SUBCELLULAR LOCALIZATION OF AND PHOTOSENSITIZATION BY PROTOPORPHYRIN IX IN HUMAN KERATINOCYTES AND FIBROBLASTS CULTIVATED WITH 5-AMINOLEVULINIC ACID

Abstract— The subcellular localization of protoporphyrin (PP) has been studied by microspectrofluo-rometric techniques in NCTC 2544 keratinocytes incubated with 5-aminolevulinic acid (ALA) for times up to 42 h. Whereas the plasma membrane shows strong staining, fluorescent spots are observed within the cytoplasm especially in the perinuclear region. Although the topographic pattern of the PP distribution does not change much with the incubation time with ALA, the fluorescence spectra suggest that the PP microenvironments are quite different at short and long incubation times. Addition of 18 uJW desferoxamine almost doubles the ALA-induced PP concentration. Colocalization experiments with rhodamine 123, a mitochondrial probe, and lucifer yellow (LY) or neutral red (NR), two lysosome probes, demonstrate that at least some of these spots are of lysosomal origin. Study of the time evolution of the NR fluorescence under irradiation with visible light in the presence and absence of ALA demonstrates that lysosomes are damaged in cells that have synthesized PP. No PP fluorescence can be detected in mitochondria after incubation with ALA. However, photosensitization of mitochondria occurs under irradiation with visible light. Very little formation of lipofuscins by photosensitization with exogenous PP or ALA-induced PP is observed with the NCTC 2544 keratinocytes, as compared to normal human fibroblasts.     

Detection of Early Stages of Carcinogenesis in Adenomas of Murine Lung by 5-Aminolevulinic Acid-Induced Protoporphyrin IX Fluorescence

Abstract— Administration of the heme precursor 5-aminolevulinic acid (ALA) leads to the selective accumulation of the photosensitizer protoporphyrin IX (PpIX) in certain types of normal and abnormal tissues. This phenomenon has been exploited clinically for detection and treatment of a variety of malignant and nonmalignant lesions. The present preclinical study examined the specificity of ALA-induced porphyrin fluorescence in chemically induced murine lung tumors in vivo. During the early stages of tumorigenesis, ALA-induced PpIX fluorescence developed in hyperplastic tissues in the lung and later in early lung tumor foci. In early tumor foci, maximum PpIX fluorescence occurred 2 h after the administration of ALA and returned to background levels after 4 h. There was approximately a 20-fold difference in PpIX fluorescence intensity between tumor foci and the adjacent normal tissue. The specificity of ALA-induced fluorescence for hyperplastic tissues and benign tumors in lung during tumorigenesis suggests a possible use for this fluorochrome in the detection of premalignant alterations in the lung by fluorescence endoscopy. Two non-small cell lung cancer cell lines developed ALA-induced PpIX fluorescence in vitro . These lines exhibited a light-dose-dependent phototoxic response to ALA photodynamic therapy (PDT) in vitro . Because PpIX is a clinically effective photosensitizer for a wide variety of malignancies, these results support the possible use of ALA-induced PpIX PDT for lung cancer.     

Roles of porphyrin and iron metabolisms in the δ-aminolevulinic acid (ALA)-induced accumulation of protoporphyrin and photodamage of tumor cells

δ-Aminolevulinic acid (ALA)-induced porphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy. To clarify the mechanisms of the tumor-preferential accumulation of protoporphyrin, we examined the effect of the expression of heme-biosynthetic and -degradative enzymes on the ALA-induced accumulation of protoporphyrin as well as photodamage. The transient expression of heme-biosynthetic enzymes in HeLa cells caused variations of the ALA-induced accumulation of protoporphyrin. When ALA-treated cells were exposed to white light, the extent of photodamage of the cells was dependent on the accumulation of protoporphyrin. The decrease of the accumulation of protoporphyrin was observed in the cells treated with inducers of heme oxygenase (HO)-1. The ALA-dependent accumulation of protoporphyrin was decreased in HeLa cells by transfection with HO-1 and HO-2 cDNA. Conversely, knockdown of HO-1/-2 with siRNAs enhanced the ALA-induced protoporphyrin accumulation and photodamage. The ALA effect was decreased with HeLa cells expressing mitoferrin-2, a mitochondrial iron transporter, whereas it was enhanced by the mitoferrin-2 siRNA transfection. These results indicated that not only the production of porphyrin intermediates but also the reuse of iron from heme and mitochondrial iron utilization control the ALA-induced accumulation of protoporphyrin in cancerous cells.     

