chlorophyll-a and pheophorbide-a

Chlorophyll breakdown products are usually studied for their antioxidant and anti-inflammatory activities. The chlorophyll derivative Pheophorbide

Topics: Antineoplastic Agents; Cell Line, Tumor; Chlorophyll; Humans

Pheophorbide a is a clorophyll catabolite that recently has drawn the attention of several investigators for its potential in photodynamic therapy. In this review we summarize its photophysical properties, phototoxicity, cellular localization, biodistribution and PDT activity as a free or conjugated molecule.

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carrier Proteins; Cell Survival; Chlorophyll; Humans; Neoplasms; Photochemotherapy; Photosensitizing Agents

Chlorophyll breakdown is an important catabolic process of leaf senescence and fruit ripening. Structure elucidation of colorless linear tetrapyrroles as (final) breakdown products of chlorophyll was crucial for the recent delineation of a chlorophyll breakdown pathway which is highly conserved in land plants. Pheophorbide a oxygenase is the key enzyme responsible for opening of the chlorin macrocycle of pheophorbide a characteristic to all further breakdown products. Degradation of chlorophyll was rationalized by the need of a senescing cell to detoxify the potentially phototoxic pigment, yet recent investigations in leaves and fruits indicate that chlorophyll catabolites could have physiological roles. This review updates structural information of chlorophyll catabolites and the biochemical reactions involved in their formation, and discusses the significance of chlorophyll breakdown. This article is part of a Special Issue entitled: Regulation of Electron Transport in Chloroplasts.

Topics: Chlorophyll; Chlorophyll A; Oxygenases; Plants

Attempts have been made to develop dye-sensitized solar cells based on the principles and materials of photosynthesis: We first tested photosynthetic pigments, carotenoids (Cars), chlorophylls (Chls) and their derivatives, to find sensitizers showing reasonable performance (photocurrent and conversion efficiency). We then tried to introduce the principles of photosynthesis, including electron transfer and energy transfer from Car to Phe a. Also, we tried co-sensitization using the pheophorbide (Phe) a and Chl c(2) pair which further enhanced the performance of the component sensitizers as follows: J(sc) = 9.0 + 13.8 --> 14.0 mA cm(-2) and eta = 3.4 + 4.6 --> 5.4%.

Topics: Carotenoids; Chlorophyll; Coloring Agents; Energy Transfer; Photosynthesis; Polyenes; Quantum Theory; Solar Energy

The cooperative binding of a novel water-soluble cationic derivative of pheophorbide-a (CatPheo-a) to inorganic polyphosphate (PPS) in buffered aqueous solutions was studied by means of polarized fluorescence spectroscopy in a wide range of molar phosphate-to-dye ratios (P/D). Under low P/D values, CatPheo-a forms extended stacking associates on the PPS matrix, while under high P/D the dye binds to PPS in the dimer form. The CatPheo-a self-association is accompanied by 40-fold dye fluorescence quenching and a substantial increase in the fluorescence polarization degree. The fluorescent titration data were used for determination of cooperative binding parameters by Schwarz's method.

Topics: Absorption; Cations; Chlorophyll; Dimerization; Fluorescent Dyes; Humans; Kinetics; Microscopy, Fluorescence; Models, Chemical; Photochemistry; Polymers; Polyphosphates; Solubility; Static Electricity

Topics: Animals; Antithyroid Agents; Bacteria; Chlorophyll; Female; Glycine max; Heart; Humans; Intestines; Iodine; Lipid Metabolism; Male; Mice; Oxidation-Reduction; Photosensitivity Disorders; Rats; Vitamins

Lipid-porphyrin conjugates are considered nowadays as promising building blocks for the conception of drug delivery systems with multifunctional properties such as photothermal therapy (PTT), photodynamic therapy (PDT), phototriggerable release, photoacoustic and fluorescence imaging. For this aim, we have recently synthesized a new lipid-porphyrin conjugate named PhLSM. This was obtained by coupling pheophorbide-a (Pheo-a), a photosensitizer derived from chlorophyll-a, to egg lyso-sphingomyelin. The pure PhLSMs were able to self-assemble into vesicle-like structures that were however not stable and formed aggregates with undefined structures due to the mismatch between the length of the alkyl chain in sn-1 position and the adjacent porphyrin. Herein, stable PhLSMs lipid bilayers were achieved by mixing PhLSMs with cholesterol which exhibits a complementary packing parameter. The interfacial behavior as well as the fine structures of their equimolar mixture was studied at the air/buffer interface by the mean of Langmuir balance and x-ray reflectomerty (XRR) respectively. Our XRR analysis unraveled the monolayer thickening and the increase in the lateral ordering of PhLSM molecules. Interestingly, we could prepare stable vesicles with this mixture that encapsulate hydrophilic fluorescent probe. The light-triggered release kinetics and the photothermal conversion were studied. Moreover, the obtained vesicles were photo-triggerable and allowed the release of an encapsulated cargo in an ON-OFF fashion.

Topics: Chlorophyll; Cholesterol; Drug Delivery Systems; Humans; Hydrophobic and Hydrophilic Interactions; Kinetics; Light; Lipid Bilayers; Lipids; Liposomes; Phospholipids; Photochemotherapy; Photosensitizing Agents; Photothermal Therapy; Porphyrins

The lymphatic vascular system is crucial for maintaining tissue fluid homeostasis and immune surveillance. Promoting lymphatic function represents a new strategy to treat several diseases including lymphedema, chronic inflammation and impaired wound healing. By screening a plant extract library, a petroleum ether extract from the aerial parts of Eupatorium perfoliatum (E. perfoliatum) was found to possess lymphangiogenic properties. With the aid of HPLC activity profiling the active compound was identified as pheophorbide a. Both plant extract and pheophorbide a induced the sprouting and tube formation of human primary lymphatic endothelial cells (LECs). The proliferation of the LECs was increased upon treatment with pheophorbide a but not the E. perfoliatum extract. Treatment with the MEK1/2 inhibitor U0126 reduced the LEC sprouting activity, indicating a potential mechanism of action. These studies suggest that pheophorbide a could represent novel natural therapeutic agent to treat human lymphatic vascular insufficiencies.

Topics: Butadienes; Cell Line; Chlorophyll; Endothelial Cells; Eupatorium; Humans; Lymphangiogenesis; Lymphatic Vessels; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Nitriles; Plant Extracts

The effects of low temperature (LT) on chlorophyll (Chl) degradation in peel of apple fruit during ripening were investigated. Apples collected at commercial maturity were stored at 4 ± 0.5°C. Our data indicated that LT treatment reduced respiration rate and ethylene production and slowed down softening of apple fruit during ripening. The LT treatment delayed increase in L*, a*, and b* values and decrease in Chl content compared with controls. The LT treatment reduced hydrogen peroxide (H

Topics: Chlorophyll; Fruit; Malus; Oxygenases; Plant Proteins; Temperature

In this study, we designed photo-triggered reactive oxygen species (ROS)-generating pheophorbide A and ROS-cleavable thioketal-SN38 conjugated hyaluronan-cholesterol nanoparticles (PheoA-SN38-HC NPs). And we observed the combined therapeutic effects of PheoA-SN38-HC NPs against HEY-T30 human ovarian cancer (OC) model. Clinical Proteomic Tumor Analysis Consortium (CPTAC) data showed that the expression of cancer stem cell (CSC) markers (CD44, ALDH1A1, and CD117) is highly associated with poor clinical outcomes in OC patients. We proved that HEY-T30 cells overexpress CSC markers and much more invasive than other cancer cells. Flow cytometry (FACS) and microscopic analysis revealed the active targeting property of PheoA-SN38-HC NPs to CD44+ HEY-T30 cells. Moreover, the combination therapeutic effect of PheoA-SN38-HC NPs was clearly demonstrated against in vitro HEY-T30 cells and an in vivo xenograft mouse model. In particular, the paracrine cytotoxic effect of SN38 probably compensates the locoregional therapeutic limitation of photodynamic therapy.

Topics: Animals; Cell Line, Tumor; Chlorophyll; Female; Humans; Hyaluronic Acid; Mice; Nanoparticles; Ovarian Neoplasms; Proteomics; Reactive Oxygen Species

Photodynamic therapy (PDT) and photothermal therapy (PTT) have attracted research interest for their noninvasive nature and selective treatment of tumor tissues. They are effective through the generation of reactive oxygen species (ROS) or heat. Nevertheless, several problems, including low bioavailability and long-lasting cutaneous photosensitivity, have limited their clinical application. In this study, we reported an in situ self-assembly strategy that could improve various biological properties of the photosensitizer

Topics: Chlorophyll; Colorectal Neoplasms; Humans; Nanoparticles; Peptides; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species

The wild bitter gourd (WBG) is a commonly consumed vegetable in Asia that has antioxidant and hypoglycemic properties. The present study aimed to investigate the anti-adipogenic activities of isolated compounds from WBG on 8-day differentiated cultures of 3 T3-L1 adipocytes that were then stained with Oil Red O (ORO) or diamidino-2-phenylindole (DAPI).. ORO stains of the methanol extracts of de-seeded HM86 cultivar of WBG (WBG-M) and the ethyl acetate fractions (WBG-M-EA) showed anti-adipogenic activities against differentiated adipocytes. Two chlorophyll-degraded compounds, pheophorbide a (1) and pyropheophorbide a (2), were isolated from WBG-M-EA. Treatments with 1 (5, 10, and 20 μmol L. Both 1 and 2 showed anti-adipogenic activities in cell models. These chlorophyll-degraded compounds commonly exist in several vegetables during storage or edible seaweeds, which will provide resources for further investigations aiming to test anti-obesity in animal studies. © 2022 Society of Chemical Industry.

Topics: Animals; Antioxidants; Chlorophyll; Hypoglycemic Agents; Lactate Dehydrogenases; Lipids; Methanol; Momordica charantia; Plant Extracts

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; Biological Transport; Cell Proliferation; Chlorophyll; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Flow Cytometry; Furocoumarins; HCT116 Cells; Heterografts; High-Throughput Screening Assays; Humans; Irinotecan; Mice; Neoplasm Proteins; Neoplasms

High-efficient vectors for the co-delivery of photosensitizers and chemotherapeutics were urgently needed for the combination therapy. In this work, a redox-responsive micelle (PCL-SS-CMC-GA) was prepared for the co-delivery of doxorubicin (DOX) and pheophorbide A (PHA). Poly-ε-caprolactone was linked to carboxymethyl chitosan through a disulfide bond, which was easily broken in the reductive solution to release the payloads. The charge conversion property and glycyrrhetinic acid (GA) targeting ligand of the micelles effectively extended the average residence time (up to 18 times) in circulation and improved their intracellular uptake by HepG2 cells. The micelles exhibited an enhanced tumor accumulation and near infrared (NIR) imaging performance. More interestingly, this nanoplatform could fully exert the synergistic effect of DOX and PHA to improve the inhibition efficiency (with an inhibitory rate of 80.5 %) in vivo. With impressive photo-chemo theranostic and NIR imaging capability, PCL-SS-CMC-GA@DOX/PHA showed great potential in image-guided treatment of liver cancer.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Cell Survival; Chitosan; Chlorophyll; Doxorubicin; Drug Carriers; Drug Combinations; Drug Liberation; Drug Synergism; Female; Hep G2 Cells; Humans; Infrared Rays; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Micelles; Nanostructures; Optical Imaging; Oxidation-Reduction; Photochemotherapy; Radiation-Sensitizing Agents; Tumor Burden; Xenograft Model Antitumor Assays

In combination therapy, synergetic effects of drugs and their efficient delivery are essential. Herein, we screened 12 anticancer drugs for combination with photodynamic therapy (PDT) using pheophorbide a (Pba). On the basis of combination index (CI) values in cell viability tests, we selected tirapazamine (TPZ) and developed self-assembled gelatin nanoparticles (NPs) containing both Pba and TPZ. The resulting TPZ-Pba-NPs showed a synergetic effect to kill tumor cells because TPZ was activated under the hypoxic conditions that originated from the PDT with Pba and laser irradiation. After they were injected into tumor-bearing mice via the tail vein, TPZ-Pba-NPs showed 3.17-fold higher blood concentration and 4.12-fold higher accumulation in tumor tissue 3 and 24 h postinjection, respectively. Upon laser irradiation to tumor tissue, TPZ-Pba-NPs successfully suppressed tumor growth by efficient drug delivery and synergetic effects

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Chlorophyll; Drug Carriers; Drug Screening Assays, Antitumor; Drug Synergism; Drug Therapy; Gelatin; Light; Mice, Inbred C3H; Nanoparticles; Neoplasms; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species; Tirapazamine

Topics: Chlorophyll; Glycation End Products, Advanced; Humans; Mallotus Plant; Myanmar; Phytochemicals; Plant Leaves; Receptor for Advanced Glycation End Products

Photodynamic therapy (PDT) has been extensively explored as a promising alternative therapeutic approach for many malignant tumors. However, the PDT system generally involves unsatisfactory tumor specificity and nonspecific accumulation of photosensitizers around the target cancer cells, leading to phototoxic damage to adjacent healthy normal cells. In this study, we developed pheophorbide a (Pheo a)/human epidermal growth factor receptor 2 (HER2) targeting peptide (epitope form, HLTV, PEG2-LTVSPWY)-co-conjugated methoxy poly(ethylene glycol)-block-poly(L-lysine hydrochloride) (PEG-PLL)/hyaluronic acid (HA) (P3H2) polymeric micelles via a self-assembly method for HER2-targeted PDT treatment for breast cancer, thereby enhancing the PDT efficacy. The synthesized P3H2 polymeric micelles were spherical, with an average diameter of 125.7 ± 21.2 nm in an aqueous solution. The results of

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Chlorophyll; Female; Humans; Hyaluronic Acid; Micelles; Photochemotherapy; Photosensitizing Agents; Polyethylene Glycols; Polylysine; Receptor, ErbB-2

This study tried to quantitatively clarify the usefulness of supercritical fluid extraction for removal of chlorophyll and pheophorbide from Chlorella pyrenoidosa. C. pyrenoidosa powder was subjected to supercritical fluid extraction, and chlorophyll a and pheophorbide a in its extracted fractions were measured by HPLC-UV. Chlorophyll a and pheophorbide a in residue after supercritical fluid extraction became below of detection limit.

Topics: Chlorella; Chlorophyll; Chromatography, High Pressure Liquid; Chromatography, Supercritical Fluid; Proteins; Spectrophotometry, Ultraviolet

Leaf senescence, the last stage of leaf development, is a well-regulated and complex process for investigation. For simplification, dark-induced leaf senescence has frequently been used to mimic the natural senescence of leaves because many typical senescence symptoms, such as chlorophyll (Chl) and protein degradation, also occur under darkness. In this study, we compared the phenotypes of leaf senescence that occurred when detached leaves or intact plants were incubated in darkness to induce senescence. We found that the symptoms of non-programmed cell death (non-PCD) with remaining green coloration occurred more heavily in the senescent leaves of whole plants than in the detached leaves. The pheophorbide

Topics: Arabidopsis; Arabidopsis Proteins; Cell Death; Chlorophyll; Darkness; Gene Expression Regulation, Plant; Oxidoreductases; Phenotype; Photosynthesis; Plant Cells; Plant Leaves; Protein Stability; Proteolysis; Soil; Water

Combination therapy provides an efficient way to overcome the potential multidrug resistance and enhance anticancer efficacy. In this work, a biodegradable pH-responsive hollow mesoporous silica nanoparticle (HMSN-GM-CS-FA) was developed for co-delivery of pheophorbide a (PA) and doxorubicin (DOX). This drug delivery system possessed controlled particle size and larger inner hollow core, which endowed the nanoparticle with excellent encapsulation capacities. The uptake efficiency of drug loaded nanoparticles HMSNs-GM-CS-FA@DOX/PA in cancer cells was greatly improved by folic acid-mediated endocytosis. The nanocarrier showed excellent drug controlled release properties based on the pH-dependent swelling effect of the coating layer. More importantly, the nanoplatform could fully combine photothermal-, photodynamic- and chemotherapies to develop synergistic antitumor efficacy. This strategy of integrating multi-therapeutic functions in one single formulation promised a powerful route to construct intelligent co-delivery carriers for efficient combinational clinical application.

Topics: Antineoplastic Agents; Chitosan; Chlorophyll; Combined Modality Therapy; Doxorubicin; Drug Delivery Systems; Drug Liberation; Drug Resistance, Multiple; Endocytosis; Humans; Hydrogen-Ion Concentration; Metal Nanoparticles; Neoplasms; Silicon Dioxide

Bacteriochlorophyll c molecules self-aggregate to form large oligomers in the core part of chlorosomes, which are the main light-harvesting antenna systems of green photosynthetic bacteria. In the biosynthetic pathway of bacteriochlorophyll c, a BciC enzyme catalyzes the removal of the C13

Topics: Bacterial Proteins; Bacteriochlorophylls; Biosynthetic Pathways; Carbon Radioisotopes; Catalysis; Chlorobi; Chlorophyll; Esters; Metals; Substrate Specificity

To evaluate the effect of drug hydrophobicity on nanoparticle delivery in vivo, we conducted a comparative study using different photosensitizer-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). Chlorin e6 (Ce6) and pheophorbide a (Pba) with similar structure but different hydrophobicity were loaded into PLGA-NPs separately. We observed release profiles and photodynamic effects in vitro from the resulting Ce6- and Pba-PLGA-NPs. After intravenous injection into SCC7 tumor-bearing mice, biodistribution and accumulation of two drugs in tumor tissue were observed by real-time fluorescence imaging. Finally, in vivo photodynamic therapy with Ce6- and Pba-PLGA-NPs provided different therapeutic results according to imaging data. The results demonstrated that drug hydrophobicity is an important factor in nanoparticle drug delivery and should be considered for efficient drug delivery in vivo.

Topics: Animals; Cell Line, Tumor; Cell Survival; Chlorophyll; Chlorophyllides; Drug Delivery Systems; Flow Cytometry; Hydrophobic and Hydrophilic Interactions; Mice; Nanoparticles; Photochemotherapy; Photosensitizing Agents; Polylactic Acid-Polyglycolic Acid Copolymer; Porphyrins

ABCG2 is one of a triumvirate of human multidrug ATP binding cassette (ABC) transporters that are implicated in the defense of cells and tissues against cytotoxic chemicals, but these transporters can also confer chemotherapy resistance states in oncology. Understanding the mechanism of ABCG2 is thus imperative if we are to be able to counter its deleterious activity. The structure of ABCG2 and its related family members (ABCG5/G8) demonstrated that there were two interfaces between the nucleotide binding domains (NBD). In addition to the canonical ATP "sandwich-dimer" interface, there was a second contact region between residues at the C-terminus of the NBD. We investigated this second interface by making mutations to a series of residues that are in close interaction with the opposite NBD. Mutated ABCG2 isoforms were expressed in human embryonic kidney (HEK) 293T cells and analysed for targeting to the membrane, drug transport, and ATPase activity. Mutations to this second interface had a number of effects on ABCG2, including altered drug specificity, altered drug transport, and, in two mutants, a loss of ATPase activity. The results demonstrate that this region is particularly sensitive to mutation and can impact not only direct, local NBD events (i.e., ATP hydrolysis) but also the allosteric communication to the transmembrane domains and drug transport.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; Biological Transport; Chick Embryo; Chlorophyll; Drug Resistance, Multiple; HEK293 Cells; Humans; Hydrolysis; Mitoxantrone; Mutation; Pharmaceutical Preparations; Protein Domains; Topoisomerase II Inhibitors

Bicontinuous nanospheres (BCNs) are underutilized self-assembled nanostructures capable of simultaneous delivery of both hydrophilic and hydrophobic payloads. Here, we demonstrate that BCNs assembled from poly(ethylene glycol)-block-poly(propylene sulfide) (PEG-b-PPS), an oxidation-sensitive copolymer, are stably retained within cell lysosomes following endocytosis, resisting degradation and payload release for days until externally triggered. The oxygen scavenging properties and enhanced stability of the bicontinuous PEG-b-PPS nanoarchitecture significantly protected cells from typically cytotoxic application of pro-apoptotic photo-oxidizer pheophorbide A and chemotherapeutic camptothecin. The photo-oxidation triggered transition from a bicontinuous to micellar morphology overcame this stability, allowing on-demand cytosolic delivery of camptothecin for enhanced control over off-on cytotoxicity. These results indicate that inducible transitions in the nanostructure morphology can influence intracellular stability and toxicity of self-assembled nanotherapeutics.

Topics: Animals; Antineoplastic Agents, Phytogenic; Camptothecin; Cell Survival; Chlorophyll; Cytosol; Drug Carriers; Endocytosis; Light; Lysosomes; Mice; Micelles; Nanostructures; Oxidation-Reduction; Polyethylene Glycols; RAW 264.7 Cells; Singlet Oxygen; Sulfides

Sodium pheophorbide a (SPA) is a natural photosensitizer. The present study investigated the antifungal activity and mechanism of SPA against Botrytis cinerea in vitro and in vivo. Its inhibitory effect was studied on the spore germination and mycelial growth of B. cinerea. The effects of SPA on cell wall integrity, cell membrane permeability, and mycelial morphology of B. cinerea were also determined. Additionally, how SPA effected B. cinerea in vivo was evaluated using cherry tomato fruit. The results showed that SPA effectively inhibited the spore germination and mycelial growth of B. cinerea under light conditions (4000 lx). SPA significantly affected both cell wall integrity and cell membrane permeability (P < .05). In addition, SEM analysis suggested that B. cinerea treated with SPA (12.134 mg/mL) showed abnormal mycelial morphology, including atrophy, collapse, flattening, and mycelial wall dissolution. In vivo tests showed that SPA could increase the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) significantly (P < .05); however, SPA had no significant effect on phenylalanine ammonia lyase (PAL) activity. In short, SPA could destroy the fungal cell structure and enhance disease resistance-related enzyme activity in cherry tomatoes, thereby controlling cherry tomato gray mold.

Topics: Botrytis; Chlorophyll; Disease Resistance; Fruit; Humans; Sodium; Solanum lycopersicum

Tumor microenvironment (TME)-responsive nanocarrier systems that keep the photosensitizer (PS) inactive during systemic circulation and then efficiently release or activate the PS in response to unique TME conditions have attracted much attention. Herein, we report novel TME-responsive, self-quenched polysaccharide nanoparticles (NPs) with a reactive oxygen species (ROS)-sensitive cascade. The PS, pheophorbide A (PhA), was conjugated to a water-soluble glycol chitosan (GC) through an ROS-sensitive thioketal (TK) linker. The amphiphilic GC-TK-PhA conjugates could arrange themselves into NPs and remain photoinactive due to their self-quenching effects. Upon reaching the ROS-rich hypoxic core of the tumor tissue, the NPs release the PS in a photoactive form by efficient, ROS-sensitive TK bond cleavage, thus generating potent phototoxic effects. Following near-infrared irradiation, the increase in locoregional ROS levels further accelerates the release and activation of PS. These cascade reactions caused a significant reduction in the tumor volume, demonstrating good antitumor potential.

Topics: Chitosan; Chlorophyll; Nanoparticles; Photochemotherapy; Polysaccharides; Reactive Oxygen Species

Recently, photodynamic therapy (PDT) and photoactivated pesticides have attracted considerable research attention. In the present study, we aimed to investigate the photodynamic activity of a chlorophyllous derivative, sodium pheophorbide a (SPA), and to evaluate its potential as a photoactivated fungicide. The singlet oxygen quantum yield, the photoreaction process, the anti-photobleaching ability in sterile water (H

Topics: Antifungal Agents; Chlorophyll; Photochemotherapy; Photosensitizing Agents; Sodium

Sodium pheophorbide a (SPA) is a new alternative fungicide with low toxicity and high efficiency, which has high fungicidal activity against Pestalotiopsis neglecta, a pathogen that causes black spot needle blight of Pinus sylvestris var. mongolica. To utilize SPA for plant disease control, understanding its antifungal mechanism is essential. Six cDNA libraries were constructed from 3 d-old P. neglecta mycelia (three SPA-infected and three untreated groups) and 29,850 expressed genes were obtained by Illumina HiSeq4000 sequencing. Compared with controls, 3268 differentially expressed genes (DEGs) were identified in SPA-treated groups, including 1879 upregulated and 1389 downregulated genes. Most DEGs were involved in the metabolism of amino acids, carbohydrates, and lipids, as well as cell structure and genetic information processing. These findings were further confirmed by decreased conductivity, RNA and protein content, and activities of nicotinamide adenine dinucleotide-dependent malate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and succinate dehydrogenase. Moreover, qRT-PCR verified the reliability of the transcriptome results. After treatment with SPA at different concentrations for 60 min, the expressions of three cell wall degrading enzyme-related genes (PnEG, PnBG, and PnPG) were all suppressed. Overall, this study provided insights into the molecular mechanisms through which SPA inhibits P. neglecta, increasing the possibility of developing SPA into an effective fungicide in the future.

Topics: Cell Wall; Chlorophyll; Gene Expression Profiling; Gene Expression Regulation, Plant; Reproducibility of Results; Sodium; Transcriptome

Immune checkpoint inhibitors become a standard therapy for malignant melanoma. As immune checkpoint inhibitor monotherapies proved to have limited efficacy in significant portion of patients, it is envisaged that combination with other therapeutic modalities may improve clinical outcomes. We investigated the effect of combining photodynamic therapy (PDT) and TLR5 agonist flagellin-adjuvanted tumor-specific peptide vaccination (FlaB-Vax) on the promotion of PD-1 blockade-mediated melanoma suppression using a mouse B16-F10 implantation model. Using a bilateral mouse melanoma cancer model, we evaluated the potentiation of PD-1 blockade by the combination of peritumoral FlaB-Vax delivery and PDT tumor ablation. A photosensitizing agent, pheophorbide A (PhA), was used for laser-triggered photodynamic destruction of the primary tumor. The effect of combination therapy in conjunction with PD-1 blockade was evaluated for tumor growth and survival. The effector cytokines that promote the activation of CD8

Topics: Adjuvants, Immunologic; Animals; Cancer Vaccines; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cell Proliferation; Chlorophyll; Combined Modality Therapy; Cross-Priming; Flagellin; Humans; Immunologic Memory; Interferon-gamma; Liposomes; Melanoma, Experimental; Mice, Inbred C57BL; Nanoparticles; Photochemotherapy; Programmed Cell Death 1 Receptor

An amphiphilic polymer (CmPOX) based on poly(2-methyl-2-oxazoline) linked to a hydrophobic part composed of an aliphatic chain ending with a photo-active coumarin group has been synthesized. It exhibits the ability of forming small polymeric self-assemblies, typically of ca. 10 nm in size, which were characterized by TEM, cryo-TEM and DLS. The nanocarriers were further formulated to yield photo-crosslinked systems by dimerization of coumarin units of coumarin-functionalized poly(methyl methacrylate) (CmPMMA) and CmPOX. The formed vectors were used to encapsulate Pheophorbide a, a known photosensitizer for photodynamic therapy. Cytotoxicity as well as phototoxicity experiments performed in vitro on human tumor cells revealed the great potential of these nanovectors for photodynamic therapy.

