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.     

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.     

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.     

Quinolone compounds enhance delta-aminolevulinic acid-induced accumulation of protoporphyrin IX and photosensitivity of tumour cells

Exogenous δ-aminolevulinic acid (ALA)-induced photodynamic therapy (PDT) has been used in the treatment of cancer. To obtain a high efficacy of ALA-PDT, we have screened various chemicals affecting ALA-induced accumulation of protoporphyrin in cancerous cells. When HeLa cells were treated with quinolone chemicals including enoxacin, ciprofloxacin or norfloxacin, the ALA-induced photodamage accompanied by the accumulation of protoporphyrin was stronger than that with ALA alone. Thus, quinolone compounds such as enoxacin, ciprofloxacin and norfloxacin enhanced ALA-induced photodamage. The increased ALA-induced photodamage in enoxacin-treated HeLa cells was decreased by haemin or ferric-nitrilotriacetate (Fe-NTA), suggesting that an increase in iron supply cancels the accumulation of protoporphyrin. On the other hand, the treatment of the cells with ALA plus an inhibitor of haem oxygenase, Sn-protoporphyrin, led to an increase in the photodamage and the accumulation of protoporphyrin compared with those upon treatment with ALA alone, indicating that the cessation of recycling of iron from haem augments the accumulation. The use of quinolones plus Sn-protoporphyrin strongly enhances ALA-induced photodamage. To examine the mechanisms involved in the increased accumulation of protoporphyrin, we incubated ferric chloride with an equivalent amount of quinolones. Iron-quinolone complexes with visible colours with a maximum at 450 nm were formed. The levels of iron-metabolizing proteins in enoxacin- or ciprofloxacin-treated cells changed, indicating that quinolones decrease iron utilization for haem biosynthesis. Hence, we now propose that the use of quinolones in combination with ALA may be an extremely effective approach for the treatment modalities for PDT of various tumour tissues in clinical practice.     

Synthesis and in vitro cellular uptake of 11C-labeled 5-aminolevulinic acid derivative to estimate the induced cellular accumulation of protoporphyrin IX

Protoporphyrin IX (PpIX) accumulation induced by exogenous 5-aminolevulinic acid (ALA) in tumors affects the therapeutic efficacy of ALA-based photodynamic and sonodynamic therapies. To develop a new imaging probe to estimate the ALA-induced PpIX accumulation, ¹¹C-labeled ALA analog (4), an ALA-dehydratase inhibitor, was radiosynthesized via ¹¹C-methylation of a Schiff-base-activated precursor in the presence of tetrabutylammonium fluoride, followed by the hydrolysis of ester and imine groups. The cellular uptake of 4 linearly increased with time and was inhibited by ALA and other transporter competitors. Monitoring analog 4 with positron emission tomography might be useful to estimate the ALA-induced PpIX accumulation in tumors.     

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。     

The role of nitric oxide in delta-aminolevulinic acid (ALA)-induced photosensitivity of cancerous cells

Application of delta-aminolevulinic acid (ALA) results in the endogenous accumulation of protoporphyrin IX and is a useful approach in the photodynamic therapy (PDT) of cancers. To investigate the role of nitric oxide (NO) in the specific accumulation of protoporphyrin and ALA-induced PDT of cancerous cells, we transfected inducible-nitric oxide synthase (NOS2) cDNA into human embryonic kidney (HEK) 293T cells and examined the ALA-induced photo-damage as well as the accumulation of porphyrin in the cells. When the NOS2-expressing HEK293T cells were treated with ALA and then exposed to visible light, they became more sensitive to the light with accumulating porphyrins, as compared with the ALA-treated control cells. An increase in the generation of NO in transfected cells led to the accumulation of protoporphyrin with a concomitant decrease of ferrochelatase, the final step enzyme of heme biosynthesis. When mouse macrophage-like RAW264.7 cells were cultured with lipopolysaccharide and interferon-gamma, the expression of NOS2 was induced. The addition of ALA to these cells led to the accumulation of protoporphyrin and cell death upon exposure to light. The treatment of cells with an NOS inhibitor, NG-monomethyl-L-arginine acetate, resulted in the inhibition of protoporphyrin accumulation and cell death. The levels of mitochondrial ferrochelatase and rotenone-sensitive NADH dehydrogenase in the NOS2-induced cells decreased. These results indicated that the generation of NO augments the ALA-induced accumulation of protoporphyrin IX and subsequent photo-damage in cancerous cells by decreasing the levels of mitochondrial iron-containing enzymes. Based on the fact that the production of NO in cancerous cells is elevated, NO in the cells is responsible for susceptibility with ALA-induced PDT.     

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.     

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.     

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.     

Evaluation of dipeptide-derivatives of 5-aminolevulinic acid as precursors for photosensitizers in photodynamic therapy

N-terminal-blocked and N-terminal-free pseudotripeptide Gly-Gly and Gly-Pro derivatives of 5-aminolevulinic acid (ALA) esters were synthesized as potential specific substrates for cellular peptidases and precursors for the production of the photosensitizer protoporphyrin IX (PpIX). These precursors were evaluated using human cell lines of either carcinoma or endothelial origin. N-blocked or N-free dipeptides-ALA-ethyl esters, but not tripeptides-ALA-ethyl esters (or dipeptides-ALA-ethyleneglycols,) were substrates for cellular peptidases and were metabolized to ALA. The precursors were hydrolyzed intracellularly involving serine-proteases and metalloproteases. Cell selectivity for human endothelial or carcinoma cells was observed for some of these dipeptides-ALA. Thus drugs coupled to Gly-Gly-/Gly-Pro-derivatives may selectively target defined cells in human cancer, depending on specific cellular activating pathways expressed by the cells.     

