Accumulation Of Protoporphyrin Research Articles

Liver transplantation for acute-on-chronic liver failure from erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is an inherited disorder of the heme metabolic pathway that is characterized by accumulation of protoporphyrin in the blood, erythrocytes, and tissues, and cutaneous manifestations of photosensitivity, all resulting from abnormalities in ferrochelatase (FECH) activity due to mutations in the FECH gene. Protoporphyrin is excreted by the liver, and excess protoporphyrin leads to cholelithiasis with obstructive episodes and chronic liver disease, finally progressing to liver cirrhosis. Patients with end-stage EPP-associated liver disease require liver transplantation. We describe here a 31-year-old male patient with EPP who experienced acute-on-chronic liver failure and underwent deceased-donor liver transplantation. Surgical and postoperative care included specific shielding from exposure to ultraviolet radiation to prevent photosensitivity-associated adverse effects. The patient recovered uneventfully and was doing well 24 months after transplantation. Future prevention and treatment of liver disease are discussed in detail.

Plasma and red cell exchange transfusions for erythropoietic protoporphyria: A case report and review of the literature

Erythropoietic protoporphyria (EPP) is a rare and usually autosomal dominant disorder characterized by ferrochelatase deficiency and accumulation of protoporphyrin in red blood cells (RBCs), skin, and liver. A small minority of patients develop severe liver dysfunction for which optimum treatment is lacking. Therapeutic plasma exchange (TPE) and RBC exchange (RCE) have been anecdotally reported to benefit patients with EPP and liver failure. A 50-year-old female with EPP developed severe liver dysfunction after knee replacement surgery and high-dose acetaminophen use. Liver biopsy showed cholestatic liver injury without fibrosis. A total of 20 TPE procedures, six RCE procedures, and then 14 more TPE procedures were performed as adjunctive therapy with the purpose of preventing progression to end-stage liver failure. After initial TPE, the plasma and RBC protoporphyrin levels decreased from 834.9 to 180.4 μg/dL (normal, ≤1 μg/dL), and from 3,905 to 2,879 μg/dL (normal, ≤80 μg/dL), respectively, without liver function improvement. RCE decreased RBC protoporphyrin levels from 2,879 to 1,225 μg/dL but plasma protoporphyrin increased from 180.4 to 1,044.1 μg/dL, and liver function failed to improve. Additional TPE again stabilized plasma protoporphyrin and improved RBC protoporphyrin levels but the patient ultimately died owing to end-stage liver disease complications. This case illustrates that TPE and RCE may improve the plasma and RBC biochemical markers of EPP activity but liver function abnormalities may persist and patients may still progress to liver failure either because of irreversible liver injury or independent pathobiological factors unrelated to EPP-induced hepatotoxicity.

An explorative study of non‐invasive ultra‐weak photon emission and the anti‐oxidative influence of oral zinc sulphate in light‐sensitive patients with erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is a rare, inherited disorder of haem biosynthesis owing to deficient ferrochelatase (FECH) and accumulation of protoporphyrin IX (PPIX). This results in acute cutaneous photosensitivity upon light exposure with production of reactive oxygen species (ROS) and ultra-weak photon emission (UPE) as a by-product. We investigated if UPE evaluated the light sensitivity in EPP patients and influence of zinc treatment. Fourteen EPP patients took zinc sulphate (3 × 200mg/day) during spring and summer. Using a photomultiplier (PM), UPE was measured from the buttock skin and dorsal hand before and after solar-simulated light (SUN) exposure. Blood samples were analysed routinely for plasma zinc, iron, ferritin, transferrin, haemoglobin, erythrocyte PPIX and Zn-PPIX. UPE in EPP patients resembled that seen in healthy individuals. Without treatment, a seasonal decrease was seen from spring to summer in four control patients. However, oral zinc treatment reduced ROS formation significantly regardless of SUN exposure. After SUN exposure, the initial burst was correlated to plasma iron and erythrocyte PPIX. During treatment, an inverse correlation was found between plasma zinc concentration and the initial burst. Measurements of UPE can be used for monitoring UVA-induced oxidative processes in vivo in the skin of EPP patients.

