Epidemiologic evidence suggests that high dietary intake of Brassica vegetables, such as broccoli, cabbage, and Brussels sprouts, protects against tumorigenesis in multiple organs. 3,3V -Diindolylmethane, one of the active products derived from Brassica vegetables, is a promising antitumor agent. Previous studies in our laboratory showed that 3,3V -diindolylmethane induced a G 1 cell cycle arrest in human breast cancer MCF-7 cells by a mechanism that included increased expression of p21. In the present study, the upstream events leading to p21 overexpression were further investigated. We show for the first time that 3,3V -diindolylmethane is a strong mitochondrial H + -ATPase inhibitor (IC 50 f20 Mmol/L). 3,3V -Diindolylmethane treatment induced hyperpolarization of mitochondrial inner membrane, decreased cellular ATP level, and significantly stimulated mitochondrial reactive oxygen species (ROS) production. ROS production, in turn, led to the activation of stress-activated pathways involving p38 and c-Jun NH 2 -terminal kinase. Using specific kinase inhibitors (SB203580 and SP600125), we showed the central role of p38 and c-Jun NH 2 -terminal kinase (JNK) pathways in 3,3V -diindolylmethaneinduced p21 mRNA transcription. In addition, antioxidants significantly attenuated 3,3V -diindolylmethane-induced activation of p38 and JNK and induction of p21, indicating that oxidative stress is the major trigger of these events. To further support the role of ROS in 3,3V -diindolylmethane-induced p21 overexpression, we showed that 3,3V -diindolylmethane failed to induce p21 overexpression in mitochondrial respiratory chain deficient R 0 MCF-7 cells, in which 3,3V -diindolylmethane did not stimulate ROS production. Thus, we have established the critical role of enhanced mitochondrial ROS release in 3,3V -diindolylmethane-induced p21 up-regulation in human breast cancer cells. (Cancer Res 2006; 66(9): 4880-7)

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To investigate the effect of C-glycosylation at different positions of luteolin, the structure-activity relationships of luteolin and a pair of isomeric C-glycosylated derivatives orientin and isoorientin, were evaluated. We investigated the effects of C-glycosylation on the antioxidant, anti-Alzheimer's disease (AD), anti-diabetic and anti-inflammatory effects of luteolin and its two C-glycosides via in vitro assays of peroxynitrite (ONOO(-)), total reactive oxygen species (ROS), nitric oxide (NO), 1,1-diphenyl-2-picrylhydraxyl (DPPH), aldose reductase, protein tyrosine phosphatase 1B (PTP1B), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-site amyloid precursor cleaving enzyme 1 (BACE1), and cellular assays of NO production and inducible nitric oxide synthase (iNOS)/cyclooxygenase-2 expression in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Of the three compounds, isoorientin showed the highest scavenging activity against DPPH, NO, and ONOO(-), while luteolin was the most potent inhibitor of ROS generation. In addition, luteolin showed the most potent anti-AD activity as determined by its inhibition of AChE, BChE, and BACE1. With respect to anti-diabetic effects, luteolin exerted the strongest inhibitory activity against PTP1B and rat lens aldose reductase. Luteolin also inhibited NO production and iNOS protein expression in LPS-stimulated macrophages, while orientin and isoorientin were inactive at the same concentrations. The effects of C-glycosylation at different positions of luteolin may be closely linked to the intensity and modulation of antioxidant, anti-AD, anti-diabetic, and anti-inflammatory effects of luteolin and its C-glycosylated derivatives.

Aim: The effect of coumarin derivatives on melanogenesis was investigated in B16 murine melanoma cells. Methods: Melanin content and tyrosinase activity were analyzed spectrophotometrically. The expression of tyrosinase, tyrosinaserelated protein-1 (TRP-1) and tyrosinase-related protein-2 (TRP-2) were measured either by reverse transcription-polymerase chain reaction (RT-PCR) or Western blot. Results: Among the coumarin derivatives studied, scoparone (6,7-dimethoxycoumarin) was the most potent; the 6-or 7-methoxy group was found to be essential for the stimulation of melanogenesis. The melanin content was greatly increased by scoparone in a dose-dependent manner; there was no cytotoxicity at the effective concentrations. Scoparone increased enzyme activity as well as protein and mRNA expression of tyrosinase. In addition, mRNA of TRP-1 and TRP-2 were also increased after treatment with scoparone. H-89, an inhibitor of protein kinase A (PKA), completely inhibited the scoparone-induced increase of melanogenesis and the tyrosinase protein. Conclusion: These results suggest that scoparone-induced stimulation of melanogenesis is likely to occur at the transcriptional level of melanogenesis-related enzymes through PKA signaling.