Combined Bezafibrate and Medroxyprogesterone Acetate: Potential Novel Therapy for Acute Myeloid Leukaemia

Background

The majority of acute myeloid leukaemia (AML) patients are over sixty years of age. With current treatment regimens, survival rates amongst these, and also those younger patients who relapse, remain dismal and novel therapies are urgently required. In particular, therapies that have anti-leukaemic activity but that, unlike conventional chemotherapy, do not impair normal haemopoiesis.

Principal Findings

Here we demonstrate the potent anti-leukaemic activity of the combination of the lipid-regulating drug bezafibrate (BEZ) and the sex hormone medroxyprogesterone acetate (MPA) against AML cell lines and primary AML cells. The combined activity of BEZ and MPA (B/M) converged upon the increased synthesis and reduced metabolism of prostaglandin D₂ (PGD₂) resulting in elevated levels of the downstream highly bioactive, anti-neoplastic prostaglandin 15-deoxy Δ^12,14 PGJ₂ (15d-PGJ₂). BEZ increased PGD₂ synthesis via the generation of reactive oxygen species (ROS) and activation of the lipid peroxidation pathway. MPA directed prostaglandin synthesis towards 15d-PGJ₂ by inhibiting the PGD₂ 11β -ketoreductase activity of the aldo-keto reductase AKR1C3, which metabolises PGD₂ to 9α11β-PGF_2α. B/M treatment resulted in growth arrest, apoptosis and cell differentiation in both AML cell lines and primary AML cells and these actions were recapitulated by treatment with 15d-PGJ₂. Importantly, the actions of B/M had little effect on the survival of normal adult myeloid progenitors.

Significance

Collectively our data demonstrate that B/M treatment of AML cells elevated ROS and delivered the anti-neoplastic actions of 15d-PGJ₂. These observations provide the mechanistic rationale for the redeployment of B/M in elderly and relapsed AML.     

Preferential involvement of both ROS and ceramide in fenretinide-induced apoptosis of HL60 rather than NB4 and U937 cells

Leukemic cells responding to apoptosis-inducing drugs can be varied in terms of the mechanisms of action. Fenretinide, a synthetic retinoid, is worth of study as a promising candidate for apoptosis-based therapy of leukemia. Yet, it remains unclear whether this drug exerts the similar mechanisms on different leukemic cells. Here, we report a comparative analysis of fenretinide-induced apoptosis in three acute myeloid leukemic (AML) cell lines including HL60, NB4 and U937. Through a series of antagonist assays, we revealed similarities and differences of mechanisms involved in these three cell lines. Antioxidant vitamin C completely abrogated fenretinide-induced apoptosis in all cell lines, demonstrating that ROS is an essential and common mediator. However, the apoptotic effects of fenretinide could be blocked by ceramide synthase inhibitor fumonisin B₁ only in HL60 rather than the other two. Moreover, fumonisin B₁ was unable to inhibit the generation of ROS in fenretinide-treated HL60 cells, indicating that ROS may function as upstream stimulus of ceramide-mediated apoptosis. These comparative results strongly suggest that the apoptotic response induced by fenretinide in HL60 involves both ROS and ceramide, whereas drug-induced apoptosis in NB4 and U937 requires ROS but is independent of ceramide. Differentiated modes of action exerting on AML may guide the use of this apoptosis-inducing drug, and hence advance our knowledge about the nature of cancer-specific responses to this drug.     

