9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol, from Eulophia ochreata, inhibits inflammatory signalling mediated by Toll-like receptors

Background and purpose:

9,10-Dihydro-2,5-dimethoxyphenanthrene-1,7-diol (RSCL-0520) is a phenanthrene isolated from Eulophia ochreata, one of the Orchidaceae family, known by local tradition to exhibit medicinal properties. However, no anti-inflammatory activity or any molecular mechanisms involved have been reported or elucidated. Here, for the first time, we evaluate the anti-inflammatory properties of RSCL-0520 on responses induced by lipopolysaccharide (LPS) and mediated via Toll-like receptors (TLRs).

Experimental approach:

The in vitro anti-inflammatory activities of RSCL-0520 were investigated in LPS-stimulated monocytic cells, measuring activation of cytokine and inflammatory genes regulated by nuclear factor-κB (NF-κB). Tumour necrosis factor (TNF)-α levels in serum following LPS stimulation in mice and carrageenan-induced paw oedema in rats were used as in vivo models.

Key results:

Pretreatment with RSCL-0520 effectively inhibited LPS-induced, TLR4-mediated, NF-κB-activated inflammatory genes in vitro, and reduced both LPS-induced TNF-α release and carrageenan-induced paw oedema in rats. Treatment with RSCL-0520 reduced LPS-stimulated mRNA expression of TNF-α, COX-2, intercellular adhesion molecule-1, interleukin (IL)-8 and IL-1β, all regulated through NF-κB activation. RSCL-0520, however, did not interfere with any cellular processes in the absence of LPS.

Conclusions and implications:

RSCL-0520 blocked signals generated by TLR4 activation, as shown by down-regulation of NF-κB-regulated inflammatory cytokines. The inhibitory effect involved both MyD88-dependent and -independent signalling cascades. Our data elucidated the molecular mechanisms involved, and support the search for plant-derived TLR antagonists, as potential anti inflammatory agents.     

Nuclear factor-kappaB is central to the expression of truncated neurokinin-1 receptor in breast cancer: implication for breast cancer cell quiescence within bone marrow stroma

Breast cancer is a leading cause of mortality among women in the United States. Tac1 and neurokinin-1 (NK1) are involved in autocrine stimulation of breast cancer cells (BCCs). The single NK1 gene produces full-length (NK1-FL) and truncated (NK1-Tr) forms. NK1-Tr mediates malignancy in breast cells. We now report a critical role for nuclear factor-kappaB (NF-kappaB) in the expression of NK1-Tr, but not NK1-FL, in human BCCs. By Western and Northern blot analyses, NK1-FL and NK1-Tr were coexpressed in BCCs but were undetectable in nontumorigenic cells. Loss of repressive activity within the 5' flanking region of the NK1 partly accounts for constitutive expression of NK1 in BCCs but could not account for the presence of NK1-Tr. Transient transfections with dominant-negative and wild-type IkappaB show that activation of NF-kappaB is required for the expression of NK1-Tr. Tac1 gene was linked to the generation of NK1-Tr because its overexpression in BCCs led to the production of multiple cytokines that can activate NF-kappaB to mediate NK1-Tr expression. Studies with Tac1 knockdown BCCs and Tac1-expressing nontumorigenic breast cells verified a role for NF-kappaB in the expression of NK1-Tr. The quiescent phenotype of BCCs on contact with bone marrow stroma was partly explained by decreased NF-kappaB activation and undetectable NK1-Tr. In summary, this study shows a role for NF-kappaB in the expression of NK1-Tr in BCCs, which seems to be reversed by bone marrow stromal cells.     

