J.C. Mazière

In vitro interaction of the photoactive anticancer porphyrin derivative photofrin II with low density lipoprotein, and its delivery to cultured human fibroblasts

Low density lipoprotein (LDL) doped with the anticancer mixture of hematoporphyrin derivatives Photofrin II (P2) competes with native LDL for binding to fibroblast receptors, despite a slight increase in the negative net charge related to the presence of acidic residues of porphyrins. P2 delivery to fibroblasts can be achieved by LDL, HDL3 or albumin doped with P2 (LDL‐P2, HDL‐P2 or A‐P2, respectively). P2 delivery to cells assessed by fluorescence measurement, is much more efficient, at low protein concentrations (10–20 ) by LDL‐P2 than by HDL‐P2 or A‐P2. Moreover, P2 delivery to cells by LDL‐P2 as a function of protein concentration is a saturable process, whereas P2 delivery by HDL‐P2 or A‐P2 is a linear process. Finally, reduction of the LDL‐receptor number by preincubation of fibroblasts in medium supplemented with lipoproteins results in a decrease of P2 delivery by LDL‐P2. These results suggest a special role of the LDL‐receptor pathway in P2 delivery to cells and could be of interest in cancer phototherapy by porphyrins.

In situ degradation of sphingomyelin by cultured normal fibroblasts and fibroblasts from patients with Niemann-Pick disease type A and C

Abstract Cultured normal human skin fibroblasts actively degraded sphingomyelin [choline-methyl-14C] introduced in ethanolic solution in the culture medium. After 17 h incubation, about 65 to 80 % of the cellular radioactivity was recovered in phosphatidylcholine. In fibroblasts from Niemann-Pick disease type A the in situ degradation of sphingomyelin was less than 2 % of controls, which was in good agreement with the strong decrease of the sphingomyelinase activity measured in vitro by conventional methods. In the two cases of Niemann-Pick disease type C studied, the in situ degradation of sphingomyelin was significantly but not dramatically decreased compared to controls.

PHOTOSENSITIZATION BY PORPHYRINS DELIVERED TO L CELL FIBROBLASTS BY HUMAN SERUM LOW DENSITY LIPOPROTEINS. A MICROSPECTROFLUOROMETRIC STUDY

Human serum LDL were used as vehicles to deliver protoporphyrin and hematoporphyrin dimer to L cell mouse fibroblasts. Topographic analysis by microspectrofluorometry on single living cells shows that after digestion of LDL, protoporphyrin is localized in cytoplasmic areas. Protoporphyrin and hematoporphyrin dimer are readily bleached by 420 ± 60 nm radiations at the high fluence rate used. Complex bleaching kinetics are observed. Spectral studies using the same technique demonstrate that an intense fluorescent emission (λmax= 450 nm) is produced immediately after the onset of irradiation with 365 ± 2 nm or 420 ± 60 nm radiations using LDL loaded with protoporphyrin or Photofrin II. These fluorescent products have been previously identified as lipofuscin‐like pigments formed by reaction of lipid photoperoxides with amino groups. The permeation of lysosomal membranes is also induced after delivery of the porphyrins by LDL. This permeation can be strongly inhibited not only by the lysosomal inhibitors chloroquine and monensin but also by a‐tocopherol. On the other hand, neither α‐tocopherol nor chloroquine or monensin inhibit the lipofuscin‐like pigment formation.

MODIFICATION OF ε‐AMINO GROUP OF LYSINES, CHOLESTEROL OXIDATION AND OXIDIZED LIPID‐APOPROTEIN CROSS‐LINK FORMATION BY PORPHYRIN‐PHOTOSENSITIZED OXIDATION OF HUMAN LOW DENSITY LIPOPROTEINS

Abstract— When incorporated into LDL, protoporphyrin and the porphyrin mixture constituting the trade mark Photofrin II photosensitize peroxidations of cholesterol and lipids. Not only 5a‐hydroperoxicholesterol, the specific product of cholesterol oxidation by 1O2 but also the epimeric 6‐hydroperoxicholesterols are produced as shown by HPLC. In addition to malonaldehyde‐like substances, the formation of 4‐hydroxynonenal, a highly reactive and toxic aldehyde resulting from lipid peroxide breakdown is detected. These products are also formed by dark radical chain reactions of lipid photoperoxides induced by photosensitization. The involvement of lipid photoperoxidation in LDL apoprotein modification is demonstrated by following the derivatization of e‐NH2 groups of Lys residues which are necessary to binding to the LDL receptor. As a result, photosensitized LDL cannot bind to their receptors on fibroblasts. Lys residues are not sensitive to direct photodynamic reaction as confirmed by delipidation of LDL and solubilization of apoLDL in 1% SDS which totally inhibit Lys derivatization without affecting the degradation of Trp residues susceptible to the photodynamic attack. Another consequence of lipid peroxidation at the protein level is the formation of cross‐links between apoLDL and photooxidized lipids as shown using LDL loaded with radioactive arachidonic acid. On the other hand, cholesterol photoperoxidation does not lead to protein‐oxidized cholesterol cross‐links. These reactions between peroxidized lipids and LDL proteins are also responsible for the formation of lipofuscin‐like fluorescent pigments encountered in all aging processes. The biological and biomedical consequences of these results are discussed.