Characterization of Endogenous Protoporphyrin IX Induced by δ-Aminolevulinic Acid in Resting and Activated Peripheral Blood Lymphocytes by Four-Color Flow Cytometry

Lymphocytes treated with δ-aminolevulinic acid (ALA) can accumulate the photoactive, fluorescent heme precursor, protoporphyrin IX (PpIX). With visible light illumination, PpIX can be used in photodynamic therapy (ALA-PDT) to kill or functionally alter cells. The aim of this study was to characterize the effects of ALA and ALA-PDT on resting and activated human peripheral blood T lymphocytes. Accumulation of PpIX depends inversely on the rate of its iron-dependent conversion into heme. Activated, replicating lymphocytes have low intracellular iron levels, with corresponding increases in the transferrin receptor (CD71). Thus, we expected activated lymphocytes would preferentially accumulate PpIX. Using four-color flow cytometry, we examined ALA-induced PpIX levels in T-cell subsets of resting and activated human peripheral blood mononuclear cells and the relationship between CD71 and PpIX. Peripheral blood mononuclear cells stimulated by phytohemagglutinin (PHA) were simultaneously phenotyped for PpIX, CD71 and the T-cell markers CD3 and CD4 or CDS. In activated cells treated with 0-6mM ALA for 4 h, PpIX fluorescence was maximal at 1 mM ALA. On a single cell basis, there was a strong correlation between PpIX ac-cumulation and CD71 expression. The ALA-treated, PHA-stimulated, CD71⁺ lymphocytes had an eight-fold greater mean PpIX fluorescence than nonactivated, CD71- cells. Approximately 87% of the CD4* and 85% of the CD8⁺ T cells accumulated PpIX. The PpIX levels of CDS⁺ cells were about 5% greater than CD4⁺ cells. In addition, mixed lymphocyte reaction-stimulated cells treated with ALA accumulated more PpIX than controls. Thus, activated cells preferentially accumulate endogenous PpIX when exogenous ALA is administered. Cytotoxicity studies showed that the majority of the activated cells following ALA-PDT were killed but resting cells were spared. Also, in examining activation markers by flow cytometry the number of cells that were positive for activation markers CD38 or CD71 dramatically decreased after ALA and light treatment in activated populations. The data suggest a role for ALA-PDT as an immunomodulator or photocytotoxic agent targeting activated lymphocytes.     

Selective Examination of Plasma Membrane–Associated Photosensitizers Using Total Internal Reflection Fluorescence Spectroscopy: Correlation between Photobleaching and Photodynamic Efficacy of Protoporphyrin IX

Fluorescence spectra, fluorescence decay kinetics, photobleaching kinetics and photodynamic efficacy of protoporphyrin IX (PP) were investigated in endothelial cells in vitro after different incubation times. Fluorescence spectra and photobleaching kinetics were determined during total internal reflection (TIR) illumination or epiillumination. Because penetration depth of the excitation light during TIR illumination was limited to about 100 nm, plasma membrane-associated PP was almost selectively examined. Spectra obtained by TIR fluorescence spectroscopy (FS) showed a very low background, where-as spectra obtained by epi-illumination exhibited considerable background by autofluorescence and scattered light. For photobleaching kinetics during TIR illumination after 1 h or 24 h incubation, a biexponential fluorescence decrease was observed with a rapidly and a slowly bleaching portion. After 1 h incubation, the rapidly bleaching portion was the predominant fraction, whereas after 24 h incubation comparable relative amounts of the rapidly and slowly bleaching portion were determined. The rapidly and slowly bleaching portion were assigned to PP monomers and aggregated species in close vicinity to the plasma membrane. Fluorescence decay measurements after epi-illumination support the decrease of PP monomers within the whole cell with increasing incubation time. In contrast to TIR illumination, photobleaching of PP during epi-illumination was characterized by slow monoexponential fluorescence decrease after 1 h or 24 h incubation. Photodynamic efficacy of PP using epi-illumination was found to depend strongly on incubation time. Considerable cell inactivation was determined for short incubation times (1 h or 3 h), whereas photodynamic efficacy was diminished for longer incubation times. Reduced photodynamic efficacy after long incubation times was assigned to the lower amount of photodynamically active monomers determined close to the plasma membrane as well as within the whole cell. In conclusion, TIRFS measurements are suggested to be an appropriate tool for the examination of the plasma membrane-associated photosensitizer fraction in living cells.     

