Showing papers on "Protoporphyrin IX published in 2009"
TL;DR: Simple pretreatments or additions to the regular practice of PDT, aimed to optimize intralesional PpIX content, improve the clinical outcome.
Abstract: The response rates of photodynamic therapy (PDT) vary widely. Limited uptake of topically applied 5-aminolaevulinic acid (ALA), or its methyl ester (MAL), and suboptimal production of protoporphyrin IX (PpIX) may account for these differences. Recently, we demonstrated that hyperkeratosis is an important negative factor in ALA uptake. This review has its focus on pretreatment of the skin in order to improve the clinical outcome of ALA/MAL PDT. Pretreatment of hyperkeratosis can be achieved with keratolytics, curettage/debulking, tape stripping, microdermabrasion or laser ablation. Penetration enhancers may alter the composition or organization of the intercellular lipids of the stratum corneum. Several studies have been performed on the use of dimethyl sulfoxide, azone, glycolic acid, oleic acid and iontophoresis to increase the penetration of ALA. As PpIX production is also dominated by temperature-dependent processes, elevating skin temperature during ALA application may also improve treatment results. Another approach is the use of additives that interact with the heme biosynthetic pathway, e.g. by removing ferrous iron with iron-chelating substances such as: ethylenediaminetetraacetic acid; 3-hydroxypyridin-4-ones; 1,2-diethyl-3-hydroxypyridin-4-one-hydrochloride; and desferrioxamine. In conclusion, simple pretreatments or additions to the regular practice of PDT, aimed to optimize intralesional PpIX content, improve the clinical outcome.
107 citations
TL;DR: In vivo relevance was established by showing that methotrexate preconditioning enhances PpIX accumulation in three models: organotypic cultures of immortalized keratinocytes, chemically induced skin tumors in mice; and human A431 squamous cell tumors implanted subcutaneously in mice.
Abstract: Purpose: To improve treatment efficacy and tumor cell selectivity of δ-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) via pretreatment of cells and tumors with methotrexate to enhance intracellular photosensitizer levels. Experimental Design: Skin carcinoma cells, in vitro and in vivo , served as the model system. Cultured human SCC13 and HEK1 cells, normal keratinocytes, and in vivo skin tumor models were preconditioned with methotrexate for 72 h and then incubated with ALA for 4 h. Changes in protoporphyrin IX (PpIX) levels and cell survival after light exposure were assessed. Results: Methotrexate preconditioning of monolayer cultures preferentially increased intracellular PpIX levels 2- to 4-fold in carcinoma cells versus normal keratinocytes. Photodynamic killing was synergistically enhanced by the combined therapy compared with PDT alone. Methotrexate enhancement of PpIX levels was achieved over a broad methotrexate concentration range (0.0003-1.0 mg/L; 0.6 nmol/L-2 mmol/L). PpIX enhancement correlated with changes in protein expression of key porphyrin pathway enzymes, ∼4-fold increase in coproporphyrinogen oxidase and stable or slightly decreased expression of ferrochelatase. Differentiation markers (E-cadherin, involucrin, and filaggrin) were also selectively induced by methotrexate in carcinoma cells. In vivo relevance was established by showing that methotrexate preconditioning enhances PpIX accumulation in three models: ( a ) organotypic cultures of immortalized keratinocytes, ( b ) chemically induced skin tumors in mice; and ( c ) human A431 squamous cell tumors implanted subcutaneously in mice. Conclusion: Combination therapy using short-term exposure to low-dose methotrexate followed by ALA-PDT should be further investigated as a new combination modality to enhance efficacy and selectivity of PDT for epithelial carcinomas.
104 citations
TL;DR: It is demonstrated that intracellular heme is the physiologically relevant mediator of the cytotoxic effects of artemisinins and indicates that cellular heme may serve a general target for the development of both anti-parasitic and anti-cancer therapeutics.
