Ella Born

Polyunsaturated fatty acids decrease the expression of sterol regulatory element-binding protein-1 in CaCo-2 cells: effect on fatty acid synthesis and triacylglycerol transport.

Regulation of sterol regulatory element-binding proteins (SREBPs) by fatty acid flux was investigated in CaCo-2 cells. Cells were incubated with 1 mM taurocholate with or without 250 microM 18:0, 18:1, 18:2, 20:4, 20:5 or 22:6 fatty acids. Fatty acid synthase (FAS) and acetyl-CoA carboxylase mRNA levels and gene and protein expression of SREBPs were estimated. 18:2, 20:4, 20:5 and 22:6 fatty acids decreased the amount of mature SREBP-1 and mRNA levels of SREBP-1c, SREBP-1a, FAS and acetyl-CoA carboxylase. SREBP-2 gene or mature protein expression was not altered. Liver X receptor (LXR) activation by T0901317 increased gene expression of SREBP-1c, SREBP-1a, FAS and acetyl-CoA carboxylase without altering SREBP-2. 20:5, but not 18:1, prevented the full expression of SREBP-1c mRNA by T0901317. T0901317 increased SREBP-1 mass without altering the mass of mature SREBP-2. Although only 18:2, 20:4, 20:5 and 22:6 suppressed SREBP-1, acetyl-CoA carboxylase and FAS expression, all fatty acids decreased the rate of fatty acid synthesis. T0901317 increased endogenous fatty acid synthesis yet did not increase secretion of triacylglycerol-rich lipoproteins. In CaCo-2 cells, polyunsaturated fatty acids decrease gene and protein expression of SREBP-1 and FAS mRNA, probably through interference with LXR activity. Since all fatty acids decreased fatty acid synthesis, mechanisms other than changes in SREBP-1c expression must be entertained. Increased endogenous fatty acid synthesis does not promote triacylglycerol-rich lipoprotein secretion.

LXR/RXR ligand activation enhances basolateral efflux of β-sitosterol in CaCo-2 cells

To examine whether intestinal ABCA1 was responsible for the differences observed between cholesterol and β-sitosterol absorption, ABCA1-facilitated β-sitosterol efflux was investigated in CaCo-2 cells following liver X receptor/retinoid X receptor (LXR/RXR) activation. Both the LXR agonist T0901317 and the natural RXR/LXR agonists 22-hydroxycholesterol and 9-cis retinoic acid enhanced the basolateral efflux of β-sitosterol without altering apical efflux. LXR-mediated enhanced β-sitosterol efflux occurred between 6 h and 12 h after activation, suggesting that transcription, protein synthesis, and trafficking was likely necessary prior to facilitating efflux. The transcription inhibitor actinomycin D prevented the increase in β-sitosterol efflux by T0901317. Glybenclamide, an inhibitor of ABCA1 activity, and arachidonic acid, a fatty acid that interferes with LXR activation, also prevented β-sitosterol efflux in response to the LXR ligand activation. Influx of β-sitosterol mass did not alter the basolateral or apical efflux of the plant sterol, nor did it alter ABCA1, ABCG1, ABCG5, or ABCG8 gene expression or ABCA1 mass. Similar to results observed with intestinal ABCA1-facilitated cholesterol efflux, LXR/RXR ligand activation enhanced the basolateral efflux of β-sitosterol without affecting apical efflux. The results suggest that ABCA1 does not differentiate between cholesterol and β-sitosterol and thus is not responsible for the selectivity of sterol absorption by the intestine. ABCA1, however, may play a role in β-sitosterol absorption.

Liver-X-receptor-mediated increase in ATP-binding cassette transporter A1 expression is attenuated by fatty acids in CaCo-2 cells: effect on cholesterol efflux to high-density lipoprotein.

The effect of fatty acids on LXR (liver X receptors)-mediated enhancement of ABCA1 (ATP-binding cassette transporter A1) expression and cholesterol efflux was investigated in human intestinal cells CaCo-2. LXR activation by T0901317 increased basolateral cholesterol efflux to lipoprotein particles isolated at a density of 1.21 g/ml or higher. Oleic and arachidonic acids attenuated the amount of cholesterol isolated from these particles. Stearic, linoleic and docosahexaenoic acids also decreased cholesterol efflux from basolateral membranes, with the polyunsaturated fatty acids being the most potent. Although oleic, arachidonic and docosahexaenoic acids modestly decreased ABCA1 mRNA levels in response to LXR activation, stearic and linoleic acids did not. Except for oleic acid, all fatty acids substantially attenuated an increase in ABCA1 mass secondary to LXR activation. Inhibiting acyl-CoA:cholesterol acyltransferase activity prevented the decrease in cholesterol efflux caused by oleic acid. Thus, in response to LXR activation, all fatty acids decreased the efflux of cholesterol from the basolateral membrane of CaCo-2 cells. Although modest suppression of ABCA1 gene expression by oleic, arachidonic and docosahexaenoic acids cannot be completely excluded as a mechanism, the predominant effect of fatty acids on ABCA1 expression and cholesterol efflux is at a post-transcriptional level.