Protoporphyrin IX-sensitized photoinactivation of 5-aminolevulinate-treated leukemia cells: effects of exogenous iron

Photodynamic therapy with 5-aminolevulinic acid (ALA) is based on metabolism of ALA to a photosensitizing agent, protoporphyrin IX (PpIX), in tumor cells. Photosensitivity of target cells may be influenced by mitochondrial iron levels because ferrochelatase-catalyzed insertion of Fe2+ into PpIX converts it to heme, a nonsensitizer. To investigate this prospect, we exposed L1210 cells (approximately 10(6)/mL in 1% serum-containing medium) to a lipophilic iron chelate, ferric-8-hydroxyquinoline (Fe[HQ]2, 0.5 microM), prior to treating with ALA (0.2 mM, 4 h) and irradiating with broadband visible light. When Fe(HQ)2 was added to cells immediately or 1 h before ALA, the initial rate of photokilling, as measured by thiazolyl blue (mitochondrial dehydrogenase) assay, was markedly less than that of non-iron controls. The HPLC analysis of cell extracts indicated that ALA-induced PpIX was at least 50% lower after this Fe(HQ)2 treatment, presumably explaining the drop in photolethality. By contrast, cells treated with ALA and light 20 h after being exposed to Fe(HQ)2 contained the same amount of PpIX as non-iron controls and were photoinactivated at nearly the same rate. The 20 h delayed cells contained approximately 12 times more immunodetectable ferritin heavy subunit than controls or 1 h counterparts, which could account for the disappearance of iron's antisensitization effects in the former. Consistent with this idea, the short-term effects of Fe(HQ)2 on ALA-induced sensitization were found to be blunted significantly in ferritin-enriched cells. The Fe(HQ)2 produced strikingly different results when cells were sensitized with exogenous PpIX, stimulating photokilling after short-term contact but inhibiting it after long-term contact while having no significant effect on the level of cell-associated PpIX in either case. Thus, iron can have diverse effects on PpIX-mediated photokilling, depending on contact time with cells and whether the porphyrin is metabolically derived or applied as such.     

Effect of mammalian cell differentiation on response to exogenous 5-aminolevulinic acid

Many different types of mammalian cells accumulate fluorescing and photosensitizing concentrations of protoporphyrin IX (PpIX) when exposed to exogenous 5-aminolevulinic acid (ALA) in vivo or in vitro. Most types of malignant cells accumulate substantially more ALA-induced PpIX than do the normal cells from which they arose. Most types of malignant cells also are less differentiated than their normal counterparts. We therefore considered the possibility that malignant cells demonstrate a malignant ALA phenotype (accumulate abnormally large amounts of PpIX when exposed to exogenous ALA) as a direct consequence of their less differentiated state. Human promyelocyte cell line HL-60 and mouse preadipocyte cell line 3T3 L1 were induced to differentiate by exposing them to inducing agents in vitro. The HL-60 cells accumulated less ALA-induced PpIX when differentiated, but the 3T3 L1 cells accumulated more. It appears then that changes in the ALA phenotype with changes in the state of differentiation are cell-type specific. The decreased accumulation of ALA-induced PpIX that accompanied differentiation of the promyelocytic leukemia cells may have clinical application for rapid quantitation of the response of myelocytic leukemia patients to differentiation therapy.     

Preparation of a water-soluble fluorinated zinc phthalocyanine and its effect for photodynamic therapy

An amphiphilic fluorinated phthalocyanine, zinc tetracarboxyoctafluorophthalocyanine (ZnC4F8Pc) was synthesized and characterized. Its photodynamic efficiency for HeLa cells was compared with hydrophilic zinc octacarboxyphthalocyanine (ZnC8Pc) and hydrophobic zinc hexadecafluorophthalocyanine (ZnF16Pc). ZnC4F8Pc had a remarkable photodynamic effect among the phthalocyanines used. The effect is apparently caused by the fact that ZnC4F8Pc is mainly accumulated in the hydrophobic lipid membrane and is in the photoactive monomer form in HeLa cells.     

