Photodynamic Efficiency of Protoporphyrin IX: Comparison of Endogenous Protoporphyrin IX Induced by 5-Aminolevulinic Acid and Exogenous Porphyrin IX

Abstract— Photodynamic efficiency of protoporphyrin IX (PP) accumulated in HeLa cells by the incubation of PP with HeLa cells was compared with that of accumulated PP formed from 5-aminolevulinic acid (ALA) as a precursor. The ALA-induced PP was photodynamically more efficient than exogenous PP. The difference is caused by monomelic PP concentration and PP localization site in HeLa cells. Exogenous PP was accumulated mainly in plasma membrane, and the membrane was strongly damaged by irradiation. The ALA-induced PP was selectively accumulated in mitochondria and inactivated the mitochondrial function by irradiation.     

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.     

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.     

New 5-aminolevulinic acid esters--efficient protoporphyrin precursors for photodetection and photodynamic therapy

Photodetection (PD) and photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) accumulation are approaches to detect and treat dysplasia and early cancer in the gastrointestinal tract and in the urinary bladder. Because ALA-induced PPIX production is limited, we synthesized ALA ester hydrochlorides 3-22 and tested them in two different in vitro models (gastrointestinal tract: HT29-CCD18; urinary bladder: J82-UROTSA). PPIX accumulation after incubation with 0.12 mmol/L for 3 h and PPIX accumulation as a function of different incubation times were measured using flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed to check cellular dark toxicity. Phototoxicity after irradiation was tested. ALA nonafluorohexylester hydrochloride 11, ALA thiohexylester hydrochloride 13 and ALA dibenzyldiester dihydrochloride 19 induced appreciably increased PPIX levels and showed improved phototoxicity compared with the references ALA hydrochloride 1, ALA hexylester hydrochloride 3 and ALA benzylester hydrochloride 4. Thus, the new compounds 11, 13 and 19 are promising compounds for PD and PDT.     

HepG2 human hepatocarcinoma cells: an experimental model for photosensitization by endogenous porphyrins

Endogenous protoporphyurin IX (PpIX) synthesis after δ-aminolaevulinic acid (ALA) administration occurs in cancer cells in vivo; PpIX, which has a short half-life, may thus constitute a good alternative to haematoporphyrin derivative (HPD) (or Photofrin). This study assesses the ability of the human hepatocarcinoma cell line HepG2 to synthesize PpIX in vitro from exogenous ALA, and compares ALA-induced toxicity and phototoxicity with the photodynamic therapy (PDT) effects of HPD on this cell line.

ALA induced a dose-dependent dark toxicity, with 79% and 66% cell survival for 50 and 100 μg ml⁻¹ ALA respectively after 3 h incubation; the same treatment, followed by laser irradiation (λ = 632 nm, 25 J cm⁻²), induced a dose-dependent phototoxicity, with 54% and 19% cell survival 24 h after PDT. Whatever the incubation time with ALA, a 3 h delay before light exposure was found to be optimal to reach a maximum phototoxicity.

HPD induced a slight dose-dependent toxicity in HepG2 cells and a dose- and time-dependent phototoxicity ten times greater than that of ALA-PpIX PDT. After 3 h incubation of 2.5 and 5 μg ml⁻¹ HPD, followed by laser irradiation (λ = 632 nm, 25 J cm⁻²), cell survival was 59% and 24% respectively at 24 h.

Photoproducts induced by light irradiation of porphyrins absorb light in the red spectral region at longer wavelengths than the original porphyrins. The possible enhancement of PDT effects after HepG2 cell incubation with ALA or HPD was investigated by irradiating cells successively with red light (λ = 632 nm) and light (λ = 650 nm). The total fluence was kept constant at 25 J cm⁻². For both HPD and ALA-PpIX PDT, phototoxicity was lower when cells were irradiated for increased periods with λ = 650 nm light than with λ = 632 nm light alone. This suggests that any photoproducts involved either have a short life or are poorly photoreactive.

Not all cell lines can synthesize PpIX after ALA incubation. HepG2 cells, which can synthesize enzymes and precursors of endogenous porphyrin synthesis, represent a good in vitro model for experiments using ALA-PpIX PDT. In addition, ALA-PpIX PDT may represent a new, specific treatment for hepatocarcinomas.     

