Evidence of cell damage induced by major components of a diet-compatible mixture of oxysterols in human colon cancer CaCo-2 cell line.

Cholesterol oxidation products, termed oxysterols, have been shown to be more reactive than unoxidized cholesterol, possessing marked pro-inflammatory and cytotoxic effects in a number of cells and tissues. Oxysterols, absorbed with the diet as products of cholesterol auto-oxidation, have recently been suggested to potentially interfere with homeostasis of the mucosal intestinal epithelium, by promoting and sustaining irreversible damage. However, the treatment of colon cancer cells with a diet-compatible mixture of oxysterols does not elicit the same responses than individual components added to the cells at the same concentrations at which they are present in the mixture. Sixty µM oxysterol mixture showed a slight pro-apoptotic effect on human colon cancer CaCo-2 cell line, evaluated in terms of caspase-3 and caspase-7 activation; conversely, 7α-hydroxycholesterol, 7ß-hydroxycholesterol and 5α,6α-epoxycholesterol were identified to be able to induce a significant pro-apoptotic effect if added to cell culture singly; 7ß-hydroxycholesterol had stronger action than other compounds. The enhanced production of reactive oxygen species through up-regulation of the colonic NADPH-oxidase isoform NOX1 appeared to be the key event in oxysterol-induced apoptosis in these colon cancer cells. As regards pro-inflammatory effects of oxysterols, IL-8 and MCP-1 were evaluated for their chemotactic activity. Only MCP-1 production was significantly induced by 7ß-hydroxycholesterol, as well as by cholesterol and oxysterol mixture. However, oxysterol-induced inflammation appeared to be NOX1-independent, suggesting a secondary role of this enzyme in inducing inflammation in colon cancer cells. A selective cell death induced by specific oxysterols against colon cancer cells, mainly exploiting their ability to activate NOX1 in generating oxidative reactions, might represent a promising field of investigation in colorectal cancer, and might bring new insights on strategies in anticancer therapy.

Proinflammatory effect of cholesterol and its oxidation products on CaCo-2 human enterocyte-like cells: effective protection by epigallocatechin-3-gallate.

Cholesterol and its oxidation products, namely oxysterols, have very recently been shown to potentially interfere with homeostasis of the human digestive tract, by promoting and sustaining irreversible damage of the colonic epithelial layer. This report concerns the strong proinflammatory action that a dietary oxysterol mixture and, to a lesser extent, an identical concentration of unoxidized cholesterol exert on CaCo-2 colonic epithelial cells by up-regulating both expression and synthesis of interleukin 8. The oxysterol mixture and its most effective component, 7ß-hydroxycholesterol, are also shown to markedly enhance the expression of key inflammatory and chemotactic cytokines in colonic epithelial cells, more efficiently than unoxidized cholesterol. The sterols' proinflammatory effect seems to be mediated by enhanced activation of NOX1, because it is prevented by pretreatment of the cells with DPI, a selective inhibitor of this oxidase. Importantly, NOX1 hyperactivation by the oxysterol mixture or cholesterol was fully prevented by CaCo-2 cell preincubation with epigallocatechin-3-gallate. Consistently, supplementation with this compound fully protected colonic epithelial cells against overexpression of inflammatory and chemotactic genes induced by the sterols investigated.

An inter-laboratory validation of methods of lipid peroxidation measurement in UVA-treated human plasma samples.

Lipid peroxidation products like malondialdehyde, 4-hydroxynonenal and F(2)-isoprostanes are widely used as markers of oxidative stress in vitro and in vivo. This study reports the results of a multi-laboratory validation study by COST Action B35 to assess inter-laboratory and intra-laboratory variation in the measurement of lipid peroxidation. Human plasma samples were exposed to UVA irradiation at different doses (0, 15 J, 20 J), encoded and shipped to 15 laboratories, where analyses of malondialdehyde, 4-hydroxynonenal and isoprostanes were conducted. The results demonstrate a low within-day-variation and a good correlation of results observed on two different days. However, high coefficients of variation were observed between the laboratories. Malondialdehyde determined by HPLC was found to be the most sensitive and reproducible lipid peroxidation product in plasma upon UVA treatment. It is concluded that measurement of malondialdehyde by HPLC has good analytical validity for inter-laboratory studies on lipid peroxidation in human EDTA-plasma samples, although it is acknowledged that this may not translate to biological validity.

Combined methotrexate and coenzyme Q10 therapy in adjuvant-induced arthritis evaluated using parameters of inflammation and oxidative stress.