CELLULAR FLUORESCENCE OF THE ENDOGENOUS PHOTOSENSITIZER PROTOPORPHYRIN IX FOLLOWING EXPOSURE TO 5-AMINOLEVULINIC ACID

Abstract— Supplying 5-aminolevulinic acid (ALA), a precursor in the biosynthetic pathway to heme from an external source leads to an accumulation of the endogenous fluorescent photosensitizer protoporphyrin IX (PPIX). Following instillation of ALA in the urinary bladder neoplastic tissue can be discerned by fluorescence cystoscopy or treated by illumination with light of an appropriate wavelength. In order to provide a biological rationale for the clinical findings, we have analyzed the capacity of three different cell lines to accumulate PPIX by flow cytometry. Three different urothelial cell lines, normal fibroblasts and endothelial cells were exposed to ALA under varying conditions. Urothelial cell lines J82 and RT4, derived from malignancies of the bladder displayed fluorescence intensities 9- and 16-fold, respectively, above the fluorescence level of the normal urothelial cell line HCV29. Human umbilical cord endothelial cells fluoresced moderately while the fibroblast cell line Nl exhibited a fluorescence level comparable to those of the cancer cells. Fluoresence increased with increasing cell density and was also dependent on the growth of cells as monolayers or multicellular spheroids. Increasing ALA concentrations led to saturation of fluorescence after 4 h of incubation at cell type-specific fluorescence levels obtained at different ALA concentrations. Continuous incubation in medium containing serum resulted in a linear rise of fluorescence during the first 4 h, which was followed by a saturation period (8–24 h) and a renewed rise. In the case of serum depletion, fluorescence intensities were significantly higher and increased linearly during the entire 48 h incubation period. By replacing serum with albumin, it could be shown that the emission of PPIX into the medium in the presence of serum is mainly caused by this protein. The ALA-induced fluorescence was predominantly perinuclear after 4 h of incubation and relocated toward the cell membrane after prolonged incubation. This study demonstrated the complexity of factors influencing the ALA-induced fluorescence and should stimulate further research in this field.     

CELL SURFACE DAMAGE IN CULTURED MAMMALIAN CELLS WITH SYNCHROTRON RADIATION AT 160 nm

Cultured mammalian cells (HeLa) were allowed to attach onto a membrane filter and were irradiated with 160 nm synchrotron radiation. The cells then were rinsed with medium, fixed, and stained. Some of the cells became detached from the membrane filter during irradiation before post-irradiation incubation at 37°C. The cells remaining attached to the membrane filter were released by trypsinization, collected and examined for dye-exclusion ability with eosin Y. The number of stained cells was increased when the cells were irradiated at 160 nm, while no such increase was observed in cells irradiated with synchrotron radiation at 220 nm, with a low pressure Hg lamp (predominantly 254 nm), or with gamma-rays of ⁶⁰Co. These results indicated that the cell surface was injured by irradiation with synchrotron radiation at 160 nm.     

Subcellular localization pattern of protoporphyrin IX is an important determinant for its photodynamic efficiency of human carcinoma and normal cell lines

Photodynamic therapy (PDT) is a combination of light with a lesion-localizing photosensitizer or its precursor to destroy the lesion tissue. PDT has recently become an established modality for several malignant and non-malignant conditions, but it can be further improved through a better understanding of the determinants affecting its therapeutic efficiency. In the present investigation, protoporphyrin IX (PpIX), an efficient photosensitizer either endogenously induced by 5-aminolevulinic acid (ALA) or exogenously administered, was used to correlate its subcellular localization pattern with photodynamic efficiency of human oesophageal carcinoma (KYSE-450, KYSE-70) and normal (Het-1A) cell lines. By means of fluorescence microscopy ALA-induced PpIX was initially localized in the mitochondria, whereas exogenous PpIX was mainly distributed in cell membranes. At a similar amount of cellular PpIX PDT with ALA was significantly more efficient than photodynamic treatment with exogenous PpIX at killing all the 3 cell lines. Measurements of mitochondrial membrane potential and intracellular ATP content, and electron microscopy showed that the mitochondria were initially targeted by ALA-PDT, consistent with intracellular localization pattern of ALA-induced endogenous PpIX. This indicates that subcellular localization pattern of PpIX is an important determinant for its PDT efficiency in the 3 cell lines. Our finding suggests that future new photosensitizers with mitochondrially localizing properties may be designed for effective PDT.     