An insight into the mechanisms of the phototoxic response induced by cyamemazine in cultured fibroblasts and keratinocytes

The phototoxicity of cyamemazine (CMZ, Tercian), a neuroleptic of the phenothiazine family, has recently been reported in humans. CMZ has an absorbance maximum at 267 nm (molar absorptivity, 25,800 M(-1) cm(-1)) but a weaker molar absorptivity in the ultraviolet A (UV-A) region. CMZ exhibits a fluorescence with maximum emission at 535 nm and a quantum yield of 0.11. CMZ is a powerful photosensitizing agent toward HS 68 human skin fibroblasts and NCTC 2544 keratinocytes. At a UV-A radiation dose of 10 J/cm2, innocuous to cells in the absence of CMZ, the LD50 (lethal dose corresponding to 50% killing) are 0.5 and 1 microM for the fibroblasts and the keratinocytes, respectively, after overnight incubation with the drug. Short incubation times do not significantly alter the LD50. The CMZ-induced phototoxicity is accompanied by lipid membrane peroxidation consistent with the amphiphilic character of this photosensitizer. Keratinocytes are an order of magnitude less sensitive to the photosensitized lipid peroxidation than fibroblasts. Microspectrofluorometry reveals that lysosomal membranes are major sites of CMZ incorporation into the two cell lines because a Forster-type resonance energy transfer process occurs from CMZ to LysoTracker Red DND99 (LTR), a specific fluorescent probe of lysosomal membranes. The CMZ-photosensitized destruction of LTR demonstrates that CMZ retains its photosensitizing capacity after its lysosomal uptake.     

DISTRIBUTION OF 5-AMINOLEVULINIC ACID-INDUCED PORPHYRINS IN NODULOULCERATIVE BASAL CELL CARCINOMA

Abstract— Microscopic fluorescence photometry incorporating a light-sensitive thermo-electrically cooled charge-coupled device (CCD) camera was employed to investigate the fluorescence distribution of 5-aminolevulinic acid (ALA)-induced porphyrins in 22 patients with a total number of 52 noduloul-cerative basal cell carcinomas (BCC) after topical ALA application with or without dimethylsulfoxide (DMSO)/ethylenediaminetetraacetic acid (EDTA) or after intravenous administration of ALA. Both localization patterns and amounts of ALA-induced porphyrins in the BCC were studied. The ALA-induced porphyrins were localized only in the superficial layers of the noduloulcerative BCC lesions after topical application of 20% ALA alone for 3 h. However, both the penetration of ALA into deep lesions and the production of the ALA-induced porphyrin fluorescence were increased after topical administration of 20% ALA and 20% DMSO/4% EDTA for 3 h. Prior treatment with 99% DMSO for 15 min further enhanced the ALA penetration into the BCC lesions after topical application of the ALA/DMSO/EDTA mixture and produced more ALA-induced porphyrins by a factor of about three compared with those treated with ALA alone. The penetration of ALA into the deep BCC lesions could also be increased by prolonging the time of topical application of 20% ALA/4% EDTA to 29–48 h (without DMSO). Intravenous injection of ALA led to a more homogeneous distribution of the ALA-derived porphyrins in the whole noduloulcerative BCC lesions.     

Photodynamic therapy with endogenous protoporphyrin IX: basic principles and present clinical experience

5-Aminolaevulinic acid (ALA) is a precursor of protoporphyrin IX (Pp IX) in the biosynthetic pathway for haem. Certain types of cells have a large capacity to synthesize Pp IX when exposed to an adequate concentration of exogenous ALA. Since the conversion of Pp IX into haem is relatively slow, such cells tend to accumulate photosensitizing concentrations of Pp IX. Pp IX photosensitization can be induced in cells of the epidermis and its appendages, but not in the dermis. Moreover, since ALA in aqueous solution passes readily through abnormal keratin, but not through normal keratin, the topical application of ALA in aqueous solution to actinic keratoses or superficial basal cell or squamous cell carcinomas induces Pp IX photosensitization that is restricted primarily to the abnormal epithelium. Subsequent exposure to photoactivating light selectively destroys such lesions. In our ongoing clinical trial of ALA-induced Pp IX photodynamic therapy, the response rate for basal cell carcinomas following a single treatment has been 90% complete response and 7.5% partial response for the first 80 lesions treated. The cosmetic results have been excellent, and patient acceptance has been very good.     

