Marirosa Putzolu

Role of cholesterol ester pathway in the control of cell cycle in human aortic smooth muscle cells

Cholesterol esterification by acyl‐CoA:cholesterol acyltransferase (ACAT) and proliferation of vascular smooth muscle cells (VSMC) are key events in vascular proliferative diseases. Here we performed experiments to ascertain the role of cholesterol ester pathway in the control of human aortic VSMC cycle progression. Results showed that serum‐induced VSMC proliferation was preceded by an increased ability of the cells to esterify cholesterol as well as by an increased expression of ACAT and multidrug resistance (MDR1) mRNAs and extracellular related kinases 1/2 (ERK1/2), whereas caveolin‐1 levels were markedly decreased. Cell cycle analyses performed in the presence of two inhibitors of cholesterol esterification, directly inhibiting ACAT (Sandoz 58–035) or the transport of cholesterol substrate from plasma membrane to endoplasmic reticulum (progesterone), indicate that each inhibitor suppressed the serum‐induced DNA synthesis by accumulation of VSMCs in the G1 phase. The effect was associated with a rapid inhibition of ERK1/2 mitogenic signaling pathway; a down‐regulation of cyclin D1, ACAT, and MDR1 mRNA; and an up‐regulation of caveolin‐1. These data provide a plausible link between cholesterol esterification and control of cell cycle G1/S transition, supporting the hypothesis that cholesterol esterification may accelerate the progression of human vascular proliferative diseases by modulating the rate of the VSMC proliferation.

CORRELATION BETWEEN CHOLESTEROL ESTERIFICATION, MDR1 GENE EXPRESSION AND RATE OF CELL PROLIFERATION IN CEM AND MOLT4 CELL LINES

Abstract. A positive correlation between cholesterol esterification and growth rate potential was previously found in our laboratory during the growth of CEM and MOLT4 lymphoblastic cells. In the current study, we investigated whether the rates of cholesterol esters synthesis correlate with changes of acyl‐CoAcholesterol acyltransferase (ACAT) mRNA levels and of other genes implied in cholesterol biosynthesis and uptake, such as 3‐hydroxy‐3‐methylglutaryl‐CoA (HMGCoA) reductase and low density lipoprotein (LDL) receptor. The results showed that the more rapid growing CEM cells had lower levels of expression of HMGCoA‐reductase and LDL receptors compared to MOLT4. By contrast, ACAT mRNA levels were higher in CEM cells, further supporting the concept of a possible involvement of cholesterol esters in the regulation of cell growth and division. In this study, high levels of cholesterol esterification and of expression of ACAT gene were also associated with a markedly increased expression of multidrug resistance (MDR1) gene, suggesting that MDR1 activity might contribute to regulate the rate of cell growth and division by modulating intracellular cholesterol ester levels.

Cell growth and cholesterol metabolism in human glucose-6-phosphate dehydrogenase deficient lymphomononuclear cells

Abstract. Atherosclerosis is an inflammatory‐fibroproliferative response of the arterial wall involving a complex set of interconnected events where cell proliferation (lymphomonocytes, and endothelial and smooth‐muscle cells) and substantial perturbations of intracellular cholesterol metabolism are considered to be among the main features. Glucose‐6‐phosphate dehydrogenase (G6PD), the key enzyme of the hexose‐monophosphate shunt pathway, is an essential enzyme involved in both cell growth and cholesterol metabolism, raising the question as to whether G6PD deficiency may have metabolic and growth implications in a deficient population. In the present study, we investigated cell growth and cholesterol metabolism in peripheral blood lymphomononuclear cells (PBMC) from G6PD‐normal (n = 5) and ‐deficient (n = 5) subjects stimulated with lectins (phytohaemoagglutinin and Concanavalin A). G6PD activity, DNA ([3H]‐thymidine incorporation) cholesterol synthesis and esterification ([14C]‐acetate and [14C]‐oleate incorporation), and G6PD, HMGCoA reductase and low density lipoprotein (LDL) receptor mRNA levels (RT‐PCR) all increased following lectin stimulation in both normal and G6PD‐deficient cells. However, these parameters were significantly lower in G6PD‐deficient cells (P < 0.05). It is of interest that G6PD‐deficient PBMC, which showed lower expression of G6PD and higher expression of the LDL receptor gene than normal PBMC under basal conditions, exhibited an opposite pattern after stimulation: G6PD and HMGCoA reductase being expressed at significantly higher levels in deficient than in normal cells (P < 0.05). We conclude that the reduced capability of G6PD‐deficient cells to respond to mitogenic stimuli and to synthesize cholesterol esters may represent favourable conditions for reducing the risk of cardiovascular diseases.