Topics: Chlorophyll; Drug Carriers; HCT116 Cells; Humans; Hydrophobic and Hydrophilic Interactions; Oxazoles; Photochemotherapy; Photosensitizing Agents; Polymers; Polymethyl Methacrylate

Photodynamic therapy has become a feasible direction for the treatment of both malignant and non-malignant diseases. It has been in the spotlight since FDA regulatory approval was granted to several photosensitizers worldwide. Nevertheless, there are still strong limitations in the targeting specificity that is vital to prevent systemic toxicity. Here, we report the synthesis and biological evaluation of a novel bimodal oxime conjugate composed of a photosensitizing drug, red-emitting pheophorbide a, and nandrolone (NT), a steroid specifically binding the androgen receptor (AR) commonly overexpressed in various tumors. We characterized the physico-chemical properties of the NT-pheophorbide a conjugate (NT-Pba) and singlet oxygen generation. Because light-triggered therapies have the potential to provide important advances in the treatment of hormone-sensitive cancer, the biological potential of this novel specifically-targeted photosensitizer was assessed in prostatic cancer cell lines in vitro using an AR-positive (LNCaP) and an AR-negative/positive cell line (PC-3). U-2 OS cells, both with and without stable AR expression, were used as a second cell line model. Interestingly, we found that the NT-Pba conjugate was not only photodynamically active and AR-specific, but also that its phototoxic effect was more pronounced compared to pristine pheophorbide a. We also examined the intracellular localization of NT-Pba. Live-cell fluorescence microscopy provided clear evidence that the NT-Pba conjugate localized in the endoplasmic reticulum and mitochondria. Moreover, we performed a competitive localization study with the excess of nonfluorescent NT, which was able to displace fluorescent NT-Pba from the cell interior, thereby further confirming the binding specificity. The oxime ether bond degradation was assayed in living cells by both real-time microscopy and a steroid receptor reporter assay using AR U-2 OS cells. Thus, NT-Pba is a promising candidate for both the selective targeting and eradication of AR-positive malignant cells by photodynamic therapy.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Chlorophyll; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Microscopy, Fluorescence; Molecular Structure; Optical Imaging; Oximes; Particle Size; Photochemotherapy; Photosensitizing Agents; Structure-Activity Relationship; Surface Properties; Testosterone

Chemoimmunotherapy by systemic administration of individual regimens suffers from inconsistent pharmacokinetics profiles, low tumor specificity, and severe side effects. Despite promising nanoparticle-based codelivery approaches in therapeutics, the pathophysiological barriers of solid tumors are a hurdle for tumor accumulation and deep penetration of the drug-loaded nanoparticles. A light-inducible nanocargo (LINC) for immunotherapy is reported. LINC is composed of a reduction-responsive heterodimer of photosensitizer pheophorbide A (PPa) and indoleamine 2,3-dioxygenase 1 (IDO-1) inhibitor, i.e., NLG919, and a light-activatable prodrug of oxaliplatin (OXA). LINC administrated through intravenous injection is passively accumulated at the tumor site to generate near-infrared (NIR) fluorescence signal. Under fluorescence imaging guidance, the first-wave of NIR laser irradiation induce reactive oxygen species (ROS) generation, trigger cleavage of the polyethylene glycol (PEG) corona, and thus promote tumor retention and deep penetration of LINC. When exposed to the second-wave NIR laser illumination, LINC efficiently elicits the immune response and promotes intratumoral infiltration of cytotoxic T lymphocytes (CTLs). Furthermore, NLG919 delivered by LINC reverses the immunosuppressive tumor microenvironment by suppressing IDO-1 activity. Chemoimmunotherapy with LINC inhibit the tumor growth, lung metastasis, and tumor recurrence. The light-inducible self-amplification strategy for improved drug delivery and immunotherapy shows potential.

Topics: Animals; Chlorophyll; Dimerization; Drug Carriers; Immunotherapy; Light; Mice; Mice, Inbred BALB C; Nanostructures; Neoplasms; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species; T-Lymphocytes, Cytotoxic

Photodynamic therapy has attracted significant attention due to its localized treatment advantage. However, the non-specific distribution of photosensitizers and the subsequent potential toxicity caused by sunshine exposure hinder its wide adoption in cancer treatment. To minimize these unwanted effects and improve its efficacy, we developed a bioactivatable self-quenched nanogel, which remains in its inactive state in healthy tissues. Anti-EGFR Affibody decorated nanogels can effectively target head and neck cancer and release activated pheophorbide A in a reducing environment, such as in the tumor stroma and cytoplasm. Consequently, the EGFR targeted nanogel coupled with NIR irradiation alleviates tumor burden by 94.5% while not inducing systemic toxicity.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chlorophyll; ErbB Receptors; Head and Neck Neoplasms; HeLa Cells; Humans; Ligands; Mice; Molecular Targeted Therapy; Nanogels; Photochemotherapy; Radiation-Sensitizing Agents; Squamous Cell Carcinoma of Head and Neck; Xenograft Model Antitumor Assays

This study was designed to determine the inhibitory effects of pheophorbide A on carbohydrate digesting enzymes and its ability to improve postprandial hyperglycemia in streptozotocin (STZ)-induced diabetic mice. Pheophorbide A caused noticeable inhibitory effects on α-glucosidase and α-amylase, with half-maximal inhibitory concentrations (IC

Topics: Animals; Blood Glucose; Chlorophyll; Diabetes Mellitus, Experimental; Glycoside Hydrolase Inhibitors; Hyperglycemia; Male; Mice; Mice, Inbred ICR; Rhodophyta; Streptozocin

A virtual screening protocol with combination of similarity search and pharmacophore modeling was applied to virtually screen a large compound library to gain new scaffolds regarding ABCC1 inhibition. Biological investigation of promising candidates revealed four compounds as ABCC1 inhibitors, three of them with scaffolds not associated with ABCC1 inhibition until now. The best hit molecule-a thienopyrimidine-was a moderately potent, competitive inhibitor of the ABCC1-mediated transport of calcein AM which also sensitized ABCC1-overexpressing cells toward daunorubicin. Further evaluation showed that it was a moderately potent, competitive inhibitor of the ABCB1-mediated transport of calcein AM, and noncompetitive inhibitor of the ABCG2-mediated pheophorbide A transport. In addition, the thienopyrimidine could also sensitize ABCB1- as well as ABCG2-overexpressing cells toward daunorubicin and SN-38, respectively, in concentration ranges that qualified it as one of the ten best triple ABCC1/ABCB1/ABCG2 inhibitors in the literature. Besides, three more new multitarget inhibitors were identified by this virtual screening approach.

Topics: ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; Cell Line, Tumor; Chlorophyll; Daunorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Fluoresceins; Humans; Irinotecan; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Pyrimidines; Small Molecule Libraries; Thiophenes

Herein, we report the development of self-assembled nanoparticles using rhamnolipid, a biosurfactant. Rhamnolipid is produced by Pseudomonas aeruginosa, and has an amphiphilic structure that is suitable for the formation of a nanoparticle shell. These rhamnolipid nanoparticles were loaded with pheophorbide a (Pba), a hydrophobic photosensitizer. The resulting nanoparticles had about 136.1-nm-diameter spherical shapes and had excellent water solubility without aggregation for one month. These nanoparticles showed fast uptake into SCC7 tumor cells and induced photodynamic damage upon laser irradiation. After intravenous injection to SCC7 tumor-bearing mice, their long blood circulation time and high accumulation in tumor tissue were observed in real-time fluorescence imaging. Upon laser irradiation, these rhamnolipid nanoparticles showed complete tumor suppression by photodynamic therapy in vivo. These promising results demonstrate the potential of rhamnolipid nanoparticles for drug delivery, and suggest that further attention to rhamnolipid research would be fruitful.

Topics: Animals; Cell Death; Cell Line, Tumor; Cell Survival; Chlorophyll; Drug Delivery Systems; Glycolipids; Mice; Nanoparticles; Photochemotherapy; Singlet Oxygen; Tissue Distribution

Interaction between foods and drugs is an important consideration in pharmaceutical therapy. Therefore, here, we examined the suppressive effects of the extracts from seven edible herbs on the induction of CYP3A4 gene expression in rifampicin-treated HepG2 cells. We evaluated the structure and suppressive activity of the most effective active compound isolated from dried peppermint (

Topics: Antibiotics, Antitubercular; Cell Survival; Chlorophyll; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP3A; Hep G2 Cells; Humans; Mentha piperita; Molecular Structure; Plant Extracts; Proton Magnetic Resonance Spectroscopy; Rifampin; RNA, Messenger; Structure-Activity Relationship

Combination chemotherapy now becomes the most standard cancer treatment protocol. Here, we present a core-shell type polymeric microgel (CSPM) which combines photodynamic and chemo therapeutic modalities in one-pot system. CSPM localizes in the malignant lesion after intratumoral injection, releases reactive oxygen species (ROS) and anticancer drug (5'-deoxy-5-fluorocytidine; DFCR) under the near-infrared (NIR) laser treatment. Pheophorbide A (PheoA)-linked poly(hydroxyethyl methacrylate) (poly-HEMA) was designated to a ROS-generating core, and chemically covered with a chitosan shell. In addition, phenylboronic acid was employed in chitosan shells and linked to DFCR to form an ROS cleavable boronic ester. The core-shell structure of CSPM was determined by transmission electron microscopy. NIR-responsive photodynamic ROS generation was confirmed by the oxidative reduction of 9,10-dimethylanthracene (a fluorescent dye), and the cascadic release of DFCR by ROS was confirmed by a release study and a live and dead cell imaging study. Typically, poly-HEMA cored microgel increased its volume by 48.9-fold after absorption of body fluid. This swelling property ensured CSPM was retained in tumor tissues after subtumoral injection and the suitability of CSPM for locoregional phototherapy. The therapeutic effect of CSPM was attributed to the combined, cascadic deliveries of cytotoxic ROS and DFCR and confirmed by growth inhibition studies in in vitro pancreatic cancer cells and in vivo colon cancer mouse model.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Chlorophyll; Combined Modality Therapy; Delayed-Action Preparations; Deoxycytidine; Humans; Infrared Rays; Laser Therapy; Mice, Inbred BALB C; Microgels; Neoplasms; Polyhydroxyethyl Methacrylate; Reactive Oxygen Species; Water

Photodynamic therapy has been efficiently applied for cancer therapy. Here, we have fabricated the folic acid (FA)- and pheophorbide A (PA)-conjugated FA/PA@Fe

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Chlorophyll; Folate Receptors, GPI-Anchored; Folic Acid; Humans; Light; Magnetite Nanoparticles; Mice; Nanocomposites; Photochemotherapy; Photosensitizing Agents; Singlet Oxygen

Sodium pheophorbide a (SPA) is a natural photosensitizer. To explore its antifungal activity and mechanism, we studied its inhibitory effects on spore germination and mycelial growth of Pestalotiopsis neglecta. We used sorbitol, 2-thiobarbituric acid (TBA) and electron microscopy to determine its effects on cell wall integrity, cell membrane lipid peroxidation and mycelial morphology. Finally, the effects of SPA on enzyme activity in mycelia were determined. The results showed that SPA effectively inhibited spore germination and mycelial growth of P. neglecta under light conditions (4000 lx, 24 h). Scanning electron microscopy (SEM) revealed that SPA treatment resulted in a roughened, twisted and knotted mycelial surface and abnormal mycelial growth. SPA influenced cell wall integrity, and the content of MDA, a cell membrane lipid peroxidation product was significantly increased (P < 0.05). SPA also significantly inhibited SOD, POD and PG activity, but enhanced PPO activity (P < 0.05). In conclusion, SPA may have potential to become a biological pesticide.

Topics: Antifungal Agents; Chlorophyll; Lipid Peroxidation; Microbial Sensitivity Tests; Microscopy, Electron, Scanning; Mycelium; Radiation-Sensitizing Agents

Targeted drug delivery has been an important issue for tumor therapy including photodynamic therapy (PDT). The purpose of our study is to increase the targeting efficiency of photosensitizer (PS) using folate-modified nanoparticles (NPs) to tumor site in vivo. Folate receptor is over-expressed on the surface of many human cancer cells. We prepared poly (lactic-co-glycolic acid) (PLGA) NPs containing pheophorbide a (Pba), a PS that is used in PDT and generates free radical for killing cancer cells. The surface of NPs was composed of phospholipids modified with polyethylene glycol (PEG) and folate (FA). The size of the resulting FA-PLGA-Pba NPs was about 200 nm in PBS at pH 7.4 and they were stable for long time. They showed faster cellular uptake to MKN28 human gastric cancer cell line than control PLGA-Pba NPs by high-affinity binding with folate receptors on cell surface. In MTT assay, FA-PLGA-Pba NPs also showed enhanced tumor cell killing compared to control PLGA-Pba NPs. In vivo and ex vivo imaging showed high accumulation of FA-PLGA-Pba NPs in tumor site during 24 h after intravenous injection to MKN28 tumor-bearing mice model. These results demonstrate that our FA-PLGA-Pba NPs are useful for tumor-targeted delivery of PS for cancer treatment by PDT.

Topics: Animals; Cell Line, Tumor; Chlorophyll; Drug Carriers; Drug Delivery Systems; Folic Acid; Humans; Lactic Acid; Mice, Nude; Nanoparticles; Photochemotherapy; Photosensitizing Agents; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Stomach Neoplasms

In recent years, photodynamic therapy (PDT) was considered to be a promising cancer treatment modality, however, the therapeutic efficiency was often attenuated by the intrinsic antioxidant defense systems. Herein, a kind of novel glutathione-induced amino-activatable micelle was designed, which was expected to weaken the antioxidant capacity and in the meantime release the photosensitizer by the exhaustion of intracellular glutathione (GSH). The amphiphilic poly(ethylene glycol)-(2-((2,4-dinitro-N-(ethyl) phenyl)sulfonamido) ethyl methacrylate) copolymers were synthesized and assembled into a core-shell nano structure in aqueous media. The nano structure demonstrated high sensitivity and selectivity to bio-thiols in vitro and in vivo. Subsequently, pheophorbide a (PhA) was encapsulated as the model photosensitizer. Upon internalization by HepG2 cells, the strongly electron-withdrawing 2,4-dinitrobenzenesulfonyl groups on the PADEE segments were readily cleaved by GSH, during which time the secondary amino groups (pKb = 11.32) were recovered and completely protonated, leading to disassembly of the micelles and rapid release of PhA. Importantly, the consumption of GSH weakened the intracellular antioxidant capacity, resulting in the synergetic accumulation of reactive oxygen species (ROS) under laser irradiation. As a result, this micellar photosensitization system could overcome the antioxidant capacity of advanced stage tumors through a simultaneous extrinsic and intrinsic strategy, facilitating therapeutic efficiency. These results demonstrate that the as-designed micelles provide a versatile photosensitization platform for on-demand PDT.

Topics: Acrylic Resins; Animals; Antineoplastic Agents; Carcinoma, Hepatocellular; Chlorophyll; Drug Synergism; Female; Glutathione; Hep G2 Cells; Humans; Mice; Mice, Inbred BALB C; Micelles; Oxidation-Reduction; Photochemotherapy; Photosensitizing Agents; Polyethylene Glycols

In this study, PPA was conjugated with JM-phage by EDC/NHS crosslinking. UV-Vis spectra was used to determine the optimum conjugation percentages of PPA and JM-phage complex for photodynamic inactivation. After photodynamic inactivation, the efficacy of PPA-JM-phage was assessed by performing in vitro experiments, such as MTS assay, scanning electron microscopy, measurement of dysfunctional mitochondria, ROS accumulation, S cell arrest and apoptotic pathway.. A single-chain variable-fragment phage (JM) with high affinity to MP65 was screened from human single-fold single-chain variable-fragment libraries and designed as a binding target for. PPa-JM-phage may induce

Topics: Apoptosis; Bacteriophages; Candida albicans; Caspases; Chlorophyll; Fungal Proteins; Humans; Membrane Potential, Mitochondrial; Microscopy, Electron, Scanning; Mitochondria; Peptide Library; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species

We previously reported that pheophorbide a (PhA), excited by 630 nm light, significantly inhibited the growth of prostate cancer cells. In this study, we employed whole-cell proteomics to investigate photodynamic treatment (PDT)-related proteins.. Two-dimensional gel electrophoresis (2-DE) coupled with tandem mass spectrometry was employed to reveal the proteins involved in PhA-mediated PDT in LNCaP and PC-3 prostate cancer cells.. After PhA-PDT treatment, decreased expression of translationally-controlled tumor protein (TCTP) was found in both PC-3 and LNCaP whole-cell proteomes. In contrast, human rab GDP dissociation inhibitor (GDI) in LNCaP cells and ras-related homologs GDI in PC-3 cells were up-regulated.. GDP-GTP exchange is an underlying target of photodynamic treatment in prostate cancer cells.

Topics: Cell Line, Tumor; Chlorophyll; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Male; PC-3 Cells; Photochemotherapy; Photosensitizing Agents; Prostatic Neoplasms; Proteomics; ras Proteins; Tandem Mass Spectrometry; Tumor Protein, Translationally-Controlled 1; Two-Dimensional Difference Gel Electrophoresis

Although the PAO/phyllobilin pathway of chlorophyll breakdown is active in grass leaf senescence, the abundance of phyllobilins is far below the amount of degraded chlorophyll. The yellowing of fully developed leaves is the most prominent visual symptom of plant senescence. Thereby, chlorophyll is degraded via the so-called pheophorbide a oxygenase (PAO)/phyllobilin pathway to a species-specific set of phyllobilins, linear tetrapyrrolic products of chlorophyll breakdown. Here, we investigated the diversity and abundance of phyllobilins in cereal and forage crops, i.e. barley, rice, ryegrass, sorghum and wheat, using liquid chromatography-mass spectrometry. A total of thirteen phyllobilins were identified, among them four novel, not yet described ones, pointing to a rather high diversity of phyllobilin-modifying activities present in the Gramineae. Along with these phyllobilins, barley orthologs of known Arabidopsis thaliana chlorophyll catabolic enzymes were demonstrated to localize in the chloroplast, and two of them, i.e. PAO and pheophytin pheophorbide hydrolase, complemented respective Arabidopsis mutants. These data confirm functionality of the PAO/phyllobilin pathway in grasses. Interestingly, when comparing phyllobilin abundance with amounts of degraded chlorophyll in senescent leaves, in most analyzed grass species only minor fractions of chlorophyll were recovered as phyllobilins, opposite to A. thaliana where phyllobilin quantities match degraded chlorophyll rather well. These data show that, despite the presence and activity of the PAO/phyllobilin pathway in barley (and other cereals), phyllobilins do not accumulate stoichiometrically, implying possible degradation of chlorophyll beyond the phyllobilin level.

Topics: Bile Pigments; Chlorophyll; Genes, Reporter; Hordeum; Metabolic Networks and Pathways; Mutation; Oxygenases; Plant Leaves; Poaceae; Recombinant Fusion Proteins; Time Factors

The dietary intake of chlorophylls is estimated to be ≈50 mg d. A group of 30 mice are fed a diet rich in chlorophylls, while 10 mice received a standard diet without chlorophylls (control group). Liver extracts are analyzed every 15 days by HPLC-ESI(+)/APCI(+)-hrTOF- MS/MS to measure the accretion of specific chlorophyll metabolites. The chlorophyll profile found in the livers of mice fed a chlorophyll-rich diet shows that the formation and/or absorption of pheophorbides, pyro-derivatives, and phytyl-chlorin e. Pheophorbide a absorption is, at least partly, protein-mediated through SR-BI. This active absorption process could explain the specific accumulation of pheophorbide a in the livers of animals fed a chlorophyll-rich diet. A complementary mechanism could be the de-esterification of pheophytin a in the liver, yielding pheophorbide a and phytol, which can explain the origin of phytol in the liver. Hence, the results suggest two molecular mechanisms responsible for the accumulation of the health-promoting compounds pheophorbide and phytol.

Topics: Animals; Caco-2 Cells; Chlorophyll; Cyclopentanes; Diet; Feces; Humans; Liver; Male; Mice, Inbred C57BL; Pigments, Biological; Scavenger Receptors, Class B; Spirulina; Thiosemicarbazones; Tissue Distribution

We investigated whether pheophorbide A (PhA) could serve as a selective breast cancer resistance protein (BCRP) substrate (victim) to screen in vivo using fluorescent live animal imaging for transporter-mediated interactions with orally administered inhibitors (perpetrators), and whether that could be coupled with serum sampling to measure the systemic concentration of PhA with a fast-throughput in vitro fluorescent assay. PhA is a breakdown product of chlorophyll and is highly fluorescent in the near-infrared (NIR) spectrum. Whole-body NIR fluorescence was greater in the Bcrp KO compared with wild-type (WT) mice fed a regular diet containing chlorophyll and PhA, with fluorescence in WT mice confined to the intestine. PhA intestinal enterocyte fluorescence, after removing lumen contents, was greater in Bcrp knockout (KO) mice versus WT mice due to PhA enterocyte absorption and lack of PhA efflux by Bcrp. This difference was eliminated by maintaining the mice on an alfalfa (chlorophyll/PhA)-free diet. The area under the fluorescence ratio-time curve up to 6 hours (AUC

Topics: Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; Biological Transport; Cell Line; Chlorophyll; Dogs; Drug Interactions; Fluorescence; Madin Darby Canine Kidney Cells; Male; Mice; Mice, Knockout

To improve the therapeutic efficacy of gemcitabine (GEM) as an anticancer drug for bile duct cancer, GEM-loaded liposomes (GDPPL) prepared from a photosensitizer-conjugated lipid were investigated regarding the drug release kinetics, photodynamic therapy (PDT) efficacy, and immunomodulatory effects. The release rate of GEM from the liposomes was improved approximately 2-fold compared to non-laser irradiation groups due to lipid disruption by reactive oxygen species produced from the activated photosensitizer upon laser irradiation. Through in vitro testing using a human liver bile duct carcinoma cell line (HuCCT-1), the cytotoxicity of GDPPL with laser irradiation was enhanced due to rapid GEM release and PDT effects. Furthermore, the results of in vivo tests using a HuCCT-1 tumor-bearing xenograft mice model showed that GDPPL exhibited approximately 3-fold antitumoral effects compared to control group. Additionally, immunohistochemical analysis demonstrated the recruitment of immunostimulatory cells in tumor tissues. IHC tests in BALB/c mice indicated that GDPPL under laser irradiation dramatically enhanced the quantities of various immune cells for effective antitumoral immunotherapy against biliary tract cancer. From these results, it was concluded that GDPPL with rapid drug release behavior, PDT efficacy, and immunomodulatory effects upon laser irradiation has potential as an antitumor therapeutic agent for biliary tract cancer.

Topics: Animals; Antineoplastic Agents; Bile Duct Neoplasms; Cell Line, Tumor; Cell Survival; Chlorophyll; Cholangiocarcinoma; Combined Modality Therapy; Deoxycytidine; Gemcitabine; Heterografts; Humans; Immunomodulation; Lasers; Liposomes; Mice, Inbred BALB C; Mice, Nude; Phosphatidylethanolamines; Photochemotherapy; Photosensitizing Agents; Polyethylene Glycols

Herein, we report the synthesis of a new prodrug system consisting of regioselectively addressable functionalized templates bearing multiple pheophorbide A moieties for use in photodynamic therapy. These coupling reactions were achieved using copper-free "click" chemistry, namely a strain-promoted azide-alkyne cycloaddition. This new design was used to obtain well-defined quenched photosensitizer prodrugs with perfect knowledge of the number and position of loaded photosensitizers, providing structures bearing up to six photosentitizers and two PEG chains. These conjugates are ideally quenched in their native state regarding their fluorescence emission (up to 155 ± 28 times less fluorescent for an hexasubstituted conjugate than a monosubstituted non-quenched reference compound) or singlet oxygen production (decreased 8.7-fold in the best case) when excited. After 2 h of proteolytic activation, the fluorescence emission of a tetrasubstituted conjugate was increased 17-fold compared with the initial fluorescence emission.

Topics: Chlorophyll; Drug Delivery Systems; Fluorescence; Molecular Structure; Peptides, Cyclic; Photochemotherapy; Photosensitizing Agents; Prodrugs; Proteolysis; Stereoisomerism; Trypsin; Urokinase-Type Plasminogen Activator

Previously, we have shown that the use of a cyclopeptidic carrier could be of great interest for the design of fully characterized prodrugs for further use in photodynamic therapy. In order to further optimize the design, we decided to modify the highly quenched conjugate uPA-cPPP

Topics: Chlorophyll; Drug Delivery Systems; Fluorescence; Humans; Molecular Structure; Peptides, Cyclic; Photochemotherapy; Photosensitizing Agents; Prodrugs; Proteolysis

Compared to conventional carrier-assistant drug delivery systems (DDSs), drug self-delivery systems (DSDSs) have advantages of unprecedented drug loading capacity, minimized carrier-related toxicity and ease of preparation. However, the colloidal stability and blood circulation time of DSDSs still need to be improved. Here we report on the development of a novel biomimicry drug self-delivery system by the integration of a top-down cell membrane complexing technique into our self-delivery multifunctional nano-platform made from bottom-up approach that contains 100% active pharmaceutical ingredients (API) of Pheophorbide A and Irinotecan conjugates (named PI). Compared to conventional cell membrane coated nanoparticles with polymer framework as core and relatively low drug loading, this system consisting of red blood cell membrane vesicles complexed PI (RBC-PI) is polymer-free with up to 50% API loading. RBC-PI exhibited 10 times higher area under curve in pharmacokinetic study and much lower macrophage uptake compared with the parent PI nanoparticles. RBC-PI retained the excellent chemophototherapeutic effects of the PI nanoparticles, but possessed superior anti-cancer efficacy with prolonged blood circulation, improved tumor delivery, and enhanced photothermal effects in animal models. This system represents a novel example of using cell membrane complexing technique for effective surface modification of DSDSs. This is also an innovative study to form a polymer-free cell membrane nanoparticle complexing with positive surface charged materials. This biomimicry DSDS takes advantages of the best features from both systems to make up for each other's shortcomings and posed all the critical features for an ideal drug delivery system.

Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Chlorophyll; Drug Carriers; Drug Liberation; Erythrocyte Membrane; Female; Humans; Irinotecan; Light; Mice, Nude; Nanoparticles; Photochemotherapy; Photosensitizing Agents; Rats, Sprague-Dawley

Photo-triggerable liposomes are considered nowadays as promising drug delivery devices due to their potential to release encapsulated drugs in a spatial and temporal manner. In this work, we have investigated the photopermeation efficiency of three photosensitizers (PSs), namely verteporfin, pheophorbide a and m-THPP when incorporated into liposomes with well-defined lipid compositions (SOPC, DOPC or SLPC). By changing the nature of phospholipids and PSs, the illumination of the studied systems was shown to significantly alter their lipid bilayer properties via the formation of lipid peroxides. The system efficiency depends on the PS/phospholipid association, and the ability of the PS to peroxidize acyl chains. Our results demonstrated the possible use of these three clinically approved (or under investigation) PSs as potential candidates for photo-triggerable liposome conception.

