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.     

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.     

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.     

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.     

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.     

THE ROLE OF TRANSFERRIN RECEPTOR (CD71) IN PHOTODYNAMIC THERAPY OF ACTIVATED AND MALIGNANT LYMPHOCYTES USING THE HEME PRECURSOR δ-AMINOLEVULINIC ACID (ALA)

Endogenously generated protoporphyrin IX (PpIX) from exogenous ALA can be an effective photosensitizer. PpIX accumulation is inversely dependent on available intracellular iron, which is required for the conversion of PpIX to heme. Iron also is necessary for cell replication. Since iron can be toxic, intracellular iron levels are tightly controlled. Activated and proliferating cells respond to the demand for intracellular iron by upregulating membrane expression of the transferrin receptor (CD71) which is needed for iron uptake. We predicted that activated lymphocytes (CD71 +) would preferentially accumulate PpIX because of their lower intracellular iron levels and because of competition for iron between ALA-induced heme production and cellular growth processes. Thus, the CD71+ cells could serve as PDT targets. Stimulation of human peripheral blood lymphocytes (PBL) with the mitogens, phytohemagglutinin A, concanavalin A and pokeweed prior to incubation with ALA results in PpIX accumulation correlating with level of activation. Activated lymphocytes expressing high levels of surface CD71 transferrin receptors generated more PpIX than those with low CD71 expression. Incubating activated cells in transferrin depleted medium (thereby decreasing the iron availability) further increased PpIX levels. Malignant, CD71 + T lymphocytes from a patient with cutaneous T-cell lymphoma (CTCL)/Sezary syndrome also accumulated increased PpIX levels in comparison to norma] lymphocytes. PDT of activated lymphocytes and Sezary cells after ALA incubation demonstrated preferential killing compared to normal, unstimulated PBL. These findings suggest a possible mechanism for the selectivity of ALA PDT for activated CD71+ cells. They also indicate a clinical use for ALA-PDT in therapy directed towards the malignant lymphocytes in leukemias and lymphomas, and as animmunomodulatory agent.     

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.     

Comparison of the pharmacokinetics and phototoxicity of protoporphyrin IX metabolized from 5-aminolevulinic acid and two derivatives in human skin in vivo

Our novel approach was to compare the pharmacokinetics of 5-aminolevulinic acid (ALA), ALA-n-butyl and ALA-n-hexylester induced protoporphyrin IX (PpIX), together with the phototoxicity after photodynamic therapy (PDT) in human skin in vivo, using iontophoresis as a dose-control system. A series of four increasing doses of each compound was iontophoresed into healthy skin of 10 volunteers. The kinetics of PpIX metabolism (n = 4) and the response to PDT (n = 6) performed 5 h after iontophoresis, were assessed by surface PpIX fluorescence and post-irradiation erythema. Whilst ALA-induced PpIX peaked at 7.5 h, highest PpIX fluorescence induced by ALA-n-hexylester was observed at 3-6 h and no clear peak was seen with ALA-n-butylester. With ALA-n-hexylester, more PpIX was formed after 3 (P < 0.05) and 4.5 h, than with ALA or ALA-n-butylester. All compounds showed a linear correlation between logarithm of dose and PpIX fluorescence/phototoxicity at 5 h, with R-values ranging from 0.87 to 1. In addition, the ALA-n-hexylester showed the tendency to cause greater erythema than ALA and ALA-n-butylester. Fluorescence microscopy (n = 2) showed similar PpIX distributions and penetration depths for the three drugs, although both ALA esters led to a more homogeneous PpIX localization. Hence, ALA-n-hexylester appears to have slightly more favorable characteristics for PDT than ALA or ALA-n-butylester.     

