Protoporphyrin IX Accumulation Research Articles

Potential involvement of the 18 kDa translocator protein and reactive oxygen species in apoptosis of THP-1 macrophages induced by sonodynamic therapy.

Sonodynamic therapy (SDT) with exogenous protoporphyrin IX (PpIX) or endogenous PpIX derived from 5-aminolevulinic acid (ALA) has been carried out to produce apoptotic effects on macrophages, indicating a potential treatment methodology for atherosclerosis. Our previous studies have found that mitochondria damage by reactive oxygen species (ROS) plays a major role in the SDT-induced apoptosis. This study aimed at investigating the potential involvement of the mitochondrial 18 kDa translocator protein (TSPO) and ROS in the pro-apoptotic effects of SDT on THP-1 macrophages. THP-1 macrophages were divided into control and SDT groups, and went through pretreatment of the specific TSPO ligand PK11195 and ROS scavengers N-Acetyl Cysteine (NAC), then compared with groups without pretreatment. Application of PK11195 reduced intracellular accumulation of endogenous PpIX. PK11195 and NAC reduced the generation of intracellular ROS and oxidation of cardiolipin induced by SDT, respectively. PK11195 and NAC also reduced SDT-induced mitochondrial membrane potential (ΔΨm) loss, the translocation of cytochrome c and cell apoptosis. PpIX accumulation, ROS generation and cell apoptosis were also attenuated by siTSPO. Our findings indicate the pivotal role of TSPO and ROS in SDT-induced cardiolipin oxidation, ΔΨm collapse, cytochrome c translocation and apoptosis in THP-1 macrophages.

Photodynamic diagnosis with methyl-5-aminolevulinate in squamous intraepithelial lesions of the vulva: Experimental research.

The incidence of the High-grade Squamous Intraepithelial Lesion of the vulva, formerly vulvar intra-epithelial neoplasia is progressively increasing. Today, an early detection and a precise localization of vulvar lesions are still problematic issues, due to the lack of accuracy of the available diagnostic tool. A new approach is the photodynamic diagnosis based on the fluorescence detection of protoporphyrin IX (PpIX) in cancer cells after topical application of a cream of methyl amino-levulinic acid. This study aimed to evaluate the effectiveness of photodiagnosis in order to discriminate the intensity of PpIX fluorescence between vulvar tumor and healthy skin. After topical application of the cream, the fluorescence on xenografted A431 tumor and adjacent skin was non-invasively measured with optical fiber. The tumor to skin fluorescence ratios were 1.38 and 1.41 at respectively 3h and 6h after application, which were significantly higher compared to those observed before application. PpIX accumulation at different depths of the tumor was investigated by spectrofluorimetry after PpIX chemical extraction from tumor sections at 3h and 6h post-application. It was noticed at both application times that the concentration of PpIX within the tumor progressively decreased. However PpIX fluorescence was always detectable up to 2.5 mm, a depth equivalent to more than three quarters of the tumor. The tumor to exposed skin ratios of PpIX fluorescence showed a good selectivity up to1mm depth at 3h post-application and up to 1.5mm at 6h post-m-ALA. Thus, the photodynamic diagnosis using in vivo topical methyl amino-levulinic acid appears to be a promising way to detect the intraepithelial lesions of the vulva. Our results open the possibility for implementation of topical methyl amino-levulinic acid in clinical settings for recognition of vulvar high-grade squamous intraepithelial lesions.

The background and philosophy behind daylight photodynamic therapy.

Conventional photodynamic therapy (PDT) is associated with side effects, primarily related to the waiting time between pretreatment with application of photosensitizer and illumination. Pain during illumination is a major issue for the patients and options for effective pain relief are limited. Post-treatment inflammation can often be severe and cause inconvenient down-time for the patients and their employers. To avoid the problems of pain and patients crowding in the clinic we eliminated red light treatment of high PpIX concentration and introduced illumination in daylight which may be performed at home. We also investigated if protoporphyrin IX (PpIX) could be activated continuously during its formation which might reduce pain and inflammation. Continuous activation of PpIX during its formation turned out to minimize pain as single PpIX molecules are activated continuously without accumulation of PpIX in the skin. PpIX molecules are formed in the mitochondria and the photodynamic effect only takes place in the mitochondria when continuously activated. This results primarily in apoptosis with little inflammation. Continuous activation of PpIX can be obtained by performing photodynamic therapy in daylight, as well as with daylight-emitting light sources of appropriate wavelengths. Use of daylight prevents the patients from crowding in the clinic. Daylight-PDT completely fulfils the purpose of minimizing pain and inflammation, as well as limiting the strain on the clinic treating the patients.

