Human BCL2A1 blocking peptide

Thymoquinone (TO), an active ingredient of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against a variety of experimental tumors. However, the precise mechanism of action of TO is not known. We investigated the mechanism of action of TO in androgen receptor (AR)-independent (C4-2B) and AR naive (PC-3) prostate cancer cells, as models of aggressive prostate cancers. Exposure (24-48 h) to TO (25-150 mu mol/L) inhibited the growth of both C4-2B and PC-3 cells, with IC(50) values of approximately 50 and 80 mu mol/L, respectively. Within one hour, TO increased reactive oxygen species (ROS) levels (3-fold) and decreased glutathione (GSH) levels (60%) in both cell types. Pretreatment with N-acetylcysteine (NAC) inhibited both TO-induced ROS generation and growth inhibition. TO did not increase the activity of caspases and the caspase inhibitor, z-VAD-FMK did not decrease TO-induced apoptosis. Furthermore, although TO treatment resulted in the activation of Jun kinase (JNK), pretreatment with the JNK inhibitor, SP600125, did not protect cells from TO. However, TO significantly up-regulated the expressions of growth arrest and DNA damage inducible gene (GADD45 alpha) and apoptosis-inducing factor-1 and down-regulated the expressions of several Bc12-related proteins, such as BAG-1, Bcl2, Bcl2A1, Bcl2L1 and BID. In C4-2B cells, TO dose dependently inhibited both total and nuclear AR levels (4-5 fold) and AR-directed transcriptional activity (10-12 fold). Interestingly, this suppressive effect on AR was not prevented by NAC, which clearly suggested that TO-induced cytotoxicity is not due to changes in AR regulation. These data suggest that TO-induced cell death is primarily due to increased ROS generation and decreased GSH levels, and is independent of AR activity.

Background. Cell free DNA (cfDNA) circulates throughout the bloodstream of both healthy people and patients with various diseases. CfDNA is substantially enriched in its GC-content as compared with human genomic DNA. Principal Findings. Exposure of haMSCs toGC-DNAinduces short-termoxidative stress (determined with H2DCFH-DA) and results in both single-and doublestrand DNA breaks (comet assay and gamma H2AX, foci). As a result in the cells significantly increases the expression of repair genes (BRCA1 (RT-PCR), PCNA (FACS)) and antiapoptotic genes (BCL2 (RT-PCR and FACS), BCL2A1, BCL2L1, BIRC3, and BIRC2 (RT-PCR)). Under the action of GC-DNA the potential of mitochondria was increased. Here we show that GC-rich extracellular DNA stimulates adipocyte differentiation of human adipose-derived mesenchymal stem cells (haMSCs). Exposure to GC-DNA leads to an increase in the level of RNAPPARG2 and LPL (RT-PCR), in the level of fatty acid binding protein FABP4 (FACS analysis) and in the level of fat (Oil Red O). Conclusions. GC-rich fragments in the pool of cfDNA can potentially induce oxidative stress and DNA damage response and affect the direction of mesenchymal stemcells differentiation in human adipose-derivedmesenchymal stem cells. Such a response may be one of the causes of obesity or osteoporosis.