additionally, metabolic reprogramming of IDH1-mutant cells increased their sensitivity to metformin in assays of cell proliferation, clonogenic potential, and mammosphere formation. Targeted metabolomics studies revealed that the ability of metformin to interfere with the anaplerotic entry of glutamine into the tricarboxylic acid cycle could explain the hypersensitivity of IDH1-mutant cells to biguanides. Moreover, synergistic interactions occurred when metformin treatment was combined with the selective R132H-IDH1 inhibitor AGI-5198. Together, these results suggest that therapy involving the simultaneous targeting of metabolic vulnerabilities with metformin, and 2HG overproduction with mutant-selective inhibitors (AGI-5198-related AG-120 [Agios]), might represent a worthwhile avenue of exploration in the treatment of IDH1-mutated tumors. PMID:25980580

[The use of natural and synthetic hydrophilic polymers in the formulation of metformin hydrochloride tablets with different profile release].

PubMed

Kołodziejczyk, Michał Krzysztof; Kołodziejska, Justyna; Zgoda, Marian Mikołaj

2012-01-01

Metformin hydrochloride after buformin and phenformin belongs to the group of biguanid derivatives used as oral anti-diabetic drugs. The object of the study is the technological analysis and the potential effect of biodegradable macromolecular polymers on the technological and therapeutic parameters of oral anti-diabetic medicinal products with metformin hydrochloride: Siofor, Formetic, Glucophage, Metformax in doses of 500mg and 1000mg and Glucophage XR in a dose of 500 mg of modified release. Market therapeutic products containing 500 and 1000 mg of metformin hydrochloride in a normal formulation and 500 mg of metformin hydrochloride in a formulation of modified release were analyzed. Following research methods were used: technological analysis of tablets, study of disintegration time of tablets, evaluation of pharmaceutical availability of metformin hydrochloride from tested therapeutic products, mathematical and kinetic analysis of release profiles of metformin hydrochloride, statistical analysis of mean differences of release coefficients. The percentage of excipients in the XR formulation is higher and constitutes 50.5% of a tablet mass. However, in standard formulations the percentage is lower, between 5.5% and 12.76%. On the basis of the results of disintegration time studies, the analysed therapeutic products can be divided into two groups, regardless the dose. The first one are preparations with faster (not fast!) disintegration: Glucophage i Metformax. The second group are preparations with slower disintegration, more balanced in the aspect of a high dose of the biologically active substance: Formetic and Siofor. Products with a lower content of excipients (Metformax, Glucophage) disintegrate in a faster way. The disintegration rate of the products with a higher content of excipients (Formetic, Siofor) is slower. The appearance of metformin hydrochloride concentration in the gastrointestinal contents, balanced in time, caused by a slower disintegration-dissolving of a tablet, is conducive to the reduction of gastrointestinal side effects and better tolerance of the therapeutic product by a patient. The study on pharmaceutical availability indicated relevant kinetic differences between tested therapeutic products. They are particularly visible between standard formulations and the one with prolonged release (Glucophage XR500). Its release profile bears features of kinetics similar to zero-order reactions. Tested therapeutic products contain a large amount of the biologically active substance in relation to the content of excipients. A higher content of excipients in a single tablet mass distinguishes Siofor in comparison with Glucophage i Metformax. The excipients used in the formulations of tested preparations are comparable. A higher percentage of binding agents (HPMC, PVP) is observed, but there is a lack of typical disintegrants which results in a longer disintegration time up to 15 minutes. Siofor disintegrates at the same time as Formetic, but longer than Glucophage i Metformax. Considering the large content of the active substance and pharmacological properties of metformin hydrochloride, such a disintegration might have beneficial consequences, because the amount of the free active substance in the gastrointestinal tract will increase over the longer time period what will reduce the level of gastrointestinal side effects. The release profiles of metformin hydrochloride from tested therapeutic products are comparable. The Glucophage XR 500 formulation with the release kinetics of metformin hydrochloride similar to the zero-order kinetics is completely different from the others. The above is confirmed by the mathematical analysis of release profiles of metformin hydrochloride from tested preparations where equations of lines describing the release profile are characterized by similar values of correlation coefficients.

[A 50-year history of new drugs in Japan-the development and progress of anti-diabetic drugs and the epidemiological aspects of diabetes mellitus].