Topics: Chlorophyll; Drug Liberation; Fluoresceins; Fluorescent Dyes; Hydrophobic and Hydrophilic Interactions; Light; Lipid Bilayers; Lipid Peroxidation; Liposomes; Mesoporphyrins; Molecular Dynamics Simulation; Permeability; Phosphatidylcholines; Photosensitizing Agents; Porphyrins; Transition Temperature; Verteporfin

MicroRNAs (miRNAs) regulate key biological processes, and their aberrant expression has been related to cancer development. Photodynamic therapy (PDT) has emerged as one of the most promising modalities for cancer treatment. However, the application of PDT has been limited to superficially localized human cancerous and precancerous lesions. To increase the usefulness of both PDT and miRNAs in cancer therapy, this study investigated whether apoptosis-related miRNA expression is influenced by PDT in oral cancer and whether miRNAs can enhance PDT efficacy. To achieve this goal, we performed a miRNA array-based comparison of apoptosis-related miRNA expression patterns following PDT using pheophorbide a (Pa) as a photosensitizer. After Pa-PDT, 13.1% of the miRNAs were down-regulated, and 16.7% of the miRNAs were up-regulated. Representative miRNAs were selected according to expression difference: miR-9-5p, miR-32-5p, miR-143-3p, miR-145-5p, miR-192-5p, miR-193a-5p, miR-204-5p, miR-212-3p, miR-338-3p, and miR-451a. Among them, only miR-145-5p showed the consistent reduction repeatedly in all cell lines after Pa-PDT. Further, the combined treatment of a miR-145-5p mimic and Pa-PDT increased phototoxicity, reactive oxygen species generation, and apoptotic cell death, suggesting that miRNAs expression could be a useful marker for enhancing the therapeutic effect of Pa-PDT. This study will provide a promising strategy for introducing miRNA as cancer therapy.

Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Chlorophyll; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Mouth Neoplasms; Oligonucleotide Array Sequence Analysis; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species

Cobra neurotoxin (CNT), a peptide isolated from snake venom of

Topics: Analgesics; Animals; Blood-Brain Barrier; Brain; Chlorophyll; Cobra Neurotoxin Proteins; Drug Delivery Systems; Drug Evaluation, Preclinical; Injections, Intraperitoneal; Light; Mice; Nanocapsules; Pain; Particle Size; Photosensitizing Agents

Approximately 6-7 million people are infected with Trypanosoma cruzi, the etiological agent of Chagas' disease. Only two therapeutic compounds have been found to be useful against this disease: nifurtimox and benznidazole. These drugs have been effective in the acute phase of the disease but less effective in the chronic phase; they also have many side effects. Thus, the search for new compounds with trypanocidal action is necessary. Natural products can be the source of many important substances for the development of drugs to treat this infection. The present study evaluated the biological activity of an extract and fractions of Arrabidaea chica against T. cruzi and observed morphological and ultrastructural characteristics of parasites exposed to the isolated compound pheophorbide a.. The crude hydroethanolic extract of A. chica was prepared. Fractions were obtained by partition and separated by liquid chromatography.. We observed a progressive increase in activity against epimastigote, trypomastigote, and amastigote forms of the parasite over the course of the fractionation process. Interestingly, we isolated a compound known as a photosensitizer that is used in photodynamic therapy. This method of treatment involving a photosensitizer, activation light and molecular oxygen is of great importance due to its selectivity. Pheophorbide a had activity against the protozoan in the presence of light and caused morphological and ultrastructural changes, demonstrating its potential in photodynamic therapy.. Based on the ability of pheophorbide a to eliminate bloodstream forms of T. cruzi, we suggest its use in blood banks for hemoprophylaxis.

Topics: Animals; Cell Line; Chlorophyll; Haplorhini; Photochemotherapy; Photosensitizing Agents; Plant Extracts; Trypanosoma cruzi

Leishmaniasis is a disease caused by hemoflagellate protozoa, affecting millions of people worldwide. The difficulties of treating patients with this parasitosis include the limited efficacy and many side effects of the currently available drugs. Therefore, the search for new compounds with leishmanicidal action is necessary. Photodynamic therapy has been studied in the medical field because of its selectivity, utilizing a combination of visible light, a photosensitizer compound, and singlet oxygen to reach the area of treatment. The continued search for selective alternative treatments and effective targets that impact the parasite and not the host are fundamentally important for the development of new drugs. Pheophorbide a is a photosensitizer that may be promising for the treatment of leishmaniasis. The present study evaluated the in vitro biological effects of pheophorbide a and its possible mechanisms of action in causing cell death in L. amazonensis. Pheophorbide a was active against promastigote and amastigote forms of the parasite. After treatment, we observed ultrastructural alterations in this protozoan. We also observed changes in promastigote macromolecules and organelles, such as loss of mitochondrial membrane potential [∆Ψ

Topics: Apoptosis; Caspase 3; Caspase 7; Cell Membrane; Chlorophyll; DNA Fragmentation; Hydrogen Peroxide; Intracellular Space; Leishmania; Light; Lipid Peroxidation; Membrane Potential, Mitochondrial; Nitric Oxide; Oxidative Stress; Photosensitizing Agents; Vacuoles

To observe the effects of pheophorbide a-mediated photodynamic therapy (Pa-PDT) on the in vitro proliferation, apoptosis, invasion and metastasis of human prostate cancer PC-3 cells and to investigate its possible mechanism.. Pa-PDT in gradient concentrations (0 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM, and 4 μM) were used to act on PC-3 cells; the cell proliferation in each group was detected via methyl thiazolyl tetrazolium (MTT) assay and clone formation assay, and the cell apoptosis was detected via Hochst33258 staining and Annexin V/propidium iodide (PI) double labeling. Moreover, the effects of Pa-PDT on invasion and proliferation of PC-3 cells were observed via wound healing assay and transwell chamber assay. Finally, the expressions of apoptosis-related proteins, epithelial-mesenchymal transition (EMT)-related proteins and matrix metalloproteinases (MMPs) in each group were detected after treatment by Western blotting.. MTT and clone formation assays showed that Pa-PDT could inhibit the proliferation of PC-3 cells in a dose-dependent manner. The results of apoptosis assay revealed that Pa-PDT could significantly promote the apoptosis of PC-3 cells, obviously up-regulate the expressions of pro-apoptotic proteins, such as B-cell lymphoma-2-associated X protein (BAX), Caspase-3 and poly adenosine diphosphate-ribose polymerase (PARP), and inhibit the expression of Bcl-2. Besides, the wound healing assay and Transwell chamber assay showed that Pa-PDT could inhibit the invasion and metastasis capacities of PC-3 cells, whose relevant mechanisms were related to the fact that Pa-PDT inhibited the EMT process and down-regulated the expressions of MMPs in PC-3 cells.. Pa-PDT can inhibit the proliferation and promote the apoptosis of PC-3 cells. Moreover, it can also inhibit the invasion and metastasis capacities of PC-3 cells via inhibiting the EMT process and down-regulating the expressions of MMPs.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Chlorophyll; Epithelial-Mesenchymal Transition; Humans; Male; Neoplasm Metastasis; Photochemotherapy; Prostatic Neoplasms

Photodynamic therapy (PDT) is a therapeutic alternative for malignant tumors that uses a photosensitizer. Our group recently synthesized photosensitizer pheophorbide a (Pa) from chlorophyll-a. The present study investigated the therapeutic effect of PDT using intratumoral administration of the synthetic photosensitizer Pa in an in vivo murine oral squamous cell carcinoma (OSCC) animal model. Pa accumulation was measured using the fluorescence spectrum and imaging in living C3H mice. Intratumoral treatment of Pa-PDT (IT Pa-PDT) significantly inhibited the growth of transplanted OSCC cells. Histopathological examination of tumor tissues showed that PCNA expression was significantly decreased, while TUNEL-stained cells were markedly increased in the IT Pa-PDT group compared to controls. IT Pa-PDT-induced apoptosis was confirmed by immunoblot. Reduction of Bcl-2 and cleavage of caspase 3 and PARP were observed in IT Pa-PDT. These data demonstrate that IT Pa-PDT inhibited tumor cell proliferation and induced apoptosis, which is correlated with the anticancer activity of IT Pa-PDT. These potent antitumor activities of IT Pa-PDT were observed in both the immunohistochemistry and Western blot experiments. Our findings suggest the intratumoral therapeutic potential of Pa-PDT on OSCC. Additionally, demonstrated detection of Pa using a fluorescence spectroscopy system or molecular imaging system provides a means for simultaneous diagnosis and treatment of OSCC.

Topics: Animals; Cell Line, Tumor; Chlorophyll; Dose-Response Relationship, Drug; Mice; Mice, Inbred C3H; Mouth Neoplasms; Photochemotherapy; Radiation-Sensitizing Agents

Photodynamic therapy (PDT) has drawn considerable attention for its efficacy against certain types of cancers. It shows however limits in the case of deep cancers, favoring tumor recurrence under suboptimal conditions. More insight into the molecular mechanisms of PDT-induced cytotoxicity and cytoprotection is essential to extend and strengthen this therapeutic modality. As PDT induces iNOS/NO in both tumor and microenvironment, we examined the role of nitric oxide (NO) in cytotoxicity and cytoprotection. Our findings show that NO mediates its cellular effects by acting on the NF-κB/YY1/RKIP loop, which controls cell growth and apoptosis. The cytoprotective effect of PDT-induced NO is observed at low NO levels, which activate the pro-survival/anti-apoptotic NF-κB and YY1, while inhibiting the anti-survival/pro-apoptotic and metastasis suppressor RKIP. In contrast, high PDT-induced NO levels inhibit NF-κB and YY1 and induce RKIP, resulting in significant anti-tumor activity. These findings reveal a critical role played by NO in PDT and suggest that the use of bifunctional PDT agents composed of a photosensitizer and a NO-donor could enhance the photo-treatment effect. A successful application of NO in anticancer therapy requires control of its concentration in the target tissue. To address this issue we propose as PDT agent, a bimolecular conjugate called DR2, composed of a photosensitizer (Pheophorbide a) and a non-steroidal anti-androgen molecule capable of releasing NO under the exclusive control of light. The mechanism of action of DR2 in prostate cancer cells is reported and discussed.

Topics: Cell Line, Tumor; Cell Survival; Chlorophyll; Humans; Male; NF-kappa B; Nitric Oxide; Nitric Oxide Donors; Phosphatidylethanolamine Binding Protein; Photochemotherapy; Photosensitizing Agents; Prostatic Neoplasms; Triazenes; YY1 Transcription Factor

The objective of this study was to develop a mitochondria-targeted photosensitizer (PS) for photodynamic therapy (PDT). Herein, a porphyrin-derivative photosensitizer, pheophorbide-a (PheoA), was conjugated to carboxybutyltriphenylphosphonium (TPP) via a carbodiimide linkage to enhance mitochondrial targeting and TPP-PheoA conjugate was further loaded into folate-cholesteryl albumin (FA-chol-BSA) nanoparticles (NPs) to improve its biocompatibility. Cellular uptake results showed that TPP-PheoA and TPP-PheoA@FA-chol-BSA NPs were readily taken up by B16F10 and HeLa cells. Further in vitro studies exhibited that TPP-PheoA and its nanoparticle primarily accumulate in the mitochondria, greatly generate ROS, lead mitochondrial disruption and cell apoptosis, and have higher phototoxicity against cancer cells. In vivo bioimaging and the in vivo antitumor studies indicated that TPP-PheoA@FA-chol-BSA NP greatly accumulated in the tumor area and significantly suppress the tumor growth as compared to PheoA@FA-chol-BSA NP in tumor-bearing mice. Taken together, TPP-PheoA@FA-chol-BSA NP could be a promising mitochondria-targeted PS for image-guided PDT.

Topics: Albumins; Animals; Chlorophyll; Folic Acid; Humans; Mice; Nanoparticles; Photochemotherapy; Photosensitizing Agents

Tailor-made drug solubilizers are studied based on peptide-poly(ethylene glycol) conjugates, which exhibit peptide segments constituting binding motifs for the small-molecule drugs of interest to render them water-soluble. Suitable 7mer peptides are selected via combinatorial means by screening large one-bead-one-compound (OBOC) peptide libraries. The capability of the screening method to read out structural detail of the drugs is investigated by comparing three related photosensitizers (Chlorin E6 (Ce6), Pheophorbide A (Pba) and meta-tetra(hydroxyphenyl)chlorin (m-THPC), which are applicable for photodynamic cancer therapy. The screening procedure delivers de novo solubilizers that show the best solubilization efficiency for the drug the screening is performed with. While molecular recognition events between peptide and drug are not expected to be found, significant binding capacity differences of, e.g., the Ce6-solubilizer for Pba are suggesting selectivity in drug binding, even among structurally closely related drugs. Cyro-Electron microscopy revealed the formation of colloidal aggregates between drug moieties and peptide conjugates. Insights into relevant amino acids in the identified peptide sequences are gained by studying capacities of systematic point mutations (alanine scans), enabling understanding of drug-binding motifs. These reveal the importance of sequence positioning of appropriate H-bonding between polar functional groups of the peptide and the drugs, which agrees well with computational binding studies performed on drug/peptide model complexes.

Topics: Amino Acid Sequence; Chlorophyll; Chlorophyllides; Mesoporphyrins; Models, Molecular; Peptides; Photochemotherapy; Photosensitizing Agents; Polyethylene Glycols; Porphyrins; Solubility

Mitochondria-targeting drug carriers have considerable potential because of the presence of many molecular drug targets in the mitochondria and their pivotal roles in cellular viability, metabolism, maintenance, and death. To compare the mitochondria-targeting abilities of triphenylphosphonium (TPP) and pheophorbide a (PhA) in nanoparticles (NPs), this study prepared mitochondria-targeting NPs using mixtures of methoxy poly(ethylene glycol)-(SS-PhA)

Topics: Cell Line, Tumor; Cell Survival; Chlorophyll; Diagnostic Imaging; Drug Carriers; Humans; Mitochondria; Nanoparticles; Organophosphorus Compounds; Particle Size; Photochemotherapy; Photosensitizing Agents; Polyesters; Polyethylene Glycols

The clinical application of intracellular gene delivery via nanosized carriers is hindered by intracellular multistep barriers that limit high levels of gene expression. To solve these issues, four different intracellular or external stimuli that can efficiently activate a gene carrier, a gene, or a photosensitizer (pheophorbide A [PhA]) were assessed in this study. The designed nanosized polymeric gene complexes were composed of PhA-loaded thiol-degradable polycation (PhA@RPC) and cytomegalovirus (CMV) promoter-equipped pDNA. After cellular internalization of the resulting PhA@RPC/pDNA complexes, the complexes escaped endosomal sequestration, owing to the endosomal pH-induced endosomolytic activity of RPC in PhA@RPC. Subsequently, intracellular thiol-mediated polycation degradation triggered the release of PhA and pDNA from the complexes. Late exposure to light (for example, 12 h post-treatment) activated the released PhA and resulted in the production of reactive oxygen species (ROS). Intracellular ROS successively activated NF-κB, which then reactivated the CMV promoter in the pDNA. These sequential, stimuli-responsive chemical and biological reactions resulted in high gene expression. In particular, the time-point of light exposure was very significant to tune efficient gene expression as well as negligible cytotoxicity: early light treatment induced photochemical internalization but high cytotoxicity, whereas late light treatment influenced the reactivation of silent pDNA via PhA-generated ROS and activation of NF-κB. In conclusion, the quadruple triggers, such as pH, thiol, light, and ROS, successively influenced a gene carrier (RPC), a photosensitizer, and a genetic therapeutic, and the tempo-spatial activation of the designed quadruple stimuli-activatable nanosized gene complexes could be potential in gene delivery applications.

Topics: Cell Line, Tumor; Chlorophyll; DNA; Endosomes; Gene Expression; Gene Transfer Techniques; HeLa Cells; Humans; Hydrogen-Ion Concentration; Nanoparticles; NF-kappa B; Photosensitizing Agents; Plasmids; Polyamines; Polyelectrolytes; Polymers; Reactive Oxygen Species; Transfection

Photodynamic therapy (PDT) is one of the most promising methods of specific cancer treatment. However, commercially available photosensitizers (PSs) show significant drawbacks, such as side toxicity, low penetration ability, low blood solubility, low tumor selectivity etc. In addition, as was shown previously, a conjugation of polyamines with several toxic agents led to an increased toxicity to cancer cells. Here, we synthesized conjugates of two chlorine photosensitizers, purpurin 18 and pheophorbide a, with spermine in natural and Boc-protected form. Using specialized software, we calculated octanol-water partition coefficients for single protonation state (logP) of single PSs and PS/spermine conjugates. We found that the addition of spermine to chlorine PSs shifted the logP towards higher hydrophilicity in comparison to logP of single chlorines. In vitro studies on several cancer cells indicated that conjugation of purpurin 18 with spermine increased its retention in cancer cells. Using various concentrations of this conjugate, we found that lower concentrations (under 0.2μM) of purpurin 18/spermine conjugate launched apoptosis in HeLa cells. This combined with its high phototoxicity makes the purpurin 18/spermine conjugate a promising photosensitizer for PDT. Obtained results might serve as a basis for further studies of this potential third-generation PS on mammalian models in vivo.

Topics: Animals; Apoptosis; Cell Line, Tumor; Chlorophyll; HeLa Cells; Humans; Hydrophobic and Hydrophilic Interactions; Photochemotherapy; Photosensitizing Agents; Porphyrins; Spermine

Phytoplasmoses such as apple proliferation (AP) and European stone fruit yellows (ESFY) cause severe economic losses in fruit production. A common symptom of both phytoplasma diseases is early yellowing or leaf chlorosis. Even though chlorosis is a well-studied symptom of biotic and abiotic stresses, its biochemical pathways are hardly known. In particular, in this context, a potential role of the senescence-related pheophorbide a oxygenase/phyllobilin (PaO/PB) pathway is elusive, which degrades chlorophyll (Chl) to phyllobilins (PBs), most notably to colorless nonfluorescent Chl catabolites (NCCs). In this work, we identified the Chl catabolites in extracts of healthy senescent apple and apricot leaves. In extracts of apple tree leaves, a total of 12 Chl catabolites were detected, and in extracts of leaves of the apricot tree 16 Chl catabolites were found. The seven major NCC fractions in the leaves of both fruit tree species were identical and displayed known structures. All of the major Chl catabolites were also found in leaf extracts from AP- or ESFY-infected trees, providing the first evidence that the PaO/PB pathway is relevant also for pathogen-induced chlorosis. This work supports the hypothesis that Chl breakdown in senescence and phytoplasma infection proceeds via a common pathway in some members of the Rosaceae family.

Topics: Chlorophyll; Malus; Oxygenases; Phytoplasma; Plant Diseases; Plant Leaves; Plant Proteins; Prunus armeniaca; Trees

During hyperglycemia, the first step toward the formation of advanced glycation end products is the nonenzymatic glycation between the carbonyl group of a sugar and the primary amino group of a protein. Advanced glycation end products are then produced through more complex reactions. Reactive oxygen species derived from advanced glycation end products may play a key role in inflammation of the endothelium, leading to the complications seen in diabetes. Glycolaldehyde-induced advanced glycation end products have been reported to express proinflammatory cytokines, such as tumor necrosis factor-α and interleukin-1β. This study focused on Capsosiphon fulvescens, a Capsosiphonaceae type of green algae that has shown potential as a functional food material. Pheophorbide a, an anti-glycation compound, was isolated from C. fulvescens by extraction using a mixture of ethanol and water, followed by column fractionation of the resulting extract. The compound separated from C. fulvescens was identified by means of high-performance liquid chromatography combined with mass spectrometry. Pheophorbide a showed scavenging activity of the intracellular reactive oxygen species as well as monocyte adhesiveness inhibitory activity on the human myelomonocytic cell line (THP-1) and human umbilical vein endothelial cells cocultivation system. The mRNA levels of inflammation-related genes such as monocyte chemoattractant protein-1 and interleukin-6 were significantly decreased by pheophorbide a, and advanced glycation end products-stimulated tumor necrosis factor-α and interleukin-1β were downregulated as well. These results indicate that pheophorbide a has significant reactive oxygen species-scavenging activity, monocyte adhesive inhibitory activity, and downregulatory activity of cytokines related to inflammation affecting the endothelium. Pheophorbide a could therefore be a promising candidate for modulating endothelial cell dysfunction.

Topics: Atherosclerosis; Cell Adhesion; Chlorophyll; Chlorophyta; Cytokines; Endothelial Cells; Glycation End Products, Advanced; Human Umbilical Vein Endothelial Cells; Humans; Inflammation; Monocytes; Reactive Oxygen Species

Light-triggered drug delivery is among the most investigated stimulus-response strategies and has been widely explored in cancer treatment. However, the limited specificity of light-triggered drug delivery reduces the therapeutic efficacy and causes considerable undesirable side effects. In this work, we demonstrate a highly tumor-specific light-triggerable drug carrier (H-LTDC) induced by a combination of internal (i.e., tumor hypoxia) and external stimuli (i.e., light). The doxorubicin (DOX)-loaded H-LTDC was self-assembled from type-1-reactive oxygen species (ROStype1)-mediated degradable chondroitin sulfate (CS) conjugated with a photosensitizer (PS), Pheophorbide-a, which has a spherical shape and a uniform size distribution. Under hypoxic conditions, ROSType1 was mainly generated due to the electron-rich sulfate groups in the polysaccharide backbone. The ROStype1 generated by H-LTDC allowed laser-triggered drug release at low oxygen concentrations. From the in vitro cytotoxicity tests with colon cancer cells (HCT-116), under laser irradiation, DOX-loaded H-LTDCs showed higher toxicity under hypoxic conditions than that under normoxic conditions. In vivo and ex vivo biodistribution studies demonstrated that H-LTDCs selectively accumulated in the tumor tissues. As a result, drug-loaded H-LTDCs exhibited high anti-tumor activity in vivo. Overall, we believe that this approach could represent a promising platform for the treatment of tumor and hypoxia-associated diseases without undesirable side effects.

Topics: Animals; Antibiotics, Antineoplastic; Carbohydrate Conformation; Cell Hypoxia; Chlorophyll; Chondroitin Sulfates; Doxorubicin; Drug Carriers; Drug Delivery Systems; Energy Transfer; HCT116 Cells; Humans; Hyaluronic Acid; Lasers, Semiconductor; Light; Mice; Mice, Nude; Nanoparticles; Optical Fibers; Oxidation-Reduction; Photochemotherapy; Photosensitizing Agents; Polysaccharides; Random Allocation; Reactive Oxygen Species; Singlet Oxygen; Xenograft Model Antitumor Assays

Carica papaya leaf decoction, an Australian Aboriginal remedy, has been used widely for its healing capabilities against cancer, with numerous anecdotal reports. In this study we investigated its in vitro cytotoxicity on human squamous cell carcinoma cells followed by metabolomic profiling of Carica papaya leaf decoction and leaf juice/brewed leaf juice to determine the effects imparted by the long heating process typical of the Aboriginal remedy preparation. MTT assay results showed that in comparison with the decoction, the leaf juice not only exhibited a stronger cytotoxic effect on SCC25 cancer cells, but also produced a significant cancer-selective effect as shown by tests on non-cancerous human keratinocyte HaCaT cells. Furthermore, evidence from testing brewed leaf juice on these two cell lines suggested that the brewing process markedly reduced the selective effect of Carica papaya leaf on SCC25 cancer cells. To tentatively identify the compounds that contribute to the distinct selective anticancer activity of leaf juice, an untargeted metabolomic approach employing Ultra High Performance Liquid Chromatography-Quadrupole Time of Flight-Mass Spectrometry followed by multivariate data analysis was applied. Some 90 and 104 peaks in positive and negative mode respectively were selected as discriminatory features from the chemical profile of leaf juice and >1500 putative compound IDs were obtained via database searching. Direct comparison of chromatographic and tandem mass spectral data to available reference compounds confirmed one feature as a match with its proposed authentic standard, namely pheophorbide A. However, despite pheophorbide A exhibiting cytotoxic activity on SCC25 cancer cells, it did not prove to be the compound contributing principally to the selective activity of leaf juice. With promising results suggesting stronger and more selective anticancer effects when compared to the Aboriginal remedy, Carica papaya leaf juice warrants further study to explore its activity on other cancer cell lines, as well as investigation to confirm the identity of compounds contributing to its selective effect, particularly those compounds altered by the long heating process applied during the traditional Aboriginal remedy preparation.

Topics: Carcinoma, Squamous Cell; Carica; Cell Death; Cell Line, Tumor; Cell Survival; Chlorophyll; Chromatography, Liquid; Humans; Keratinocytes; Mass Spectrometry; Metabolomics; Multivariate Analysis; Native Hawaiian or Other Pacific Islander; Plant Extracts; Plant Leaves; Reference Standards

Five series of pyrrolo[3,2-d]pyrimidines were synthesized and evaluated with respect to potency and selectivity toward multidrug resistance-associated protein 1 (MRP1, ABCC1). This transport protein is a major target to overcome multidrug resistance in cancer patients. We investigated differently substituted pyrrolopyrimidines using the doxorubicin selected and MRP1 overexpressing small cell lung cancer cell line H69 AR in a calcein AM and daunorubicin cell accumulation assay. New compounds with high potency and selectivity were identified. Piperazine residues at position 4 bearing large phenylalkyl side chains proved to be beneficial for MRP1 inhibition. Its replacement by an amino group led to decreased activity. Aliphatic and aliphatic-aromatic variations at position 5 and 6 revealed compounds with IC50 values in high nanomolar range. All investigated compounds had low affinity toward P-glycoprotein (P-gp, ABCB1). Pyrrolopyrimidines with small substituents showed moderate inhibition against breast cancer resistance protein (BCRP, ABCG2).

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line, Tumor; Chlorophyll; Daunorubicin; Dogs; Doxorubicin; Drug Evaluation, Preclinical; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Fluoresceins; Humans; Inhibitory Concentration 50; Madin Darby Canine Kidney Cells; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Pyridines; Pyrroles; Structure-Activity Relationship

Chlorophyll degradation naturally occurs during plant senescence. However, in fruit such as citrus, it is a positive characteristic, as degreening is an important colour development contributing to fruit quality. In the present work, Citrus sinensis Osbeck, cv. Newhall fruit was used as a model for chlorophyll degradation. An ethylene response factor, CitERF13, was isolated and its transcriptional changes were closely correlated with fruit peel degreening during development or in response to ethylene. Dual-luciferase and yeast one-hybrid assays, as well as motif mutation, indicated that CitERF13 directly binds to the CitPPH promoter and enhances its activity. Transient and stable over-expression of CitERF13 resulted in rapid chlorophyll degradation in Nicotiana tabacum leaves and led to accumulation of pheophorbide (Pheide) a, a metabolite of pheophorbide hydrolase (PPH). Similar results were observed from transient transformation of CitERF13 in citrus fruit peel. Moreover, this function of CitERF13 was conserved within Arabidopsis and tomato, as the homologs AtERF17 and SlERF16 similarly acted as activators of PPH genes and accelerators of chlorophyll degradation.