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.     

Simulations on the selectivity of 5-aminolaevulinic acid-induced fluorescence in vivo

The knowledge of the exact time course of a photosensitizer in tumour and surrounding host tissue is fundamental for effective photodynamic therapy (PDT) and fluorescence-based diagnosis. In this study the time course of porphyrin fluorescence following topical application of 5-aminolaevulinic acid (ALA) using different formulations, concentrations and incubation times has been measured in amelanotic melanomas (A-Mel-3) (n = 54) grown in transparent dorsal skinfold chambers of Syrian golden hamsters and in human basal cell carcinomas (BCCs) (n = 40) in vivo. To simulate the accumulation of ALA-induced protoporphyrin IX (Pp IX), a three-compartment model has been developed and rate constants have been determined. The kinetics of both the A-Mel-3 tumours and the BCCs show a significantly higher fluorescence intensity in tumour as compared to normal surrounding host tissue. Maximal fluorescence intensity in A-Mel-3 tumours as a percentage of the reference standard used occurs 150 min post incubation (p.i.) using a 1, 3 or 10% (vol.) ALA solution buffered to pH 7.4 and 1 h incubation time. After a 4 h incubation time maximal fluorescence intensity in tumour is measured shortly p.i. A concentration of 10% ALA does not increase the fluorescence intensity as compared to 3% ALA following 4 h incubation, but either 3 or 10% ALA yields a significantly higher fluorescence after 4 h incubation time as compared to 1 h. The fluorescence intensity following an 8 h incubation reaches its maximum directly p.i. for all concentrations and then decreases exponentially. The fluorescence intensity in the surrounding host tissue shows no statistically significant difference regarding concentration or incubation time. At least during the first hour p.i., the fluorescence intensity measured in the surrounding tissue is lower as compared to that in the tumour in all groups. 24 h after topical application hardly any fluorescence is detectable in tumour or surrounding host tissue in all experimental groups. Incubating human BCCs with a 20% ALA cream (water-in-oil emulsion) or a 20% ALA gel (containing 40% dimethyl sulfoxide) for approximately 2 h yields a similar fluorescence intensity directly after incubation for either cream or gel. However, while yielding a maximum 120 min p.i. with cream, the fluorescence intensity increases for a longer time (about 2-3 h p.i.) and up to higher values using the gel formulation. In surrounding normal skin, cream as well as gel formulation yields a similar fluorescence intensity directly after incubation. Afterwards the fluorescence intensity decreases slowly using the cream whereas a further increase of the fluorescence intensity is measured in the normal skin with a maximum 240 min p.i. using the gel formulation. The results of the proposed three-compartment model indicate that the observed selectivity of accumulated porphyrins following topical application of ALA is mainly governed by an increased ALA penetration of the stratum corneum of the skin, an accelerated ALA uptake into the cell and a higher porphyrin formation in tumour as compared to normal skin tissue, but not by a reduced ferrocheletase activity.     

Is δ‐Aminolevulinic Acid Dehydratase Rate Limiting in Heme Biosynthesis Following Exposure of Cells to δ‐Aminolevulinic Acid?¶