Continuous ultra‐low‐intensity artificial daylight is not as effective as red LED light in photodynamic therapy of multiple actinic keratoses

Daylight-mediated photodynamic therapy (PDT) is a simple and tolerable treatment of nonmelanoma skin cancer. It is of interest which light intensity is sufficient to prevent accumulation of protoporphyrin IX (PpIX) and effectively treat actinic keratoses (AKs). We compared the efficacy of PDT with light-emitting diode (LED) to daylight-mediated PDT with very low-intensity artificial daylight ('daylight') in the treatment of multiple AKs in the face or scalp. Twenty patients were treated with conventional methyl aminolevulinate (MAL) PDT in one area. Another area was, after half an hour of occlusive treatment with MAL, illuminated for 2.5 h with low-intensity 'daylight' (0.5 mW/cm(2) -3.7 mW/cm(2)) that corresponds to midday outdoor intensity in the Scandinavian winter. After 3 months, with a response rate of 52%, low-dose artificial daylight was less effective than conventional LED-PDT (63%) (P = 0.0017). The mean PpIX light dose during 'daylight' exposure was 2.23 J/cm(2) and the lower the PpIX light intensity, the higher the accumulation of PpIX (P = 0.003). Even very low-intensity/dose artificial daylight-mediated PDT of multiple AKs resulted in a response rate of more than 50%. However, to ensure efficacies equivalent to conventional LED-PDT, the treatment should not be conducted on very overcast days.

The Escherichia coli Protein YfeX Functions as a Porphyrinogen Oxidase, Not a Heme Dechelatase

ABSTRACTThe protein YfeX from Escherichia coli has been proposed to be essential for the process of iron removal from heme by carrying out a dechelation of heme without cleavage of the porphyrin macrocycle. Since this proposed reaction is unique and would represent the first instance of the biological dechelation of heme, we undertook to characterize YfeX. Our data reveal that YfeX effectively decolorizes the dyes alizarin red and Cibacron blue F3GA and has peroxidase activity with pyrogallal but not guiacol. YfeX oxidizes protoporphyrinogen to protoporphyrin in vitro. However, we were unable to detect any dechelation of heme to free porphyrin with purified YfeX or in cellular extracts of E. coli overexpressing YfeX. Additionally, Vibrio fischeri, an organism that can utilize heme as an iron source when grown under iron limitation, is able to grow with heme as the sole source of iron when its YfeX homolog is absent. Plasmid-driven expression of YfeX in V. fischeri grown with heme did not result in accumulation of protoporphyrin. We propose that YfeX is a typical dye-decolorizing peroxidase (or DyP) and not a dechelatase. The protoporphyrin reported to accumulate when YfeX is overexpressed in E. coli likely arises from the intracellular oxidation of endogenously synthesized protoporphyrinogen and not from dechelation of exogenously supplied heme. Bioinformatic analysis of bacterial YfeX homologs does not identify any connection with iron acquisition but does suggest links to anaerobic-growth-related respiratory pathways. Additionally, some genes encoding homologs of YfeX have tight association with genes encoding a bacterial cytoplasmic encapsulating protein.

Prodrugs in Photodynamic Anticancer Therapy

Photodynamic therapy (PDT), the concept of cancer treatment through the selective uptake of a light-sensitive agent followed by exposure to a specific wavelength, is limited by the transport of a photosensitizer (PS) to the tumor tissue. Porphyrin, an important PS class, can be used in PDT in the form of its prodrug molecule 5-aminolevulinic acid (5-ALA). Unfortunately, its poor pharmacokinetic properties make this compound difficult to administer. Two different methods for eliminating this problem can be distinguished. The first approach is to play with its formulation in order to improve the drug's applicability. The second approach, which is to find possible 5- ALA prodrugs, is an example of the double-prodrug method, a strategy often used in modern drug design. In this approach, the biological mechanisms in a long biosynthetic pathway involving several steps must be completed before the active drug appears. Recently, an idea of enhancing PDT sensitization using the so-called iron chelators seemed to increase the accumulation of protoporphyrin in cells. At the same time, iron chelators can destroy tumor cells by producing active oxygen after the formation of an active drug by chelating iron in the cancer cells. Thus, in the latter case, the therapy resembles a prodrug strategy. The mechanism can be explained by the Fenton reaction. Vitamin C is another example of a potential anticancer agent of this type.

