ar-c155858 and Breast-Neoplasms

In 1930, Otto Warburg reported that "aerobic glycolysis" is the intrinsic property of all tumor cells' fermentation of glucose to L-Lactate by lactate dehydrogenase A (LDHA) activity. This only produces per mole of glucose two moles of adenosine triphosphate (ATP), compared with 32 moles of ATP in a normal cell. Thus, tumor cells have to uptake 30 folds more glucose, the resulting accumulated lactate are then transported by a monocarboxylate transporter (MCT) with the participation of a CD147 molecule. Inhibition of MCT1 by RNA interference (RNAi) disrupted the unique metabolism of the tumor and caused tumor cell death. However, the effectiveness of the strategies depends on the targeted delivery of the therapeutics.. In this study, a synergistic approach was used to target LDHA and MCT1 with small molecule inhibitors FX11 and AR-C155858, respectively. Cell cytotoxicity assays (AlamarBlue assay), and Mitochondria Membrane Potential (JC-1) dye assays were performed on human breast cancer cells MCF-7 and colorectal cancer cells HCT116. To achieve this aim, the following objectives were proposed: the effect of metabolic inhibitors on tumor glycolytic metabolite environment, and the efficacy of metabolite inhibitors on human breast and colorectal cancer cells in vitro. Then, gene expression analysis was performed using Qiagen RT2 Profiler PCR array for apoptosis. All these assays were performed on human breast cancer cells MCF-7 and colorectal cancer cells HCT116. Normal human fibroblasts were used as control cells under normal and hypoxic culture conditions.. In this study, the use of FX-11 inhibitors under normoxia or hypoxia in two or more cancer and normal cell lines has a direct effect on LDHA, whereby it inhibits its production, and this reduces the growth and cell proliferation of tumors. One of the more significant findings to emerge from this study is that using AR-C155858 inhibitor alone has increased the cell proliferation and showed no significant changes compared with the control. The other major finding was that combination of the two inhibitors, FX-11 and AR-C155858, under normoxia or hypoxia in two different cell lines MCF-7 and HCT-116 measured a decrease in the cells proliferative and red/green ratio.. We successfully demonstrated that a combination of MCT1 inhibitor and LDHA inhibitor led to better outcomes. Indeed, this makes LDHA an ideal metabolic therapeutic target.

Topics: Adenosine Triphosphate; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Female; Glucose; Glycolysis; Humans; Lactate Dehydrogenase 5; Lactates; Monocarboxylic Acid Transporters

AR-C155858 and AZD3965, pyrrole pyrimidine derivatives, represent potent monocarboxylate transporter 1 (MCT1) inhibitors, with potential immunomodulatory and chemotherapeutic properties. Currently, there is limited information on the inhibitory properties of this new class of MCT1 inhibitors. The purpose of this study was to characterize the concentration- and time-dependent inhibition of L-lactate transport and the membrane permeability properties of AR-C155858 and AZD3965 in the murine 4T1 breast tumor cells that express MCT1. Our results demonstrated time-dependent inhibition of L-lactate uptake by AR-C155858 and AZD3965 with maximal inhibition occurring after a 5-min pre-incubation period and prolonged inhibition. Following removal of AR-C155858 or AZD3965 from the incubation buffer, inhibition of L-lactate uptake was only fully reversed after 3 and 12 h, respectively, indicating that these inhibitors are slowly reversible. The uptake of AR-C155858 was concentration-dependent in 4T1 cells, whereas the uptake of AZD3965 exhibited no concentration dependence over the range of concentrations examined. The uptake kinetics of AR-C155858 was best fitted to a Michaelis-Menten equation with a diffusional clearance component, P (K

Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Coumaric Acids; Drug Screening Assays, Antitumor; Female; Humans; Hydrogen-Ion Concentration; Lactic Acid; Mice; Monocarboxylic Acid Transporters; Pyrimidinones; Symporters; Thiophenes; Uracil

AZD3965, a pyrole pyrimidine derivative, is a potent and orally bioavailable inhibitor of monocarboxylate transporter 1 (MCT1), currently in a Phase I clinical trial in UK for lymphomas and solid tumors. There is currently no published assay for AZD3965. The objectives of this study were to develop and validate a LC/MS/MS assay for quantifying AZD3965 in mouse plasma and tumor tissue. Protein precipitation with 0.1% formic acid in acetonitrile was used for sample preparation. Chromatographic separation was achieved on a C18 column followed by tandem mass spectrometry detection in multiple reaction monitoring mode with utilizing Atmospheric Pressure Chemical Ionization. AR-C155858 was used as the internal standard. The inter-day and intra-day precision and accuracy of quality control samples evaluated in plasma and tumor tissue were less than ±7% of the nominal concentrations. The extraction recovery, matrix effect and stability values were all within acceptable levels. Sample dilution integrity, accessed by diluting plasma spiked with AZD3965 10-fold with blank plasma, was 101%. The lower limit of quantification (LLOQ) and upper limit of quantification (ULOQ) were 0.15 ng/mL and 12 μg/mL, respectively, in plasma. The assay of AZD3965 in tumor tissue was also validated with good precision and accuracy. The LLOQ was 0.15 ng/mL in tumor tissue. This assay was successfully applied to pharmacokinetic and murine 4T1 breast tumor xenograft studies of AZD3965 in mice.