Understanding the regulation and control of heme/porphyrin biosynthesis is critical for the optimization of the delta-aminolevulinic-acid (ALA)-mediated photodynamic therapy of cancer, in which endogenously produced protoporphyrin IX (PPIX) is the photosensitizer. The human breast cancer cell line MCF-7, the rat mammary adenocarcinoma cell line R3230AC, the mouse mammary tumor cell line EMT-6 and the human mesothelioma cell line H-MESO-1 were used to study ALA-induced PPIX levels and their relationship to delta-aminolevulinic acid dehydratase (ALA-D) activity in vitro. Incubation of these cell lines with 0.5 mM ALA for 3 h resulted in a significant increase in PPIX accumulation, compared with control cells, but there was no significant change in ALA-D activity. Exposure of cells incubated with ALA to 30 mJ/cm2 of fluorescent light, a dose that would cause a 50% reduction in cell proliferation, did not significantly alter the activity of ALA-D. Increasing the activity of porphobilinogen deaminase (PBGD), the enzyme immediately subsequent to ALA-D, by four- to seven-fold via transfection of cells with PBGD complementary DNA did not alter the activity of ALA-D. However, incubation of cells with various concentrations of succinyl acetone, a potent inhibitor of ALA-D, caused a concomitant decline in both PPIX accumulation and ALA-D activity. These data imply that when cells are exposed to exogenous ALA, ALA-D is an important early-control step in heme/porphyrin biosynthesis and that regulation of PPIX synthesis by this dehydratase may impact the effectiveness of ALA-mediated photosensitization.     

Fluorescence spectroscopy of normal mouse skin exposed to 5-aminolaevulinic acid and red light

Photobleaching and phototransformation of protoporphyrin IX (PpIX) was investigated in normal mouse skin. The PpIX was induced by topical application of 5-aminolaevulinic acid (ALA). Exposure to laser light (635 nm) caused photobleaching of PpIX fluorescence and formation of fluorescent products. Analysis of the fluorescence spectra revealed appearance of new fluorescent photoproducts during light exposure. The main photoproduct, supposedly chlorin-type photoprotoporphyrin (PPp), exhibited fluorescence with an emission maximum at 675 nm. The other products exhibited main fluorescence peaks at around 588 and 623 nm that can presumably be attributed to an endogenous metallo-porphyrin and water-soluble porphyrin(s), respectively. Our results indicate that light exposure causes alterations in the enzymatic pathway of PpIX synthesis from ALA and leads to accumulation of intermediate water-soluble porphyrins. ALA-induced porphyrins are transported away from the treated area and partly deposited in remote skin sites.     

Topical use of liposomal copper palmitate formulation blocks porphyrin-induced photosensitivity in rats

Photodynamic therapy (PDT) is a new treatment modality that uses porphyrin derivatives and visible light, especially for the treatment of cancer. However, PDT with certain photosensitisers can cause prolonged skin photosensitization. This is particularly true for Photofrin II (Photofrin)-mediated PDT where patients are required to avoid direct exposure to sunlight for a period of 4-6 weeks. This is the only long-term adverse reaction to the drug. Recent studies have shown that topical copper treatment avoids this type of inflammatory reaction. In this study, we have tested the efficiency of the liposomal formulation of copper palmitate on porphyrin-photosensitized rats. Initially, adult male Sprague-Dawley rats were rendered photosensitive either by administration of Photofrin or aminolevulinic acid (ALA), a precursor of protoporphyrin IX (PpIX). Prior to this, their dorsal skin was shaved and treated topically with a cream consisting of either empty or copper palmitate-encapsulated liposomal formulation. After being kept in a dimmed light environment, the rats were exposed to visible light, and inflammatory responses were inspected. Histological studies revealed that no inflammatory cells were present at the skin sites treated with liposomal cream containing copper palmitate in the Photofrin-sensitized group while no reduction in the number of inflammatory cells was observed at the skin samples treated with the empty liposomes. In conclusion, the data demonstrate the significant protective effect of topically-applied liposome-encapsulated copper palmitate against both Photofrin and ALA-induced PpIX photosensitivity.     

Protein changes in HL60 leukemia cells associated with 5-aminolevulinic acid-based photodynamic therapy. Early effects on endoplasmic reticulum chaperones

Using two-dimensional electrophoresis we investigated the effect of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT; induction with 1 mM ALA for 4 h followed by blue light dose of 18 J/cm2) on the protein expression in HL60 leukemia cells. ALA-PDT resulted in extensive qualitative and quantitative changes in the protein pattern of HL60 cell lysates. Of more than 1350 protein spots recognized on the protein maps of ALA-induced cells, seven proteins were enhanced and 17 suppressed following irradiation. Three of these, calreticulin precursor, p58 microsomal protein (ERp57) and protein disulfide isomerase (p55) have been identified by matrix-assisted laser desorption and ionization-mass spectrometry and the pI/molecular weight parameters of the affected proteins were estimated by computer analysis. The findings suggest participation of endoplasmic reticulum Ca(2+)-binding chaperones and/or Ca2+ signaling in ALA-PDT mediated cytotoxicity.     