Abstract: Heme (Fe2+ protoporphyrin IX) is an essential molecule that has been implicated the potent antimalarial action of artemisinin and its derivatives, although the source and nature of the heme remain controversial. Artemisinins also exhibit selective cytotoxicity against cancer cells in vitro and in vivo. We demonstrate that intracellular heme is the physiologically relevant mediator of the cytotoxic effects of artemisinins. Increasing intracellular heme synthesis through the addition of aminolevulinic acid, protoporphyrin IX, or transferrin-bound iron increased the cytotoxicity of dihydroartemisinin, while decreasing heme synthesis through the addition of succinyl acetone decreased its cytotoxic activity. A simple and robust high throughput assay was developed to screen chemical compounds that were capable of interacting with heme. A natural products library was screened which identified the compound coralyne, in addition to artemisinin, as a heme interacting compound with heme synthesis dependent cytotoxic activity. These results indicate that cellular heme may serve a general target for the development of both anti-parasitic and anti-cancer therapeutics.
89 citations
TL;DR: The results suggested that FECH siRNA improved the phototoxicity of ALA‐PDT, overexpression of HO‐1 was associated with shorter (GT)n repeat of the promoter region, and siRNA‐mediated knockdown ofHO‐1 could suppress the growth of bladder cancer cells overexpressing HO‐ 1.
Abstract: Photodynamic therapy mediated by 5-aminolevulinic acid (ALA-PDT) has been developed as a therapeutic modality for refractory superficial bladder cancers. Here, in experiments using urothelial cancer cell lines, we investigated the effects of siRNA modulating heme-synthetic and degradation pathways for ALA-PDT. Targeted knockdown of ferrochelatase (FECH) suppressed heme synthesis and significantly increased intracellular protoporphyrin IX (PpIX) accumulation, leading to enhanced phototoxicity in four of five cell lines. Heme oxygenase-1 (HO-1) is recognized as important for cytoprotection against oxidative stress such as PDT. Targeted knockdown of HO-1 leads to decreased intracellular PpIX accumulation, resulting in a failure to enhance ALA-PDT effect in four cell lines. Knockdown of HO-1 caused marked growth inhibition in UM-UC-2 overexpressing HO-1, whereas no inhibitory effect was observed in UM-UC-3 lacking HO-1 expression. Moreover, HO-1 protein levels and (GT)n repeat polymorphism of the HO-1 gene promoter region were examined with the implication that the constitutive expressions of HO-1 protein were associated with a shorter (GT)n repeat. Our results suggested that (1) FECH siRNA improved the phototoxicity of ALA-PDT, (2) overexpression of HO-1 was associated with shorter (GT)n repeat of the promoter region, and (3) siRNA-mediated knockdown of HO-1 could suppress the growth of bladder cancer cells overexpressing HO-1.
72 citations
TL;DR: Fluorescence induction of protoporphyrin IX by a new 5‐aminolevulinic acid nanoemulsion used for photodynamic therapy in a full‐thickness ex vivo skin model is described.
Abstract: An ex vivo porcine skin model was utilized to analyse the penetration of 5-aminolevulinic acid (5-ALA) contained in a nanoemulsion-based formulation BF-200 ALA (10% 5-ALA-hydrochloride) versus 16% aminolevulinate methyl ester-hydrochloride in a commercially cream (MAL cream) by fluorescence microscopy of their common metabolite protoporphyrin IX (PpIX) after 3, 5, 8 and 12 h. Fluorescence signals of PpIX in pig skin treated with BF-200 ALA were stronger than those for MAL cream. At 8 and 12 h, the PpIX fluorescence signals were 4.8- and 5.0-fold higher than those measured after MAL cream application. Fluorescence signals of PpIX after application of BF-200 ALA were detected in deeper tissue layers of the epidermis than after application of MAL cream (97.2 +/- 5.7 microm for BF-200 ALA vs 42.0 +/- 4.2 microm for MAL cream). These data implicate that BF-200 ALA in photodynamic therapy might lead to a superior therapeutically effect of intraepidermal (in situ) squamous cell carcinomas.
69 citations
61 citations
TL;DR: PpIX-encapsulated colloidal mesoporous silica nanoparticles were uptaken by tumor cells in vitro, and the effect of photon-induced toxicity was demonstrated after comparison with some control experiments.
Abstract: Protoporphyrin IX (PpIX)-encapsulated mesoporous silica nanoparticles were synthesized, characterized, and utilized for photodynamic therapy (PDT) of cancer. Silica encapsulation is relatively transparent for activated light and can protect the PpIX against denaturation induced by the extreme bioenvironment. The mesoporous silica can also ensure that the encapsulated PpIX can be well-contacted with oxygen, stimulated, and released. PpIX-encapsulated colloidal mesoporous silica nanoparticles were uptaken by tumor cells in vitro, and the effect of photon-induced toxicity was demonstrated after comparison with some control experiments. The surface of PpIX-encapsulated silica nanoparticles can be grafted with appropriate functionalized groups and conjugated with certain biomolecules for specific targeting.
