5-氨基γ-酮戊酸及其己酯衍生物对几种细胞株的光动力作用

5 氨基γ 酮戊 (ALA)及其己酯 (He ALA)具有内源生成光敏剂的特点 ,在肿瘤光动力探测及治疗中显示出了优势。ALA及He ALA对神经母细胞瘤、肝癌细胞及成纤维细胞的光动力作用被研究比较。由特征荧光光谱证实 ,经ALA或He ALA培养后 ,三种细胞内均可生成原卟啉 (PpIX)产物。激光扫描荧光显微镜显示 ,在经ALA或He ALA培养后的神经母细胞瘤中 ,PpIX均以弥散方式分布在细胞质中。PpIX在三种细胞中的积聚动力学过程不同 ,随着ALA或He ALA培育时间的增长 ,PpIX在肝癌细胞及成纤维原细胞中的积累增加 ,而在神经母细胞瘤中PpIX在 8h后已达到饱和。此外 ,在同样的培育条件下 ,神经母细胞瘤中PpIX的生成浓度明显高于肝癌细胞及成纤维细胞。经ALA培养及光照射后 ,可使近 90 %的神经母细胞瘤失活 ;而在同样条件下却只能杀伤 5 0 %左右的肝癌细胞及成纤维细胞。揭示了神经母细胞瘤对ALA光动力作用有极高的敏感性 ,并适于光动力治疗。与ALA相比 ,He ALA可在三种细胞内造成与ALA相近的杀伤率 ,但所用的药物浓度却比ALA低 10倍 ,显示He ALA具有极高的光动力灭活效率。因此在内源光动力治疗中 ,He ALA是一种极具开发前景的新药物。     

5—氨乙酰丙酸与氨乙酰丙酸己酯对肝癌细胞的光动力作用比较

5 氨乙酰丙酸 (ALA)可在肿瘤内诱导原卟啉区 (PpIX)光敏剂形成 ,但其亲脂性极差 ,进入细胞的能力有限。脂化的ALA衍生物能增强其进入细胞的能力 ,增进细胞中PpIX的合成。比较了氨乙酰丙酸己酯 (He ALA)与ALA对肝癌细胞中PpIX的生成及光动力损伤作用。细胞的荧光显微图象显示 ,经He ALA培育后 ,细胞中生成了PpIX。PpIX分布在细胞质中 ;细胞的荧光光谱显示出PpIX的特征荧光峰 ,证实细胞中PpIX的生成。实验发现 ,0 .2mmol/L的He ALA药物浓度与 2mmol/LALA的药物浓度在细胞中生成的PpIX含量相当 ;予以相同剂量的光照射后 ,两者对细胞的光敏损伤程度相近 ,反映He ALA对癌细胞有更高的光动力损伤功效。因此在光动力治疗的应用中 ,He ALA是一种极有开发前景的新药物     

Production of protoporphyrin IX from 5-aminolevulinic acid and two of its esters in cells in vitro and tissues in vivo.

5-Aminolevulinic acid (ALA) and two of its esters were studied in cells in vitro and in vivo on skin of healthy hairless mice. In vitro, both esters, which are more lipophilic than ALA, induced higher PpIX fluorescence at lower concentrations compared with ALA. In vivo, ALA induced PpIX fluorescence more efficiently than the esters. The difference between ALA and the esters may be related to structures in the stratum corneum or to rate of penetration through this skin layer. The stratum corneum may bind the esters temporarily, and slow down their penetration into the living cells where PpIX is formed.     