Deferoxamine photosensitizes cancer cells in vitro

Effect of the iron chelator deferoxamine (DF) on the production of endogenous porphyrins was studied in adenocarcinoma WiDr cells and erythroid K562 cells in vitro. Porphyrin fluorescence was observed in the cells in vitro incubated with DF. The fluorescence spectra recorded in the cells were similar to that of protoporphyrin IX (PpIX). The amount of PpIX generated by DF was around 5% of the ALA effect. Around 90% of the WiDr cells incubated in vitro with DF (0.5 mM, 24 h) and then exposed to light (400-460 nm, 20 min) were photodynamically inactivated. In conclusion, the present study describes a novel approach of using iron chelating agents without 5-aminolevulinic acid (ALA) to photosensitize cancer cells.     

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.     

Regulation of Magnesium Chelatase Activity during Excessive Accumulation of Porphyrins in Green Barley Leaves

Activity of magnesium chelatase was studied in green barley leaves treated with 5-aminolevulinic acid (ALA). After this treatment, leaves accumulated excessive amounts of porphyrinic precursors of chlorophyll : protoporphyrin IX (PP), magnesium-protoporphyrin IX (MgPP), its monomethyl ester (MgPPE), and protochlorophyllide. The enzyme activity was found to be inversely dependent on the amount of MgPP formed from exogenous ALA. A conclusion was drawn about the existence of a mechanism for the regulation of the enzyme activity in vivo via its inhibition by the reaction product.     

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.     

Role of mitochondrial cardiolipin peroxidation in apoptotic photokilling of 5-aminolevulinate-treated tumor cells

In 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT), ALA taken up by tumor cells is metabolized to protoporphyrin IX (PpIX), which sensitizes photodamage leading to apoptotic or necrotic cell death. Since lipophilic PpIX originates in mitochondria, we postulated that photoperoxidation of highly unsaturated cardiolipin (CL), which anchors cytochrome c (cyt c) to the inner membrane, is an early proapoptotic event. As initial evidence, PpIX-sensitized photooxidation of liposomal CL to hydroperoxide (CLOOH) species precluded cyt c binding, but this could be reinstated by GSH/selenoperoxidase (GPX4) treatment. Further support derived from site-specific effects observed using (i) a mitochondrial GPX4-overexpressing clone (7G4) of COH-BR1 tumor cells, and (ii) an ALA treatment protocol in which most cellular PpIX is either inside (Pr-1) or outside (Pr-2) mitochondria. Sensitized cells were exposed to a lethal light dose, and then analyzed for death mechanism and lipid hydroperoxide (LOOH) levels. Irradiated Pr-1 vector control (VC) cells died apoptotically following cyt c release and caspase-3 activation, whereas 7G4 cells were highly resistant. Irradiated Pr-2 VC and 7G4 cells showed negligible cyt c release or caspase-3 activation, and both types died via necrosis. CLOOH (detected long before cyt c release) accumulated approximately 70% slower in Pr-1 7G4 cells than in Pr-1 VC, and this slowdown exceeded that of all other LOOHs. These and related findings support the hypothesis that CL is a key upstream target in mitochondria-dependent ALA-PDT-induced apoptosis.     

5-aminolevulinic acid enhances cell death under thermal stress in certain cancer cell lines

5-aminolevulinic acid (5-ALA) is contained in all organisms and a starting substrate for heme biosynthesis. Since administration of 5-ALA specifically leads cancer cells to accumulate protoporphyrin IX (PpIX), a potent photosensitizer, we tested if 5-ALA also serves as a thermosensitizer. 5-ALA enhanced heat-induced cell death of cancer cell lines such as HepG2, Caco-2, and Kato III, but not other cancer cell lines including U2-OS and normal cell lines including WI-38. Those 5-ALA-sensitive cancer cells, but neither U2-OS nor WI-38, accumulated intracellular PpIX and exhibited an increased reactive oxygen species (ROS) generation under thermal stress with 5-ALA treatment. In addition, blocking the PpIX-exporting transporter ABCG2 in U2-OS and WI-38 cells enhanced their cell death under thermal stress with 5-ALA. Finally, a ROS scavenger compromised the cell death enhancement by 5-ALA. These suggest that 5-ALA can sensitize certain cancer cells, but not normal cells, to thermal stress via accumulation of PpIX and increase of ROS generation.     

Improving the use of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) for the gastrointestinal tract by esterification--an in vitro study.

Possible approaches to improve the diagnostic and therapeutic effects of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) are the esterification of ALA for enhanced uptake and the choice of wavelength for irradiation. The human colonic cell lines HT29 [G2] and CCD18 (fibroblasts) were incubated with 0.6 mM ALA, ALA-hexylester or -benzylester respectively, and for further assays with hypotaurine, in addition. PPIX-accumulation was analyzed by flow cytometry and fluorescence spectroscopy. PPIX formation kinetics were continuously recorded. Incubated cells were irradiated with an incoherent light source lambda = 400-700 nm or lambda = 590-700 nm, respectively. After PDT treatment, clonogenicity assays were performed to determine cell viability. Esterification leads to increased PPIX-accumulation, decreased time for production of detectable amounts of PPIX as well as increased response to PDT. Tumor specificity is always maintained or exceeds values for ALA alone. ALA enters the cells via beta transporter whereas esters by passive diffusion. Altering irradiation wavelengths showed the independence of wavelength rather than light dose. Results emphasize the role of heme metabolism for generating tumor specificity rather than the process of ALA-uptake, an important detail for future clinical application.     

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     

Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation

Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol.