George Notas

Antiproliferative and apoptotic effects of selective phenolic acids on T47D human breast cancer cells: potential mechanisms of action

IntroductionThe oncoprotective role of food-derived polyphenol antioxidants has been described but the implicated mechanisms are not yet clear. In addition to polyphenols, phenolic acids, found at high concentrations in a number of plants, possess antioxidant action. The main phenolic acids found in foods are derivatives of 4-hydroxybenzoic acid and 4-hydroxycinnamic acid.MethodsThis work concentrates on the antiproliferative action of caffeic acid, syringic acid, sinapic acid, protocatechuic acid, ferulic acid and 3,4-dihydroxy-phenylacetic acid (PAA) on T47D human breast cancer cells, testing their antioxidant activity and a number of possible mechanisms involved (interaction with membrane and intracellular receptors, nitric oxide production).ResultsThe tested compounds showed a time-dependent and dose-dependent inhibitory effect on cell growth with the following potency: caffeic acid > ferulic acid = protocatechuic acid = PAA > sinapic acid = syringic acid. Caffeic acid and PAA were chosen for further analysis. The antioxidative activity of these phenolic acids in T47D cells does not coincide with their inhibitory effect on tumoral proliferation. No interaction was found with steroid and adrenergic receptors. PAA induced an inhibition of nitric oxide synthase, while caffeic acid competes for binding and results in an inhibition of aryl hydrocarbon receptor-induced CYP1A1 enzyme. Both agents induce apoptosis via the Fas/FasL system.ConclusionsPhenolic acids exert a direct antiproliferative action, evident at low concentrations, comparable with those found in biological fluids after ingestion of foods rich in phenolic acids. Furthermore, the direct interaction with the aryl hydrocarbon receptor, the nitric oxide synthase inhibition and their pro-apoptotic effect provide some insights into their biological mode of action.

Potent inhibitory action of red wine polyphenols on human breast cancer cells.

Breast cancer (one of the most common malignancy in Western societies), as well as esophagus, stomach, lung, bladder, and prostate cancer, depend on environmental factors and diet for growth and evolution. Dietary micronutriments have been proposed as effective inhibitory agents for cancer initiation, progression, and incidence. Among them, polyphenols, present in different foods and beverages, have retained attention in recent years. Red wine is a rich source of polyphenols, and their antioxidant and tumor arresting effects have been demonstrated in different in vitro and in vivo systems. In the present study, we have measured the antiproliferative effect of red wine concentrate, its total polyphenolic pool, and purified catechin, epicatechin, quercetin, and resveratrol, which account for more than 70% of the total polyphenols in red wine, on the proliferation of hormone sensitive (MCF7, T47D) and resistant (MDA‐MB‐231) breast cancer cell lines. Our results indicate that polyphenols, at the picomolar or the nanomolar range, decrease cell proliferation in a dose‐ and a time‐dependant manner. In hormone sensitive cell lines, a specific interaction of each polyphenol with steroid receptors was observed, with IC50s lower than previously described. Interaction of polyphenols with steroid receptors cannot fully explain their inhibitory effect on cell proliferation. In addition, discrete antioxidant action on each cell line was detected under the same concentrations, both by modifying the toxic effect of H2O2, and the production of reactive oxygen species (ROS), after phorbol ester stimulation. Our results suggest that low concentrations of polyphenols, and consecutively, consumption of wine, or other polyphenol‐rich foods and beverages, could have a beneficial antiproliferative effect on breast cancer cell growth. J. Cell. Biochem. 78:429–441, 2000.

Wine antioxidant polyphenols inhibit the proliferation of human prostate cancer cell lines.