Triazole-based inhibitors of geranylgeranyltransferase II.

A small set of triazole bisphosphonates has been prepared and tested for the ability to inhibit geranylgeranyltransferase II (GGTase II). The compounds were prepared through use of click chemistry to assemble a central triazole that links a polar head group to a hydrophobic tail. The resulting compounds were tested for their ability to inhibit GGTase II in an in vitro enzyme assay and also were tested for cytotoxic activity in an MTT assay with the human myeloma RPMI-8226 cell line. The most potent enzyme inhibitor was the triazole with a geranylgeranyl tail, which suggests that inhibitors that can access the enzyme region that holds the isoprenoid tail will display greater activity.

Geranyl and neryl triazole bisphosphonates as inhibitors of geranylgeranyl diphosphate synthase

When inhibitors of enzymes that utilize isoprenoid pyrophosphates are based on the natural substrates, a significant challenge can be to achieve selective inhibition of a specific enzyme. One element in the design process is the stereochemistry of the isoprenoid olefins. We recently reported preparation of a series of isoprenoid triazoles as potential inhibitors of geranylgeranyl transferase II but these compounds were obtained as a mixture of olefin isomers. We now have accomplished the stereoselective synthesis of these triazoles through the use of epoxy azides for the cycloaddition reaction followed by regeneration of the desired olefin. Both geranyl and neryl derivatives have been prepared as single olefin isomers through parallel reaction sequences. The products were assayed against multiple enzymes as well as in cell culture studies and surprisingly a Z-olefin isomer was found to be a potent and selective inhibitor of geranylgeranyl diphosphate synthase.

Effect of okadaic acid on apo B and apo A-I secretion by CaCo-2 cells

The effect of protein phosphorylation on the synthesis and secretion of apo B and apo A-I by CaCo-2 cells was investigated. Okadaic acid, a potent inhibitor of protein serine/threonine phosphatases 1 and 2A, caused a significant increase in total cellular protein phosphorylation. Apo B-48 was phosphorylated in control cells and this was increased significantly in the presence of okadaic acid. Under the experimental conditions, the phosphorylation of apo B-100 or apo A-I was not observed. No evidence of tyrosine phosphorylation of apo B-100, B-48, or apo A-I was found. Okadaic acid did not change the amount of apo B mass within cells but apo B mass secreted into the basolateral medium was decreased by 40%. Apo A-I mass within cells or in the basolateral medium was unaffected by okadaic acid. Despite causing an 18% decrease in total protein synthesis, okadaic acid did not alter the rate of synthesis of apo B-100, apo B-48, or apo A-I. Cellular turnover of labeled apo B-100 in cells incubated with okadaic acid was similar to controls, whereas apo B-48 and apo A-I turnover were slowed by okadaic acid. Compared to controls, however, 1 microM okadaic acid caused a 75% and 50% decrease in the secretion of newly synthesized apo B-100 and apo B-48, respectively, while decreasing labeled apo A-I secretion by 35%. In contrast to apo A-I mRNA levels, which were not altered by okadaic acid, apo B mRNA levels were significantly decreased by the polyether fatty acid. Despite differences observed in the phosphorylation state of apo B-100 and apo B-48, okadaic acid decreased the secretion of both forms of apo B without altering their synthesis. Okadaic acid, by increasing cellular protein phosphorylation, significantly disrupts the secretory processing of apo B by CaCo-2 cells.