Susceptibility to 5-aminolevulinic acid based photodynamic therapy in WHO I meningioma cells corresponds to ferrochelatase activity

5-Aminolevulinic acid (5-ALA) is a natural precursor of protoporphyrin IX (PpIX), which can be used as a photosensitizer in photodynamic therapy (PDT). Accumulation of PpIX in benign meningioma cells has been observed previously, its exploitation for PDT, however, was discouraged by inconsistent results. To evaluate PDT for benign meningiomas, we investigated PpIX synthesis in two human meningioma cell lines (HBL-52 and BEN-MEN-1), their respective extracellular loss of PpIX and corresponding ferrochelatase (FECH) activity as well as their susceptibility to PDT. We demonstrated PpIX production after 5-ALA administration and minor loss to the extracellular space in both cell lines. However, significantly more (up to five times) PpIX was accumulated in BEN-MEN-1 as compared with HBL-52 cells. FECH activity was 2.7-fold higher in HBL-52 compared with BEN-MEN-1 cells and accordingly higher FECH levels could be shown in HBL-52 cells by Western blot analysis. BEN-MEN-1 cells were much more sensitive to PDT and cells could be almost completely killed by irradiation doses of 2 J cm(-2) , whereas HBL-52 showed only an insufficient response at this irradiation dose. We conclude that differences in intracellular PpIX concentrations between HBL-52 and BEN-MEN-1 benign meningioma cells were mainly due to differences in FECH activity and that these differences correspond to their susceptibility to 5-ALA-induced PDT.     

Effects of Silencing Heme Biosynthesis Enzymes on 5‐Aminolevulinic Acid‐mediated Protoporphyrin IX Fluorescence and Photodynamic Therapy

Aminolevulinic acid (ALA)‐mediated protoporphyrin IX (PpIX) production is being explored for tumor fluorescence imaging and photodynamic therapy (PDT). As a prodrug, ALA is converted in heme biosynthesis pathway to PpIX with fluorescent and photosensitizing properties. To better understand the role of heme biosynthesis enzymes in ALA‐mediated PpIX fluorescence and PDT efficacy, we used lentiviral shRNA to silence the expression of porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD) and ferrochelatase (FECH) in SkBr3 human breast cancer cells. PBGS and PBGD are the first two cytosolic enzymes involved in PpIX biosynthesis, and FECH is the enzyme responsible for converting PpIX to heme. PpIX fluorescence was examined by flow cytometry and confocal fluorescence microscopy. Cytotoxicity was assessed after ALA‐mediated PDT. Silencing PBGS or PBGD significantly reduced ALA‐stimulated PpIX fluorescence, whereas silencing FECH elevated basal and ALA‐stimulated PpIX fluorescence. However, compared with vector control cells, the ratio of ALA‐stimulated fluorescence to basal fluorescence without ALA was significantly reduced in all knockdown cell lines. PBGS or PBGD knockdown cells exhibited significant resistance to ALA‐PDT, while increased sensitivity to ALA‐PDT was found in FECH knockdown cells. These results demonstrate the importance of PBGS, PBGD and FECH in ALA‐mediated PpIX fluorescence and PDT efficacy.     

Photodynamic Priming Overcomes Per- and Polyfluoroalkyl Substance (PFAS)-Induced Platinum Resistance in Ovarian Cancer†

Per- and polyfluoroalkyl substances (PFAS) are widespread environmental contaminants linked to adverse outcomes, including for female reproductive biology and related cancers. We recently reported, for the first time, that PFAS induce platinum resistance in ovarian cancer, potentially through altered mitochondrial function. Platinum resistance is a major barrier in the management of ovarian cancer, necessitating complementary therapeutic approaches. Photodynamic therapy (PDT) is a light-based treatment modality that reverses platinum resistance and synergizes with platinum-based chemotherapy. The present study is the first to demonstrate the ability of photodynamic priming (PDP), a low-dose, sub-cytotoxic variant of PDT, to overcome PFAS-induced platinum resistance. Comparative studies of PDP efficacy using either benzoporphyrin derivative (BPD) or 5-aminolevulinic acid-induced protoporphyrin IX (PpIX) were conducted in two human ovarian cancer cell lines (NIH:OVCAR-3 and Caov-3). BPD and PpIX are clinically approved photosensitizers that preferentially localize to, or are partly synthesized in, mitochondria. PDP overcomes carboplatin resistance in PFAS-exposed ovarian cancer cells, demonstrating the feasibility of this approach to target the deleterious effects of environmental contaminants. Decreased survival fraction in PDP + carboplatin treated cells was accompanied by decreased mitochondrial membrane potential, suggesting that PDP modulates the mitochondrial membrane, reducing membrane potential and re-sensitizing ovarian cancer cells to carboplatin.     

