Glucose-6-phosphate-dehydrogenase deficiency as a risk factor in proliferative disorder development

Glucose-6-phosphate dehydrogenase (G6PD) is an important site of metabolic control in the pentose phosphate pathway (PPP) which provides reducing power (NADPH) and pentose phosphates. The former is mainly involved in the detoxification of chemical reactive species; the latter in the regulation of cell proliferation. G6PD deficiency is the most common enzymopathy in the human population, characterized by decreased G6PD activity, mainly in red blood cells, but actually also in nucleated cells. This decreased activity is not due to enzyme synthesis impairment, but rather to reduced enzyme stability, which leads to a shortening of its half-life. Therefore, a major problem is to understand the underlying mechanisms linking G6PD deficiency to oxidative stress and cell proliferation. In order to address this issue, in the present study we utilized, as an experimental model, fibroblasts isolated from pterygium, an ocular proliferative lesion, from G6PD normal and deficient (PFs+ and PFs-, respectively) patients. Our choice was determined by the fact that pterygium is believed to be caused by chronic oxidative stress induced by UV exposure, and that pterygium fibroblasts resemble a tumorigenic phenotype. As controls we utilized fibroblasts isolated from conjunctiva from G6PD normal and deficient patients (NCFs+ and NCFs-, respectively) who had undergone cataract surgery. 
Growth rate analysis revealed that PFs grow faster than NCFs, but while NCFs- grow more slowly than NCFs+, PFs- and PFs+ grow at the same rate. This was associated with significantly lower G6PD activity in NCFs+ compared to NCFs-, while no significant differences in the G6PD activity of PFs+ and PFs- were noted. This result was supported by the finding that in PFs-, G6PD mRNA levels were significantly higher than in PFs+. Another interesting finding of this study was increased green autofluorescence in both NCFs- and PFs- compared to corresponding positive cells, indicative of pronounced oxidative stress in deficient cells. Finally, abnormal accumulation of neutral lipids, mainly cholesterol esters was observed both in PFs- and PFs+ compared to NCFs- and NCFs+. Though further studies are necessary for better understanding the exact mechanism which links G6PD to oxidative stress and cell proliferation, our data allow to speculate on the role of G6PD on tumorigenesis, and to consider G6PD-deficient subjects at major risk to develop common and dreaded proliferative disorders, such as atherosclerosis and cancer. 
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Modulation of cholesterol homeostasis by antiproliferative drugs in human pterygium fibroblasts.

PURPOSE. The authors have previously shown that the growth of cultured fibroblasts obtained from primary pterygia was associated with an increase in cholesterol esterification, suggesting that alterations of cholesterol homeostasis may be involved in the development and progression of this disorder. This investigation was conducted to determine whether antiproliferative agents such as pioglitazone (PIO) and everolimus (EVE) may inhibit proteins involved in the cholesterol ester cycle and the proliferation of pterygium fibroblasts (PF). METHODS. Quiescent normal conjunctival fibroblasts and PFs were treated with or without inhibitors of cell proliferation (PIO and EVE) or with inhibitors of cholesterol esterification— progesterone (Pg) and Sandoz compound (SaH)—and then were stimulated to growth by 10% fetal calf serum (FCS). Cell proliferation was assessed by counting cells. Trypan blue uptake was used to determine cell viability. mRNA and protein levels were determined by reverse transcription‐polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. RESULTS. PIO and EVE significantly abolished the increase in cholesterol esters, acyl-coenzyme A cholesterol acyltransferase (ACAT1), and multidrug resistance protein (MDR1) mRNA observed in growing cells. Each inhibitor upregulated ATP-binding cassette-A1 (ABCA1), neutral cholesterol ester hydrolase (NCEH) mRNA, and caveolin-1 expression in a manner similar to that of specific inhibitors of cholesterol esterification such as Pg and SaH. CONCLUSIONS. Intracellular modifications of cholesterol homeostasis may be relevant to pterygium development. Moreover, antiproliferative agents such as PIO and EVE may represent a potential topical medication in the prevention and inhibition of pterygium growth at an early stage, probably by modulation of cholesterol ester metabolism. (Invest Ophthalmol Vis Sci. 2007;48:3450‐3458) DOI:10.1167/iovs.06-105

acat1 cav 1 and prp expression in brains and skin fibroblasts from sarda breed sheep with scrapie resistant and scrapie susceptible genotype