Mechanisms for autophagy modulation by isoprenoid biosynthetic pathway inhibitors in multiple myeloma cells

Multiple myeloma (MM) is characterized by the production of monoclonal protein (MP). We have shown previously that disruption of the isoprenoid biosynthetic pathway (IBP) causes a block in MP secretion through a disruption of Rab GTPase activity, leading to an enhanced unfolded protein response and subsequent apoptosis in MM cells. Autophagy is induced by cellular stressors including nutrient deprivation and ER stress. IBP inhibitors have been shown to have disparate effects on autophagy. Here we define the mechanisms underlying the differential effects of IBP inhibitors on autophagic flux in MM cells utilizing specific pharmacological inhibitors. We demonstrate that IBP inhibition induces a net increase in autophagy as a consequence of disruption of isoprenoid biosynthesis which is not recapitulated by direct geranylgeranyl transferase inhibition. IBP inhibitor-induced autophagy is a cellular defense mechanism as treatment with the autophagy inhibitor bafilomycin A1 enhances the cytotoxic effects of GGPP depletion, but not geranylgeranyl transferase inhibition. Immunofluorescence microscopy studies revealed that IBP inhibitors disrupt ER to Golgi trafficking of monoclonal light chain protein and that this protein is not a substrate for alternative degradative pathways such as aggresomes and autophagosomes. These studies support further development of specific GGTase II inhibitors as anti-myeloma agents.

Regulation of LDL receptor expression by luminal sterol flux in CaCo-2 cells.

The regulation of expression of the intestinal low density lipoprotein (LDL) receptor by luminal (apical) sterol flux was investigated in the human intestinal cell line CaCo-2. Cells were cultured on semipermeable micropore filters, which separated an upper and lower well. To the apical media were added solutions containing either taurocholate micelles alone or micelles containing sterols. Because of an efflux of cholesterol, which occurred from cells incubated with micelles alone, LDL receptor mRNA levels increased threefold. With an influx of micellar sterols, receptor mRNA levels decreased in a dose-dependent manner. Synthesis and degradation of the LDL receptor were addressed by pulse-chase experiments. In cells incubated with micelles containing 25-hydroxycholesterol, the rate of receptor synthesis was significantly decreased, whereas the rate of receptor turnover remained unchanged. As assessed by immunoblots and steady-state labeling of proteins followed by immunoprecipitation of the LDL receptor, cells incubated with micellar 25-hydroxycholesterol contained substantially less receptor protein. These cells also bound and degraded less LDL. In contrast, in cells incubated with micelles alone, the rate of receptor synthesis was increased and cells contained more LDL receptor protein, although this was not reflected in an increased in LDL binding. The results suggest that LDL receptor expression in CaCo-2 cells is regulated by luminal sterol flux and that this regulation occurs at the level of transcription.

Targeting HSP90 and monoclonal protein trafficking modulates the unfolded protein response, chaperone regulation and apoptosis in myeloma cells

Multiple myeloma is characterized by the production of substantial quantities of monoclonal protein. We have previously demonstrated that select inhibitors of the isoprenoid biosynthetic pathway (IBP) induce apoptosis of myeloma cells via inhibition of Rab geranylgeranylation, leading to disruption of monoclonal protein trafficking and induction of the unfolded protein response (UPR) pathway. Heat-shock protein 90 (HSP90) inhibitors disrupt protein folding and are currently under clinical investigation in myeloma. The effects of combining IBP and HSP90 inhibitors on cell death, monoclonal protein trafficking, the UPR and chaperone regulation were investigated in monoclonal protein-producing cells. An enhanced induction of cell death was observed following treatment with IBP and HSP90 inhibitors, which occurred through both ER stress and non-ER stress pathways. The HSP90 inhibitor 17-AAG abrogated the effects of the IBP inhibitors on intracellular monoclonal protein levels and localization as well as induction of the UPR in myeloma cells. Disparate effects on chaperone expression were observed in myeloma vs amyloid light chain cells. Here we demonstrate that the novel strategy of targeting MP trafficking in concert with HSP90 enhances myeloma cell death via a complex modulation of ER stress, UPR, and cell death pathways.

N-Oxide derivatives of 3-(3-pyridyl)-2-phosphonopropanoic acids as potential inhibitors of Rab geranylgeranylation

The N-oxide derivatives of [2-(3-pyridinyl)-1-hydroxyethylidene-1,1-phosphonocarboxylic acid (or PEHPC) and [2-(3-pyridinyl)-1-ethylidene-1,1-phosphonocarboxylic acid (or PEPC) have been prepared and evaluated for their activity against several enzymes which utilize isoprenoids. The parent pyridines are known inhibitors of GGTase II, but the N-oxide derivatives show no improvement in biological activity in assays with the isolated enzyme. However, the PEHPC N-oxide did induce significant accumulation of intracellular light chain in myeloma cells, consistent with inhibition of Rab geranylgeranylation.