Regulation of 5-aminolevulinic acid-dependent protoporphyrin IX accumulations in human histiocytic lymphoma U937 cells

The aim of the present work is to clarify the mechanism(s) that regulates the accumulation of protoporphyrin IX (PpIX) in human histiocytic lymphoma cell line U937 incubated with 5-aminolevulinic acid (ALA). Biosynthesis and accumulation of PpIX in the cells was determined after incubation with 0.1-5 mM ALA using a flow cytometric technique. The synthesized endogenous PpIX was found to localize predominantly in the mitochondrial region of the cells. The ALA-enhanced PpIX synthesis was suppressed by the presence of either beta-alanine, a competitive inhibitor of beta-transporters on cell membranes, or carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, an uncoupler of mitochondrial oxidative phosphorylation. In contrast, cellular accumulation of PpIX was enhanced by the presence of either deferoxamine (an iron chelater), MnCl2 (a ferrochelatase inhibitor), or Sn-mesoporphyrin (heme oxygenase inhibitor). These results suggest that ALA-enhanced accumulation of PpIX in U937 cells was regulated by cellular uptake and conversion of ALA to PpIX and by degradation of Heme.     

Photobleaching kinetics, photoproduct formation, and dose estimation during ALA induced PpIX PDT of MLL cells under well oxygenated and hypoxic conditions.

Fluorescence photobleaching and photoproduct formation were investigated during delta-aminolevulinic acid (ALA) induced protoporphyrin IX (PpIX) PDT of MLL cells in vitro. Cells were incubated in either 0.1 or 1.0 mM ALA for 4 h and were treated with 532 nm or 635 nm light under well oxygenated or hypoxic conditions. Fluorescence spectra were acquired during treatment. Photobleaching and photoproduct formation were quantified using singular value decomposition fitting of fluorescence spectra to experimentally determined basis spectra for PpIX, photoprotoporphyrin (Ppp), product II (peak at 655 nm), and product III (peak at 618 nm). PpIX photobleaching occurred under both normal and hypoxic conditions. The photobleaching kinetics could not be explained by purely first- or second-order photobleaching kinetics, and were attributed to differences in PpIX binding at the two ALA incubation concentrations. Ppp was the main photoproduct and accumulated in higher levels in the absence of oxygen, likely a result of reduced Ppp photobleaching under hypoxia. Increases in product II fluorescence occurred mainly in the presence of oxygen. To assess potential fluorescence based PDT dose metrics, cell viability was measured at select times during treatment using a colony formation assay. Cell survival correlated well to changes in product II fluorescence, independent of oxygenation, sensitizer concentration, and treatment wavelength, suggesting that this product is primarily a result of singlet oxygen mediated reactions and may potentially be useful to quantify singlet oxygen dose during PDT.     

The Effect of Iron Ion on the Specificity of Photodynamic Therapy with 5-Aminolevulinic Acid

Recently, photodynamic therapy using 5-aminolevulinic acid (ALA-PDT) has been widely used in cancer therapy. ALA administration results in tumor-selective accumulation of the photosensitizer protoporphyrin IX (PpIX) via the heme biosynthetic pathway. Although ALA-PDT has selectivity for tumor cells, PpIX is accumulated into cultured normal cells to a small extent, causing side effects. The mechanism of tumor-selective PpIX accumulation is not well understood. The purpose of the present study was to identify the mechanism of tumor-selective PpIX accumulation after ALA administration. We focused on mitochondrial labile iron ion, which is the substrate for metabolism of PpIX to heme. We investigated differences in iron metabolism between tumor cells and normal cells and found that the amount of mitochondrial labile iron ion in cancer was lower than that in normal cells. This finding could be because of the lower expression of mitoferrins, which are the mitochondrial iron transporters. Accordingly, we added sodium ferrous citrate (SFC) with ALA as a source of iron. As a result, we observed the accumulation of PpIX only in tumor cells, and only these cells showed sensitivity to ALA-PDT. Taken together, these results suggest that the uptake abilities of iron ion into mitochondria play a key role in tumor-selective PpIX accumulation. Using SFC as a source of iron might thus increase the specificity of ALA-PDT effects.     

Protoporphyrin IX fluorescence kinetics in C6 glioblastoma cells after delta-aminolevulinic acid incubation: effect of a protoporphyrinogen oxidase inhibitor.

