Introduction

Approximately 80% of lung cancer belongs to non-small-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[9–12].

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 C2-ceramide induced cell death[22]. On the contrary, the above study also found that C2-ceramide induces cell death and activation of NFκB in lung cancer H1299 cells[22]. The effects of C2-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 C2-ceramide as well as its possible apoptosis mechanism, especially inhibiting Akt and NFκB pathways.

Materials and methods

Cell cultures

The H1299 lung cancer cells were maintained in DMEM medium (Invitrogen, Carlsbad, CA USA) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 0.03% glutamine and 1 mM sodium pyruvate[24] and kept at 37°C in a humidified atmosphere with 5% CO2.

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 × 105 cells per well. After 24 h of incubation, the cells were treated with C2-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 IC50 was calculated by the slope and intercept accordingly to two concentrations of C2-ceramide between the half-maximal proliferative inhibition.

Apoptosis assay

Apoptosis was detected by annexin/PI staining (Pharmingen, San Diego, CA, USA) as previously described[27]. Briefly, cells were treated with C2-ceramide at concentrations of 0, 10, 20, and 50 μM for 24 h. After collection, cells were treated with 10 μg/ml of annexin V-fluorescein isothiocyanate and 5 μg/ml of PI for analysis with a FACSCalibur™ flow cytometer (Becton-Dickinson).

Chromatin condensation assay

5 × 105 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 C2-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).

Western blotting

Western blot assay was performed as described previously[27]. Briefly, cells were collected for lysate preparation. After centrifugation, and protein concentrations of lysates were determined. Protein lysates for 40 μg were loaded and electrophoresed by 10% SDS-polyacrylamide gel (PAGE) and then transferred to membrane. The membranes were blocked with 5% non-fat milk. Subsequently, it was reacted with primary antibodies against t-Akt (#1081), p-NFκB (Ser536, #2220) and Bax (#1063, Epitomics, CA, USA); t-NFκB (sc-8008), β-catenin (sc-7963) and p-Akt (Ser473, sc-7985, Santa Cruz Biotech, Santa Cruz, CA, USA); Cyclin A2 (GeneTex Co., Cat No. GTX103042); survivin (AnaSpec, San Jose, CA, USA) and β-actin (#sc-8432, Santa Cruz Biotech), and their corresponding secondary antibodies. The ECL™ (Amersham Piscataway, NJ, USA) chemiluminescence detection kit was applied.

Statistical analysis

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

Results

Anti-proliferative effects of C2-Ceramide-Treated H1299 lung cancer cells

In the trypan blue assay (Figure 1), the proliferation rates at various concentrations of C2-ceramide (0, 10, 20, and 50 μM) after 24 h were 100.0 ± 2.3, 90.1 ± 3.2, 69.2 ± 2.8, 5.0 ± 3 (n = 3). The proliferation rate of C2-ceramide-treated H1299 lung cancer cells significantly decreased in a dose–response manner (P < 0.001). The 50% inhibitory concentration (24 h, IC50) of C2-ceramide for H1299 cells was 22.9 μM.

Figure 1
figure 1

Effect of C 2 -Ceramide on proliferation of H1299 cells. The cell proliferation assay showed the inhibition of cell growth at high dose of treatment for 24 h.

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G1 arrest of C2-Ceramide-treated H1299 lung cancer cells

The role of cell cycle interference in the C2-ceramide-induced apoptosis of H1299 lung cancer cells was examined by the flow cytometry-based PI assay (Figure 2). The G1 percentages were increased at concentration of 50 μM C2-ceramide for 24 h (Figure 2a). Apparently, the G1 arrest activities in cells treated with C2-ceramide showed a significant increase at higher concentration (P < 0.001) (Figure 2b).

Figure 2
figure 2

Treatment with C 2 -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.

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Apoptosis induction of C2-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 C2-ceramide for 24 h. After 24 h C2-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).

Figure 3
figure 3

Treatment with C 2 -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).

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Chromatin condensation of C2-Ceramide-treated H1299 lung cancer cells

Chromatin condensation is one of the most important markers for apoptotic cells[29]. The apoptotic effect of C2-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 C2-ceramide for 24 h were shown (Figure 4a). The DAPI-positive percentages of C2-ceramide-treated H1299 lung cancer cells were significantly reduced in a dose–response manner (P < 0.001) (Figure 4b).

Figure 4
figure 4

C 2 -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).

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Modulation of p-Akt and p-NFκB in C2-Ceramide-treated H1299 lung cancer cells

The role of C2-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 C2-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 down-regulated dramatically. On the contrary, the protein level of pro-apoptotic Bax was increased significantly following C2-Ceramide treatment) (Figure 5b).

Figure 5
figure 5

p-Akt and p-NFκB levels of C 2 -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.

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Discussion

The modulations of ceramide as the strategy for many kinds of cancer therapies have been reported[18–21]. 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 C2-ceramide is not very harmful to normal cells. For example, the IC50 (24 h) of human dermal neonatal fibroblast (HDNF) cells for C2-ceramide was 66.5 μM[30], suggesting the moderately selective anti-proliferative effect of C2-ceramide toward cancer cells. In the current study, we found that the C2-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-G1 accumulation may not appear in concert with the apoptosis in terms of Annexin V/PI staining. For example, no sub-G1 accumulation was found in C2-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-G1 accumulation before 48 h incubation, however, the (-)-Anonaine ends up increasing apoptosis at 72 h treatment[31]. Therefore, the absence of sub-G1 accumulation in C2-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 C2-ceramide caused stress-induced chromatin condensation, which may explain the reason that C2-ceramide induces anti-proliferation without apoptosis (Figure 4). Eventually, a higher dose of 50 μM C2-ceramide causes the apoptotic chromatin condensation, resulting in cell death of H1299 cells.

Previously, C2-ceramide-induced H1299 cells was investigated[22, 23]. Demarchi’s work indicated that C2-ceramide triggers the NFκB-dependent survival pathway. However, our study showed that C2-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 C2-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. C2-ceramide also causes the down-regulation of survivin and cyclin A2 (Figure 5b), and the up-regulation of pro-apoptotic 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 C2-ceramide treatment exerts anti-growth potential against human non-small cell lung cancer cells H1299 in a dose-responsive manner. C2-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 C2-ceramide may be a promising reagent for lung cancer treatment or adjuvant therapy in future.

Figure 6
figure 6

Schematic diagram of hypothesized mechanism of C 2 -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.

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