58 citations
TL;DR: It is shown that the suppression of ferrochelatase increased PpIX accumulation in UC cells using small amount of ALA, thus leading to an improved clinical practicability of photodynamic diagnosis and therapy.
Abstract: Purpose: The purpose of this study was to clarify the regulatory mechanism of protoporphyrin IX (PpIX) synthesis mediated by 5-aminolevulinic acid (ALA) in human urothelial carcinom
57 citations
TL;DR: In vivo and in vitro, the conjugation of ALA to a second-generation dxcendrimer for enhancing porphyrin synthesis in vitro and in vivo in a murine tumour model using systemic i.p. administration of 5-aminolaevulinic acid (ALA) showed good correlation with higher cellular ALA dendrimer accumulation.
57 citations
TL;DR: Results indicated that ALA‐based‐PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA.
Abstract: Photodynamic therapy (PDT) for tumors is based on the tumor-selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5-aminolevulinic acid (ALA)-based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl-xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase-3 activation, phosphatidylserine (PS) externalization. PDT-induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA-based-PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA.
52 citations
TL;DR: Findings reveal a previously unidentified class of PPO enzymes that do not utilize oxygen as an electron acceptor, thereby allowing gamma-proteobacteria to synthesize heme in both aerobic and anaerobic environments.
Abstract: Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) catalyzes the six-electron oxidation of protoporphyrinogen IX to the fully conjugated protoporphyrin IX. Eukaryotes and Gram-positive bacteria possess an oxygen-dependent, FAD-containing enzyme for this step, while the majority of Gram-negative bacteria lack this oxygen-dependent PPO. In Escherichia coli, PPO activity is known to be linked to respiration and the quinone pool. In E. coli SASX38, the knockout of hemG causes a loss of measurable PPO activity. HemG is a small soluble protein typical of long chain flavodoxins. Herein, purified recombinant HemG was shown to be capable of a menadione-dependent conversion of protoporphyrinogen IX to protoporphyrin IX. Electrochemical analysis of HemG revealed similarities to other flavodoxins. Interestingly, HemG, a member of a class of the long chain flavodoxin family that is unique to the γ-proteobacteria, possesses a 22-residue sequence that, when transferred into E. coli flavodoxin A, produces a chimera that will c...
TL;DR: In this paper, the contribution of protoporphyrin IX (PpIX) to enhanced generation of reactive oxygen species (ROS) under X-ray and UV irradiations was examined.
TL;DR: Findings show that at least in the cases of Mn, Pb, Cd, and Hg, metal "inhibition" of ferrochelatase is not due to the inability of the enzyme to insert the metal into the macrocycle or by binding to a second metal binding site as has been previously proposed.
TL;DR: Results indicate that PPIX, PPIXDE, and Ce6 bind to HSA efficiently, evidence that porphyrins bind strongly to H SA if they have a hydrophobic and hydrophilic side.
Abstract: The efficacy of porphyrin binding to human serum albumin (HSA) is critical to clinical use in photodynamic therapy (PDT). Several porphyrins were utilized to measure the effect of porphyrin structure on its binding to HSA. Two categories of porphyrins were utilized: porphyrins with a hydrophobic and hydrophilic side: Protoporphyrin IX (PPIX), Protoporphyrin IX dimethylester (PPIXDE), and Chlorin e6 (Ce6) and porphyrins with hydrophilic substituents on both sides: Hematoporphyrin IX (Hme), Hematoporphyrin IX dimethylester (HmeDE), and Deuteroporphyrin IX dimethylester (DPIXEG). The following methods were used for the analysis: Stern–Volmer quenching, fluorescence lifetimes, anisotropy, fluorescence binding, and homogeneous studies. The results indicate that PPIX, PPIXDE, and Ce6 bind to HSA efficiently, evidence that porphyrins bind strongly to HSA if they have a hydrophobic and hydrophilic side. Hme is thought to bind to HSA but likely to a lesser degree than the aforementioned three porphyrins. HmeDE and DPIXEG seem not to bind to HSA probably due to the lack of hydrophobic substituents.
