Osteosarcoma remains the most common primary malignant bone cancer affecting children and adolesc... more 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 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 confirm 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
The results showed a highly significant reduction of proliferation rate in leukemia cells treated... more The results showed a highly significant reduction of proliferation rate in leukemia cells treated with each cholesterol esterification inhibitor. This effect was not caused by cell toxicity, as cell morphology and viability were unaffected at the concentrations of inhibitors used. In addition, it appears that changes in the cholesterol biosynthetic pathway are not responsible for the antiproliferative effect observed, being cholesterol synthesis unaffected in leukemia cells with reduced activity of cholesterol esterification. The inhibition of cell proliferation was associated with a reduction of ACAT and MDR1 mRNA expression and conversely with an up-regulation of p-53 protein expression (p53 not shown). Our results imply that cholesterol esters and MDR1 could play a role in the dynamic processes of lipid transport inside the cells. ACAT inhibitors, by reducing cellular cholesterol esters and MDR1 gene expression and through these pathways the proliferation rate of leukemia cells, may warrant consideration as innovative targeted anticancer agents
Recent studies in both animal and cell models of Alzheimer's disease (AD) indicated that sub-... more 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 l...
Scrapie is an infective ovine neurodegenerative disease; the only identified component of the inf... more 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 i...
Background. Previous epidemiological and experimental studies indicated cholesterol as a central ... more Background. Previous epidemiological and experimental studies indicated cholesterol as a central player in Alzheimer disease (AD). Here, we utilized skin fibroblasts and PBMC as possible ex vivo models for the study of dysfunctions of cholesterol homeostasis which may be related to AD development. Methods. We analyzed cholesterol homeostasis using colorimetric, thin layer chromatography (TLC), and histologic technique in ex vivo cultures of skin fibroblasts and PBMCs from patients with probable AD and their first-degree relatives. Additionally, healthy age-matched individuals served as controls. Findings. As compared to controls, skin fibroblasts and PBMCs from AD patients, displayed an evident alteration of cholesterol metabolism; namely an anomalous accumulation of cholesterol esters in their cytoplasm. No change in intracellular free cholesterol was observed. Cellular overloading of cholesterol esters was dramatically increased after specific growth stimulation of the different c...
CEM and MOLT4 are human T-cell lines isolated from patients with acute cell leukaemia. In culture... more CEM and MOLT4 are human T-cell lines isolated from patients with acute cell leukaemia. In culture they show important differences in cholesterol metabolism, CEM being less efficient at synthesizing cholesterol and having a lower activity of 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase. To investigate further the relationship between regulation of intracellular cholesterol metabolism at various steps and rate of cell growth, cholesterol synthesis, esterification and efflux were evaluated in CEM and MOLT4 cells at different times during exponential and stationary growth in vitro. It was shown that, although CEM cells have a lower rate of cholesterol synthesis, they grow at a faster rate than MOLT4 cells. However, CEM cells exhibit an increased capacity to esterify cholesterol associated with a decreased efflux of newly synthesized cholesterol into the medium. These results provide evidence for an association between the capability to synthesize and retain cell cholesterol esters ...
Osteosarcoma remains the most common primary malignant bone cancer affecting children and adolesc... more 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 MD...
The effects of ageing on the metabolism of cholesterol were examined in three different organs (l... more The effects of ageing on the metabolism of cholesterol were examined in three different organs (liver, aorta and brain) of 6-, 12- and 24-month-old male Sprague-Dawley rats. Ageing was associated with a significant increase in intracellular cholesterol esters in all three organs. Steady state mRNA levels of multidrug resistance protein (MDR) and acylCoA:cholesterol acyl transferase (ACAT), enzymes involved in cholesterol import and esterification, were also increased. By contrast, expression of mRNA for neutral cholesterol ester hydrolase (nCEH) and caveolin-1, proteins involved in cholesterol ester hydrolysis and export, were significantly reduced. Dietary restriction is the only intervention shown to extend lifespan and retard age-related declines in function in mammals. To further explore the possible correlation between changes in cholesterol esterification and ageing, we analysed cholesterol metabolism in liver, aorta, and brain of aged rats exposed to two dietary restriction regimens: intermittent (alternate-day) fasting (IF) and food intake restriction (60% of ad libitum feeding). Both dietary regimens attenuated the age-related changes in cholesterol esters and in the expression of genes involved in cholesterol metabolism. These results provide evidence that distinctive age-associated changes in intracellular cholesterol metabolism occur in rats. Furthermore, these modifications can be partially reversed by dietary restriction, a condition known to affect the ageing process. Age-related changes in cholesterol metabolism may play a role in triggering and/or aggravating senescence-related disorders characterized by altered cholesterol homeostasis.
Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnorm... more 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 fr...
