Capsicodendrin (CPCD, 1), an epimeric mixture of a dimeric drimane-type sesquiterpene, is one of the major compounds present in the three endemic species of Madagascan traditional chemopreventive plants: Cinnamosma species ( C. fragrans, C. macrocarpa, and C. madagascariensis). Despite the popular use of Cinnamosma in Madagascan traditional medicine and the reported antiproliferative properties of CPCD, elucidation of its mechanism(s) of action is still to be accomplished. In the present study, CPCD at low micromolar concentrations was cytotoxic and induced apoptosis in human myeloid leukemia cells in a time- and concentration-dependent manner. The activity of CPCD in HL-60 and K562 cells was modulated by glutathione (GSH), since depletion of this intracellular thiol-based antioxidant with buthionine sulfoximine resulted in significantly ( p < 0.05) greater potency in antiproliferation assays. GSH depletion also significantly potentiated the cytotoxic activity in CPCD-treated human HL-60 cells. Single-cell gel electrophoresis (Comet) assays revealed that GSH depletion in HL-60 cells enhanced the formation of DNA strand breaks in the presence of CPCD. Although CPCD does not contain an obvious Michael acceptor in its structure, H NMR analyses indicated that cinnamodial (2), a monomer of CPCD, was formed within a few hours when dissolved in DMSO- d and interacts with GSH to form a covalent bond via Michael addition at the C-7 carbon. Together the results strongly suggest that 2 is responsible for the DNA-damaging, pro-apoptotic, and cytotoxic effects of CPCD and that depletion of GSH enhances overall activity by diminishing covalent interaction between GSH and this 2-alkenal decomposition product of CPCD.

E. coli endotoxin (LPS) combined with interferon‐γ (IFN) induces inducible nitric oxide synthase (iNOS), with production of nitric oxide (NO) and reactive nitrogen species in endothelial cells. Appropriate manipulation of iNOS during onset or resolution of inflammation may be useful. Since transcription is required for induction of iNOS by LPS/IFN, we hypothesized that depletion of cyclin dependent kinase (CDK) 7, a promoter of transcription, would antagonize induction. Mouse aortic endothelial cells (MAEC) were transfected with Non‐targeted (NT) control RNA or CDK7 siRNA to deplete the kinase, followed by stimulation with LPS/IFN for 12 h. Rather than suppressing, CDK7 depletion increased induction of iNOS, and its dimerization and NO production. The effect was post‐transcriptional in that mRNA induction was unaffected, while iNOS protein turnover was reduced in the absence of CDK7. Cellular calpain activity, which is required for iNOS protein degradation in MAEC, was unaffected by CDK7 depletion, suggesting an effect on iNOS itself. Indeed, iNOS in lysates of kinase‐deficient cells was resistant to in vitro digestion with exogenous calpain. In conjunction with protease resistance, the association of iNOS with calmodulin, known to inhibit its digestion by calpain, was increased in cells lacking CDK7. These results suggest that CDK7 normally restrains iNOS induction by limiting calmodulin binding and maintaining calpain‐mediated turnover. By regulating iNOS enzyme complexes, CDK7 may serve as a novel target for manipulation of vascular NO. Support or Funding Information Support was received from the Popat Patil Fellowship (SS).

Endothelial cell prostaglandins modulate the response to inflammatory stimuli. Combined treatment with E. coli endotoxin and interferon‐γ (LPS/IFN) induces cyclooxygenase‐2 (COX2) mRNA and protein. Modulation of COX2 expression may be therapeutically useful in different inflammatory states. Given the induction of COX2 mRNA by LPS/IFN, it was hypothesized that depletion of the transcription‐stimulating enzyme, cyclin‐dependent kinase 7 (CDK7), would antagonize induction of COX2. Here, mouse aortic endothelial cells were treated with CDK7 or non‐targeted siRNA, followed by LPS/IFN. Contrary to our expectations, knocking down CDK7 super‐induced the expression of COX2, without affecting mRNA or turnover of the protein. This suggested that CDK7 depletion increased protein synthesis. Here, signaling mechanisms known to regulate translation were investigated. CDK7 knockdown increased phosphorylation at Serine 209 of the mRNA cap‐binding translation initiation factor, eIF4E. Knockdown also increased cap‐binding activity of eIF4E. CDK7 depletion increased phosphorylation of the eIF4E repressor, 4EBP1, and decreased its association with eIF4E. Finally, increased phosphorylation of ribosomal protein S6 (RPS6), a component of the 40S ribosomal subunit, was also seen in the absence of CDK7, indicating increased translation. The results suggest that CDK7 represses COX2 expression by limiting translation through effects on RPS6, eIF4E and/or 4EBP1. CDK7 may serve as a target for the manipulation of COX2 expression in endothelial cells. Support or Funding Information Support was received from the Popat Patil Fellowship (SS).