Neurotransmitter Transporter Family Including SLC6A6 and SLC6A13 Contributes to the 5‐Aminolevulinic Acid (ALA)‐Induced Accumulation of Protoporphyrin IX and Photodamage,through Uptake of ALA by Cancerous Cells

δ‐Aminolevulinic acid (ALA)‐induced protoporphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy (PDT). To clarify the mechanisms of ALA uptake by tumor cells, we have examined the ALA‐induced accumulation of protoporphyrin by the treatment of colon cancer DLD‐1 and epithelial cancer HeLa cells with γ‐aminobutyric acid (GABA)‐related compounds. When the cells were treated with GABA, taurine and β‐alanine, the level of protoporphyrin was decreased, suggesting that plasma membrane transporters involved in the transport of neurotransmitters contribute to the uptake of ALA. By transfection with neurotransmitter transporters SLC6A6, SLC6A8 and SLC6A13 cDNA, the ALA‐ and ALA methylester‐dependent accumulation of protoporphyrin markedly increased in HEK293T cells, dependent on an increase in the uptake of ALA. When ALA‐treated cells were exposed to white light, the extent of photodamage increased in SLC6A6‐ and SLC6A13‐expressing cells. Conversely, knockdown of SLC6A6 or SLC6A13 with siRNAs in DLD‐1 and HeLa cells decreased the ALA‐induced accumulation. The expression of SLC6A6 and SLC6A13 was found in some cancer cell lines. Immunohistochemical studies revealed that the presence of these transporters was elevated in colon cancerous cells. These results indicated that neurotransmitter transporters including SLC6A6 and SLC6A13 mediate the uptake of ALA and can play roles in the enhancement of ALA‐induced accumulation of protoporphyrin in cancerous cells.     

Effects of 5-aminolaevulinic acid on human ovarian cancer cells and human vascular endothelial cells in vitro.

Results are reported on the cellular effects and the sensitivity of cultured tumor epithelial cells (TEC) derived from human ovarian cystadenocarcinoma and human umbilical vein-derived endothelial cells (HUVEC) to exogenous 5-aminolaevulinic acid (ALA) and ALA-induced photodynamic therapy (PDT). Cellular alterations and PDT efficiency were evaluated using colorimetric thiazolyl blue (MTT) assay, trypan blue exclusion assay, electron microscopy, and gel electrophoresis. ALA-induced protoporphyrin IX (PpIX) accumulation in TEC was associated with a concentration and time-dependent significant decrease in mitochondrial activity, increase in cell membrane permeability, and dark toxicity. Maximum PpIX loaded TEC demonstrated a high sensitivity to PDT. Neither cellular alterations nor PDT effects were observed in HUVEC under identical experimental conditions. These results indicate a potential clinical value for the use of ALA-mediated PDT to treat minimal residual disease in mucinous ovarian carcinoma. In addition, the ALA-induced PpIX cytotoxicity may be exported to a new chemotherapeutic regimen via a conventionally viewed photochemotherapeutic agent.     

Influence of diamino acid derivatives of protoporphyrin IX on mouse immunological system: preliminary results

Immunological response related to photodynamic therapy (PDT) is one of the basic elements that influence on the efficiency of this cancer treatment method. Diamino acid derivatives of protoporphyrin IX are promising photosensitizing agents that are intended to be the components of new anti-tumor drug. The influence of three derivatives - PP(Ser)(2)Arg(2), PP(Ala)(2)Arg(2), PP(Phe)(2)Arg(2) and a mixture of these compounds called Sensyphyrine on mouse immunological system was investigated where animals were exposed and unexposed to the laser irradiation. Porphyrins solutions were injected intravenously, mice were irradiated with the red diode laser at lambda=632 nm. Cells and blood samples were taken at time intervals after irradiation. The levels of interleukin-6, interleukin-1beta and the production of reactive forms of nitrogen by macrophages were determined. The results show that all of the diamino acid derivatives of protoporphyrin IX indicate an immunological response when the light is applied. Each of the porphyrin revealed different impact on mice immunological system. The most potent stimulant properties disclosed PP(Phe)(2)Arg(2) derivative for which the highest values of IL-1beta, IL-6 and NO(2)(-) were noticed. The weakest immunological activation revealed PP(Ser)(2)Arg(2) derivative.     

Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy.