The effect of different wine antioxidant polyphenols (catechin, epicatechin, quercetin, and resveratrol) on the growth of three prostate cancer cell lines (LNCaP, PC3, and DU145) was investigated. A dose- and time-dependent inhibition of cell growth by polyphenols was found at nanomolar concentrations. The proliferation of LNCaP and PC3 cells was preferentially inhibited by flavonoids (catechin, epicatechin, and quercetin), whereas resveratrol was the most potent inhibitor of DU145 cell growth. Possible mechanisms of action were investigated: 1) The competition of polyphenols for androgen binding in LNCaP cells revealed significant interaction only in the case of high concentrations of quercetin, at least at five orders of magnitude higher than the concentrations needed for cell growth inhibition. All other phenols showed low interactions. 2) Oxygen species production after mitogen stimulation and H²O²2 sensitivity of these cell lines did not correlate with the observed antiproliferative effects, ruling out such a mode of action. 3) NO production revealed two different patterns: LNCaP and DU145 cells produced high concentrations of NO, whereas PC3 cells produced low concentrations. Phorbol ester stimulation of cells did not reveal any additional effect in LNCaP and DU145 cells, whereas it enhanced the secretion of NO in PC3 cells. Polyphenols decreased NO secretion. This effect correlates with their antiproliferative action and the inhibition of inducible NO synthase. It is therefore proposed that the antiproliferative effect of polyphenols is mediated through the modulation of NO production. In conclusion, our data show a direct inhibitory effect of low concentrations of antioxidant wine phenols on the proliferation of human prostate cancer cell lines mediated by the production of NO, further suggesting potential beneficial effects of wine and other phenol-containing foods or drinks for the control of prostate cancer cell growth.

Polyphenols and cancer cell growth

Polyphenols constitute an important group of phytochemicals that gained increased research attention since it was found that they could affect cancer cell growth. Initial evidence came from epidemiologic studies suggesting that a diet that includes regular consumption of fruits and vegetables (rich in polyphenols) significantly reduces the risk of many cancers. In the present work we briefly review the effects of polyphenols on cancer cell fate, leading towards growth, differentiation and apoptosis. Their action can be attributed not only to their ability to act as antioxidants but also to their ability to interact with basic cellular mechanisms. Such interactions include interference with membrane and intracellular receptors, modulation of signaling cascades, interaction with the basic enzymes involved in tumor promotion and metastasis, interaction with oncogenes and oncoproteins, and, finally, direct or indirect interactions with nucleic acids and nucleoproteins. These actions involve almost the whole spectrum of basic cellular machinery--from the cell membrane to signaling cytoplasmic molecules and to the major nuclear components--and provide insights into their beneficial health effects. In addition, the actions justify the scientific interest in this class of compounds, and provide clues about their possible pharmaceutical exploitation in the field of oncology.

A new automated method for the determination of the Total Antioxidant Capacity (TAC) of human plasma, based on the crocin bleaching assay

BackgroundAntioxidant molecules, which scavenge free radical species to prevent or delay oxidative damage of important macromolecules, membrane lipids and lipoproteins, are prevalent in plasma and other biological fluids. Among them, bilirubin, uric acid and protein thiols are the major endogenous antioxidants, while vitamins C and E, as well as a number of food-derived (poly)aromatic substances, belonging to stilbens, flavonoids and phenolic acids, are the main classes of nutritional antioxidants. Assays for total antioxidant capacity in plasma differ in their type of oxidation source, target and measurement used to detect the oxidized product.MethodsIn the present work we present an automated assay for the estimation of blood total antioxidant capacity (TAC assay), based on the crocin bleaching (oxidation) method. This method was adapted on a modern autoanalyzer, was linear over a wide range of values (0–3 mmol/L), and performed using an end point measurement.ResultsThe TAC method presented a linear correlation with another automated commercial Total Antioxidant Status (TAS) test. Detection of the interference of different metabolites revealed a significant participation of TAC from uric acid, bilirubin, albumin, a minor interference from ascorbic acid, and no interference from hemoglobin. TAC was not modified by two freeze/thawing cycles, and was stable in samples stored at room temperature for 4 hours. K-EDTA and heparin were the best anticoagulants, while citrate decreased TAC by 20%. Reference values derived from samples of normal blood donors was 1.175 ± 0.007 mmol/L (mean ± SEM), while a diet rich in antioxidants more than doubled this value.ConclusionsThe proposed TAC assay, is fully automated, stable and reliable, and could be of value in the estimation of the AC of plasma. It is further proposed to calculate the antioxidant capacity of plasma after a subtraction of all interference deriving from endogenous and/or exogenous metabolites. The antioxidant capacity of plasma thus calculated can be used as a useful indicator of the antioxidant value of foods and beverages in the daily diet.

