Polyphenolic extracts from cowpea (Vigna unguiculata) protect colonic myofibroblasts (CCD18Co cells) from lipopolysaccharide (LPS)-induced inflammation--modulation of microRNA 126.

Cowpea (Vigna unguiculata) is a drought tolerant crop with several agronomic advantages over other legumes. This study evaluated varieties from four major cowpea phenotypes (black, red, light brown and white) containing different phenolic profiles for their anti-inflammatory property on non-malignant colonic myofibroblasts (CCD18Co) cells challenged with an endotoxin (lipopolysaccharide, LPS). Intracellular reactive oxygen species (ROS) assay on the LPS-stimulated cells revealed antioxidative potential of black and red cowpea varieties. Real-time qRT-PCR analysis in LPS-stimulated cells revealed down-regulation of proinflammatory cytokines (IL-8, TNF-α, VCAM-1), transcription factor NF-κB and modulation of microRNA-126 (specific post-transcriptional regulator of VCAM-1) by cowpea polyphenolics. The ability of cowpea polyphenols to modulate miR-126 signaling and its target gene VCAM-1 were studied in LPS-stimulated endothelial cells transfected with a specific inhibitor of miR-126, and treated with 10 mg GAE/L black cowpea extract where the extract in part reversed the effect of the miR-126 inhibitor. This suggests that cowpea may exert their anti-inflammatory activities at least in part through induction of miR-126 that then down-regulate VCAM-1 mRNA and protein expressions. Overall, Cowpea therefore is promising as an anti-inflammatory dietary component.

The drug resistance suppression induced by curcuminoids in colon cancer SW-480 cells is mediated by reactive oxygen species-induced disruption of the microRNA-27a-ZBTB10-Sp axis.

SCOPE: Mechanisms involving the curcuminoids effects in decreasing the prooncogenic specificity protein (Sp) transcription factors, and Sp-regulated genes in SW-480 colon cancer cells and how the multidrug resistance protein (MDR1) inhibition is mediated by Sp suppression. METHODS AND RESULTS: HT-29 and SW-480 colon cancer and normal CCD-18Co colon fibroblast cells were treated with curcuminoids previously analyzed by HPLC. Gene and protein expression regulation were assessed by RT-PCR, transfections with expression constructs, and Western blots. Curcuminoids (2.5-10 µg/mL) suppressed preferentially the growth of SW-480 and HT-29 compared to CCD-18Co cells and enhanced the anticancer activity of the chemotherapeutic drug 5-fluorouracil due to the suppression of MDR1. Additionally, Sp1, Sp3, and Sp4 and Sp-regulated genes were downregulated by curcuminoids in SW-480 and this was accompanied by suppression of microRNA-27a (miR-27a) and induction of ZBTB10, an mRNA target of miR-27a and a transcriptional repressor of Sp expression. This mechanism was mediated by the induction of ROS. RNA-interference and transfection with ZBTB10-expression plasmid demonstrated that MDR1 was regulated by Sp1 and Sp3 and the disruption of the miR-27a-ZBTB10-Sp axis. CONCLUSION: Colon cancer treatment with curcuminoids will enhance the therapeutic effects of drugs in patients who have developed drug resistance.

Betulinic acid decreases ER-negative breast cancer cell growth in vitro and in vivo: role of Sp transcription factors and microRNA-27a:ZBTB10.

Betulinic acid (BA), a pentacyclic triterpenoid isolated from tree bark is cytotoxic to cancer cells. There is evidence that specificity proteins (Sps), such as Sp1, Sp3, and Sp4, are overexpressed in tumors and contribute to the proliferative and angiogenic phenotype associated with cancer cells. The objective of this study was to determine the efficacy of BA in decreasing the Sps expression and underlying mechanisms. Results show that BA decreased proliferation and induced apoptosis of estrogen-receptor-negative breast cancer MDA-MB-231 cells. The BA-induced Sp1, Sp3, and Sp4 downregulation was accompanied by increased zinc finger ZBTB10 expression, a putative Sp-repressor and decreased microRNA-27a levels, a microRNA involved in the regulation of ZBTB10. Similar results were observed in MDA-MB-231 cells transfected with ZBTB10 expression plasmid. BA induced cell cycle arrest in the G2/M phase and increased Myt-1 mRNA (a microRNA-27a target gene), which causes inhibition in G2/M by phosphorylation of cdc2. The effects of BA were reversed by transient transfection with a mimic of microRNA-27a. In nude mice with xenografted MDA-MB-231 cells, tumor size and weight were significantly decreased by BA treatment. In tumor tissue, ZBTB10 mRNA was increased while mRNA and protein of Sp1, Sp3 and Sp4, as well as mRNA of vascular endothelial growth factor receptor (VEGFR), survivin and microRNA-27a were decreased by BA. In lungs of xenografted mice, human ß2-microglobulin mRNA was decreased in BA-treated animals. These results show that the anticancer effects of BA are at least in part based on interactions with the microRNA-27a-ZBTB10-Sp-axis causing increased cell death.

Standardized curcuminoid extract (Curcuma longa l.) decreases gene expression related to inflammation and interacts with associated microRNAs in human umbilical vein endothelial cells (HUVEC).