Cinnamtannin B-1 as an antioxidant and platelet aggregation inhibitor

Cinnamtannin B-1 is a naturally occurring trimeric A-type proanthocyanidin, present in a limited number of plants, which exhibits a large number of cellular actions mostly derived from its antioxidant properties. Cinnamtannin B-1 modulates several biological processes such as changes in cytosolic free Ca(2+) concentration, endogenous reactive oxygen species generation, protein tyrosine phosphorylation and platelet aggregation. Proanthocyanidins, such as cinnamtannin B-1, have been reported to exert antitumoral activity mediated by a selective proapoptotic action in a number of tumoral cell lines associated with antiapoptotic activity in normal cells. The opposite effects of proanthocyanidins in normal and tumoral cells suggest that these compounds might be the base for therapeutic strategies directed selectively against tumoral cells. In addition, cinnamtannin B-1 shows antithrombotic actions through inhibition, in platelets, of endogenous ROS generation, Ca(2+) mobilization and, subsequently, aggregation. This has been reported to be especially relevant in platelets from diabetic patients, where cinnamtannin B-1 reverses both platelet hypersensitivity and hyperactivity. Considering the large number of cellular effects of cinnamtannin B-1 the development of therapeutic strategies for thrombotic disorders or certain types of cancer deserves further studies. This review summarizes the current knowledge on the actions and relevance of the signalling pathways modulated by cinnamtannin B-1.     

High Field Proton NMR Investigations of the Metabolic Profiles of Multidrug-Sensitive and -Resistant Leukaemic Cell Lines: Evidence for Diminished Taurine Levels in Multidrug-Resistant Cells

High field proton (¹H) nuclear magnetic resonance (NMR) spectroscopy has for the first time been employed to investigate and compare the metabolic profiles of vinblastine-sensitive and -resistant T-lymphoid leukaemic cell lines (CCRF-CEM and CEM/VLB₁₀₀ respectively) and evidence is presented for a significantly lower taurine content in the CEM/VLB₁₀₀ resistant subline when expressed relative to that of its drug-sensitive parental counterpart. These data suggest differences in the nature and relative involvements of taurine biosynthetic pathways between the two cell lines, a phenomenon that may be related to their differing sensitivities towards chemotherapeutic agents such as adriamycin which promote the generation of cytotoxic reactive oxygen species (ROS) in vivo. However, the ¹H NMR data obtained provided no evidence for an increased metabolic consumption of hypotaurine (a metabolic precursor of taurine with powerful ·OH radical scavenging properties) in CCRF-CEM cells since differences observed in the hypotaurine: taurine concentration ratio between the drug-sensitive and -resistant cell lines were not statistically significant. Furthermore, hypotaurine is unlikely to compete with alternative endogenous ·OH radical scavengers present such as lactate since its level in either of the two cell lines investigated (ca. 6.0 × 10^-8mol./10⁸ cells) is insufficient for it to act as an antioxidant in this context. The biochemical and therapeutic significance of these results are discussed.     

The role of thioredoxin 1 in the mycophenolic acid-induced apoptosis of insulin-producing cells

Mycophenolic acid (MPA) is one of many effective immunosuppressive drugs. However, MPA can induce cellular toxicity and impair cellular function in β-cells. To explore the effects of MPA and the relation between MPA and Trx-1, we used various methods, including an Illumina microarray, to identify the genes regulated during pancreatic β-cell death following MPA treatment. INS-1E cells (a pancreatic β-cell line) and isolated rat islets were treated with MPA for 12, 24, or 36 h, and subsequent microarray analysis showed that (Trx1) gene expression was significantly reduced by MPA. Further, Trx1 overexpression increased the cell viability, decreased the activations of c-jun N-terminal kinase (JNK) and caspase-3 by MPA, and attenuated ROS upregulation by MPA. Furthermore, siRNA knockdown of Trx1 increased MPA-induced cell death and the activations of p-JNK and caspase-3, and MPA significantly provoked the apoptosis of insulin-secreting cells via Trx1 downregulation. Our findings suggest that the prevention of Trx1 downregulation in response to MPA is critical for successful islet transplantation.     