Scrapie is an infective ovine neurodegenerative disease; the only identified component of the infectious agent being an aberrant isoform (PrPSc) of the cellular prion protein (PrPC). So far, no means for ante-mortem diagnosis are available for Scrapie as well as for any other mammal Transmissible Spongiform Encephalopaties. We recently found a strong relationship between cell susceptibility to scrapie-infection and intracellular cholesterol homeostasis alterations. In brain tissues as well as in ex vivo cultures of skin fibroblasts and PBMCs from healthy and scrapie-affected sheep carrying a scrapie-susceptible (ARQ/ARQ) genotype, the levels of cholesterol esters were consistently higher than in tissues and cultures derived from animals with a scrapie-resistant (ARR/ARR) genotype. Moreover, both uninfected and scrapie-affected ARQ/ARQ sheep showed abnormally low levels of high density lipoprotein-cholesterol (HDL-C) in their plasma, as compared to ARR/ARR animals. We now show that intracellular accumulation of cholesterol esters in fibroblasts derived from scrapie-susceptible sheep was accompanied by parallel alterations in the expression level of genes and gene products (ACAT1 and Cav-1) that are involved in the pathways leading to intracellular cholesterol esterification and trafficking. Comparative analysis of PrPc mRNA, showed an higher expression level in cells from animals carrying susceptible genotype, with or without Scrapie. Preliminary experiments also revealed the presence of PK-resistant PrP isoforms in the latter cultures. The data reported in the present paper suggest that accumulation of cholesterol esters in peripheral cells, together with the altered expression of some proteins implicated in intracellular cholesterol homeostasis, might serve to identify a distinctive lipid metabolic profile associated with increased susceptibility to develop prion disease following infection

Altered cholesterol ester cycle in ex vivo skin fibroblasts from Alzheimer patients

Recent studies in both animal and cell models of Alzheimer's disease (AD) indicated that sub-cellular cholesterol distribution seems to regulate amyloid-beta (A[beta]) generation in the brain. In particular, cholesterol-esters (CE), rather than total cholesterol levels, appear directly correlated with A[beta] production. Here we observed that, similarly to brain cells, skin fibroblasts obtained from AD patients produce and accumulate more CE than skin fibroblasts from age-matched healthy controls do. AD fibroblasts also exhibited a 2 fold increase in the expression of ACAT1, in addition to lower levels of SREBP2, nCEH, Caveolin-1 and ABCA1 mRNA levels, all of which are involved in the CE cycle. HMGCoA-reductase and LDL-receptor mRNAs levels did not show statistically significant changes in AD, compared to non-AD, cells. Furthermore, although APP mRNA did not significantly vary, neprilysin (NEP), the most important enzyme in the proteolysis of A[beta], was expressed at very low levels in skin fibroblasts of sporadic AD patients. Our results contribute to the concept that AD may be the consequence of a basic and systemic defect in the CE cycle. Moreover, our results identify new possible targets for the diagnosis, prevention, and cure or, at least, amelioration of the symptoms of AD

In vitro synergistic anti-prion effect of cholesterol ester modulators

Background. Our studies on the role of cholesterol in prion infection/replication showed that brains and peripheral cells of sheep susceptible to or suffering from Scrapie were characterized by an altered cholesterol homeostasis compared to animals with a scrapie-resistant genotype, and that drugs influencing cholesterol esterification were endowed with selective anti-prion activity in N2a cell lines infected with the 22L and RML prion strains. Results. In prion-infected N2a cell lines we now report increased anti-prion activity of dual-drug combinations consisting of cholesterol ester modulators associated with prion inhibitors Synergism was obtained with the cholesterol ester modulators everolimus, pioglitazone, progesterone, and verapamil associated with the anti-prion chlorpromazine, and with everolimus and pioglitazone associated with the anti-prion quinacrine. Comparative lipid analyses in prion-infected and non-infected N2a cells by colorimetric, enzymatic, and chemical means, clearly demonstrated a derangement of type and distribution of cholesterol esters, free cholesterol, and triglycerides in the infected N2a cells. Although single-drug treatments influenced lipid syntheses, only the combined-drug treatments appeared to restore a lipid profile similar to that of untreated-uninfected cells. Conclusions. We conclude that the anti-prion synergistic effect of cholesterol ester modulators with the cholesterol metabolism interfering anti-prion drugs chlorpromazine and quinacrine may arise from the ability of combined drugs to re-establish the intracellular lipid profile of untreated-uninfected cells. Overall, these data suggest that inhibition of prion replication can be readily potentiated by combinatorial drug treatments, and that steps of cholesterol/cholesterol ester metabolism may represent suitable targets

accumulation of cholesterol esters in ex vivo lymphocytes from scrapie susceptible sheep and in scrapie infected mouse neuroblastoma cell lines

Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie in sheep, revealed abnormal accumulation of cholesterol esters in brains and in ex vivo skin fibroblasts from genetically scrapie-susceptible, as compared to sheep with resistant genotype. We now report that PBMCs isolated from scrapie-susceptible sheep, as well as mouse neuroblastoma cell lines persistently infected with two different mouse-adapted strains of scrapie, showed similar alterations with up to 3-fold higher cholesterol ester levels than their resistant or uninfected counterparts. Treatments with drugs that interfere with intracellular cholesterol metabolism strongly reduced accumulation of cholesterol esters in scrapie-infected cell lines, whereas had significantly lower, or no effect, in uninfected cell line. These data add support to our hypothesis that accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection and a potential molecular target for prion inhibitors