5-Aminolevulinic Acid Photosensitization of Dysplastic Barrett's Esophagus: A Pharmacokinetic Study

Photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) may have a role in the treatment of dysplastic Barrett's esophagus. Before ALA-induced PDT can be used clinically, optimum treatment parameters must be established. In this study of 35 patients, the issues of drug dosage, time interval between drug and light delivery and side effects of oral ALA administration are addressed. Spectrofluorometric analysis of tissue samples demonstrates that oral ALA administration induces porphyrin accumulation in esophageal tissues, with maximum levels at 4-6 h. High-performance liquid chromatography confirms the identity of this porphyrin as PpIX, and fluorescence microscopy analysis demonstrates that it preferentially accumulates in the esophageal mucosa, rather than in the underlying stroma. Side effects of ALA administration included malaise, headache, photosensitivity, alopecia, transient derangement of liver function, nausea and vomiting. Fewer side effects and less hepatic toxicity was seen with 30 mg/kg than 50 mg/kg ALA. In conclusion, oral ALA administration induces preferential PpIX accumulation in the esophageal mucosa, with peak PpIX fluorescence noted at 4 h and minimal systemic toxicity at a dose of 30 mg/kg.     

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.     

In vivo pharmacokinetics of 8-aminolevulinic acid-induced protoporphyrin IX during pre- and post-photodynamic therapy in 7,12-dimethylbenz(a)nthracene-treated skin carcinogenesis in Swiss mice: a comparison by three-compartment model

Delta-aminolevulinic acid-photodynamic therapy (ALA-PDT) has emerged as a useful technique in the treatment of superficial basal cell carcinoma, actinic keratosis, squamous cell carcinoma and tumors of other organs. Earlier reports mention that there is reappearance of protoporphyrin IX (PpIX) after photoirradiation of tumors. This property of reappearance of PpIX is being utilized to treat nodular tumors by fractionated light dose delivery. However, there is still no unanimously accepted reason for this reappearance phenomenon and the rate of resynthesis after PDT. On account of this, studies are carried out on the estimation of the pharmacokinetics of the ALA-induced PpIX in mice tumor models and the surrounding normal tissues before and after PDT. Further, a mathematical model based on a multiple compartment system is proposed to estimate the rate parameter for the diffusion of PpIX from the surrounding normal tissues into the tumor tissue (km) caused by photobleaching during PDT with irradiating fluences of 36.0 and 57.6 J/cm2. The km value at two different fluences, 36.0 and 57.6 J/cm2, are estimated as 3.0636+/-0.7083 h(-1) and 6.9231+/-2.17651 h(-1), respectively. Further, the rate parameter for the cleavage and efflux of ALA (k1) and the rate parameter for the evasion of PpIX from the tumor tissues after PDT (kt) were also estimated by fitting the experimental data to the developed mathematical model. The statistical significance of the estimated parameters was determined using Student's t-test. The experimental results and the rate parameters obtained using the proposed compartment model suggest that in addition to the earlier reported reasons, the invasion or diffusion of PpIX from the surrounding tissues to the tumor tissues after photoirradiation might also contribute to the reappearance of PpIX after PDT.     