Granzyme B induction signalling pathway in acute myeloid leukemia cell lines stimulated by tumor necrosis factor alpha and Fas ligand

Acute myeloid leukemia (AML) cell lines treated by genotoxic agents or by Tumor Necrosis Factor alpha (TNFalpha) acquire potent cytotoxicity towards myeloid cells through activation of granzyme B (GrB)/perforin (PFN) system. Here we first extend this observation to another death receptor activator, Fas Ligand (FasL). Moreover, we analyzed GrB induction signalling pathway in TNFalpha- and FasL-stimulated AML cells. The effects of TNFalpha and FasL on GrB expression were specifically mediated by p38MAPK (Mitogen-activated-protein-kinase) activation. Otherwise, TNFalpha and FasL stimulation led to radical oxygen species (ROS) generation and ASK1 (Apoptosis-signal-regulating-kinase-1) activation. Endogenous activation of ASK1 by either H2O2 or thioredoxin (Trx) reductase inhibition had the same effects as TNFalpha and FasL on GrB up regulation. Altogether, our results suggest that TNFalpha- and FasL-stimulated AML cell lytic induction is regulated by a signalling pathway involving sequentially, ROS generation, Trx oxidation, ASK1 activation, p38MAPK stimulation and GrB induction at mRNA and protein levels.     

A Comparison of Azacitidine and Decitabine Activities in Acute Myeloid Leukemia Cell Lines

Background

The cytidine nucleoside analogs azacitidine (AZA) and decitabine (DAC) are used for the treatment of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). Few non-clinical studies have directly compared the mechanisms of action of these agents in a head-to-head fashion, and the agents are often viewed as mechanistically similar DNA hypomethylating agents. To better understand the similarities and differences in mechanisms of these drugs, we compared their in vitro effects on several end points in human AML cell lines.

Methodology/Principal Findings

Both drugs effected DNA methyltransferase 1 depletion, DNA hypomethylation, and DNA damage induction, with DAC showing equivalent activity at concentrations 2- to 10-fold lower than AZA. At concentrations above 1 µM, AZA had a greater effect than DAC on reducing cell viability. Both drugs increased the sub-G1 fraction and apoptosis markers, with AZA decreasing all cell cycle phases and DAC causing an increase in G2-M. Total protein synthesis was reduced only by AZA, and drug-modulated gene expression profiles were largely non-overlapping.

Conclusions/Significance

These data demonstrate shared mechanisms of action of AZA and DAC on DNA-mediated markers of activity, but distinctly different effects in their actions on cell viability, protein synthesis, cell cycle, and gene expression. The differential effects of AZA may be mediated by RNA incorporation, as the distribution of AZA in nucleic acid of KG-1a cells was 65∶35, RNA∶DNA.     

Metabolomic approach to evaluating adriamycin pharmacodynamics and resistance in breast cancer cells

Continuous exposure of breast cancer cells to adriamycin induces high expression of P-gp and multiple drug resistance. However, the biochemical process and the underlying mechanisms for the gradually induced resistance are not clear. To explore the underlying mechanism and evaluate the anti-tumor effect and resistance of adriamycin, the drug-sensitive MCF-7S and the drug-resistant MCF-7Adr breast cancer cells were used and treated with adriamycin, and the intracellular metabolites were profiled using gas chromatography mass spectrometry. Principal components analysis of the data revealed that the two cell lines showed distinctly different metabolic responses to adriamycin. Adriamycin exposure significantly altered metabolic pattern of MCF-7S cells, which gradually became similar to the pattern of MCF-7Adr, indicating that metabolic shifts were involved in adriamycin resistance. Many intracellular metabolites involved in various metabolic pathways were significantly modulated by adriamycin treatment in the drug-sensitive MCF-7S cells, but were much less affected in the drug-resistant MCF-7Adr cells. Adriamycin treatment markedly depressed the biosynthesis of proteins, purines, pyrimidines and glutathione, and glycolysis, while it enhanced glycerol metabolism of MCF-7S cells. The elevated glycerol metabolism and down-regulated glutathione biosynthesis suggested an increased reactive oxygen species (ROS) generation and a weakened ability to balance ROS, respectively. Further studies revealed that adriamycin increased ROS and up-regulated P-gp in MCF-7S cells, which could be reversed by N-acetylcysteine treatment. It is suggested that adriamycin resistance is involved in slowed metabolism and aggravated oxidative stress. Assessment of cellular metabolomics and metabolic markers may be used to evaluate anti-tumor effects and to screen for candidate anti-tumor agents.     