The tissue photosensitizer protoporphyrin IX (PpIX) is an immediate precursor of heme in the biosynthetic pathway for heme. In certain types of cells and tissues, the rate of synthesis of PpIX is determined by the rate of synthesis of 5-aminolevulinic acid (ALA), which in turn is regulated via a feedback control mechanism governed by the concentration of free heme. The presence of exogenous ALA bypasses the feedback control, and thus may induce the intracellular accumulation of photosensitizing concentrations of PpIX. However, this occurs only in certain types of cells and tissues. The resulting tissue-specific photosensitization provides a basis for using ALA-induced PpIX for photodynamic therapy. The topical application of ALA to certain malignant and non-malignant lesions of the skin can induce a clinically useful degree of lesion-specific photosensitization. Superficial basal cell carcinomas showed a complete response rate of approximately 79% following a single exposure to light. Recent preclinical studies in experimental animals and human volunteers indicate that ALA can induce a localized tissue-specific photosensitization if administered by intradermal injection. A generalized but still quite tissue-specific photosensitization may be induced if ALA is administered by either subcutaneous or intraperitoneal injection or by mouth. This opens the possibility of using ALA-induced PpIX to treat tumors that are too thick or that lie too deep to be accessible to either topical or locally injected ALA.     

The Temperature Dependence of Protoporphyrin IX Production in Cells and Tissues

The formation of protoporphyrin IX (PpIX) in human skin during topical application of 5-aminolevulinic acid (ALA) was found to be strongly temperature dependent, with an activation energy of about 17 kcal/mol. This temperature dependence is mainly related to porphyrin production and not to ALA penetration into the skin. The penetration of ALA into mouse and human skin was almost temperature independent. The activation energy of PpIX production in mouse skin was practically identical with that in human skin. The activation energy of ALA uptake by cells in vitro was about 10 kcal/mol and that for PpIX production was about 13 kcal/mol. The latter activation energy was within the error limits similar to that for the activity of the enzyme porphobilinogen deaminase, suggesting that this enzyme might represent a rate-limiting step for PpIX production in living tissue.     

5-Aminolevulinic Acid-induced Protoporphyrin IX Levels in Tissue of Human Malignant Brain Tumors

Protoporphyrin IX (PpIX) produced from exogenous, orally administered 5-aminolevulinic acid (ALA) displays high tumor-selective uptake and is being successfully employed for fluorescence-guided resection (FGR) of human malignant gliomas. Furthermore, the phototoxicity of PpIX can be utilized for photodynamic therapy (PDT) of brain tumors, which has been shown previously. Here, the absolute PpIX concentration in human brain tissue was investigated following oral ALA administration (20 mg kg⁻¹ b.w.). An extraction procedure was used to quantify PpIX in macroscopic tissue samples, weighing 0.013–0.214 g, obtained during FGR. The PpIX concentration was significantly higher in vital grade IV tumors (5.8 ± 4.8 μm , mean ± SD, range 0–28.2 μm , n = 8) as compared with grade III tumors (0.2 ± 0.4 μm , mean ± SD, range 0–0.9 μm , n = 4). There was also a large heterogeneity within grade IV tumors with PpIX displaying significantly lower levels in infiltration zones and necrotic regions as compared with vital tumor parts. The average PpIX concentration in vital grade IV tumor parts was in the range previously shown sufficient for PDT-induced tissue damage following irradiation. However, the feasibility of PDT for grade III brain tumors and for grade IV brain tumors displaying mainly necrotic tissue areas without solid tumor parts needs to be further investigated.     

Photochemical properties of a potential photosensitiser indocyanine green in vitro

The present study investigates the photochemical properties of a potential photosensitiser, indocyanine green (ICG), in an in vitro HeLa cell system. Cell proliferation was studied after a combined effect of ICG, at a concentration range of 24-94 microM, and therapeutic laser irradiation at several different energy densities. In addition, ICG cytotoxicity was evaluated in HeLa cells and V79 Chinese hamster by the MTT assay. Phototoxicity was evaluated at 1, 24, and 48 h after irradiation. No phototoxic effect was detected 1h after irradiation. The maximum phototoxic effect of ICG on HeLa cells was detected for an ICG concentration of 94 microM, a laser output of 360 mW, and an energy density of 99 J/cm(2) at 24h after irradiation. Potentiation of the ICG phototoxic effect was achieved by adding 20 microM H(2)O(2), which was a non-toxic concentration for HeLa cells in this experimental design. At 48 h after laser irradiation a statistically significant difference was found between the toxicity of ICG plus peroxide, as compared to ICG alone. The addition of H(2)O(2) at a concentration of 20 microM caused a significant increase in phototoxicity of ICG for HeLa cells. Our results confirm that ICG could be a perspective agent for use in photodynamic therapy and that its phototoxic effect can be potentiated by addition of an oxidative agent.     