Subcellular Localization of Photofrin and Aminolevulinic Acid and Photodynamic Cross-Resistance in Vitro in Radiation-Induced Fibrosarcoma Cells Sensitive or Resistant to Photofrin-Mediated Photodynamic Therapy

Abstract— The subcellular and, specifically, mitochondrial localization of the photodynamic sensitizers Photofrin and aminolevulinic acid (ALA)-induced protoporphyrin-IX (PpIX) has been investigated in vitro in radiation-induced fibrosarcoma (RIF) tumor cells. Comparisons were made of parental RIF-1 cells and cells (RIF-8A) in which resistance to Photofrin-mediated photodynamic therapy (PDT) had been induced. The effect on the uptake kinetics of Photofrin of coincubation with one of the mitochondria-specific probes 10N-Nonyl acridine orange (NAO) or rhodamine-123 (Rh-123) and vice versa was examined. The subcellular colocalization of Photofrin and PpIX with Rh-123 was determined by double-label confocal fluorescence microscopy. Clonogenic cell survival after ALA-mediated PDT was determined in RIF-1 and RIF-8A cells to investigate cross-resistance with Photofrin-mediated PDT. At long (18 h) Photofrin incubation times, stronger colocalization of Photofrin and Rh-123 was seen in RIF-1 than in RIF-8A cells. Differences between RIF-1 and RIF-8A in the competitive mitochondrial binding of NAO or Rh-123 with Photofrin suggest that the inner mitochondrial membrane is a significant Photofrin binding site. The differences in this binding may account for the PDT resistance in RIF-8A cells. With ALA, the peak accumulations of PpIX occurred at 5 h for both cells, and followed a diffuse cytoplasmic distribution compared to mitochondrial localization at 1 h ALA incubation. There was rapid efflux of PpIX from both RIF-1 and RIF-8A. As with Photofrin, ALA-induced PpIX exhibited weaker mitochondrial localization in RIF-8A than in RIF-1 cells. Clonogenic survival demonstrated cross-resistance to incubation in PpIX but not to ALA-induced PpIX, implying differences in mitochondrial localization and/or binding, depending on the source of the PpIX within the cells.     

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.     

5-aminolevulinic acid-based photodynamic therapy in leukemia cell HL60

A study to explore the optimal experimental parameters and the photosensitization of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) in promyelocytic leukemia cell HL60 has been conducted, in which HL60 cells and their control groups, peripheral blood mononuclear cell (PBMC), first are incubated with different concentrations of ALA in dark for different periods of time and then followed by irradiating with different wavebands for different fluences. Fluorescence microscope and spectrofluorometer have been used to detect the fluorescence of protoporphyrin IX (PpIX) endogenously produced by ALA. The response of the cells to ALA-PDT was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2-5-diphenyl-2H-tetrazolium bromide (MTT) assay (interval between irradiation and the MTT assay is 24 h) and by flow cytometry (the length of time between irradiation and the flow assay is 30 min). MTT results will reflect the relative number of metabolically active mitochondria in the population. Propidium iodide uptake in flow cytometry will test for membrane damage. The results of parameter experiments were obtained: 1 x 10(5)/mL HL60 cell was first incubated with 1 mmol/L ALA in dark for 4 h and the maximum fluorescence of PpIX level appeared; then irradiated with 410 nm (4 mW/cm2) for 14.4 J/cm2 and maximum photodamage to membrane and mitochondrial function of HL60 cell resulted. With the normal granulocytes, such response was not detected. Therefore a hypothetical idea can be brought forward that ALA-based PDT can be used for inactivation of leukemia cell HL60 and these optimal parameters may be useful for clinical application.     