The anti-inflammatory effects of curcuminoids have been extensively investigated. However, few studies investigate the mechanistic involvement of microRNAs (miRNAs) in their activity. The objective of this study was to examine the protective effects of standardized curcuminoid extract (SCE) in vascular inflammation of human umbilical vein endothelial cells (HUVEC) and the potential involvement of miRNA-126 and miRNA-146a. Escherichia coli lipopolysacharides (LPS) were used to induce inflammation. LPS-challenge increased gene-expression of toll-like receptor-4 (TLR-4) and downstream genes IL-1 receptor-associated kinase 1 (IRAK-1) and tumor necrosis factor receptor-associated factor 6 (TRAF-6) up to 2.58-, 2.39-, and 3.73-fold, respectively, relative to DMSO-treated controls that were not challenged with LPS. LPS up-regulated TLR-4, IRAK-1, and TRAF-6 in SCE pretreated cells (5 mg L(-1)), only up to 0.69-, 1.28-, and 1.15-fold, respectively. miRNA-146a can be up-regulated by transcription nuclear factor kappa B (NF-κB) and acts as a negative feedback loop regulator involving IRAK-1 and TRAF-6 downregulation. In this study, the down-regulation of NF-κB was accompanied by reduced miRNA-146a expression. LPS-challenge induced mRNA levels of vascular cell adhesion molecule-1 (VCAM-1) and intracellular cell adhesion molecule-1 (ICAM-1) up to 5.65- and 10.65-fold, respectively. SCE prevented this effect and increases of up to only 2.92- and 5.26-fold of DMSO-treated controls not challenged with LPS were observed. miRNA-126 regulates endothelial expression of VCAM-1, but was not inversely correlated to the expression of its target gene VCAM-1 upon SCE treatment; therefore, miRNA-126 does not appear to be involved in the down-regulation of VCAM-1. Overall, curcuminoids are confirmed to have anti-inflammatory properties in HUVEC; however, neither miRNA-146a nor miRNA-126 seem to be involved in the SCE-induced down-regulation of the NF-κB-target genes IRAK-1, TRAF-6, and VCAM-1.

Red wine polyphenolics reduce the expression of inflammation markers in human colon-derived CCD-18Co myofibroblast cells: potential role of microRNA-126.

Chronic intestinal inflammation is an established risk factor for colon cancer. Polyphenolic compounds from fruit and vegetables have been shown to have anti-inflammatory properties in several cell lines and tissues. However, their anti-inflammatory mechanisms, involving microRNAs in the regulation of inflammation, have not been extensively investigated. The goal of this research was to assess the chemopreventive potential of polyphenolics extracted from red wine made with Lenoir grapes (Vitis aestivalis hybrid) in human colon-derived CCD-18Co myofibroblasts cells, and to assess the potential involvement of microRNA-126 (miR-126) in the underlying mechanisms. The results show that the polyphenolic red wine extract (WE) decreased mRNA expression of lipopolysaccharide (LPS)-induced inflammatory mediators NF-kB, ICAM-1, VCAM-1, and PECAM-1 by 1.95-, 1.98-, 1.52-, and 1.84-fold respectively, in a dose dependent manner (0-100 µg of gallic acid equivalent (GAE) mL(-1)) down to 0.80-, 0.79-, 0.66-, and 0.68-fold in DMSO-treated control cells not challenged with LPS, respectively. Correspondingly, miR-126, which has a target region within the 3'-UTR of VCAM-1 mRNA, was increased 2.79-fold by the WE at 100 µg GAE mL(-1). The potential role of miR-126 was confirmed by transfecting cells with a specific miR-126-antagomir, as-miR-126. Transfection with as-miR-126 down-regulated miR-126 to 0.71-fold in the control cells and up-regulated mRNA levels of NF-kB, ICAM-1, VCAM-1, and PECAM-1 to 1.80-, 1.49-, 2.30-, and 1.95-fold of controls, respectively. WE at 100 µg GAE mL(-1) partially reversed the effects of the as-miR-126 to 1.02-, 1.01-, 1.04-, and 1.05-fold, for mRNA levels of NF-kB, ICAM-1, VCAM-1, and PECAM-1 respectively. This indicates the potential role of miR-126 in the anti-inflammatory properties of polyphenolics from red wine in CCD-18Co myofibroblasts cells.

Polyphenolics from açaí ( Euterpe oleracea Mart.) and red muscadine grape (Vitis rotundifolia ) protect human umbilical vascular Endothelial cells (HUVEC) from glucose- and lipopolysaccharide (LPS)-induced inflammation and target microRNA-126.

Endothelial anti-inflammatory effects of açaí (Ac) and red muscadine grape (Gp) polyphenolics have not been extensively investigated. It was hypothesized that polyphenolics from Ac and Gp exert comparable protective effects in human vascular endothelial cells (HUVEC) upon inflammatory stress. Furthermore, this study investigated whether microRNAs relevant to endothelial function might be regulated by Ac and Gp. Results showed that Ac and Gp (5-20 mg gallic acid equivalent/L) protected HUVEC against glucose-induced oxidative stress and inflammation. Glucose-induced expression of interleukin-6 and -8 was down-regulated by Ac and Gp at mRNA and protein levels. Upon lipopolysaccharide (LPS; 1 µg/L)-induced inflammation, Ac and Gp inhibited gene expression of adhesion molecules and NF-κB activation to similar extents, although Gp was more effective in decreasing PECAM-1 and ICAM-1 protein. Of the screened microRNAs, only microRNA-126 expression was found to be modulated by Ac and Gp as the underlying mechanism to inhibit gene and protein expression of VCAM-1.