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

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.


Introduction
Approximately 80% of lung cancer belongs to nonsmall-cell lung cancer (NSCLC) and the other is SCLC histologically [1]. Although smoking is one of the main risk factors for lung cancer [2], about 10% patients are non-smokers [3]. NSCLC is generally detected and diagnosed at late stage and its prognosis is poor [4]. Therefore, the anticancer drugs for NSCLC treatment remain a challenge.
Akt, a serine/threonine kinase, regulates cell growth, cell cycle progression, survival and anti-apoptosis. Dysregulation of Akt was reported to be observed in various cancers including breast cancer [5] and lung cancer cells [6]. Furthermore, the constitutive activation of Akt has been shown to cause chemoresistant of cancer cells. Similarly, NFκB, an inflammatory-associated transcription factor, is also found to be constitutively activated or aberrantly expressed in lung cancer [7,8]. Therefore, targeting of Akt and NFκB signaling seems to be a promising strategy for lung cancer therapy [9][10][11][12].
Ceramides are important components composed of lipid molecules and can form into sphingolipids when added functional groups on their hydroxyl group such as phosphate choline or monosaccharide. When cells are triggered by certain stimuli, an enzyme called sphingomyelinase [13] would hydrolyze sphingolipids and cause the release of ceramides into cytoplasm, which can undergo many biological processes, such as differentiation, proliferation, growth arrest and apoptosis [14]. Ceramide was reported to act as an important mediator in apoptosis pathways. Exogenous ceramides have been demonstrated to induce anti-proliferation and apoptosis in many cancers [15]. Furthermore, accumulating evidence showed that ceramide inhibits proliferation of cancer cells via inhibiting Akt and NFκB signal pathways [16,17].
The ceramide-mediated anticancer effects have been reported in many types of cancers such as pancreatic [18], breast [19], gastric [20], hematologic [21] cancer. However, the final outcome of ceramide treatment may depend on the context of cell types. Previous studies showed that knockout of NFκB p65 sensitizes embryonic fibroblasts toward C 2 -ceramide induced cell death [22]. On the contrary, the above study also found that C 2 -ceramide induces cell death and activation of NFκB in lung cancer H1299 cells [22]. The effects of C 2 -ceramide on apoptosis of H1299 cells were investigated previously [22,23]. In the current study, we examined the growth inhibitory property to NSCLC H1299 cells by C 2 -ceramide as well as its possible apoptosis mechanism, especially inhibiting Akt and NFκB pathways.

Cell survival assay
Cell survival was determined by the trypan blue dye exclusion assay as previously described [25,26]. In brief, Cells were seeded at a density of 1 × 10 5 cells per well. After 24 h of incubation, the cells were treated with C 2 -ceramide (#A7191, Sigma) at concentrations of 0, 10, 20, and 50 μM for 24 h, then 0.2% trypan blue were added to wells. Finally, the viable cells we are calculated by the Countess® Automated Cell Counter (Invitrogen, Diego, CA, USA). The assay was triplicated and the IC 50 was calculated by the slope and intercept accordingly to two concentrations of C 2 -ceramide between the half-maximal proliferative inhibition.
Chromatin condensation assay 5 × 10 5 H1299 cells were seeded onto a 6-well plate. After 24 h, cells were treated with indicated concentrations (0 to 50 μM) of ceramide for 24 h. After wards, cells were stained with 5 μg/ml of DAPI for 3 mins at 37°C. The level of chromatin condensation was determined by a flow cytometry (Becton-Dickinson). At least 10,000 stained cells were counted and calculated as percentage of chromatin condensation compared to those of the control cells.

Cell cycle distribution
Propidium iodide (PI, Sigma, St. Louis, MO, USA) staining for DNA content measurement was performed as described previously [28]. Briefly, cells were treated with 0, 10, 20, and 50 μM of C 2 -ceramide for 24 h. After collection, cells were washed twice with PBS before 70% ethanol fixation. After centrifugation, the cells were incubated with 10 μg/ml PI and 10 μg/ml RNase A in PBS for 15 min at room temperature in the dark. Cell cycle analyses were performed using a FACSCalibur flowcytometer (Becton-Dickinson, Mansfield, MA, USA).

Statistical analysis
All data are presented as mean ± S.D. Comparison between experimental groups and vehicle controls was assessed by one-way ANOVA test.

Apoptosis induction of C 2 -Ceramide-treated H1299 lung cancer cells
In Figure 2a, the profiles of PI/annexin V-positive percentages were shown for the treatments with vehicle control or 0, 10, 20, and 50 μM of C 2 -ceramide for 24 h. After 24 h  C 2 -ceramide treatment, the annexin V-positive percentages of H1299 lung cancer cells were significantly increased in a dose-response manner for most concentrations (P < 0.05) (Figure 3).