PpIX synthesis after incubation with delta-aminolevulinic acid (ALA) is highly variable from one cell to another within a single cell population and in human glioblastomas in vivo. To improve PpIX synthesis, we attempted to modify the PpIX synthesis pathway in a C6 glioma cell model. To perform this experiment we used confocal microspectrofluorometry to analyse the effects of a highly purified form of sulfentrazone (FP846) on the kinetics of PpIX synthesis after ALA administration to living C6 cells. Our results show that PpIX fluorescence was maximal (seven-fold higher than basal values) 3 to 4 hrs. after the beginning of incubation with ALA. FP846 depressed this increase in fluorescence nearly to basal levels not only in C6 cells but also in HT29 and HepG2 cells. Fluorescence spectra shape were not affected by FP846, except for intensity. ALA/PpIX-induced photocytoxicity was perfectly correlated with fluorescence intensity recorded in cell cytoplasm. ALA alone (100 microg/ml) did not induce a significant decrease in cell survival, but irradiation of 25 J/cm2 leading to an overall cell death of 60%. FP846 added together with ALA suppressed ALA/PpIX-induced phototoxicity. The fact that the FP846-induced decrease in PpIX synthesis was not the same in animal and plant cells suggests that the porphyrin metabolic pathway differs due to the relative amounts of substrate or the effect of inhibitor and that another chemical would be needed alone or in combination with FP846 to improve PpIX synthesis.     

Glycoside esters of 5-aminolevulinic acid for photodynamic therapy of cancer

Aliphatic and ethylene glycol esters of 5-aminolevulinic acid (ALA) are very efficient precursors of the photosensitizer protoporphyrin IX (PpIX) for photodynamic therapy; however, they diffuse passively across the cell membrane and thus lack cell selectivity. We evaluated whether alpha-glucose, alpha-mannose, or beta-galactose esters of ALA would present improved properties as precursors of PpIX. Esterification was performed either at the position O-1 or O-6 of the sugars with or without an ethylene glycol linker, and these glycoside esters of ALA were evaluated in human cells. The results demonstrated that glycoside esters of ALA are efficient precursors of PpIX in human cancer and angiogenic endothelial cells, comparable to free ALA, but not in normal human fibroblasts. PpIX production was confirmed by fluorescence microscopy and photodynamic treatment of cells. The O-1 or O-6 positions of functionalization and the nature of the sugar moiety did not influence PpIX production. The presence of the ethylene glycol linker generally resulted in decreased PpIX production. The uptake of these glycoside esters of ALA by cells was not decreased in the presence of high concentrations of the related sugars. Inhibitors of alpha-glucosidases or alpha-mannosidases did not decrease PpIX production. These results suggest the involvement of active non-glycoside-specific membrane transporter(s) for uptake and of esterases rather than glycosidases in the release of ALA from the glycoside esters of ALA.     

Effect of 5-aminolevulinic acid on kinetics of protoporphyrin IX production in CHO cells

5-aminolevulinic acid (ALA) is utilized in a photodynamic therapy as a compound capable of augmenting intracellular pool of protoporphyrin IX (PpIX), which exhibits properties of a photosensitizer. The studies were aimed at monitoring accumulation of endogenous protoporphyrin IX in CHO cells under effect of various concentrations of ALA in culture medium and following removal of the compound from the culture medium. Cell content of PpIX was determined following incubation of the cells for 72 h in a culture medium containing different concentration of ALA. Moreover, the cells were preincubated for 2 h in ALA at various concentrations and separated from the compound by medium change and their PpIX content was monitored following incubation. PpIX content was defined by a fluorescent technique under the confocal microscope. In the course of continuous incubation of cells with ALA, biphasic alterations were noted in cellular PpIX concentration. Removal of ALA from the incubation medium resulted at first in a decrease in PpIX content in cells, which was followed by an evidently augmented accumulation of the compound in the cells. The results suggested that in the case of CHO cells, exogenous ALA was not an exclusive source of PpIX synthesis and that alterations in enzyme activities were responsible for production of PpIX.     

