OBJECTIVE To investigate the effects of ceramide on apoptosis and its difference in colon carcinoma LoVo cells which were
induced by the major
catechins in green tea,including (-)epigallocatechin3gallate (EGCG),(-)epigallocatechin (EGC),and (-)epicatechin (EC).METHODS After
treatment of LoVo cells with EGCG,EGC and EC,as well as C 2 and C 6ceramide,the apoptosisassociated alterations in morphology and biochemistry were examined qualitatively and quantitatively usig agarose gel electrophoresis,staining with the DNAbinding fluorochrome bisbenzimide,Hoechst 33342,and flow cytometry (FCM).The changes of intracellular ceramide content after treatment of LoVo cells with EGCG,EGC,or EC were determined by diacylglycerol kinase assay.RESULTS Treatment of LoVo cells with EGCG or EGC resulted in the apoptosisassociated alterations such as the formation of internucleosomal DNA fragements on agarose gel electrophoresis and hypodiploid peaks on the histogram of FCM.EC,however,did not have the effects,indicating that there were different effects of the three individual catechins on the induction of apoptosis in LoVo cells.In addition,the addition of membranepermeable C 2and C 6ceramide to LoVo cells mimicked EGCG or EGC in leading to the apoptotic alterations.Treatment of LoVo cells with EGCG or EGC resulted in a transient increase in ceramide content at about 20 min,which was 2 to 3 times higher than that in the control cells,and then back gradually to base line level.In contrast,EC,resulted in a first decrease within 30 min and then a gradual increase to base line level.CONCLUSION Ceramide takes part in the regulation of the catechinsinduced apoptosis in LoVo cells.The differences in the effects of the individual catechins on the induction of apoptosis in LoVo cells may be associated with the differences in the generation of intracellular ceramide.