5-Aminolevulinic-acid-mediated Photodynamic Diagnosis Enhances the Detection of Peritoneal Metastases in Biliary Tract Cancer in Mice.

Previous studies on the accuracy of 5-aminolevulinic-acid-mediated photodynamic diagnosis (5-ALA PDD) have been reported for various cancers and brain surgery. However, biliary tract cancer is rare. Therefore, 5-ALA PDD has not been fully evaluated in biliary tract cancers. Small biliary tract cancer lesions such as peritoneal dissemination, liver metastases, and lymph node metastases are negative prognosticators in patients with biliary cancer. The purpose of this exploratory study was to determine if 5-ALA PDD could detect small biliary tract cancer lesions in murine models of biliary cancers. Biliary cancer cell lines (TFK-1, HuCCT-1, G415, HuH28, SSP25, RBE, KKU055 and KKU100) and Normal human dermal fibroblast cells were used to evaluate protoporphyrin IX (PpIX) accumulation in vitro. Subcutaneous tumor mice were established using two cell lines (TFK-1 and HuCCT-1). 5-ALA (250 mg/kg) was administered intraperitoneally, and fluorescent 5ALA-PDD was performed 3 h later to evaluate tumoral PpIX accumulation. A murine peritoneal disseminated nodule model was established by intraperitoneal injection of TFK-1 cells. Four weeks later, 5-ALA was administered intraperitoneally, and 5-ALA-PDD was performed 3 h post administration to evaluate PpIX accumulation in the disseminated nodules. The presence of tumor cells in tumors and nodules was confirmed by haematoxylin and eosin staining. Compared TO non-cancerous cell lines, PpIX accumulation was increased in biliary tract cancer cell lines. PpIX accumulation led to a strong fluorescent signal in all subcutaneous tumors. In the murine model of peritoneal dissemination, microdisseminated nodules (<1 mm) that could not be detected under white light were clearly visible using 5-ALA-PDD. 5-ALA PDD was useful for diagnosis of biliary tract cancer and detection of small peritoneal metastatic lesions in murine models of biliary cancers. Clinical studies and applications of 5-ALA PDD for biliary tract cancer are expected in the future.

An experimental investigation of a novel iron chelating protoporphyrin IX prodrug for the enhancement of photodynamic therapy.

ObjectivesNon‐melanoma skin cancers are the most frequently occurring type of cancer worldwide. They can be effectively treated using topical dermatological photodynamic therapy (PDT) employing protoporphyrin IX (PpIX) as the active photosensitising agent as long as the disease remains superficial. Novel iron chelating agents are being investigated to enhance the effectiveness and extend the applications of this treatment modality, as limiting free iron increases the accumulation of PpIX available for light activation and thus cell kill.MethodsHuman lung fibroblasts (MRC‐5) and epithelial squamous carcinoma (A431) cells were treated with PpIX precursors (aminolaevulinic acid [ALA] or methyl‐aminolevulinate [MAL]) with or without the separate hydroxypyridinone iron chelating agent (CP94) or alternatively, the new combined iron chelator and PpIX producing agent, AP2‐18. PpIX fluorescence was monitored hourly for 6 hours prior to irradiation. PDT effectiveness was then assessed the following day using the lactate dehydrogenase and neutral red assays.ResultsGenerally, iron chelation achieved via CP94 or AP2‐18 administration significantly increased PpIX fluorescence. ALA was more effective as a PpIX‐prodrug than MAL in A431 cells, corresponding with the lower PpIX accumulation observed with the latter congener in this cell type. Addition of either iron chelating agent consistently increased PpIX accumulation but did not always convey an extra beneficial effect on PpIX‐PDT cell kill when using the already highly effective higher dose of ALA. However, these adjuvants were highly beneficial in the skin cancer cells when compared with MAL administration alone. AP2‐18 was also at least as effective as CP94 + ALA/MAL co‐administration throughout and significantly better than CP94 supplementation at increasing PpIX fluorescence in MRC5 cells as well as at lower doses where PpIX accumulation was observed to be more limited.ConclusionsPpIX fluorescence levels, as well as PDT cell kill effects on irradiation can be significantly increased by pyridinone iron chelation, either via the addition of CP94 to the administration of a PpIX precursor or alternatively via the newly synthesized combined PpIX prodrug and siderophore, AP2‐18. The effect of the latter compound appears to be at least equivalent to, if not better than, the separate administration of its constituent parts, particularly when employing MAL to destroy skin cancer cells. AP2‐18 therefore warrants further detailed analysis, as it may have the potential to improve dermatological PDT outcomes in applications currently requiring enhancement. Lasers Surg. Med. 50:552–565, 2018. © 2018 The Authors. Lasers in Surgery and Medicine Published by Wiley Periodicals, Inc.