Oxygen‐dependent delayed fluorescence of protoporphyrin IX measured in the stomach and duodenum during upper gastrointestinal endoscopy

Protoporphyrin IX‐triplet state lifetime technique (PpIX‐TSLT) is a method used to measure oxygen (PO₂) in human cells. The aim of this study was to assess the technical feasibility and safety of measuring oxygen‐dependent delayed fluorescence of 5‐aminolevulinic acid (ALA)‐induced PpIX during upper gastrointestinal (GI) endoscopy. Endoscopic delayed fluorescence measurements were performed 4 hours after oral administration of ALA in healthy volunteers. The ALA dose administered was 0, 1, 5 or 20 mg/kg. Measurements were performed at three mucosal spots in the gastric antrum, duodenal bulb and descending duodenum with the catheter above the mucosa and while applying pressure to induce local ischemia and monitor mitochondrial respiration. During two endoscopies, measurements were performed both before and after intravenous administration of butylscopolamine. Delayed fluorescence measurements were successfully performed during all 10 upper GI endoscopies. ALA dose of 5 mg/kg showed adequate signal‐to‐noise ratio (SNR) values >20 without side effects. All pressure measurements showed significant prolongation of delayed fluorescence lifetime compared to measurements performed without pressure (P < .001). Measurements before and after administration of butylscopolamine did not differ significantly in the duodenal bulb and descending duodenum. Measurements of oxygen‐dependent delayed fluorescence of ALA‐induced PpIX in the GI tract during upper GI endoscopy are technically feasible and safe.     

Methadone enhances the effectiveness of 5‐aminolevulinic acid‐based photodynamic therapy for squamous cell carcinoma and glioblastoma in vitro

Although having shown promising clinical outcomes, the effectiveness of 5‐aminolevulinic acid‐based photodynamic therapy (ALA‐PDT) for squamous cell carcinoma (SCC) and glioblastoma remains to be improved. The analgesic drug methadone is able to sensitize various tumors to chemotherapy. In this in vitro study, the influence of methadone to the effectiveness of ALA‐PDT for SCC (FADU) and glioblastoma (A172) was investigated on the protoporphyrin IX (PpIX) fluorescence, survival rates, apoptosis, and cell cycle phase, each with or without the presence of methadone. The production of PpIX was increased by methadone in FADU cells while it was decreased in A172 cells. The survival rates of both cell lines treated by ALA‐PDT were significantly reduced by the combination with methadone (P < .05). Methadone also significantly increased the percentage of apoptotic cells and improved the effect of ALA‐PDT on the cell cycle phase arrest in the G0/G1 phase (P < .05). This study demonstrates the potential of methadone to influence the cytotoxic effect of ALA‐PDT for both SCC and glioblastoma cell lines.      

Accumulation of protoporphyrin-IX (PpIX) in leukemic cell lines following induction by 5-aminolevulinic acid (ALA)

We investigated the amounts of protoporphyrin IX (PpIX) accumulated in noninduced cells and following 5-aminolevulinic acid (ALA)-induction. Following ALA administration PpIX increased in all leukemic cell lines under investigation (HEL 26-fold, HL60 6-fold, Jurkat 3-fold, ML2 2-fold) but not in lymphocytes. Compared to other cell lines studied, HEL cells showed the lowest basal level of PpIX and the largest relative increase in PpIX. Despite a high increase following ALA treatment, the PpIX level in HEL cells is almost as low as in lymphocytes. It is in agreement with their relatively low sensitivities of ALA-induced photodynamic therapy (ALA-PDT) shown previously [(Grebenová, D., Cajthamlová, H., Bartosová, J., Marinov, J., Klamová, H., Fuchs, O., Hrkal, Z., 1998. Selective destruction of leukemic cells by photo-activation of 5-aminolevulinic acid induced protoporphyrin IX. J. Photochem. Photobiol. B: Biol. 47, 74-81)]. The ferrochelatase activities in the individual cell lines are in good inverse correlation with PpIX amounts accumulated in the ALA-induced cells, but not with the relative increase (ratio) of PpIX levels from basal to ALA-induced ones. This is most apparent in HEL cells and lymphocytes. There is probably different regulation of heme biosynthesis in erythroid cells, which are therefore not suitable for the studies of ALA-PDT mechanism. PpIX was accumulated more extensively in absence of fetal calf serum than in its presence. The amounts of PpIX accumulated in cells decreased exponentially with increasing fetal calf serum concentration.     