Topics: Chlorophyll; Citrus sinensis; Ethylenes; Fruit; Gene Expression Regulation, Plant; Nicotiana; Plant Growth Regulators; Plant Proteins; Receptors, Cell Surface

Photosensitization, also known as photodermatitis, occurs when phototoxic or photoactive substances accumulate in the skin and interact with sunlight to result in an often severe, crusting, itching or painful dermatitis in unpigmented and/or lightly haired areas of the skin. Primary photosensitization, caused by direct ingestion of photosensitizing agents, has been reported anecdotally in horses after ingestion of alfalfa hay. Between 2004 and 2014, several large outbreaks of primary photosensitization in horses fed primarily alfalfa hay were investigated in California. Alfalfa hay samples were collected and carefully examined for the presence of known photosensitizing plants and pesticide residues but none were identified. Select hay samples were evaluated for unusual fungal infestation and for phototoxicity assay using a specific Candida albicans assay; results were negative. In the 2004 outbreak, a feeding study was conducted with three horses exclusively fed alfalfa hay that was suspected to have caused the outbreak. Two weeks after ingestion of alfalfa hay, two horses developed several lesions in non-pigmented skin characterized as chronic ulcerative and necrotizing dermatitis with superficial vasculitis, which was consistent with photosensitization. In the 2014 outbreak, seven different implicated alfalfa hay samples were analyzed for chlorophyll a and b, and pheophorbide a. These compounds had been suspected to play a role in alfalfa-induced primary photosensitization. The chlorophyll contents ranged from 0.90 to 2.30 mg/g in the alfalfa hay samples, compared to 1.37 and 2.94 mg/g in locally grown alfalfa and orchard grass hay. The pheophorbide a levels ranged from 3.36 to 89.87 µg/g in alfalfa samples compared to 81.39 and 42.33 µg/g in control alfalfa and orchard grass hay samples. These findings eliminate chlorophyll a, chlorophyll b, and pheophorbide a as possible causes for alfalfa-hay induced primary photosensitization.

Topics: Animal Feed; Animals; California; Chlorophyll; Chlorophyll A; Diet; Disease Outbreaks; Female; Horse Diseases; Horses; Male; Medicago sativa; Photosensitivity Disorders; Silage

The genus Carpinus of Betulaceae is the most widely distributed in the European landscape. This study reports a comparative study based on the pheophorbide a and flavonoid content from the two main species of the genus Carpinus, Carpinus betulus and Carpinus turczaninowii, respectively, in Nanjing, China. The pheophorbide a and flavonoid content depends on the organ, species, and season. HPLC analysis showed that the pheophorbide a and flavonoid levels were the highest in May and June, respectively, from the leaves of C. betulus 'Fastigiata.' In contrast, the content of pheophorbide a and flavonoid in the stems of C. betulus 'Fastigiata' or in other species was low. The immunosuppressive effects of the ethyl acetate extracts and methanol extracts from the two Carpinus species were also evaluated. The ethyl acetate extracts of C. betulus 'Fastigiata' in May and the methanol extracts of C. betulus 'Fastigiata' in June showed better immunosuppressive activity than in other seasons, which coincided with the content of pheophorbide a and flavonoid, respectively. Our findings indicated that C. betulus 'Fastigiata' can serve as a medicinal plant against inflammation because of its pheophorbide a and flavonoid content.

Topics: Animals; Betulaceae; Cell Line; Cell Survival; China; Chlorophyll; Chromatography, High Pressure Liquid; Flavonoids; Immunosuppressive Agents; Lymphocyte Activation; Mice, Inbred BALB C; Plant Extracts; Seasons; Species Specificity; T-Lymphocytes

The objective of this study was to develop an active-targeted, pH-responsive albumin-photosensitizer-incorporated graphene oxide nanocomplex as an image-guided theranostic agent for dual therapies. Herein, bovine serum albumin (BSA)-cis-aconityl pheophorbide-a (c-PheoA) conjugate was complexed with graphene oxide (GO) at ratios of 1:1, 1:0.5, and 1:0.1 with the mean hydrodynamic diameter of the resulting complex being 100-200nm. Further, with the 1:0.5 ratio, we developed a folate-BSA-c-PheoA conjugate:GO complex incorporated free PheoA (PheoA+GO:FA-BSA-c-PheoA NC) with a mean hydrodynamic diameter of 182.0±33.2nm. The release study showed that the photosensitizer from the nanocomplex was released rapidly at pH5.5 compared to that at pH7.4 when incubated for 24h. Cellular uptake results showed that the PheoA+GO:FA-BSA-c-PheoA NCs was readily taken up by B16F10 and MCF7 cancer cells. In vitro phototoxicity results showed that PheoA+GO:FA-BSA-c-PheoA NC has a higher efficacy against cancer cells than free PheoA, thereby demonstrating the synergistic effect of PS and GO in response to a single laser of 670nm. In vivo and ex vivo bioimaging results showed that fluorescence signals of higher intensity were observed in the tumor area of mice treated with PheoA+GO:FA-BSA-c-PheoA NC than those in the tumor of mice treated with free PheoA, thereby suggesting that the targeted nanocomplex selectively accumulated in the tumor area compared to free PheoA. Through antitumor study, PheoA+GO:FA-BSA-c-PheoA NC showed a synergistic effect in tumor-bearing mice by a single 671nm laser treatment. These results demonstrate that our prepared PheoA+GO:FA-BSA-c-PheoA NC can be used as a theranostic agent in phototherapies and for the photodiagnosis of cancer.

Topics: Aconitic Acid; Animals; Chlorophyll; Drug Liberation; Folic Acid; Graphite; Humans; Lasers; MCF-7 Cells; Melanoma, Experimental; Mice; Mice, Nude; Microscopy, Confocal; Nanoconjugates; Oxides; Photochemotherapy; Photosensitizing Agents; Serum Albumin, Bovine; Xenograft Model Antitumor Assays

Photodynamic therapy (PDT) has been considered to be a possible candidate approach for the treatment of multidrug-resistant (MDR) cancer. To investigate the photocytotoxicity of pheophorbide a-based PDT on MDR cells, the intracellular pathways were studied using the human oral cancer FaDu cell line and its paclitaxel-selected subline FaDu-PTX. Pheophorbide a (Pa)-PDT induced significant photocytotoxicity in both FaDu and FaDu-PTX cell lines with cell apoptosis greater in FaDu cells compared to FaDu-PTX cells. We found that Hoxc6 and MDR-1 expression was strongly detected in FaDu-PTX cells compared to FaDu cells. Intriguingly, Pa-PDT effectively reduced Hoxc6 and MDR-1 expression in FaDu-PTX cells. The siRNA for HOXC6 can inhibit the intracellular MDR-1 levels in FaDu-PTX cells and induce the phototoxic effects of Pa-PDT. Furthermore, our in vivo studies showed that the Pa-PDT and HOXC6 siRNA significantly reduce the growth of FaDu-PTX xenograft tumors in C3H mice compared with control- and PTX-treated tumors. Histopathology was also used to confirm this antitumor effect. Pa-PDT may be a potential therapeutic modality for multidrug-resistant cancer, and Hoxc6, as a possible contributor to MDR, may reduce the therapeutic potential in multidrug-resistant oral malignancies.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; Cell Line, Tumor; Chlorophyll; Combined Modality Therapy; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Homeodomain Proteins; Humans; Mice; Mice, Inbred C3H; Paclitaxel; Pharyngeal Neoplasms; Pharynx; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Xenograft Model Antitumor Assays

Drug delivery by nanovectors involves numerous processes, one of the most important being its release from the carrier. This point still remains unclear. The current work focuses on this point using poly(ethyleneglycol-b-ε-caprolactone) micelles containing either pheophorbide-a (Pheo-a) as a fluorescent probe and a phototoxic agent or fluorescent copolymers. This study showed that the cellular uptake and the phototoxicity of loaded Pheo-a are ten times higher than those of the free drug and revealed a very low cellular penetration of the fluorescence-labeled micelles. Neither loaded nor free Pheo-a displayed the same cellular localization as the labeled micelles. These results imply that the drug entered the cells without its carrier and probably without a disruption, as suggested by their stability in cell culture medium. These data allowed us to propose that Pheo-a directly migrates from the micelle to the cell without disruption of the vector. This mechanism will be discussed.

Topics: Cell Survival; Chlorophyll; Drug Carriers; Drug Evaluation, Preclinical; Drug Liberation; HCT116 Cells; Humans; Hydrophobic and Hydrophilic Interactions; Kinetics; Lactones; Micelles; Photosensitizing Agents; Polyethylene Glycols

Photodynamic therapy (PDT) with photosensitizer is one of the promising modalities for cancer treatment. For clinical use of PDT, screening process should be preceded to enhance sensitivity to PDT. Thus, we investigated a molecular biomarker to determine the sensitivity to pheophorbide a (Pa)-PDT in immortalized human oral keratinocytes (IHOK) and oral squamous cell carcinoma (OSCC) cell lines. Two IHOK and several OSCC cell lines were used. After Pa-PDT, cell viability was reduced by more than 50%, and reactive oxygen species were generated in IHOK and OSCC cell lines. Additionally, apoptosis occurred in PDT-treated cells. IHOK(S) and IHOK(P), the two IHOK cell lines derived from the same source, showed a difference in cytotoxicity after Pa-PDT. To explain this difference in cytotoxicity, we looked at the expression of Wnt signaling-related genes in these two cell lines, for the morphology of IHOK(S) which was spindle like and elongated and distinct from IHOK(P) and the parent cell. Among the relevant genes, runt-related transcription factor 3 (RUNX3), an apoptosis-related gene, was selected as a potential marker that confers sensitivity to PDT. We found that the cytotoxicity by Pa-PDT was proportional to RUNX3 expression in OSCC cell lines. Additionally, knockdown of RUNX3 expression reduced cytotoxicity by Pa-PDT, suggesting that RUNX3 might be a biomarker to determine sensitivity to Pa-PDT. This was the first study to find a new target molecule that enhances Pa-PDT effects in IHOK and OSCC cell lines. Hence, the development of a PDT-dependent biomarker could provide a novel approach to improve the effects of PDT on oral precancerous and cancerous lesions.

Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Line, Tumor; Chlorophyll; Core Binding Factor Alpha 3 Subunit; Gene Expression Regulation, Neoplastic; Humans; Keratinocytes; Mouth Neoplasms; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species; Signal Transduction; Wnt Proteins

Pheophorbide a (Pa) is a chlorine-based photosensitizer, and Pa-mediated photodynamic therapy (PDT) reportedly exhibits antitumor activity against various malignancies. The aim of our study was to investigate the therapeutic effect of Pa-mediated PDT on 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorobol-13-acetate (TPA)-induced mouse papillomas. Thirty mice received a topical application of DMBA/TPA on their backs to induce mouse papillomas. One week after two sessions of Pa-mediated PDT, immunohistochemical stains and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were performed to evaluate the apoptotic effects thereof on the papillomas. Among 63 mouse papillomas treated with Pa-mediated PDT, 17.5% of the lesions were completely removed 1 week after the first treatment, while 31.7% disappeared 1 week after the second treatment. Statistical analyses revealed significant differences in therapeutic outcomes for the Pa-mediated PDT group in comparison to a solvent-PDT group and a Pa group. Additionally, a marked downregulation of proliferating cell nuclear antigen expression, as well as upregulation of cleaved caspase 3 and cleaved poly(ADP-ribose) polymerase expression, was noted in the Pa-PDT group, compared to the solvent-PDT group and Pa group. TUNEL assay revealed higher apoptotic cell counts in the Pa-PDT group, although the difference was not statistically significant. Our data demonstrated that Pa-mediated PDT is effective in treating DMBA/TPA-induced mouse papillomas.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Apoptosis; Carcinogenesis; Caspase 3; Chlorophyll; Female; Immunohistochemistry; In Situ Nick-End Labeling; Mice, Hairless; Papilloma; Photochemotherapy; Photosensitizing Agents; Proliferating Cell Nuclear Antigen; Tetradecanoylphorbol Acetate

Runt-related transcription factor 3 (RUNX3) has recently been reported to be a possible predictor of sensitivity of cancer cells for photodynamic therapy (PDT), a promising therapeutic modality for keloids. In this study, we aimed to elucidate the implications of RUNX3 for keloid pathogenesis and sensitivity to pheophorbide a-based PDT (Pa-PDT). RUNX3 and proliferating cell nuclear antigen (PCNA) expression were examined in 6 normal skin samples and 32 keloid tissue samples by immunohistochemistry. We found that RUNX3 expression was detected more often in keloid tissues than in dermis of normal skin. In keloid tissues, RUNX3 expression was significantly increased in patients presenting with symptoms of pain or pruritus, and was also significantly related to PCNA expression. The therapeutic effect of Pa-PDT was comparatively investigated in keloid fibroblasts (KFs) with and without RUNX3 expression. Significant differences were found after Pa-PDT between KFs with and without RUNX3 expression in cell viability, proliferative ability, type I collagen expression, generation of reactive oxygen species (ROS), and apoptotic cell death. In addition, RUNX3 expression was significantly decreased after Pa-PDT in KFs, and KFs with downregulation of RUNX3 showed significantly increased cell viability after Pa-PDT. Pa-PDT may be a potential therapeutic modality for keloids, and RUNX3, as a possible contributor to keloid pathogenesis, may improve sensitivity to Pa-PDT in KFs.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Apoptosis; Cell Proliferation; Cell Survival; Child; Chlorophyll; Collagen Type I; Core Binding Factor Alpha 3 Subunit; Down-Regulation; Female; Fibroblasts; Humans; Keloid; Male; Middle Aged; Photochemotherapy; Proliferating Cell Nuclear Antigen; Reactive Oxygen Species; Skin; Young Adult

It has been reported that intestinal urate excretion is increased at chronic kidney disease (CKD) state. In this report, whether uremic toxins are involved in the upregulation of intestinal breast cancer resistance protein (BCRP), an intestinal urate exporter, was examined.. Uremic toxins that were increased at least 15-fold at CKD state were selected for investigation. Caco-2 cells were exposed to these uremic toxins at clinically relevant concentrations. mRNA was quantified by real-time PCR, and flow cytometry was utilized to measure BCRP protein and function in Caco-2 cells. Transcellular secretory transport of [(14) C]urate was determined utilizing Transwell studies after uremic toxin exposure.. Indoxyl sulfate (IS) treatment alone resulted in ∼ 3-fold increase in BCRP mRNA in Caco-2 cells. Membrane protein expression of BCRP in Caco-2 cells also was increased by 1.8-fold after treatment with IS. Intracellular accumulation of pheophorbide A, a selective BCRP substrate, was decreased by 22% after IS treatment for 3 days. Consistent with these findings, transcellular secretory transport of urate across Caco-2 cell monolayers was increased by 22%.. Intestinal urate secretion may be increased at CKD state partially by upregulation of intestinal BCRP by uremic toxins such as IS.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Transport; Breast Neoplasms; Caco-2 Cells; Chlorophyll; Humans; In Vitro Techniques; Indican; Intestinal Mucosa; Intestinal Secretions; Kidney; Neoplasm Proteins; Real-Time Polymerase Chain Reaction; Renal Insufficiency, Chronic; RNA, Messenger; Up-Regulation; Uric Acid

A polyelectrolyte nanocomplex was prepared via the ionic interaction between the anionic heparin-pheophorbide a (HPhA) conjugate, which served as a water-soluble polysaccharide photosensitizer (PS), and the cationic polyethylenimine (PEI)-β-carotene (PCAR) conjugate, which served as a polymeric scavenger. This nanocomplex was designed to improve the water solubility and tumor specificity of PhA and to only release singlet oxygen at the tumor cell. A stable 150 nm-sized nanocomplex could be formed in the weight ratio range (PCAR/HPhA) of 0.3-0.5 in an aqueous environment. The PCAR scavenger significantly diminished the generation of active singlet oxygen from HPhA in a buffer solution. Singlet oxygen scavenging activity was lost only when HPhA and PCAR were separated from each other due to the dissociation of the complex nanostructures. It was confirmed that HPhA itself has neither colloidal properties nor a decrease in its ability to produce singlet oxygen. At the same time, the HPhA/PEI complex produced singlet oxygen in response to light. In a cell culture system, the cytotoxicity of the HPhA/PCAR nanocomplex toward cancer cells was greatly enhanced due to the efficient generation of singlet oxygen under light irradiation; this finding implies that the scavenging activity of PCAR can be restricted to intracellular environments. These results suggest that the HPhA/PCAR nanocomplex could provide a new activatable PS platform that facilitates more accurate and reliable photodynamic therapy (PDT) with site-specific and controllable production of singlet oxygen to be used for the treatment of cancer.

Topics: beta Carotene; Chlorophyll; Free Radical Scavengers; Heparin; Humans; MCF-7 Cells; Nanoparticles; Particle Size; Photochemotherapy; Photosensitizing Agents; Polyethyleneimine; Singlet Oxygen; Solubility; Water

Photodynamic therapy (PDT) is a clinically approved treatment that causes a selective cytotoxic effect in cancer cells. In addition to the production of singlet oxygen and reactive oxygen species, PDT can induce the release of nitric oxide (NO) by up-regulating nitric oxide synthases (NOS). Since non-optimal PDT often causes tumor recurrence, understanding the molecular pathways involved in the photoprocess is a challenging task for scientists. The present study has examined the response of the PC3 human metastatic prostate cancer cell line following repeated low-dose pheophorbide a treatments, mimicking non-optimal PDT treatment. The analysis was focused on the NF-kB/YY1/RKIP circuitry as it is (i) dysregulated in cancer cells, (ii) modulated by NO and (iii) correlated with the epithelial to mesenchymal transition (EMT). We hypothesized that a repeated treatment of non-optimal PDT induces low levels of NO that lead to cell growth and EMT via the regulation of the above circuitry. The expressions of gene products involved in the circuitry and in EMT were analyzed by western blot. The findings demonstrate the cytoprotective role of NO following non-optimal PDT treatments that was corroborated by the use of L-NAME, an inhibitor of NOS.

Topics: Cell Line, Tumor; Chlorophyll; Dose-Response Relationship, Drug; Epithelial-Mesenchymal Transition; Humans; Male; Models, Biological; Nitric Oxide; Nitric Oxide Synthase Type II; Photochemotherapy; Prostatic Neoplasms; Radiation-Sensitizing Agents

In this study, photosensitizer conjugated iron oxide nanoparticles were strategically designed and prepared for simultaneous PDT and dual-mode fluorescence/MR imaging. The MRI contrast agent Fe3O4 was modified by APTES to functionalize the surface and further to link with heparin-pheophorbide-A conjugates.

Topics: Cell Death; Cell Survival; Chlorophyll; Contrast Media; Ferric Compounds; Fluorescence; Heparin; Humans; KB Cells; Magnetic Resonance Imaging; Nanoparticles; Photochemotherapy; Photosensitizing Agents

Pheophorbide-a, a non-selective photosensitizer, was conjugated with cancer-targeting moieties, such as folic acid, the CRGDLASLC peptide, the cRGDfK peptide and leuprorelin, for the purpose of targeted photodynamic cancer therapy. The cellular uptake of pheophorbide-a conjugates in cancer cells overexpressing the corresponding receptors of the targeting moieties was largely enhanced compared with that in the receptor-negative cells. In the study of in vitro photodynamic activity and selectivity of pheophorbide-a conjugates in the receptor-positive and receptor-negative cells, a pheophorbide-a conjugate, (14) with an αvβ6 ligand (CRGDLASLC) exhibited the highest selectivity in the positive FaDu cells. Targeted PDT with 14 induced cell death through apoptosis and morphological apoptosis-like characteristics. These results suggest that pheophorbide-a conjugate 14 could be utilized in selective photodynamic therapy for oral cancers primarily expressing the αvβ6 receptor.

Topics: Cell Line, Tumor; Cell Survival; Chlorophyll; Dose-Response Relationship, Drug; Drug Delivery Systems; Humans; Photochemotherapy; Photosensitizing Agents

The accurate diagnosis and proper therapy for cancer are essential to improve the success rate of cancer treatment. Here, we demonstrated that the vitamin Bc -bearing hydrophilic photosensitizer conjugate folic acid-polyethylene glycol-pheophorbideA (FA-PEG-PheoA) has been synthesized for the intracellular diagnosis and photodynamic therapy of a tumor. The synthesized vitamin Bc -bearing hydrophilic photosensitizer conjugate has been characterized for the folic acid receptor expressing the ability to target tumor cells, which is facilitated by the chemical conjugation with folic acid. The vitamin Bc -bearing hydrophilic photosensitizer conjugate internalization mechanism was identified through a competitive inhibition test with free folic acid. We optimized the laser-sensitive, cytotoxicity changeable, vitamin Bc -bearing hydrophilic photosensitizer conjugate concentration, which is non-cytotoxic under normal conditions and specifically cytotoxic toward cancer cells (maximum 69.15%) under laser irradiation conditions used for theranostic agents. The cancer therapeutic and diagnosis effects of synthesized conjugate were confirmed in MDA-MB-231 cells and MDA-MB-231-bearing mice. As a result, the vitamin Bc -bearing hydrophilic photosensitizer conjugate exhibited a highly photodynamic therapeutic effect, which enabled the selective detection of a folic acid receptor expressing cancer using optical imaging.

Topics: Animals; Cell Line, Tumor; Chlorophyll; Folic Acid; Humans; Hydrophobic and Hydrophilic Interactions; Mice; Nanoparticles; Neoplasms; Photochemotherapy; Photosensitizing Agents; Polyethylene Glycols; Theranostic Nanomedicine; Thiamine; Xenograft Model Antitumor Assays

Overcoming multidrug resistance (MDR) in cancer is a major challenge and efforts are on-going to develop inhibitors against the most characterized and ubiquitous MDR transporters: P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP1) and breast cancer resistance protein (BCRP). Recently reported, two 4-anilinoquinazolines (compounds 1 and 2), demonstrate potential MDR reversal activity against BCRP and to a lesser extent, P-gp. In this work, we formulated the compounds as polymeric nanoparticles (NPs) and assessed their MDR inhibitory activity in relevant BCRP and P-gp over-expressing cell line models. Particles in the size range 300-365nm with a loading efficiency of 69% (compound 1 NP) and 77% (compound 2 NP) respectively were obtained. BCRP inhibition was observed in Hoechst 33342 and pheophorbide A assays while P-gp inhibition was evaluated in calcein AM and rhodamine-123 assays. In cytotoxicity studies, while BCRP expressing cells showed complete reversal of drug resistance in nearly all treatment groups (both compounds and their respective NP); a higher reversal in NP treated group was obtained as compared with inhibitory compound treated group in P-gp expressing cells. These results demonstrate promising inhibitory activity of both formulations, especially against P-gp expressing cells; which is possibly due to a prolonged presence of encapsulated compounds in NPs and consequently a prolonged sensitization of transmembrane drug transporter. These formulations can therefore be considered as dual-transporter inhibitors and it is imperative to investigate both inhibitors in animal models of MDR owing to the presence of multiple efflux transporters in several cancer models.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzimidazoles; Camptothecin; Chemistry, Pharmaceutical; Chlorophyll; Dogs; Doxorubicin; Irinotecan; Madin Darby Canine Kidney Cells; Microscopy, Confocal; Nanoparticles; Neoplasm Proteins; Rhodamine 123

Fluorescence spectra and fluorescence lifetime images of talaporfin sodium and sodium-pheophorbide a, which can be regarded as photosensitizers for photodynamic therapy, were measured in normal and cancer cells. The reduction of the fluorescence intensity by photoirradiation was observed for both photosensitizers in both cells, but the quenching rate was much faster in cancer cells than in normal cells. These results are explained in terms of the excessive generation of reactive oxygen species via photoexcitation of these photosensitizers in cancer cells. The fluorescence lifetimes of both photosensitizers in cancer cells are different from those in normal cells, which originates from the different intracellular environments around the photosensitizers between normal and cancer cells.

Topics: Animals; Cell Line, Tumor; Chlorophyll; Photosensitizing Agents; Porphyrins; Rats; Spectrometry, Fluorescence

Prostate cancer (PC) represents the most common type of cancer among males and is the second leading cause of cancer death in men in Western society. Current options for PC therapy remain unsatisfactory, since they often produce uncomfortable long-term side effects, such as impotence (70%) and incontinence (5-20%) even in the first stages of the disease. Light-triggered therapies, such as photodynamic therapy, have the potential to provide important advances in the treatment of localized and partially metastasized prostate cancer. We have designed a novel molecular conjugate (DR2) constituted of a photosensitizer (pheophorbide a, Pba), connected to a nonsteroidal anti-androgen molecule through a small pegylated linker. This study aims at investigating whether DR2 represents a valuable approach for PC treatment based on light-induced production of single oxygen and nitric oxide (NO) in vitro. Besides being able to efficiently bind the androgen receptor (AR), the 2-trifluoromethylnitrobenzene ring on the DR2 backbone is able to release cytotoxic NO under the exclusive control of light, thus augmenting the general photodynamic effect. Although DR2 is similarly internalized in cells expressing different levels of androgen receptor, the AR ligand prevents its efflux through the ABCG2-pump. In vitro phototoxicity experiments demonstrated the ability of DR2 to kill cancer cells more efficiently than Pba, while no dark toxicity was observed. Overall, the presented approach is very promising for further development of AR-photosensitizer conjugates in the multimodal photodynamic treatment of prostate cancer.

Topics: Androgen Antagonists; Antineoplastic Agents; Chlorophyll; Humans; In Vitro Techniques; Male; Photochemotherapy; Photosensitizing Agents; Prostatic Neoplasms; Receptors, Androgen; Tumor Cells, Cultured

Integrated diagnosis and therapy systems that can offer traceable cancer therapy are in high demand for personalized medicine. Herein, a pH-responsive polymeric probe containing tetraphenylsilole (TPS) with aggregation-induced emission characteristics and pheophorbide A (PheA) photosensitizer (PS) with aggregation-caused quenching property for tracing the whole process of cancer therapy is reported. At physiological conditions (pH 7.4), the probe self-assembles into nanoparticles (NPs), which show weak fluorescence of PheA with low phototoxicity, but strong green fluorescence from TPS for probe self-tracking. Upon uptake by cancer cells and entrapment in lysosomes (pH 5.0), the NPs disassemble to yield weak emission of TPS but strong red fluorescence of PheA with restored phototoxicity for PS activation monitoring. Upon light irradiation, the generated reactive oxygen species can cause lysosomal disruption to trigger cell apoptosis. Meanwhile, the probe leaks to the cytoplasm (pH 7.2), where the TPS fluorescence is restored for in situ visualization of the therapeutic response. The probe design thus represents a novel strategy for traceable cancer therapy.

Topics: Apoptosis; Cell Line, Tumor; Chlorophyll; Cytoplasm; Cytosol; Fluorescent Dyes; Green Fluorescent Proteins; HEK293 Cells; Humans; Hydrogen-Ion Concentration; Lysosomes; MCF-7 Cells; Microscopy, Fluorescence; Nanoparticles; Neoplasms; Photosensitizing Agents; Polylysine; Polymers; Precision Medicine; Reactive Oxygen Species

In recent years, the photodynamic therapy studies have gained considerable attention as an alternative method to surgery, chemotherapy and radiotherapy which is commonly used to fight cancer. In this study, biological potentials of a benzyloxy bearing zinc(II) pheophorbide-a (Zn-PH-A) were investigated via in vivo and in vitro experiments. Zn-PH-A was labeled with (131)I with high efficiency (95.3 ± 2.7%) and its biodistribution studies were investigated on female Albino Wistar rats. The radiolabeled photosensitizer had been intravenously injected into the tail vein, and then the animals were sacrificed at 30, 60 and 120 min post injection. The percent of radioactivity per gram of organs (%ID/g) was determined. The radiolabeled Zn-PH-A showed high uptake in ovary. In addition, photodynamic therapy studies of the photosensitizer were conducted in EMT6, murine mammary carcinoma and HeLa, human cervix carcinoma cell lines. For the photodynamic therapy studies, the cells with Zn-PH-A were exposed to red light (650 nm) at the doses of 10-30 J/cm(2). The results showed that Zn-PH-A has stronger PDT effect in EMT6 than HeLa cell. Our present work demonstrates (131)I-labeled photosensitizer as a bifunctional agent (PDT and nuclear imaging) which could be improved in future by using EMT6 growing tumor in nude mice.

Topics: Animals; Cell Line, Tumor; Chemistry, Pharmaceutical; Chlorophyll; Female; HeLa Cells; Humans; Hydrogen-Ion Concentration; Iodine Radioisotopes; Mice; Photochemotherapy; Photosensitizing Agents; Rats; Rats, Wistar; Zinc

Block-polymer nanoparticles are now well-known candidates for the delivery of various non-soluble drugs to cells. The release of drugs from these nanoparticles is a major concern related to their efficiency as nanovectors and is still not completely deciphered. Various processes have been identified, depending of both the nature of the block-polymer and those of the drugs used. We focused our interest on an amphiphilic photosensitizer studied for photodynamic treatments of cancer, Pheophorbide-a (Pheo). We studied the transfer of Pheo from poly(ethyleneglycol-b-ϵ-caprolactone) nanoparticles (I) to MCF-7 cancer cells and (II) to models of membranes. Altogether, our results suggest that the delivery of the major part of the Pheo by the nanoparticles occurs via a direct transfer of Pheo from the nanoparticles to the membrane, by collision. A minor process may involve the internalization of a small amount of the nanoplatforms by the cells. So, this research illustrates the great care necessary to address the question of the choice of such nanocarriers, in relation with the properties - in particular the relative hydrophobicity - of the drugs encapsulated, and gives elements to predict the mechanism and the efficiency of the delivery.

Topics: Chemistry, Pharmaceutical; Chlorophyll; Drug Carriers; Drug Delivery Systems; Drug Liberation; Humans; Hydrophobic and Hydrophilic Interactions; Lactones; MCF-7 Cells; Nanoparticles; Polyethylene Glycols; Radiation-Sensitizing Agents; Solubility

In a hybrid mixture of organic (dye) and inorganic (metallic nanoparticles) components, the optical properties of a dye can be easily controlled by tailoring the shape or the concentration of the noble metal nanoparticles (NPs). The influences of multiexcitation (multiwavelength excitation) of photosensitizers (pheophorbide a and hematoporphyrin) on the interactions with pegylated Au-NPs and on the photophysical parameters of the dyes are studied. Detailed, systematic fluorescence quenching studies were performed in the mixtures of different contents of Au-NPs, and interpreted together with the results of quantum singlet oxygen yield examinations. According to the results, the fluorescence of the two dyes studied was effectively quenched in the presence of Au-NPs, mainly because of the resonance energy transfer between the donor (dye) and the acceptor (Au-NPs). Stern-Volmer quenching constants were determined by a few orders of magnitude higher than those describing the photochemical quenching process. In hybrid mixtures analyzed, the mechanism of energy transfer between the donor and the acceptor was nanometal surface energy transfer. Furthermore, different behavior of the mixtures on excitation with the wavelengths from the Soret and Q bands of the dyes and with those corresponding to the surface plasmon resonance band of Au-NPs was analyzed. Moreover, for certain concentrations of Au-NPs and for certain excitation wavelengths, an increase in singlet oxygen generation was observed. The results obtained indicate the significance of further studies of photosensitizers in hybrid mixtures with NPs.

Topics: Chlorophyll; Fluorescent Dyes; Gold; Hematoporphyrins; Metal Nanoparticles; Optical Phenomena; Photosensitizing Agents; Singlet Oxygen

Compositions and contents of sedimentary pigments were examined using high performance liquid chromatography in order to discuss the spatial distributions of phytoplankton primary production, phytoplankton functional type and the preservation efficiency of phytoplankton pigments and their influencing factors. The results showed that: chloropigments [Chlorins, including chlorophyll-a (Chl-a) and pheopigments (Pheo-a), such as pheophytin-a (PHtin-a), pheophorbide-a (PHide-a), pPheophytin-a (pPHtin-a), sterol chlorin esters (SCEs) and carotenol chlorin esters (CCEs)] were the major type of sedimentary pigments. The nutrients inputs from Changjiang Diluted Water and upwelling in the Zhe-Min coastal mud area were the major cause for the patchy distribution with high sedimentary chloropigment contents. Carotenoid contents showed no trending changes and exhibited high values in the Changjiang Estuary and Zhe-Min Coasts. Based on the relative proportions of each diagnostic carotenoid to the total diagnostic carotenoids in the sediments, the relative contributions of diatoms, dinoflagellates, prymnesiophytes, prasinophytes, cryptophytes and cyanobacterias in the phytoplankton fuctional types were 48.8% +/- 17.4%, 10.7% +/- 11.5%, 8.1% +/- 7.2%, 18.6% +/- 8.2%, 9.4% +/- 6.4% and 4.3% +/- 3.2%, respectively. The preference for external environmental conditions (e.g., nutrient level and water salinity) was the main cause for the decreasing trends of diatoms and dinoflagellates proportions and the increasing trends of prasinophytes, cryptophytes and cyanobacterias seawards. Based on the spatial distribution of Chl-a/Pheo-a ratios, the higher preservation efficiencies of sedimentary pigments in the coastal regions (e.g., outer edge of maximum turbidity zone in the Changjiang Estuary, mouth of the Hangzhou Bay and upwelling region in the Zhe-Min Coast) were mainly due to the higher sedimentation rate and seasonal occurrences of hypoxia in bottom water, and these regions with higher sedimentary pigment preservation efficiencies were probably ideal areas for the marine eco-environmental evolutions. The bad sedimentary environment caused by the water exchange inside and outside of Hangzhou Bay was the dominant reason for the low sedimentary pigment contents and preservation efficiencies in this region.

Topics: Carotenoids; China; Chlorophyll; Chlorophyll A; Cryptophyta; Diatoms; Dinoflagellida; Estuaries; Geologic Sediments; Haptophyta; Phytoplankton; Porphyrins; Water

Pheophorbide a (Pa), a chlorophyll derivative, is a photosensitizer that can induce significant antitumor effects in several types of tumor cells. The present study investigated the mechanism of Pa-mediated photodynamic therapy (Pa-PDT) in the human skin cancer cell lines A431 and G361. PDT significantly inhibited the cell growth in a Pa-concentration-dependent manner. We observed increased expression of Beclin-1, LC3B and ATG5, which are markers of autophagy, after PDT treatment in A431 cells but not in G361 cells. In G361 cells, Pa-PDT strongly induced PARP cleavage and subsequent apoptosis, which was confirmed using Annexin V/Propidium iodide double staining. Pa-PDT predominantly exhibited its antitumor effects via activation of ERK1/2 and p38 in A431 and G361 cells, respectively. An in vivo study using the CAM xenograft model demonstrated that Pa-PDT strongly induced autophagy and apoptosis in A431-transplanted tumors and/or apoptosis in G361-transplanted tumors. These results may provide a basis for understanding the underlying mechanisms of Pa-PDT and for developing Pa-PDT as a therapy for skin cancer.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; Beclin-1; Cell Line, Tumor; Cell Proliferation; Chlorophyll; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Humans; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Membrane Proteins; Mice; Microtubule-Associated Proteins; p38 Mitogen-Activated Protein Kinases; Photochemotherapy; Photosensitizing Agents; Poly(ADP-ribose) Polymerases; Skin Neoplasms; Xenograft Model Antitumor Assays

The chlorophyll-related compound pheophorbide a (Pa) was successively purified from an edible red seaweed, Grateloupia elliptica, using silica, octadecyl silica column chromatography and reversed phase-high-performance liquid chromatography, as well as the cell cycle inhibitory and apoptotic effects of Pa being investigated in U87MG glioblastoma cells. The Pa exhibited strong anticancer effects in the absence of direct photo-irradiation against various cancer cell lines, including U87MG, SK-OV-3, and HeLa cells. Among the cancer cells, the strongest anticancer activity of Pa exhibited on U87MG cells with IC50 values of 2.8 μg/ml. In addition, Pa specifically had cytostatic activity on glioblastoma cells rather than human umbilical vein endothelial cells. Analysis of the cell cycle distribution showed that Pa induced G0/G1 arrest of U87 MG cells. In addition, arrested cells induced late apoptosis and DNA degradation under dark condition. These results suggest that Pa isolated from G. elliptica is a potential glioblastoma-specific anticancer agent without side effects on normal cells.

Topics: Antineoplastic Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; Chlorophyll; Cytostatic Agents; DNA Fragmentation; Endothelial Cells; Epithelial Cells; Humans; Inhibitory Concentration 50; Neurons; Rhodophyta

The ATP-binding cassette transporter ABCG2 plays a prominent role in cardiovascular and cancer pathophysiology, is involved in the pathogenesis of gout, and affects pharmacokinetics of numerous drugs. Telmisartan, a widely used AT1 receptor antagonist, inhibits the transport capacity of ABCG2 and may cause drug-drug interactions, especially in individuals carrying polymorphism that facilitate the telmisartan-ABCG2 interaction. Thus, the aim of this study was to identify ABCG2 polymorphisms and somatic mutations with relevance for the telmisartan-ABCG2 interaction. For this purpose, a cellular system for the conditional expression of ABCG2 was established. ABCG2 variants were generated via site-directed mutagenesis. Interaction of telmisartan with these ABCG2 variants was investigated in HEK293-Tet-On cells using the pheophorbide A efflux assay. Moreover, expression of ABCG2 variants was studied in these cells. Importantly, protein levels of the Q141K and F489L variant were significantly reduced, a phenomenon that was partly reversed by pharmacological proteasome inhibition. Moreover, basal pheophorbide A efflux capacity of S248P, F431L, and F489L variants was significantly impaired. Interestingly, inhibition of ABCG2-mediated pheophorbide A transport by telmisartan was almost abolished in cells expressing the R482G variant, whereas it was largely increased in cells expressing the F489L variant. We conclude that the arginine residue at position 482 of the ABCG2 molecule is of major importance for the interaction of telmisartan with this ABC transporter. Furthermore, individuals carrying the F489L polymorphism may be at increased risk of developing adverse drug reactions in multi-drug regimens involving ABCG2 substrates and telmisartan.

Topics: Angiotensin II Type 1 Receptor Blockers; Arginine; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzimidazoles; Benzoates; Binding Sites; Chlorophyll; Genetic Variation; HEK293 Cells; Humans; Mutagenesis, Site-Directed; Mutation; Neoplasm Proteins; Polymorphism, Genetic; Recombinant Proteins; Telmisartan

The development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still needed. Antiviral compounds in medicinal plants are potentially good targets to study. Morinda citrifolia is a common plant distributed widely in Indo-Pacific region; its fruits and leaves are food sources and are also used as a treatment in traditional medicine. In this study, using a HCV cell culture system, it was demonstrated that a methanol extract, its n-hexane, and ethyl acetate fractions from M. citrifolia leaves possess anti-HCV activities with 50%-inhibitory concentrations (IC(50)) of 20.6, 6.1, and 6.6 μg/mL, respectively. Bioactivity-guided purification and structural analysis led to isolation and identification of pheophorbide a, the major catabolite of chlorophyll a, as an anti-HCV compound present in the extracts (IC(50) = 0.3 μg/mL). It was also found that pyropheophorbide a possesses anti-HCV activity (IC(50) = 0.2 μg/mL). The 50%-cytotoxic concentrations (CC(50)) of pheophorbide a and pyropheophorbide a were 10.0 and 7.2 μg/mL, respectively, their selectivity indexes being 33 and 36, respectively. On the other hand, chlorophyll a, sodium copper chlorophyllin, and pheophytin a barely, or only marginally, exhibited anti-HCV activities. Time-of-addition analysis revealed that pheophorbide a and pyropheophorbide a act at both entry and the post-entry steps. The present results suggest that pheophorbide a and its related compounds would be good candidates for seed compounds for developing antivirals against HCV.

Topics: Antiviral Agents; Chlorophyll; Hepacivirus; Hepatitis C; Humans; Morinda; Plant Extracts; Plant Leaves

Various polymeric micelles were formed from amphiphilic block copolymers, namely, poly(ethyleneoxide-b-ε-caprolactone), poly(ethyleneoxide-b-d,l-lactide), and poly(ethyleneoxide-b-styrene). The micelles were characterized by static and dynamic light scattering, electron microscopy, and asymmetrical flow field-flow fractionation. They all displayed a similar size close to 20 nm. The influence of the chemical structure of the block copolymers on the stability upon dilution of the polymeric micelles was investigated to assess their relevance as carriers for nanomedicine. In the same manner, the stability upon aging was assessed by FRET experiments under various experimental conditions (alone or in the presence of blood proteins). In all cases, a good stability over 48 h for all systems was encountered, with PDLLA copolymer-based systems being the first to release their load slowly. The cytotoxicity and photocytotoxicity of the carriers were examined with or without their load. Lastly, the photodynamic activity was assessed in the presence of pheophorbide a as photosensitizer on 2D and 3D tumor cell culture models, which revealed activity differences between the 2D and 3D systems.

Topics: Cell Culture Techniques; Chlorophyll; Drug Carriers; Drug Stability; Fluorescence Resonance Energy Transfer; HCT116 Cells; Humans; Lactones; Light; Micelles; Photochemotherapy; Photosensitizing Agents; Polyesters; Polyethylene Glycols; Polymers; Scattering, Radiation; Structure-Activity Relationship

In the study presented here, we developed a bioreducible biarmed methoxy poly(ethylene glycol)-(pheophorbide a)2 (mPEG-(ss-PhA)2) conjugate for cancer-cell-specific photodynamic therapy (PDT). PhA molecules were chemically conjugated with biarmed linkages at one end of the mPEG molecule via disulfide bonds. Under aqueous conditions, the amphiphilic mPEG-(ss-PhA)2 conjugate self-assembled to form core-shell-structured nanoparticles (NPs) with good colloidal stability. The mPEG-(ss-PhA)2 NPs exhibited intramolecular and intermolecular self-quenching effects that enabled the NPs to remain photoinactive in a physiological buffer. However, the dissociation of the NP structure was effectively induced by the cleavage of the disulfide bonds in response to intracellular reductive conditions, triggering the rapid release of PhA molecules in a photoactive form. In cell-culture systems, in addition to significant phototoxicity and intracellular uptake, we observed that the dequenching processes of PhA in the mPEG-(ss-PhA)2 NPs highly depended on the expression of intracellular thiols and that supplementation with glutathione monoethylester facilitated more rapid PhA release and enhanced the PhA phototoxicity. These findings suggest that the bioreducible activation mechanism of mPEG-(ss-PhA)2 NPs in cancer cells can maximize the cytosolic dose of active photosensitizers to achieve high cytotoxicity, thereby enhancing the treatment efficacy of photodynamic cancer treatment.

Topics: Chlorophyll; Drug Carriers; Drug Delivery Systems; HeLa Cells; Humans; MCF-7 Cells; Photochemotherapy; Polyethylene Glycols

Poly(ethylene glycol)-b-poly(caprolactone) (PEG-b-PCL) micelles dually loaded with both pheophorbide a (PhA) as a photosensitizer and β-carotene (CAR) as a singlet oxygen ((1)O2) scavenger were designed to control photodynamic therapy (PDT) activity in cancer treatment. The CAR in the PhA/CAR micelles significantly diminished PhA-generated (1)O2 through direct (1)O2 scavenging, whereas the CAR molecules lost their (1)O2 scavenging activity when the PhA and CAR were spatially isolated by the disintegration of the PEG-b-PCL micelles. In cell-culture systems, light irradiation at a post-treatment time that corresponded to the presence of the micelles in the blood environment induced negligible phototoxicity, whereas light irradiation at a post-treatment time that corresponded to the presence of the micelles in the intracellular environment induced remarkable phototoxicity. In addition, a longer post-treatment time induced greater internalization of PhA/CAR micelles, which resulted in higher phototoxicity, suggesting an increase in photo killing activity against the tumor cells of interest. Thus, the co-loading of a (1)O2 generator and a (1)O2 scavenger into a single micelle is a potential strategy that may be useful in facilitating more accurate and reliable PDT with site-specific controllable production of singlet oxygen species for cancer treatment.

Topics: beta Carotene; Cell Survival; Chlorophyll; Delayed-Action Preparations; Drug Carriers; Drug Combinations; Drug Compounding; Ethylene Glycols; Free Radical Scavengers; HeLa Cells; Humans; MCF-7 Cells; Particle Size; Photochemotherapy; Photosensitizing Agents; Polyesters; Reactive Oxygen Species; Surface Properties

Photodynamic therapy (PDT) is a new therapeutic approach for the palliative treatment of malignant bile duct obstruction. In this study, we designed photosensitizer-embedded self-expanding nonvascular metal stent (PDT-stent) which allows repeatable photodynamic treatment of cholangiocarcinoma without systemic injection of photosensitizer. Polymeric photosensitizer (pullulan acetate-conjugated pheophorbide A; PPA) was incorporated in self-expanding nonvascular metal stent. Residence of PPA in the stent was estimated in buffer solution and subcutaneous implantation on mouse. Photodynamic activity of PDT-stent was evaluated through laserexposure on stent-layered tumor cell lines, HCT-116 tumor-xenograft mouse models and endoscopic intervention of PDT-stent on bile duct of mini pigs. Photo-fluorescence imaging of the PDT-stent demonstrated homogeneous embedding of polymeric Pheo-A (PPA) on stent membrane. PDT-stent sustained its photodynamic activities at least for 2 month. And which implies repeatable endoscopic PDT is possible after stent emplacement. The PDT-stent after light exposure successfully generated cytotoxic singlet oxygen in the surrounding tissues, inducing apoptotic degradation of tumor cells and regression of xenograft tumors on mouse models. Endoscopic biliary in-stent photodynamic treatments on minipigs also suggested the potential efficacy of PDT-stent on cholangiocarcinoma. In vivo and in vitro studies revealed our PDT-stent, allows repeatable endoscopic biliary PDT, has the potential for the combination therapy (stent plus PDT) of cholangiocarcinoma.

Topics: Animals; Bile Ducts; Chlorophyll; Cholangiocarcinoma; Endoscopy; Fluorescence; HeLa Cells; Humans; Metals; Mice; NIH 3T3 Cells; Photochemotherapy; Photosensitizing Agents; Polymers; Stents; Subcutaneous Tissue; Swine; Swine, Miniature; Time Factors; Xenograft Model Antitumor Assays

The purpose of this study was to investigate the effectiveness of photodynamic therapy (PDT) with Na-pheophorbide A in anticancer treatment, using osteosarcoma cells in vitro.. It has been reported that PDT with chlorophyll derivatives inhibits the proliferation of various cancer cells. However, there have been no reports that have evaluated the effectiveness of PDT in suppressing osteosarcoma cells.. Uptake of Na-pheophorbide A into Hu09 cells (osteosarcoma cells) was assayed using fluorescence microscopy following incubation of the cells with 28 μmol/L of Na-pheophorbide A. The viability of Hu09 cells after PDT treatment was assessed using trypan blue dye staining and MTS assays. PDT-induced apoptosis was determined by evaluation of the activity of selected members of the caspase family and by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining of cells.. Na-pheophorbide A uptake by cells was rapid, being observed after 60 min of treatment, and Na-pheophorbide A persisted in cells for >24 h. PDT treatment decreased cell viability compared with the control group, indicating high cytocidal activity of PDT. This cytocidal effect was dependent upon drug concentration, light dose, and the number of irradiation times. An increase in the number of cells positive for TUNEL staining and increases in the activity of caspases-3, -8 and -9 were observed in the first 2 h after PDT treatment.. A cytotoxic effect of PDT with Na-pheophorbide A on an osteosarcoma cell line in vitro was shown. Caspase activity assays suggested that PDT with Na-pheophorbide A induced an apoptotic change in HuO9 cells, mainly via activation of mitochondrial caspase -9 and -3 pathways.

Topics: Apoptosis; Bone Neoplasms; Caspase 3; Cell Line, Tumor; Cell Survival; Chlorophyll; Humans; In Situ Nick-End Labeling; In Vitro Techniques; Lasers, Semiconductor; Microscopy, Fluorescence; Osteosarcoma; Photochemotherapy; Radiation-Sensitizing Agents

Branched-type hydrophilic polyethyleneimine derivatives (i.e., bPEI derivatives) are developed polymeric carriers for photodynamic therapy. Their chemical structures which contain pH-tunable hydrophobic/hydrophilic cavities enable efficient loading of hydrophobic drugs in basic pH environments. Intracellular stimuli trigger the release of the loaded drugs in bPEI derivatives. As expected, the hydrophobic photosensitizer known as pheophorbide A (PheoA) is solubilized by physical loading in the inner hydrophobic spaces of bPEI derivatives in environments with basic pH values. Interestingly, acidic pH environments induce aggregation, resulting in poor release of the loaded PheoA as well as in quenched photo-activity of the PheoA-loaded polymers. However, when reducible polycation derivatives of bPEI are used (i.e., RPC-bPEI), intracellular thiols degrade the disulfide linkages in the polymers, resulting in rapid PheoA release. Particularly, a RPC-bPEI containing 6 wt% PheoA (i.e., RPC-bPEI(0.8 kDa)-PheoA(6%)) respond remarkably well to light exposure and display large differences between dark toxicity and light-induced toxicity. Cellular uptake of RPC-bPEI(0.8 kDa)-PheoA(6%) is approximately sevenfold to ninefold lower than that of free PheoA. Nevertheless, the photo-toxicity of RPC-bPEI(0.8 kDa)-PheoA(6%) was only two- to sixfold less potent than that of free PheoA. These results suggest that reducible bPEI materials may act as potential solubilizers and carriers for low-molecular-weight hydrophobic anti-cancer drugs.

Topics: Antibiotics, Antineoplastic; Cell Survival; Chlorophyll; Doxorubicin; Drug Carriers; Female; HeLa Cells; Humans; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Light; MCF-7 Cells; Oxidation-Reduction; Photochemotherapy; Photosensitizing Agents; Polyethyleneimine

Photodynamic therapy (PDT) has emerged as an effective treatment for various solid tumors. The transcription factor NRF2 is known to protect against oxidative and electrophilic stress; however, its constitutive activity in cancer confers resistance to anti-cancer drugs. In the present study, we investigated NRF2 signaling as a potential molecular determinant of pheophorbide a (Pba)-based PDT by using NRF2-knockdown breast carcinoma MDA-MB-231 cells. Cells with stable NRF2 knockdown showed enhanced cytotoxicity and apoptotic/necrotic cell death following PDT along with increased levels of singlet oxygen and reactive oxygen species (ROS). A confocal microscopic visualization of fluorogenic Pba demonstrated that NRF2-knockdown cells accumulate more Pba than control cells. A subsequent analysis of the expression of membrane drug transporters showed that the basal expression of BCRP is NRF2-dependent. Among measured drug transporters, the basal expression of breast cancer resistance protein (BCRP; ABCG2) was only diminished by NRF2-knockdown. Furthermore, after incubation with the BCRP specific inhibitor, differential cellular Pba accumulation and ROS in two cell lines were abolished. In addition, NRF2-knockdown cells express low level of peroxiredoxin 3 compared to the control, which implies that diminished mitochondrial ROS defense system can be contributing to PDT sensitization. The role of the NRF2-BCRP pathway in Pba-PDT response was further confirmed in colon carcinoma HT29 cells. Specifically, NRF2 knockdown resulted in enhanced cell death and increased singlet oxygen and ROS levels following PDT through the diminished expression of BCRP. Similarly, PDT-induced ROS generation was substantially increased by treatment with NRF2 shRNA in breast carcinoma MCF-7 cells, colon carcinoma HCT116 cells, renal carcinoma A498 cells, and glioblastoma A172 cells. Taken together, these results indicate that the manipulation of NRF2 can enhance Pba-PDT sensitivity in multiple cancer cells.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Breast Neoplasms; Cell Line, Tumor; Chlorophyll; Colonic Neoplasms; Female; Gene Knockdown Techniques; Gene Silencing; Genetic Vectors; Humans; Laser Therapy; Lasers; Lentivirus; Neoplasm Proteins; Neoplasms; NF-E2-Related Factor 2; Peroxiredoxin III; Photochemotherapy; Radiation-Sensitizing Agents; Reactive Oxygen Species; RNA, Small Interfering; Transduction, Genetic

Anti-inflammatory activity of Saccharina japonica and its active components was evaluated via in vitro inhibitory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) expression in RAW 264.7 murine macrophage cells. Since the methanolic extract of S. japonica showed strong anti-inflammatory activity, it was fractionated with several solvents. Among the fractions, the ethyl acetate fraction demonstrated the highest inhibition of LPS-induced NO production (IC50=25.32μg/mL), followed by the CH2Cl2 fraction (IC50=75.86μg/mL). Considering the yield and anti-inflammatory potential together, the CH2Cl2 fraction was selected for chromatographic separation to yield two active porphyrin derivatives, pheophorbide a and pheophytin a, together with an inactive fucoxanthin. In contrast to fucoxanthin, pheophorbide a and pheophytin a showed dose-dependent inhibition against LPS-induced NO production at nontoxic concentrations in RAW 264.7 cells. Both compounds also suppressed the expression of iNOS proteins, while they did not inhibit the COX-2 expression in LPS-stimulated macrophages. These results indicate that pheophorbide a and pheophytin a are two important candidates of S. japonica as anti-inflammatory agents which can inhibit the production of NO via inhibition of iNOS protein expression. Thus, these compounds hold great promise for use in the treatment of various inflammatory diseases.

Topics: Animals; Anti-Inflammatory Agents; Cell Line; Chlorophyll; Cyclooxygenase 2; Lipopolysaccharides; Macrophage Activation; Macrophages; Magnetic Resonance Spectroscopy; Mice; Nitric Oxide Synthase Type II; Phaeophyceae; Pheophytins

Nitric oxide (NO) has been known to preserve the level of chlorophyll (Chl) during leaf senescence. However, the mechanism by which NO regulates Chl breakdown remains unknown. Here we report that NO negatively regulates the activities of Chl catabolic enzymes during dark-induced leaf senescence. The transcriptional levels of the major enzyme genes involving Chl breakdown pathway except for RED CHL CATABOLITE REDUCTASE (RCCR) were dramatically up-regulated during dark-induced Chl degradation in the leaves of Arabidopsis NO-deficient mutant nos1/noa1 that exhibited an early-senescence phenotype. The activity of pheide a oxygenase (PAO) was higher in the dark-induced senescent leaves of nos1/noa1 compared with wild type. Furthermore, the knockout of PAO in nos1/noa1 background led to pheide a accumulation in the double mutant pao1 nos1/noa1, which retained the level of Chl during dark-induced leaf senescence. The accumulated pheide a in darkened leaves of pao1 nos1/noa1 was likely to inhibit the senescence-activated transcriptional levels of Chl catabolic genes as a feed-back inhibitory effect. We also found that NO deficiency led to decrease in the stability of photosynthetic complexes in thylakoid membranes. Importantly, the accumulation of pheide a caused by PAO mutations in combination with NO deficiency had a synergistic effect on the stability loss of thylakoid membrane complexes in the double mutant pao1 nos1/noa1 during dark-induced leaf senescence. Taken together, our findings have demonstrated that NO is a novel negative regulator of Chl catabolic pathway and positively functions in maintaining the stability of thylakoid membranes during leaf senescence.

Topics: Apoptosis Regulatory Proteins; Arabidopsis; Arabidopsis Proteins; Blotting, Western; Chlorophyll; Chloroplasts; Darkness; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Microscopy, Electron, Transmission; Mutation; Nitric Oxide; Nitric Oxide Synthase; Oxidoreductases; Oxygenases; Photosynthesis; Plant Leaves; Reverse Transcriptase Polymerase Chain Reaction; Thylakoids; Time Factors

Prolonged cancer chemotherapy is associated with the development of multidrug resistance (MDR), which is a major cause of treatment failure. Photodynamic therapy (PDT) has been applied as anticancer therapy and a means of circumventing MDR. The antiproliferative effect of pheophorbide a-mediated photodynamic therapy (Pa-PDT) has been demonstrated in several human cancer cell lines, including the uterine sarcoma cell line, MES-SA. This study set out to evaluate, first, the therapeutic potential of Pa-PDT on MES-SA/Dx5 uterine sarcoma cells and, subsequently, the effectiveness of combination therapy using Pa-PDT with doxorubicin (Dox). Our results showed that Pa-PDT was able to circumvent MDR in the P-glycoprotein (P-gp) overexpressing human uterine sarcoma cell line, MES-SA/Dx5. Intracellular accumulation of Pa and Pa-PDT-induced cell death was not abrogated by MDR phenotype, when compared to the parental cell line, MES-SA. Combined therapy using Pa-PDT and Dox, a common chemotherapeutic drug, was found to be synergistic in the cell line, MES-SA/Dx5. Both activity and expression of MDR1 and P-gp were reduced by Pa-PDT treatment and such reductions were attenuated by α-tocopherol, the scavenger of reactive oxygen species (ROS), suggesting that the effect of Pa-PDT was mediated by the generation of intracellular ROS. In conclusion, our findings demonstrated the therapeutic potential of Pa-PDT alone or in combination with Dox in combating multidrug-resistant malignancies.

Topics: alpha-Tocopherol; Analysis of Variance; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Cell Proliferation; Chlorophyll; DNA Fragmentation; DNA Primers; Doxorubicin; Drug Resistance, Multiple; Drug Synergism; Female; Flow Cytometry; Humans; Photochemotherapy; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Sarcoma; Uterine Neoplasms

Three new natural products, designated trehangelins A, B and C, were isolated by solvent extraction, silica gel and octadecylsilyl silica gel column chromatographies and subsequent preparative HPLC from the cultured broth of an endophytic actinomycete strain, Polymorphospora rubra K07-0510. The trehangelins consisted of a trehalose moiety and two angelic acid moieties. Trehangelins A (IC50 value, 0.1 mg ml(-1)) and C (IC50 value, 0.4 mg ml(-1)), with symmetric structures, showed potent inhibitory activity against hemolysis of red blood cells induced by light-activated pheophorbide a. However, trehangelin B, with an asymmetric structure, displayed only a slight inhibition (IC50 value, 1.0 mg ml(-1)).

Topics: Actinobacteria; Biological Products; Cell Survival; Chemical Phenomena; Chlorophyll; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Endophytes; Erythrocytes; HEK293 Cells; Hemolysis; HT29 Cells; Humans; Inhibitory Concentration 50; Molecular Conformation; Orchidaceae; Oxidative Stress; Plant Roots; Radiation-Protective Agents; Radiation-Sensitizing Agents; Trehalose

Substrate-induced upregulation of ATP-binding cassette subfamily G member 2 (ABCG2) has been well studied in cancer cells, but it is also important to understand whether ABCG2 is upregulated by its substrates in tissues in which it is constitutively expressed. In the present study, we aimed to clarify the regulatory mechanism of Abcg2 expression by its substrate, mitoxantrone, in placental cells. Abcg2 mRNA expression in rat placental TR-TBT 18d-1 cells treated with 10 μM mitoxantrone for 24 h was increased, compared with that in nontreated cells, whereas 10 μM pheophorbide-a had no effect. Methylated CpG level in the promoter region of the Abcg2 gene was low and was not altered by mitoxantrone. On the contrary, mitoxantrone markedly increased the expression of estrogen receptor (ER) α and progesterone receptor (PR) B. Fulvestrant, an ER antagonist, attenuated the mitoxantrone-induced increase of Abcg2 mRNA expression, whereas mifepristone, a PR antagonist, had little effect. 17β-estradiol, an ER ligand, positively regulated the mitoxantrone-induced increase of Abcg2 expression. DNA demethylation by 5-aza-2-deoxycytidine treatment increased ERα expression, but mitoxantrone failed to facilitate the demethylation of ERα promoter in TR-TBT 18d-1 cells. In conclusion, Abcg2 expression is induced by mitoxantrone via the induction of ERα in TR-TBT 18d-1 cells.

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line; Chlorophyll; DNA Methylation; Estrogen Receptor alpha; Female; Mitoxantrone; Promoter Regions, Genetic; Rats; Rats, Wistar; Trophoblasts; Up-Regulation

The characterization of ABCI4, a new intracellular ATP-binding cassette (ABC) half-transporter in Leishmania major, is described. We show that ABCI4 is involved in heavy metal export, thereby conferring resistance to Pentostam, to Sb(III), and to As(III) and Cd(II). Parasites overexpressing ABCI4 showed a lower mitochondrial toxic effect of antimony by decreasing reactive oxygen species production and maintained higher values of both the mitochondrial electrochemical potential and total ATP levels with respect to controls. The ABCI4 half-transporter forms homodimers as determined by a coimmunoprecipitation assay. A combination of subcellular localization studies under a confocal microscope and a surface biotinylation assay using parasites expressing green fluorescent protein- and FLAG-tagged ABCI4 suggests that the transporter presents a dual localization in both mitochondria and the plasma membrane. Parasites overexpressing ABCI4 present an increased replication in mouse peritoneal macrophages. We have determined that porphyrins are substrates for ABCI4. Consequently, the overexpression of ABCI4 confers resistance to some toxic porphyrins, such as zinc-protoporphyrin, due to the lower accumulation resulting from a significant efflux, as determined using the fluorescent zinc-mesoporphyrin, a validated heme analog. In addition, ABCI4 has a significant ability to efflux thiol after Sb(III) incubation, thus meaning that ABCI4 could be considered to be a potential thiol-X-pump that is able to recognize metal-conjugated thiols. In summary, we have shown that this new ABC transporter is involved in drug sensitivity to antimony and other compounds by efflux as conjugated thiol complexes.

Topics: Animals; Antimony; Antimony Sodium Gluconate; Antiprotozoal Agents; ATP-Binding Cassette Transporters; Biological Transport; Cadmium; Chlorophyll; Drug Resistance; Green Fluorescent Proteins; Leishmania major; Macrophages; Male; Membrane Potential, Mitochondrial; Mice; Mitochondria; Mitochondrial Membranes; Protein Multimerization; Protoporphyrins; Protozoan Proteins; Sulfhydryl Compounds

We designed a cancer-cell specific photosensitizer nano-carrier by synthesizing pheophorbide a (PheoA) conjugated glycol chitosan (GC) with reducible disulfide bonds (PheoA-ss-GC). The amphiphilic PheoA-ss-GC conjugates self-assembled in aqueous condition to form core-shell structured nanoparticles (PheoA-ss-CNPs) with good colloidal stability and switchable photoactivity. The photoactivity of PheoA-ss-CNPs in an aqueous environment was greatly suppressed by the self-quenching effect, which enabled the PheoA-ss-CNPs to remain photo-inactive and in a quenched state. However, after the cancer cell-specific uptake, the nanoparticular structure instantaneously dissociated by reductive cleavage of the disulfide linkers, followed by an efficient dequenching process. Compared to non-reducible PheoA-conjugated GC-NPs with stable amide linkages (PheoA-CNPs), PheoA-ss-CNPs rapidly restored their photoactivity in response to intracellular reductive conditions, thus presenting higher cytotoxicity with light treatment. In addition, the PheoA-ss-CNPs presented prolonged blood circulation in vivo compared to free PheoA, demonstrating enhanced tumor specific targeting behavior through the enhanced permeation and retention (EPR) effect. The enhanced tumor accumulation of PheoA-ss-CNPs enabled tumor therapeutic efficacy that was more efficient than free PheoA in tumor-bearing mice. Based on the enhanced intracellular release for cytosolic high dose and switchable photoactivity mechanism for reduced side effects, these results suggest that PheoA-ss-CNPs have good potential for photodynamic therapy (PDT) in cancer treatment.

Topics: Animals; Chitosan; Chlorophyll; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Nanoparticles; Neoplasms; Oxidation-Reduction; Photochemotherapy; Radiation-Sensitizing Agents

Lung adenocarcinoma is a leading human malignancy with fatal prognosis. Ninety percent of the deaths, however, are caused by metastases. The model of subcutaneous tumor xenograft in nude mice was adopted to study the growth of control and photodynamically treated tumors derived from the human A549 lung adenocarcinoma cell line. As a side-result of the primary studies, observations on the metastasis of these tumors to the murine lungs were collected, and reported in the present paper. The metastasizing primary tumors were drained by a prominent number of lymphatic vessels. The metastatic tissue revealed the morphology of well-differentiated or trans-differentiated adenocarcinoma. Further histological and histochemical analyses demonstrated the presence of golden-brown granules in the metastatic tissue, similar to these found in the tumor tissue. In contrast to the primary tumors, the electron paramagnetic resonance spectroscopy revealed no nitric oxide - hemoglobin complexes (a source of intense paramagnetic signals), in the metastases. No metastases were found in other murine organs; however, white infarctions were identified in a single liver. Taken together, the A549-derived tumors growing subcutaneously in nude mice can metastasize and grow on site in the pulmonary tissue. Thus, they can represent an alternative for the model of induced metastatic nodule formation, following intravenous administration of the cancerous cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Cell Line, Tumor; Chlorophyll; Electron Spin Resonance Spectroscopy; Humans; Injections, Subcutaneous; Light; Liver Neoplasms; Lung Neoplasms; Male; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Experimental; Photosensitizing Agents; Skin Neoplasms; Transplantation, Heterologous

In contrast to the success in artificial DNA- and peptide-based nanostructures, the ability of polysaccharides to self-assemble into one-, two-, and three-dimensional nanostructures are limited. Here, we describe a strategy for designing and fabricating nanorods using a regioselectively functionalized cellulose derivative at the air-water interface in a stepwise manner. A semisynthetic chlorophyll derivative, pyro-pheophorbide a, was partially introduced into the C-6 position of the cellulose backbone for the design of materials with specific optical properties. Remarkably, controlled formation of cellulose nanorods can be achieved, producing light-harvesting nanorods that display a larger bathochromic shift than their solution counterparts. The results presented here demonstrate that the self-assembly of functionalized polysaccharides on surfaces could lead the nanostructures mimicking the naturally occurring chloroplasts.

Topics: Aluminum Silicates; Bioelectric Energy Sources; Cellulose; Chlorophyll; Electrodes; Hydrogen Bonding; Nanotubes; Surface Properties

In this study, we developed a new photosensitizer (PS)-conjugated hybrid nanoparticle comprised of gold nanoparticle (AuNP) as an efficient energy quencher, polysaccharide heparin and a second generation PS, pheophorbide a (PhA) for PDT. The hybrid nanoparticles (PhA-H/AuNPs) with an average size of 40nm were prepared by surface coating of AuNPs with PhA conjugated heparins via gold-thiol interaction. The glutathione (GSH)-mediated switchable photoactivity of the PhA-H/AuNPs was observed by fluorescence quenching and dequenching behaviors in the absence and presence of GSH. The photoactivity was significantly suppressed in aqueous media, but instantaneously restored at the GSH-rich intracellular environment to generate a strong fluorescence signal together with active production of singlet oxygen species with light treatment. In vitro cell tests revealed marked phototoxicity and high intracellular uptake of PhA-H/AuNPs in contrast with free PhA. The PhA-H/AuNPs also exhibited a prolonged circulation characteristic, enhanced tumor specificity, and improved photodynamic therapeutic efficacy compared with free PhA in tumor-bearing mice. As a result, the PhA-H/AuNPs may serve as an effective smart nanomedicine platform for PDT and have great potential for the clinical treatment of various tumors.

Topics: Animals; Cell Line, Tumor; Chlorophyll; Drug Carriers; Glutathione; Gold; Heparin; Humans; Light; Metal Nanoparticles; Mice; Mice, Nude; Neoplasms; Photochemotherapy; Photosensitizing Agents; Tumor Burden

This study aimed to evaluate correlations between fermentation characteristics and end products of selected fermentable fibres (three types of fructans, citrus pectin, guar gum), incubated with faecal inocula from donor cats fed two diets, differing in fibre and protein sources and concentrations. Cumulative gas production was measured over 72 h, fermentation end products were analysed at 4, 8, 12, 24, 48 and 72 h post-incubation, and quantification of lactobacilli, bifidobacteria and bacteroides in fermentation liquids were performed at 4 and 48 h of incubation. Partial Pearson correlations, corrected for inoculum, were calculated to assess the interdependency of the fermentation characteristics of the soluble fibre substrates. Butyric and valeric acid concentrations increased with higher fermentation rates, whereas acetic acid declined. Concentrations of butyric acid (highest in fructans) and propionic acid were inversely correlated with protein fermentation end products at several time points, whereas concentrations of acetic acid (highest in citrus pectin) were positively correlated with these products at most time points. Remarkably, a lack of clear relationship between the counts of bacterial groups and their typically associated products after 4 h of incubation was observed. Data from this experiment suggest that differences in fibre fermentation rate in feline faecal inocula coincide with typical changes in the profile of bacterial fermentation products. The observed higher concentrations of propionic and butyric acid as a result of fibre fermentation could possibly have beneficial effects on intestinal health, and may be confounded with a concurrent decrease in the production of putrefactive compounds. In conclusion, supplementing guar gum or fructans to a feline diet might be more advantageous compared with citrus pectin. However, in vivo research is warranted to confirm these conclusions in domestic cats.

Topics: Amino Acids; Ammonia; Animal Feed; Animal Nutritional Physiological Phenomena; Animals; Cats; Cellulose; Chlorophyll; Diet; Dietary Fiber; Fatty Acids, Volatile; Feces; Female; Fermentation; Kinetics; Male; Time Factors

Our previous study has shown a prolonged retention and accumulation of Zn-pheophorbide a, a water-soluble derivative of chlorophyll a, in tumor tissue (Szczygiel et al. [19]). This prompted us to further evaluate the phototherapeutic potential of this photosensitizer of excellent physicochemical properties.. Cellular uptake of Zn-pheophorbide, its localization in cells, cytotoxicity, phototoxicity and cell death mechanisms were studied in human adenocarcinoma cell lines: A549, MCF-7 and LoVo. The PDT efficacy was tested against A549 tumors growing in nude mice.. Zn-pheophorbide a even at very low concentrations (∼1×10(-6)M) and at low light doses (5J/cm(2)) causes a strong photodynamic effect, leading to 100% cell mortality. Confocal microscopy showed that in contrast to most derivatives of chlorophyll, Zn-pheophorbide a does not localize to mitochondria. The photodynamic effects and the cell death mechanisms of Zn-pheophorbide a, its Mg analog (chlorophyllide a) and Photofrin were compared on the A549 cells. Zn-pheophorbide a showed the strongest photodynamic effect, at low dose killing all A549 cells via apoptosis and necrosis. The very high anti-cancer potential of Zn-pheophorbide was confirmed in a photodynamic treatment of the A549 tumors. They either regressed or were markedly inhibited for up to 4 months after the treatment, resulting, on average, in a 5-fold decrease in tumor volume.. These results show that Zn-pheophorbide a is a very promising low-cost, synthetically easily accessible, second generation photosensitizer against human cancer.

Topics: Adenocarcinoma; Animals; Cell Line, Tumor; Chlorophyll; Disease Models, Animal; Humans; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Photochemotherapy; Photosensitizing Agents; Treatment Outcome; Zinc

The introduction of photodynamic therapy (PDT) to the treatment of advanced prostate cancer can accomplish the eradication of local neoplasm and distant metastases with minimized damage to the adjacent structures. The evidence of PDT efficacy for androgen-refractory prostate cancer will be especially meaningful for the patients resistant to hormone therapy.. Pheophorbide a (PhA) as a photosensitizer was employed to evaluate the photodynamic efficacy in androgen-insensitive PC-3 prostate cancer cells in culture by cell viability assay, reactive oxygen species (ROS) measurement and cell cycle test. Characteristics of apoptosis and autophagy were investigated via DNA fragmentation electrophoresis and immune-fluorescence staining, acidic vesicle determination and detection of LC3B in puncta form by fluorescence microscopy, Western blotting of autophagy-related (Atg) proteins and detailed phenotype shown by electron microscopy.. PhA exerted significant photo-cytotoxicity toward androgen-insensitive prostate cancer PC-3 cells in photosensitizer-dose and light-dose dependent manners. The photoactivation immediately initiated hyperproduction of ROS, the depolarization of mitochondrial membrane potential and the arrest of the cell cycle in the G0/G1 phase. Autophagy was revealed in PhA-PDT treated PC-3 cells by a significant high amount of acidic vesicular organelles with acridine orange staining, recruitment of LC3B on the membrane of autophagosomes by fluorescent microscopy, double membrane-bound vesicles suggesting autophagosomes by electron microscopy, significant increased Atg proteins such as beclin-1, Atg12-Atg5 conjugation, Atg7 and the conversion of LC3B-I to LC3B-II by Western blot analysis.. PhA-mediated PDT induced significant autophagy in hormone-refractory prostate cancer PC-3 cells.

Topics: Apoptosis; Autophagy; Cell Cycle; Cell Line, Tumor; Chlorophyll; Humans; Male; Photosensitizing Agents; Prostatic Neoplasms, Castration-Resistant; Treatment Outcome

Multidrug resistance (MDR), usually mediated by overexpression of efflux transporters such as P-gp, ABCG2 and/or MRP1, remains a major obstacle hindering successful cancer chemotherapy. There has been great interest in the development of inhibitors towards these transporters to circumvent resistance. However, since the inhibition of transporter is not specific to cancer cells, a decrease in the cytotoxic drug dosing may be needed to prevent excess toxicity, thus undermining the potential benefit brought about by a drug efflux inhibitor. The design of potent MDR modulators specific towards resistant cancer cells and devoid of drug-drug interactions will be needed to effect MDR reversal.. Recent evidence suggests that the PTEN/PI3K/Akt pathway may be exploited to alter ABCG2 subcellular localization, thereby circumventing MDR. Three PPARγ agonists (telmisartan, pioglitazone and rosiglitazone) that have been used in the clinics were tested for their effect on the PTEN/PI3K/Akt pathway and possible reversal of ABCG2-mediated drug resistance.. The PPARγ agonists were found to be weak ABCG2 inhibitors by drug efflux assay. They were also shown to elevate the reduced PTEN expression in a resistant and ABCG2-overexpressing cell model, which inhibit the PI3K-Akt pathway and lead to the relocalization of ABCG2 from the plasma membrane to the cytoplasma, thus apparently circumventing the ABCG2-mediated MDR.. Since this PPARγ/PTEN/PI3K/Akt pathway regulating ABCG2 is only functional in drug-resistant cancer cells with PTEN loss, the PPARγ agonists identified may represent promising agents targeting resistant cells for MDR reversal.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzimidazoles; Benzoates; Biological Transport; Breast Neoplasms; Chlorophyll; Drug Resistance, Neoplasm; Endocytosis; Female; HEK293 Cells; Humans; Kinetics; MCF-7 Cells; Neoplasm Proteins; Phosphatidylinositol 3-Kinase; Pioglitazone; PPAR gamma; Protein Transport; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; RNA Interference; Rosiglitazone; Signal Transduction; Telmisartan; Thiazolidinediones; Transfection; Up-Regulation

Pheophorbide a (Pba) is a chlorophyll catabolite that has been proposed as photosensitizer in photodynamic therapy. In a previous study we conjugated Pba to monomethoxy-polyethylene glycol (mPEG-Pba), to increase its solubility and pharmacokinetics. Here, we compare the photodynamic therapy efficacy of free Pba and mPEG-Pba to cure a subcutaneous amelanotic melanoma transplanted in C57/BL6 mice. The photosensitizers, i.p. injected (30 mg/kg), showed no toxicity when the animals were kept in the dark. But, after photoactivation with a 660 nm laser (fluence of 193 J/cm(2)), both photosensitizers, in particular mPEG-Pba, showed a strong efficacy to cure the tumor, both in terms of tumor growth delay and increase of Kaplan-Meier median survival time. Together, our in vivo data demonstrate that mPEG-conjugated Pba is a promising photosensitizer for the photodynamic therapy of cancer.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Chlorophyll; Female; Light; Melanoma, Amelanotic; Mice; Mice, Inbred C57BL; Photochemotherapy; Polyethylene Glycols; Radiation-Sensitizing Agents

Pheophorbide a (Pa) is a chlorine-based photosensitizer derived from an ethnopharmacological herb, and our group recently synthesized Pa by the removal of a magnesium ion and a phytyl group from chlorophyll-a. In this study, the effect of photodynamic therapy (PDT) with synthesized Pa was examined in a human oral squamous cell carcinoma (OSCC) cells.. Cells were treated with PDT with Pa, and reactive oxygen species (ROS) and mitochondrial membrane potential [ΔΨ (m)] were examined. Apoptosis was measured using annexin V staining and immunoblot. Autophagy was characterized by the increase in LC3B-II and the formation of autophagosome and acidic vesicular organelles (AVOs).. Pa-PDT inhibited the proliferation of OSCC cells in a dose-dependent manner. Pa-PDT increased the number of apoptotic cells by inactivating ERK pathway. Pa-PDT also induced autophagy in OSCC cells evidenced by the increased levels of LC3 type II expression and the accumulation of AVOs. The inhibition of autophagy enhanced Pa-PDT-mediated cytotoxicity through an increase in necrosis.. These results suggest that synthesized Pa-PDT exerts anti-tumor effects by inducing apoptosis and autophagy and provide novel evidence that Pa-PDT induces autophagy, and autophagy inhibition enhances Pa-PDT-mediated necrosis in OSCC cells.

Topics: Apoptosis; Autophagy; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Chlorophyll; Humans; MAP Kinase Signaling System; Microtubule-Associated Proteins; Mouth Neoplasms; Necrosis; Phagosomes; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species

Tumor necrosis factor (TNF) receptor 1-associated periodic syndrome (TRAPS) is an autoinflammatory disorder caused by autosomal dominantly inherited mutations in the TNF receptor superfamily 1A (TNFRSF1A) gene. The D12E substitution has been described only once to date, in a 4-year-old boy with fever.. For DNA sequence analysis of the TNFRSF1A gene, genomic DNA was isolated, amplified by PCR, purified, and sequenced.. We describe 3 families (8 subjects) with the TNFRSF1A D12E substitution and TRAPS-related symptoms, in 4 cases associated with the autoimmune diseases multiple sclerosis and rheumatoid arthritis.. The clinical phenotype might be associated with the TNFRSF1A D12E mutation. There is a close pathophysiological relationship between TNF signaling and autoimmune disorders.

Topics: Adult; Arthritis, Rheumatoid; Child; Chlorophyll; Female; Fever; Hereditary Autoinflammatory Diseases; Humans; Male; Middle Aged; Multiple Sclerosis; Mutation; Receptors, Tumor Necrosis Factor, Type I

Cell recurrence in cancer photodynamic therapy (PDT) is an important issue that is poorly understood. It is becoming clear that nitric oxide (NO) is a modulator of PDT. By acting on the NF-κB/Snail/RKIP survival/anti-apoptotic loop, NO can either stimulate or inhibit apoptosis. We found that pheophorbide a/PDT (Pba/PDT) induces the release of NO in B78-H1 murine amelanotic melanoma cells in a concentration-dependent manner. Low-dose PDT induces low NO levels by stimulating the anti-apoptotic nature of the above loop, whereas high-dose PDT stimulates high NO levels inhibiting the loop and activating apoptosis. When B78-H1 cells are treated with low-dose Pba/PDT and DETA/NO, an NO-donor, intracellular NO increases and cell growth is inhibited according to scratch-wound and clonogenic assays. Western blot analyses showed that the combined treatment reduces the expression of the anti-apoptotic NF-κB and Snail gene products and increases the expression of the pro-apoptotic RKIP gene product. The combined effect of Pba and DETA/NO was also tested in C57BL/6 mice bearing a syngeneic B78-H1 melanoma. We used pegylated Pba (mPEG-Pba) due to its better pharmacokinetics compared to free Pba. mPEG-Pba (30 mg/Kg) and DETA/NO (0.4 mg/Kg) were i.p. injected either as a single molecule or in combination. After photoactivation at 660 nM (fluence of 193 J/cm(2)), the combined treatment delays tumor growth more efficiently than each individual treatment (p<0.05). Taken together, our results showed that the efficacy of PDT is strengthened when the photosensitizer is used in combination with an NO donor.

Topics: Animals; Cell Line, Tumor; Chlorophyll; Female; Flow Cytometry; Melanoma, Amelanotic; Mice; Mice, Inbred C57BL; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitroso Compounds; Photochemotherapy; Photosensitizing Agents; Skin Neoplasms; Wound Healing

Amphiphilic heparin-retinoic acid (HR) and heparin-folate-retinoic acid bioconjugates (HFR) were synthesized by chemical conjugation of a hydrophobic anticancer agent all-trans-retinoic acid (RA) and a targeting ligand, folic acid (FA), to the high molecular weight heparin backbone. The HR and HFR bioconjugates had a high RA content (22%, w/w) and could self-assemble into nanoparticles with efficient encapsulation of a hydrophobic photosensitizer, pheophorbide a (PhA). The HFR bioconjugate demonstrated higher PhA loading content and loading efficiency compared to HR bioconjugate. The PhA-loaded HR and HFR nanoparticles had an average diameter of about 70 nm, a negatively charged surface, a sustained release pattern and self-quenching effect in a buffered solution. Furthermore, the cellular uptake of PhA-loaded HFR nanoparticles in folate receptor-positive HeLa cells was higher than that of PhA-loaded HR nanoparticles. Upon irradiation, HFR nanoparticles selectively enhanced the phototoxicity of PhA in HeLa cells while the dark-toxicity of the nanoparticles was minimal without light treatment. HFR nanoparticles also demonstrated targeted anti-cancer effect, improving the cytotoxicity of RA in HeLa cells compared to HR nanoparticles at RA concentration ≥50 μg/mL. The targeting effect of HFR and PhA-loaded HFR nanoparticles was not observed in folate receptor-negative HT-29 cells. The results indicated that HFR nanoparticles may be useful for targeted delivery of hydrophobic PDT agents and as a potential nanocarrier for dual chemo-and photodynamic therapies.

