Showing papers on "Protoporphyrin IX published in 2016"
TL;DR: Because PPIX is a hydrophobic molecule, its disposition is by hepatic rather than renal excretion, and large amounts of PPIX are toxic to the liver and can cause cholestatic liver injury.
Abstract: Protoporphyrin IX (PPIX) is ubiquitously present in all living cells in small amounts as a precursor of heme. PPIX has some biologic functions of its own, and PPIX-based strategies have been used for cancer diagnosis and treatment (the good). PPIX serves as the substrate for ferrochelatase, the final enzyme in heme biosynthesis, and its homeostasis is tightly regulated during heme synthesis. Accumulation of PPIX in human porphyrias can cause skin photosensitivity, biliary stones, hepatobiliary damage, and even liver failure (the bad and the ugly). In this work, we review the mechanisms that are associated with the broad aspects of PPIX. Because PPIX is a hydrophobic molecule, its disposition is by hepatic rather than renal excretion. Large amounts of PPIX are toxic to the liver and can cause cholestatic liver injury. Application of PPIX in cancer diagnosis and treatment is based on its photodynamic effects.
144 citations
TL;DR: This study elucidates the underlying molecular transduction mechanism and identifies potential targets of therapy, and suggests a therapeutic use of the pertinent antagonists in clinical development.
Abstract: Photosensitization, an exaggerated sensitivity to harmless light, occurs genetically in rare diseases, such as porphyrias, and in photodynamic therapy where short-term toxicity is intended. A common feature is the experience of pain from bright light. In human subjects, skin exposure to 405 nm light induced moderate pain, which was intensified by pretreatment with aminolevulinic acid. In heterologous expression systems and cultured sensory neurons, exposure to blue light activated TRPA1 and, to a lesser extent, TRPV1 channels in the absence of additional photosensitization. Pretreatment with aminolevulinic acid or with protoporphyrin IX dramatically increased the light sensitivity of both TRPA1 and TRPV1 via generation of reactive oxygen species. Artificial lipid bilayers equipped with purified human TRPA1 showed substantial single-channel activity only in the presence of protoporphyrin IX and blue light. Photosensitivity and photosensitization could be demonstrated in freshly isolated mouse tissues and led to TRP channel-dependent release of proinflammatory neuropeptides upon illumination. With antagonists in clinical development, these findings may help to alleviate pain during photodynamic therapy and also allow for disease modification in porphyria patients. SIGNIFICANCE STATEMENT Cutaneous porphyria patients suffer from burning pain upon exposure to sunlight and other patients undergoing photodynamic therapy experience similar pain, which can limit the therapeutic efforts. This study elucidates the underlying molecular transduction mechanism and identifies potential targets of therapy. Ultraviolet and blue light generates singlet oxygen, which oxidizes and activates the ion channels TRPA1 and TRPV1. The disease and the therapeutic options could be reproduced in models ranging from isolated ion channels to human subjects, applying protoporphyrin IX or its precursor aminolevulinic acid. There is an unmet medical need, and our results suggest a therapeutic use of the pertinent antagonists in clinical development.
65 citations
TL;DR: Performing a photodynamic diagnosis (PDD) and photodynamic therapy (PDT) using 5-ALA have given rise to a new strategy for tumor diagnosis and therapy.
60 citations
TL;DR: In this paper, a two-dimensional covalent organic polymer (COP) containing fully conjugated multiple porphyrin macrocycles with sulfonic acid side groups was synthesized.
Abstract: Photodynamic therapy (PDT) is attractive for treatment of various cancers, with a high selectivity, minimal long-term effect, and excellent cosmetic appeal. Well water-dispersive photosensitizers with strong optical absorption within the tissue transparency window (700–1000 nm) are needed for efficient PDT. However, clinically used PDT agents based on oligomeric porphyrin units (e.g., protoporphyrin IX) are effective at 532 nm irradiation only. Herein, we synthesized a two-dimensional covalent organic polymer (COP) containing fully conjugated multiple porphyrin macrocycles with sulfonic acid side groups. The resultant COP-P-SO3H is well water-dispersive, showing strong optical absorption within the desired therapeutic window and a high quantum yield of reactive oxygen species, especially singlet oxygen (1O2), for efficiently killing tumor cells upon near-infrared light irradiation. Our first-principles calculations revealed that the observed high yield 1O2 resulted from the unique side-on parallel diatomi...