An In Vitro Comparison of the Effects of the Iron‐Chelating Agents, CP94 and Dexrazoxane, on Protoporphyrin IX Accumulation for Photodynamic Therapy and/or Fluorescence Guided Resection

Photodynamic therapy (PDT) utilizes the combined interaction of a photosensitizer, light and molecular oxygen to ablate tumor tissue. Maximizing the accumulation of the photosensitizer protoporphyrin IX (PpIX) within different cell types would be clinically useful. Dermatological PpIX-induced PDT regimes produce good clinical outcomes but this currently only applies when the lesion remains superficial. Also, as an adjuvant therapy for the treatment of primary brain tumors, fluorescence guided resection (FGR) and PDT can be used to highlight and destroy tumor cells unreachable by surgical resection. By employing iron chelators PpIX accumulation can be enhanced. Two iron-chelating agents, 1,2-diethyl-3-hydroxypyridin-4-one hydrochloride (CP94) and dexrazoxane, were individually combined with the porphyrin precursors aminolevulinic acid (ALA), methyl aminolevulinate (MAL) and hexyl aminolevulinate (HAL). Efficacies of the iron-chelating agents were compared by recording the PpIX fluorescence in human squamous epithelial carcinoma cells (A431) and human glioma cells (U-87 MG) every hour for up to 6 h. Coincubation of ALA/MAL/HAL with CP94 resulted in a greater accumulation of PpIX compared to that produced by coincubation of these congeners with dexrazoxane. Therefore the clinical employment of iron chelation, particularly with CP94 could potentially increase and/or accelerate the accumulation of ALA/MAL/HAL-induced PpIX for PDT or FGR.

A Heme Oxygenase Isoform is Essential for Aerobic Growth in the Cyanobacterium Synechocystis sp. PCC 6803: Modes of Differential Operation of Two Isoforms/Enzymes to Adapt to Low Oxygen Environments in Cyanobacteria

Heme oxygenase (HO) catalyzes the oxygen-dependent cleavage of heme to produce biliverdin IXα in phycobilin biosynthesis. In the genome of the cyanobacterium Synechocystis sp. PCC 6803 there are two genes, ho1 (sll1184) and ho2 (sll1875), encoding HO isoforms. Reverse transcription-PCR indicated that ho1 is constitutively expressed, and ho2 is induced under micro-oxic conditions. A mutant lacking ho1 (Δho1) failed to grow under aerobic conditions while it did grow at a significantly slower rate than the wild type under anaerobic (micro-oxic) conditions. When micro-oxically grown Δho1 was incubated under aerobic conditions, the cells underwent chlorosis with a significant decrease in phycocyanin accompanied by anomalous accumulation of protoporphyrin IX. These results suggested that HO1 is essential for aerobic growth as the sole HO and is dispensable under micro-oxic conditions. A mutant lacking ho2 (Δho2) grew under both aerobic and micro-oxic conditions like the wild type at low light intensity (50 μmol(photon) m⁻² s⁻¹). At higher light intensity (120 μmol(photon) m⁻² s⁻¹) the Δho2 mutant showed significant growth retardation under micro-oxic conditions. It is suggested that HO2 operates as a dominant HO under high light and micro-oxic environments and acts as an accessory HO at low light intensity. Constitutive expression of HO2 in a neutral site of the chromosome restored aerobic growth of Δho1, suggesting that HO2 has an activity high enough to substitute for HO1 under aerobic conditions. The differential operation of two isoforms/enzymes in cyanobacterial tetrapyrrole biosynthesis to adapt to low oxygen environments is discussed, including three other reactions.