Topics: Antineoplastic Agents; Biological Transport; Chlorophyll; Drug Carriers; Folic Acid; HeLa Cells; Heparin; HT29 Cells; Humans; Hydrophobic and Hydrophilic Interactions; Nanoparticles; Photosensitizing Agents; Singlet Oxygen; Tretinoin

Methyl pheophorbide-a/a' derivatives covalently linked with oligomethylene chains at the 3-CH(2)OCO- and 13(2)-COO- moieties in a molecule were prepared by modifying chlorophyll-a through intramolecular ring-closing metathesis of vinyl groups. At least, a C10-length between the 3(3)- and 13(4)-positions was necessary for the cyclization and connection of a C12-strap was the most suitable to achieve the highest closure yield. The oligomethylene chain in 13(2) R-epimers derived from methyl pheophorbide-a covered the α-face of the chlorin π-plane and the strap in the corresponding 13(2) S-epimers protected the β-face. Synthetic 13(2) R-epimer with a dodecamethylene chain gave a flat chlorin π-plane, while the decamethylene chain in the 13(2) R-epimer distorted the π-system due to its shorter linkage. The distortion by strapping in the 13(2) R-epimer induced a slight blue-shift of Qy peak in dichloromethane. CD spectra of the 13(2) R-epimers were similarly dependent on the chain length, i.e., the distortion of π-plane. Visible absorption and CD spectra of all the strapped 13(2) S-epimers were almost identical and only slightly different from those of the unstrapped. The strapping in the 13(2) S-epimers shifted the Qy peak bathochromically.

Topics: Chlorophyll; Chlorophyll A; Circular Dichroism; Models, Molecular; Molecular Conformation; Optics and Photonics; Solutions; Spectrophotometry, Atomic; Spirulina; Stereoisomerism

As part of our continuing research for anticancer compounds from the Walloon Region forest, EtOAc extract from Carpinus betulus leaves was phytochemically studied, leading to the bioguided isolation of pheophorbide a, which is responsible of anticancer properties of C. betulus young leaves. This compound was identified using nuclear magnetic resonance and mass spectrophotometric data and comparison with a commercial standard. Evaluation of the growth inhibitory activities of pheophorbide a using MTT colorimetric assay and phase-contrast microscopy in various human cancer cell lines confirmed the photoactivable properties of this compound. Our research showed, for the first time, the presence of pheophorbide a, a chlorophyll derived compound, which we quantified in high quantities in young leaves of C. betulus. This is in contrast with the literature which generally describes pheophorbide a as a catabolic product of chlorophyll, then preferentially present in old leaves.

Topics: Acetates; Antineoplastic Agents, Phytogenic; Betulaceae; Cell Line, Tumor; Chemical Fractionation; Chlorophyll; Chromatography, High Pressure Liquid; Humans; Inhibitory Concentration 50; Magnetic Resonance Spectroscopy; Microscopy, Phase-Contrast; Plant Extracts; Plant Leaves

ATP-binding cassette (ABC) drug transporters ABCB1 [P-glycoprotein (Pgp)] and ABCG2 are expressed in many tissues including those of the intestines, the liver, the kidney and the brain and are known to influence the pharmacokinetics and toxicity of therapeutic drugs. In vitro studies involving their functional characteristics provide important information that allows improvements in drug delivery or drug design. In this study, we report use of the BacMam (baculovirus-based expression in mammalian cells) expression system to express and characterize the function of Pgp and ABCG2 in mammalian cell lines. BacMam-Pgp and BacMam-ABCG2 baculovirus-transduced cell lines showed similar cell surface expression (as detected by monoclonal antibodies with an external epitope) and transport function of these transporters compared to drug-resistant cell lines that overexpress the two transporters. Transient expression of Pgp was maintained in HeLa cells for up to 72 h after transduction (48 h after removal of the BacMam virus). These BacMam-baculovirus-transduced mammalian cells expressing Pgp or ABCG2 were used for assessing the functional activity of these transporters. Crude membranes isolated from these cells were further used to study the activity of these transporters by biochemical techniques such as photo-cross-linking with transport substrate and adenosine triphosphatase assays. In addition, we show that the BacMam expression system can be exploited to coexpress both Pgp and ABCG2 in mammalian cells to determine their contribution to the transport of a common anticancer drug substrate. Collectively, these data demonstrate that the BacMam-baculovirus-based expression system can be used to simultaneously study the transport function and biochemical properties of ABC transporters.

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Baculoviridae; Biological Transport; Cell Line, Tumor; Cell Membrane; Chlorophyll; Doxorubicin; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Genetic Vectors; Humans; Mammals; Mitoxantrone; Neoplasm Proteins; Neoplasms; Radiation-Sensitizing Agents; Recombinant Proteins; Transduction, Genetic

Breast cancer is conventionally treated by surgery and radiotherapy, with adjuvant chemotherapy and hormonotherapy as supplementary treatments. However, such treatments are associated with adverse side effects and drug resistance. In this study, Pheophorbide a (Pa), a photosensitizer isolated from Scutelleria barbata, was analysed for its antiproliferative effect on human breast tumour cells. The IC (inhibitory concentration)(50) of the combined treatment of Pa and photodynamic therapy (Pa-PDT) on human breast tumour MCF-7 cells was 0.5 µm. Mechanistic studies in MCF-7 cells demonstrated that Pa was localized in the mitochondria, and reactive oxygen species were found to be released after Pa-PDT. Apoptosis was the major mechanism responsible for the tumour cell death, and mitochondrial membrane depolarization and cytochrome c release highlighted the role of mitochondria in the apoptotic mechanism. Up-regulation of tumour suppressor protein p53, cleavage of caspase-9 and poly (ADP-ribose) polymerase suggested that the caspase-dependent pathway was induced, while the release of apoptosis-inducing factors demonstrated that the apoptosis was also mediated by the caspase-independent mechanism. In vivo study using the mouse xenograft model showed a significant inhibition of MCF-7 tumour growth by Pa-PDT. Together, the results of this study provide a basis for understanding and developing Pa-PDT as a cure for breast cancer.

Topics: Animals; Apoptosis; Breast Neoplasms; Caspase 9; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chlorophyll; Cytochromes c; DNA Fragmentation; Female; Humans; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Photochemotherapy; Photosensitizing Agents; Plant Extracts; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Scutellaria; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

In this study, we reported on the synthesis and biological evaluation of radiolabeled fluorescent dye conjugated bovine serum albumin nanoparticles within the size range 190-210 nm. The bovine serum albumin nanoparticles (BSANPs) were prepared using a desolvation method, and chemical cross-linking was performed using gluteraldehyde. Furthermore, pheophorbide-a (PH-A) was loaded on the BSANPs. The results obtained from dynamic light scattering and electron microscopy have proved that nanoparticles are highly monodisperse and near-spherical shaped. The photo-physical properties of the PH-A-BSANPs were obtained using the spectrophotometric techniques. According to the results, PH-A and BSANPs show high non-covalent interaction. PH-A loaded nanoparticles were labeled with (99m)Tc and the radio-labeling efficiency was determined as 90 ± 1.2%. Biodistribution studies of (99m)Tc labeled PH-A-BSANPs and PH-A were carried out using female Albino Wistar rats, and (99m)Tc-PH-A-BSANPs showed a significantly higher uptake in the breast and uterus than (99m)Tc-PH-A. Cell culture study was carried out in MCF-7 cell line (human breast adenocarcinoma cell line). According to the cell culture studies, (99m)Tc-PH-A-BSANPs showed a higher uptake than (99m)Tc-PH-A. Moreover, PH-A-BSANPs demonstrated good photo-physical properties and BSANPs increased the uptake of PH-A on to the MCF-7 cell line. These results confirm that (99m)Tc labeled PH-A-BSANPs could be utilized for radioimaging.

Topics: Adenocarcinoma; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Chlorophyll; Cross-Linking Reagents; Drug Carriers; Female; Glutaral; Humans; Light; Magnetic Resonance Spectroscopy; Mass Spectrometry; Microscopy, Electron; Microscopy, Fluorescence; Molecular Imaging; Nanoparticles; Particle Size; Radiation-Sensitizing Agents; Radiopharmaceuticals; Rats; Rats, Wistar; Scattering, Radiation; Serum Albumin, Bovine; Spectrophotometry, Ultraviolet; Technetium; Tissue Distribution

Photoreaction of zinc methyl 20-substituted meso(pyro)pheophorbide-a prepared by modifying naturally occurring chlorophyll-a in the presence of oxygen molecules gave its C19-C20 oxidative cleavage (1-carbonyl-19-oxo-bilatrienes) as the major products and the regioisomeric C1-C20 cleavage (19-carbonyl-1-oxo-bilatrienes) as the minor products. The resulting zinc complexes of linear tetrapyrroles took a helical conformation and the P-conformers were preferential over the M-stereoisomers due to the presence of their 17S,18S-chiral centers. The helical conformers (diastereomers) of the corresponding nickel complexes were separated by reverse-phase or chiral HPLC and their conformational changes were observed in solution.

Topics: Chlorophyll; Chlorophyll A; Coordination Complexes; Crystallography, X-Ray; Light; Molecular Conformation; Oxidation-Reduction; Stereoisomerism; Tetrapyrroles; Zinc

Photodynamic therapy (PDT) with several photosensitizers is a promising modality for the treatment of cancer. In this study, the therapeutic effect of PDT using the synthetic photosensitizer pheophorbide a (Pa-PDT) was examined in AT-84 murine oral squamous cell carcinoma (OSCC) cells. The MTT assay revealed that Pa-PDT induced cell growth inhibition in a dose- and time-dependent manner. Pa-PDT treatment significantly induced intracellular ROS generation, which is critical for cell death induced by Pa-PDT. Cell cycle analysis showed the increased sub-G1 proportion of cells in Pa-PDT-treated cells. Induction of apoptotic cell death was confirmed by DAPI staining and the reduction of mitochondrial membrane potential (ΔΨm) on Pa-PDT-treated cells. The changes in apoptosis-related molecules were next examined using western blotting. Cytochrome c release and cleavage of caspase-3 and PAPR were observed in AT-84 cells, whereas Bcl-2 protein levels were decreased. To determine the therapeutic effect of Pa-PDT in vivo, a murine OSCC animal model was used. Treatment of mice with Pa-PDT significantly inhibited tumor growth, especially PDT with Pa intravenous administration (i.v. Pa-PDT), and increased proliferative cell nuclear antigen (PCNA) levels and TUNEL-stained apoptotic cells compared to vehicle-treated controls. The data demonstrate that the in vitro effects of Pa-PDT on the inhibition of tumor cell proliferation and induction of apoptosis correlate to the anticancer activity of Pa-PDT in vivo. Our findings suggest the therapeutic potential of Pa-PDT in OSCC.

Topics: Animals; Apoptosis; Carcinoma, Squamous Cell; Caspase 3; Cell Line, Tumor; Cell Proliferation; Chlorophyll; Cytochromes c; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C3H; Mitochondria; Mouth Neoplasms; Photochemotherapy; Photosensitizing Agents; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species

The autofluorescence of fingermarks is used for their detection. The components responsible for this autofluorescence are largely unknown. Thin layer chromatography and fluorescence spectroscopy were used to identify autofluorescent components and evaluate their forensic value. Based on our results, tryptophan is hypothesized to be a major contributor to the autofluorescence when part of peptides or proteins, id est, not in its free form. Part of the autofluorescence could be assigned to a kynurenine derivative. Pheophorbide A, a metabolite of chlorophyll, is inferred as a red fluorescent fingermark component. Chlorophyll is a plant pigment which implies that dietary information can potentially be retrieved from fingermarks.

Topics: beta Carotene; Bilirubin; Chlorophyll; Chromatography, Thin Layer; Dermatoglyphics; Flavin-Adenine Dinucleotide; Fluorescence; Humans; Kynurenine; Light; Phenylalanine; Pheophytins; Protoporphyrins; Riboflavin; Sebum; Spectrometry, Fluorescence; Sweat; Thiamine; Tryptophan; Tyrosine; Ultraviolet Rays; Vitamin B 6; Xanthurenates

Ponatinib is a novel tyrosine kinase inhibitor with potent activity against BCR-ABL with mutations, including T315I, and also against fms-like tyrosine kinase 3. We tested interactions between ponatinib at pharmacologically relevant concentrations of 50 to 200 nmol/L and the MDR-associated ATP-binding cassette (ABC) proteins ABCB1, ABCC1, and ABCG2. Ponatinib enhanced uptake of substrates of ABCG2 and ABCB1, but not ABCC1, in cells overexpressing these proteins, with a greater effect on ABCG2 than on ABCB1. Ponatinib potently inhibited [(125)I]-IAAP binding to ABCG2 and ABCB1, indicating binding to their drug substrate sites, with IC(50) values of 0.04 and 0.63 μmol/L, respectively. Ponatinib stimulated ABCG2 ATPase activity in a concentration-dependent manner and stimulated ABCB1 ATPase activity at low concentrations, consistent with it being a substrate of both proteins at pharmacologically relevant concentrations. The ponatinib IC(50) values of BCR-ABL-expressing K562 cells transfected with ABCB1 and ABCG2 were approximately the same as and 2-fold higher than that of K562, respectively, consistent with ponatinib being a substrate of both proteins, but inhibiting its own transport, and resistance was also attenuated to a small degree by ponatinib-induced downregulation of ABCB1 and ABCG2 cell-surface expression on resistant K562 cells. Ponatinib at pharmacologically relevant concentrations produced synergistic cytotoxicity with ABCB1 and ABCG2 substrate chemotherapy drugs and enhanced apoptosis induced by these drugs, including daunorubicin, mitoxantrone, topotecan, and flavopiridol, in cells overexpressing these transport proteins. Combinations of ponatinib and chemotherapy drugs warrant further testing.

Topics: Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Carbocyanines; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Chlorophyll; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Synergism; fms-Like Tyrosine Kinase 3; Fusion Proteins, bcr-abl; Humans; Imidazoles; Inhibitory Concentration 50; Mitoxantrone; Neoplasm Proteins; Protein Binding; Pyridazines; Rhodamine 123; Topotecan

Efflux pumps are vital bacterial components, and research has demonstrated that some plant compounds such as pheophorbide a (php) possess efflux pump inhibitor (EPI) activity. This study determined the quantity of php present in feces as an indicator of EPI activity. Feces were collected from different species of animals fed a variety of feeds. The chlorophyll metabolites php and pyropheophorbide a (pyp) were determined using fluorescense spectroscopy. The average concentrations [μg/g dry matter (DM) feces] of pyp/php in feces were as follows: guinea pig, 180; goat, 150; rabbit, 150; dairy cow, 120; feedlot cattle, 60; rat, <1; pig, <1; chicken, <1. These data indicate that animals consuming "green" diets will excrete feces with concentrations of php/pyp that exceed levels demonstrated to be inhibitory to bacterial efflux pumps (0.5 μg/mL). The natural presence EPIs in the gastrointestinal tract may modulate the activity of microbial efflux pumps and exert selection pressure upon resident microbial populations.

Topics: Animals; Chlorophyll; Feces; Spectrometry, Fluorescence

Singlet oxygen plays a crucial role in photo-dermatology and photodynamic therapy (PDT) of cancer. Its direct observation by measuring the phosphorescence at 1270 nm, however, is still challenging due to the very low emission probability. It is especially challenging for the time-resolved detection of singlet oxygen kinetics in vivo which is of special interest for biomedical applications. Photosensitized generation of singlet oxygen, in pig ear skin as model for human skin, is investigated here. Two photosensitizers (PS) were topically applied to the pig ear skin and examined in a comparative study, which include the amphiphilic pheophorbide-a and the highly hydrophobic perfluoroalkylated zinc phthalocyanine (F64PcZn). Fluorescence microscopy indicates the exclusive accumulation of pheophorbide-a in the stratum corneum, while F64PcZn can also accumulate in deeper layers of the epidermis of the pig ear skin. The kinetics obtained with phosphorescence measurements show the singlet oxygen interaction with the PS microenvironment. Different generation sites of singlet oxygen correlate with the luminescence kinetics. The results show that singlet oxygen luminescence detection can be used as a diagnostic tool, not only for research, but also during treatment. The detection methodology is suitable for the monitoring of chemical quenchers’ oxidation as well as saturation at singlet oxygen concentration levels relevant to PDT treatment protocols.

Topics: Animals; Chlorophyll; Humans; Indoles; Isoindoles; Luminescent Measurements; Microscopy, Fluorescence, Multiphoton; Photochemotherapy; Photolysis; Photosensitizing Agents; Singlet Oxygen; Skin; Swine

The ATP-binding cassette transporter breast cancer resistance protein (BCRP/ABCG2) is supposed to be a major determinant of the multidrug resistance phenotype of tumors by extruding chemically diverse cytostatic drugs out of tumor cells. BCRP physically and possibly also functionally interacts with caveolin-1 (CAV1, encoded by Cav1), an integral membrane protein of lipid rafts important for signal transduction and membrane trafficking. Moreover, Cav1 is linked to an aggressive phenotype of cancer cells in various tumors. We therefore investigated whether Cav1 plays a functional role in the regulation of BCRP transport activity and in the resistance against chemotherapeutics that are BCRP substrates. As a cell model, we used the BCRP overexpressing cell line MDCKII-BCRP and the corresponding parental cell line MDCKII as a control. Cav1 expression was down-regulated using retrovirus-mediated RNA interference technology. BCRP activity was assessed by pheophorbide A efflux assay and the resistance towards cytostatic drugs was measured by proliferation assays. Efficient knockdown of Cav1 reduced Cav1 expression by 85-95% and BCRP activity by 35%. Concurrently, it reduced resistance towards the BCRP substrate mitoxantrone but not towards vincristine, a chemotherapeutic that is not extruded by BCRP. Western blot analysis of gradient ultracentrifugation fractions and immunofluorescence demonstrates that BCRP localization within the plasma membrane was largely unaltered in Cav1-deficient cells compared to controls. The diminished BCRP function after Cav1 knockdown is, thus, likely mediated by alterations in protein-protein interactions and suggests a positive regulation of BCRP function by CAV1.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Transport; Caveolin 1; Cell Line; Cell Membrane; Cell Proliferation; Chlorophyll; Dogs; Drug Resistance, Neoplasm; Epithelial Cells; Gene Expression; Gene Knockdown Techniques; Humans; Membrane Microdomains; Mitoxantrone; Neoplasm Proteins; Radiation-Sensitizing Agents; Transduction, Genetic; Transfection; Vincristine

Extraction of the leaves of Zanthoxylum ailanthoides Sieb. & Zucc. affords extracts and four isolated compounds which exhibit activities against leukemia cells. The chloroform-soluble fraction (ZAC) of the crude extract of this plant showed cytotoxic activity against human promyelocytic leukemia (HL-60) and myelomonocytic leukemia (WEHI-3) cells with IC(50) values of 73.06 and 42.22 μg/mL, respectively. The active ZAC was further separated to yield pheophorbide-a methyl ester (1), pheophorbide-b methyl ester (2), 13(2)-hydroxyl (13(2)-S) pheophorbide-a methyl ester (3) and 13(2)-hydroxyl (13(2)-R) pheophorbide-b methyl ester (4) whose structures were confirmed by spectroscopic methods. Compounds 2-4 showed cytotoxic activities against both leukemia cells with IC(50) value in the range of 46.76-79.43 nM, whereas compound 1 exhibited only weak cytotoxic activity. The extracts and compounds 1-4 also induced apoptosis and DNA damage in leukemia cells after treatment. The results suggested that the Z. ailanthoides is biologically active against leukemia cells.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Cell Cycle; Cell Line, Tumor; Cell Survival; Chloroform; Chlorophyll; DNA Damage; HL-60 Cells; Humans; Inhibitory Concentration 50; Leukemia, Promyelocytic, Acute; Mice; Plant Extracts; Plant Leaves; Radiation-Sensitizing Agents; Zanthoxylum

In this study, we examined the therapeutic effect of photodynamic therapy (PDT) using the photosensitizer Na-Pheophorbide a (Na-Phde a) on osteomyelitis models in rats.. Osteomyelitis is one of the most serious infectious problems in the orthopedic field. Recently, as a new clinical approach against septic arthritis, an experimental in vivo and in vitro model for the inactivation of methicillin-resistant-Staphylococcus aureus by PDT using Na-Phde a has been developed.. Methicillin-sensitive Staphylococcus aureus (MSSA) was injected into the tibia of the rats to create osteomyelitis models (n = 10, 10 legs). A total of 560 μmol/l of Na-Phde a solution was injected into five of these tibial osteomyelitis models (five legs) 48 h after the initial MSSA infection. Sixty minutes after the Na-Phde a injection, a semiconductor laser (125 mW, 670 nm) was used to irradiate the models for 10 min with a total energy of 93.8 J/mm(2). As a control group, five rats (five legs) were treated with a phosphate buffered saline injection at 48 h after MSSA infection. Weight and leg perimeter changes were plotted. Bacterial growth, histological examination and radiological examination were evaluated at 14 days after initial treatment.. PDT with Na-Phde a significantly prevented leg swelling. In the PDT group, bone destruction owing to osteomyelitis was inhibited not only histologically but also radiographically.. The results in these experiments show that PDT using Na-Phde a improved osteomyelitis in rats. This suggests that PDT using Na- Phde a can be a useful treatment for osteomyelitis.

Topics: Animals; Biopsy, Needle; Chlorophyll; Disease Models, Animal; Female; Immunohistochemistry; Osteomyelitis; Photochemotherapy; Photosensitizing Agents; Random Allocation; Rats; Rats, Sprague-Dawley; Reference Values; Staphylococcal Infections; Statistics, Nonparametric; Tibia; Treatment Outcome

Electronic excitation energy transfer (EET) is described theoretically for the chromophore complex P(4) formed by a butanediamine dendrimer to which four pheophorbide-a molecules are covalently linked. To achieve a description with atomic resolution, and to account for the effect of an ethanol solvent, a mixed quantum-classical methodology is utilized. Room-temperature molecular dynamics simulations are used to describe the nuclear dynamics, and EET is accounted for in utilizing a mixed quantum-classical formulation of the transition rates. Therefore, the full quantum expression of the EET rates is given and the change to a mixed quantum-classical version is briefly explained. The description results in the calculation of transition rates which coincide rather satisfactory with available experimental data on P(4). It is also shown that different assumptions of classical Förster theory are not valid for P(4). The temporal behavior of EET deduced from the rate equations is confronted with that following from the solution of the time-dependent Schrödinger equation entering the mixed quantum-classical description of EET. From this we can conclude that EET in flexible chromophore complexes such as P(4) can be rather satisfactory estimated by single transition rates. A correct description, however, is only achievable by using a sufficiently large set of rates that correspond to the various possible equilibrium configurations of the complex.

Topics: Algorithms; Chlorophyll; Dendrimers; Fluorescence Resonance Energy Transfer; Molecular Dynamics Simulation; Quantum Theory

In our screening program for new photosensitizers from Malaysian biodiversity for photodynamic therapy (PDT) of cancer, MeOH extracts of ten terrestrial plants from Cameron Highlands in Pahang, Peninsular Malaysia, were tested. In a short-term 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, 20 μg/ml each of these extracts were incubated in a pro-myelocytic leukemia cell-line, HL60, with or without irradiation with 9.6 J/cm(2) of a broad spectrum light. Three samples, Labisia longistyla, Dichroa febrifuga, and Piper penangense, were photocytotoxic by having at least twofold lower cell viability when irradiated compared to the unirradiated assay. The extract of the leaves of Piper penangense, a shrub belonging to the family Piperaceae and widely distributed in the tropical and subtropical regions in the world, was subsequently subjected to bioassay-guided fractionation using standard chromatography methods. Eight derivatives of pheophorbide-a and -b were identified from the fractions that exhibited strong photocytotoxicity. By spectroscopic analysis, these compounds were identified as pheophorbide-a methyl ester (1), (R,S)-13(2) -hydroxypheophorbide-a methyl ester (2 and 3), pheophorbide-b methyl ester (4), 13(2) -hydroxypheophorbide-b methyl ester (5), 15(2) -hydroxylactone pheophorbide-a methyl ester (6), 15(2) -methoxylactone pheophorbide-a methyl ester (7), 15(2) -methoxylactone pheophorbide-b methyl ester (8).

Topics: Antineoplastic Agents, Phytogenic; Cell Survival; Chlorophyll; Drug Screening Assays, Antitumor; HL-60 Cells; Humans; Molecular Structure; Photosensitizing Agents; Piperaceae; Plant Extracts; Plant Leaves; Stereoisomerism; Structure-Activity Relationship

Pheophorbide a (Pa) has been proposed to be a potential photosensitizer for the photodynamic therapy of human cancer. However, the immunomodulatory effect of Pa, in the absence of irradiation, has not yet been investigated. The present study revealed that Pa possessed immunostimulating effect on a murine macrophages cell line RAW 264.7. Pa could significantly stimulate the growth of RAW 264.7 cells with the maximum effect at 1.0μM after 24, 48 and 72 h of treatment (all p<0.05). Besides, intracellular mitogen activated protein kinases (MAPK) including extracellular signal-regulated kinase (ERK) and p38 MAPK were activated by Pa treatment in a dose-dependent manner. The activation of ERK and p38 MAPK was found to be related to the Pa-induced intracellular reactive oxygen species. Furthermore, Pa could significantly induce the release of interleukine-6 and tumour necrosis factor-α, and enhance the phagocytic activity of RAW 264.7 cells (all p<0.05). The present work is the first report to demonstrate the potential immunomodulatory effect of Pa on macrophages, apart from its well-studied anti-tumour activity.

Topics: Adjuvants, Immunologic; Animals; Blotting, Western; Cell Cycle; Cell Proliferation; Chlorophyll; Flow Cytometry; Humans; Macrophages; Mice; Mitogen-Activated Protein Kinases; Radiation-Sensitizing Agents

The unbiased and comprehensive analysis of metabolites in any organism presents a major challenge if proper peak annotation and unambiguous assignment of the biological origin of the peaks are required. Here we provide a comprehensive multi-isotope labelling-based strategy using fully labelled (13) C, (15) N and (34) S plant tissues, in combination with a fractionated metabolite extraction protocol. The extraction procedure allows for the simultaneous extraction of polar, semi-polar and hydrophobic metabolites, as well as for the extraction of proteins and starch. After labelling and extraction, the metabolites and lipids were analysed using a high-resolution mass spectrometer providing accurate MS and all-ion fragmentation data, providing an unambiguous readout for every detectable isotope-labelled peak. The isotope labelling assisted peak annotation process employed can be applied in either an automated database-dependent or a database-independent analysis of the plant polar metabolome and lipidome. As a proof of concept, the developed methods and technologies were applied and validated using Arabidopsis thaliana leaf and root extracts. Along with a large repository of assigned elemental compositions, which is provided, we show, using selected examples, the accuracy and reliability of the developed workflow.

Topics: Arabidopsis; Carbon Isotopes; Chlorophyll; Databases, Factual; Fourier Analysis; Isotope Labeling; Lipids; Mass Spectrometry; Metabolomics; Nitrogen Isotopes; Plant Extracts; Plant Leaves; Plant Roots; Proteomics; Reproducibility of Results; Sulfur Isotopes

Pheophorbide a oxygenase (PAO) and red chlorophyll catabolite reductase (RCCR) catalyze key steps in chlorophyll degradation by opening the porphyrin macrocycle of pheophorbide a and forming the primary non-photoreactive fluorescent chlorophyll catabolite. These genes strongly participate in senescence and reportedly involved in plants' responses to physical wounding and pathogens. In this report, a single PAO gene (OsPAO) and two RCCR genes (OsRCCR1 and OsRCCR2) have been isolated from rice. Expression analysis by semi-quantitative PCR or quantitative real-time PCR showed that OsRCCR1 transcripts were much more abundant than OsRCCR2, and all of these genes were upregulated during senescence and following wound treatment. RNA interference knockdown of OsPAO led to pheophorbide a accumulation in leaves (especially dark-induced senescent leaves) and leaf death from regeneration stage onwards, even transgenic plants inviability after transplantation. While, knockdown of OsRCCR1 resulted in lesion-mimic spots generation in older leaves which died off early in the transgenic plants. These results suggest that OsPAO and OsRCCR1 play key roles in senescence and are involved in wound responses.