45 citations
TL;DR: It is demonstrated that cancer cells exhibited dormancy in a cell density-dependent manner not only in spheroids but also in 2D culture, and ALA-PDT would be an effective approach for dormant cancer cells and can be enhanced by combining with a cell-growth inhibitor.
Abstract: Photodynamic therapy (PDT) and diagnosis (PDD) using 5-aminolevulinic acid (ALA) to drive the production of an intracellular photosensitizer, protoporphyrin IX (PpIX), are in common clinical use. However, the tendency to accumulate PpIX is not well understood. Patients with cancer can develop recurrent metastatic disease with latency periods. This pause can be explained by cancer dormancy. Here we created uniformly sized PC-3 prostate cancer spheroids using a 3D culture plate (EZSPHERE). We demonstrated that cancer cells exhibited dormancy in a cell density-dependent manner not only in spheroids but also in 2D culture. Dormant cancer cells accumulated high PpIX levels and were sensitive to ALA-PDT. In dormant cancer cells, transporter expressions of PEPT1, ALA importer, and ABCB6, an intermediate porphyrin transporter, were upregulated and that of ABCG2, a PpIX exporter, was downregulated. PpIX accumulation and ALA-PDT cytotoxicity were enhanced by G0/G1-phase arrestors in non-dormant cancer cells. Our results demonstrate that ALA-PDT would be an effective approach for dormant cancer cells and can be enhanced by combining with a cell-growth inhibitor.
42 citations
TL;DR: The assessment of implicit photodynamic dose was a more significant predictor of efficacy for ALA-PDT skin cancer treatments than prescription of an enhanced treatment strategy, likely because of high individual variation in response between subjects.
Abstract: Aminolevulinic acid (ALA)-based photodynamic therapy (PDT) provides selective uptake and conversion of ALA into protoporphyrin IX (PpIX) in actinic keratosis and squamous cell carcinoma, yet large response variations in effect are common between individuals. The aim of this study was to compare pre-treatment strategies that increase the therapeutic effect, including fractionated light delivery during PDT (fPDT) and use of iron chelator desferrioxamine (DFO), separately and combined. Optical measurements of fluorescence were used to quantify PpIX produced, and the total amount of PpIX photobleached as an implicit measure of the photodynamic dose. In addition, measurements of white light reflectance were used to quantify changes in vascular physiology throughout the PDT treatment. fPDT produced both a replenishment of PpIX and vascular re-oxygenation during a 2 h dark interval between the first and second PDT light fractions. The absolute photodynamic dose was increased 57% by fPDT, DFO and their combination, as compared with PDT group (from 0.7 to 1.1). Despite that light fractionation increased oedema and scab formation during the week after treatment, no significant difference in long-term survival has been observed between treatment groups. However, outcomes stratified on the basis of measured photodynamic dose showed a significant difference in long-term survival. The assessment of implicit photodynamic dose was a more significant predictor of efficacy for ALA-PDT skin cancer treatments than prescription of an enhanced treatment strategy, likely because of high individual variation in response between subjects.
40 citations
TL;DR: In this article, photosynthetically active chlorophyll molecules were biosynthesized from 5-aminolevulinic acid through protoporphyrin IX, and the multistep enzymatic transformations were well investigated.
Abstract: Photosynthetically active chlorophyll molecules are biosynthesized from 5-aminolevulinic acid through protoporphyrin IX. The multistep enzymatic transformations were well investigated, but their pa...
35 citations
TL;DR: The current fluorescence-guided malignant gliomas surgery was started 15 years ago and had become a standard of care in many countries and has a very high specificity but still lower sensibility, particularly regarding the zones with poor tumoral infiltration.
Abstract: The current first-line treatment of malignant gliomas consists in surgical resection (if possible) as large as possible. The existing tools don’t permit to identify the limits of tumor infiltration, which goes beyond the zone of contrast enhancement on MRI. The fluorescence-guided malignant gliomas surgery was started 15 years ago and had become a standard of care in many countries. The technique is based on fluorescent molecule revelation using the filters, positioned within the surgical microscope. The fluorophore, protoporphyrin IX (PpIX), is converted in tumoral cells from 5-aminolevulinic acid (5-ALA), given orally before surgery. Many studies have shown that the ratio of gross total resections was higher if the fluorescence technique was used. The fluorescence signal intensity is correlated to the cell density and the PpIX concentration. The current method has a very high specificity but still lower sensibility, particularly regarding the zones with poor tumoral infiltration. This book reviews the principles of the technique and the results (extent of resection and survival).
