6). survival, against serum deprivation-induced apoptosis. Their effects were time- and dose-dependent, with EC50 1.8, 1.1, and 1.5 nM, respectively. The antiapoptotic effect of DHEA DHEAS and Allo was compared to that of a long list of structurally related compounds and was found to be structure-specific, confined mainly to conformation 3-OH-5 for androstenes and 3-OH for pregnanes. Indeed, 3-keto, 4, or C7 hydroxylated androstenes and 3 pregnanes were ineffective. The prosurvival effect of DHEA(S) and Allo was for their action because Bcl-2 antisense oligonucleotides reversed their effects. Finally, DHEA(S) and Allo activated cAMP response element-binding protein and NF-B, upstream effectors of antiapoptotic Bcl-2 protein expression. They also activated the antiapoptotic kinase PKC/, CK-666 a posttranslational activator of Bcl-2 protein. Our findings suggest that decline of DHEA(S) and Allo during aging or stress may leave the adrenal medulla unprotected against proapoptotic challenges. The neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester DHEA sulfate (DHEAS) and allopregnanolone (Allo) are produced in the brain and the adrenals (1C3). Their production rate and levels in serum and adrenals decrease gradually with advancing age (4C7). Physical or emotional stress may decrease them, characteristic paradigms being depressive disorder (8) and chronic inflammation (9). The decline of their levels is associated with neuronal dysfunction and degeneration (10C12), most probably because these steroids safeguard CNS neurons against noxious brokers (13C15). Indeed, both DHEA and DHEAS [DHEA(S)] protects rat hippocampal neurons against = 3, 0.001). For comparison, serum supplementation for 12 h showed an apoptosis rate of 0.61 0.04. Inhibition of apoptosis in chromaffin cells was retained for at least 48 h. Open in a separate window Fig. 1. DHEA(S) and Allo guarded rat chromaffin cells in culture against serum deprivation-induced apoptosis. Freshly isolated rat chromaffin cells were cultured either in complete or serum-free media made up of 10C7 CK-666 M DHEA, DHEAS, or Allo for various time periods (2C48 h). Apoptosis was quantified by the APOPercentage assay. Values represent mean SD of three impartial experiments. Each measurement was performed in triplicate (*, 0.05). Based on these data, additional experiments were carried out by using the well established model of chromaffin cell apoptosis, the PC12 rat pheochromocytoma cell line (20). As expected, CK-666 serum deprivation had a deleterious effect on PC12 cell cultures. FACS analysis revealed that 25% of PC12 cells maintained in serum-free medium underwent apoptosis within 24 h (Fig. 2 0.05). ( 0.05). (axis represents Annexin V-FITC, whereas the axis represents the number of events. A total of 10,000 cells were assigned per treatment. ( 0.05). The effects of steroids were examined in serum-deprived PC12 cells and were compared to the protective effect of serum, according to the conditions used in the case of chromaffin cells. DHEA, DHEAS, and Allo exerted a strong time-dependent Rabbit Polyclonal to TPH2 (phospho-Ser19) antiapoptotic effect (Fig. 2= 6, 0.001). Thus, all three steroids tested strongly inhibited serum deprivation-induced apoptosis by 50%, to the extent that their protective effects were also easily visualized under optical microscopy. For comparison, serum supplementation for 24 h showed an apoptosis rate of 0.047 0.008, resulting, as expected, in higher protection. The antiapoptotic effects were dose-dependent with EC50 at 1.8, 1.1, and 1.5 nM for DHEA, DHEAS, and Allo, respectively (Fig. 2depicts a mean 40% inhibition of serum deprivation-induced apoptosis in PC12 cells exposed to three steroids. Indeed, the percentage of apoptotic cells cultured in serum-free medium in the absence of steroids was 24.6%, compared to 15.1%, 16.9%, and 10.8% for DHEA, DHEAS, and Allo, respectively. This profile of FACS analysis was highly reproducible in at least three impartial experiments. The Antiapoptotic Effect of DHEA(S) and Allo Was Structure-Specific. To assess the specificity of the cytoprotective action of DHEA, DHEAS, and Allo, a host of structurally related compounds were also CK-666 tested in parallel to our steroids. StructureCactivity analysis revealed the following data. (depicts CK-666 their effect on the transcriptional level. Open in a separate window Fig. 3. DHEA(S) and Allo induced the expression of the antiapoptotic Bcl-2 proteins in serum-deprived PC12 cells. Cells were cultured for 2C12 h either in complete or serum-free media made up of 10C7 M DHEA, DHEAS, or Allo. Cellular extracts made up of total mRNA or total proteins were collected, and levels of Bcl-2.