ABSTRACT
Multidrug resistance may be achieved by the activation of membrane transporters, detoxification, alterations in DNA repair or failure in apoptotic pathways. Recent data have suggested an involvement of mitogenic signalling pathways mediated by Ras and phosphoinositol-3-kinase (PI3K/Akt) in controlling multidrug resistance. Since these pathways are important targets for therapeutic interference, we sought to investigate whether blocking effectors kinases by specific inhibitors would result in a sensitization toward cytotoxic drugs. We found that cotreatment of drug-resistant HT29RDB colon cancer cells with the topoisomerase inhibitor doxorubicin and the PI3K-inhibitor LY294002 resulted in massive apoptosis, while cotreatment with the Mek inhibitors PD98059 or U0126 had no effect. This suggested that the PI3K-pathways controls cell survival and drug resistance in these cells. Besides blocking Akt phosphorylation, the PI3K-inibitor increased the intracellular doxorubicin concentration threefold. LY294002 inhibits drug export in a competitive manner as revealed by measuring drug efflux in the presence and the absence of inhibitor. The efficacy of drug efflux inhibition by LY294002 was similar to that achieved by the MRP1 inhibitors MK571 and genistein. We conclude that the PI3K inhibitor LY294002 may have therapeutic potential when combined with doxorubicin in the treatment of MRP1-mediated drug resistance.
Subject(s)
Chromones/pharmacology , Colonic Neoplasms/drug therapy , Drug Resistance, Neoplasm/drug effects , Enzyme Inhibitors/pharmacology , Morpholines/pharmacology , Multidrug Resistance-Associated Proteins/metabolism , Antibiotics, Antineoplastic/metabolism , Antibiotics, Antineoplastic/pharmacology , Apoptosis/drug effects , Apoptosis/physiology , Biological Transport/drug effects , Biological Transport/physiology , Blotting, Western , Cell Line, Tumor , Doxorubicin/metabolism , Doxorubicin/pharmacology , Drug Resistance, Neoplasm/physiology , Fluorescent Antibody Technique , Humans , Phosphatidylinositol 3-Kinases/drug effectsABSTRACT
The human Y-box binding protein, YB-1, is a multifunctional protein that regulates gene expression. Nuclear expression of YB-1 has been associated with chemoresistance and poor prognosis of tumour patients. Representative samples from autopsied material of primary tumours from 77 patients with NSCLC were investigated by immunohistochemistry for subcellular distribution of YB-1 and p53, in order to evaluate the prognostic role of nuclear expression of YB-1. Cytoplasmic YB-1 expression was found in all tumour samples, whereas nuclear expression was only observed in 48%. There was no correlation with histological classification, clinical parameters or tumour size, stage and metastasis status. However, patients with positive nuclear YB-1 expression in tumours showed reduced survival times when compared with patients without nuclear expression. Including information about the histology and mutational status for p53 increased the prognostic value of nuclear YB-1. Patients with nuclear YB-1 expression and p53 mutations had the worst prognosis (median survival 3 months), while best outcome was found in patients with no nuclear YB-1 and wildtype p53 (median survival 15 months). This suggests that the combined analysis of both markers allows a better identification of subgroups with varying prognosis. Nuclear expression of Y-box binding protien seems to be an independent prognostic marker.
Subject(s)
Biomarkers, Tumor/analysis , CCAAT-Enhancer-Binding Proteins/analysis , Carcinoma, Non-Small-Cell Lung/mortality , Cell Nucleus/chemistry , DNA-Binding Proteins , Lung Neoplasms/mortality , Transcription Factors , Aged , Aged, 80 and over , Carcinoma, Non-Small-Cell Lung/chemistry , Cytoplasm/chemistry , Female , Follow-Up Studies , Humans , Immunohistochemistry , Lung Neoplasms/chemistry , Male , Middle Aged , Mutation , NFI Transcription Factors , Nuclear Proteins , Prognosis , Tumor Suppressor Protein p53/analysis , Y-Box-Binding Protein 1ABSTRACT
Genotoxic stress leads to nuclear translocation of the Y-box transcription factor YB-1 and enhanced expression of the multidrug resistance gene MDR1. Because hyperthermia is used for the treatment of colon cancer in combination with chemoradiotherapy, we investigated the influence of hyperthermia on YB-1 activity and the expression of multidrug resistance-related genes. Here we report that hyperthermia causes YB-1 translocation from the cytoplasm into the nucleus of human colon carcinoma cells HCT15 and HCT116. Nuclear translocation of YB-1 was associated with increased MDR1 and MRP1 gene activity, which is reflected in strong efflux pump activity. However, a combination of hyperthermia and drug treatment effectively reduced cell survival of the HCT15 and HCT116 cells. These results demonstrate that activation of MDR1 and MRP1 gene expression and increased efflux pump activity after hyperthermia were insufficient to cause an increase in drug resistance in colon cancer cell lines. The ability of hyperthermia to abrogate drug resistance in the presence of an increase in functional MDR proteins may provide an explanation for the efficacious results seen in the clinic in colon cancer patients treated with a combination of hyperthermia and chemotherapy.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/biosynthesis , Active Transport, Cell Nucleus , CCAAT-Enhancer-Binding Proteins/metabolism , DNA-Binding Proteins , Fever , Transcription Factors , ATP-Binding Cassette Transporters/biosynthesis , Antineoplastic Agents/pharmacology , Cell Line , Cell Nucleus/metabolism , Cell Survival/drug effects , Chloramphenicol O-Acetyltransferase/metabolism , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , Cytoplasm/metabolism , DNA, Complementary/metabolism , Doxorubicin/pharmacology , Flow Cytometry , Fluorescent Antibody Technique, Indirect , Genes, Reporter , Humans , Microscopy, Confocal , Microscopy, Fluorescence , Multidrug Resistance-Associated Proteins , NFI Transcription Factors , Nuclear Proteins , Plasmids/metabolism , RNA/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Rhodamines/metabolism , Temperature , Time Factors , Transfection , Tumor Cells, Cultured , Y-Box-Binding Protein 1ABSTRACT
Regulated mRNA turnover is a highly important process, but its mechanism is poorly understood. Using interleukin-2 (IL-2) mRNA as a model, we described a role for the JNK-signaling pathway in stabilization of IL-2 mRNA during T-cell activation, acting via a JNK response element (JRE) in the 5' untranslated region (UTR). We have now identified two major RNA-binding proteins, nucleolin and YB-1, that specifically bind to the JRE. Binding of both proteins is required for IL-2 mRNA stabilization induced by T-cell activation signals and for JNK-induced stabilization in a cell-free system that duplicates essential features of regulated mRNA decay. Nucleolin and YB-1 are required for formation of an IL-2 mRNP complex that responds to specific mRNA stabilizing signals.
Subject(s)
CCAAT-Enhancer-Binding Proteins , DNA-Binding Proteins/metabolism , Interleukin-2/genetics , Lymphocyte Activation , Mitogen-Activated Protein Kinases/metabolism , Phosphoproteins/metabolism , RNA Stability , RNA-Binding Proteins/metabolism , T-Lymphocytes/metabolism , Transcription Factors , Amino Acid Sequence , Base Sequence , Cytoplasm/chemistry , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , Enzyme Activation , Gene Expression Regulation/genetics , Half-Life , Humans , JNK Mitogen-Activated Protein Kinases , Jurkat Cells , MAP Kinase Signaling System , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogen-Activated Protein Kinases/genetics , Molecular Sequence Data , Mutation/genetics , NFI Transcription Factors , Nuclear Proteins , Phosphoproteins/chemistry , Phosphoproteins/genetics , Phosphoproteins/isolation & purification , Precipitin Tests , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/isolation & purification , Response Elements/genetics , T-Lymphocytes/cytology , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , Y-Box-Binding Protein 1 , NucleolinABSTRACT
Breast cancers are either primarily resistant to chemotherapy (intrinsic resistance), or respond to chemotherapy but later recur with a multidrug-resistant phenotype because of overexpression of the multidrug transporter P-glycoprotein. The MDR1 gene encoding P-glycoprotein may be transcriptionally regulated by a Y-box transcription factor. We now report that, in multidrug-resistant MCF-7 breast cancer cells, nuclear localization of YB-1 is associated with MDR-1 gene expression. In drug-sensitive MCF-7 cells, however, YB-1 was localized to the cytoplasm. Regulated overexpression of YB-1 in drug-sensitive diploid breast epithelial cells induced MDR-1 gene expression and multidrug resistance. In 27 out of 27 untreated primary breast cancers, YB-1 protein was expressed in the cytoplasm although it was undetectable in normal breast tissue of these patients. In a subgroup of tumors (9/27), however, YB-1 was also localized to the nucleus and, in these cases, high levels of P-glycoprotein were present. These results show that in a subset of untreated primary breast cancers, nuclear localization of YB-1 protein is associated with intrinsic multidrug resistance. Our data show that YB-1 has an important role in controlling MDR1 gene transcription and this finding provides a basis for the analysis of molecular mechanisms responsible for intrinsic multidrug resistance in human breast cancer.