34 citations
TL;DR: It is reported for the first time that glioblastoma stem-like cells accumulate PPIX when subjected to5-aminolaevulinic acid and are sensitive to 5-aminolevulinc acid based photodynamic therapy.
Abstract: Glioblastoma (GBM) is the most frequent and lethal primary brain tumor in adults. Despite multimodal therapy combining resection, radio- and alkylating chemotherapy, disease recurrence is universal and prognosis of patients is poor. Glioblastoma stem-like cells (GSC), which can be grown as neurospheres from primary tumors in vitro, appear to be resistant to the established therapies and are suspected to be the driving force for disease recurrence. Thus, efficacy of emerging therapies may depend on targeting GSC. 5-aminolaevulinic acid-mediated photodynamic therapy (5-ALA/PDT) is a promising therapeutic approach in GBM. It utilizes the selective accumulation of the photosensitizer protoporphyrin IX (PPIX) in GBM cells after application of 5-ALA. When exposed to laser light of 635nm wavelength, PPIX initiates a photochemical reaction resulting in the generation of reactive oxygen species, which kill the tumor cells. Whether GSC accumulate PPIX and are sensitive to 5-ALA/PDT is currently unknown. Therefore, human GSC were derived from primary tumors and grown as neurospheres under serum free conditions. When subjected to exogenous 5-ALA, a dose- and time-dependent accumulation of PPIX in GSC was observed by flow cytometry, which varied between individual GSC preparations. Subsequent exposure to laser light of 635nm wavelength substantially killed GSC, whereas treatment with 5-ALA or exposure to laser light only had no effect. LD50 values differed between GSC preparations, but were negatively correlated with PPIX accumulation in GSC. In summary, we report for the first time that glioblastoma stem-like cells accumulate PPIX when subjected to 5-aminolaevulinic acid and are sensitive to 5-aminolaevulinc acid based photodynamic therapy.
33 citations
TL;DR: In vivo animal studies reveal that protoporphyrin IX fluorescence is strongly correlated with both MRI and histological staining, confirming that the fluorescence signals are highly specific to tumor cells, and ex vivo spectroscopic studies of excised brain tissues demonstrate that the hand-held spectroscopy device is capable of detecting diffuse tumor margins with low fluorescence contrast that are not detectable with current systems in the operating room.
Abstract: Intraoperative cancer imaging and fluorescence-guided surgery have attracted considerable interest because fluorescence signals can provide real-time guidance to assist a surgeon in differentiating cancerous and normal tissues. Recent advances have led to the clinical use of a natural fluorophore called protoporphyrin IX (PpIX) for image-guided surgical resection of high-grade brain tumors (glioblastomas). However, traditional fluorescence imaging methods have only limited detection sensitivity and identification accuracy and are unable to detect low-grade or diffuse infiltrating gliomas (DIGs). Here we report a low-cost hand-held spectroscopic device that is capable of ultrasensitive detection of protoporphyrin IX fluorescence in vivo, together with intraoperative spectroscopic data obtained from both animal xenografts and human brain tumor specimens. The results indicate that intraoperative spectroscopy is at least 3 orders of magnitude more sensitive than the current surgical microscopes, allowing ultr...
33 citations
TL;DR: Results show that purified GUN4 and oxidatively damaged ChlH increase the rate of PPIX-generated singlet oxygen production in the light, by a factor of 5 and 10, respectively, when compared with PPIX alone.
TL;DR: The results show that the nanocarrier developed is suitable for use in topical PDT with PpIX and did not cause skin irritation after application for two consecutive days.
TL;DR: The results of this study demonstrated the effective application of Ppix-loaded MLs for in vitro photodynamic therapy at nanomolar concentrations and indicated that an LED light source provided sufficient energy to stimulate the PpIX molecules.