NK and NKT cells in liver injury and fibrosis.

The innate immune mechanisms of the liver represent an important first line of defense against bacterial products, toxins, and food antigens coming from the intestine. Natural Killer (NK) and Natural Killer T cells (NKT) are components of the innate immune system with increased presence in the liver compared to other organs and have been reported to participate in the inflammatory processes during hepatic diseases. However significant confusion has been noted in this field mainly due to changes in the characterization of these cells as new knowledge accumulates and due to differences in the approaches used for their study. Both cell types can mediate hepatic injury in several models but studies in human liver diseases have not managed to fully explain their functions. However accumulating evidence supports an antifibrotic role of NK cells mainly via an inhibitory effect on hepatic stellate cells by inducing apoptosis and via production of interferon-gamma. Therefore, downregulation of NK cells during most types of liver injury may facilitate liver fibrosis. Data about the role of NKT cells in liver fibrosis are limited. This review will summarize the studies about the role of NK and NKT cells in liver diseases with a special interest in hepatic injury and liver fibrosis.

Adipocytes as Immune Cells: Differential Expression of TWEAK, BAFF, and APRIL and Their Receptors (Fn14, BAFF-R, TACI, and BCMA) at Different Stages of Normal and Pathological Adipose Tissue Development

Adipose tissue represents a rich source of multipotent stem cells. Mesenchymal cells, isolated from this source, can differentiate to other cell types in vitro and therefore can be used for a number of regenerative therapies. Our view of adipose tissue has recently changed, establishing adipocytes as new members of the immune system, as they produce a number of proinflammatory cytokines (such as IL-6 and TNFα and chemokines, in addition to adipokines (leptin, adiponectin, resistin) and molecules associated with the innate immune system. In this paper, we report the differential expression of TNF-superfamily members B cell activating factor of the TNF Family (BAFF), a proliferation inducing ligand (APRIL), and TNF-like weak inducer of apoptosis (TWEAK) in immature-appearing and mature adipocytes and in benign and malignant adipose tissue-derived tumors. These ligands act through their cognitive receptors, BAFF receptor, transmembrane activator and calcium signal-modulating cyclophilic ligand (TACI), B cell maturation Ag (BCMA), and fibroblast growth factor-inducible 14 (Fn14), which are also expressed in these cells. We further report the existence of functional BCMA, TACI, and Fn14 receptors and their ligands BAFF, APRIL, and TWEAK on adipose tissue-derived mesenchymal cells, their interaction modifying the rate of adipogenesis. Our data integrate BAFF, APRIL, and TWEAK and their receptors BCMA, TACI, and Fn14 as novel potential mediators of adipogenesis, in addition to their specific role in immunity, and define immature and mature adipocytes as source of immune mediators.

Secretion of inflammatory mediators by isolated rat Kupffer cells: the effect of octreotide

AIMS We studied the production of inflammatory mediators by rat KC and the possible in vitro effect of the somatostatin analogue octreotide. METHODS Primary KC cultures were incubated with LPS added alone or with different concentrations of octreotide. The production of TNFalpha, IL-6, IL-10, IL-12 and IL-13 was assessed in culture supernatants by ELISA and that of nitric oxide (NO) by a modification of the Griess reaction. RESULTS Isolated KC produced a basal amount of TNFalpha, IL-6, IL-12, IL-13, and NO but not IL-10. LPS-stimulated KC secreted significantly increased amounts of TNFalpha (P < 0.001), IL-6 (P < 0.01), IL-10 (P < 0.001), IL-12 (P < 0.01), and NO (P < 0.001) whereas IL-13 production remained constant. Octreotide reduced IL-12 (P < 0.05) and increased IL-13 (P < 0.05) production by unstimulated KC. Furthermore, octreotide suppressed TNFalpha production (P < 0.05), without modifying TNFalpha mRNA expression and decreased iNOS expression and NO (P approximately 0.05) production by LPS-activated KC. These effects were reversed with Wortmannin pre-treatment suggesting that octreotide may act via interference with phosphatidylinositol 3-kinase pathways. CONCLUSIONS These data demonstrate that KC is a source of multiple inflammatory mediators, indicating a critical role in liver inflammatory disorders. Octreotide modulates inflammatory mediator production by isolated KC, suggesting that it might have immunoregulatory and anti-inflammatory effects in liver diseases.

