The anti-proliferative and pro-apoptotic effects of the trans9, trans11 conjugated linoleic acid isomer on MCF-7 breast cancer cells are associated with LXR activation.

Conjugated linoleic acids (CLA), naturally found in dairy products and ruminant meat, are positional and geometric isomers (trans: t or cis: c) of linoleic acid, and have been widely reported to possess anti-tumoral activity against breast cancer both in vitro and in vivo. CLA isomer t9,t11 was recently proposed as an agonist of the transcriptional factor LXR, which is known for inducing genes implicated in cholesterol efflux. In this study, the growth inhibitory effect of three CLA isomers (c9,t11-CLA, t9,t11-CLA and t10,c12-CLA) was investigated on MCF-7 breast cancer cells, as well as their effect on LXR target genes. Our results revealed that t9,t11-CLA was the most efficient isomer by decreasing MCF-7 proliferation, inhibiting migration, and inducing apoptosis after 24h of treatment. t9,t11-CLA treatment led to an increase in the mRNA levels of LXR target genes involved in cholesterol efflux (ABCG1 and ARL7), as well as an increase of HMG-CoA-reductase which is the rate-limiting step of cholesterol biosynthesis. Interestingly, confocal microscopy analysis showed that t9,t11-CLA treatment remarkably reduced the intracellular and membrane-associated cholesterol levels. LXR activation through t9,t11-CLA isomer could lead to cholesterol cell deprivation by stimulating its efflux, which results in the inhibition of cell proliferation and stimulation of apoptosis.

Effect of LPS on basal and induced apo E secretion by 25-OH chol and 9cRA in differentiated CaCo-2.

The infection and inflammation process is associated with disturbances in lipid and lipoprotein metabolism. The apolipoprotein E (apo E) plays an important role in the lipoprotein metabolism and has been linked to inflammatory disease such as atherosclerosis and Alzheimer disease. An anti-inflammatory effect has also been suggested. The heterodimer nuclear receptor Liver-X-Receptor(alpha)/Retinoid-X-Receptor (LXR(alpha)/RXR) is considered to be a transcription factor for apo E. The aim of this study was to determine whether lipopolysaccharide (LPS) (principal component of the outer membrane Gram-negative bacteria) has an effect on apo E secretion by intestinal mucosa cells, using the Caco-2 cell line. Differentiated Caco-2 cells grown on filter inserts were incubated apically with LPS and/or 25-hydroxycholesterol (25-OH chol) and 9 cis retinoic acid (9cRA), ligands of LXR and RXR, respectively. The apical and basolateral media were separately collected. Apo E was detected by specific antibodies after protein separation by Two-dimensional nondenaturing gradient gel electrophoresis and apo E secreted in the cell culture media was measured by enzyme linked immunosorbent assay (ELISA). Apo E mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). LXR(alpha) and RXR mass was analyzed by Western Blot. We demonstrate here that CaCo-2 cells secrete apo E, by either apical or basolateral sides, associated with a high-density like lipoprotein, with a stoke's diameter comprised between 7.10 and 8.16 nm. We show that only apical secretion is decreased by LPS in a dose and time dependent manner. This is associated with a decrease in apo E gene expression contrasting with an increase of Il-8, a chemokine factor. Moreover, we demonstrate that only basolateral apo E secretion by CaCo-2 is significantly increased by 25-OH chol and 9cRA while apical secretion remains unchanged. LPS does not decrease the 25-OH chol and 9cRA mediated apo E secretion in basolateral compartment, while apical secretion is diminished under these circumstances. Our results provide evidence for the polarized secretion of apo E by intestinal epithelium. They also demonstrate that apo E secretion by CaCo-2 cell line is decreased by LPS through an LXR(alpha)/RXR independent signaling pathway.

Adenovirus-mediated gene transfer into isolated mouse adult pancreatic islets: normal beta-cell function despite induction of an anti-adenovirus immune response.

The in vitro purification of pancreatic islets offers an opportunity for their modification by ex vivo gene transfer. We investigated the efficiency and functional consequences of adenovirus-mediated gene transfer into adult murine pancreatic islets with a recombinant adenovirus encoding for the beta-galactosidase (beta-Gal) reporter gene. At 10(6) pfu/islet, almost all of the islets were transduced, but maximal transduction was obtained at 10(7) pfu/islet. Histochemical analysis of frozen islet sections showed that transduced cells were only located at the periphery of islets. Transduced islets showed normal insulin secretion in vitro, and were able to normalize in vivo the glycemia of streptozotocin-induced diabetes in syngeneic and allogeneic mice. beta-Gal expression in transduced islets was observed for at least 6 weeks in naive normal recipients and in immunodeficient mice, but was shortened in mice preimmunized to adenovirus. Nevertheless, islets maintained normal control of glycemia in all mice. An early leukocyte infiltrate was observed in syngeneic grafts of transduced islets, but no acceleration in rejection of fully MHC-incompatible islet grafts occurred. In summary, adenovirus-mediated gene transfer in adult mouse islets, although sparing most of the beta-cells, was highly efficient and did not impair insulin secretion by islets. The immune response to the adenovirus and/or to the transgene might be only partially responsible for the decreased expression over time of the transduced gene. Accordingly, adenovirus-mediated gene transfer might allow efficient expression of vectorized sequences with potential immunosuppressive effects in the islet microenvironment.

Intact pig pancreatic islet function in the presence of human xenoreactive natural antibody binding and complement activation.

BACKGROUND: The expression of xenogeneic epitopes and the activation of human complement by adult pig islets after prolonged culture have hitherto not been described. MATERIALS AND METHODS: Freshly isolated and cultured islets were analyzed by fluorescence-activated cell sorter analysis, fluorescence microscopy, and immunohistology for expression of Gal(alpha1,3)Gal epitopes, binding of human xenoreactive natural antibodies (XNA), and complement deposition. RESULTS: Freshly isolated and cultured islets showed detectable Gal(alpha1,3)Gal expression and human XNA binding limited to intraislet capillary endothelial cells. No significant modification in Gal(alpha1,3)Gal expression and human XNA binding levels was detected in adult pig islets cultured for up to 4 days compared with freshly isolated islets. Incubation of pig islets with human serum demonstrated the deposition of C3, C4, and membrane attack complex, but not factor B with a similar pattern to XNA. However C3 and C4 showed a more widespread deposition. Despite complement activation, no cytotoxic effect on islets was detected after 4 hr of incubation with human serum capable of killing porcine endothelial cells. Even after 4 days of culture in 50% intact human serum, pig islets retained both their normal morphology and a normal insulin response to glucose stimulation. CONCLUSIONS: Neither islet cell lysis nor, more importantly, any alteration in beta cell function occurred, which suggests that adult pig islets may not be directly damaged by serum after xenotransplantation in humans. Nevertheless, complement activation in vivo could trigger rapid cellular rejection mechanisms through islet cell opsonization and release of bioactive fragments.