Journal Article•
TL;DR: Findings suggest that mitochondria are one of the target organelles of 5-ALA-SDT, which showed an antitumor effect in mouse mammary tumor EMT6 cells through oxidation of the mitochondrial membrane via ROS production.
Abstract: Background/aim 5-Aminolevulinic acid (5-ALA), a precursor of protoporphyrin IX (PpIX), is now used for photodynamic therapy (PDT) of pre-cancers of the skin and photodynamic diagnosis (PDD) of brain tumors. Sonodynamic therapy (SDT) of cancers with ultrasound has been studied using 5-ALA as a sonosensitizer. In this article, we evaluated the sonosensitizing activity and mode of action of 5-ALA/PpIX by using mouse mammary tumor EMT6 cells. Results 5-ALA-SDT showed significant antitumor effects toward EMT6 cells in vitro and in vivo. The fluorescence of MitoSOX Red, an indicator specific for mitochondrial superoxide, was significantly increased by 5-ALA-SDT. Moreover, the fluorescence derived from JC-1, an indicator of mitochondrial membrane potential, was also significantly increased by 5-ALA-SDT. These findings suggest that mitochondria are one of the target organelles of 5-ALA-SDT. PpIX enhanced reactive oxygen species (ROS) production from tert-butyl hydroperoxide (tBHP), suggesting that PpIX might stabilize or promote ROS generation from tBHP. Conclusion 5-ALA-SDT showed an antitumor effect in mouse mammary tumor EMT6 cells through oxidation of the mitochondrial membrane via ROS production.
TL;DR: The expression of ferrochelatase has a strong correlation in protoporphyrin IX accumulation with photodynamic detection of bladder cancer, and was significant factors to predict positive 5-aminolevulinic acid-induced fluorescent cytology.
TL;DR: This study illustrated that chronic treatment with INH causes PPIX accumulation in mouse liver in part through ALAS1 induction and FECH downregulation, and highlights that drugs can disrupt the metabolic pathways of endobiotics and increase the risk of liver damage.
Abstract: Isoniazid (INH) can cause hepatotoxicity. In addition, INH is contraindicated in patients suffering from porphyrias. Our metabolomic analysis revealed that chronic treatment with INH in mice causes a hepatic accumulation of protoporphyrin IX (PPIX). PPIX is an intermediate in the heme biosynthesis pathway, and it is also known as a hepatotoxin. We further found that INH induces delta-aminolevulinate synthase 1 (ALAS1), the rate-limiting enzyme in heme biosynthesis. We also found that INH downregulates ferrochelatase (FECH), the enzyme that converts PPIX to heme. In summary, this study illustrated that chronic treatment with INH causes PPIX accumulation in mouse liver in part through ALAS1 induction and FECH downregulation. This study also highlights that drugs can disrupt the metabolic pathways of endobiotics and increase the risk of liver damage.
TL;DR: A general method for the detection of metal ions and bioactive molecules but also presents a way to investigate their synergistic functions in the regulation of various biological processes to improve the imaging and therapeutic efficacy of PPIX and 5-ALA.
Abstract: Parallel monitoring of K+ and protoporphyrin IX (PPIX) is of vital importance because they are not only involved in a variety of biological processes but also closely linked to each other in numerous cellular pathways. However, there are currently no existing methods that can meet the requirements for parallel and in vivo detection of K+ and PPIX in living organisms. Herein, we demonstrated a functional nucleic acid (FNA)-based technique for parallel monitoring of K+ and PPIX in living animals. Specifically, the selected G-rich FNA probe was selectively induced to form a parallel G-quadruplex by K+. The parallel G-quadruplex then remarkably enhanced the fluorescence of PPIX. Thus, by modulating the fluorescence “turn on” with the G-quadruplex and K+/PPIX, both K+ and PPIX could be detected. After validating the developed method for selective and sensitive detection of K+ and PPIX in vitro, their dynamic changes in living organisms (i.e., living brains and tumors) following various physiological and pathol...
TL;DR: These compounds display an excellent stability under acidic, basic and physiological conditions and display reduced acute toxicity compared to 5-ALA-Hex with superior dose response profiles of protoporphyrin IX synthesis and fluorescence intensity in human glioblastoma cells in vitro.