The photosensitizing effect of the photoproduct of protoporphyrin IX

The photodynamic effect of a photoproduct of protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) was investigated in WiDr cells, a human adenocarcinoma cell line. The fluorescence excitation and emission spectra of PpIX and the photoproduct were measured. After 1, 3 or 5 min exposure of the ALA-incubated cells to 140 mW/cm² light at 635 nm, the photoproduct — the chlorin photoprotoporphyrin (Ppp), had an emission band around 670 nm. The Ppp excitation peak at 670 nm is well separated from the PpIX peak at 635 nm. The outcome of photodynamic therapy (PDT) was determined by measuring intracellular fluorescence intensity of propidium iodide (PI) 2 h following PDT and methylene blue (MB) staining 24 h following PDT. A significant increase in the fluorescence intensity of PI was noted when the ALA-loaded cells were exposed to 670 nm light after exposure to 635 nm, indicating enhanced cell membrane inactivation induced by the photodynamic action of the photoproduct. However, the fraction of the cells that survived following the same treatment as measured by MB staining was not significantly affected based on an analysis of variance. The fluorescence of PpIX decayed significantly during 635 nm light exposure. Exposure to light at 670 nm does not lead to any photodegradation of PpIX. The fluorescence of Ppp was bleached during 670 nm light exposure. Exposure of Ppp at 670 nm gives no PpIX back. Thus, the phototransformation of PpIX to Ppp is probably not a reversible process.     

The role of the p53 tumor suppressor in the response of human cells to photofrin-mediated photodynamic therapy

Although there is evidence that the p53 tumor suppressor plays a role in the response of some human cells to chemotherapy and radiation therapy, its role in the response of human cells to photodynamic therapy (PDT) is less clear. In order to examine the role of p53 in cellular sensitivity to PDT, we have examined the clonogenic survival of normal human fibroblasts that express wild-type p53 and immortalized Li-Fraumeni syndrome (LFS) cells that express only mutant p53, following Photofrin-mediated PDT. The LFS cells were found to be more resistant to PDT compared to normal human fibroblasts. The D37 (LFS cells)/D37 (normal human fibroblasts) was 2.8 +/- 0.3 for seven independent experiments. Although the uptake of Photofrin per cell was 1.6 +/- 0.1-fold greater in normal human fibroblast cells compared to that in LFS cells over the range of Photofrin concentrations employed, PDT treatment at equivalent cellular Photofrin levels also demonstrated an increased resistance for LFS cells compared to normal human fibroblasts. Furthermore, adenovirus-mediated transfer and expression of wild-type p53 in LFS cells resulted in an increased sensitivity to PDT but no change in the uptake of Photofrin per cell. These results suggest a role for p53 in the response of human cells to PDT. Although normal human fibroblasts displayed increased levels of p53 following PDT, we did not detect apoptosis or any marked alteration in the cell cycle of GM38 cells, despite a marked loss of cell viability. In contrast, LFS cells exhibited a prolonged accumulation of cells in G2 phase and underwent apoptosis following PDT at equivalent Photofrin levels. The number of apoptotic LFS cells increased with time after PDT and correlated with the loss of cell viability. A p53-independent induction of apoptosis appears to be an important mechanism contributing to loss of clonogenic survival after PDT in LFS cells, whereas the induction of apoptosis does not appear to be an important mechanism leading to loss of cell survival in the more sensitive normal human fibroblasts following PDT at equivalent cellular Photofrin levels.     