ALA and its clinical impact, from bench to bedside.

ALA-induced protoporphyrin IX (PpIX) is used for fluorescence diagnosis (ALA-FD) and for fluorescence-guided resection of both (pre)malignant and non-malignant diseases. ALA is also applied in photodynamic therapy (ALA-PDT) of superficial (pre)malignant lesions in dermatology, urology, neurosurgery, otorhinolaryngology, gynecology and gastroenterology. Today, ALA is approved as Levulan for actinic keratoses, the ALA-methyl ester Metvix for actinic keratoses and basal cell carcinoma, the ALA-hexyl ester Hexvix for the diagnosis of bladder cancer and Gliolan for malignant glioma. The use of ALA for PDT and FD was established around 25 years ago, with most of the fundamental knowledge gained at the "bench" and implemented at the "bedside" due to the diligence of a few researchers within the first 10 years of research. After 1993 ALA research was taken up by many groups. For patient treatment, several factors are relevant. Administered mainly in a topical or oral form, ALA penetrates tissue in a sub-optimal way, which is currently improved by special techniques and the use of ALA-esters. PpIX accumulation is elevated in many malignant tissues, several tissue abnormalities, and in mucosa. It is also found at elevated levels in macrophages, dendritic cells and activated lymphocytes. Following sufficient PpIX accumulation in the target cells, irradiation is carried out which may be accompanied by a burning sensation at the treatment site. Due to a saturation process of PpIX formation and rapid photobleaching during irradiation the risk of overtreatment is relatively low. Pharmacokinetical studies have demonstrated a low systemic photosensitivity and excretion of PpIX via natural routes.     

Serum-dependent export of protoporphyrin IX by ATP-binding cassette transporter G2 in T24 cells

Accumulation of protoporphyrin IX (PpIX) in cancer cells is a basis of 5-aminolevulinic acid (ALA)-induced photodymanic therapy. We studied factors that affect PpIX accumulation in human urothelial carcinoma cell line T24, with particular emphasis on ATP-binding cassette transporter G2 (ABCG2) and serum in the medium. When the medium had no fetal bovine serum (FBS), ALA induced PpIX accumulation in a time- and ALA concentration-dependent manner. Inhibition of heme-synthesizing enzyme, ferrochelatase, by nitric oxide donor (Noc18) or deferoxamine resulted in a substantial increase in the cellular PpIX accumulation, whereas ABCG2 inhibition by fumitremorgin C or verapamil induced a slight PpIX increase. When the medium was added with FBS, cellular accumulation of PpIX stopped at a lower level with an increase of PpIX in the medium, which suggested PpIX efflux. ABCG2 inhibitors restored the cellular PpIX level to that of FBS(-) samples, whereas ferrochelatase inhibitors had little effects. Bovine serum albumin showed similar effects to FBS. Fluorescence microscopic observation revealed that inhibitors of ABC transporter affected the intracellular distribution of PpIX. These results indicated that ABCG2-mediated PpIX efflux was a major factor that prevented PpIX accumulation in cancer cells in the presence of serum. Inhibition of ABCG2 transporter system could be a new target for the improvement of photodynamic therapy.     

Increase in protoporphyrin IX after 5-aminolevulinic acid based photodynamic therapy is due to local re-synthesis.