Visualizing protoporphyrin IX formation in the dura tail of meningiomas by mass spectrometry imaging.

The advantages of 5-aminolevulinacid (5-ALA)-induced fluorescence-guided surgery in meningiomas are increasingly discussed. In this context, despite detectable tumor tissue in histopathologial analyses, no fluorescence was shown at the dura tail using the standard operating microscope. Thus, 5-ALA metabolism in this surgically important site remains unknown but needs to be elucidated when further evaluating indications of fluorescence-guided surgery in meningiomas. We here present the spatially resolved identification of protoporphyrin IX (PpIX) in sphenoid ridge meningioma cryosections from a patient who underwent fluorescence-guided microsurgery using molecular imaging analysis by matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). Despite a strong fluorescence of the main tumor, no fluorescence could be detected at the dura tail using the standard operating microscope (blue-light, 405nm). However, histopathological analyses clearly showed meningioma tissue. Remarkably, MALDI-MS/MS analysis revealed PpIX formation also at the non-fluorescing dura tail. However, no PpIX was detected in the tumor free dura mater. MALDI-MS/MS visualized a selective accumulation of PpIX within the tumor tissue including the dura tail. Thus, absence of fluorescence in the dura tail as visualized by the operating microscope is not caused by the lack of PpIX formation.

Enhancement of Cancer-Specific Protoporphyrin IX Fluorescence by Targeting Oncogenic Ras/MEK Pathway.

Protoporphyrin IX (PpIX) is an endogenous fluorescent molecule that selectively accumulates in cancer cells treated with the heme precursor 5-aminolevulinic acid (5-ALA). This cancer-specific accumulation of PpIX is used to distinguish tumor from normal tissues in fluorescence-guided surgery (FGS) and to destroy cancer cells by photodynamic therapy (PDT). In this study, we demonstrate that oncogenic Ras/mitogen-activated protein kinase kinase (MEK) pathway can modulate PpIX accumulation in cancer cells.Methods: To identify Ras downstream elements involved in PpIX accumulation, chemical inhibitors were used. To demonstrate the increase of PpIX accumulation by MEK inhibition, different human normal and cancer cell lines, BALB/c mice bearing mammary 4T1 tumors and athymic nude mice bearing human tumors were used. To identify the mechanisms of PpIX regulation by MEK, biochemical and molecular biological experiments were conducted.Results: Inhibition of one of the Ras downstream elements, MEK, promoted PpIX accumulation in cancer cells treated with 5-ALA, while inhibitors against other Ras downstream elements did not. Increased PpIX accumulation with MEK inhibition was observed in different types of human cancer cell lines, but not in normal cell lines. We identified two independent cellular mechanisms that underlie this effect in cancer cells. MEK inhibition reduced PpIX efflux from cancer cells by decreasing the expression level of ATP binding cassette subfamily B member 1 (ABCB1) transporter. In addition, the activity of ferrochelatase (FECH), the enzyme responsible for converting PpIX to heme, was reduced by MEK inhibition. Finally, we found that in vivo treatment with MEK inhibitors increased PpIX accumulation (2.2- to 2.4-fold) within mammary 4T1 tumors in BALB/c mice injected with 5-ALA without any change in normal organs. Similar results were also observed in a human tumor xenograft model.Conclusion: Our study demonstrates that inhibition of oncogenic Ras/MEK significantly enhances PpIX accumulation in vitro and in vivo in a cancer-specific manner. Thus, suppressing the Ras/MEK pathway may be a viable strategy to selectively intensify PpIX fluorescence in cancer cells and improve its clinical applications in FGS.

Novel potential photodynamic therapy strategy using 5-Aminolevulinic acid for ovarian clear-cell carcinoma.

