The synthetic triterpenoid CDDO-Im inhibits fatty acid synthase expression and has antiproliferative and proapoptotic effects in human liposarcoma cells

Abstract

Liposarcomas constitute a rare group of tumors of mesenchymal origin that are often poorly responsive to therapy. This study characterizes a novel human liposarcoma cell line (LiSa-2) and defines the mechanism of its response to a synthetic triterpenoid. Fatty acid synthase (FAS) is a key enzyme of de-novo fatty acid synthesis and is highly expressed in both human liposarcoma tissue specimens and LiSa-2 cells. Treatment of the LiSa-2 cell line with the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic imidazolide (CDDO-Im) markedly inhibited FAS mRNA expression, FAS protein production and FAS gene promoter activity. As expected, fatty acid synthesis was down regulated, but there was no effect on cellular fatty acid uptake or glycerol-3-phosphate synthesis suggesting a selective inhibition of endogenous fatty acid synthesis. Importantly, CDDO-Im produced a dose-dependent apoptotic effect in the LiSa-2 cell line, and simultaneous treatment with CDDO-Im and the fatty acid synthase inhibitor Cerulenin produced a synergistic cytotoxic effect. Thus, CDDO-Im and Cerulenin act at different loci to inhibit long chain fatty acid synthesis in liposarcoma cells. This study's demonstration of CDDO-Im inhibition of FAS and Spot 14 (S14) expression is the first report of triterpenoid compounds affecting the fatty acid synthesis pathway. The observed dependence of liposarcomas on lipogenesis to support their growth and survival provides a novel approach to the treatment of liposarcomas with agents that target fatty acid production.

Figure 1

Immunohistochemical detection of lipogenesis-related proteins in human liposarcoma tissue. Detection was with horseradish peroxidase (brown pigment), and slides were counterstained with hematoxylin (blue). Asterisks mark fat droplets in normal adipocytes adjacent to tumors. Specimens from two additional subjects showed similar staining patterns. Panel A: Staining with a polyclonal goat-antihuman FAS antibody yields a primarily cytoplasmic signal (arrow) in tumor and adipocytes. Panel B: Staining with a mouse monoclonal anti-human Spot 14 antibody reveals a primarily nuclear signal (arrow) in tumor and an adipocyte.

Figure 2

CDDO-Im down-regulates FAS expression in LiSa-2 cells. Panel A: Quantitative real-time PCR demonstrates a 70% decrease in FAS mRNA after 15 day exposure to 100 nM CDDO-Im compared to control plates. Data (mean ± SEM, n=6–10/group) are expressed as the ratio of the FAS signal to that of cyclophilin. Panel B: Quantitative real-time PCR demonstrates an 85% decrease in S14 mRNA after 15 day exposure to 100 nM CDDO-Im compared to control plates. Data (mean ± SEM, n = 6/group) are expressed as the ratio of the S14 signal to that of cyclophilin. Panel C: Western blot (20 μg protein/lane) demonstrates reduced FAS protein in cells treated with CDDO-Im, as opposed to diluent, for 15 days. Serum day 0 indicates subconfluent LiSa-2 cells in serum-containing medium. T47D human breast cancer cells were used as a (+) control. The FAS band migrates at ~ 206 kD. Panel D: Diagram of the 178 bp fragment of the proximal human FAS gene promoter used to drive luciferase expression in transfection experiments. SREBP-BS denotes the binding site for SREBP-1c; the position of binding sites for transcription factors NF-Y and SP1 are indicated, as are a non-essential sterol response element sequence (SRE) and the TATA box. Panel E: Luciferase activity (mean ± SEM, n= 3/group) measured 4 days after transfection is shown. FAS gene promoter activity is reduced by ~ 60% in cells exposed to 100 nM CDDO-Im. Non-transfected LiSa-2 cells acted as negative controls.

Figure 3

Metabolic effects of CDDO-Im in LiSa-2 cells. Panel A: CDDO-Im inhibits incorporation of [14]-C-acetate into lipids. Cells were labeled for 3 hours, at which time total lipids were extracted, and incorporation was measured in a liquid scintillation counter. Data are mean cpm (×10²)± SEM (n = 4/group). Panel B: CDDO-Im does not exert a significant effect on the activity of the major enzyme of triglyceride synthesis. GPDH enzyme activity (mean ± SEM, 8 wells/group) was measured before and after 15 day exposure to vehicle or 100 nM CDDO-Im. Panel C: The uptake of exogenous fatty acids is unaffected by exposure to CDDO-Im. Cells were incubated in the presence of the indicated culture media spiked with [14]-C-palmitic acid for 1 hour, and cell-associated radioactivity was determined. Data are mean cpm (x 10⁴)± SEM (n = 4/group).

Figure 4

CDDO-Im-treated LiSa-2 cells exhibit dose-dependent apoptosis and increased sensitivity to the cytotoxic effect of Cerulenin. Panel A: Net cell accumulation after 5 day exposure to CDDO-Im, determined by DNA content/well is shown (mean ± SEM, n = 4 wells/data point). Panel B: Cerulenin inhibits LiSa-2 cell growth. Cells were incubated in the indicated concentrations of Cerulenin x 5 days, and assayed as in panel A. Panel C: Cells exposed to Cerulenin exhibit reduced growth at a sublethal concentration of CDDO-Im. Cells were exposed to 100 nM CDDO-Im combined with the indicated concentrations of Cerulenin for 5 days, and analyzed for DNA content/well. Panel D: CDDO-Im causes enhanced sensitivity to Cerulenin-induced apoptosis. Cells were grown x 5 days in 100 nM CDDO-Im in the presence of the indicated concentrations of Cerulenin or diluent were analyzed in a TUNEL assay. Addition of a submaximally-lethal concentration of Cerulenin (5 μg/mL) to cells exposed to 100 nM CDDO-Im caused a 5-fold increase in apoptosis. Note that the interaction is obscured at a maximally cytotoxic level of Cerulenin (10 μg/mL).

Figure 5

Fatty acid synthase (FAS) gene expression in human liposarcoma. Relative level of FAS expression from global gene expression microarray of 181 human sarcomas using web-based database at http://watson.nhgri.nih.gov/sarcoma/ [3]. An average 0.5 log over expression of FAS is seen in liposarcomas, with down regulation seen in other adult sarcomas (mean ± SEM, n=17–38/data point) MFH = malignant fibrous histiocytoma.