Octreotide regulates CC but not CXC LPS-induced chemokine secretion in rat Kupffer cells

Kupffer cells (KC) and lipopolysaccharide (LPS) interaction is the initial event leading to hepatic inflammation and fibrosis in many types of liver injury. We studied chemokine secretion by KC activated with LPS and the possible effect of the somatostatin analogue octreotide, in the regulation of this process. KC isolated from Sprague–Dawley rats were cultured in the presence of LPS added alone or with different concentrations of octreotide for 24 and 48 h, and chemokine production was assessed in culture supernatants by ELISA. CC chemokine mRNA expression was assessed by semiquantitative RT–PCR. Vehicle‐stimulated KC produced a basal amount of CC and CXC chemokines. LPS‐stimulated KC secreted significantly increased amounts of IL‐8 (GRO/CINC‐1) (P<0.001), MIP‐2 (P<0.001), MCP‐1 (P<0.001), and RANTES (P<0.01). Octreotide inhibited LPS‐induced secretion of the CC chemokines MCP‐1 (P<0.05) and RANTES (P<0.05), but not the CXC chemokines IL‐8 (GRO/CINC‐1) and MIP‐2, in a concentration‐dependent manner. Downregulation of basal and LPS‐induced mRNA expression of the CC chemokines was also observed in the presence of octreotide. Pretreatment with phosphatidylinositol 3 (PI3)‐kinase inhibitors reduced chemokine production by LPS‐treated KC in both the mRNA and protein level. Furthermore, it prevented the octreotide inhibitory effect on LPS‐induced chemokine secretion, indicating a possible involvement of the PI3‐kinase pathway. In conclusion, these data demonstrate that chemokine secretion by KC can be differentially regulated by octreotide, and suggest that this somatostatin analogue may have immunoregulatory effects on resident liver macrophages.

Apoptosis and apoptosis related proteins in chronic viral liver disease

AbstractBackground: Apoptosis may be an important mechanism of hepatocyte death in chronic viral liver disease. Methods: We studied apoptosis in liver biopsies from 30 patients with chronic viral hepatitis and 8 patients with viral cirrhosis by the TUNEL method. 12 cases of non-alcoholic steatohepatitis and 12 cases of primary biliary cirrhosis were used as non-viral disease controls. Immunohistochemical expression of p53, p21/waf1, bcl-2 and mdm-2 proteins was also studied in the same patients. Results: A statistically significant increase of apoptotic liver cells was found in severe chronic viral hepatitis (5.3 ± 0.3%), cirrhosis (3.4 ± 0.5%) and PBC (4.4 ± 0.4%) cases compared to patients with non-alcoholic steatohepatitis (0.8 ± 0.3%). The expression of p53 protein was increased in the cases of viral cirrhosis and in chronic severe viral hepatitis whereas in the cases of chronic mild hepatitis, PBC and non-alcoholic steatohepatitis we found no expression of p53. P21/waf1 expression was increased in severe chronic hepatitis, cirrhosis and PBC cases compared to mild hepatitis and non-alcoholic steatohepatitis cases. However no induction of mdm-2 was observed in the subgroups of chronic liver disease. Bcl-2 was expressed only in epithelium of bile ducts and mononuclear cells of the portal tracts and liver lobules. A weaker Bcl-2 expression was noted in the epithelium of bile ducts of 7/12 PBC cases. Conclusion: Our results provide evidence of increased apoptosis in severe chronic viral liver disease, suggesting that apoptotic cell death might be involved in the pathogenesis of hepatocellular damage of viral hepatitis and cirrhosis. Furthermore we analysed part of the apoptotic pathways implicated in the above process and found an increased expression of p21/waf1, probably p53 mediated, without overexpression of the apoptosis inhibiting bcl-2 and mdm-2 proteins. By contrast p21/waf1 overexpression in PBC seems to be propagated by a p53 independent mechanism.