Rheumatoid arthritis is a common severe joint disease that affects all age groups, it is thus of great importance to develop new strategies for its treatment. The aim of the present study was to examine the combined effect of coenzyme Q10 (CoQ10) and methotrexate (MTX) on the progression of adjuvant-induced arthritis in rats. Adjuvant arthritis (AA) was induced by a single intradermal injection of heat-inactivated Mycobacterium butyricum in incomplete Freund's adjuvant. The experiments included healthy animals, arthritic animals not treated, arthritic animals treated with CoQ10, with methotrexate, and with a combination of CoQ10 and methotrexate. The two latter groups received a daily oral dose of 20 mg/kg b.w. of CoQ10, either alone or with methotrexate in an oral dose of 0.3 mg/kg b.w. twice a week. We found that CoQ10 potentiated both the antiarthritic (decrease of hind paw volume) and the antioxidant effect of methotrexate on the level of oxidation of proteins (suppression of protein carbonyl level in plasma) as well as lipoperoxidation (suppression of levels of HNE-adducts and MDA-adducts to plasma proteins). The same effect was observed for plasmatic levels of CoQ9 and IL-1α, and partially also for γ-glutamyltransferase activity assessed in joints and spleen. Moreover, the combination therapy improved the functionality of peripheral blood neutrophils in AA, with a balancing effect on the immunosuppression caused by MTX monotherapy. In summary, combined administration of CoQ10 and methotrexate suppressed arthritic progression in rats more effectively than did MTX alone. This finding may help improve treatment of rheumatoid arthritis.

Reduction of oxidative stress in adjuvant arthritis. Comparison of efficacy of two pyridoindoles: stobadine dipalmitate and SMe1.2HCl.

The aim of this study was to evaluate the therapeutic potential of oxidative stress (OS) reduction by using pyridoindole (PI) antioxidants in adjuvant arthritis (AA). The substances tested were stobadine dipalmitate (STB) and SMe1. AA was used as animal model. The experiments included healthy animals, control arthritic animals and arthritic animals with administration of PI in the oral daily dose of 15 mg/kg b.m during 28 experimental days. The rats were sacrificed on day 28. Clinical and biochemical parameters were determined. The effect of PI administration was evaluated on the basis of the following parameters: (a) arthritis (volume of hind paws - HPW, change of animal body mass - CBM), (b) OS (chemiluminescence of whole blood - CWB, levels of thiobarbituric acid reacting substance - TBARS and of HNE- and MDA-protein adducts in plasma and activity of gamma-glutamyltransferase (GGT) in hind paw joint homogenates). The PI studied significantly increased the CBM of animals and corrected the HPW. STB also significantly decreased the activity of GGT in joint homogenates. SMe1 was more effective in decreasing plasmatic TBARS levels, but STB was more effective in reducing plasmatic HNE- and MDA-protein adducts. The assay for HNE- and MDA-adducts in plasma as a function of time was applied for the first time in AA. STB markedly decreased spontaneous and PMA-stimulated CWB and reduced neutrophil count. In summary, STB was more effective than SMe1 in reducing OS in AA. Our results showed that the reduction of OS in arthritis also corrected the clinical manifestations of the disease.

Pro-oxidant and proapoptotic effects of cholesterol oxidation products on human colonic epithelial cells: a potential mechanism of inflammatory bowel disease progression.

With the aim of investigating whether cholesterol oxidation products could contribute to the pathogenesis of the intestinal epithelial barrier dysfunction that occurs in human inflammatory bowel disease (IBD), differentiated versus undifferentiated CaCo-2 cells, an accepted model for human intestinal epithelial cells, were challenged with a dietary-representative mixture of oxysterols. Only differentiated colonic cells were susceptible to the proapoptotic action of the oxysterol mixture, checked both by enzymatic and by morphological methods, mainly because of a very low AKT phosphorylation pathway compared to the undifferentiated counterparts. Enhanced production of reactive oxygen species by a colonic NADPH oxidase hyperactivation seemed to represent the key event in oxysterol-induced up-regulation of the mitochondrial pathway of programmed death of differentiated CaCo-2 cells. These in vitro findings point to the pro-oxidant and cytotoxic potential of cholesterol oxidation products, of both dietary and endogenous origin, as an important mechanism of induction and/or worsening of the functional impairment of enteric mucosa that characterizes IBD.

The contribution of animal fat oxidation products to colon carcinogenesis, through modulation of TGF-beta1 signaling.

