GENE EXPRESSION PROFILING OF HUMAN LIVER CARCINOMA (HepG2) CELLS EXPOSED TO THE MARINE TOXIN OKADAIC ACID

Abstract

The marine toxin, okadaic acid (OA) is produced by dinoflagellates of the genera Prorocentrum and Dinophysis and is the causative agent of the syndrome known as diarrheic shellfish poisoning (DSP). In addition, OA acts as both a tumor promoter, attributed to OA-induced inhibition of protein phosphatases as well as an inducer of apoptosis. To better understand the potentially divergent toxicological profile of OA, the concentration dependent cytotoxicity and alterations in gene expression on the human liver tumor cell line HepG2 upon OA exposure were determined using RNA microarrays, DNA fragmentation, and cell proliferation assays as well as determinations of cell detachment and cell death in different concentrations of OA. mRNA expression was quantified for approximately 15,000 genes. Cell attachment and proliferation were both negatively correlated with OA concentration. Detached cells displayed necrotic DNA signatures but apoptosis also was broadly observed. Data suggest that OA has a concentration dependent effect on cell cycle, which might explain the divergent effects that at low concentration OA stimulates genes involved in the cell cycle and at high concentrations it stimulates apoptosis.

Fig. 1

Dose-response relationship between [OA] in spent media and HepG2 cell parameters. A. Detachment from substrate after 48 hr in OA, with slope of 0.98 and (EC₅₀) of 25.4 nM. B. Normalized cell proliferation during the last 24 hr of 48 hr exposure to OA, with slope of 1.0 and (EC₅₀) of 5.03 nM. Fifty-four percent of control cells (“10e-10” M) proliferated in 24 hr.

Fig. 2

Agarose gel of DNA fragmentation in HepG2 cells by OA. A. Time course of DNA fragmentation via 607 nM OA (total cells). B. DNA fragmentation in adherent vs. detached cell fractions after 48 hrs at the indicated [OA] from 200–607 nM.

Fig. 3

Pattern of significant gene expression in HepG2 cells exposed to OA. Relative level of mRNA expression for gene significantly different among OA concentrations (0=Control, 1=2.4, 2=37, 3=51, 4= 200 and 5=607 nM OA). A. All 389 genes that differ from control (Dunnett’s test, p <0.01). B. Fifty-two genes with significant differences in expression and associated with cell cycle or replication. C. Sixty-five genes with significant differences in expression and associated with extracellular matrix or secretion. D. Colorbar indicating fold changes.

Fig. 4

Regression: OA dose versus mRNA levels in HepG2 cells. Four out of 169 significant regressions of log₁₀ transformed OA empirically derived doses. A & B. Two of the 65 linear regressions. C & D. Two of the 150 non-linear regressions. Dashed gray line is the best fit (but non-significant) linear equation. Y-axis are the normalized log2 expression data and x-axis is the log₁₀ OA doses.