sodium-taurodeoxycholate and Liver-Neoplasms

This study is to investigate the mechanism underlying the anti-apoptotic effects of freeze-dried grape powder (FDGP) and identify the polyphenolic compounds involved. We examined apoptotic signaling pathways affected by FDGP and by its active components, including epicatechin, cyanidin, quercetin, and resveratrol, in human Huh7 hepatoma cells by assaying cell viability assays, the activities of caspase 3 and caspase 7, and the expression of endoplasmic reticulum stress-associated proteins. FDGP dramatically decreased taurodeoxycholic acid (TDCA)-induced production of reactive oxygen species (ROS). Assessment of individual active components revealed that at concentrations corresponding to 300 μg/mL FDGP, only quercetin demonstrated cytoprotective effects against mitochondrial-mediated apoptosis. In contrast, increased concentrations of other individual polyphenolic compounds were required to produce measurable cytoprotective effect. Only combinations of all four polyphenolic compounds (epicatechin, cyanidin, quercetin, and resveratrol) restored a degree of the anti-apoptotic effects seen with FDGP. The pretreatment of FDGP at 30 μg/mL concentration could reverse the thapsigargin-induced effects on the expression of endoplasmic reticulum stress-associated proteins. In conclusion, FDGP reduced oxidative stress, endoplasmic reticulum stress, and apoptosis. The mechanisms involved in the anti-apoptotic effects of FDGP included reduced generation of ROS, and reduced processing of certain caspases. We demonstrated that quercetin, epicatechin, and cyanidin are active compounds within FDGP that attenuate apoptosis. These findings contribute to our understanding of the molecular mechanisms of anti-apoptotic and anti-oxidant effects of grape and are expected to assist in developing clinical protocols to treat a variety of stress-mediated conditions.

Topics: Anthocyanins; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 7; Catechin; Cell Death; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum; Food, Preserved; Freeze Drying; Fruit; Humans; Liver Neoplasms; Mitochondria, Liver; Oxidative Stress; Quercetin; Reactive Oxygen Species; Resveratrol; Stilbenes; Taurodeoxycholic Acid; Vitis

The beneficial effects of grape consumption have been attributed to the antioxidant activity of its polyphenols. This study was conducted to investigate the cytoprotective effects of a freeze-dried grape powder (FDGP) on liver cells. FDGP treatment of primary hepatocytes and hepatoma cells revealed increased metabolic activity of cells and phosphorylation of Akt and IkappaBalpha, as well as up-regulation of proliferating cell nuclear antigen (PCNA) level. To study the molecular mechanisms of FDGP effects, cells were treated with TNF-related apoptosis-inducing ligand (TRAIL); taurodeoxycholic acid (TDCA); thapsigargin (TG), to induce cell apoptosis through death receptor-, mitochondria-, or ER-mediated pathway; and H(2)O(2), to induce oxidative stress, respectively. TDCA-induced activation of caspase-3, caspase-7, caspase-9, and Bax was dramatically decreased with cotreatment of FDGP. Furthermore, FDGP reduced levels of annexin V positive cells by 4-fold. Also, FDGP pretreatment restored cellular glutathione content by 71% in cells treated with H(2)O(2). However, FDGP did not inhibit ER-mediated apoptosis. In conclusion, FDGP increased the viability and metabolic activity of liver cells and attenuated oxidative stress- and mitochondria-mediated apoptosis. These data may contribute to the understanding of the mechanisms involved in protective effects of grape in a variety of liver conditions associated with cellular stress.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Flavonoids; Food, Preserved; Freeze Drying; Fruit; Hepatocytes; Humans; Hydrogen Peroxide; Liver Neoplasms; Mice; Mitochondria, Liver; Oxidative Stress; Phenols; Polyphenols; Taurodeoxycholic Acid; Vitis

To investigate the effect of Tauroursodeoxycholic acid (TUDCA) on Taurodeoxycholic acid (TDCA)-induced HepG2 cell apoptosis and to clarify the molecular mechanism of its anti-apoptosis effect of TUDCA.. Morphologic evaluation of apoptotic cells was performed by Hoechst 33258 staining and electron microscope. DNA fragment was detected by electrophoresis on 1.5% agarose gels. Apoptosis rate was measured by flow cytometry using PI dye. Following incubation of HepG2 cells either with TDCA alone, or coincubation with TUDCA and TDCA, the releasing level of cytochrome c from mitochondria into cytosol was determined by western blot, also the activity of caspase-3, 8, 9.. Incubating the cells with 400 micromol/L TDCA for 12 h induced the cells apoptosis significantly. The apoptotic rate decreased from 50.35% +/- 2.20% to 13.78% +/- 0.84% after coincubation with TUDCA, and this anti-apoptotic effect of TUDCA was confirmed by morphological and DNA ladder detection. TUDCA significantly inhibited the release of cytochrome C from mitochondria into cytosol, and the activity of caspase-9, 3 (t > or = 13.00, P < 0.01), especially at 12 h, caspase-3 activity decreased by 54.9% (t = 16.88, P < 0.01) and 52.5%, however it had no obvious effect on the activity of caspase-8 (t = 1.94, P > 0.05).. TUDCA prevents HepG2 cells apoptosis induced by TDCA through modulating mitochondrial membrane stability, inhibiting the release of cytochrome c and the activation of procaspase-9 and 3. Anti-apoptotic mechanism of TUDCA may be considered to be one of the most important reasons that TUDCA exerts significant efficacy in the treatment of cholestatic liver diseases.

Topics: Apoptosis; Carcinoma, Hepatocellular; Caspase 3; Caspase 9; Caspases; Cytochromes c; Humans; Liver Neoplasms; Taurochenodeoxycholic Acid; Taurodeoxycholic Acid; Tumor Cells, Cultured

In vitro treatment of isolated rat hepatocytes with brominated taurodehydrocholic acid (BTC) reduced their sensitivity against phalloidin and inhibited the uptake of phalloidin as well as of cholate in an irreversible and concentration dependent manner. BTC was taken up itself by liver cells; this process was inhibited by 4,4'-diisothiocyano 2,2'-stilbene disulfonate (DIDS). When hepatocytes were incubated with 35S-BTC their plasma membranes contained five labeled protein species with molecular weights of 67,000, 49,000, 38,000, 32,000 and 24,000 as shown by SDS-electrophoresis. No marked difference was observed when isolated plasma membranes from livers were directly treated with the affinity label. DIDS suppressed covalent binding of 35S-BTC to membrane components drastically. Incubation of phalloidin insensitive AS-30D ascites hepatoma cells with 35S-BTC did not result in a chemical modification of the above five proteins. This agrees with an earlier observation that hepatoma cells are unable to take up phalloidin and bile acids (Petzinger et al. 1979; Rufeger and Grundmann 1977; Kroker et al. 1978).

Topics: Affinity Labels; Animals; Carcinoma, Hepatocellular; Cholic Acids; Deoxycholic Acid; In Vitro Techniques; Liver; Liver Neoplasms; Membrane Proteins; Oligopeptides; Phalloidine; Rats; Steroids, Brominated; Sulfur Radioisotopes; Taurodeoxycholic Acid