Role of reactive oxygen species and Cr(VI) in Ras-mediated signal transduction

Previous studies have shown that a constitutively active isoform of Ras is able to produce superoxide radical (O2(-)). The present study investigate the mechanisms by which O2(-) radical mediates signals from Ras protein to the nucleus, leading to cellular responses such as apoptosis in Cr(VI)-stimulated cells. Two human prostate tumor cell lines, Ras(+), which overexpresses Ras, and Ras(-), which has a normal Ras level, were utilized. Compared to Ras(-) cells, Ras(+) cells exhibited higher susceptibility to apoptosis induced by Cr(VI). Catalase, sodium formate, and deferoxamine inhibited Cr(VI)-induced apoptosis. Similar differences were observed in both cellular DNA damage and the activation of p53 protein. The differences in Cr(VI)-induced cell responses in Ras(+) and Ras(-) cells were due to differences in the generation of free radicals between these two cells. ESR spin trapping measurements showed that Ras(+) cells generated more hydroxyl radical ((.)OH), O2(-) radical, and Cr(V) than Ras(-) cells following Cr(VI) stimulation. The generation of the reactive oxygen species (ROS) can be abolished by the addition of superoxide dismutase (SOD) or if the experiment were carried out in an argon atmosphere. Catalase inhibited spin adduct signals but was much less potent than SOD. The mechanism of ROS generation in Cr(VI)-stimulated Ras(+) cells involves the reduction of molecular oxygen to O2(-) radical by a flavoenzyme-containing NADPH oxidase complex as shown by oxygen consumption and diphenylene iodonium (DPI) inhibition. Results shown above support the following conclusions: (a) Ras protein mediates O2(-) radical generation through reduction of molecular oxygen by NADPH oxidase in Cr(VI)-stimulated cells. (b) The O2(-) radical and Cr(VI) produce other reactive species, including H2O2, OH radical, and Cr(V) through O2(-) dismutation and Haber-Weiss type of reactions. (c) Among these reactive species, (.)OH radical is responsible for the further transduction of signals from Ras to the nucleus, leading to various cell responses.     

The Bcl-2 inhibitor venetoclax inhibits Nrf2 antioxidant pathway activation induced by hypomethylating agents in AML

Induction of reactive oxygen species (ROS), an important process for the cytotoxicity of various acute myeloid leukemia (AML) therapies including hypomethylating agents (HMAs), concurrently activates the NF-E2-related factor 2 (Nrf2) antioxidant response pathway which in turn results in induction of antioxidant enzymes that neutralize ROS. In this study, we demonstrated that Nrf2 inhibition is an additional mechanism responsible for the marked antileukemic activity in AML seen with the combination of HMAs and venetoclax (ABT-199). HMA and venetoclax combined treatment augmented mitochondrial ROS induction and apoptosis compared with treatment HMA alone. Treatment of AML cell lines as well as primary AML cells with venetoclax disrupted HMA decitabine-increased nuclear translocation of Nrf2 and induction of downstream antioxidant enzymes including heme oxygenase-1 and NADP-quinone oxidoreductase-1. Venetoclax treatment also leads to dissociation of B-cell lymphoma 2 from the Nrf2/Keap-1 complex and targets Nrf2 to ubiquitination and proteasomal degradation. Thus, our results here demonstrated an undiscovered mechanism underlying synergistic effect of decitabine and venetoclax in AML cells, elucidating for impressive results in antileukemic activity against AML in preclinical and early clinical studies by combined treatment of these drugs.     