Roles of Porphyrin and Iron Metabolisms in the δ‐Aminolevulinic Acid (ALA)‐induced Accumulation of Protoporphyrin and Photodamage of Tumor Cells

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

Photodynamic therapy based on 5‐aminolevulinic acid and its use as an antimicrobial Agent

Exogenous 5-aminolevulinic acid (ALA) is taken up directly by bacteria, yeasts, fungi, and some parasites, which then induces the accumulation of protoporphyrin IX (PPIX). Subsequent light irradiation of PPIX leads to the inactivation of these organisms via photodamage to their cellular structures. ALA uptake and light irradiation of PPIX produced by host cells leads to the inactivation of other parasites, along with some viruses, via the induction of an immune response. ALA-mediated PPIX production by host cells and light irradiation result in the inactivation of other viruses via either the induction of a host cell response or direct photodynamic attack on viral particles. This ALA-mediated production of light-activated PPIX has been extensively used as a form of photodynamic therapy (PDT) and has shown varying levels of efficacy in treating conditions that are associated with microbial infection, ranging from acne and verrucae to leishmaniasis and onychomycosis. However, for the treatment of some of these conditions by ALA-based PDT, the role of an antimicrobial effect has been disputed and in general, the mechanisms by which the technique inactivates microbes are not well understood. In this study, we review current understanding of the antimicrobial mechanisms used by ALA-based PDT and its role in the treatment of microbial infections along with its potential medical and nonmedical applications.

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.

Erythropoietic protoporphyria: a family study and report of a novel mutation in the FECH gene

Erythropoietic protoporphyria (EPP) is a rare inherited disorder of heme biosynthesis mostly caused by a deficient activity of the enzyme ferrochelatase (FECH), and consequent accumulation of protoporphyrin (PP) in various tissues. Clinical manifestations include a childhood onset, cutaneous photosensitivity and, sometimes, hepatobiliary disease. We report a 16-year-old male with EPP characterized by acute episodes of painful photosensitivity since early infancy, permanent changes in the photoexposed skin, microcytic anemia, thrombocytopenia, and mild hepatic dysfunction. His 18-year-old sister presented less acute symptoms with no chronic changes. Lesional biopsy disclosed perivascular deposition of PAS positive hyaline material. Rimington-Cripps test was positive and PP erythrocyte levels were >9,000 μg/L (N<1,600), but normal in their parents and younger brother. Genetic studies in both patients and their mother revealed heterozygosity for a novel mutation (c.1052delA) in FECH gene of both children, and heterozygosity for the hypomorphic allele IVS3-48T>C in all of them. This confirms the "pseudodominant" inheritance pattern usually observed, explained by the combined presence of a disabling FECH mutation and a common intronic polymorphism affecting the counterpart allele (IVS3-48T>C). Phenotypic heterogeneity for this genotype explains the divergent clinical presentation. This is the first description of a Portuguese family with EPP characterized at the molecular level.

PROTOPORFIRIA ERITROPOIÉTICA – Caracterização de uma Família Portadora de uma Nova Mutação no Gene FECH

A protoporfiria eritropoiética (PPE), é uma genodermatose rara causada, na maioria dos doentes, pela redução da actividade da ferroquelatase (FECH), a última enzima da biossíntese do heme, e consequente acumulação de protoporfirina (PP) em vários tecidos. Os autores descrevem um adolescente de 16 anos (caso índice) que sofria de PPE desde a infância, caracterizada por episódios agudos de fotossensibilidade dolorosa nas áreas foto-expostas, alterações crónicas, anemia microcítica, trombocitopenia e disfunção hepática ligeira. A irmã de 18 anos apresentava sintomas agudos mais ligeiros e não evidenciava alterações crónicas. Os progenitores e o irmão mais novo eram assintomáticos. A biópsia lesional efectuada no caso índice mostrou deposição de material hialino, PAS-positivo, em localização perivascular. O teste de Rimington-Cripps foi positivo em ambos os doentes sintomáticos e os níveis de PPs eritrocitárias encontravam-se elevados, mas normais nos pais e no irmão mais novo. O estudo genético, efectuado em ambos os doentes e na mãe (amostra do pai não disponível por paradeiro desconhecido), revelou heterozigotia para uma nova mutação no gene FECH (c.1052delA) nos filhos e heterozigotia para o alelo hipomórfico IVS3-48T&gt;C em todos os elementos testados. Confirmou-se, assim, o padrão de hereditariedade “pseudodominante” observado na maioria das famílias, explicado pela presença simultânea de uma mutação no gene FECH e de um polimorfismo frequente, afectando o alelo oposto (IVS3-48T&gt;C). Neste caso, o alelo mutante foi provavelmente herdado do pai. Do nosso conhecimento, esta é a primeira descrição de uma família portuguesa com PPE caracterizada do ponto de vista molecular, tendo-se descrito uma nova mutação, não referida na literatura.

