ABSTRACT
Irinotecan is a topoisomerase I inhibitor widely used as an anticancer agent in the treatment of metastatic colon cancer. However, its efficacy is often limited by the development of resistance. We have isolated a colon carcinoma cell line, HCT116-SN6, which displays a 6-fold higher resistance to SN38, the active metabolite of irinotecan. In this paper, we studied the molecular mechanisms that cause resistance to SN38 in the HCT116-SN6 cell line. First, we analyzed proliferation, cell cycle distribution, apoptosis, topoisomerase I expression and activity in SN38-resistant (HCT116-SN6) and sensitive (HCT116-s cells). We showed that the SN38-induced apoptosis and the SN38-activated cell cycle checkpoints leading to G(2)/M cell cycle arrest were similar in both cell lines. Topoisomerase I expression and catalytic activity were also unchanged. Then, we compared mRNA expression profiles in the two cell lines using the Affymetrix Human Genome GeneChip arrays U133A and B. Microarray analysis showed that among the genes, which were differentially expressed in HCT116-s and HCT116-SN6 cells, 27% were related to cell proliferation suggesting that proliferation might be the main target in the development of resistance to SN38. This result correlates with the phenotypic observation of a reduced growth rate in HCT116-SN6 resistant cells. Furthermore, 29% of the overexpressed genes were Interferon Stimulated Genes and we demonstrate that their overexpression is, at least partially, due to endogenous activation of the p38 MAP kinase pathway in SN38 resistant cells. In conclusion, a slower cell proliferation rate may be a major cause of acquired resistance to SN38 via a reduction of cell cycle progression through the S phase which is mandatory for the cytotoxic action of SN38. This lower growth rate could be due to the endogenous activation of p38.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Camptothecin/analogs & derivatives , Cell Proliferation , Colonic Neoplasms/drug therapy , Colonic Neoplasms/pathology , Drug Resistance, Neoplasm , Interferons/metabolism , Apoptosis , Camptothecin/pharmacology , Cell Cycle , Cell Line, Tumor , Cell Survival , DNA Topoisomerases, Type I/metabolism , Humans , Inhibitory Concentration 50 , Irinotecan , Oligonucleotide Array Sequence Analysis , Time FactorsABSTRACT
DNA microarray technology enables investigators to measure the expression of several 1000 mRNA species simultaneously in a biological specimen. However, the reliability of the microarray technology to detect transcriptional differences representative of the original samples is affected by the quality of the extracted RNA. Thus, it is of critical importance to standardize sample-handling protocols and to perform a quality assessment of RNA preparations. In this report, 59 human tissue samples were used to evaluate the relationships between RNA quality and gene expression. From Affymetrix GeneChip array data analysis of these samples, we compared the performance of the 28S/18S ratio, two computer methods (RIN and degradometer) and our in-house RNA quality scale (RQS) in assessing RNA quality. The optimal RNA reliability threshold was determined for each method using statistical discrimination measures. We showed that RQS, RIN and degradometer have a similar capacity to detect reliable RNA samples whereas the 28S/18S ratio leads to a misleading categorization. Furthermore, we developed a new approach, based on clustering analyses of full chip expression, to control RNA quality after hybridization experiments. The combination of these methods, allowing monitoring of RNA quality prior to and after the hybridization experiments, ensured reliable and reproducible microarray data.
Subject(s)
Gene Expression Profiling/methods , RNA Stability , RNA/chemistry , RNA/standards , Cluster Analysis , Humans , Nucleic Acid Hybridization , Oligonucleotide Array Sequence Analysis , Quality Control , RNA/genetics , Reproducibility of ResultsABSTRACT
Overcoming drug resistance has become an important issue in cancer chemotherapy. Among all known mechanisms that confer resistance, active efflux of chemotherapeutic agents by proteins from the ATP-binding cassette family has been extensively reported. The aim of the present study was to determine the involvement of ABCG2 in resistance to SN38 (the active metabolite of irinotecan) in colorectal cancer. By progressive exposure to increasing concentrations of SN38, we isolated 2 resistant clones from the human colon carcinoma cell line HCT116. These clones were 6- and 53-fold more resistant to SN38 than the HCT116-derived sensitive clone. Topoisomerase I expression was unchanged in our resistant variants. The highest resistance level correlated with an ABCG2 amplification. This overexpression was associated with a marked decrease in the intracellular accumulation of SN38. The inhibition of ABCG2 function by Ko143 demonstrated that enhanced drug efflux from resistant cells was mediated by the activity of ABCG2 protein and confirmed that ABCG2 is directly involved in acquired resistance to SN38. Furthermore, we show, for the first time in clinical samples, that the ABCG2 mRNA content in hepatic metastases is higher after an irinotecan-based chemotherapy than in irinotecan-naive metastases. In conclusion, this study supports the potential involvement of ABCG2 in the development of irinotecan resistance in vivo.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Adenocarcinoma/drug therapy , Camptothecin/analogs & derivatives , Camptothecin/therapeutic use , Colonic Neoplasms/drug therapy , Drug Resistance, Neoplasm , Liver Neoplasms/drug therapy , Neoplasm Proteins/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/secondary , Antineoplastic Agents, Phytogenic/therapeutic use , Colon/metabolism , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , DNA Topoisomerases, Type I/metabolism , Gene Expression Regulation, Neoplastic , Humans , Irinotecan , Liver Neoplasms/metabolism , Liver Neoplasms/secondary , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/genetics , RNA, Messenger/metabolism , RNA, Neoplasm , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
To investigate acquired resistance to oxaliplatin, we selected two resistant clones from the HCT116 cell line. We found that the resistant phenotype was associated with resistance to oxaliplatin-induced apoptosis as demonstrated by FACS analysis and by Western blotting of caspase 3 activation. In addition, the resistant phenotype showed a concomitant resistance to lonidamine and arsenic trioxide which are inducers of mitochondrial apoptosis. Furthermore, a complete loss of Bax expression due to a frameshift mutation was observed in the most resistant clone. Taken together, these findings suggest that altered mitochondrial-mediated apoptosis could play a role in oxaliplatin resistance.