Topics: Acetonitriles; Animals; Atmospheric Pressure; Breast Neoplasms; Cell Line, Tumor; Chromatography, Liquid; Female; Heterografts; Mice; Plasma; Pyrimidines; Pyrimidinones; Reproducibility of Results; Tandem Mass Spectrometry; Thiophenes; Uracil

High lactate concentration in tumors is associated with bad prognosis. Lactate is released by glycolytic cells in tumors and recaptured by oxidative cancer cells to feed the tricarboxylic acid (TCA) cycle after conversion into pyruvate. Monocarboxylate transporters (MCT) mediate these fluxes of proton-linked lactate and represent attractive targets to interrupt lactate shuttle and to inhibit tumor growth. Here, we investigated the properties of 7-aminocarboxycoumarins (7ACC) developed to selectively interfere with lactate fluxes in the lactate-rich tumor microenvironment. The pharmacologic properties of two compounds of this family, including their effects on lactate influx and efflux and antitumor activity, were investigated using human cancer cell lines and mouse xenograft models. Contrary to the reference MCT1 inhibitor AR-C155858, 7ACC unexpectedly inhibited lactate influx but not efflux in tumor cells expressing MCT1 and MCT4 transporters. 7ACC delayed the growth of cervix SiHa tumors, colorectal HCT116 tumors, and orthoptopic MCF-7 breast tumors. MCT target engagement was confirmed by the lack of activity of 7ACC on bladder UM-UC-3 carcinoma that does not express functional MCT. 7ACC also inhibited SiHa tumor relapse after treatment with cisplatin. Finally, we found that contrary to AR-C155858, 7ACC did not prevent the cell entry of the substrate-mimetic drug 3-bromopyruvate (3BP) through MCT1, and contributed to the inhibition of tumor relapse after 3BP treatment. In conclusion, our results indicate that 7ACC selectively affects a single part of the MCT symporter translocation cycle, leading to strict inhibition of lactate influx. This singular activity is associated with antitumor effects less prone to resistance and side effects.

Topics: Animals; Breast Neoplasms; Colorectal Neoplasms; Coumarins; Female; HCT116 Cells; Humans; Lactic Acid; MCF-7 Cells; Mice; Neoplasm Recurrence, Local; Prognosis; Thiophenes; Uracil

Here, we used quantitative proteomics analysis to identify novel therapeutic targets in cancer stem cells and/or progenitor cells. For this purpose, mammospheres from two ER-positive breast cancer cell lines (MCF7 and T47D) were grown in suspension using low-attachment plates and directly compared to attached monolayer cells grown in parallel. This allowed us to identify a subset of proteins that were selectively over-expressed in mammospheres, relative to epithelial monolayers. We focused on mitochondrial proteins, as they appeared to be highly upregulated in both MCF7 and T47D mammospheres. Key mitochondrial-related enzymes involved in beta-oxidation and ketone metabolism were significantly upregulated in mammospheres, as well as proteins involved in mitochondrial biogenesis, and specific protein inhibitors of autophagy/mitophagy. Overall, we identified >40 "metabolic targets" that were commonly upregulated in both MCF7 and T47D mammospheres. Most of these "metabolic targets" were also transcriptionally upregulated in human breast cancer cells in vivo, validating their clinical relevance. Based on this analysis, we propose that increased mitochondrial biogenesis and decreased mitochondrial degradation could provide a novel mechanism for the accumulation of mitochondrial mass in cancer stem cells. To functionally validate our observations, we utilized a specific MCT1/2 inhibitor (AR-C155858), which blocks the cellular uptake of two types of mitochondrial fuels, namely ketone bodies and L-lactate. Our results indicate that inhibition of MCT1/2 function effectively reduces mammosphere formation, with an IC-50 of ~1 µM, in both ER-positive and ER-negative breast cancer cell lines. Very similar results were obtained with oligomycin A, an inhibitor of the mitochondrial ATP synthase. Thus, the proliferative clonal expansion of cancer stem cells appears to require oxidative mitochondrial metabolism, related to the re-use of monocarboxylic acids, such as ketones or L-lactate. Our findings have important clinical implications for exploiting mitochondrial metabolism to eradicate cancer stem cells and to prevent recurrence, metastasis and drug resistance in cancer patients. Importantly, a related MCT1/2 inhibitor (AZD3965) is currently in phase I clinical trials in patients with advanced cancers: http://clinicaltrials.gov/show/NCT01791595.

Topics: Breast Neoplasms; Cell Line, Tumor; Female; Humans; MCF-7 Cells; Mitochondria; Mitochondrial Proteins; Molecular Targeted Therapy; Monocarboxylic Acid Transporters; Neoplastic Stem Cells; Proteomics; Symporters; Thiophenes; Uracil