TL;DR: This demonstrated use of endogenous fluorescence stimulation from aminolevulinic acid (ALA) and provides the first in vivo demonstration of deep tissue tomographic imaging with protoporphyrin IX, which is demonstrated using a noncontact single-photon counting fan-beam acquisition system interfaced with microCT imaging.
Abstract: Tomographic imaging of a glioma tumor with endogenous fluorescence is demonstrated using a noncontact single-photon counting fan-beam acquisition system interfaced with microCT imaging. The fluorescence from protoporphyrin IX (PpIX) was found to be detectable, and allowed imaging of the tumor from within the cranium, even though the tumor presence was not visible in the microCT image. The combination of single-photon counting detection and normalized fluorescence to transmission detection at each channel allowed robust imaging of the signal. This demonstrated use of endogenous fluorescence stimulation from aminolevulinic acid (ALA) and provides the first in vivo demonstration of deep tissue tomographic imaging with protoporphyrin IX.
TL;DR: In this paper, 27 dipeptide derivatives of general structure Ac-Xaa-ALA-OR were synthesized as potential prodrugs for 5aminolaevulinic acid-based photodynamic therapy (ALA)-PDT.
Abstract: Twenty-seven dipeptide derivatives of general structure Ac-Xaa-ALA-OR were synthesized as potential prodrugs for 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT). Xaa is an (x-amino acid, chosen to provide a prodrug with appropriately tailored lipophilicity and water solubility. Although no simple correlation is observed between downstream production of protoporphyrin IX (PpIX) in PAM212 keratinocytes and HPLC-derived descriptors of compound lipophilicity, quantification of prodrug uptake reveals that most of the dipeptides are actually more efficiently accumulated than ALA in PAM212 and also A549 and Caco-2 cell lines. Subsequent ALA release is the limiting factor, which emphasizes the importance of decoupling prodrug uptake and intracellular metabolization when assessing the efficacy of ALA derivatives for PDT. In agreement with PpIX fluorescence studies, at,I concentration of 0.1 MM, L-Phe derivatives 4m and 4o, and L-Leu, L-Met, and L-Glu derivatives 4f, 4k, and 4u, exhibit significantly enhanced photoxicity in PAM212 cells compared to ALA.
TL;DR: This is the first work to report vascular cell photosensitisation employing alkyl-esters of ALA, and it is demonstrated that these derivatives could exert the same effect as ALA and under certain conditions enhance photosensItisation of vasculature.
Abstract: Photodynamic therapy (PDT) may cause tumour cell destruction by direct toxicity or by inducing microcirculatory shutdown. Protoporphyrin IX generated from 5-aminolevulinic acid (ALA) has been widely used as an endogenous photosensitiser in PDT. However, the hydrophilic nature of the ALA molecule limits its penetration through the stratum corneum of the skin and cell membranes and thus, ALA alkyl-esters have been developed to improve ALA permeation. The aim of this work was to study Protoporphyrin IX synthesis from ALA and its derivatives ALA methyl ester (Me-ALA) and ALA hexyl ester (He-ALA) in the microvascular endothelial cell line HMEC-1 derived from normal skin, and to evaluate their response to PDT. We found that lower light doses are required to photosensitise HMEC-1 endothelial cells than to photosensitise PAM212 transformed keratinocytes, showing some possible selectivity of ALA-PDT for vascularisation in skin. Employed at concentrations leading to equal Protoporphyrin IX synthesis, ALA, He-ALA and Me-ALA presented the same efficacy of HMEC-1 photosensitisation. However, He-ALA was a promising compound for the use in the enhancement of Protoporphyrin IX in HMEC-1 cells employed at low concentrations at both short and long time exposures whereas Me-ALA should be employed at high concentrations and longer time periods in order to surpass the Protoporphyrin IX levels obtained with ALA. The advantage of Me-ALA over ALA was based on its lower dark toxicity. This is the first work to report vascular cell photosensitisation employing alkyl-esters of ALA, and we demonstrated that these derivatives could exert the same effect as ALA and under certain conditions enhance photosensitisation of vasculature.