TL;DR: A human ex vivo model suitable to explore drug permeation in human skin is developed and significantly higher PpIX concentrations than the 20% ALA formulation are triggered, indicating that clinical efficacy with BF-200 ALA may be higher.
TL;DR: Systemic administration of the heme precursors PP‐IX or ALA+DFO into zebrafish larvae provides a new model of acute protoporphyria with consequent hepatocyte protein aggregation and proteotoxic multiorganelle alterations and stress.
Abstract: Protoporphyria is a metabolic disease that causes excess production of protoporphyrin IX (PP-IX), the final biosynthetic precursor to heme. Hepatic PP-IX accumulation may lead to end-stage liver disease. We tested the hypothesis that systemic administration of porphyrin precursors to zebrafish larvae results in protoporphyrin accumulation and a reproducible nongenetic porphyria model. Retro-orbital infusion of PP-IX or the iron chelator deferoxamine mesylate (DFO), with the first committed heme precursor α-aminolevulinic acid (ALA), generates high levels of PP-IX in zebrafish larvae. Exogenously infused or endogenously produced PP-IX accumulates preferentially in the liver of zebrafish larvae and peaks 1 to 3 d after infusion. Similar to patients with protoporphyria, PP-IX is excreted through the biliary system. Porphyrin accumulation in zebrafish liver causes multiorganelle protein aggregation as determined by mass spectrometry and immunoblotting. Endoplasmic reticulum stress and induction of autophagy were noted in zebrafish larvae and corroborated in 2 mouse models of protoporphyria. Furthermore, electron microscopy of zebrafish livers from larvae administered ALA + DFO showed hepatocyte autophagosomes, nuclear membrane ruffling, and porphyrin-containing vacuoles with endoplasmic reticulum distortion. In conclusion, systemic administration of the heme precursors PP-IX or ALA + DFO into zebrafish larvae provides a new model of acute protoporphyria with consequent hepatocyte protein aggregation and proteotoxic multiorganelle alterations and stress.-Elenbaas, J. S., Maitra, D., Liu, Y., Lentz, S. I., Nelson, B., Hoenerhoff, M. J., Shavit, J. A., Omary, M. B. A precursor-inducible zebrafish model of acute protoporphyria with hepatic protein aggregation and multiorganelle stress.
TL;DR: The aPDT with MB significantly reduces (but does not sterilize) the amount of CFU after 10min of irradiation, and it is not dose-dependent, and the lack of effect of the protoporphyrin is likely because the negative charges of the proteoglycans present in the extracellular matrix repel the negativeCharges of the PS, thus preventing its diffusion in the cells.
TL;DR: Testing the efficacy of aminolevulinic acid-mediated photodynamic therapy against the human osteosarcoma cell line MG-63 found this therapy could represent a less invasive, but effective, treatment for this disease.
Abstract: Objective: The aim of this study was to test the efficacy of aminolevulinic acid-mediated photodynamic therapy (PDT) against the human osteosarcoma cell line MG-63. Background data: Osteosarcoma is the most common type of primary malignant bone tumor diagnosed in the United States among adolescents and children. Treatments for osteosarcoma often result in diminished limb use or amputation. Because ALA-mediated PDT exhibits dual specificity in the context of tumor killing, this therapy could represent a less invasive, but effective, treatment for this disease. Materials and methods: To assess ALA dark toxicity in MG-63 cells, cells were incubated with varying concentrations of ALA, and cell viability was determined by crystal violet assay. Protoporphyrin IX (PpIX) accumulation was assessed subsequent to ALA incubation at various concentrations using spectrofluorometry. Cell death subsequent to ALA-PDT was determined by illuminating cells at a wavelength of 635 nm at various light intensities subse...
TL;DR: It is demonstrated that transfection of NeuT (a mutated Her2/Neu) oncogene in MCF10A human breast epithelial cells significantly enhanced ALA-induced PpIX fluorescence by elevating some enzymes involved in Ppix biosynthesis, and this results support the use of ALA for imaging and photodynamic targeting Her2 /Neu-positive tumors.