MDR1 gene expression in normal and atherosclerotic human arteries(1).

Recent studies have shown that a membrane p-glycoprotein, encoded by MDR1 gene, is involved in the transport of free cholesterol from the plasma membrane to endoplasmic reticulum, the site of cholesterol esterification by acyl-CoA:cholesterol acyltransferase (ACAT). Moreover, results deriving from our previous studies have shown that the rate of cell proliferation was positively correlated with cholesteryl ester levels as well as with ACAT and MDR1 gene expression. In this study, lipid content and the expression of the genes involved in cholesterol metabolism such as hydroxy-methylglutaryl coenzyme A reductase (HMGCoA-R), low-density lipoprotein receptor (LDL-R), ACAT and MDR1 have been investigated in control and atherosclerotic arteries. The results have shown that the levels of cholesteryl ester increase with the age of cadaveric donors in arteries prone to atherosclerosis (abdominal aorta, superficial femoral artery) and become predominant in advanced atherosclerotic lesions. The mRNA levels of ACAT and MDR1 showed the same age correlation, reaching the highest values in atherosclerotic specimens. These results suggest that MDR1 may be involved in the accumulation of intracellular cholesterol ester levels found in atherosclerotic lesions. Moreover, the levels of HMGCoA-R, LDL-R and ACAT gene expressions progressively increased with the age of cadaveric donors; conversely, in atherosclerotic specimens, the mRNA levels of HMGCoA-R and LDL-R drastically decreased while ACAT gene expression reached its maximum. These findings suggest a reactivation of normal homeostatic regulation of cholesterol in advanced and complicated lesions.

Antiprion Activity of Cholesterol Esterification Modulators: a Comparative Study Using Ex Vivo Sheep Fibroblasts and Lymphocytes and Mouse Neuroblastoma Cell Lines

ABSTRACT Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared to sheep with a resistant genotype. Similar alterations were observed in mouse neuroblastoma N2a cell lines persistently infected with mouse-adapted 22L and RML strains of scrapie that showed up to threefold-higher cholesterol ester levels than parental N2a cells. We now report that proteinase K-resistant prion protein (PrPres)-producing cell populations of subclones from scrapie-infected cell lines were characterized by higher cholesterol ester levels than clone populations not producing PrPres. Treatments with a number of drugs known to interfere with different steps of cholesterol metabolism strongly reduced the accumulation of cholesterol esters in ex vivo PBMCs and skin fibroblasts from scrapie-affected sheep but had significantly less or no effect in their respective scrapie-resistant or uninfected counterparts. In scrapie-infected N2a cells, inhibition of cholesterol esters was associated with selective antiprion activity. Effective antiprion concentrations of cholesterol modulators (50% effective concentration [EC50] range, 1.4 to 40 μM) were comparable to those of antiprion reference compounds (EC50 range, 0.6 to 10 μM). These data confirm our hypothesis that abnormal accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection/replication and a novel molecular target of potential clinical importance.

Abstract C84: Inhibition of cholesterol esterification by fenretinide in osteosarcoma in vitro

Osteosarcoma remains the most common primary malignant bone cancer affecting children and adolescents. Although the combination of systemic chemotherapy and surgery enables long‐term survival in most cases, the poor prognosis of patients with metastatic or recurrent disease and the lack of establishment of second‐line chemotherapy suggest that novel therapeutic strategies for this tumor are needed. Here we show that fenretinide, a synthetic derivative of all‐trans‐retinoic acid, is active against osteosarcoma in vitro, at concentrations identical or lower to those detectable in breast cancer patients plasma during chemopreventive clinical trials. Cell lines were HOS and MG63, known to be resistant to methotrexate and cyclophosphamide, drugs in use in osteosarcoma treatment. We demonstrate for the first time that fenretinide is active in this tumor and that the molecular basis of fenretinide activity is the inhibition of cholesterol esterification, with down‐regulation of ACAT and MDR1 mRNA, followed by downregulation of caveolin‐1 protein expression. This result is in line with our recent results obtained in VSMC and in leukemia cells. The confirmation and extention of these data to different human tumors will provide evidence of a novel mechanism of either cancer or human vascular proliferation disease control by the inhibition of cholesterol esterification. Grants from MIUR, Carisbo Foundation, Pallotti9s Legacy for Cancer Research and University of Bologna. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C84.