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The medicinal plant Withania somniferais widely researched for its anti-inflammatory, cardioactive and central nervous system effects. In Ayurveda, the major Traditional Indian medicine system, extracts from W. somniferaare distinctively employed for the treatment of arthritis and menstrual disorders. Because these conditions involve angiogenic processes we hypothesized that the W. somniferaextracts might contain angiogenesis inhibitors. We employed an endothelial cell-sprouting assay to monitor the purification of substances from W. somniferaroot extracts and isolated as the active principle the previously known natural product withaferin A. We show that withaferin A inhibits human umbilical vein endothelial cell (HUVEC) sprouting in three-dimensional collagen-I matrix at doses which are relevant to NF-kappa B-inhibitory activity. Withaferin A inhibits cell proliferation in HUVECs (IC50=12 nM) at doses that are significantly lower than those required for tumor cell lines through a process associated with inhibition of cyclin D1 expression. We propose that the inhibition of NF-kappa B by withaferin A in HUVECs occurs by interference with the ubiquitin-mediated proteasome pathway as suggested by the increased levels of poly-ubiquitinated proteins. Finally, withaferin A is shown to exert potent anti-angiogenic activity in vivoat doses that are 500-fold lower than those previously reported to exert anti-tumor activity in vivo. In conclusion, our findings identify a novel mode of action of withaferin A, which highlights the potential use of this natural product for cancer treatment or prevention.

Matrix metalloproteinases (MMPs) drive normal tissue remodeling and are implicated in a wide range of pathologies. Although MMP activity is controlled at multiple levels, the primary regulation of MMP activity is transcriptional. The transcriptional promoter elements required for MMP gene expression in cultured cells have been defined, but this has not been extended to the in vivo situation. In this paper, we show that the DNA sequences between -522 and +19 of the rabbit gelatinase B gene (MMP-9) (as characterized in the transgenic mouse line 3445) constitute a minimal promoter that drives appropriate developmental and injury-induced reporter gene expression in transgenic mice. We further show that the expression and activity of three transcription factors (NF-kappaB, AP-2, and Sp1) that control the activity of the gelatinase B promoter are selectively induced in the epithelium migrating to heal a wound. Although promoter activity parallels expression of the endogenous gene in cell cultures, we show by several criteria that cell cultures cannot model many aspects of promoter regulation in vivo. This study reveals that the transgenic mouse line 3445 might be a useful model for investigating the regulation of gelatinase B expression in vivo and for identifying and characterizing new drugs that can control gelatinase B gene transcription.

Matrix metalloproteinases (MMPs) drive normal tissue remodeling and are implicated in a wide range of pathologies. Although MMP activity is controlled at multiple levels, the primary regulation of MMP activity is transcriptional. The transcriptional promoter elements required for MMP gene expression in cultured cells have been defined, but this has not been extended to the in vivosituation. In this paper, we show that the DNA sequences between −522 and +19 of the rabbit gelatinase B gene (MMP-9) (as characterized in the transgenic mouse line 3445) constitute a minimal promoter that drives appropriate developmental and injury-induced reporter gene expression in transgenic mice. We further show that the expression and activity of three transcription factors (NF-κB, AP-2, and Sp1) that control the activity of the gelatinase B promoter are selectively induced in the epithelium migrating to heal a wound. Although promoter activity parallels expression of the endogenous gene in cell cultures, we show by several criteria that cell cultures cannot model many aspects of promoter regulation in vivo. This study reveals that the transgenic mouse line 3445 might be a useful model for investigating the regulation of gelatinase B expressionin vivoand for identifying and characterizing new drugs that can control gelatinase B gene transcription.