Abstract: // Xue Yang 1 , Pratheeba Palasuberniam 1 , Kenneth A. Myers 2 , Chenguang Wang 3 , Bin Chen 1 1 Department of Pharmaceutical Sciences, Philadelphia College of Pharmacy, University of The Sciences, Philadelphia, Pennsylvania, USA 2 Department of Biological Sciences, Misher College of Arts and Sciences, University of The Sciences, Philadelphia, Pennsylvania, USA 3 Key Laboratory of Tianjin Radiation and Molecular Nuclear Medicine, Institute of Radiation Medicine, Peking Union Medical College and Chinese Academy of Medical Sciences, Tianjin, China Correspondence to: Bin Chen, email: b.chen@usciences.edu Keywords: human epidermal growth receptor 2 (Her2), aminolevulinic acid (ALA), protoporphyrin IX (PpIX), photodynamic therapy (PDT), heme biosynthesis Received: April 04, 2016 Accepted: July 19, 2016 Published: August 04, 2016 ABSTRACT Enhanced protoporphyrin IX (PpIX) production in tumors derived from the administration of 5-aminolevulinic acid (ALA) enables the use of ALA as a prodrug for photodynamic therapy (PDT) and fluorescence-guided tumor resection. Although ALA has been successfully used in the clinic, the mechanism underlying enhanced ALA-induced PpIX production in tumors is not well understood. Human epidermal growth receptor 2 (Her2, Neu, ErbB2) is a driver oncogene in human cancers, particularly breast cancers. Here we showed that, in addition to activating Her2/Neu cell signaling, inducing epithelial-mesenchymal transition and upregulating glycolytic enzymes, transfection of NeuT (a mutated Her2/Neu) oncogene in MCF10A human breast epithelial cells significantly enhanced ALA-induced PpIX fluorescence by elevating some enzymes involved in PpIX biosynthesis. Furthermore, NeuT-transformed and vector control cells exhibited drastic differences in the intracellular localization of PpIX, either produced endogenously from ALA or applied exogenously. In vector control cells, PpIX displayed a cell contact-dependent membrane localization at high cell densities and increased mitochondrial localization at low cell densities. In contrast, no predominant membrane localization of PpIX was observed in NeuT cells and ALA-induced PpIX showed a consistent mitochondrial localization regardless of cell density. PDT with ALA caused significantly more decrease in cell viability in NeuT cells than in vector cells. Our data demonstrate that NeuT oncogene transformation enhanced ALA-induced PpIX production and altered PpIX intracellular localization, rendering NeuT-transformed cells increased response to ALA-mediated PDT. These results support the use of ALA for imaging and photodynamic targeting Her2/Neu-positive tumors.
TL;DR: It is suggested that FC1 contributes to mitochondrial heme synthesis in mitochondria through its role in ferrochelatase function in plant organs.
Abstract: In plants, two genes encode ferrochelatase (FC), which catalyzes iron chelation into protoporphyrin IX at the final step of heme biosynthesis. FERROCHELATASE1 (FC1) is continuously, but weakly expressed in roots and leaves, while FC2 is dominantly active in leaves. As a continuation of previous studies on the physiological consequences of FC2 inactivation in tobacco, we aimed to assign FC1 function in plant organs. While reduced FC2 expression leads to protoporphyrin IX accumulation in leaves, FC1 down-regulation and overproduction caused reduced and elevated FC activity in root tissue, respectively, but were not associated with changes in macroscopic phenotype, plant development or leaf pigmentation. In contrast to the lower heme content resulting from a deficiency of the dominant FC2 expression in leaves, a reduction of FC1 in roots and leaves does not significantly disturb heme accumulation. The FC1 overexpression was used for an additional approach to re-examine FC activity in mitochondria. Transgenic FC1 protein was immunologically shown to be present in mitochondria. Although matching only a small portion of total cellular FC activity, the mitochondrial FC activity in a FC1 overexpressor line increased 5-fold in comparison with wild-type mitochondria. Thus, it is suggested that FC1 contributes to mitochondrial heme synthesis.
TL;DR: The selective photodamage of MUC1 expressing HeLa cervical cancer cells through ROS generation in the presence of the QD-PpIX FRET probe upon irradiation is successfully demonstrated.
