Abstract 4112: Lysophosphatidic acid up-regulates expression of TNF-alpha in human liver cancer cell lines

Abstract

Abstract Background: Lysophosphatidic acid (LPA) is an important intercellular signaling molecule involved in a number of physiologic and pathophysiological processes. Elevated serum LPA was reported to be associated with liver fibrosis in patients with chronic hepatitis C. We have previously demonstrated that the enzyme mediated LPA generation- autotaxin(ATX) was overexpressed in hepatitis and hepatitis associated HCC. But the exact mechanism of the enhanced LPA signaling contributed to the initiation or progression of tumorigenesis in human liver cancer remains unclear. Methods: Human liver cancer cell lines Hep3B, HepG2 and Huh7 were serum-starved overnight and stimulated with LPA (18:1, Avanti Polar-Lipids, Inc., Alabaster, AL, USA) by different doses (0, 2.5, 5, 10 and 20 uM) and different time intervals (3, 8 and 16 hours). RNA was extracted using Qiagen kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. Quantitative real-time PCR was applied to determine the expression of interleukin 1, beta (IL-1B), IL-6, tumor necrosis factor alpha(TNF-alpha), transforming growth factor alpha 1 (TGF-alpha1) and transforming growth factor beta1 (TGF-beta1) using standard commercially available TaqMAN probes (Applied Biosystems, Foster City, CA). The amount of target gene was normalized to the internal standard 18S rRNA (Hs99999901_s1) levels and reported as a relative value. Results: TNF-alpha mRNA level was rapidly induced more than 3-fold after stimulating with 2.5 uM LPA for 3 hours in Hep3B cells (P<0.01), but this induction did not show significant change in spite of increasing treatment time or LPA doses. The curves of TNF-alpha expression in Huh7 and HepG2 cells were nearly identical to that of Hep3B cells, Treatment with 2.5uM LPA for 3 hours induced more than 5- and 2- fold TNF-alpha mRNA in Huh7 and HepG2 cells respectively(p<0.001). Different dose or treatment time did not result in a significant increase of induction of TNF-alpha mRNA in these two liver cancer cell lines. Interestingly, LPA treatment had no effect on the mRNA expression of TGF-alpha1, TGF-beta1, IL-1 or IL-6 in these three liver cancer cell lines in spite of increasing LPA doses or time intervals. Conclusions: Our study demonstrates that LPA transcriptionally activates TNF-alpha expression in human liver cancer cell lines. TNF-alpha is known to induce hepatocytes apoptosis and is one of the few critical factors that prime liver regeneration. LPA-induced TNF-alpha production in liver cancer cells may be important not only for tumor behavior, but also has potential damage to normal tissues which in turn fuel the liver tumor progression. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4112.