Transcriptomic and functional pathways analysis of ascorbate-induced cytotoxicity and resistance of Burkitt lymphoma

Abstract

Ascorbate is a pro-oxidant that generates hydrogen peroxide-dependent cytotoxity in cancer cells without adversely affecting normal cells. To determine the mechanistic basis for this phenotype, we selected Burkitt lymphoma cells resistant to ascorbate (JLPR cells) and their ascorbate-sensitive parental cells (JLPS cells). Compared with JLPS cells, the increased glucose uptake in JLPR cells (with upregulated glucose transporters, increased antioxidant enzyme activity, and altered cell cycling) conferred ascorbate-induced cytotoxicity and resistance. Transcriptomic profiles and function pathway analysis identified differentially expressed gene signatures for JLPR cells and JLPS cells, which differential expression levels of five genes (ATF5, CD79B, MHC, Myosin, and SAP18) in ascorbate-resistant cells were related to phosphoinositide 3 kinase, cdc42, DNA methylation and transcriptional repression, polyamine regulation, and integrin-linked kinase signaling pathways. These results suggested that coordinated changes occurred in JLPR cells to enable their survival when exposed to the cytotoxic pro-oxidant stress elicited by pharmacologic ascorbate treatment.

Keywords: Burkitt lymphoma cells; ascorbate; drug resistance; pathway analysis; transcriptomic profiles.

Figure 1. Cytotoxicity analysis of ascorbate and H₂O₂ with the measurement of intracellular ascorbate in JLPR cells and JLPS cells

(A) Viability rates of JLPS cells, JLPS cells pretreated with DHA (JLPS+DHA), JLPR cells, and JLPS cells pretreated with CAT following treatment with increasing concentrations of ascorbate. (B) Viability rates of JLPS cells, JLPS+DHA, JLPR cells, and JLPR cells that had not been maintained in ascorbate (JLPR−) following treatment with increasing concentrations of H₂O₂. (C) Posttreatment concentrations of ascorbate in JLPS cells, JLPS+DHA cells, JLPR cells, and JLPR− cells.

Figure 2. Metabolomic assay and qRT -PCR analysis in JLPR cells and JLPS cells

(A) 2-DG uptake at different times. (B) Real-time PCR analysis of Glut1 and Glut3 mRNA. (C) 2-DG uptake in cells treated with increasing concentrations of ascorbate. (D) 2-DG uptake in cells treated with increasing concentrations of H₂O₂. Antioxidant enzyme analysis in JLPS and JLPR cells. (E) qRT-PCR analysis of CAT and GPX4 gene expression. (F) Western blot analysis of CAT protein expression levels. (G) CAT activity. (H) GPX4 activity.

Figure 3. Cellular DNA content in JLPS and JLPR cells following treatment with ascorbate and H₂O₂

(A) Cells following treatment with 0 μM or 1000 μM ascorbate. (B) Cells following treatment with 0 μM, 20 μM or 50 μM H₂O₂. (C) H₂O₂ production in RPMI 1640 medium alone, containing JLPS cells, or containing JLPR cells. Ascorbate (1 mM) was added and medium was incubated at room temperature for up to 120 min. Data are means ± standard deviations of three independent experiments.

Figure 4. Real-time PCR and western blot analyses of differentially expressed genes and protein expression levels of JLPS and JLPR cells treated with ascorbate

(A) Real-time PCR analysis of HMGB1, HIST1H4, CD74, HSPH1, c-Myc, TCL1A, CD79B, SSX3, ATF5, ASNS, FTL, TOP2B, HLA-A, XRCC5, GNB1, ATP5, and GPX4. Results were normalized using 18S ribosomal RNA. (B) Western blot analysis of CD74, CD79B, ATF5, TOP2B, HSPH1, HIS2AE, JNK, p44/p42, p-p44/p42, PARP, and c-Myc expression in JLPS and JLPR cells. β-actin was used as the loading control.