Protoporphyrin IX (PpIX) fluorescence that is bleached during aminolevulinic acid (ALA) mediated photodynamic therapy (PDT) increases again in time after treatment. In the present study we investigated if this increase in PpIX fluorescence after illumination is the result of local re-synthesis or of systemic redistribution of PpIX. We studied the spatial distribution of PpIX after PDT with and without cooling using the skin-fold observation chamber model. We were unable to show a correlation between the local PpIX fluorescence increase and the distance from a blood vessel. The spatial distribution of PpIX fluorescence within normal tissue or tumour is not changed in response to the illumination. These observations suggest that there is no diffusion of PpIX into the treated tissue. Cooling the tissue to 12 degrees C, a temperature at which PpIX synthesis is inhibited, inhibited the PpIX fluorescence increase normally observed after illumination. We also found a strong correlation between local PpIX photobleaching during illumination and the fluorescence intensity 1 h after illumination similar to what we have observed in patients treated with ALA-PDT. Therefore we conclude that the increase in PpIX fluorescence after illumination is due to local cellular re-synthesis.     

Cell specific effects of polyunsaturated fatty acids on 5-aminolevulinic acid based photosensitization.

The purpose of this study was to examine whether the dietary components n-6 and n-3 polyunsaturated fatty acids (PUFAs) may potentiate the effect of photodynamic therapy (PDT) in human cancer cell lines by enhancing the lipid peroxidation. The effects of the porphyrin precursor 5-aminolevulinic acid (5-ALA) and light (320 < lambda < 440 nm, 33 W m(-2)), with or without docosahexaenoic acid (DHA) or arachidonic acid (AA), were tested in the colon carcinoma cell lines SW480 and WiDr, the glioblastoma cell line A-172 and the lung adenocarcinoma cell line A-427. The production of endogenous protoporphyrin IX (PpIX) varied substantially between the cell lines and was approximately 4-fold higher in WiDr as compared with SW480. Cell killing by 5-ALA-PDT also varied between the cell lines, but without clear correlation with PpIX levels. Treatment with DHA or AA (10 or 70 microM, 48 or 72 h) in combination with 5-ALA-PDT (1 or 2 mM) enhanced the cytotoxic effect in A-172 and A-427 cells, but not in SW480 and WiDr cells. While 5-ALA-PDT alone increased the lipid peroxidation in A-172 and WiDr cells only, 5-ALA-PDT plus PUFAs increased the lipid peroxidation substantially in all four cell lines. Interestingly, alpha-tocopherol (50 microM, 48 h) strongly reduced lipid peroxidation after all treatments in all cell lines, while cytotoxicity was only reduced substantially in A-427 cells. This demonstrates that induction of lipid peroxidation is not a general mechanism responsible for the cytotoxicity of 5-ALA-PDT, although it may be important in cell lines with an inherent sensitivity to lipid peroxidation products. Thus, the mechanisms of cell growth inhibition/cell killing by PDT are complex and cell specific.     

Photosensitizing effect of protoporphyrin IX in pigmented melanoma of mice

No fluorescence of protoporphyrin IX (PpIX) was measured using a fiber optic probe in pigmented B16F10 melanoma in mice after topical application of 5-aminolevulinic acid methylester (ALA-Me). However, chemical extraction of tissues excised from mice after intratumoral administration of ALA-Me or its parent compound ALA revealed that this tumor had the capability to produce PpIX. Small amounts of endogenous porphyrins, mainly PpIX, were found in the melanoma not treated with these drugs. Topical application of ALA-Me followed by exposure with laser light (633nm) delayed the growth of the tumors slightly. Light alone also had a significant effect on the tumor growth.     

Modulating ALA-PDT efficacy of mutlidrug resistant MCF-7 breast cancer cells using ALA prodrug

Multi-drug resistance of breast cancer is a major obstacle in chemotherapy of cancer treatments. Recently it was suggested that photodynamic therapy (PDT) can overcome drug resistance of tumors. ALA-PDT is based on the administration of 5-aminolevulinic acid (ALA), the natural precursor for the PpIX biosynthesis, which is a potent natural photosensitizer. In the present study we used the AlaAcBu, a multifunctional ALA-prodrug for photodynamic inactivation of drug resistant MCF-7/DOX breast cancer cells. Supplementation of low doses (0.2mM) of AlaAcBu to the cells significantly increased accumulation of PpIX in both MCF-7/WT and MCF-7/DOX cells in comparison to ALA, or ALA + butyric acid (BA). In addition, our results show that MCF-7/DOX cells are capable of producing higher levels of porphyrins than MCF-7/WT cells due to low expression of the enzyme ferrochelatase, which inserts iron into the tetra-pyrrol ring to form the end product heme. Light irradiation of the AlaAcBu treated cells activated efficient photodynamic killing of MCF-7/DOX cells similar to the parent MCF-7/WT cells, depicted by low mitochondrial enzymatic activity, LDH leakage and decreased cell survival following PDT. These results indicate that the pro-drug AlaAcBu is an effective ALA derivative for PDT treatments of multidrug resistant tumors.     

