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Transcriptomic comparison of chemosensitive and chemoresistant cells

ABSTRACT: First, in a model of xenografts induced with HT-29 human colon cancer cells, we demonstrated that the transcription factor NF-kB was involved in the resistance to CPT-11. Then, from one tumor treated with CPT-11 plus a NF-kB inhibitor, we established a resistant cell line to CPT-11. The aim of this study was to compare the expression of genes involved in the signaling pathway of NF-kB between the sensitive and the resistant cell lines and to identify other genes (target or not of NF-kB) which the expression was modified when the resistant phenotype was acquired.

ORGANISM(S): Homo sapiens

PROVIDER: GSE23433 | GEO | 2012/08/23

SECONDARY ACCESSION(S): PRJNA131103

REPOSITORIES: GEO

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Project description:Metastasis and drug-resistance are major problems in cancer chemotherapy. The purpose of this work was to analyze the molecular mechanisms underlying the invasive potential of drug-resistant colon carcinoma cells. Cellular models included the parental HT-29 cell line and its drug-resistant derivatives selected after chronic treatment with either 5-fluorouracil (5-FU), methotrexate (MTX), doxorubicin (DOX) or oxaliplatin (OXA). Drug-resistant invasive cells were compared to non invasive cells using cDNA microarray, qRT-PCR, flow cytometry, immunoblots and ELISA. Functional and cellular signaling analyses were undertaken using pharmacological inhibitors, function-blocking antibodies, and silencing by retrovirus-mediated RNA interference. 5-FU- and MTX-resistant HT-29 cells expressing an invasive phenotype in collagen type I and a metastatic behaviour in immunodeficient mice exhibited high expression of the chemokine receptor CXCR4. Macrophage migration inhibitory factor (MIF) was identified as the critical autocrine CXCR4 ligand promoting invasion in drug-resistant colon carcinoma HT-29 cells. Silencing of CXCR4 and impairing the MIF-CXCR4 signaling pathways by ISO-1, pAb FL-115, AMD-3100, mAb 12G5, and BIM-46187 abolished this aggressive phenotype. Induction of CXCR4 is associated with up-regulation of two genes encoding transcription factors previously shown to control CXCR4 expression (HIF-2a and ASCL2) and maintenance of intestinal stem cells (ASCL2). Enhanced CXCR4 expression was detected in liver metastases resected from colon cancer patients treated by the standard FOLFOX regimen. Combination therapies targeting the CXCR4-MIF axis can potentially counteract the emergence of the invasive metastatic behaviour in clonal derivatives of drug-resistant colon cancer cells. Samples: HT-29 cell clones were obtained by limiting dilution from HT-29 subpopulations resistant to methothrexate (HT-29 5M21 cell clone) and or to 5-Fluoro-uracil (HT-29 5F7 and HT-29 5F31). Samples were provided by Dr. T Lesuffleur. Xenografts: HT-29 5M21, HT-29 5F7, and HT-29 5F31 xenografts were obtained by a first subcutaneous inoculation of cells in Nude mice and then fragments of subcutaneous tumors were reimplantated in SCID mice. First experiment of microarrays: HT-29 5M21 and HT-29 5F7 cells (that are invasive in in vitro assays on type I collagen) were compared to HT-29 5F31 cells (that are not invasive in vitro on type I collagen). Samples were co-hybridized on Agilent Human 44K GEP arrays (respectively 5F31 vs 5M21, 5F31 vs 5F7, 5M21 vs 5F31 and 5F7 vs 5F31). Second experiment of microarrays: HT-29 5M21 and HT-29 5F7 xenografts (that develop metastases in immuno-deficient mice lungs) were compared to HT-29 5F31 xenografts (that do not develop metastases in immuno-deficient mice lungs). Samples were consequently co-hybridized on human and mouse 4x44K GEP arrays (respectively AG41_5F31-5M21-138869, AG39_5F31-5F7-138867, AG42_5M21-5F31-138870 and AG40_5F7-5F31-138868 for human microarrays; 5M21_5F31_27033_4, 5F31_5M21_27033_3, 5F7_5F31_27033_2 and 5F31_5F7_27033_1_1 for mouse microarrays).

Project description:A commercially available product containing three probiotic bacterial strains (Lactobacillus helveticus R0052, Bifidobacterium longum subsp. infantis R0033, and Bifidobacterium bifidum R0071) was previously shown in animal trials to modulate both TH1 and TH2 immune responses. Clinical studies on this combination of bacteria have also shown positive health effects against seasonal winter diseases and rotavirus infection. The goal of this study was to use a well-established in vitro intestinal epithelial (HT-29) cell model that has been shown to constitutively express double-stranded RNA (dsRNA) sensors (Toll-like receptor 3[TLR3], retinoic acid-inducible gene I, melanoma differentiation-associated gene 5, and dsRNA-activated protein kinase). By using the HT-29 cell model, we wanted to evaluate whether or not this combination of three bacteria had the capacity to immune modulate the host cell response to a dsRNA ligand, poly(IM-bM-^@M-"C). Using a custom-designed, two-color expression microarray targeting genes of the human immune system, we investigated the response of HT-29 cells challenged with poly(IM-bM-^@M-"C) both in the presence and in the absence of the three probiotic bacteria. We observed that the combination of the three bacteria had a major impact on attenuating the expression of genes connected to proinflammatory TH1 and antiviral innate immune responses compared to that obtained by the poly(IM-bM-^@M-"C)-only challenge. Major pathways through which the multistrain combination may be eliciting its immune-modulatory effect include the TLR3-TRIF, mitogen-activated protein kinase, and NF-KB signaling pathways.Such a model may be useful for selecting potential biomarkers for the design of future clinical trials The overall design consisted of 3 samples of HT-29 cells treated with poly (I:C)-only, probiotic combination (PC) and poly (I:C) + PC versus unchallenged HT-29 cells. A minimum of four dye-swap hybridizations (4 biological replicates) were performed for each of the 3 samples analyzed. Unchallenged HT-29 cells were controls and challenged HT-29 cells with poly (I:C)-only, PC-only and poly (I:C) + PC were treated samples.

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Transcriptomic comparison of chemosensitive and chemoresistant cells

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