Anti-proliferative effect of LXR agonist T0901317 in ovarian carcinoma cells

Abstract

Background: Ovarian cancer is the most common cause of cancer related death from gynecologic tumors in the United States. The insidious nature of the disease precludes early diagnosis, therefore surgical debulking and chemotherapy are considered as standard treatment modalities for advanced stages. We investigated the effect of the LXR agonist, T0901317, on ovarian cancer cell proliferation and apoptosis as a potential therapeutic agent.

Results: T0901317 treatment resulted in a significant (P <0.001) inhibition of cell proliferation in a time- and dose-dependent manner in CaOV3, SKOV3 and A2780 cells. Western blot analysis demonstrated an induction of p21 and p27 with a concominant reduction in phospho-RB protein levels. Cell cycle analysis demonstrated a significant (P <0.001) arrest in the G1 cell cycle phase. Significant induction of Caspase-3 and BAX gene expression occurred with treatment. Induction of apoptosis was confirmed by significant (P < 0.001) elevation of caspase activity on FACS analysis, caspase-glo assay, BAX protein induction and decreased caspase 3 precursor protein expression on Western blot analysis. LXR alpha/beta knockdown experiments did not reverse the anti-proliferative and cytotoxic effects of T0901317.

Conclusions: The LXR agonist, T0901317, significantly suppresses cell proliferation and induces programmed cell death in a dose- and time-dependent manner. Our results indicate that T0901317 induces its anti-proliferative and cytotoxic effects via an LXR-independent mechanism.

Figure 1

Expression levels of LXRα/β proteins in human ovarian carcinoma cell lines. Whole-cell lysates of A2780, CaOV3 and SKOV3 cells were obtained and subjected to immunoblotting. Forty micrograms of lysate were loaded per lane. LXRα primary antibody was used in (A) and LXRβ primary antibody was used in (B).

Figure 2

Characterization of antiproliferative effects of T0901317 treatment in ovarian carcinoma cells. A2780, CaOV3 and SKOV3 cells were cultured and treated with DMSO (♦ blue) or T0901317 at a concentration of 5 μM (■ pink), 10 μM (▲ yellow), 20 μM (X, light blue), 40 μM (X, purple) or 50 μM (● red) for 24 h, 48 h or 72 h (A-C). Proliferation status was determined by the CyQuant proliferation assay. T0901317 significantly inhibits cellular proliferation in all cell lines in a dose-dependent and time-dependent manner. Each value is the mean ± SD of three independent experiments, and the proliferation value is expressed as percentage of vehicle-treated cells (DMSO). (*P < 0.0001 vs. untreated cells). After culturing with vehicle (DMSO) or with T0901317 for the indicated time-points at a concentration of 10 μM, cells were stained with propidium iodide as detailed in Material and Methods and examined by flow cytometry to determine cell cycle phase distribution (D). After 24, 48 or 72 hours of treatment, the LXR agonist T0901317 decreased the percentage of cells in S phase and increased the percentage of cells in the G0/G1 phase, indicating a cell cycle arrest at the G1-S checkpoint. The percentage of cells in G0/G1 phase increases in a time-dependent manner. Results are the mean of three independent experiments and are expressed as percentage of cells, presented as mean ± SD. *P < 0.001. CaOV3 cells were grown in media supplemented with 10% FBS for 48 hours in presence of vehicle (DMSO) or the indicated concentrations of T0901317 (5 μM to 40 μM). Whole-cell extract was obtained and 60-90 μg of protein was analyzed for phospho-pRb (E), p21 (F) or p27 (G) protein levels by Western blot analysis.

Figure 3

Effect of the LXR agonist T0901317 on cellular morphology. CaOV3 cells were cultured and treated with DMSO (1%, A) or T0901317 at a concentration of 5 μM (B), 10 μM (C), 20 μM (D), 40 μM (E) or 50 μM (F) for a total of 48 hours. Cells were visualized microscopically (10X) and pictures taken. The pictures clearly demonstrate a significant effect on cellular morphology. At increasing doses of the LXR agonist, the cells appeared to have a decreased amount of cytoplasm with a concomitant decrease in cell cumber. At the doses of 40 μM and 50 μM, the cells appeared apoptotic with necrotic debris present in the media.

Figure 4

Induction of apoptosis with T0901317 treatment. Flow cytometric analysis of apoptosis was utilized for determination of caspase-3 and -7 activities. CaOV3 cells were treated with either vehicle (DMSO) or T0901317 at the indicated doses (10 μM to 40 μM) for 24 hours and then stained with Vybrant FAM dye, and 7-AAD strictly following manufacturer's instruction. Data are mean ± SD of three different experiments (A). Caspase 3/7 activity was also measured in CaOV3 cells after 12 hours of treatment with vehicle (DMSO) or 5 μM, 10 μM, 20 μM, 40 μM or 50 μM. A luminescent assay was used, as detailed in Material and Methods. T0901317 significantly increases Caspase 3/7 activation. Results are the mean ± SD of three independent experiments and are expressed as percentage of negative control (DMSO). (* p < 0.006 vs. negative control, (B). The activation of caspase 3 was confirmed by Western Blot analysis. LXR agonist treatment enhances caspase 3 activation, resulting in increased caspase 3 precursor cleavage rate and decreased caspase 3 precursor protein levels. Decreased caspase-3 precursor protein levels occur in a concentration dependent manner (C). β-actin expression was determined by Western blot analysis and used as an endogenous control.

Figure 5

Effect of T0901317 treatment on apoptotic gene and BAX protein expression. After a 24 hour treatment, cells were harvested for isolation of mRNA as detailed in the methods section. A significant induction of BAX and caspase gene induction was demonstrated, especially at the 30 μM dose. Upregulation of the anti-apoptotic Bcl-2 gene expression was demonstrated with the 30 μM concentration (A-C), *P < 0.05, **P < 0.001). CaOV3 cells were grown in media supplemented with 10% FBS for 24 hours in presence of vehicle (DMSO) or the indicated concentrations of T0901317 (5 μM to 50 μM). Whole-cell extract was obtained and 60 μg of protein was analyzed for BAX protein levels by Western blot analysis. β-actin expression was used as an endogenous control (D).

Figure 6

Effect of LXRα/β inhibition on cell proliferation in T0901317 treated CaOV3 cells. (A) Evaluation of LXRα and (B) LXRβ expression in siRNA transfected cells by quantitative real time RT-PCR. (C) siRNA transfected CaOV3 cells were cultured and treated with DMSO or 20 μM T0901317 for 24 hours. The cell growth of was assessed by the Cyquant proliferation assay. Each value is the mean ± SD of three independent experiments (* p < 0.05 vs control siRNA).

Figure 7

Effect of T0901317 Treatment on an FXR-dependent gene, short heterodimer partner (SHP) in ovarian carcinoma cells. (A) Whole-cell lysates of HS68, A2780, CaOV3 and SKOV3 cells were obtained and subjected to immunoblotting. Fifty micrograms of lysate were loaded per lane and the blot was probed with anti-FXR antibody. (B) CaOV3 cells were treated with T0901317 for 24 hours and SHP gene mRNA expression was examined by real time RT-PCR. (*P < 0.001)