TL;DR: Evidence of the damage effects of sonodynamic therapy (SDT) on a novel intracellular target, cytoskeletal F-actin, and the observed effect on F- actin and the subsequent bleb formation mainly due to apoptosis formation due to the treatment were reported.
TL;DR: Results indicated that the ultrasonically-induced cell killing effect could be enhanced by PpIX, and that the mitochondrial pathway might be involved in the cell damage process.
Abstract: Sonodynamic therapy (SDT) is a potential cancer therapy based on the synergistic interactions of ultrasound and certain chemical compounds (sono-sensitizers). It has become evident that the direct sonochemical and subsequent redox reactions induced by SDT treatment can lead to apoptotic cell death. However, the detailed mechanisms are not well understood. This study sought to identify the cytotoxic effects of ultrasound-activated protoporphyrin IX (PpIX) on Hepatoma-22 cells (H22). The fluorescence microscope was used to detect the generation of mitochondrial reactive oxygen species (ROS), mitochondrial swelling and release of mitochondrial protein cytochrome c. Several distinct sonochemical effects were found after SDT treatment, including changes of cell viability (decreased to 82.6% in SDT-treated group), generation of mitochondrial ROS, mitochondrial swelling and release of cytochrome c (19.16% in SDT-treated group). These results indicated that the ultrasonically-induced cell killing effect could be enhanced by PpIX, and that the mitochondrial pathway might be involved in the cell damage process.
TL;DR: In photodynamic therapy the endogenous photosensitizer protoporphyrin IX is synthesized following topical application of aminolaevulinic acid, but different tissues have distinct PpIX‐accumulating properties, due to differences in penetration of ALA through the stratum corneum and/or alterations in the haem cycle.
Abstract: BACKGROUND: In photodynamic therapy the endogenous photosensitizer protoporphyrin IX (PpIX) is synthesized following topical application of aminolaevulinic acid (ALA). However, different tissues have distinct PpIX-accumulating properties, due to differences in penetration of ALA through the stratum corneum and/or alterations in the haem cycle. Preferential PpIX accumulation has been reported in terminally differentiated cell cultures but ex vivo data are lacking. OBJECTIVES: To study the intrinsic PpIX-accumulating capabilities of skin explants from lesional and nonlesional skin in psoriasis and actinic keratosis, with normal skin serving as a control, and to study PpIX accumulation in relation to differentiation status in normal skin. METHODS: Skin explants from patients with psoriasis, actinic keratosis and normal skin were incubated with ALA and PpIX was measured spectrofluorometrically. PpIX was measured in basal (beta1-integrin-positive) and suprabasal (beta1-integrin-negative) keratinocytes in normal skin. In addition, PpIX accumulation was studied in cell cultures at different levels of confluence and after induction of terminal differentiation. RESULTS: No significant differences in PpIX content were found between the different tissues. However, increased PpIX was found in beta1-integrin-negative compared with beta1-integrin-positive cells. In addition, in subconfluent cell cultures less PpIX was found compared with confluent cell cultures. Induction of terminal differentiation in vitro, however, resulted in less PpIX, which was likely to be related to cell volume. CONCLUSIONS: As no significant differences in PpIX synthesis could be found between the different tissue types, these data emphasize the importance of the penetration route of ALA through the stratum corneum. Preferential PpIX accumulation observed in suprabasal epidermal keratinocytes and confluent cell cultures points towards a terminal differentiation-specific effect.
TL;DR: The red light bleaching is found to increase the ability to accurately quantify PpIX fluorescence in vivo, but decreases the specificity of detection between tumor-bearing and nontumor-bearing groups.
Abstract: Aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence is studied as a contrast agent for noninvasive detection of murine glioma, using the fluorescence-to-transmission ratio measured through the cranium Signals measured prior to administration of ALA are very similar between control animals, 9L-GFP, and U251 tumor-bearing animals However, 2 h after ALA administration, the PpIX signal from both tumor-bearing groups is significantly higher than the control group (9L-GFP group p-value=0016, and U251 group p-value=0004, relative to the control group) The variance in signal from the 9L-GFP group is much larger than either the control group or the U251 group, which is consistent with higher intrinsic PpIX fluorescence heterogeneity as seen in situ at ex vivo analysis Decreasing the skin PpIX fluorescence via intentional photobleaching using red light (635 nm) is examined as a tool for increasing PpIX contrast between the tumor-bearing and control groups The red light bleaching is found to increase the ability to accurately quantify PpIX fluorescence in vivo, but decreases the specificity of detection between tumor-bearing and nontumor-bearing groups
23 Feb 2009
TL;DR: A compact and portable fiber optic based fluorescence spectroscopy system is developed to allow objective detection of glioblastoma and selectiveness of GBM to 5-aminolevulinic acid induced protoporphyrin IX (PpIX) is reported by other researchers to visualize tumor margins under blue light microscopy.