Phototoxicity of protoporphyrin IX, diarginine diprotoporphyrinate and N,N-diphenylalanyl protoporphyrin toward human fibroblasts and keratinocytes in vitro: effect of 5-methoxypsoralen

The phototoxicity of two new porphyrin photosensitizers, diarginine diprotoporphyrinate (PP(Arg)2) and N,N-diphenylalanyl protoporphyrin (PP(Phe)2), and the synergistic effect of 5-methoxyposralen (5-MOP) have been studied in comparison with that of protoporphyrin IX (PPIX). Under ultraviolet-A (UV-A) irradiation (lambda=365 nm), the phototoxicity of the porphyrins toward cultured human fibroblasts and keratinocytes decreases in the order: PPIX > PP(Arg)2 > PP(Phe)2. A synergistic effect of 5-MOP on the phototoxicity of PPIX, PP(Arg)2 and PP(Phe)2 has been observed. The combination of PPIX, PP(Arg)2 and PP(Phe)2 with 0.1-0.5 microM 5-MOP significantly potentiates the phototoxicity of the three porphyrins. The most effective potentiation was observed with the water-soluble PP(Arg)2 and 5-MOP concentrations lower than 0.75 microM. Above this 5-MOP concentration this potentiation is abolished. The intracellular concentration of PPIX and PP(Phe)2 is independent of the presence of 5-MOP. On the other hand, the intracellular content of PP(Arg)2 is decreased in a concentration-dependent manner by the psoralen. Illumination with red light, not absorbed by 5-MOP, leads to a weak potentiation of the PP(Arg)2 phototoxic effect in the presence of 5-MOP, suggesting that dark interaction of 5-MOP with cell membranes aggravated by porphyrin photosensitization is involved in the observed phenomena. The results are tentatively explained by differences in hydrophobicity and molecular structures of the examined photosensitizers. PPIX, which is barely soluble in water, has a significantly higher affinity for cell membranes and simultaneously exerts a stronger phototoxic effect than PP(Arg)2 whose solubility in water is high. On the other hand, the weak phototoxicity of PP(Phe)2 could be explained by the steric hindrance brought by the phenylalanyl substituents on the pyrrole ring. The loss in the PP(Arg)2 cell content probably explains the inhibition of the synergistic effect of 5-MOP on the PP(Arg)2 phototoxicity at high 5-MOP concentration. This study suggests that PP(Arg)2 in combination with 5-MOP might reveal a strong phototoxic effect when applied to skin cancer treatment.     

5-Aminolaevulinic acid-mediated photodynamic therapy in multidrug resistant leukemia cells

To verify if photodynamic therapy (PDT) could overcome multidrug resistance (MDR) when it it applied to eradicate minimal residual disease in patients with leukemia, we investigated the fluorescence kinetics of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) and the effect of subsequent photodynamic therapy on MDR leukemia cells, which express P-glycoprotein (P-gp), as well as on their parent cells. Evaluation of PpIX accumulation by flow cytometry showed that PpIX accumulated at higher levels in mdr-1 gene-transduced MDR cells (NB4/MDR) and at lower levels in doxorubicin-induced MDR cells (NOMO-1/ADR) than in their parent cells. A P-gp inhibitor could not increase PpIX accumulation. Measurement of extracellular PpIX concentration by fluorescence spectrometry showed that P-gp did not mediate the fluorescence kinetics of ALA-induced PpIX production. Assessment of ferrochelatase activity using high-performance liquid chromatography indicated that PpIX accumulation in drug-induced MDR cells was probably regulated by this enzyme. Assessment of phototoxicity of PDT using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that PDT was effective in NB4, NB4/MDR, NOMO-1 and NOMO-1/ADR cells, which accumulated high levels of PpIX, but not effective in K562 and K562/ADR cell lines, which accumulated relatively low levels of PpIX. These findings demonstrate that P-gp does not mediate the ALA-fluorescence kinetics, and multidrug resistant leukemia cells do not have cross-resistance to ALA-PDT.