Photosensitization of Wi26-VA4 transformed human fibroblasts by low density lipoprotein loaded with the anticancer porphyrin mixture photofrin II: evidence for endoplasmic reticulum alteration

Wi26 VA4 cells (SV40-transformed human lung fibroblasts) were incubated with low density lipoprotein (LDL) loaded with the anticancer porphyrin mixture photofrin II (P2). After labelling with 51Cr sodium chromate, cells were exposed to near UV light. After light exposure, kinetics of 51Cr release by cells were studied as a probe of cell damage. The activity of acyl-coenzyme A:cholesterol-O-acyltransferase, a key enzyme of cholesterol metabolism localized in endoplasmic reticulum (ER), was decreased as a function of the irradiation time in cells pre-incubated with P2-LDL. This result suggests that ER alteration occurred during cell photosensitization by P2-LDL.

Inhibition of human fibroblasts sphingomyelinase by cholesterol and 7-dehydrocholesterol.

Abstract An inhibition of human fibroblast sphingomyelinase by cholesterol and 7-dehydrocholesterol is shown. This effect is obtained for cholesterol and 7-dehydrocholesterol/sphingomyelin molar ratios above 0.1. Diffusion measurements performed on mixed liposomes demonstrated for cholesterol/sphingomyelin and 7-dehydrocholesterol/sphingomyelin molar ratios above 0.1 a sharp increase in diffusion intensity. The mechanism of the inhibition of sphingomyelinase by sterols is discussed in relation to the physical state of the substrate. A possible involvement of this phenomenon in sphingomyelin accumulation observed in aging or in atheroma is discussed.

Alterations in cholesterol metabolism in cultured fibroblasts from patients with Niemann-Pick disease type C.

SummaryCholesterol synthesis, esterification and efflux were investigated in cultured fibroblasts from patients with Niemann-Pick disease type C. Sterol synthesis from sodium acetate was markedly increased in the two Niemann-Pick disease type C strains as compared to controls, either in the presence or absence of exogenous cholesterol supply by low-density lipoproteins. By contrast, cholesterol esterification was about 2–3-fold reduced when measured by oleic acid incorporation into cholesteryl esters and 10–15-fold reduced when measured with labelled free cholesterol as precursor, although acylcoenzyme-A: cholesterol acyltransferase activity was normal when studiedin vitro on cell homogenates. Chase experiments with14C-cholesterol demonstrated that the rate of cholesterol efflux was decreased by about 3–4-fold in fibroblasts from patients with Niemann-Pick disease type C. These results provide further evidence for alterations of sterol metabolism in Niemann-Pick disease type C and support the hypothesis of a trapping of exogenous cholesterol, which cannot enter the regulatory intracellular pools.

Effect of cyclic AMP on low density lipoprotein binding and internalization by cultured human fibroblasts

Pretreatment of cultured human fibroblasts by cyclic AMP resulted in a marked decrease in the binding and internalization of the low density lipoproteins (about 55% of controls for cyclic AMP 2.10(-3) M). This effect was dose dependent and increased by theophyllin. DL propranolol, an inhibitor of adenylcyclase, had an opposite effect. Isoproterenol, which stimulates adenylcyclase, reproduced the effect of cyclic AMP. The cholesterol synthesis from [2-14C] acetate was decreased by cyclic AMP, theophyllin and isoproterenol, and increased by propranolol. The incorporation of [1-14C] oleate into cholesteryl esters was reduced by cyclic AMP, theophyllin, isoproterenol and propranolol.

Epinephrine decreases low density lipoprotein processing and lipid synthesis in cultured human fibroblasts

The effect of epinephrine on 125I-low density lipoprotein (LDL) uptake and cholesterol metabolism was investigated after a 24 hours pretreatment of cultured human fibroblasts. Epinephrine decreased LDL uptake (binding + internalization) and degradation in a dose-dependent manner. Cholesterol synthesis from 14C sodium acetate and cholesterol esterification measured by 14C oleic acid incorporation into cholesteryl esters were also decreased. These results are in agreement with the general view that epinephrine increases cyclic AMP intracellular level, as it was previously demonstrated that dibutyryl cyclic AMP or isoproterenol treatment of cultured fibroblasts had similar effect on these pathways. The decrease in LDL processing induced by epinephrine could be involved in the worsening effect of epinephrine on atherosclerosis.

Cholesterol and 7 dehydrocholesterol inhibit the in situ degradation of sphingomyelin by cultured human fibroblasts

Feeding cultured human fibroblasts with cholesterol and 7-dehydrocholesterol resulted in a strong decrease of the in situ degradation of sphingomyelin (about 20 and 40 fold reduction for cholesterol and 7-dehydrocholesterol respectively, at 50 micrograms/ml of medium, and for 24 h incubation). Measurement performed on cell homogenates showed a slight decrease of the sphingomyelinase activity (about 75% of controls), whereas the activities of other lysosomal enzymes (beta glucosaminidase, beta galactosidase) were not significantly affected.