Curcumin-induced inhibition of cellular reactive oxygen species generation: Novel therapeutic implications

There is evidence for increased levels of circulating reactive oxygen species (ROS) in diabetics, as indirectly inferred by the findings of increased lipid peroxidation and decreased antioxidant status. Direct measurements of intracellular generation of ROS using fluorescent dyes also demonstrate an association of oxidative stress with diabetes. Although phenolic compounds attenuate oxidative stress-related tissue damage, there are concerns over toxicity of synthetic phenolic antioxidants and this has considerably stimulated interest in investigating the role of natural phenolics in medicinal applications. Curcumin (the primary active principle in turmeric,Curcuma longa Linn.) has been claimed to represent a potential antioxidant and antiinflammatory agent with phytonutrient and bioprotective properties. However there are lack of molecular studies to demonstrate its cellular action and potential molecular targets. In this study the antioxidant effect of curcumin as a function of changes in cellular ROS generation was tested. Our results clearly demonstrate that curcumin abolished both phorbol-12 myristate-13 acetate (PMA) and thapsigargin-induced ROS generation in cells from control and diabetic subjects. The pattern of these ROS inhibitory effects as a function of dose-dependency suggests that curcumin mechanistically interferes with protein kinase C (PKC) and calcium regulation. Simultaneous measurements of ROS and Ca²⁺ influx suggest that a rise in cytosolic Ca²⁺ may be a trigger for increased ROS generation. We suggest that the antioxidant and antiangeogenic actions of curcumin, as a mechanism of inhibition of Ca²⁺ entry and PKC activity, should be further exploited to develop suitable and novel drugs for the treatment of diabetic retinopathy and other diabetic complications.     

A systematic investigation of <Emphasis Type="Italic">Escherichia coli</Emphasis> central carbon metabolism in response to superoxide stress

Background  

The cellular responses of bacteria to superoxide stress can be used to model adaptation to severe environmental changes. Superoxide stress promotes the excessive production of reactive oxygen species (ROS) that have detrimental effects on cell metabolic and other physiological activities. To antagonize such effects, the cell needs to regulate a range of metabolic reactions in a coordinated way, so that coherent metabolic responses are generated by the cellular metabolic reaction network as a whole. In the present study, we have used a quantitative metabolic flux analysis approach, together with measurement of gene expression and activity of key enzymes, to investigate changes in central carbon metabolism that occur in Escherichia coli in response to paraquat-induced superoxide stress. The cellular regulatory mechanisms involved in the observed global flux changes are discussed.     

Bortezomib enhances cytotoxicity of ex vivo-expanded gamma delta T cells against acute myeloid leukemia and T-cell acute lymphoblastic leukemia

Engagement between the natural killer group 2, member D (NKG2D) receptor and its ligands is one of the main mechanisms used by immune cells to target stressed cells for cell death. NKG2D ligands are known markers of cellular stress and are often upregulated on tumor cells. Certain drugs can further increase NKG2D ligand levels, thereby making tumor cells more susceptible to immune cell detection and destruction. However, the effectiveness of this approach appears to be limited with drug treatment alone, possibly due to immune dysregulation in the setting of malignancies. We hypothesized that a more effective approach would be a combination of NKG2D ligand-inducing drugs, such as the proteasome inhibitor bortezomib, and ex vivo-expanded peripheral blood γδ T cells (i.e., Vγ9Vδ2 T cells). Acute myeloid leukemia (AML) is a high-risk hematologic malignancy, and treatment has shown limited benefit with the addition of bortezomib to standard chemotherapy regimens. Two AML cells lines, Nomo-1 and Kasumi-1, were treated with increasing concentrations of bortezomib, and changes in NKG2D ligand expression were measured. Bortezomib treatment significantly increased expression of the NKG2D ligand UL16 binding protein (ULBP) 2/5/6 in both cell lines. Vγ9Vδ2 T cells were expanded and isolated from peripheral blood of healthy donors to generate a final cellular product with a mean of 96% CD3⁺/γδ T-cell receptor-positive cells. Combination treatment of the AML cell lines with γδ T cells and bortezomib resulted in significantly greater cytotoxicity than γδ T cells alone, even at lower effector-to-target ratios. Based on the positive results against AML and the generalizable mechanism of this combination approach, it was also tested against T-cell acute lymphoblastic leukemia (T-ALL), another high-risk leukemia. Similarly, bortezomib increased ULBP 2/5/6 expression in T-ALL cell lines, Jurkat and MOLT-4 and improved the cytotoxicity of γδ T cells against each line. Collectively, these results show that bortezomib enhances γδ T-cell-mediated killing of both AML and T-ALL cells in part through increased NKG2D ligand-receptor interaction. Furthermore, proof-of-concept for the combination of ex vivo-expanded γδ T cells with stress ligand-inducing drugs as a therapeutic platform for high-risk leukemias is demonstrated.     