PubMed

Ozawa, Hikaru; Murai, Yuriko; Ozawa, Terutaka

2003-01-01

The development and progress of antidiabetic drugs (e.g., insulin preparations and hypoglycemic drugs) are retrospectively investigated in Japan. Their influences on the treatment of diabetes mellitus (DM) and its epidemiological aspects are also discussed. 1) Insulin preparations: Insulin was introduced for DM therapy in 1925, two or three years after its discovery in Canada. The preparations were raw extracts of bovine or porcine pancreas. These did not prevail widely in Japan because of the low incidence of DM before World Wan II. After the war, a shortage of mammalian materials compelled the use of fish pancreatic tissues such as bonito and/or tuna for insulin production. Insulin infection, so-called regular insulin, was first promoted in the 6th "Pharmacopoeia Japonica" (JP6) in 1951 and has been maintained to the present edition (JP14, 2001). Although depot-type insulin preparations were developed in the USA and Europe during the war, the introduction of those preparations to Japan was delayed until 1951, when Protamine zinc insulin appeared. Globin zinc insulin and Isophane insulin were introduced for clinical use in 1952 and 1955, respectively. These were also adopted for JP7 (1961). Biphasic-type insulin, which has a rapid onset and long duration of activity, appeared in 1965. Purified preparations from bovine or porcine sources have been available since 1980, which might be a strong reason for the decrease in insulin allergy. Insulin from animal origin has been supplied for almost 60 years since its discovery. Amino acid sequences of insulins from various species of animals were determined by the pioneering studies of Sanger and his associates. Human insulin, which differs from porcine insulin by only one amino acid, was produced by Novo researchers in 1982 using a semi-synthetic method. Then the Lilly group soon succeeded in obtaining human insulin by recombinant DNA technology in the same year. Both products were introduced to Japan in 1985, and the recombinant products prevailed throughout the 1990s. Human insulin analogues (i.e., Insulin lispro and Insulin aspart) appeared in 2001. These are applied for after-meal glycosmia owing to their ultrarapid onset of activity. Self-injection by DM patients was legalized in 1981. To make the infection technique sure and easy, cartridge (pen-type) and disposable kit-type needles were devised in the 1990s. 2) Oral hypoglycemic drugs: Instead of the exclusive parenteral usage of insulins, there was also demand for oral dosage forms. The first of the sulfonyrlurea (SU) group, BZ-55, was used for DM clinically in 1955 in Germany. But it was soon withdrawn because of its antibacterial action. This led to the development of various SU groups. Tolbutamide (1956), chlorpropamide (1959), acetohexamide (1964) and tolazamide (1961) were introduced to Japan as first-generation SUs. Then glyclopyramide (Kyorin, 1965), glybenclamide (1971), gliclazide (1984) and glimepiride (1999) appeared as the second-generation SUs. These were used orally for Type 2 diabetes. Biguanide (BG) group, phenformin HC1 (1959), metformin HC1 (1961) and buformin HC1 (1961) had also been in use by oral treatment of Type 2 diabetes. SU appears to act by increasing the sensitivity of b-cells, which secrete insulin. BG probably exerts by increasing glucose transport across the membranes of target organs. 3) New types of antidiabetic drugs: a-Glucosidase inhibitors (i.e., acarbose: Bayer, 1993; and voglibose: Takeda, 1994) act on hyperglycemia after meals by decreasing glucose absorption. Thiazolidinedione compounds, such as troglitazone (Sankyo, 1995) and pioglitazone HC1 (Takeda, 1994) act by increasing the insulin sensitivity of the target tissues. These are useful for Type 2 DM patients when SUs are ineffective. Nevertheless, troglitazone was discontinued in 2000 due to severe liver damage. Nateglinide (Ajinomoto Co., 1999), which is a D-phenylalanine derivative acting similar to SUs, is useful orally for after-meal hyperglycemia of Type 2 diabetes. Epalrestat (Ono Yakuhin Co., 1992) is effective for diabetic neuropathy by reducing the formation of sorbitol. These anti-DM drugs were recently studied and developed in Japan. 4) The Japan Diabetes Society proposed a guideline on diagnostic criteria and treatment of diabetes mellitus (DM) in 1999 and revised it in 2002. DM is classified as insulin-dependent DM (Type l) and non-insulin dependent DM (Type 2). Type 1, juvenile onset DM, requires insulin therapy to prevent ketosis and to sustain life. Treatment of type 2, adult onset DM, is recommended as a step-by-step method, starting with dietary-exercise therapy, followed by oral hypoglycemic drugs and then insulin therapy. DM patients with complications should have a therapy devised to match their circumstances. 5) Epidemiological aspects: The mortality rate of DM compared to the time of drug appearance was traced from 1920 to 2000. The curve goes down slowly in the time frame of World War II, but rises from 1950 to 1970. The elevation could not be suppressed by the appearance of SUs, BGs or improved insulin preparations. The curve runs flat from 1980 to 1990, which might be related to the use of purified insulin or human insulin therapy. The mortality rate of DM indicates that death by hyperglycemic coma and other deaths resulting from complications are excluded. The survey of the principal cause of death by DM during the period of 1981-1990 indicates that the death rate due to hyperglycemic coma is only 1.7% of the total deaths caused by DM. The effect of drug therapy on all of the death resulting from DM is not detected. Hospital visitation and admission rates of the DM patients have been recorded since 1952 in Japan. This curve is rising continuously, and none of the antidiabetic drugs has been able to suppress it. These data show that the antidiabetic drugs relieve DM symptoms through their effective hypoglycemic actions, but that they cannot suppress the mortality rate of DM. It is possible that none of the drugs currently available can suppress the increasing tendency of DM patients.