Topical applications of iron chelators in photosensitization.

Generation of the reactive oxygen species (ROS) in skin by exposure to ultraviolet (UV) radiation induces a number of cutaneous pathologies such as skin cancer, photosensitization, and photoaging among others. Skin iron catalyzes UV generation of ROS. Topical application of iron chelators reduces erythema, epidermal and dermal hypertrophy, wrinkle formation, tumour appearance. It has been proposed that iron chelators can be useful agents against damaging effects of both short- and long-term UV exposure. A better understanding of the action mechanisms of iron chelators, might be useful to developing effective anticancer and antiphotoaging cosmetic products. Iron chelators may lead to accumulation of protoporphyrin IX (PpIX), a strong photosensitizer. The action of iron chelators in skin, related to PpIX increase has not yet been thoroughly studied. Therefore, we have investigated the formation of PpIX in normal mouse skin after topical application of creams containing metal chelators. The amount and distribution of porphyrins formed was determined by means of non-invasive fluorescence spectroscopy. Deferoxamine (DF), ethylenediaminetetraacetic acid (EDTA), 1,2-diethyl-3-hydroxypyridin-4-one (CP94), but not meso-2,3-dimercaptosuccinic acid (DMSA), caused increased accumulation of endogenous porphyrins in the skin. Fluorescence excitation and emission spectroscopy confirmed that PpIX was the main fluorescent species. The amount of PpIX accumulated in skin under the present conditions was not large enough to produce any significant erythema after light exposure. Further studies are needed to evaluate the role of PpIX induced by iron chelators used, against photoaging and cancer prevention.     

Hyperresistance to photosensitized lipid peroxidation and apoptotic killing in 5-aminolevulinate-treated tumor cells overexpressing mitochondrial GPX4

Antitumor photodynamic therapy (PDT) with administered 5-aminolevulinic acid (ALA) is based on metabolism of ALA to protoporphyrin IX (PpIX), which acts as a sensitizer of photo-oxidative damage leading to apoptotic or necrotic cell death. An initial goal of this study was to ascertain how the PpIX-sensitized death mechanism for a breast tumor line (COH-BR1 cells) might be influenced by the conditions of ALA exposure in vitro. Two different treatment protocols were developed for addressing this question: (i) continuous incubation with 1 mM ALA for 90 min; and, (ii) discontinuous incubation, i.e., 15 min with 1 mM ALA followed by 225 min without it. Following exposure to 2 J/cm2 of visible light, cell viability, death mechanism, and lipid hydroperoxide (LOOH) level were evaluated for each protocol using thiazolyl blue, Hoechst staining, and HPLC with electrochemical detection assays, respectively. PpIX was found to sensitize apoptosis when it existed mainly in mitochondria (protocol-1), but necrosis when it diffused to other sites, including plasma membrane (protocol-2). Experiments with a transfectant clone, 7G4, exhibiting approximately 85 times greater activity of the LOOH-detoxifying selenoenzyme GPX4 than parental cells, provided additional information about death mechanism. Located predominantly in mitochondria of 7G4 cells, GPX4 strongly inhibited both LOOH accumulation and apoptosis under protocol-1 conditions, but had no significant effect under protocol-2 conditions. These findings support the hypothesis that LOOHs produced by attack of photogenerated singlet oxygen on mitochondrial membrane lipids play an important early role in the apoptotic death cascade.