Project description:A summary of the work associated to these microarrays is the following: The need for an integrated view of all data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize phenomena in terms of how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed Biological Association Networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). Seven cell lines representative of different types of cancer including colon cancer (HT29 and Caco2), breast cancer (MCF7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by microarrays covering the whole human genome and analyzed with the GeneSpring GX software package, v.7.3.1. Genes deregulated in common in the two colon cancer cell lines studied, were subject of Biological Association Networks construction. Dikkopf homolog-1 (DKK1) was a clear node of this network, and functional validations of this target using a siRNA showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of differentially expressed genes in the two breast cancer cell lines studied. siRNA treatment against UGT1A showed also an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was a gene overexpressed in common among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. Biological Association Networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using iRNA technology against these three genes show chemosensitization toward MTX. Two cell lines are compared, which are MCF7 breast cancer cells sensitive to methotrexate and MCF7 cells resistant to 10e-6M methotrexate. Six samples are provided which correspond to tripicates of each cell line. The samples provided were analyzed using the specific software GeneSpring GX.

Project description:The cannabinoid, cannabidiol (CBD), is part of the plant's natural defense system that when given to animals has many useful medicinal properties, including activity against cancer cells, modulation of the immune system, and efficacy in epilepsy. Although there is no consensus on its precise mode of action as it affects many cellular targets, CBD does appear to influence mitochondrial function. This would suggest that there is a cross-kingdom ability to modulate stress resistance systems that enhance homeostasis. As NAD(P)H autofluorescence can be used as both a metabolic sensor and mitochondrial imaging modality, we assessed the potential of this technique to study the in vitro effects of CBD using 2-photon excitation and fluorescence lifetime imaging microscopy (2P-FLIM) of NAD(P)H against more traditional markers of mitochondrial morphology and cellular stress in MCF7 breast cancer cells. 2P-FLIM analysis revealed that the addition of CBD induced a dose-dependent decrease in bound NAD(P)H, with 20 µM treatments significantly decreased the contribution of bound NAD(P)H by 14.6% relative to the control (p < 0.001). CBD also increased mitochondrial concentrations of reactive oxygen species (ROS) (160 ± 53 vs. 97.6 ± 4.8%, 20 µM CBD vs. control, respectively, p < 0.001) and Ca2+ (187 ± 78 vs. 105 ± 10%, 20 µM CBD vs. the control, respectively, p < 0.001); this was associated with a significantly decreased mitochondrial branch length and increased fission. These are all suggestive of mitochondrial stress. Our results support the use of NAD(P)H autofluorescence as an investigative tool and provide further evidence that CBD can modulate mitochondrial function and morphology in a dose-dependent manner, with clear evidence of it inducing oxidative stress at higher concentrations. This continues to support emerging data in the literature and may provide further insight into its overall mode of action, not only in cancer, but potentially its function in the plant and why it can act as a medicine.

Project description:A summary of the work associated to these microarrays is the following: The need for an integrated view of all data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize phenomena in terms of how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed Biological Association Networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). Seven cell lines representative of different types of cancer including colon cancer (HT29 and Caco2), breast cancer (MCF7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by microarrays covering the whole human genome and analyzed with the GeneSpring GX software package, v.7.3.1. Genes deregulated in common in the two colon cancer cell lines studied, were subject of Biological Association Networks construction. Dikkopf homolog-1 (DKK1) was a clear node of this network, and functional validations of this target using a siRNA showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of differentially expressed genes in the two breast cancer cell lines studied. siRNA treatment against UGT1A showed also an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was a gene overexpressed in common among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. Biological Association Networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using iRNA technology against these three genes show chemosensitization toward MTX. Two cell lines are compared, which are MCF7 breast cancer cells sensitive to methotrexate and MCF7 cells resistant to 10e-6M methotrexate. Six samples are provided which correspond to tripicates of each cell line. The samples provided were analyzed using the specific software GeneSpring GX.

Project description:IntroductionResistance to anti-estrogen therapies is a major cause of disease relapse and mortality in estrogen receptor alpha (ER?)-positive breast cancers. Tamoxifen or estrogen withdrawal increases the dependence of breast cancer cells on Notch signalling. Here, we investigated the contribution of Nicastrin and Notch signalling in endocrine-resistant breast cancer cells.MethodsWe used two models of endocrine therapies resistant (ETR) breast cancer: tamoxifen-resistant (TamR) and long-term estrogen-deprived (LTED) MCF7 cells. We evaluated the migratory and invasive capacity of these cells by Transwell assays. Expression of epithelial to mesenchymal transition (EMT) regulators as well as Notch receptors and targets were evaluated by real-time PCR and western blot analysis. Moreover, we tested in vitro anti-Nicastrin monoclonal antibodies (mAbs) and gamma secretase inhibitors (GSIs) as potential EMT reversal therapeutic agents. Finally, we generated stable Nicastrin overexpessing MCF7 cells and evaluated their EMT features and response to tamoxifen.ResultsWe found that ETR cells acquired an epithelial to mesenchymal transition (EMT) phenotype and displayed increased levels of Nicastrin and Notch targets. Interestingly, we detected higher level of Notch4 but lower levels of Notch1 and Notch2 suggesting a switch to signalling through different Notch receptors after acquisition of resistance. Anti-Nicastrin monoclonal antibodies and the GSI PF03084014 were effective in blocking the Nicastrin/Notch4 axis and partially inhibiting the EMT process. As a result of this, cell migration and invasion were attenuated and the stem cell-like population was significantly reduced. Genetic silencing of Nicastrin and Notch4 led to equivalent effects. Finally, stable overexpression of Nicastrin was sufficient to make MCF7 unresponsive to tamoxifen by Notch4 activation.ConclusionsETR cells express high levels of Nicastrin and Notch4, whose activation ultimately drives invasive behaviour. Anti-Nicastrin mAbs and GSI PF03084014 attenuate expression of EMT molecules reducing cellular invasiveness. Nicastrin overexpression per se induces tamoxifen resistance linked to acquisition of EMT phenotype. Our finding suggest that targeting Nicastrin and/or Notch4 warrants further clinical evaluation as valid therapeutic strategies in endocrine-resistant breast cancer.

Dataset Information

The redox state of cytochrome c modulates resistance to methotrexate in human MCF7 breast cancer cells.

Background

Results

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Publications

The redox state of cytochrome c modulates resistance to methotrexate in human MCF7 breast cancer cells.

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