Abstract: The highly malignant brain tumor, glioblastoma multiforme (GBM), is difficult to fully delineate during surgical resection due to its infiltrative ingrowth and morphological similarities to surrounding functioning brain tissue. Selectiveness of GBM to 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PpIX) is reported by other researchers to visualize tumor margins under blue light microscopy. To allow objective detection of GBM, a compact and portable fiber optic based fluorescence spectroscopy system is developed. This system is able to deliver excitation laser light (405 nm) in both the continuous and pulsed mode. PpIX fluorescence peaks are detected at 635 and 704 nm, using a fiber-coupled spectrometer. It is necessary to optimize the detection efficiency of the system as the PpIX quickly photobleaches during the laser illumination. A light dose of 2.5 mJ (fluence rate = 9 mJ/mm 2 ) is experimentally approved to excite an acceptable level of fluourescence signal arising from glioblastoma. In pulsed illumination mode, an excitation dose of 2.5 mJ, with a dark interval of 0.5 s (duty cycle 50%) shows a significantly shorter photobleaching time in comparison to the continuous illumination mode with the same laser power (p < 0.05). To avoid photobleaching (the remaining signal is more than 90% of its initial value) when measuring with 2.5 mJ delivered energy, the time for continuous and pulsed illumination should be restricted to 2.5 and 1.1 s, respectively.
TL;DR: Aclonifen was shown to act on two different biochemical pathways including carotenoid synthesis on the one hand, as well as protoporphyrinogen oxidase, in the chlorophyll synthesis pathway, on the other for the same range of concentrations.
TL;DR: The amount of ZPP in Parma ham was larger than that of heme, and ZPP accounted for 60-70% of all porphyrins.
TL;DR: It is demonstrated that treatment of endometrial cancer cells with both 5-ALA and hypericin followed by illumination with white light induced a significantly higher phototoxicity as revealed by colony formation.
TL;DR: The dppCBDF gene cluster constitutes part of the periplasmic heme-acquisition systems of H. influenzae and its potential role in heme utilization is investigated.
Abstract: Haemophilus influenzae requires a porphyrin source for aerobic growth and possesses multiple mechanisms to obtain this essential nutrient This porphyrin requirement may be satisfied by either heme alone, or protoporphyrin IX in the presence of an iron source One protein involved in heme acquisition by H influenzae is the periplasmic heme binding protein HbpA HbpA exhibits significant homology to the dipeptide and heme binding protein DppA of Escherichia coli DppA is a component of the DppABCDF peptide-heme permease of E coli H influenzae homologs of dppBCDF are located in the genome at a point distant from hbpA The object of this study was to investigate the potential role of the H influenzae dppBCDF locus in heme utilization An insertional mutation in dppC was constructed and the impact of the mutation on the utilization of both free heme and various proteinaceous heme sources as well as utilization of protoporphyrin IX was determined in growth curve studies The dppC insertion mutant strain was significantly impacted in utilization of all tested heme sources and protoporphyin IX Complementation of the dppC mutation with an intact dppCBDF gene cluster in trans corrected the growth defects seen in the dppC mutant strain The dppCBDF gene cluster constitutes part of the periplasmic heme-acquisition systems of H influenzae
TL;DR: It is shown that down regulation of ALA dehydratase (ALAD) expression and activity by specific shRNA induced a marked decrease in PpIX synthesis in K562 erythroleukemic cells, indicating that mitochondria were protected in the silenced cells.