Abstract: Non-targeted photosensitizers lack selectivity that undermines the potential use of photodynamic therapy (PDT). Herein, we report the DNA mediated assembly of a ZnSe/ZnS quantum dot (QD)–photosensitizer (PS)–Mucin 1(MUC1) aptamer conjugate for targeting the MUC1 cancer biomarker and simultaneous generation of reactive oxygen species (ROS). A photosensitizer, protoporphyrin IX (PpIX), was conjugated to a single stranded DNA and self-assembled to a complementary strand that was conjugated to a QD and harboring a MUC1 aptamer sequence. A multistep fluorescence resonance energy transfer (FRET) is shown that involves the QD, PpIX and covalently linked CF™ 633 amine dye (CF dye) to the MUC1 peptide that tracks the potency of the aptamer to attach itself with the MUC1 peptide. Since the absorption spectra of the CF dye overlap with the emission spectra of PpIX, the former acts as an acceptor to PpIX forming a second FRET pair when the dye labeled MUC1 binds to the aptamer. The binding of the QD–PpIX nanoassemblies with MUC1 through the aptamer was further confirmed by gel electrophoresis and circular dichroism studies. The selective photodamage of MUC1 expressing HeLa cervical cancer cells through ROS generation in the presence of the QD–PpIX FRET probe upon irradiation is successfully demonstrated.
TL;DR: The investigation on the binding mode of protogen IX in the Nicotiana tabacum PPO (mtPPO) is carried out by performing a computational docking followed by molecular simulations, quantum mechanics calculations, and an integrated analysis, and the proposed binding mode was consistent with experimental studies.
Abstract: Protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) catalyzes the oxidation of protoporphyrinogen IX (protogen IX) to protoporphyrin IX (proto IX) in the haem/chlorophyll biosynthetic pathway. Although extensive studies of PPO have already afforded many insights into its biological function and its significance to agriculture and medicine, details of the enzymatic mechanism as well as the nature of the specific amino acids involved in substrate binding still remain largely unknown due to the lack of structural information about protogen IX binding to PPO. In this study, we carried out a detailed and systematic investigation on the binding mode of protogen IX in the Nicotiana tabacum PPO (mtPPO) by performing a computational docking followed by molecular simulations, quantum mechanics calculations, and an integrated analysis. The proposed binding mode was consistent with experimental studies, and several potential key residues that have not been investigated in previous studies, such as Thr70, Arg233, Ser235, Ser474 and Lys477, were identified. In addition, we compared the binding modes of protogen IX in mtPPO and Homo sapiens PPO, and found their differences. Considering the low sequence identity and the differences of biochemical properties among the PPOs from various species, the investigation could provide a valuable basis for the design of PPO inhibitors with high potency and species-selectivity.
TL;DR: Using absorption and fluorescence spectroscopy, it has been demonstrated that PpIX forms complexes with HSA and creates a strong ‘sandwich-type’ complex between its highly conjugated porphine system and aromatic side chains of tryptophan and tyrosine.
Abstract: Human serum albumin (HSA) is the main plasma protein responsible for a distribution of drugs in the human circulatory system. The binding to HSA is one of the factors that determines both the pharmacological actions and the side effects of drugs. The derivative of heme, protoporphyrin IX (PpIX), is a hydrophobic photosensitizer widely used in photodynamic diagnosis and therapy of various malignant disorders. Using absorption and fluorescence spectroscopy, it has been demonstrated that PpIX forms complexes with HSA. Its binding sites in the tertiary structure of HSA were found in the subdomains IB and IIA. PpIX binds to HSA in one class of binding sites with the association constant of 1.68 × 105 M−1 and 2.30 × 105 M−1 for an excitation at wavelength λex = 280 nm and 295 nm, respectively. The binding interactions between HSA and PpIX have been studied by means of molecular docking simulation using the CLC Drug Discovery Workbench (CLC DDWB) computer program. PpIX creates a strong ‘sandwich-type’ complex between its highly conjugated porphine system and aromatic side chains of tryptophan and tyrosine. In summary, fluorescent studies on binding interactions between HSA and PpIX have been confirmed by the results of computer simulation.
TL;DR: It is demonstrated that Ppix-mediated SDT could induce apoptosis on HL-60 cells, suggesting that apoptosis is an important mechanism of cell death induced by PpIX- mediated SDT.
