Butyrate-induced proapoptotic and antiangiogenic pathways in EAT cells require activation of CAD and downregulation of VEGF

Butyrate, a short-chain fatty acid produced in the colon, induces cell cycle arrest, differentiation, and apoptosis in transformed cell lines. In this report, we study the effects of butyrate (BuA) on the growth of Ehrlich ascites tumor (EAT) cells in vivo. BuA, when injected intraperitoneally (i.p) into mice, inhibited proliferation of EAT cells. Further, induction of apoptosis in EAT cells was monitored by nuclear condensation, annexin-V staining, DNA fragmentation, and translocation of caspase-activated DNase into nucleus upon BuA-treatment. Ac-DEVD-CHO, a caspase-3 inhibitor, completely inhibited BuA-induced apoptosis, indicating that activation of caspase-3 mediates the apoptotic pathway in EAT cells. The proapoptotic effect of BuA also reflects on the antiangiogenic pathway in EAT cells. The antiangiogenic effect of BuA in vivo was demonstrated by the downregulation of the secretion of VEGF in EAT cells. CD31 immunohistochemical staining of peritoneum sections clearly indicated a potential angioinhibitory effect of BuA in EAT cells. These results suggest that BuA, besides regulating other fundamental cellular processes, is able to modulate the expression/secretion of the key angiogenic growth factor VEGF in EAT cells.     

Structural features of a polysaccharide from the mucin of water hyacinth

The mucin found in the nodal region of the weed, water hyacinth (Eichhornia crassipes), is a heteropolysaccharide composed of d-xylose, l-galactose and l-arabinose in the mol ratio of 1.3:1.2:1.0. Partial hydrolysis with acid gave four oligosaccharides which were characterized as: d-Xylp-(1 → 3)-l-Ara, l-Galp-(1 -→ 2)-l-Ara, d-Xylp-(1 → 3)-l-Galp-(1 → 2)-l-Ara, and d-Xylp-(1 → 2)-d-Xylp-(1 → 3)-l-Galp-(1 → 2)-l-Ara. These, together with the results of methylation analysis using GC and GC/MS and periodate oxidation, indicated that the trisaccharide repeating unit, → 4)-d-Xylp-(1 → 3)-l-Galp-(1 → 2)-l-Araf-(1 →, constitutes the backbone of the polysaccharide. Further, all the d-xylopyranosyl residues of the backbone are substituted at O-2 and, in addition, one out of seven such residues is also substituted at O-3; the substituents being l-Araf-(1 →, d-Xylp-(1 →, l-Galp-(1 →, d-Xylp-(1 → 3)-l-Araf-(1 →, residues.     

Molecular mechanisms of anti-angiogenic effect of curcumin

Modulation of pathological angiogenesis by curcumin (diferuloylmethane), the active principle of turmeric, seems to be an important possibility meriting mechanistic investigations. In this report, we have studied the effect of curcumin on the growth of Ehrlich ascites tumor cells and endothelial cells in vitro. Further, regulation of tumor angiogenesis by modulation of angiogenic ligands and their receptor gene expression in tumor and endothelial cells, respectively, by curcumin was investigated. Curcumin, when injected intraperitoneally (i.p) into mice, effectively decreased the formation of ascites fluid by 66% in EAT bearing mice in vivo. Reduction in the number of EAT cells and human umbelical vein endothelial cells (HUVECs) in vitro by curcumin, without being cytotoxic to these cells, is attributed to induction of apoptosis by curcumin, as is evident by an increase in cells with fractional DNA content seen in our results on FACS analysis. However, curcumin had no effect on the growth of NIH3T3 cells. Curcumin proved to be a potent angioinhibitory compound, as demonstrated by inhibition of angiogenesis in two in vivo angiogenesis assay systems, viz. peritoneal angiogenesis and chorioallantoic membrane assay. The angioinhibitory effect of curcumin in vivo was corroborated by the results on down-regulation of the expression of proangiogenic genes, in EAT, NIH3T3, and endothelial cells by curcumin. Our results on Northern blot analysis clearly indicated a time-dependent (0-24h) inhibition by curcumin of VEGF, angiopoietin 1 and 2 gene expression in EAT cells, VEGF and angiopoietin 1 gene expression in NIH3T3 cells, and KDR gene expression in HUVECs. Further, decreased VEGF levels in conditioned media from cells treated with various doses of curcumin (1 microM-1mM) for various time periods (0-24h) confirm its angioinhibitory action at the level of gene expression. Because of its non-toxic nature, curcumin could be further developed to treat chronic diseases that are associated with extensive neovascularization.     

Antiangiogenic effects of butyric acid involve inhibition of VEGF/KDR gene expression and endothelial cell proliferation

The formation of new blood vessels from pre-existing ones is required for the growth of solid tumors and for metastasis. Interaction of tumor-secreted vascular endothelial growth factor (VEGF) with its receptor(s) on endothelial cells triggers endothelial cell proliferation and migration, which facilitate tumor angiogenesis. Butyric acid (BuA), a fermentation product of dietary fibers in the colon, is shown to alter gene expression and is postulated to be anticarcinogenic. The results presented in this paper indicate that BuA can be antiangiogenic in vivo by inhibiting angiogenesis in chorioallantoic membrane assay. BuA was not cytotoxic to endothelial cells but was a potent antiproliferative agent besides being proapoptotic to endothelial cells as verified by FACS analysis. Conditioned media from BuA-treated Ehrlich ascites tumor cells showed a 30% decrease in VEGF concentration when compared with untreated cells. The decrease in VEGF mRNA and its receptor, KDR mRNA levels in EAT and endothelial cells respectively, suggests that the VEGF-KDR system of angiogenesis is the molecular target for the antiangiogenic action of BuA.     

Purification and characterization of butyrate-induced protein phosphatase involved in apoptosis of Ehrlich ascites tumor cells

Short chain fatty acids including butyrate exhibit wide variety of biological effects towards cell growth, morphology and gene expression. In this report, we study the mechanism by which butyrate (BuA) modulates the expression of protein phosphatase when treated to the cells. As a model system, we used Ehrlich Ascites Tumor (EAT) cells in which BuA-treatment induces expression of a protein phosphatase enzyme. Subsequently, BuA-induced protein phosphatase has been biochemically purified and characterized. Further, pretreatment of caspase-3 inhibitor abolished the activity of BuA-induced protein phosphatase indicating the involvement of caspase-3 in the activation of BuA-induced protein phosphatase. In addition, the relationship between BuA-induced protein phosphatase and apoptosis has been verified. Activation of endonuclease-II has been shown in BuA-treated EAT cells and that activity was completely inhibited by sodium orthovanadate, a tyrosine phosphatase inhibitor suggesting that endonuclease-II may serve as a possible down-stream target for BuA-induced protein phosphatase. Together, the data suggest that activation of protein phosphatase may be an early and essential step in BuA-mediated apoptotic signaling pathway in EAT cells.