Cellular stress induced by resazurin leads to autophagy and cell death via production of reactive oxygen species and mitochondrial impairment

Mitochondrial bioenergetics and reactive oxygen species (ROS) often play important roles in cellular stress mechanisms. In this study we investigated how these factors are involved in the stress response triggered by resazurin (Alamar Blue) in cultured cancer cells. Resazurin is a redox reactive compound widely used as reporter agent in assays of cell biology (e.g. cell viability and metabolic activity) due to its colorimetric and fluorimetric properties. In order to investigate resazurin‐induced stress mechanisms we employed cells affording different metabolic and regulatory phenotypes. In HL‐60 and Jurkat leukemia cells resazurin caused mitochondrial disintegration, respiratory dysfunction, reduced proliferation, and cell death. These effects were preceded by a burst of ROS, especially in HL‐60 cells which were also more sensitive and contained autophagic vesicles. Studies in Rho⁰ cells (devoid of mitochondrial DNA) indicated that the stress response does not depend on the rates of mitochondrial respiration. The anti‐proliferative effect of resazurin was confirmed in native acute myelogenous leukemia (AML) blasts. In conclusion, the data suggest that resazurin triggers cellular ROS production and thereby initiates a stress response leading to mitochondrial dysfunction, reduced proliferation, autophagy, and cell degradation. The ability of cells to tolerate this type of stress may be important in toxicity and chemoresistance. J. Cell. Biochem. 111: 574–584, 2010. © 2010 Wiley‐Liss, Inc.     

A dual PI3 kinase/mTOR inhibitor BEZ235 reverses doxorubicin resistance in ABCB1 overexpressing ovarian and pancreatic cancer cell lines

BackgroundMulti-drug resistance (MDR) develops because cancer cells evade toxicity of several structurally unrelated drugs. Besides other mechanisms, MDR is linked to the overexpression of ATP Binding Cassette (ABC), transporters, among which ABCB1 is the best characterized one. Since overactivation of PI3K/Akt/mTOR plays a pivotal role in the growth of human cancers, we hypothesized whether dual PI3K and mTOR inhibitor, BEZ235 (BEZ, dactolisib) reverses resistance to doxorubicin (DOX).MethodsOvarian (A2780) and pancreatic (MiaPaca2) cancer cells were used to generate DOX-resistant clones by overexpressing ABCB1 or stepwise treatment of DOX. Intracellular accumulation of DOX was measured by flow cytometry after treatment with BEZ.ResultsBEZ treatment caused an increase in intracellular levels of DOX which was almost identical to the naïve parental cell lines. BEZ was found to be a weak substrate for ABCB1 as demonstrated by minimal increase in ATPase activity. BEZ treatment caused a dose-dependent decrease in cell viability in combination with DOX, which was associated with an increase in cleaved PARP expression in the drug resistant clones.ConclusionsThese results suggest that BEZ is a non-substrate inhibitor of ABCB1 and is able to effectively re-sensitize cells overexpressing ABCB1 to the effects of DOX.General significanceDual PI3 Kinase/mTOR inhibitor, BEZ, has the potential to reverse MDR in cancer patients.     