Photodynamic therapy (PDT) is known as a minimally invasive treatment for cancer. 5-Aminolevulinic acid (ALA) is a precursor of the photosensitizing agent protoporphyrin IX (PpIX). Patients with ovarian clear-cell carcinoma (CCC) have poorer prognoses than those of patients with other histological CCC types. We evaluated the efficacy of ALA-PDT on CCC cells in vitro. We used seven human CCC cell lines to measure the cytotoxicity of ALA-PDT. PpIX production in cancer cells was measured using a micro-plate reader. Quantitative real-time PCR was performed to assess the mRNA levels of genes involved in the accumulation of PpIX in cancer cells. Additionally, we measured the enhancement in cytotoxicity with the use of an ABCG2 inhibitor. We found that three cell lines were highly sensitive to ALA-PDT. In contrast, one cell line was resistant to ALA-PDT. The cytotoxicity of ALA-PDT varied among CCC cell lines. The IC50 values of ALA-PDT for the CCC cell lines had a wide range (30-882μM). The cytotoxicity of ALA-PDT was correlated with the intracellular PpIX accumulation. The cell lines sensitive to ALA-PDT expressed PEPT1 (an ALA uptake transporter). The cell line resistant to ALA-PDT expressed ABCG2 (a PpIX export transporter). In the resistant cell line, a combination treatment with both ALA and an ABCG2 inhibitor resulted in the promotion of cytotoxic sensitivity. The present study revealed the efficacy of ALA-PDT against CCC with chemoresistance in vitro.

Menthol reduces phototoxicity pain in a mouse model of photodynamic therapy.

Phototoxicity-induced pain is a major clinical problem triggered by light acting on photosensitising drugs or endogenous porphyrins, notably protoporphyrin IX (PpIX), an intermediary in heme biosynthesis. Protoporphyrin IX accumulates in individuals with erythropoietic protoporphyria and is elevated during photodynamic therapy subsequent to application of 5-aminolevulinic acid (ALA). Pain occurs during irradiation of PpIX and responds poorly to conventional analgesics. Our objective was to develop a model of PpIX phototoxicity pain and investigate the potential of menthol as an analgesic. Application of ALA to the tails of C57 black and SWISS white mice caused PpIX accumulation and nociception during irradiation (630 nm at 3.7 J/cm). Despite similar PpIX accumulation, C57 mice exhibited less pain behavior compared with SWISS mice because of light absorption by pigmentation. Irradiation of ALA-treated dorsal root ganglion neurons caused phototoxicity-evoked action potentials (APs) in both mouse strains. The antioxidant L-tryptophan increased the light dose required to elicit such APs. By contrast, the addition of keratinocytes to neuronal cultures decreased the threshold for APs, suggesting a requirement for proliferating cells. Inhibition of fatty acid amide hydrolase, selective antagonism of TRPV1 or the application of lidocaine or its quaternary derivative QX-314, reduced AP frequency, whereas antagonism of TRPA1 had no effect. These results suggest that products of singlet oxygen-mediated lipid peroxidation trigger nociceptor activation via TRPV1. Menthol inhibited phototoxicity-evoked APs and reduced pain behavior when applied topically to mice. These findings suggest that menthol might provide pain relief in patients experiencing PpIX-phototoxicity pain caused by photodynamic therapy or erythropoietic protoporphyria.

Topical calcipotriol before ablative fractional laser-assisted photodynamic therapy enhances treatment outcomes for actinic keratosis in Fitzpatrick grades III-V skin: A prospective randomized clinical trial

Topical calcipotriol before ablative fractional laser-assisted photodynamic therapy enhances treatment outcomes for actinic keratosis in Fitzpatrick grades III-V skin: A prospective randomized clinical trial

5-Aminolevulinic acid-based photodynamic therapy of chordoma: In vitro experiments on a human tumor cell line

