Screening of Vietnamese medicinal plants for NF-κB signaling inhibitors: Assessing the activity of flavonoids from the stem bark of Oroxylum indicum

Chronic and persistent inflammation contributes to cancer development and can predispose to carcinogenesis. Infection-driven inflammations are involved in the pathogenesis of approximately 15-20% of human tumors. However, even tumors that are not epidemiologically linked to pathogens are characterized by the presence of an inflammatory component in their microenvironment. Hallmarks of cancer-associated inflammation include the presence of infiltrating leukocytes, cytokines, chemokines, growth factors, lipid messengers, and matrix-degrading enzymes. Schematically, two interrelated pathways link inflammation and cancer: (1) genetic events leading to neoplastic transformation promote the construction of an inflammatory milieu; (2) tumor-infiltrating leukocytes, in particular macrophages, are prime regulators of cancer inflammation. Thus, an intrinsic pathway of inflammation (driven in tumor cells), as well as an extrinsic pathway (in tumor-infiltrating leukocytes) have been described and both contribute to tumor progression.

Polyphenols are major components of many traditional herbal remedies, which exhibit several beneficial effects including anti-inflammation. The exact mechanism of the anti-inflammatory action of polyphenols, however, has not been determined. In the present study, we examined the effects of eight different polyphenols isolated from Chinese herbs, including two flavonoids (myricitrin and oroxylin A), four ellagitannins (penta-O-galloyl-beta-glucopyranose, woodfordin C, oenothein B, and cuphiin D1), and two anthraquinones (emodin and physcion), on lipopolysaccharide (LPS)-induced nitric oxide (NO) production, and inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expression in RAW264.7 macrophages. The results indicated that only oroxylin A and emodin concentration-dependently inhibited LPS-induced NO production. The remaining compounds slightly inhibited LPS-induced NO production only at the highest concentration examined. Furthermore, oroxylin A inhibited the expression of LPS-induced iNOS and COX-2 proteins and mRNAs without an appreciable cytotoxic effect on RAW264.7 cells. Emodin also inhibited LPS-induced iNOS protein as potently as oroxylin A, but it inhibited LPS-induced iNOS mRNA expression only slightly and did not affect COX-2 mRNA and proteins. This was consistent with the findings that oroxylin A but not emodin or physcion inhibited prostaglandin E(2) synthesis induced by LPS. The inhibitory effects of oroxylin A on LPS-induced iNOS and COX-2 gene expression were also demonstrated in Bcl-2-overexpressing RAW264.7 macrophages, suggesting that oroxylin A inhibition of iNOS and COX-2 expression was not due to its antioxidant effect. Furthermore, oroxylin A but not emodin blocked nuclear factor-kappaB (NF-kappaB) binding and transcriptional activation associated with decreased p65 proteins in the nucleus induced by LPS. These results indicated that oroxylin A, an active component in Huang Qin, inhibited LPS-induced iNOS and COX-2 gene expression by blocking NF-kappaB activation, whereas emodin inhibition of LPS-induced iNOS expression may be mediated by a different transcription factor.

Water soluble fullerenes, such as the hydroxylated fullerene, fullerenol (C₆₀OHx), are currently under development for diagnostic and therapeutic biomedical applications in the field of nanotechnology. These molecules have been shown to undergo urinary clearance, yet there is limited data available on their renal biocompatibility. Here we examine the biological responses of renal proximal tubule cells (LLC-PK1) exposed to fullerenol. Fullerenol was found to be cytotoxic in the millimolar range, with viability assessed by the sulforhodamine B and trypan blue assays. Fullerenol-induced cell death was associated with cytoskeleton disruption and autophagic vacuole accumulation. Interaction with the autophagy pathway was evaluated in vitro by Lysotracker Red dye uptake, LC3-II marker expression and TEM. Fullerenol treatment also resulted in coincident loss of cellular mitochondrial membrane potential and ATP depletion, as measured by the Mitotracker Red dye and the luciferin-luciferase assays, respectively. Fullerenol-induced ATP depletion and loss of mitochondrial potential were partially ameliorated by co-treatment with the autophagy inhibitor, 3-methyladenine. In vitro fullerenol treatment did not result in appreciable oxidative stress, as measured by lipid peroxide and glutathione content. Based on these data, it is hypothesized that cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, leading to subsequent autophagy dysfunction and loss of mitochondrial capacity. As nanoparticle-induced cytoskeleton disruption, autophagic vacuole accumulation and mitochondrial dysfunction are commonly reported in the literature, the proposed mechanism may be relevant for a variety of nanomaterials. Copyright © 2010 Elsevier Inc. All rights reserved.