Apoptosis of human colon carcinoma HT-29 cells induced by ceramide.

AIM
To investigate the effect of exogenous ceramide-induced apoptosis on human colon carcinoma HT-29 cells.


METHODS
Light microscope, transmission electron microscope and fluorescence microscope were used to observe the morphology change of apoptosis in HT-29 cells. Agarose gel electrophoresis was performed to detect the DNA fragment. Mitochondrial function was detected by MTT assay. mRNA expression of Bcl-2 family gene members was determined by reverse transcription polymerase chain reaction (RT-PCR) assay.


RESULTS
After C2-ceramide treatment, typical characteristics of apoptosis, such as nuclear chromatin breakage, apoptotic body and DNA ladder, could be observed. After exposure to 50 micromol/L C2-ceramide for 12 and 24 h, cell apoptosis was 64.1% and 81.3% respectively, which had a time-and dose-effect relationship. Mitochondrial function started to decrease from 6 h after exposure to ceramide. Meanwhile, ceramide up-regulated or down-regulated the mRNA expression of Bcl-2 family gene members.


CONCLUSION
Ceramide induces apoptosis of human colon carcinoma HT-29 cells by affecting the expression of Bcl-2 family gene members and impacting the mitochondrial function.


INTRODUCTION
Ceramide (N-acetyl-sphingosine) is a fundamental unit of sphingomyelin which is a moiety of cytoplasmic membrane. Ceramide, also an important bioactive compound in vivo, can conduct specific signal transduction from cell surface receptor and environmental stress to nuclei, and then provoke multiple cytobiologic effects [1] . Meanwhile, ceramide is a common second messenger molecule participating in sphingomyelin cycle, apoptosis and differentiation of many cell types [2,3] . Apoptosis can be induced by surrounding stimulating factors, such as tumor necrosis factor a (TNF-a), endotoxin, FAS ligand, radiation, chemotherapeutics and heat shock, and is associated with ceramide. Recent studies indicate that ceramide has a close relationship with genesis and progression of digestive tract tumor. But little information about Bcl-2 family gene member and mitochondrial function is available concerning the effect of ceramideinduced apoptosis in colon carcinoma which is one of the most aggressive forms of cancer. Even ceramide could induce apoptosis of HT-29 cells. This study was to determine how ceramide induces apoptosis of human colon carcinoma cells and to discuss its mechanism.

Materials
C2-ceramide (N-acetyl-D-sphingosine) was purchased from Sigma, dissolved to 5 mmol/L in DMSO as a stock solution. Hoechest 33 258 and MTT were purchased from Sigma, dissolved in PBS solution. The two solutions were preserved at 4℃ from light. Apoptosis DNA extract kit was from Shanghai Huashun Biotechnology Company. RPMI-1640 media, DMSO and fetal bovine serum (FBS) were bought from GIBCO. Trypsin and EDTA were obtained from Amresco.

Cell culture
HT-29 cells were provided by the Institute of Tumor Prevention and Cure in Beijing, China. Cells were cultured in RPMI-1640 media supplemented with 10% FBS and 2 mmol/L glutamine. Antibiotics added to the media were 100 U/mL penicillin and 100 U/mL streptomycin. Cells maintained in a humidified atmosphere of 95 mL/L air and 50 mL/L CO2 at 37℃ were then passed at pre-confluent densities in a solution containing 0.25% trypsin and 0.02% EDTA. Exponentially growing cells were used throughout all experiments.

Light microscopy
HT-29 cells were treated with C2-ceramide at final concentrations of 12.5, 25 and 50 mmol/L for 24 and 48 h respectively. DMSO served as negative control. During the procedure, cell morphology was observed under light microscope for different time.

Transmission electron microscopy
Cells were collected and fixed with 4% glutaraldehyde in phosphate buffer overnight at 4℃. After post-fixation with 1% OsO4 in cacodylate buffer for 1h at 4℃, the pellet was dehydrated in graded ethanol solutions and embedded in Epon. Ultrathin sections of pellet were counterstained with uranyl acetate and lead citrate and observed under transmission electron microscope.

Fluorescence microscopy
Cells (4 × 10 4 ) were treated as above, suspended with FBS and incubated with Hoechest 33 258 for 30 min at 37℃ from light. Then the cells were observed under fluorescence microscope. Three hundred cells were counted and apoptotic cell rate was calculated.

Detection of DNA fragmentation
Cells (2 × 10 6 ) were treated as above and collected. Fragmented DNA was extracted according to the manufacturer's instructions of apoptosis DNA extract kit and underwent electrophoresis on 2 g/L agarose gel containing 0.5 g/L of ethidium bromide and visulized by UV transillumination.

Detection of mitochondrial function (MTT assay)
Cells (1.5 × 10 4 ) were cultured in 96-well plates and treated with C2-ceramide for 1, 3, 6, 9, 12 and 24 h as above. Then MTT and DMSO were added by turns. Absorbance was determined with a multi-well plate reader at wavelength 570 nm.