Primary prevention of colorectal cancer. The WHO Collaborating Centre for the Prevention of Colorectal Cancer

Colorectal cancer is the third most common malignant neoplasm worldwide. Epidemiological and laboratory animal studies have established a link between various nutritional factors and the etiology of this cancer. Recent studies in genetic epidemiology and molecular biology have shown that inherited genetic factors also play an important role in colorectal carcinogenesis. Thus, genetic-nutritional interactions may form the basis for the development of this cancer. Nutritional factors that appear to promote or attenuate the carcinogenic process in the colon include fat, excess calories, fibre, calcium, selenium, and various vitamins. Strategies for primary prevention of colorectal cancer should therefore be targeted to all populations who are at risk because of dietary and hereditary predisposition. Based on current knowledge, recommended nutrition guidelines for reducing the risk of colon cancer include decreased fat consumption, adequate amounts of fruits, vegetables, and calcium, and avoidance of overweight. Research to further elucidate the role of diet in colorectal carcinogenesis should include randomized studies in humans, testing of various nutritional regimens, and the use of colonic adenomas and markers of cell proliferation and differentiation as end-points.     

Autocrine proliferation of neuroblastoma cells is partly mediated through neurokinin receptors: relevance to bone marrow metastasis

Despite intensive therapy, ~60–80% of children who are diagnosed with metastatic neuroblastoma (NB) succumb to the disease. NB preferentially metastasizes to the bone marrow (BM). In the present study we used SY5Y and CHP212 (NB cell lines) to study the roles of the preprotachykinin-I (PPT-I) gene and the natural receptors for PPT-I peptides, neurokinin-1 (NK-1) and NK-2, in the biology of NB. PPT-I, NK-1 and NK-2 were constitutively expressed in the NB cells. Functional studies, with specific NK receptor antagonists, showed that PPT-I peptides mediate autocrine proliferation of the NB cells through both NK-1 and NK-2 receptors. Full-length and truncated NK-1 receptors were detected in NB cells. Since there is one copy of the NK-1 gene, we used NK-1-specific siRNA to suppress the expression of NK-1. The NK-1-deficient NB cells showed phenotypes consistent with cell differentiation. Suppression of NK-1 did not appear to cause cell death, as demonstrated by trypan blue exclusion and by undetectable active caspase. NK-1 suppression reduced the proliferation of the NB cells beginning by 10-fold at day 1 and reached a 10⁵-fold reduction by day 10. The NK-1 deficient cells did not proliferate when they were placed as cocultures with BM stroma, which suggests that NK-1 signaling is important for the survival of NB cells in the BM. The results show potential roles for NK receptors in the proliferation of NB.     

Genetic association study of age‐related macular degeneration in the Spanish population

Acta Ophthalmol. 2011: 89: e12–e22

Abstract.

Purpose: To investigate new genetic risk factors and replicate reported associations with advanced age‐related macular degeneration (AMD) in a prospective case–control study developed with a Spanish cohort. Methods: Three hundred and fifty‐three unrelated patients with advanced AMD (225 with atrophic AMD, 57 with neovascular AMD, and 71 with mixed AMD) and 282 age‐matched controls were included. Functional and tagging SNPs in 55 candidate genes were genotyped using the SNPlex^TM genotyping system. Single SNP and haplotype association analysis were performed to determine possible genetic associations; interaction effects between SNPs were also investigated. Results: In agreement with previous reports, ARMS2 and CFH genes were strongly associated with AMD in the studied Spanish population. Moreover, both loci influenced risk independently giving support to different pathways implicated in AMD pathogenesis. No evidence for association of advanced AMD with other previous reported susceptibility genes, such as CST3, CX3CR1, FBLN5, HMCN1, PON1, SOD2, TLR4, VEGF and VLDLR, was detected. However, two additional genes appear to be candidate markers for the development of advanced AMD. A variant located at the 3′ UTR of the FGF2 gene (rs6820411) was highly associated with atrophic AMD, and the functional SNP rs3112831 at ABCA4 showed a marginal association with the disease. Conclusion: We performed a large gene association study in advanced AMD in a Spanish population. Our findings show that CFH and ARMS2 genes seem to be the principal risk loci contributing independently to AMD in our cohort. We report new significant associations that could also influence the development of advanced AMD. These findings should be confirmed in further studies with larger cohorts.