The Hydroxypyridinone Iron Chelator CP94 Can Enhance PpIX-induced PDT of Cultured Human Glioma Cells

Photodynamic therapy (PDT) with the pro-drugs 5-aminolevulinic acid (ALA) or methyl aminolevulinate (MAL) utilizes the combined interaction of a photosensitizer, light and molecular oxygen to ablate tumor tissue. To potentially increase accumulation of the photosensitizer, protoporphyrin IX (PpIX), within tumor cells an iron chelator can be employed. This study analyzed the effects of ALA/MAL-induced PDT combined with the iron chelator 1, 2-diethyl-3-hydroxypyridin-4-one hydrochloride (CP94) on the accumulation of PpIX in human glioma cells in vitro. Cells were incubated for 0, 3 and 6 h with various concentrations of ALA/MAL with or without CP94 and the resulting accumulations of PpIX, which naturally fluoresces, were quantified prior to and following light irradiation. In addition, counts of viable cells were recorded. The use of CP94 in combination with ALA/MAL produced significant enhancements of PpIX fluorescence in human glioma cells. At the highest concentrations of each prodrug, CP94 enhanced PpIX fluorescence significantly at 3 h for ALA and by more than 50% at 6 h for MAL. Cells subsequently treated with ALA/MAL-induced PDT in combination with CP94 produced the greatest cytotoxicity. It is therefore concluded that with further study CP94 may be a useful adjuvant to photodiagnosis and/or PpIX-induced PDT treatment of glioma.     

Activation of the stress-activated JNK and p38 MAP kinases in human cells by Photofrin-mediated photodynamic therapy

We have examined the possible role of the stress-activated JNK and p38 protein kinases in cellular sensitivity following Photofrin-mediated photodynamic therapy (PDT). Previously we reported that immortalized Li-Fraumeni syndrome (LFS) cells are more resistant to Photofrin-mediated PDT compared to normal human fibroblasts (NHF) at equivalent cellular Photofrin levels. In the current work we report that Photofrin-mediated PDT increased the activity of JNK1 and p38 within 30 min in both cell types. However, the increased activity of JNK1 and p38 was transient in the sensitive NHF cells and returned back to near basal levels by 3 h after PDT. In contrast, the resistant LFS cells exhibited a more prolonged activation of JNK and p38, which lasted for at least 11 h and 7 h after PDT, respectively. Blocking of the p38 pathway in LFS cells by transient infection with a recombinant adenovirus expressing a dominant negative mutant of p38 or in HeLa cells by stable transfection with a dominant negative mutant of p38 had no effect on cell survival following PDT. These data suggest that although Photofrin-mediated PDT is able to induce JNK1 and p38 in human cells, the p38 pathway alone does not play a major role in the sensitivity of LFS cells to Photofrin-mediated PDT.     

Diblock copolymer micelles deliver hydrophobic protoporphyrin IX for photodynamic therapy

Polymeric micelles are emerging as an effective drug delivery system for hydrophobic photosensitizers in photodynamic therapy (PDT). The objective of this study was to investigate the formulation of hydrophobic protoporphyrin IX (PpIX) with MePEG(5000)-b-PCL(4100) [methoxy poly (ethylene glycol)-b-poly (caprolactone)] diblock copolymers and to compare their PDT response to that of free PpIX. The photophysical and photochemical properties of the polymeric PpIX micelles were studied by measuring absorbance and fluorescence spectra, PpIX-loading efficiency and stability, the micelle particle size and morphology, as well as singlet oxygen luminescence and lifetime. The spherical micelles have a high PpIX-loading efficiency of 82.4% and a narrow size distribution with a mean diameter of 52.2 +/- 6.4 nm. The cellular uptake of PpIX in RIF-1 cells using PpIX micelles was approximately two-fold higher than that for free PpIX. Free PpIX and PpIX formulated in micelles exhibited similar subcellular localization in or around the cellular plasma membrane, as demonstrated using fluorescence microscopy. In vitro PDT results showed that the PpIX micelles have markedly increased photocytotoxicity over that with free PpIX, by nearly an order of magnitude at the highest light dose used. The micelles alone had no evident phototoxicity or dark toxicity. These findings suggest that MePEG(5000)-b-PCL(4100) diblock copolymer micelles have great potential as a drug delivery system for hydrophobic photodynamic sensitizers.     

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.