The antiproliferative effect of C2-ceramide on lung cancer cells through apoptosis by inhibiting Akt and NFκB

Abstract

The anticancer effects of ceramide have been reported in many types of cancers but less in lung cancer. In this study, we used C2-ceramide to further investigate its possible anticancer effects and mechanisms on non-small cell lung cancer (NSCLC) H1299 cells. The result of cell proliferation in terms of trypan blue assay showed high dose of C2-ceramide inhibited cell survival after 24 h treatment. The flow cytometry-based assays indicated the effect of apoptosis, chromatin condensation, and G1 arrest in terms of Annexin V/propidium iodide (PI), DAPI, and PI stainings, respectively. Moreover, the decreased protein level of p-Akt, p-NFκB, survivin and cyclin A2 were detected by Western blot assay. Taken together, these results indicated the antiproliferative effect of C2-ceramide is majorly responsible for cell apoptosis in lung cancer H1299 cells.

Figure 1

Effect of C₂-Ceramide on proliferation of H1299 cells. The cell proliferation assay showed the inhibition of cell growth at high dose of treatment for 24 h.

Figure 2

Treatment with C₂-ceramide induced different accumulations of G1 population in lung cancer H1299 cells. Cells were treated with 0, 10, 20, and 50 μM C₂-ceramide for 24 h. (a) Representative cell cycle distribution in C₂-ceramide-treated H1299 cells. (b) Cell phase percentages obtained for (a) in triplicate experiments.

Figure 3

Treatment with C₂-ceramide induced different apoptotic profiles in lung cancer H1299 cells. Cells were treated with 0, 10, 20, and 50 μM C₂-ceramide for 24 h. (a) Representative apoptotic profiles obtained by Annexin V/PI double staining in C₂-ceramide-treated H1299 cells. (b) Quantification analysis results for late apoptosis population (%). Only annexin V (+)/PI (+) regions were analyzed. Data, mean ± SD (n = 3). Asterisks indicate statistically significant differences compared to control (P < 0.001).

Figure 4

C₂-ceramide increased chromatin condensation levels of H1299 lung cancer cells. (a) Flow cytometry-based DAPI profiles for C₂-ceramide-treated cells. Cells treated with different concentrations (0 to 50 μM) of C₂-ceramide for 24 h. Positive % is indicated in each panel; (b) Quantificative analysis of DAPI-positive population. Data are presented as mean ± S.D. (n = 3). Asterisks indicate statistically significant differences compared to control (P < 0.001).

Figure 5

p-Akt and p-NFκB levels of C₂-ceramide-treated H1299 lung cancer cells. Cells treated with different concentrations (0 to 50 μM) of C₂-ceramide for 24 h. After treatment, the protein lysates were resolved by SDS-PAGE, transferred onto nitrocellulose membranes and probed with specific antibodies and detected signals using an enhanced chemiluminescence kit. (a) The changes of Akt and NFκB phosphorylation. (b) The changes of protein level of survivin, cyclin A2 and Bax. β-actin as an internal control.

Figure 6

Schematic diagram of hypothesized mechanism of C₂-ceramide-induced apoptosis of lung cancer cells. C₂-ceramide inhibits the activity of both Akt and NF-κB, causing the down-regulation of pro-survival survivin and cell cycle promoter cyclin A2. On the contrary, C₂-ceramide increases the protein level of pro-apoptotic Bax. As a result, C₂-creamide treatment causes cell cycle G₁ arrest and chromatin condensation, subsequently, triggering the apoptosis of lung cancer cells.