J. Yu1, R. Damoiseaux2, S. Liu1, R. Sanchez1, F. C. Brunicardi1 1University Of California – Los Angeles,Department Of Surgery,Los Angeles, CA, USA 2University Of California – Los Angeles,Department Of Molecular And Medical Pharmacology,Los Angeles, CA, USA
Introduction:
The prostaglandins are a family of lipid compounds, found in most human tissues with diverse hormone-like effects. We have recently identified prostaglandins E1, A2, and B2 (PGE1, PGA2, and PGB2) as potential inhibitors of pancreatic cancer proliferation via high-throughput drug screening of small molecules. However, the molecular mechanisms of how prostaglandins inhibit pancreatic cancer cell proliferation are poorly understood.
Methods:
A dilution series for a final concentration from 1mM to 1pM were used to determine the concentration required for 50% inhibition (IC50) for PGA2, PGB2 and PGE1 on human pancreatic cancer cell line MiaPaca2. To further determine whether PGA2, PGB2 and PGE1 inhibit pancreatic cancer cell growth via shared molecular targets or via independent mechanisms, RNA sequencing was applied to pancreatic cancer cell line MiaPaca2 with or without treatment of 5mM PGA2, PGB2 and PGE1. There were 3 replicates per each treatment group. Total RNAs were extracted 72 h post-treatment using Qiagen RNeasy columns. Sequencing reads were obtained using the Illumina HiSeq2000 platform. Differentially expressed genes from treated versus non-treated pancreatic cancer cells were calculated using “Cuffdiff” program from the Cufflinks suites and the top differentially expressed genes were then validated using QPCR.
Results:
The dose-response curve of PGE1 showed that IC50 of PGE1 on Mia Paca2 cell proliferation was 550nM, which was lower than the IC50 for PGA2 (1260nM) and PGB2 (1600nM). Giving a cut-off at log2 fold-change>0.5 and P<0.05, 205, 237 and 284 up-regulated genes and 289, 278 and 355 down-regulated genes were identified associated with PGA2, PGB2, and PGE1 treatment, respectively. Among these genes, only 31 genes in common were up regulated after treatments of all 3 prostaglandins. Gene ontology analysis revealed that these 31 up-regulated genes were enriched in genes of DNA damage response and apoptosis (P=0.05). In contrast, 93 genes were found significantly inhibited in all 3 prostaglandin-treated cells, including Tetraspanin 1 (TSPAN1), Sulforaphane (SFN) and Keratin 15 (KRT15). Gene ontology analysis revealed that these 93 down-regulated genes were enriched in genes involved in epidermal ectoderm development (P=2.9E-6) and cell proliferation (P=0.02). QPCR proved the significant reduction of gene expression for TSPAN1, SFN and KRT15, which have been associated with epidermal ectoderm development and oncogenesis.
Conclusion:
This analysis demonstrates the inhibitory effect of PGE1, PGA2, and PGB2 on pancreatic cancer cell proliferation in vitro. Moreover, by comparing the transcriptome of pancreatic cancer cells before and after prostaglandin treatments, the RNA Sequencing data revealed a list of shared gene targets including TSPAN1, SFN and KRT15, suggesting the mechanism of the inhibitory effect of PGE1, PGA2, and PGB2 on pancreatic cancer cells.