Chemopreventive activity of fluphenazine and its analogues in human lymphocyte cultures preincubated with ceramide synthase inhibitor
Abstract
Introduction. The ceramide (cer) is generated mainly via hydrolysis of sphingomyelin by sphingomeylinase (SMase). It was previously documented that chemopreventive activity — proapoptotic and chemosensitizng of piperazine phenothiazine derivative, fluphenazine (FPh) and its newly synthesized analogues, 1b and 3f compounds, depends on the activity of lysosomal acidic sphingomyelinase (aSMase), Zn2+-independent, one of the enzymes participating in intracellular ceramide generation. Another important pathway of intracellular formation of ceramide is de novo synthesis with participation of ceramide synthase (CerS). The aim of this research is to assess the chemopreventive activity of FPh and 1b and 3f compounds after irreversible CerS blockade.
Material and methods. Chemopreventive activity of the tested compounds (10 μM, 2 h) was evaluated in human lymphocyte cultures, genotoxically damaged by benzo[a]pyrene (+B[a]P; 7,5 μM, 48 h) and preincubated with a selective inhibitor CerS — fumonisin B1 (+FB1; 20 μM, 1,5 h) and inspected with fluorescence microscopy, as well as with spectrophotometric and spectrofluorimetric methods. Student’s t-test was used for testing the statistical significance of the results.
Results. It was established that in the presence of the CerS inhibitor (+B[a]P; +FB1) the effects of 1b and 3f analogues on apoptotic cell frequency in lymphocyte cultures and on accumulation of rhodamine 123 (Rod-123), were significantly (p < 0.05) decreased when compared to the cultures carried out in the absence of the CerS inhibitor (+B[a]P; -FB1). Whereas in the case of the parent compound: the FPh, the presence of the inhibitor did not influence on FPh chemopreventive activity.
Conclusions. The proapoptotic and chemosensitizing effects of 1b and 3f compounds were determined in major part by CerS activation. Also the chemopreventive activity of the parent compound: the FPh, was independent of the cer de novo synthesis pathway conducted by CerS.