BackgroundChordomas are very rare tumors of the skull base and the sacrum. They show infiltrating and destructive growth and are known to be chemo- and radio-resistant. After surgical resection, the recurrence rate is high and overall survival limited. As current adjuvant treatments are ineffective, new treatment concepts are urgently needed. 5-aminolevulinic acid-based photodynamic therapy (5-ALA based PDT) showed promising results for malignant gliomas. However, it is unknown so far, whether chordomas accumulate protoporphyrin IX (PPIX) after application of 5-ALA and whether they are sensitive to subsequent 5-ALA based PDT. MethodsThe immortalized human chordoma cells U-CH2 were used as in vitro model. After incubation for 4h or 6h with different 5-ALA concentrations, PPIX accumulation was determined by flow cytometry. To assess sensitivity to PDT, chordoma cells were incubated at 30.000cells/well (high cell density) or 15.000cells/well (low cell density) with graded doses of 5-ALA (0–50μg/ml) in 96-well plates and subsequently exposed to laser light of 635nm wavelength (18.75J/cm2). Cell survival was measured 24h after exposure to laser light using the WST-1 assay. ResultsU-CH2 cells dose-dependently accumulated PPIX (ANOVA; p<0.0001). PPIX fluorescence was significantly higher, when cells were incubated with 5-ALA for 6h compared to 4h at higher 5-ALA concentrations (ANOVA/Bonferroni; p≤0.05 for≥30μg/ml 5-ALA). For both cell densities, a 5-ALA dose-dependent decline in viability was observed (ANOVA; p<0.0001). Viability was significantly lower at higher 5-ALA concentrations, when 30.000 cells/wells were treated compared to 15.000cells/well (ANOVA/Bonferroni; p≤0.001 for≥30μg/ml 5-ALA). LD50 was 30.25μg/ml 5-ALA. ConclusionThe human UCH-2 cell line was a very useful in vitro model to study different effects of 5-ALA based PDT. For the first time, it could be shown that human chordoma cells may be destroyed by 5-ALA/PDT.

Randomized, Prospective Double-Blinded Study Comparing 3 Different Doses of 5-Aminolevulinic Acid for Fluorescence-Guided Resections of Malignant Gliomas.

BACKGROUND: Five-aminolevulinic acid (5-ALA) is used for fluorescence-guided resections of malignant glioma at a dose of 20 mg/kg; yet, it is unknown whether lower doses may also provide efficacy. OBJECTIVE: To perform a double-blinded randomized study comparing 3 different doses of 5-ALA. METHODS: Twenty-one patients with suspected malignant glioma were randomly assigned to 0.2, 2, or 20 mg/kg 5-ALA. Investigators were unaware of dose. Intraoperatively, regions of interest were first defined in tumor core, margin, and adjacent white matter under white light. Under violet–blue illumination, the surgeon's impression of fluorescence was recorded per region, followed by spectrometry and biopsy. Plasma was collected after administration and analyzed for 5-ALA and protoporphyrin IX (PPIX) content. RESULTS: The positive predictive value of fluorescence was 100%. Visual and spectrometric fluorescence assessment showed 20 mg/kg to elicit the strongest fluorescence in tumor core and margins, which correlated with cell density. Spectrometric and visual fluorescence correlated significantly. A 10-fold increase in 5-ALA dose (2-20 mg/kg) resulted in a 4-fold increase of fluorescence contrast between marginal tumor and adjacent brain. tmax for 5-ALA was 0.94 h for 20 mg/kg (0.2 kg: 0.50 h, 2 mg/kg: 0.61 h). Integrated PPIX plasma levels were 255.8 and 779.9 mcg*h/l (2 vs 20 mg/kg). Peak plasma concentrations were observed at 1.89 ± 0.71 and 7.83 ± 0.68 h (2 vs 20 mg/kg; average ± Standard Error of Mean [SEM]). CONCLUSION: The highest visible and measurable fluorescence was yielded by 20 mg/kg. No fluorescence was elicited at 0.2 mg/kg. Increasing 5-ALA doses did not result in proportional increases in tissue fluorescence or PPIX accumulation in plasma, indicating that doses higher than 20 mg/kg will not elicit useful increases in fluorescence.

Brazilian Green Propolis Extract Synergizes with Protoporphyrin IX-mediated Photodynamic Therapy via Enhancement of Intracellular Accumulation of Protoporphyrin IX and Attenuation of NF-κB and COX-2.