Abstract: Sonodynamic therapy (SDT) is expected to be a novel therapeutic strategy for tumor. The protoporphyrin IX disodium salt (PpIX), a photosensitizer, can be activated by ultrasound. The present study aims to investigate apoptosis of HL-60 cells induced by PpIX-mediated SDT. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was adopted to examine cell toxicity. Apoptosis was detected using Annexin V-PE/7-amino-actinomycin D (7-AAD) double staining. Detection of apoptotic bodies was examined by Hoechst33342 (HO) staining. Western blotting was used to analyze the protein of caspase-3 and poly ADP-ribose polymerase (PARP). Intracellular reactive oxygen species (ROS) was detected by a flow cytometer after exposures. Compared with PpIX alone and ultrasound alone groups, the synergistic cytotoxicity of PpIX plus ultrasound were significantly boosted. In addition, as determined by Annexin V-PE/7-AAD staining, SDT significantly induced HL-60 cell apoptosis, the obvious nuclear condensation was also found with HO staining at 4 hours post-SDT treatment. Furthermore, Western blotting showed visible enhancement of caspase-3 and PARP cleavage in this process. Besides, intracellular ROS production was significantly enhanced after SDT. Our findings demonstrate that PpIX-mediated SDT could induce apoptosis on HL-60 cells, suggesting that apoptosis is an important mechanism of cell death induced by PpIX-mediated SDT.
TL;DR: This study discovered eEF1A1 as a novel photosensitizer binding protein, which may play an essential role in the enrichment of ALA-induced PpIX in cancer cells during PDT, and suggested e EF1A 1 as a molecular marker to predict the selectivity and efficiency of 5-ALA based PDT in cancer therapy.
Abstract: Photodynamic therapy (PDT) with protoporphyrin IX (PpIX), which is endogenously derived from 5-aminolevulinic acid (5-ALA) or its derivatives, is a promising modality for the treatment of both pre-malignant and malignant lesions. However, the mechanisms of how ALA-induced PpIX selectively accumulated in the tumors are not fully elucidated. Here we discovered that eukaryotic translation elongation factor 1 alpha 1 (eEF1A1) interacted with PpIX (with an affinity constant of 2.96 × 106 M−1). Microscopy imaging showed that ALA-induced PpIX was co-localized with eEF1A1 in cancer cells. eEF1A1 was found to enrich ALA-induced PpIX in cells by competitively blocking the downstream bioavailability of PpIX. Taken together, our study discovered eEF1A1 as a novel photosensitizer binding protein, which may play an essential role in the enrichment of ALA-induced PpIX in cancer cells during PDT. These suggested eEF1A1 as a molecular marker to predict the selectivity and efficiency of 5-ALA based PDT in cancer therapy.
TL;DR: It is demonstrated, for the first time, that HemY of S. aureus is able to generate both protoporphyrin IX and coproporphyr in III, and that the terminal enzyme of this pathway, HemQ, can stimulate the generation of protoporalin IX (but not copropoiryrin III).
Abstract: Bacteria require a haem biosynthetic pathway for the assembly of a variety of protein complexes including cytochromes, peroxidases, globins, and catalase. Haem is synthesised via a series of tetrapyrrole intermediates including non-metallated porphyrins such as protoporphyrin IX, which is well-known to generate reactive oxygen species (ROS) in the presence of light and oxygen. Staphylococcus aureus has an ancient haem biosynthetic pathway that proceeds via the formation of coproporphyrin III, a less reactive porphyrin. Herein, we demonstrate for the first time that HemY of S. aureus is able to generate both protoporphyrin IX and coproporphyrin III, and that the terminal enzyme of this pathway, HemQ, can stimulate the generation of protoporphyrin IX (but not coproporphyrin III). Assays with hydrogen peroxide, horseradish peroxidase, superoxide dismutase, and catalase confirm that this stimulatory effect is mediated by superoxide. Structural modelling reveals that HemQ enzymes do not possess the structural attributes that are common to peroxidases that form compound I [FeIV=O]+, which taken together with the superoxide data leaves Fenton chemistry as a likely route for the superoxide-mediated stimulation of protoporphyrinogen IX oxidase activity of HemY. This generation of toxic free radicals could explain why HemQ enzymes have not been identified in organisms that synthesise haem via the classical protoporphyrin IX pathway. This work has implications for the divergent evolution of haem biosynthesis in ancestral microorganisms and provides new structural and mechanistic insights into a recently discovered oxidative decarboxylase reaction.