Level of protoporphyrinogen oxidase activity tightly correlates with photodynamic and defense responses in oxyfluorfen-treated transgenic rice

Transgenic rice systems involving the plastidal expression of Arabidopsis protoporphyrinogen oxidase (Protox) (AP line) and the dual expression of Myxococcus xanthus Protox in chloroplasts and mitochondria (TTS line) were used to investigate the effect of Protox levels on photodynamic stress and defense responses imposed by peroxidizing herbicide oxyfluorfen. The oxyfluorfen-treated TTS line showed less photodynamic stress and antioxidant responses than AP and wild-type lines, indicating that sustained Protox activity in the TTS line during oxyfluorfen action prevented the accumulation of photodynamic protoporphyrin IX. This study suggests evidence of a tight correlation of Protox activity with herbicidal resistance in rice plants.

Fluorescence Diagnostics of Basal Cell Carcinomas Comparing Methyl-aminolaevulinate and Aminolaevulinic Acid and Correlation with Visual Clinical Tumour Size

Fluorescence diagnostics based on aminolaevulinic acid (ALA) fluorescence has been suggested as an in vivo pre-surgical tool for tumour demarcation. We performed fluorescence diagnostics of 35 basal cell carcinomas (BCCs) undergoing photodynamic therapy (PDT) using methyl-aminolaevulinate (MAL). In addition, a semi-automated thresholding algorithm was implemented to detect the potential tumour region. The mean tumour fluorescence contrast was found to be 1.65 ± 0.06 during the first MAL-PDT session, and increased to 1.84 ± 0.07 at the second treatment (p < 0.01). This could imply that disruption of the skin barrier and inflammatory responses after the first session of PDT led to higher accumulation of proto-porphyrin IX during the second session of PDT. The tumour areas detected based on fluorescence in small BCCs (< 1 cm(2)) were in general (n = 18/23) larger than the visual clinical tumour size. In addition, the fluorescence contrast using MAL (1.65 ± 0.06) was found to be significantly higher (p<10(-4)) than the contrast (data from previous study) after application of ALA (1.20 ± 0.06). Thus, MAL generally provides higher tumour contrast than ALA in BCCs, and should be preferred for use in fluorescence diagnostics. Correlation between fluorescence, lack of treatment response and/or pain was not observed.

Monitoring the accumulation and dissipation of the photosensitizer protoporphyrin IX during standard dermatological methyl-aminolevulinate photodynamic therapy utilizing non-invasive fluorescence imaging and quantification

