Transcriptomic changes induced by mycophenolic acid in gastric cancer cells

Abstract

Background: Inhibition of inosine monophosphate dehydrogenase (IMPDH) by mycophenolic acid (MPA) can inhibit proliferation and induce apoptosis in cancer cells. This study investigated the underlying molecular mechanisms of MPA's anticancer activity.

Methods: A gastric cancer cell line (AGS) was treated with MPA and gene expression at different time points was analyzed using Illumina whole genome microarrays and selected genes were confirmed by real-time RT-PCR.

Results: Transcriptomic profiling identified 1070 genes with ≥2 fold changes and 85 genes with >4 fold alterations. The most significantly altered biological processes by MPA treatment include cell cycle, apoptosis, cell proliferation and migration. MPA treatment altered at least ten KEGG pathways, of which eight (p53 signaling, cell cycle, pathways in cancer, PPAR signaling, bladder cancer, protein processing in ER, small cell lung cancer and MAPK signaling) are cancer-related. Among the earliest cellular events induced by MPA is cell cycle arrest which may be caused by six molecular pathways: 1) up-regulation of cyclins (CCND1 and CCNE2) and down-regulation of CCNA2 and CCNB1, 2) down-regulation of cyclin-dependent kinases (CDK4 and CDK5); 3) inhibition of cell division related genes (CDC20, CDC25B and CDC25C) and other cell cycle related genes (MCM2, CENPE and PSRC1), 4) activation of p53, which activates the cyclin-dependent kinase inhibitors (CDKN1A), 5) impaired spindle checkpoint function and chromosome segregation (BUB1, BUB1B, BOP1, AURKA, AURKB, and FOXM1); and 6) reduction of availability of deoxyribonucleotides and therefore DNA synthesis through down-regulation of the RRM1 enzyme. Cell cycle arrest is followed by inhibition of cell proliferation, which is mainly attributable to the inhibition of the PI3K/AKT/mTOR pathway, and caspase-dependent apoptosis due to up-regulation of the p53 and FAS pathways.

Conclusions: These results suggest that MPA has beneficial anticancer activity through diverse molecular pathways and biological processes.

Keywords: MPA; drug repurposing; microarray; regulatory networks.

Figure 1

Molecular changes related to cell cycle arrest induced by MPA treatment. A: Heatmap for genes differentially expressed after MPA treatment for 0h, 12h, 24h, 48h and 72h. Each sample is represented in a column and each gene is represented in a row. Increased expression is indicated as red and decreased expression is indicated as green. Representative genes are shown on the right panel and gene clusters are indicated on the left. B: A network illustrating the connectedness of the genes (>2-fold differences) involved in cell cycle regulation. C: Western blot for RRM1.

Figure 2

Molecular changes related to inhibition of cell proliferation induced by MPA treatment. A: Heatmap for genes differentially expressed after MPA treatment for 0h, 12h, 24h, 48h and 72h. B: A network illustrating the connectedness of the genes (>2-fold differences).

Figure 3

Confirmation of gene expression differences in AGS cells by real-time RT-PCR. Box blots are shown for each gene. Relative expression levels are shown in log 2 scale. FC (fold change) for each time point (12h, 24h and 48h) is compared to untreated controls (0h) with respective p-values.

Figure 4

Gene expression analyses in Hs746T cells by real-time RT-PCR. Data presentation is identical to Figure 4.

Figure 5

Molecular changes related to induction of apoptosis induced by MPA treatment. A: Heatmap for genes differentially expressed after MPA treatment for 0h, 12h, 24h, 48h and 72h. B: A network illustrating the connectedness of the genes (>2-fold differences).

Figure 6

Overall molecular networks and biological functions altered by MPA treatment of AGS cells. Genes in light red and proteins in dark red are up-regulated. Genes in light green and proteins in dark green are down-regulated after MPA treatment.