Functional and transcriptomic characterization of carboplatin-resistant A2780 ovarian cancer cell line

Abstract

Background: Ovarian cancer is a significant cancer-related cause of death in women worldwide. The most used chemotherapeutic regimen is based on carboplatin (CBDCA). However, CBDCA resistance is the main obstacle to a better prognosis. An in vitro drug-resistant cell model would help in the understanding of molecular mechanisms underlying this drug-resistance phenomenon. The aim of this study was to characterize cellular and molecular changes of induced CBDCA-resistant ovarian cancer cell line A2780.

Methods: The cell selection strategy used in this study was a dose-per-pulse method using a concentration of 100 μM for 2 h. Once 20 cycles of exposure to the drug were completed, the cell cultures showed a resistant phenotype. Then, the ovarian cancer cell line A2780 was grown with 100 μM of CBDCA (CBDCA-resistant cells) or without CBDCA (parental cells). After, a drug sensitivity assay, morphological analyses, cell death assays and a RNA-seq analysis were performed in CBDCA-resistant A2780 cells.

Results: Microscopy on both parental and CBDCA-resistant A2780 cells showed similar characteristics in morphology and F-actin distribution within cells. In cell-death assays, parental A2780 cells showed a significant increase in phosphatidylserine translocation and caspase-3/7 cleavage compared to CBDCA-resistant A2780 cells (P < 0.05 and P < 0.005, respectively). Cell viability in parental A2780 cells was significantly decreased compared to CBDCA-resistant A2780 cells (P < 0.0005). The RNA-seq analysis showed 156 differentially expressed genes (DEGs) associated mainly to molecular functions.

Conclusion: CBDCA-resistant A2780 ovarian cancer cells is a reliable model of CBDCA resistance that shows several DEGs involved in molecular functions such as transmembrane activity, protein binding to cell surface receptor and catalytic activity. Also, we found that the Wnt/β-catenin and integrin signaling pathway are the main metabolic pathway dysregulated in CBDCA-resistant A2780 cells.

Keywords: A2780 cell line; Carboplatin; Drug resistance; Integrin signaling pathway; Ovarian cancer; Wnt/β-catenin-signaling pathway.

Fig. 1

The EC₅₀ values for cell viability in parental A2780 cells from CBDCA-resistant A2780 cells. EC₅₀ values were calculated using mathematic function antilog of values provided by sigmoidal dose–response curves. Antilog _{EC50 A2780-parental} (0.78 log µM) = 6.05 µM; Antilog _{EC50 A2780-CBDCA} (1.29 log µM) = 19.35 µM. ***P < 0.001

Fig. 2

Morphological comparisons between parental and CBDCA-resistant A2780 cells. a Giemsa staining and ImageJ analysis for morphometric observation according to the cellular and nuclear perimeter of each cell line. b Distribution of F-actin in both conditions. No significant differences were observed according to morphological features between parental and CBDCA-resistant A2780 cells

Fig. 3

Effect of CBDCA exposure in the viability and cell death of parental and CBDCA-resistant A2780 cells. a Cell viability. b Phosphatidylserine (PS) translocation. c Caspase-3/7 cleavage. These results confirm the CBDCA resistant phenotype of CBDCA-resistant A2780 cells. *P < 0.05; **P < 0.005; ***P < 0.0005

Fig. 4

Differentially expressed genes (DEGs) in carboplatin-resistant A2780. Upregulated (green bars) and downregulated (red bars) genes are ordered according to fold-change value. Gene ontology is shown according to the three main categories: binding (purple dots), catalytic activity (light blue dots) and transporter activity (orange dots). Table shows P-value, q-value and fold-change for each gene

Fig. 5

Gene ontology (GO) analysis of the DEGs. Pie charts represent the enriched GO: term associated to DEGs. a GO categories. b Binding category and its respective ontological subcategories. c Catalytic activity category and its respective ontological subcategories. d Transporter activity category and its respective ontological subcategories

Fig. 6

Molecular pathways analysis of DEGs. The table shows the signaling pathways deregulated in CBDCA-R-A2780 and their associated DEGs. In the graph, the vertical axes show the percentage of genes (orange) and number of genes (blue) annotated for each signaling pathway. Horizontal axis shows the name of the 21 deregulated metabolic pathways

Fig. 7

Workflow used in the development of CBDCA-resistant A2780 cells