Dermatological methyl-aminolevulinate photodynamic therapy (MAL-PDT) is utilized to successfully treat dermatological conditions. This study monitored fluorescence changes attributed to the accumulation and destruction of the photosensitizer, protoporphyrin IX (PpIX), at several different stages during the first and second treatments of clinical dermatological MAL-PDT. A commercially available, non-invasive, fluorescence imaging system (Dyaderm, Biocam, Germany) was utilized to monitor fluorescence changes during the first and second MAL-PDT treatments in seventy-five lesions. The clinical data indicated statistically significant increases in fluorescence within lesions following the application of MAL for both treatments (P<0.001 and P<0.01 respectively) and subsequent statistically significant decreases in fluorescence within the lesions following light irradiation for both treatments (P<0.001 and P<0.01 respectively) whilst normal skin fluorescence remained unaltered. Lesions receiving a second treatment accumulated and dissipated significantly less PpIX (P<0.05) than during the first treatment. No significant differences were noted in PpIX accumulation or dissipation during MAL-PDT when gender, age, lesion type and lesion surface area were considered. It can therefore be concluded that PpIX fluorescence imaging can be used in real-time to assess PpIX levels during dermatological PDT. Similar observations were recorded from the three currently licensed indications indicating that the standard 'one size fits all' protocol currently employed appears to allow adequate PpIX accumulation, which is subsequently fully utilized during light irradiation regardless of patient age, gender or lesion surface area.

An integrated diagnosis and therapeutic system using intra-operative 5-aminolevulinic-acid-induced fluorescence guided robotic laser ablation for precision neurosurgery

We have developed an integrated diagnosis and therapeutic system for precision malignant gliomas resection during neurosurgery. A combination of three-dimensional (3-D) magnetic resonance imaging (MRI) navigation and 5-aminolevulinic acid (5-ALA)-induced fluorescence based intra-operative tumor diagnosis technique has been incorporated into a robotic laser ablation neurosurgery system with an automatic focusing and robotic scanning mechanism. 5-ALA is a non-fluorescent prodrug that leads to intracellular accumulation of fluorescent protoporphyrins IX (PpIX) in malignant glioma. The PpIX tends to accumulate in pathological lesions, and emits red fluorescence when excited by blue light. This fluorescence is illuminated with laser excitation, enables intra-operative identification of the position of a tumor and provides guidance for resection with laser photocoagulation. The information provided by the MRI is enhanced by the intra-operative 5-ALA fluorescence data, and this enhanced information is integrated into a robotic laser ablation system. The accuracy of the fluorescent measurement of the tumor is improved using high-precision spectral analysis. The fluorescence assists in the detection of malignant brain tumors intra-operatively and improves their removal rate.

Delayed diagnosis and diminished quality of life in erythropoietic protoporphyria: results of a cross-sectional study in Sweden

Delayed diagnosis and diminished quality of life in erythropoietic protoporphyria: results of a cross-sectional study in Sweden

Quinolone compounds enhance -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.

Comparison of Accumulation, Subcellular Location, and Sonodynamic Cytotoxicity between Hematoporphyrin and Protoporphyrin IX in L1210 Cells

Background: Sonodynamic therapy (SDT) is a promising modality for cancer treatment which requires the synergistic effect of ultrasound and tumor-localized sonosensitizers. Sonodynamic efficacy can be improved through a better understanding of the accumulation and subcellular location of sonosensitizers. Here, a comparison of the accumulation, sublocation, and sonodynamic effect of hematoporphyrin (Hp) and protoporphyrin IX (PpIX) was studied in L1210 cells. Methods: The kinetics of intracellular Hp and PpIX accumulation were detected using a fluorescence spectrophotometer. The subcellular distributions of Hp and PpIX were monitored by laser scanning confocal microscopy. The cytotoxic effects of Hp-mediated SDT (Hp-SDT) and PpIX-mediated SDT (PpIX-SDT) were evaluated by MTT assay. Results: The accumulation of Hp and PpIX presented different kinetic changes depending on the time, and was also concentration- and temperature-dependent. The intracellular PpIX content was much higher than that of Hp under the same conditions; however, there were no obvious differences in terms of their subcellular locations, and both of them mainly accumulated on the mitochondria and the plasma membrane in L1210 cells. PpIX exhibited more potential cytotoxicity than did Hp when they were irradiated with ultrasound under the same experimental conditions. Conclusion: Our results indicate that there were significant differences regarding the intracellular accumulation features between Hp and PpIX. PpIX-SDT produced a more serious cytotoxic effect than did Hp-SDT, which may be due to the higher PpIX uptake in L1210 cells compared to that of Hp at the same concentrations. Additionally, the absorption of Hp and PpIX in L1210 cells might be energy dependent.