Background: The antihyperglycemic drug metformin may have beneficial effects on the prevention and treatment of cancer. Metformin is known to activate AMP-activated protein kinase (AMPK). It has also been shown to inhibit cyclin D1 expression and proliferation of some cultured cancer cells. However, the mechanisms of action by which metformin mediates cell cycle arrest are not completely understood.

We describe a procedure for detecting highaffinity, sequence-specific DNA-binding proteins from crude nuclear extracts. The technique utilizes electrophoretic transfer of NaDodSO4/PAGE-fractionated proteins onto nitrocellulose filters. Incubation of the filters with a 5% (wt/vol) solution of nonfat dry milk effectively blocks nonspecific and low-affinity DNA-binding sites. Incubation of the blocked filters with radiolabeled DNA under optimal binding conditions and subsequent autoradiography reveals high-affinity DNA-protein interactions. We have used this procedure to identify proteins that bind specifically to the promoter region of the transferrin receptor gene.DNA sequences required for the regulation of eukaryotic gene expression have now been identified, largely through in vitro mutagenesis and subsequent analysis of the transcriptional activity of the mutagenized genes. What remains undetermined in most of these cases is the identity of specific transcription factors that interact with these DNA sequences to direct gene expression. The interaction of specific proteins with DNA regulatory sequences probably represents a fundamental process in controlling gene activity, and thus, the overall complement of sequence-specific proteins in the cell nucleus may largely determine the growth or differentiation state of the cell.The existence of site-specific DNA-protein interactions in eukaryotes has been demonstrated by several approaches. These include nitrocellulose filter-binding assays (1-4), detection of discrete DNase-hypersensitive sites in chromatin (5-7), exonuclease III digestion of chromatin (8), affinity chromatography (9, 10), and in vitro transcription (11)(12)(13)(14)(15)(16)(17)(18)(19). A limitation of these techniques is that they identify the site of protein binding within the DNA but do not identify the specific proteins involved. Only after extensive purification procedures can information be obtained about the nature of the proteins involved. Bowen et al. (20) have introduced a procedure that allows detection of DNA-binding proteins by blotting electrophoretically separated proteins on nitrocellulose. This procedure, however, has not proven to be effective for directly identifying site-specific DNA-binding proteins without partial purification (21-23) or heat-inactivation (3) of crude nuclear extracts. We describe here a procedure that uses protein blotting and that requires no prior purification steps, which has the potential for directly identifying and studying the regulation of proteins that interact with a target DNA sequence. We have used this procedure to identify a group of proteins that appear to bind with specificity and high affinity to the promoter region of the gene encoding the transferrin receptor. MATERIALS AND METHODSPreparation of Nuclear Extract. Cells were grown, at 370C in a 10% CO2 atmosphere in roller bottles, in Dulbecco's modified Eagle's medium (GIBCO) containing 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 ,ug/ml), Confluent cultur...

Phenformin (phenethylbiguanide; an anti-diabetic agent) plus oxamate [lactate dehydrogenase (LDH) inhibitor] was tested as a potential anti-cancer therapeutic combination. In in vitro studies, phenformin was more potent than metformin, another biguanide, recently recognized to have anti-cancer effects, in promoting cancer cell death in the range of 25 times to 15 million times in various cancer cell lines. The anti-cancer effect of phenformin was related to complex I inhibition in the mitochondria and subsequent overproduction of reactive oxygen species (ROS). Addition of oxamate inhibited LDH activity and lactate production by cells, which is a major side effect of biguanides, and induced more rapid cancer cell death by decreasing ATP production and accelerating ROS production. Phenformin plus oxamate was more effective than phenformin combined with LDH knockdown. In a syngeneic mouse model, phenformin with oxamate increased tumor apoptosis, reduced tumor size and 18F-fluorodeoxyglucose (FDG) uptake on positron emission tomography/computed tomography compared to control. We conclude that phenformin is more cytotoxic towards cancer cells than metformin. Furthermore, phenformin and oxamate have synergistic anti-cancer effects through simultaneous inhibition of complex I in the mitochondria and LDH in the cytosol, respectively.

Different cancer cells exhibit altered sensitivity to metformin treatment. Recent studies suggest these findings may be due in part to the common cell culture practice of utilizing high glucose, and when glucose is lowered, metformin becomes increasingly cytotoxic to cancer cells. In low glucose conditions ranging from 0 to 5 mM, metformin was cytotoxic to breast cancer cell lines MCF7, MDAMB231 and SKBR3, and ovarian cancer cell lines OVCAR3, and PA-1. MDAMB231 and SKBR3 were previously shown to be resistant to metformin in normal high glucose medium. When glucose was increased to 10 mM or above, all of these cell lines become less responsive to metformin treatment. Metformin treatment significantly reduced ATP levels in cells incubated in media with low glucose (2.5 mM), high fructose (25 mM) or galactose (25 mM). Reductions in ATP levels were not observed with high glucose (25 mM). This was compensated by enhanced glycolysis through activation of AMPK when oxidative phosphorylation was inhibited by metformin. However, enhanced glycolysis was either diminished or abolished by replacing 25 mM glucose with 2.5 mM glucose, 25 mM fructose or 25 mM galactose. These findings suggest that lowering glucose potentiates metformin induced cell death by reducing metformin stimulated glycolysis. Additionally, under low glucose conditions metformin significantly decreased phosphorylation of AKT and various targets of mTOR, while phospho-AMPK was not significantly altered. Thus inhibition of mTOR signaling appears to be independent of AMPK activation. Further in vivo studies using the 4T1 breast cancer mouse model confirmed that metformin inhibition of tumor growth was enhanced when serum glucose levels were reduced via low carbohydrate ketogenic diets. The data support a model in which metformin treatment of cancer cells in low glucose medium leads to cell death by decreasing ATP production and inhibition of survival signaling pathways. The enhanced cytotoxicity of metformin against cancer cells was observed both in vitro and in vivo.

There is substantial evidence that metformin, a drug used to treat type 2 diabetics, is potentially useful as a therapeutic agent for cancer. However, a better understanding of the molecular mechanisms through which metformin promotes cell cycle arrest and cell death of cancer cells is necessary. It will also be important to understand how the response of tumor cells differs from normal cells and why some tumor cells are resistant to the effects of metformin. We have found that exposure to metformin induces cell death in all but one line, MDA-MB-231, in a panel of breast cancer cell lines. MCF10A non-transformed breast epithelial cells were resistant to the cytotoxic effects of metformin, even after extended exposure to the drug. In sensitive lines, cell death was mediated by both apoptosis and a caspase-independent mechanism. The caspase-independent pathway involves activation of poly(ADP-ribose) polymerase (PARP) and correlates with enhanced synthesis of poly(ADP-ribose) and nuclear translocation of AIF, which plays an important role in mediating cell death. Metformin-induced PARP-dependent cell death is associated with a striking enlargement of mitochondria. Mitochondrial enlargement was observed in all sensitive breast cancer cell lines but not in non-transformed cells or resistant MDA-MB-231. Mitochondrial enlargement was prevented by inhibiting PARP activity or expression. A caspase inhibitor blocked metformin-induced apoptosis but did not affect PARP-dependent cell death or mitochondrial enlargement. Thus metformin has cytotoxic effects on breast cancer cells through two independent pathways. These findings will be pertinent to efforts directed at using metformin or related compounds for cancer therapy.

Purpose The unique metabolism of breast cancer cells provides interest in exploiting this phenomenon therapeutically. Metformin, a promising breast cancer therapeutic, targets complex I of the electron transport chain leading to an accumulation of reactive oxygen species (ROS) that eventually lead to cell death. Inhibition of complex I leads to lactate production, a metabolic byproduct already highly produced by reprogrammed cancer cells and associated with a poor prognosis. While metformin remains a promising cancer therapeutic, we sought a complementary agent to increase apoptotic promoting effects of metformin while attenuating lactate production possibly leading to greatly improve efficacy. Dichloroacetate (DCA) is a well-established drug used in the treatment of lactic acidosis which functions through inhibition of pyruvate dehydrogenase kinase (PDK) promoting mitochondrial metabolism. Our purpose was to examine the synergy and mechanisms by which these two drugs kill breast cancer cells. Methods Cell lines were subjected to the indicated treatments and analyzed for cell death and various aspects of metabolism. Cell death and ROS production was analyzed using flow cytometry, Western blot analysis, and cell counting methods. Images of cells were taken with phase contrast microscopy or confocal microscopy. Metabolism of cells was analyzed using the Seahorse XF24 analyzer, lactate assays, and pH analysis. Results We show that when DCA and metformin are used in combination, synergistic induction of apoptosis of breast cancer cells occurs. Metformin-induced oxidative damage is enhanced by DCA through PDK1 inhibition which also diminishes metformin promoted lactate production. Conclusions We demonstrate that DCA and metformin combine to synergistically induce caspase-dependent apoptosis involving oxidative damage with simultaneous attenuation of metformin promoted lactate production. Innovative combinations such as metformin and DCA show promise in expanding breast cancer therapies.

p27 is a key regulator of cell proliferation through inhibition of G 1 cyclin-dependent kinase (CDK) activity. Translation of the p27 mRNA is an important control mechanism for determining cellular levels of the inhibitor. Nearly all eukaryotic mRNAs are translated through a mechanism involving recognition of the 5 cap by eukaryotic initiation factor 4E (eIF4E). In quiescent cells eIF4E activity is repressed, leading to a global decline in translation rates. In contrast, p27 translation is highest during quiescence, suggesting that it escapes the general repression of translational initiation. We show that the 5 untranslated region (5-UTR) of the p27 mRNA mediates cap-independent translation. This activity is unaffected by conditions in which eIF4E is inhibited. In D6P2T cells, elevated cyclic AMP levels cause a rapid withdrawal from the cell cycle that is correlated with a striking increase in p27. Under these same conditions, cap-independent translation from the p27 5-UTR is enhanced. These results indicate that regulation of internal initiation of translation is an important determinant of p27 protein levels.

p27Kip1 levels increase in many cells as they leave the cell cycle and begin to differentiate. The increase in p27Kip1 levels generally precedes the expression of differentiation-specific genes. Previous studies from our laboratory showed that the overexpression of p27 Kip1 enhances myelin basic protein (MBP) promoter activity. This activation is specific to p27Kip1 . Additionally, inhibition of cyclin-dependent kinase activity alone is not sufficient to increase MBP expression. In this study, we focused on understanding how p27 Kip1 involves a novel mechanism that is mediated through the stabilization and binding of transcription factor Sp1.