RT-PCR
Cells (1 × 10 6 ) were treated for 24 h as above. Total RNA was isolated from the treated cells with Trizol and reversely transcribed into cDNA with human specific primers for Bax, Bad, Bid, Bcl-2, Bcl-xl and b-actin. Sequences of primers are shown in Table 1. Briefly, 35 cycles of PCR amplification were performed at 94℃ for 30 s, annealing at 60℃ for 30 s and extension at 72℃ for 30 s in a 25 mL reaction system. PCR products were confirmed by 1.5 g/L agarose gel electrophoresis and visualized by UV transillumination. mRNA expression of genes was assessed by correcting housekeeping gene b-actin, which served as an internal control.

Statistical analysis
All statistical analyses were performed with SPSS 10.0 statistical package for Microsoft Windows. Data were expressed as mean ± SD for all measurements. P < 0.05 was considered statistically significant.

C2-ceramide induced morphology changes of apoptosis in HT-29 cells
With increasing concentration of C2-ceramide and exposure time, HT-29 cells became round, atrophic and poorly adherent and floating cells increased under light microscope. Cells from control showed normal distribution and morphology in all cellular organelles except for a few necrotic cells under transmission electron microscope. The main ultra-microstructural changes seen in all treated groups were chromatin aggregation, mitochondrial denaturation and apoptotic body, as well as cytoplasmic compartments, swelling and disappearance of mitochondrial cristae, etc (Figure 1). The necrotic changes were more pronounced in cells treated with 50 mmol/L C2ceramide.
Apoptotic cells determined by Hoechest 33 258 assay increased in a time-and dose-dependent manner after treatment with C2-ceramide (Table 2). Normal cellular chromatin did not change and uniformly spread over the whole nuclei displaying diffusion uniformity fluorescence. However, apoptotic chromatin was identifiable by its scattered drop-like structure locating on the area of the original nuclei which displayed high brightness lump or punctiform fluorescence. The total size of apoptotic nuclei  www.wjgnet.com appeared smaller and more shrunken than the intact cells ( Figure 2).

C2-ceramide induced DNA fragmentation of HT-29 cells
DNA ladder was seen through DNA ag arose g el electrophoresis, especially pronounced in the cells treated with 50 and 25 mmol/L C2-ceramide for 12 and 24 h. Cells treated with 12.5 mmol/L C2-ceramide and DMSO kept their integrity, having no ladder appearance (Figure 3).

C2-ceramide damaged mitochondrial function of HT-29 cells
The absorbance value increased gradually with increased concentration of C2-ceramide and prolongation of exposure time. But the absorbance value after treatment with 50 and 25 mmol/L C2-ceramide was obviously lower than that in the control, showing statistical significance from 6 to 24 h, indicating that C2-ceramide could decrease mitochondrial function (Table 3).

C2-ceramide affected mRNA expression of apoptosisrelated genes
After cells were treated with C2-ceramide for 24 h, the mRNA expressions of Bax, Bad and Bid genes were upregulated. However, the expressions of Bcl-2 and Bclxl were down-regulated ( Figure 4). Moreover, the ratio of Bcl-2 to Bax in 50 mmol/L C2-ceramid group was less than 1.

DISCUSSION
Recently, the importance of ceramide in cell metabolism has been broadly investigated. The biological effect of ceramide on different cell lines is different. The role of ceramide-induced apoptosis has been confirmed [4] .
Recent studies indicate that ceramide is a common second    messenger molecule of apoptosis. Apoptosis induced by stimulating factors is mediated by ceramide from sphingomyelin circulation way [5,6] . The effect of ceramide on apoptosis has been studied extensively in neoplastic cells but rarely in solid tumor cells. Ceramide is closely related with genesis and progression of digestive tract tumor. Decreased ceramide content increases the risk of developing digestive tract tumor since ceramide content in human colon carcinoma cells is lower than that in normal colon mucosa cells [7] . To some extent, tumor chemotherapy is to induce apoptosis. In this experiment, human colon cancer cells were exposed to exogenous C2-ceramide. Results indicated that C2-ceramide could induce typical characteristics of apoptosis, such as nuclear chromatin break and apoptotic body as well as DNA ladder in a time-and dosedependent manner. Succinate dehydrogenase (SDH) in the mitochondria is an index of cellular respiration and energy, which reflects mitochondrial function. MTT assay suggested that C2-ceramide could decrease mitochondrial function. By studying Bcl-2 family gene members, we also found that C2-ceramide could up-regulate or down-regulate the mRNA expression of these genes, suggesting that exogenous C2-ceramide induces apoptosis of human colon carcinoma cells in vitro by affecting the expression of Bcl-2 family gene members and damaging the mitochondrial functions.
In conclusion, mitochondria is the target of ceramide. The importance and mechanism of mitochondria in ceramide-induced apoptosis need to be further studied.