It is now unanimously accepted that neoplastic cells tend to become less susceptible to the growth regulatory effects of transforming growth factor-beta1 (TGF-beta1), mainly because of reduced expression and/or activity of TGF-beta1-specific receptors, as reported for many human cancers including colon cancer. Consequently, a sustained increase of TGF-beta1 in the intestinal mucosa, like that caused by inflammatory processes and/or high dietary intake of animal fat, might become crucial for the progression of a neoplastic clone. In fact, this proapoptotic and prodifferentiating cytokine could eliminate neoplastic cells still susceptible to TGF-beta1's antiproliferative action (TGF-beta1 receptor-positive cells), indirectly favoring the expansion of TGF-beta1 resistant ones (TGF-beta1 receptors deficient or negative cells). The actual concentration of TGF-beta1 in the colonic mucosa undergoing neoplastic transformation is still debated, and the phase of the relevant carcinogenetic process in which a reduced susceptibility to this antiproliferative molecule first occurs has not been precisely established yet. However, no doubt that TGF-beta1 level and activity may be upregulated in cells of the macrophage lineage by animal fat oxidation products, such as oxysterols and aldehydes, as reviewed here. But phagocytes as well as fibroblasts constitutively express TGF-beta1 and are accumulating in tumor-associated stroma. Thus, upregulation of this cytokine system within colonic tumor-associated stroma by excess dietary intake of cholesterol and n-6 polyunsaturated fatty acids appears as a primary mechanism of cancer progression at least in neoplastic lesions of the digestive tract.

TGFbeta1 expression in colonic mucosa: modulation by dietary lipids.

Transforming growth factor beta1 (TGFbeta1) is fundamental to maintain the intestinal epithelial cell homeostasis through its control action on cell proliferation, differentiation and apoptosis. TGFbeta1 dysregulation has been observed in several chronic human diseases, including ulcerative colitis, Crohn's disease and colon carcinoma. In the first two conditions, a marked oxidative stress is consistently present, while in the third one, levels of reactive oxygen species tend to be significantly lower than in the surrounding normal tissue. Lipid-derived compounds such as the aldehyde 4-hydroxynonenal (HNE) or cholesterol oxidation products (oxysterols) were shown able to induce expression and synthesis of TGFbeta1, an event which can be detrimental or beneficial, essentially depending on its actual intensity. Understanding how specific dietary lipids may influence the complex molecular signaling underlying this cytokine expression, may provide new indications for therapeutic and preventive strategies in inflammatory bowel diseases and colon carcinoma.

c-Jun N-terminal kinase upregulation as a key event in the proapoptotic interaction between transforming growth factor-beta1 and 4-hydroxynonenal in colon mucosa.

Cells of colonic mucosa are sensitive to the Smad-mediated growth-inhibitory effect of transforming growth factor-beta1 (TGF-beta1). Another important cell growth inhibitor is the polyunsaturated lipid peroxidation end product, 4-hydroxynonenal (HNE), which triggers apoptosis through c-Jun N-terminal kinase (JNK) activation. Interestingly, a close association between TGF-beta1 and HNE was found in the progression of human colon cancer, with concentration of both molecules inversely related to the malignancy. We investigated the cross talk between Smads and JNK signal transduction pathways in inducing apoptosis. To this purpose TGF-beta1 and HNE were added singly or in combination to CaCo-2 human colon adenocarcinoma cells. The cotreatment induced a marked enhancement of apoptosis and of JNK and Smad4 activities much more than either individual molecule. Cell preincubation with the JNK inhibitor SP600125 significantly prevented JNK and Smad4 enhancement and, subsequently, the cooperative proapoptotic effect was abolished. The primary role of JNK activity in TGF-beta1/HNE cooperative signaling was fully confirmed in a second set of experiments by using JNKi I, a more selective kinase inhibitor. Hence, in tumor cells becoming resistant to TGF-beta1-mediated growth inhibition, increased induction of the remaining TGF-beta1 pathways by interaction with other antiproliferative molecules, such as HNE, could help in inhibiting tumor growth.

Early involvement of ROS overproduction in apoptosis induced by 7-ketocholesterol.

Cholesterol oxidation products are increasingly considered as much more bioactive than the parent compound in the multifactor and multistep process that characterizes atherosclerosis. In particular, 7-ketocholesterol has been reported to induce oxidative stress as well as a marked pro-apoptotic effect in vascular cells including macrophages. With the aim to investigate a possible pathogenic correlation between the two events, cultivated murine macrophages were challenged with a concentration of 7-ketocholesterol actually detectable in human vasculature. Conclusive proof was obtained of a primary role of NADPH-oxidase in the overproduction of reactive oxygen species within cells treated with the oxysterol. In addition, such oxidative burst occurred very early after cell intoxication and it was definitely demonstrated as able to lead cells to apoptotic death. In fact, two metabolic inhibitors of NADPH-oxidase and the antioxidant epicatechin very well counteracted 7-ketocholesterol-induced apoptosis by preventing the oxysterol pro-oxidant action.