Apoptotic Effect Of Ethanol On The Sphingomyelin Cycle In PC12 Cells

Objective In this study, we established the ethanol-induced apoptotic model of pheochromocytoma (PC 12) cells. Using the model, we examined the key enzymes activity and mRNA expression changes of the sphingomyelin cycle pathway in PC 12 cells, and assessed the role of the sphingomyelin cycle pathway in apoptosis. Our data lend support to the further study of the neuron apoptosis mechanism.Methods The inhibition effect of ethanol on PC 12 cells proliferation was examined by MTT assay. Cell apoptosis was determined by Hoechst 33258 fluorescence staining and DNA agarose gel electrophoresis mothod. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the mRNA expression changes of SMS1, SMS2 and neutral sphingomyelin enzyme (N-SMase). The sphingomyelin synthase (SMS) activity was detected by the thin-layer chromatography, and N-SMase activity was detected by fluorescence spectrophotometry.Results The ethanol with the concentration of 100 mmol/L,200 mmol/L,400mmol/L and 800 mmol/L decreased the number of PC12 cells to 87.54%,70.73%,57.89% and 51.70% of the control in serum-free media for 24 h. The result indicates the inhibition effect of ethanol on cell proliferation significantly in a dose-dependent manner (P<0.05). No change in viability was observed in cells exposed to 200 mmol/L ethanol in the presence of 10% serum (P>0.05). The nuclei morphological changes detected by using Hoechst 33258 staining and fluorescent microscopy. Addition of ethanol with different concentration to PC 12 cells, resulted in a number of morphological changes that are characteristic of apoptosis,these included cell shrinkage, chromatin compaction and nuclear fragmentation. After being treated with 100mmoL/L,200mmoL/L and 300mmoL/L ethanol in serum-free media for 24 h, the apoptosis rate was 19.21±3.2%(P<0.05),28.39±5.11% (P<0.05) and 38.68±4.28% (P<0.01). The data showed that the cell apoptosis rate was significantly increased by ethanol in does dependent manners. Agarose gel electrophoresis of the DNA isolated from ethanol(100-300mmoL/L)-treated cells maintained in the absence of serum yielded the classical DNA laddering pattern of 180 to 200 bp fragments suggestive of apoptosis. Treatment of PC 12 cells with different ethanol concerntration for 1 or 2 hours increased mRNA expression of SMS1 in does dependent manners,but did not increase mRNA expression of SMS2. After treated with different ethanol concerntration, total SMS activaty was increased in a dose-dependent manner. Although adding ethanol for 0.5-1 hours did not alter mRNA expression and activity of N-SMase, a significant increase in mRNA expression and activity of N-SMase was observed in PC 12 cells incubated for 2 hours in the presence of different concerntration ethanol.Conclusions:1. Ethanol can induce apoptosis of pheochromocytoma (PC 12) cells in a dose-dependent manner.2. Treatment of PC 12 cells with different ethanol concerntration increased mRNA expression and activity of SMS1 and N-SMase in does dependent manners.