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. 2013 Jul 16:14:480.
doi: 10.1186/1471-2164-14-480.

Oxaliplatin induces different cellular and molecular chemoresistance patterns in colorectal cancer cell lines of identical origins

Piroska Virag  1 , Eva Fischer-Fodor, Maria Perde-Schrepler, Ioana Brie, Corina Tatomir, Loredana Balacescu, Ioana Berindan-Neagoe, Bogdan Victor, Ovidiu Balacescu
Affiliations

Oxaliplatin induces different cellular and molecular chemoresistance patterns in colorectal cancer cell lines of identical origins

Piroska Virag et al. BMC Genomics. .

Abstract

Background: 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 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.

Results: Morphology 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.

Conclusions: Our 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).

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Figures

Figure 1
Figure 1
Microscopic images of Colo320 (a), Colo320R (b), HT-29 (c) and HT-29R (d) CC cell lines. Morphologic analyses revealed round (a1, c1) and polygonal (c2) types for the sensitive cells. For the resistant ones, transitional (d1) and fusiforme (b2, d2) types of cells were identified and specific alterations for epithelial-to-mesenchymal transition: presence of pseudopodia (b1; d1), loss of cell polarity and adoption of fusiforme shape (b2; d2), increased adherence for the generally non-adherent Colo320R (b3) and increased cell-to-cell distance for the HT-29R cells (d4).
Figure 2
Figure 2
Representation of lesion scores (LS) of the Colo320 and Colo320R CC cell lines. Controls (C); irradiated with doses of 2Gy (I/2) and 4Gy (I/4) of gamma irradiations; exposed to 50 μg/ml or 100 μg/ml L-OHP and irradiated with a dose of 2Gy radiations (I/2/50 and I/2/100, respectively); exposed to 100 μg/ml L-OHP and irradiated with doses of 2Gy (I/2/100) and 4Gy (I/4/100); values are means of three experiments (*** p < 0.0001, one-way analysis of variance test).
Figure 3
Figure 3
Representation of lesion scores (LS) of the HT-29 and HT-29R CC cell lines. Controls (C); irradiated with doses of 2Gy (I/2) and 4Gy (I/4) of gamma irradiations; exposed to 50 μg/ml or 100 μg/ml L-OHP and irradiated with a dose of 2Gy radiations (I/2/50 and I/2/100, respectively); exposed to 100 μg/ml L-OHP and irradiated with doses of 2Gy (I/2/100) and 4Gy (I/4/100); values are means of three experiments (* p < 0.05, ** p < 0.001 and *** p < 0.0001, one-way analysis of variance test).
Figure 4
Figure 4
Venn diagram of DE genes induced by L-OHP in tested cell lines. The overlap area indicates the common set of genes (sequences) modulated by L-OHP in Colo320R and HT-29R cell lines. In the left area is represented the number of the genes (392) modulated by L-OHP only in Colo320R, whereas in the right area is represented the number of the genes (564) uniquely modulated by the drug in HT-29R cell line.
Figure 5
Figure 5
IPA Network. The network displays the relationship between upstream regulators and their target molecules in HT-29R cell line. The colors indicate the level of mRNA expression: upregulated genes are represented in red and downregulated genes in green.
Figure 6
Figure 6
qRT-PCR validation of microarray results in Colo320 cell line. The bars represent the mean (± SD) of three biological replicates for every gene. All genes were normalized to 18 rRNA and fold regulation was calculated relative to Colo320S. (* p < 0.05, ** p < 0.01, *** p < 0.001).
Figure 7
Figure 7
qRT-PCR validation of microarray results in HT-29 cell line. The bars represent the mean (± SD) of three biological replicates for every gene. All genes were normalized to 18 rRNA and fold regulation was calculated relative to HT-29S. (* p < 0.05, ** p < 0.01, *** p < 0.001).
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