Mechanisms involved in ceramide-induced cell cycle arrest in human hepatocarcinoma cells

Figure 1 Ceramide inhibited proliferation by halt of cell cycle in Bel7402 cells. A: ceramide inhibited cell proliferation in Bel7402 cells. Cells were cultured in the medium with different concentrations of C2-ceramide for 24 h. Cell viability was analyzed by MTT assay and presented as cell proliferative rates. The results show the mean ± SE (n = 3-4); B and C: ceramide down-regulated the expression of E-cadherin. Bel7402 cells were incubated with or without 30 μmmo/L of ceramide for 24 h. Cells were then harvested and stained with anti-E-cadherin directly labeled by PE. The protein levels of E-cadherin were determined by flow cytometry. Data was represented by the mean ± SE of 3 or 4 separate experiments (B). Bel7402 cells were incubated with or without 30 μmmo/L of ceramide for 24 h. Cells were then harvested, lysed and resolved in 12% SDS-PAGE. The expression of E-cadherin was determined by Western blot. Actin was used as a control. Data represent the results of three separate experiments (C).

Figure 2 Ceramide up-regulated the expression of p21^WAF1/BIP1 and down-regulated that of cyclinD1. Bel7402 cells were incubated with different concentrations of ceramide as indicated for 24 h. Cells were harvested, lysed and resolved in 15% SDS-PAGE. The expression of p21 and cyclinD1 were determined by Western blot. Actin was used as a control. Data represent the results of three separate experiments.

Figure 3 CDK7 and PPARγ pathways involved in cell cycle arrest induced by C2-ceramide. A: activation of PPARγ transcriptional activity induced by C2-ceramide. Bel7402 cells were transiently transfected with the PPAR responsive element (PPRE) reporter construct (pAOXPPREluc) or the promoter-less control vector pAOXBluc following the treatment of different concentrations of C2-ceramide as indicated, PPARγ transcriptional activity was measured as described. The bar represents the relative fold increase of luciferase activity. The results show means ± SE (n = 3); B: C2-ceramide down-regulated the expression of CDK7, which was blocked by GW9662. Bel7402 cells were cultured with 30 μmol/L C2-ceramide in the presence or absence of GW9662, which was claimed as a specific PPARγ antagonist for 24 h. Total proteins were extracted and resolved on SDS-PAGE followed by Western blot assay using anti-CDK7 antibody. Data represents the results of three separate experiments.

Figure 4 Ceramide inhibited activation of ERK pathway in Bel7402. Bel7402 cells were incubated with different concentrations of ceramide as indicated. Total proteins were extracted and resolved on SDS-PAGE. Phospho-ERK1/2 and c-myc were normalized to total Erk1/2 and internal standard actin was determined by Western blot. Data represents the results of three separate experiments.