Osteosarcoma remains the most common primary malignant bone cancer affecting children and adolesc... more 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 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 confirm 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
The results showed a highly significant reduction of proliferation rate in leukemia cells treated... more The results showed a highly significant reduction of proliferation rate in leukemia cells treated with each cholesterol esterification inhibitor. This effect was not caused by cell toxicity, as cell morphology and viability were unaffected at the concentrations of inhibitors used. In addition, it appears that changes in the cholesterol biosynthetic pathway are not responsible for the antiproliferative effect observed, being cholesterol synthesis unaffected in leukemia cells with reduced activity of cholesterol esterification. The inhibition of cell proliferation was associated with a reduction of ACAT and MDR1 mRNA expression and conversely with an up-regulation of p-53 protein expression (p53 not shown). Our results imply that cholesterol esters and MDR1 could play a role in the dynamic processes of lipid transport inside the cells. ACAT inhibitors, by reducing cellular cholesterol esters and MDR1 gene expression and through these pathways the proliferation rate of leukemia cells, may warrant consideration as innovative targeted anticancer agents
Recent studies in both animal and cell models of Alzheimer's disease (AD) indicated that sub-... more 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 l...
Scrapie is an infective ovine neurodegenerative disease; the only identified component of the inf... more 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 i...
Background. Previous epidemiological and experimental studies indicated cholesterol as a central ... more Background. Previous epidemiological and experimental studies indicated cholesterol as a central player in Alzheimer disease (AD). Here, we utilized skin fibroblasts and PBMC as possible ex vivo models for the study of dysfunctions of cholesterol homeostasis which may be related to AD development. Methods. We analyzed cholesterol homeostasis using colorimetric, thin layer chromatography (TLC), and histologic technique in ex vivo cultures of skin fibroblasts and PBMCs from patients with probable AD and their first-degree relatives. Additionally, healthy age-matched individuals served as controls. Findings. As compared to controls, skin fibroblasts and PBMCs from AD patients, displayed an evident alteration of cholesterol metabolism; namely an anomalous accumulation of cholesterol esters in their cytoplasm. No change in intracellular free cholesterol was observed. Cellular overloading of cholesterol esters was dramatically increased after specific growth stimulation of the different c...
CEM and MOLT4 are human T-cell lines isolated from patients with acute cell leukaemia. In culture... more CEM and MOLT4 are human T-cell lines isolated from patients with acute cell leukaemia. In culture they show important differences in cholesterol metabolism, CEM being less efficient at synthesizing cholesterol and having a lower activity of 3-hydroxy-3-methylglutaryl-CoA (HMGCoA) reductase. To investigate further the relationship between regulation of intracellular cholesterol metabolism at various steps and rate of cell growth, cholesterol synthesis, esterification and efflux were evaluated in CEM and MOLT4 cells at different times during exponential and stationary growth in vitro. It was shown that, although CEM cells have a lower rate of cholesterol synthesis, they grow at a faster rate than MOLT4 cells. However, CEM cells exhibit an increased capacity to esterify cholesterol associated with a decreased efflux of newly synthesized cholesterol into the medium. These results provide evidence for an association between the capability to synthesize and retain cell cholesterol esters ...
Osteosarcoma remains the most common primary malignant bone cancer affecting children and adolesc... more 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 MD...
The effects of ageing on the metabolism of cholesterol were examined in three different organs (l... more The effects of ageing on the metabolism of cholesterol were examined in three different organs (liver, aorta and brain) of 6-, 12- and 24-month-old male Sprague-Dawley rats. Ageing was associated with a significant increase in intracellular cholesterol esters in all three organs. Steady state mRNA levels of multidrug resistance protein (MDR) and acylCoA:cholesterol acyl transferase (ACAT), enzymes involved in cholesterol import and esterification, were also increased. By contrast, expression of mRNA for neutral cholesterol ester hydrolase (nCEH) and caveolin-1, proteins involved in cholesterol ester hydrolysis and export, were significantly reduced. Dietary restriction is the only intervention shown to extend lifespan and retard age-related declines in function in mammals. To further explore the possible correlation between changes in cholesterol esterification and ageing, we analysed cholesterol metabolism in liver, aorta, and brain of aged rats exposed to two dietary restriction regimens: intermittent (alternate-day) fasting (IF) and food intake restriction (60% of ad libitum feeding). Both dietary regimens attenuated the age-related changes in cholesterol esters and in the expression of genes involved in cholesterol metabolism. These results provide evidence that distinctive age-associated changes in intracellular cholesterol metabolism occur in rats. Furthermore, these modifications can be partially reversed by dietary restriction, a condition known to affect the ageing process. Age-related changes in cholesterol metabolism may play a role in triggering and/or aggravating senescence-related disorders characterized by altered cholesterol homeostasis.
Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnorm... more 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 fr...
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