Cellular uptake and photosensitizing properties of anticancer porphyrins in cell membranes and low and high density lipoproteins.

The mechanisms of the phototoxic effect of anticancer porphyrins used in the photodynamic therapy (PDT) of tumours are not yet completely understood. Irradiation of porphyrins gives rise to singlet oxygen which reacts with key residues of proteins, polyunsaturated fatty acids and cholesterol in membranes, leading to inactivation of various enzymes and transporters. Lipoproteins, mainly low density lipoproteins (LDL), are efficient carriers of anticancer porphyrins in blood and can deliver these photosensitizers to tissues through the apolipoprotein (apo) B/E specific LDL receptor pathway. In this review, we discuss some aspects of anticancer porphyrin transport, cellular uptake and photosensitizing properties in cell membranes and lipoproteins.

Synthesis, cellular uptake of, and cell photosensitization by a porphyrin bearing a quinoline group.

A tetraphenyl porphine linked to a 7-chloroquinoline (5,10,15,20-tetraphenyl-1-3-[4-(4-aminobutyl)7-chloroquinoline] propioamidoporphine, TPPQ) was synthesized and examined as a potential photosensitizer for photodynamic therapy (PDT) of proliferative diseases. With respect to haematoporphyrin, TPPQ is a good in vitro photodynamic sensitizer producing singlet oxygen in 1% Triton X100 solutions. As with other hydrophobic porphyrins used in PDT, blood lipoproteins strongly bind TPPQ. Thus one low density lipoprotein (LDL) can incorporate 25 TPPQ molecules and 17 TPPQ molecules are taken up by one high density lipoprotein (HDL). Cell delivery of TPPQ using HDL or human serum albumin (HSA) as carrier is rather weak. However, an efficient TPPQ delivery to human skin fibroblasts is observed, partly aided by receptor-mediated endocytosis of LDL. Fluorescence spectroscopy shows that the cellular localization of TPPQ is both carrier and time dependent. During its delivery with LDL, TPPQ does not significantly impair the endocytosis of LDL-receptor complexes. After delivery with LDL, TPPQ is as efficient as other haematoporphyrin derivatives used in the PDT of cancers in photosensitizing human skin fibroblasts.

Sensitivity of tryptophan and related compounds to oxidation induced by lipid autoperoxidation. Application to human serum low- and high-density lipoproteins.

Tryptamine, serotonin and tryptophan are readily oxidized during the Cu2+-catalyzed peroxidation of arachidonic acid (AA) at neutral pH and under certain experimental conditions which determine their relative susceptibility to oxidation. Thus, in AA micelles, fluorescence spectroscopy demonstrates that positively-charged indoles interact with negatively-charged micelles while Trp remains in the aqueous phase. As a result, serotonin and tryptamine are preferentially oxidized. In egg phosphatidylcholine liposomes loaded with AA, the three substrates interact with vesicles and undergo lipid-induced oxidation. EDTA inhibits the formation of thiobarbituric-reactive substances (TBARS) and prevents the indoles from oxidation. Owing to the intricate contact between the lipidic core and the apolipoproteins, the Trp residues of human serum LDL and HDL3 are very rapidly oxidized, i.e., at least one order of magnitude faster than Tyr HDL and Lys LDL, which are believed to be involved in the binding of these lipoproteins to their cell receptors. Cupric ions are rather specific for the lipid-induced autoxidation of Trp residues of lipoproteins whereas in micelles and liposomes, Mn2+ and Fe2+ can lead to TBARS production and to oxidation of indoles. This specificity is surprising considering the known ability of Fe2+ to catalyze LDL modification (measured by TBARS production) during their incubation with various cells. Biological consequences of the easy lipid-induced oxidation of biologically important indoles 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.

[Oxidation of the indole ring induced by the auto-oxidation of unsaturated lipids. Application to tryptophan residues of high and low density lipoproteins of human serum]. / Oxydation du noyau indole induite par l'auto-oxydation des lipides insaturés. Application aux résidus tryptophane des lipoprotéines de haute et basse densité du sérum humain.

Tryptophan residues are rapidly destroyed during Cu2+-catalyzed autoperoxidation of human serum lipoproteins. The same phenomenon occurs in the peroxidation of arachidonic acid incorporated into phosphatidylcholine liposomes using tryptophan, tryptamine and serotonin as oxidizable substrates. Replacing arachidonic acid by cholesterol inhibits the indole ring degradation.

The effects of perhexiline maleate on low density lipoprotein processing and cholesterol metabolism in cultured human fibroblasts.

The effects of perhexiline maleate (PM, Pexid) on low density lipoprotein (LDL) processing and cholesterol metabolism were investigated after a 24 h pretreatment with the drug. Perhexiline maleate increased LDL uptake in the range 10(-6) to 10(-5) M (180% of control for 10(-5) M), whereas LDL binding and degradation were not affected. Sterol synthesis from sodium acetate was enhanced by perhexiline maleate (X 2.2 for 10(-5) M), while cholesterol esterification with oleic acid was decreased (40% of control for 10(-5) M). These effects of perhexiline on cholesterol metabolism are specific, since the synthesis of triacylglycerols from sodium acetate is not affected and since the incorporation of oleic acid into triacylglycerols is much less impaired. The decreased cholesterol esterification is accompanied by a reduction of acylcoenzyme A-cholesterol-O-acyltransferase (ACAT) activity measured in vitro on cell extracts.

[Phospholipid methylation in fibroblasts of patients with trisomy 21]. / Méthylation des phospholipides dans les fibroblastes de patients atteints de trisomie 21.

Comparative studies of phospholipid methylation in normal and Down syndrome (trisomy 21) fibroblasts were performed on a series of 7 pairs. Two patients showed an increased phospholipid methylation pathway as compared to the appropriate controls; one patient showed a decreased pathway; and in four patients there was no significant difference. The phospholipid methylation pathway appears to be a minor pathway, as compared to the choline pathway of phosphatidylcholine synthesis and thus does not allow the detection of an eventual disturbance of monocarbon metabolism in Down syndrome.

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 micrograms/ml) 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.