Brazilian green propolis (BGP) is noted for its impressive antitumor effects and has been used as a folk medicine in various cultures for many years. It has been demonstrated that BGP could enhance the cytotoxic effect of cytostatic drugs on tumor cells. Photodynamic therapy (PDT) is a therapeutic approach used against malignant cells. To assess the synergistic effect of BGP extract on protoporphyrin IX (PpIX)-mediated photocytotoxicity, MTT assays were performed using A431 and HeLa cells. TUNEL assay and Annexin V-FITC/PI staining were performed to confirm the induction of apoptosis. Western blotting analysis was performed to examine the pro-apoptotic proteins, anti-apoptotic proteins and inflammation related proteins in A431 cells. Intracellular accumulation of PpIX was examined by flow cytometry. The synergistic effect of BGP extract in PpIX-PDT was also evaluated with a xenograft model. Our findings reveal that BGP extract increased PpIX-mediated photocytotoxicity in A431 and HeLa cells. PpIX-PDT with BGP extract treatment resulted in a decrease in Bcl-xL and an increase in NOXA, Bax and caspase-3 cleavage. The protein expression levels of p-IKKα/β, NF-κB and COX-2 were upregulated by PpIX-PDT but significantly attenuated when in combination with BGP extract. BGP extract was also found to significantly enhance the intracellular accumulation of PpIX in A431 cells. BGP extract increased PpIX-mediated photocytotoxicity in a xenograft model as well. Our findings provide evidence for a synergistic effect of BGP extract in PpIX-PDT both in vitro and in vivo.

Increased protoporphyrin IX accumulation does not improve the effect of photodynamic therapy for actinic keratosis: a randomized controlled trial

Photodynamic therapy (PDT) with methyl aminolaevulinate (MAL) is highly effective for treating actinic keratosis (AK) on the face/scalp, but less effective on the extremities. Insufficient accumulation of protoporphyrin IX (PpIX) may cause these inferior efficacy rates. However, it is possible to increase PpIX accumulation by extending the MAL application time and/or pretreating the skin with curettage. To investigate whether increased PpIX accumulation improves the effect of MAL-PDT for AKs in a randomized intra-individual study. Twenty-two patients with 533 AKs on both hands were treated with MAL-PDT. To obtain different concentrations of PpIX, four symmetrical areas on each patient were randomly allocated to different regimens: (i) 3-h MAL application without prior curettage (3hC-); (ii) 3 h with curettage (3hC+); (iii) 21 h without curettage (21hC-); and (iv) 21 h with curettage (21hC+). Treatment efficacy was evaluated after 3 months, whereas PpIX fluorescence, pain and erythema were assessed during and after PDT. Extended MAL application with and without curettage increased PpIX accumulation significantly compared with the standard 3hC+ regimen (P = 0·001 and P = 0·002, respectively). However, the median total clearance rates did not improve accordingly: 3hC+ (55·0%), 21hC- (55·0%) and 21hC+ (53·6%). Conversely, insufficient PpIX accumulation in the 3hC- regimen led to a significantly lower clearance rate (33·3%) than the other regimens (P < 0·045). Furthermore, pain and erythema were correlated to PpIX accumulation. Increased PpIX accumulation does not improve the effect of MAL-PDT for AKs on the hands, but leads to worse adverse events. Different strategies are needed to improve PDT on the extremities.

Comparison of Physical Pretreatment Regimens to Enhance Protoporphyrin IX Uptake in Photodynamic Therapy

Skin pretreatment is recommended for adequate penetration of topical photosensitizing agents and subsequent protoporphyrin IX (PPIX) accumulation in photodynamic therapy (PDT). To compare the relative potential of different physical pretreatments to enhance PPIX fluorescence in normal skin. This intraindividual, randomized clinical trial was performed from November 28 to December 20, 2014, at Bispebjerg Hospital, Copenhagen, Denmark, among 12 healthy volunteers 18 years or older. Analysis was based on intention to treat. All participants completed the study protocol. Participants were each exposed to standardized skin preparation with curettage, microdermabrasion with abrasive pads, microneedling with dermarollers, ablative fractional laser (AFXL), non-AFXL, and no pretreatment, followed by 3 hours of methyl aminolevulinate hydrochloride incubation and subsequent red light illumination. The primary outcome measure was methyl aminolevulinate-induced PPIX fluorescence accumulation. Secondary outcome measures were PPIX photobleaching and clinical local skin reactions, supported by noninvasive reflectance measurements of percentage of skin redness, transepidermal water loss, and participant-assessed pain. Among the 12 healthy study participants (8 men; 4 women; mean [SD] age, 33 [15] years), histologic findings confirmed standardization of interventions with partial removal of the stratum corneum after curettage and microdermabrasion and similar vertical penetration depths for microneedling, AFXL, and non-AFXL (median, 125 μm). PPIX fluorescence reached highest intensities in skin pretreated with AFXL (median, 8661 arbitrary units [AU]) compared with microdermabrasion (median, 6731 AU), microneedling (median, 5609 AU), and curettage (median, 4765 AU) (P < .001), among which similar enhancement was shown. Comparatively lower fluorescence levels were demonstrated for skin pretreated with non-AFXL (median, 2898 AU), methyl aminolevulinate-treated controls (median, 2254 AU), and untreated controls (median, 239 AU) (P < .03). Increasing laser densities (2% vs 4% vs 6%) and the number of pretreatment passes (1, 2, and 3 passes) did not enhance PPIX fluorescence. Local skin reactions were most intensified in AFXL-pretreated skin and correlated with PPIX fluorescence and degree of PPIX photobleaching. Under standardized conditions, PPIX accumulation was most enhanced after AFXL pretreatment, followed by microdermabrasion, microneedling, and curettage. Increasing the number of pretreatment passes and laser densities did not further augment PPIX accumulation. These results may indicate relatively enhanced PDT response by AFXL pretreatment in diseased skin. clinicaltrials.gov Identifier: NCT02372370.

