Abstract
BackgroundCancer cells frequently adopt cellular and molecular alterations and acquire resistance to cytostatic drugs. Chemotherapy with oxaliplatin is among the leading treatments for colorectal cancer with a response rate of 50%, inducing intrastrand cross-links on the DNA. Despite of this drug’s efficiency, resistance develops in nearly all metastatic patients. Chemoresistance being of crucial importance for the drug’s clinical efficiency this study aimed to contribute to the identification and description of some cellular and molecular alterations induced by prolonged oxaliplatin therapy. Resistance to oxaliplatin was induced in Colo320 (Colo320R) and HT-29 (HT-29R) colorectal adenocarcinoma cell lines by exposing the cells to increasing concentrations of the drug. Alterations in morphology, cytotoxicity, DNA cross-links formation and gene expression profiles were assessed in the parental and resistant variants with microscopy, MTT, alkaline comet and pangenomic microarray assays, respectively.ResultsMorphology analysis revealed epithelial-to-mesenchymal transition in the resistant vs parental cells suggesting alterations of the cells’ adhesion complexes, through which they acquire increased invasiveness and adherence. Cytotoxicity measurements demonstrated resistance to oxaliplatin in both cell lines; Colo320 being more sensitive than HT-29 to this drug (P < 0.001). The treatment with oxaliplatin caused major DNA cross-links in both parental cell lines; in Colo320R small amounts of DNA cross-links were still detectable, while in HT-29R not. We identified 441 differentially expressed genes in Colo320R and 613 in HT-29R as compared to their parental counterparts (at least 1.5 -fold up- or down- regulation, p < 0.05). More disrupted functions and pathways were detected in HT-29R cell line than in Colo320R, involving genes responsible for apoptosis inhibition, cellular proliferation and epithelial-to-mesenchymal transition. Several upstream regulators were detected as activated in HT-29R cell line, but not in Colo320R.ConclusionsOur findings revealed a more resistant phenotype in HT-29R as compared to Colo320R and different cellular and molecular chemoresistance patterns induced by prolonged treatment with oxaliplatin in cell lines with identical origins (colorectal adenocarcinomas).
Highlights
Cancer cells frequently adopt cellular and molecular alterations and acquire resistance to cytostatic drugs
Cytotoxicity assessment The cytotoxicity of L-OHP on the selected CC cell lines was calculated using a sigmoid-type non-linear regression method. 2.76 (P < 0.0001) and 2.54 (P < 0.001)-fold increases of IC50 values were recorded in Colo320R and HT-29R cell lines, respectively (Table 1)
Validation of microarray results by quantitative real-time PCR (qRT-PCR) In order to assess the reliability of microarray results, we considered 9 Differentially expressed (DE) genes as candidates for validation by qRT-PCR as following: 3 common genes modulated in both cell lines (PTPRO, keratin 18 (KRT18), N-myc downstream regulated 1 (NDRG1)), 3 genes modulated exclusively in Colo320R (ID1, WNT inhibitory factor 1 (WIF1) and AVEN)
Summary
Cancer cells frequently adopt cellular and molecular alterations and acquire resistance to cytostatic drugs. Chemotherapy with oxaliplatin is among the leading treatments for colorectal cancer with a response rate of 50%, inducing intrastrand cross-links on the DNA. Despite of this drug’s efficiency, resistance develops in most metastatic patients. The last decade has brought major improvements in the treatment of cancers, but in spite of the efficacy of the cytostatic drugs, in time, cells adopt several cellular and molecular alterations, acquiring resistance. The intrastrand cross-links formed by L-OHP being the most abundant lesions capable of blocking both replication and transcription of DNA, they are considered to cause the major cytotoxic lesions and being directly involved in the cancer cells death [4,5]. Cells become resistant to platinum-based drugs through reduced cellular uptake, impaired DNA adducts formation, alterations in DNA repair genes such as ERCC1 and XRCC1 and modifications in the levels of copper transporters (ATP7A and ATP7B) [8,9,10]
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