Abstract: Synthesis of protoporphyrin IX (PpIX) by malignant cells is essential for the success of ALA-based photodynamic therapy (PDT). Two key enzymes that were described as affecting PpIX accumulation during ALA treatment are porphobilinogen deaminase (PBGD) and ferrochelatase. Here, we show that down regulation of ALA dehydratase (ALAD) expression and activity by specific shRNA induced a marked decrease in PpIX synthesis in K562 erythroleukemic cells. Photo-inactivation efficacy following ALA-PDT was directly correlated with ALAD-silencing and cellular levels of PpIX. MTT metabolism following ALA-PDT was shown to be 60% higher in ALAD-silenced cells in comparison to control cells, indicating that mitochondria were protected in the silenced cells. Morphological analysis by scanning electron microscopy (SEM) of cells treated by ALA-PDT showed no morphological changes in ALAD-silenced cells, in contrast to controls exhibiting cell deformations and lysis. Membrane integrity following ALA-PDT was kept intact and undamaged in ALAD-silenced cells as examined by Annexin V-FITC/PI staining and LDH-L leakage. We conclude that ALAD, although it is present in the cell at abundant levels, has a major and limiting role in regulating PpIX synthesis and ALA-PDT outcome.
TL;DR: The heme prosthetic group of the heme protein adsorbed at the mercury electrode surface reacts with mercury ion produced by the electrochemical oxidation of mercury and is quantitatively converted into the mercury protoporphyrin IX group using single-sweep polarography.
Abstract: We have found that in the presence of hydroxylamine, the heme prosthetic group of the heme protein adsorbed at the mercury electrode surface reacts with mercury ion produced by the electrochemical oxidation of mercury and is quantitatively converted into the mercury protoporphyrin IX group using single-sweep polarography. As a result, the small redox peak P0 of the heme prosthetic group at about −0.46 V (vs SCE) disappears and a large new reduction peak P of mercury protoporphyrin IX group at −0.89 V comes out in a pH 9.6 NaHCO3−Na2CO3 solution. Peak P is extremely sensitive to heme protein concentration. On the basis of the reduction peak P, a unique electrochemical method for heme protein assays is constructed. For the cytochrome c determination, the peak height is linearly proportional to the concentration in the range of 0.005−15 mg L−1 (correlation coefficient 0.999). The detection limit is 0.003 mg L−1. In contrast with peak P0, the detection limit of cytochrome c is only 0.6 mg L−1. The voltammogra...
TL;DR: Among investigated compounds PP(Ser)(2)Arg(2) exhibited the lowest dark toxicity and the highest PDT effectiveness towards both cell lines, and may be a potential PS for PDT.
Abstract: Photodynamic therapy (PDT) is an alternative treatment modality involving light activated drugs, called photosensitizers (PSs), to treat cancer and non-cancerous conditions. The search for new compounds which might become effective PSs is the major direction for PDT development. In the present work we have studied the dark toxicity, intracellular localization and photodynamic properties of four potential, water soluble, second generation PSs – PP(Arg)2, PP(Ser)2Arg2, PP(Ala)2Arg2, PP(Phe)2Arg2, all diamino acid derivatives of protoporphyrin IX. Human prostate cancer (DU-145) and squamous carcinoma (A431) cells were used as experimental model.
Among investigated compounds PP(Ser)2Arg2 exhibited the lowest dark toxicity and the highest PDT effectiveness towards both cell lines. Fluorescence microscopy revealed the time-dependent changes in intracellular localization of the PS which were related to the phototoxicity. The results show that PP(Ser)2Arg2 may be a potential PS for PDT.
TL;DR: The aim of this study was to find the best conditions for cell killing using ALA to temporarily increase the concentration of Ppix in two cell lines and it was shown that a considerable efflux of synthesized PpIX occurs.
Abstract: One of the major advances in PDT is the use of 5-aminolevulinic acid (ALA) to induce the production of an endogenous photosensitizer inside the cells using intracellular enzymatic pathways. ALA is the first intermediate in heme biosynthesis and a precursor of the protoporphyrin IX (PpIX). When activated by light, this efficient photosensitizer accumulated in the target cells can produce cytotoxicity. The aim of this study was to find the best conditions for cell killing using ALA to temporarily increase the concentration of PpIX in two cell lines. It was shown that a considerable efflux of synthesized PpIX occurs. Since this efflux is time-dependent, it is essential to know the optimum time for irradiation after ALA administration. So, the efflux of PpIX from the cells is an important parameter to be considered for ALA-PDT dosimetry.