Chromatin condensation of C 2 -Ceramide-treated H1299 lung cancer cells
Chromatin condensation is one of the most important markers for apoptotic cells [29]. The apoptotic effect of C 2 -ceramide-treated H1299 lung cancer cells was further examined by the flow cytometry-based DAPI assay. The profiles of DAPI-positive percentages of 0, 10, 20, and 50 μM C 2 -ceramide for 24 h were shown (Figure 4a). The DAPI-positive percentages of C 2 -ceramide-treated H1299 lung cancer cells were significantly reduced in a dose-response manner (P < 0.001) (Figure 4b).

Modulation of p-Akt and p-NFκB in C 2 -Ceramide-treated H1299 lung cancer cells
The role of C 2 -Ceramide-induced modulating the levels of p-Akt and p-NFκB in H1299 lung cancer cells was examined by Western blotting. Both p-Akt and p-NFκB levels in C 2 -Ceramide-treated H1299 cells were significantly reduced at the concentration of 20 and 50 μM (Figure 5a). Likewise, the protein levels of pro-survival survivin and the cell cycle promoter cyclin A2 were downregulated dramatically. On the contrary, the protein level of pro-apoptotic Bax was increased significantly following C 2 -Ceramide treatment) (Figure 5b).

Discussion
The modulations of ceramide as the strategy for many kinds of cancer therapies have been reported [18][19][20][21]. For example, acid ceramidase was regarded as the target for breast cancer therapy [19] because it can hydrolyze ceramide, and thus reduce its intracellular levels. Previous study showed that C 2 -ceramide is not very harmful to normal cells. For example, the IC 50 (24 h) of human dermal neonatal fibroblast (HDNF) cells for C 2− ceramide was 66.5 μM [30], suggesting the moderately selective anti-proliferative effect of C 2− ceramide toward cancer cells. In the current study, we found that the C 2 -ceramide induced apoptosis of H1299 lung cancer cells. It provides the idea that pharmacological modulation of sphingolipid metabolism can enrich the tumor cell ceramide for cancer chemotherapy.
Sometimes the degree of the sub-G 1 accumulation may not appear in concert with the apoptosis in terms of Annexin V/PI staining. For example, no sub-G 1 accumulation was found in C 2 -ceramide-treated H1299 lung cancer cells at 24 h treatment, but it still showed the apoptosis-inducible effects in terms of Annexin V/PI and DAPI-based chromatin condensation assays using flow cytometry. Similarly, (−)-Anonaine inhibits growth of H1299 cells without sub-G 1 accumulation before 48 h incubation, however, the (−)-Anonaine ends up increasing apoptosis at 72 h treatment [31]. Therefore, the absence of sub-G 1 accumulation in C 2 -ceramide-treated H1299 at 24 h treatment may be due to the detection timing. Furthermore, chromatin condensation was thought to be one of hallmarks in apoptotic cells [32,33]. However, some study indicated that certain stresses such as heat shock may induce a non-apoptotic chromosome condensation [34]. For example, Plehn-Dujowich's work found the non-apoptotic chromatin condensation [34]. Accordingly, in our study, the non-apoptosis inducing dose of 20 μM C 2 -ceramide caused stress-induced chromatin condensation, which may explain the reason that C 2 -ceramide induces anti-proliferation without apoptosis ( Figure 4). Eventually, a higher dose of 50 μM C 2 -ceramide causes the apoptotic chromatin condensation, resulting in cell death of H1299 cells.
Previously, C 2 -ceramide-induced H1299 cells was investigated [22,23]. Demarchi's work indicated that C 2ceramide triggers the NFκB-dependent survival pathway. Figure 6 Schematic diagram of hypothesized mechanism of C 2 -ceramide-induced apoptosis of lung cancer cells. C 2 -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, C 2 -ceramide increases the protein level of pro-apoptotic Bax. As a result, C 2 -creamide treatment causes cell cycle G 1 arrest and chromatin condensation, subsequently, triggering the apoptosis of lung cancer cells.
However, our study showed that C 2 -ceramide dramatically decreases the level of phosphorylated NFκB (Figure 5a). This may due to the different duration of NFκB treatment (8 h vs. 24 h for Demarchi and Lin respectively). Importantly, our study demonstrated that C 2 -ceramide potently inhibits Akt phosphorylation of H1299 cells at the of 20 and 50 μM, suggesting that it will be an advantage of treating lung cancer with constitutively phosphorylated Akt. C 2 -ceramide also causes the down-regulation of survivin and cyclin A2 (Figure 5b), and the up-regulation of proapoptotic factor Bax in H1299 cells. This may sensitize lung cancer cells towards proliferation inhibition and apoptosis ( Figure 6). The results of this study demonstrated that that C 2 -ceramide treatment exerts antigrowth potential against human non-small cell lung cancer cells H1299 in a dose-responsive manner. C 2 -ceramide also reduces the pro-survival proteins Akt and NFκB, causing the down-regulation of survivin and cyclin A2, which are reported to frequently overexpress in non-small cell lung cancer [35]. This may sensitize lung cancer cells towards proliferation inhibition and apoptosis ( Figure 6). Accordingly, the above results suggested that C 2 -ceramide may be a promising reagent for lung cancer treatment or adjuvant therapy in future.