Topical application of 5-aminolevulinic acid hexyl ester and 5-aminolevulinic acid to normal nude mouse skin: differences in protoporphyrin IX fluorescence kinetics and the role of the stratum corneum

An important limitation of topical 5-aminolevulinic acid (ALA)-based photodetection and photodynamic therapy is that the amount of the fluorescing and photosensitizing product protoporphyrin IX (PpIX) formed is limited. The reason for this is probably the limited diffusion of ALA through the stratum corneum. A solution to this problem might be found in the use of ALA derivatives, as these compounds are more lipophilic and therefore might have better penetration properties than ALA itself. Previous studies have shown that ALA hexyl ester (ALAHE) is more successful than ALA for photodetection of early (pre)malignant lesions in the bladder. However, ALA pentyl ester slightly increased the in vivo PpIX fluorescence in early (pre)malignant lesions in hairless mouse skin compared to ALA. The increased PpIX fluorescence is located in the stratum corneum and not in the dysplastic epidermal layer. In the present study, ALA- and ALAHE-induced PpIX fluorescence kinetics are compared in the normal nude mouse skin, of which the permeability properties differ from the bladder. Application times and ALA(HE) concentrations were varied, the effect of a penetration enhancer and the effect of tape stripping the skin before or after application were investigated. Only during application for 24 h, did ALAHE induce slightly more PpIX fluorescence than ALA. After application times ranging from 1 to 60 min, ALA-induced PpIX fluorescence was higher than ALAHE-induced PpIX fluorescence. ALA also induced higher PpIX production than ALAHE after 10 min of application with concentrations ranging from 0.5 to 40%. The results of experiments with the penetration enhancer and tape stripping indicated that the stratum corneum acts a barrier against ALA and ALAHE. Use of penetration enhancer or tape stripping enhanced the PpIX production more in the case of ALAHE application than in the case of ALA application. This, together with the results from the different application times and concentrations indicates that ALAHE diffuses more slowly across the stratum corneum than ALA.     

EARLY ALTERATIONS OF ACTIN IN CULTURED HUMAN KERATINOCYTES AND FIBROBLASTS EXPOSED TO LONG-WAVELENGTH RADIATIONS. POSSIBLE INVOLVEMENT IN THE UVA-INDUCED PERTURBATIONS OF ENDOCYTOTIC PROCESSES

Abstract Exposure of cultured MRC5 human fibroblasts or NCTC 2544 human keratinocytes to mild doses of ultraviolet A (UVA: 320-400 nm) radiations markedly decreased the actin reactivity with fluorescein-labeled phalloidin. This indicates a change in the degree of poly merization of actin and thus in the organization of actin filaments. Such a phenomenon might be involved in the previously reported UVA-induced inhibition of specific and nonspecific endocytotic processes.     

Noninvasive Optical Imaging of UV‐Induced Squamous Cell Carcinoma in Murine Skin: Studies of Early Tumor Development and Vitamin D Enhancement of Protoporphyrin IX Production

Better noninvasive techniques are needed to monitor protoporphyrin IX (PpIX) levels before and during photodynamic therapy (PDT) of squamous cell carcinoma (SCC) of the skin. Our aim was to evaluate (1) multispectral fluorescent imaging of ultraviolet light (UV)‐induced cancer and precancer in a mouse model of SCC and (2) multispectral imaging and probe‐based fluorescence detection as a tool to study vitamin D (VD) effects on aminolevulinic acid (ALA)‐induced PpIX synthesis. Dorsal skin of hairless mice was imaged weekly during a 24‐week UV carcinogenesis protocol. Hot spots of PpIX fluorescence were detectable by multispectral imaging beginning at 14 weeks of UV exposure. Many hot spots disappeared after cessation of UV at week 20, but others persisted or became visible after week 20, and corresponded to tumors that eventually became visible by eye. In SCC‐bearing mice pretreated with topical VD before ALA application, our optical techniques confirmed that VD preconditioning induces a tumor‐selective increase in PpIX levels. Fluorescence‐based optical imaging of PpIX is a promising tool for detecting early SCC lesions of the skin. Pretreatment with VD can increase the ability to detect early tumors, providing a potential new way to improve efficacy of ALA‐PDT.