Enhancement of 5-aminolevulinic acid-based fluorescence detection of side population-defined glioma stem cells by iron chelation

Cancer stem cells (CSCs) are dominantly responsible for tumor progression and chemo/radio-resistance, resulting in tumor recurrence. 5-aminolevulinic acid (ALA) is metabolized to fluorescent protoporphyrin IX (PpIX) specifically in tumor cells, and therefore clinically used as a reagent for photodynamic diagnosis (PDD) and therapy (PDT) of cancers including gliomas. However, it remains to be clarified whether this method could be effective for CSC detection. Here, using flow cytometry-based analysis, we show that side population (SP)-defined C6 glioma CSCs (GSCs) displayed much less 5-ALA-derived PpIX fluorescence than non-GSCs. Among the C6 GSCs, cells with ultralow PpIX fluorescence exhibited dramatically higher tumorigenicity when transplanted into the immune-deficient mouse brain. We further demonstrated that the low PpIX accumulation in the C6 GSCs was enhanced by deferoxamine (DFO)-mediated iron chelation, not by reserpine-mediated inhibition of PpIX-effluxing ABCG2. Finally, we found that the expression level of the gene for heme oxygenase-1 (HO-1), a heme degradation enzyme, was high in C6 GSCs, which was further up-regulated when treated with 5-ALA. Our results provide important new insights into 5-ALA-based PDD of gliomas, particularly photodetection of SP-defined GSCs by iron chelation based on their ALA-PpIX-Heme metabolism.

5-Aminolevulinic acid-mediated photodynamic therapy for bladder cancer.

Photodynamic therapy using 5-aminolevulinic acid is a treatment method in which the fluorescent substance of protoporphyrinIX excessively accumulated specifically in cancer cells is excited by visible red or green light irradiation, and reactive oxygen is produced and injures cancer cells. Photodynamic therapy using 5-aminolevulinic acid less markedly influences the surrounding normal cells and tissue as a result of no accumulation of protoporphyrinIX, being a low-invasive, less harmful treatment localized to cancer. Furthermore, photodynamic therapy using 5-aminolevulinic acid is painless, requiring no anesthesia because localized lesions are treated at a low-energy level, and repeatedly applicable, unlike radiotherapy, and so is expected to be a new low-invasive treatment based on a concept completely different from existing treatments. In fact, photodynamic therapy using 5-aminolevulinic acid for bladder cancer was clinically demonstrated mainly for treatment-resistant bladder carcinoma insitu, and favorable outcomes have been obtained. Photodynamic therapy using 5-aminolevulinic acid are photodynamic technologies based on the common biological characteristic of cancers, and are expected as novel therapeutic strategies for many types of cancer.