Effect of polyamines on superoxide dismutase activity under dexamethasone-induced apoptosis in rat thymocytes

The intracellular redox state is of importance for cell growth, differentiation, and apoptosis through reactive oxygen species (ROS) functioning as metabolic fine-tuner. Optimal levels of polyamines are necessary for growth, differentiation, and apoptotic cell death while they also protect cell from ROS accumulation. We have carried out studies to find out the interrelation between these two distant metabolic pathways. For that purpose, the glucocorticoid-triggered programmed cell death of rat thymocytes has been used. Our data confirm that SOD activity (which testifies both to the level of ROS generation and antioxidative defense state) changes in response to programmed cell death conditions and to alteration of intracellular polyamines level. Thymocytes death induced by dexamethasone is partially mediated by polyamines content. Our data prove that one of the molecular mechanisms of thymocytes population resistance after dexamethasone treatment is an enhanced level of antioxidant defense. It is evident that in dexamethasone-treated rat thymocytes polyamines modulate signal transduction processes to apoptosis development via changes in cellular redox status.     

Glucose promotes breast cancer aggression and reduces metformin efficacy

Metformin treatment has been associated with a decrease in breast cancer risk and improved survival. Metformin induces complex cellular changes, resulting in decreased tumor cell proliferation, reduction of stem cells, and apoptosis. Using a carcinogen-induced rodent model of mammary tumorigenesis, we recently demonstrated that overfeeding in obese animals is associated with a 50% increase in tumor glucose uptake, increased proliferation, and tumor cell reprogramming to an “aggressive” metabolic state. Metformin significantly inhibited these pro-tumorigenic effects. We hypothesized that a dynamic relationship exists between chronic energy excess (glucose by dose) and metformin efficacy/action.

Media glucose concentrations above 5 mmol/L was associated with significant increase in breast cancer cell proliferation, clonogenicity, motility, upregulation/activation of pro-oncogenic signaling, and reduction in apoptosis. These effects were most significant in triple-negative breast cancer (TNBC) cell lines. High-glucose conditions (10 mmol/L or above) significantly abrogated the effects of metformin. Mechanisms of metformin action at normal vs. high glucose overlapped but were not identical; for example, metformin reduced IGF-1R expression in both the HER2+ SK-BR-3 and TNBC MDA-MB-468 cell lines more significantly at 5, as compared with 10 mmol/L glucose. Significant changes in gene profiles related to apoptosis, cellular processes, metabolic processes, and cell proliferation occurred with metformin treatment in cells grown at 5 mmol/L glucose, whereas under high-glucose conditions, metformin did not significantly increase apoptotic/cellular death genes. These data indicate that failure to maintain glucose homeostasis may promote a more aggressive breast cancer phenotype and alter metformin efficacy and mechanisms of action.     

Adaptive stress response to menadione-induced oxidative stress in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> KNU5377

The molecular mechanisms involved in the ability of yeast cells to adapt and respond to oxidative stress are of great interest to the pharmaceutical, medical, food, and fermentation industries. In this study, we investigated the time-dependent, cellular redox homeostasis ability to adapt to menadione-induced oxidative stress, using biochemical and proteomic approaches in Saccharomyces cerevisiae KNU5377. Time-dependent cell viability was inversely proportional to endogenous amounts of ROS measured by a fluorescence assay with 2′,7′-dichlorofluorescin diacetate (DCFHDA), and was hypersensitive when cells were exposed to the compound for 60 min. Morphological changes, protein oxidation and lipid peroxidation were also observed. To overcome the unfavorable conditions due to the presence of menadione, yeast cells activated a variety of cell rescue proteins including antioxidant enzymes, molecular chaperones, energy-generating metabolic enzymes, and antioxidant molecules such as trehalose. Thus, these results show that menadione causes ROS generation and high accumulation of cellular ROS levels, which affects cell viability and cell morphology and there is a correlation between resistance to menadione and the high induction of cell rescue proteins after cells enter into this physiological state, which provides a clue about the complex and dynamic stress response in yeast cells.