Involvement of protoporphyrin IX accumulation in the pathogenesis of isoniazid/rifampicin-induced liver injury: the prevention of curcumin

Combination of isoniazid (INH) and rifampicin (RFP) causes liver injury frequently among tuberculosis patients. However, mechanisms of the hepatotoxicity are not entirely understood. Protoporphyrin IX (PPIX) accumulation, as an endogenous hepatotoxin, resulting from isoniazid and rifampicin co-therapy (INH/RFP) has been reported in PXR–humanized mice. Aminolevulinic acid synthase1 (ALAS1), ferrochelatase (FECH) and breast cancer resistance protein (BCRP) play crucial roles in PPIX synthesis, metabolism and transport, respectively. Herein, this study focused on the role of INH/RFP in these processes. We observed PPIX accumulation in human hepatocytes (L-02) and mouse livers. FECH expression was initially found downregulated both in L-02 cells and mouse livers and expression levels of ALAS1 and BCRP were elevated in L-02 cells after INH/RFP treatment, indicating FECH inhibition and ALAS1 induction might confer a synergistic effect on PPIX accumulation. Additionally, our results revealed that curcumin alleviated INH/RFP-induced liver injury, declined PPIX levels and induced FECH expression in both L-02 cells and mice. In conclusion, our data provide a novel insight in the mechanism of INH/RFP-induced PPIX accumulation and evidence for understanding pathogenesis of INH/RFP-induced liver injury, and suggest that amelioration of PPIX accumulation might be involved in the protective effect of curcumin on INH/RFP-induced liver injury.

Modeling the ferrochelatase c.315-48C modifier mutation for erythropoietic protoporphyria (EPP) in mice.

ABSTRACTErythropoietic protoporphyria (EPP) is caused by deficiency of ferrochelatase (FECH), which incorporates iron into protoporphyrin IX (PPIX) to form heme. Excitation of accumulated PPIX by light generates oxygen radicals that evoke excessive pain and, after longer light exposure, cause ulcerations in exposed skin areas of individuals with EPP. Moreover, ∼5% of the patients develop a liver dysfunction as a result of PPIX accumulation. Most patients (∼97%) have a severe FECH mutation (Mut) in trans to an intronic polymorphism (c.315-48C), which reduces ferrochelatase synthesis by stimulating the use of an aberrant 3′ splice site 63 nt upstream of the normal site for exon 4. In contrast, with the predominant c.315-48T allele, the correct splice site is mostly used, and individuals with a T/Mut genotype do not develop EPP symptoms. Thus, the C allele is a potential target for therapeutic approaches that modify this splicing decision. To provide a model for pre-clinical studies of such approaches, we engineered a mouse containing a partly humanized Fech gene with the c.315-48C polymorphism. F1 hybrids obtained by crossing these mice with another inbred line carrying a severe Fech mutation (named m1Pas) show a very strong EPP phenotype that includes elevated PPIX in the blood, enlargement of liver and spleen, anemia, as well as strong pain reactions and skin lesions after a short period of light exposure. In addition to the expected use of the aberrant splice site, the mice also show a strong skipping of the partly humanized exon 3. This will limit the use of this model for certain applications and illustrates that engineering of a hybrid gene may have unforeseeable consequences on its splicing.

5-Aminolevulinic acid enhances mitochondrial stress upon ionizing irradiation exposure and increases delayed production of reactive oxygen species and cell death in glioma cells

5-Aminolevulinic acid (5-ALA) can accumulate protoporphyrinIX (PpIX) in tumour cell mitochondria and is well known for its utility in fluorescence-guided resection of malignant gliomas as a live molecular marker. Previously, we and other authors demonstrated that 5-ALA has a radiosensitizing effect for tumours. In the present study, we aimed to investigate the mechanism underlying the radiosensitizing effect of 5-ALA by focusing on glioma cell mitochondria. Using an enhancer (ciprofloxacin) of 5-ALA-induced PpIX accumulation, we evaluated the influence of ionizing irradiation(IR) and delayed reactive oxygen species(ROS) production 12h after IR by colony-forming assay and flow cytometry(FCM) with different amounts of PpIX accumulation. The mitochondrial mass and mitochondrial electron transport chain(mtETC) activity were evaluated by FCM and western blot analysis. Cell death and delayed ROS production after IR in glioma cells were increased in proportion to 5-ALA-induced PpIX accumulation. Delayed ROS production enhanced by 5-ALA localized to the glioma cell mitochondria. Mitochondrial mass and mitochondrial complexIII activity, among mtETC factors, were also increased 12h after IR in glioma cells in proportion to 5-ALA-induced PpIX accumulation with some variation. These results suggest that 5-ALA enhances IR-induced mitochondrial oxidative stress and leads to increased cell death with mitochondrial changes, thereby acting as a targeting mitochondrial drug, and so‑called radiosensitizer in glioma cells.