Topics: Amino Acid Sequence; Cell Death; Chlorophyll; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Organ Specificity; Oryza; Oxidoreductases; Oxygenases; Phenotype; Phylogeny; Plant Leaves; Plant Proteins; Plant Roots; Plant Stems; Plants, Genetically Modified; RNA Interference; RNA, Messenger; RNA, Plant; Seeds; Sequence Alignment; Up-Regulation

Over-expression of ABCG2 is linked to multidrug resistance in cancer chemotherapy. We have previously shown that functionalized aurones effectively reduced the efflux of pheophorbide A (an ABCG2 substrate) from ABCG2 over-expressing MDA-MB-231/R ("R") cells. In the present report, we investigated the functional relevance of this observation and the mechanisms by which it occurs. Aurones and related analogs were investigated for re-sensitization of R cells to mitoxantrone (MX, a chemotherapeutic substrate of ABCG2) in cell-based assays, accumulation of intracellular MX by cell cytometry, interaction with ABCG2 by biochemical assays and in vivo efficacy in MX resistant nude mice xenografts. We found that methoxylated aurones interacted directly with ABCG2 to inhibit efflux activity, possibly by competing for occupancy of one of the substrate binding sites on ABCG2. The present evidence suggests that they are not transported by ABCG2 although they stimulate ABCG2-ATPase activity. Alteration of ABCG2 protein expression was also discounted. One member was found to re-sensitize R cells to MX in both in vitro and in vivo settings. Our study identified methoxylated aurones as promising compounds associated with low toxicities and potent modulatory effects on the ABCG2 efflux protein. Thus, they warrant further scrutiny as lead templates for development as reversal agents of multidrug resistance.

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzofurans; Cell Survival; Chalcones; Chlorophyll; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Flavones; Indans; Mice; Mice, Inbred BALB C; Mice, Nude; Mitoxantrone; Neoplasm Transplantation; Transplantation, Heterologous

Pheophorbide-a, a chlorine based photosensitizer known to be selectively accumulated in cancer cells, was conjugated with anticancer drugs, doxorubicin and paclitaxel in the purpose of selective cancer diagnosis and therapy. Pheophorbide-a was conjugated with anticancer drugs via directly and by the use of selective cleavage linkers in cancer cell. The fluorescence of pheophorbide-a and doxorubicin conjugate by excitation at 420 or 440 nm was greatly diminished possibly by the energy transfer mechanism between two fluorescent groups. However, upon treatment in cancer cells, the conjugate showed to be cleaved to restore each fluorescence of pheophorbide-a and doxorubicin after 48 h of incubation. Also, pheophorbide-a conjugates either with doxorubicin and paclitaxel inhibited the growth of various cancer cells more potently than pheophorbide-a, which displayed very weak inhibitory activity. The results indicated that the pheophorbide-a conjugates with anticancer drugs could be utilized for selective cancer therapy as well as for the fluorescence detection of cancer.

Topics: Animals; Antineoplastic Agents; Cell Proliferation; Cell Survival; Chlorophyll; Dose-Response Relationship, Drug; Doxorubicin; Drug Screening Assays, Antitumor; Fluorescence; Fluorescent Dyes; HeLa Cells; Humans; Mice; Mice, Inbred C3H; Microscopy, Confocal; Molecular Conformation; Neoplasms; Paclitaxel; Spectrometry, Fluorescence; Stereoisomerism; Structure-Activity Relationship; Tissue Distribution; Tumor Cells, Cultured

Photodynamic therapy (PDT) is a therapeutic modality whose efficacy depends on several factors including type of photosensitizer, light fluence and cellular response. Cell recurrence is one of the problems still unsolved in PDT. In this work we found that in B78-H1 murine amelanotic melanoma cells there is a correlation between cell recurrence and the NF-κB/Snail/RKIP loop.. Proliferation and migration of surviving cells were analyzed by MTT and wound-scratch assays. The levels of ROS/NO in B78-H1 melanoma cells treated with pheophorbide a (Pba) and light (Pba/PDT) were measured by FACS, while expression of NF-κB, Snail and RKIP were determined by Western blots. The mechanism of cell death was investigated by caspase and microscopy assays.. Our data show that after a low-dose Pba/PDT treatment, B78-H1 cells are able to recover. This correlates with a low level of NO production, which blocks apoptosis via NF-κB pathway. Western blot analyses showed that a low-dose Pba/PDT increases the expression of NF-κB and anti-apoptotic Snail, but reduces the expression of pro-apoptotic RKIP. The role played by NF-κB in the modulation of Snail and RKIP was investigated using DHMEQ: a NF-κB inhibitor which behaves as NO donor. DHMEQ caused a decrease of Snail and an increase of RKIP expression. When B78-H1 cells were treated with a low dose Pba/PDT and DHMEQ, the NO level strongly increased, with the result that Snail was down-regulated and RKIP was upregulated, as observed with a high-dose Pba/PDT.. One major problem in PDT is the cellular rescue occurring in tissue regions receiving a low-dose PDT. To minimize this problem and sensitize cancer cells to PDT we propose a combined treatment in which the photosensitizer is delivered with a donor of NO acting on the NF-κB/Snail/RKIP loop.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chlorophyll; Dose-Response Relationship, Drug; Melanoma, Amelanotic; Mice; NF-kappa B; Nitric Oxide; Phosphatidylethanolamine Binding Protein; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species; Skin Neoplasms; Snail Family Transcription Factors; Transcription Factors

From our modulator library an interesting inhibitor of breast cancer resistance protein (BCRP) was identified. Due to its high inhibitory potency, this compound may serve as a promising novel lead for the design of new and potent modulators. This adds a new structural class to the few known highly active BCRP inhibitors.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzamides; Benzimidazoles; Cell Line, Tumor; Chlorophyll; Humans; Molecular Conformation; Neoplasm Proteins; Phenylurea Compounds; Small Molecule Libraries

The aim of this study was to assess the ability of commonly available red- or blue-light dental sources to generate reactive oxygen species (ROS) from photosensitive chemicals that might be useful for photodynamic antimicrobial chemotherapy (PACT).. Although the use of red diode lasers is well documented, there is limited information on how useful blue-light sources might be for PACT in dental contexts.. A diode laser (Periowave; see Table 1 for material and equipment sources) emitting red light (660-675 nm) was used to activate toluidine blue; riboflavin and pheophorbide-a polylysine (pheophorbide-a-PLL) were photoactivated using an Optilux 501 curing unit emitting blue light (380-500 nm). Ozone gas (generated by OzoTop, Tip Top Tips, Rolle, Switzerland), sodium hypochlorite, and hydrogen peroxide were used for comparison. ROS production was estimated using an iodine-triiodide colorimetric assay, and ROS levels were plotted versus concentration of chemicals to determine each chemical's efficiency in ROS production. One-way ANOVA with Tukey post hoc analysis (alpha = 0.05) was used to compare the efficiencies of ROS production for the various chemicals.. Sodium hypochlorite, hydrogen peroxide, and ozone gas produced ROS spontaneously, whereas pheophorbide-a-PLL, riboflavin, and toluidine blue required light exposure. The efficiency of ROS production was higher for pheophorbide-a-PLL and toluidine blue than for ozone gas or riboflavin (p < 0.05). Hydrogen peroxide was the least efficient ROS producer.. The results of the current study support the use of blue- or red-light-absorbing photosensitizers as candidates to produce ROS for clinical applications. Blue-light photosensitizers were as efficient as red-light photosensitizers in producing ROS and more efficient than the oxidant chemicals currently used for dental disinfection.

Topics: Chlorophyll; Dental Offices; Humans; Hydrogen Peroxide; Lasers, Semiconductor; Oxidants; Ozone; Photochemotherapy; Photosensitizing Agents; Radiation-Sensitizing Agents; Reactive Oxygen Species; Riboflavin; Sodium Hypochlorite; Tolonium Chloride

The time-resolved fluorescence technique based on melanin-concentrating hormone (MCH) receptor subtype-1 (MCH-1 receptor) binding assay was adopted to carry out a bioassay-guided fractionation of the methanol extract of Morus alba leaves. This fractionation and purification led to the isolation of two compounds identified as pheophorbide a methyl ester and 13(2)(S)-hydroxypheophorbide a methyl ester. These active pheophorbides exhibited potent inhibitory activity in binding of europium-labeled MCH to the human recombinant MCH-1 receptor (IC(50) value; 4.03 and 0.33 microM, respectively). Besides binding activity, the pheophorbides inhibited MCH-mediated extracellular signal-regulated kinase (ERK) phosphorylation in Chinese hamster ovary cells expressing human MCH-1 receptor. These results suggest that pheophorbide a methyl ester and 13(2)(S)-hydroxypheophorbide a methyl ester act as modulators of MCH-1 receptor and MCH-mediated ERK signaling.

Topics: Animals; Chlorophyll; CHO Cells; Cricetinae; Cricetulus; Extracellular Signal-Regulated MAP Kinases; Humans; Molecular Structure; Morus; Phosphorylation; Plant Extracts; Plant Leaves; Receptors, Pituitary Hormone; Recombinant Proteins; Signal Transduction

Scutellaria barbata is a traditional Chinese medicine for cancer treatments. Pheophorbide-a (Pa), one of the active components isolated from this herbal medicine has been proposed to be a potential natural photosensitizer for photodynamic therapy. The anti-tumor effect of pheophorbide-a based photodynamic therapy (Pa-PDT) has been successfully demonstrated in a wide range of human malignant cell lines. However, the effectiveness of Pa-PDT has not yet been evaluated on human breast cancer, which is documented as the second common and the fifth most lethal cancer worldwide.. The cytotoxicity of Pa-PDT was evaluated by using an estrogen receptor (ER)-negative human breast adenocarcinoma cell line MDA-MB-231. The involvement of mitochondria was revealed by the change of mitochondrial membrane potential and the increase of intracellular reactive oxygen species (ROS). The hallmarks of apoptosis, ER stress and autophagy were also assessed by DNA fragmentation, Western blotting, and immunostaining assays.. Pa-PDT showed inhibitory effect on the growth of MDA-MB-231 cells with an IC(50) value of 0.5 microM at 24h. Mitogen-activated protein kinase (MAPK) pathway was found to be triggered, where activation of c-Jun N-terminal kinase (JNK) and inhibition of extracellular signal-regulated kinase (ERK) were occurred in the Pa-PDT-treated cells. Our findings suggested that Pa-PDT exhibited its anti-tumor effects by the activation of mitochondria-mediated apoptosis and the ERK-mediated autophagy in MDA-MB-231 cells.. The present study suggested Pa-PDT is a potential protocol for the late phase human breast cancer, and it is the first study to demonstrate the Pa-PDT induced autophagy contributed to the anti-tumor effects of Pa-PDT on human cancer cells.

Topics: Adenocarcinoma; Apoptosis; Autophagy; Breast Neoplasms; Cell Line, Tumor; Chlorophyll; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Plant Extracts; Protein Kinase Inhibitors; Radiation-Sensitizing Agents; Receptors, Estrogen; Scutellaria

A new class of specific breast cancer resistance protein (BCRP) inhibitors was identified, showing no inhibition of the ATP binding cassette (ABC) transporters P-gp and MRP1. Some of these modulators inhibit BCRP with high potency; they are only slightly less potent than Ko143 and could serve as promising lead structures for the design of novel effective BCRP inhibitors. These inhibitors are structurally related to tariquidar (XR9576) and belong to a library of multidrug-resistance modulators synthesized by our research group. The absence of the tetrahydroisoquinoline substructure appears to play a crucial role for specificity; we found that the presence of this substructure is not essential for interaction with BCRP. To determine the type of interaction between pheophorbide A and compounds with and without the tetrahydroisoquinoline substructure, various substrate pheophorbide A concentrations were used in enzyme kinetics assays. The resulting data show that these compounds share a noncompetitive-type interaction with pheophorbide A. Experiments with imatinib and pheophorbide A revealed a mixed-type interaction. The combination of imatinib and compounds with and without the tetrahydroisoquinoline substructure resulted in a positive cooperative effect, indicating that imatinib engages a binding site distinct from that of the new compounds on one side and distinct from that of pheophorbide A on the other side as well. The results of this study suggest that the category of BCRP-specific inhibitors, which includes only fumitremorgin C, Ko143 and analogues, and novobiocin needs to be extended by this new class of inhibitors, which possess three key characteristics: specificity, potency, and low toxicity.

Topics: Adenosine; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzamides; Binding Sites; Breast Neoplasms; Cell Line, Tumor; Chlorophyll; Diketopiperazines; Drug Resistance, Multiple; Female; Heterocyclic Compounds, 4 or More Rings; Humans; Imatinib Mesylate; Indoles; Neoplasm Proteins; Novobiocin; Piperazines; Pyrimidines; Quinolines; Structure-Activity Relationship

ABCG2 is an ATP-binding cassette (ABC) transporter preferentially expressed by immature human hematopoietic progenitors. Due to its role in drug resistance, its expression has been correlated with a protection role against protoporhyrin IX (PPIX) accumulation in stem cells under hypoxic conditions. We show here that zinc mesoporphyrin, a validated fluorescent heme analog, is transported by ABCG2. We also show that the ABCG2 large extracellular loop ECL3 constitutes a porphyrin-binding domain, which strongly interacts with heme, hemin, PPIX, ZnPPIX, CoPPIX, and much less efficiently with pheophorbide a, but not with vitamin B12. K(d) values are in the range 0.5-3.5 μm, with heme displaying the highest affinity. Nonporphyrin substrates of ABCG2, such as mitoxantrone, doxo/daunorubicin, and riboflavin, do not bind to ECL3. Single-point mutations H583A and C603A inside ECL3 prevent the binding of hemin but hardly affect that of iron-free PPIX. The extracellular location of ECL3 downstream from the transport sites suggests that, after membrane translocation, hemin is transferred to ECL3, which is strategically positioned to release the bound porphyrin to extracellular partners. We show here that human serum albumin could be one of these possible partners as it removes hemin bound to ECL3 and interacts with ABCG2, with a K(d) of about 3 μm.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Hypoxia; Chlorophyll; Hematopoietic Stem Cells; Heme; Humans; K562 Cells; Mesoporphyrins; Neoplasm Proteins; Photosensitizing Agents; Point Mutation; Protein Structure, Secondary; Protoporphyrins; Serum Albumin; Vitamin B 12; Vitamin B Complex

The physiological importance of the human ATP-binding cassette (ABC) transporter ABCG2 has been recognized with regard to porphyrin-mediated photosensitivity. Functional impairment owing to inhibition of ABCG2 by drugs or its genetic polymorphisms may lead to the disruption of porphyrin homeostasis, which in turn causes cellular toxicity.. We evaluated the impact on photosensitivity of the inhibition by cyclin-dependent kinase (CDK) inhibitors of ABCG2 function. For this purpose, we established new methods for photosensitivity assays by using Flp-In-293 cells and plasma membrane vesicles prepared from Sf9 insect cells. With the new methods, we subsequently tested CDK inhibitors, i.e., purvalanol A, WHI-P180, bohemine, roscovitine, and olomoucine.. Among CDK inhibitors tested, purvalanol A was found to be the most potent inhibitor (IC50=3.5 microM) for ABCG2-mediated hematoporphyrin transport. At a concentration of 2.5 microM, it evoked the photosensitivity of ABCG2-expressing Flp-In-293 cells treated with pheophorbide a. WHI-P180 moderately inhibited ABCG2 function, exhibiting weak phototoxicity. In contrast, the phototoxicity of bohemine, roscovitine, and olomoucine were minimal in our assay system.. It is suggested that the planar structure is an important factor for interactions with the active site of ABCG2. The present study provides a new approach to studying drug-induced phototoxicity in vitro.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line; Cell Membrane; Cell Survival; Chlorophyll; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Hematoporphyrins; Humans; Kinetin; Models, Molecular; Molecular Conformation; Molecular Structure; Neoplasm Proteins; Photosensitizing Agents; Protein Kinase Inhibitors; Purines; Quantitative Structure-Activity Relationship; Quinazolines; Roscovitine; Spodoptera; Transfection

When used at low concentrations and added to the water body, water-soluble chlorophyllin (resulting from chlorophyll after removal of the phytol) and pheophorbid (produced from chlorophyllin by acidification) are able to kill mosquito larvae and other small animals within a few hours under exposure of solar radiation. Under laboratory conditions, the use of chlorophyllin/pheophorbid as photodynamic substances for pest control in water bodies promises to be not only effective and ecologically beneficial but also cheap. The LD50 (50% of mortality in the tested organisms) value in Culex sp. larvae was about 6.88 mg/l, in Chaoborus sp. larvae about 24.18 mg/l, and in Daphnia 0.55 mg/l. The LD50 values determined for pheophorbid were 8.44 mg/l in Culex, 1.05 mg/l in Chaoborus, and 0.45 mg/l in Daphnia, respectively. In some cases, chlorophyllin and pheophorbid were also found to be (less) active in darkness. The results presented in this paper show that chlorophyllin is about a factor of 100 more effective than methylene blue or hematoporphyrine, which were tested earlier for the same purpose. It is also much cheaper and, as a substance found in every green plant, it is 100% biodegradable.

Topics: Animals; Chlorophyll; Chlorophyllides; Culex; Daphnia; Diptera; Ecosystem; Insecticides; Lethal Dose 50; Mosquito Control; Photosensitizing Agents; Water

Larval insect herbivores feeding externally on leaves are vulnerable to numerous and varied enemies. Larvae of the Neotropical herbivore, Chelymorpha alternans (Chrysomelidae:Cassidinae), possess shields made of cast skins and feces, which can be aimed and waved at attacking enemies. Prior work with C. alternans feeding on Merremia umbellata (Convolvulaceae) showed that shields offered protection from generalist predators, and polar compounds were implicated. This study used a ubiquitous ant predator, Azteca lacrymosa, in field bioassays to determine the chemical constitution of the defense. We confirmed that intact shields do protect larvae and that methanol-water leaching significantly reduced shield effectiveness. Liquid chromatography-mass spectrometry (LC-MS) of the methanolic shield extract revealed two peaks at 20.18 min and 21.97 min, both with a molecular ion at m/z 593.4, and a strong UV absorption around 409 nm, suggesting a porphyrin-type compound. LC-MS analysis of a commercial standard confirmed pheophorbide a (Pha) identity. C. alternans shields contained more than 100 microg Pha per shield. Shields leached with methanol-water did not deter ants. Methanol-water-leached shields enhanced with 3 microg of Pha were more deterrent than larvae with solvent-leached shields, while those with 5 microg additional Pha provided slightly less deterrence than larvae with intact shields. Solvent-leached shields with 10 microg added Pha were comparable to intact shields, even though the Pha concentration was less than 10% of its natural concentration. Our findings are the first to assign an ecological role for a chlorophyll catabolite as a deterrent in an insect defense.

Topics: Animals; Chlorophyll; Chromatography, High Pressure Liquid; Coleoptera; Larva; Mass Spectrometry

Topics: Carcinosarcoma; Chlorophyll; Female; Humans; Mitochondria; Radiation-Sensitizing Agents; Uterine Neoplasms

Incorporation of unaggregated monomeric molecules of pheophorbide(a) into micelles of poly(ethyleneoxide-b-epsilon-caprolactone) has been characterized by fluorescence spectroscopy, dynamic light scattering, transmission electron microscopy and asymmetric flow field flow fractionation. It was shown that the method used leads to 20 nm micelles, corresponding to approximately 200 molecules of polymer and 4 molecules of monomeric pheophorbide(a) per nano-object which was able to generate (1)O(2) in the medium. They have been used thereafter as nanocarriers for photosensitizers in photodynamic therapy against cancer cells. The encapsulation of photosensitizer has been verified and in vitro tests on human cancerous cells have revealed a ca. 2-fold enhanced photocytotoxicity and cellular uptake compared to free pheophorbide(a).

Topics: Cell Line, Tumor; Chlorophyll; Drug Compounding; Ethylene Glycols; Humans; Micelles; Microscopy, Electron, Transmission; Nanoparticles; Particle Size; Photochemotherapy; Photosensitizing Agents; Polyesters; Singlet Oxygen

Tetrapyrroles are well-known photosensitizers. In plants, various intermediate molecules of tetrapyrrole metabolism have been reported to induce cell death in a light-dependent manner. In contrast to these reports, we found that pheophorbide a, a key intermediate of chlorophyll catabolism, causes cell death in complete darkness in a transgenic Arabidopsis plant, As-ACD1. In this plant, expression of mRNA for pheophorbide a oxygenase was suppressed by expression of Acd1 antisense RNA; thus, As-ACD1 accumulated an excessive amount of pheophorbide a when chlorophyll breakdown occurred. We observed that when senescence was induced by a continuous dark period, leaves of As-ACD1 plants became dehydrated. By measuring electrolyte leakage, we estimated that >50% of the leaf cells underwent cell death within a 5 d period of darkness. Light and electron microscopic observations indicated that the cellular structure had collapsed in a large population of cells. Partially covering a leaf with aluminum foil resulted in light-independent cell death in the covered region and induced bleaching in the uncovered regions. These results indicate that accumulation of pheophorbide a induces cell death under both darkness and illumination, but the mechanisms of cell death under these conditions may differ. We discuss the possible mechanism of light-independent cell death and the involvement of pheophorbide a in the signaling pathway for programmed cell death.

Topics: Apoptosis Regulatory Proteins; Arabidopsis; Arabidopsis Proteins; Cell Death; Chlorophyll; Darkness; Gene Expression Regulation, Plant; Oxygenases; Plant Leaves; Plants, Genetically Modified; RNA, Antisense

Uterine carcinosarcoma is an aggressive neoplasm with low survival rates because of the lack of very effective chemotherapy protocol. Photodynamic therapy (PDT) is recently suggested to be an efficient protocol for this cancer. Pheophorbide a (Pa) is a chlorophyll degradation product in the green plant cells, its antitumor effect was reported on a number of human cancer cells with PDT approach. This study demonstrated that using Pa in PDT (Pa-PDT) significantly inhibited the human uterine sarcoma cell line MES-SA with an IC50 value of 0.5 μM at 24 h. Induction of apoptosis was found on the Pa-PDT treated cells according to the results of propidium iodide (PI) staining, annexin-V staining and DNA fragmentation assay. Pa was found to be localized in the mitochondria that lead to the depolarization of mitochondrial membrane potential by the rapid generation of singlet oxygen during light irradiation, where release of cytochrome c was detected and lead to the activation of intrinsic apoptotic pathway in MES-SA cells. Our findings revealed the therapeutic potential of Pa-PDT on the human uterine cancer.

Topics: Apoptosis; Biological Transport; Carcinosarcoma; Cell Line, Tumor; Cell Proliferation; Chlorophyll; Female; Humans; Mitochondria; Photochemotherapy; Radiation-Sensitizing Agents; Reactive Oxygen Species; Uterine Neoplasms

The electronic excitation spectra of both chlorophyllide a (Chl) and pheophorbide a (Pheo) molecules in solvents have been investigated by using the time-dependent density functional theory (TDDFT) along with the polarizable continuum solvation model (PCM). With increasing Hartree-Fock (HF) exchange percentage in DFT functionals, the predicted HOMO-LUMO gaps increase linearly while the excitation energies increase gradually and even strongly for excited states with partial intramolecular charge-transfer (CT) nature. On the basis of the calculated excitation energies, oscillator strengths and frontier molecular orbital analysis, we provide some new insights into the absorption spectra of Pheo and Chl both in the gas phase and in solutions, especially for the B and higher electronic absorption bands. It is shown that the experimental observed visible Q(y) and Q(x) and ultraviolet B(y) and B(x) bands are all due to singlet (1)(pi,pi*) valence excitations, with the B bands being more strongly red-shifted by solvent effects. Two (1)(pi,pi*) dark states are predicted slightly below (or near) the strong B band for both Chl and Pheo, with one related to the excitation of tetrapyrrole ring and the other related to the excitation of ring I vinyl substituent. The (1)(n,pi*) CT state from the conjugated carbonyl substituent is above B bands and further strongly blue-shifted by solvent effects. The higher eta and N bands are mainly due to (1)(pi,pi*) valence excitations with only partial CT character, which are also red-shifted in solvent.

Topics: Chloroform; Chlorophyll; Chlorophyllides; Computer Simulation; Crystallography, X-Ray; Electrons; Ethanol; Methanol; Models, Chemical; Models, Molecular; Solvents; Spectrophotometry, Ultraviolet; Time Factors; Water

Photodynamic therapy (PDT) is a treatment modality that uses a combination of a photosensitizer and light to induce a photokilling process in the tumor tissue. Recently we re-considered pheophorbide a (Pba), a second-generation photosensitizer that has not yet been thoroughly investigated. Here, we report that Pba irradiated at 14 J/cm(2) induces a strong photodynamic effect in four tumor cell lines, with IC(50) values ranging between 70 and 250 nM. The mechanism of phototoxicity has been investigated in HeLa (IC(50) = 150 nM) and HepG2 (IC(50) = 95 nM) cells. In both cell lines Pba induces lipid peroxidation, as indicated by a marked increase of malonyldialdehyde and oxidized C11 BODIPY(581/591). At high doses (>IC(50)), Pba arrests cell growth completely by activating apoptosis and/or necrosis, while at low doses (

Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Line, Tumor; Chlorophyll; Drug Screening Assays, Antitumor; Drug Synergism; Glutathione Transferase; HeLa Cells; Heme Oxygenase-1; Humans; Inhibitory Concentration 50; Lipid Peroxidation; Liver Neoplasms; Neoplasm Proteins; Oxidative Stress; Photochemotherapy; Radiation-Sensitizing Agents; RNA Interference; RNA, Small Interfering

Amide linked pyro-pheophorbide a dimers, equipped with a suitable length of linkage, assemble in non-polar solvents by internal hydrogen bonding into C2-symmetric stacked structures as evidenced by UV-vis, fluorescence, IR, CD, 1H NMR spectroscopy and DFT molecular modelling studies.

Topics: Amides; Chlorophyll; Circular Dichroism; Dimerization; Fluorescence; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Molecular Structure; Porphyrins; Solvents; X-Ray Diffraction

In this work two types of pheophorbide-HSA (Pheo-HSA) nanoparticles, PHSA40 and PHSA100, were prepared and their photophysical and photosensitizing properties were investigated. Due to intramolecular interactions the singlet oxygen quantum yield of PHSA40 and PHSA100 is very low (less than 0.1). Intracellular uptake and phototoxicity of pheophorbide a as well as of the Pheo-HSA nanoparticles were studied in Jurkat cells. The HSA nanoparticles do not influence the amount of dye accumulation in cells. After 24h incubation, PHSA40 and PHSA100 showed a higher phototoxicity than Pheo. The reason for this behavior is an efficient nanoparticle decomposition in the cellular lysosomes. The process of drug release during incubation of cells with Pheo-HSA nanoparticles was illustrated by fluorescence lifetime imaging (FLIM) and confocal laser scanning microscopy (CLSM). The final phototoxicity of Pheo-HSA is at the same scale as induced by free Pheo. The drug release ability of HSA nanoparticles shows the possibility to use such formulations as drug carriers in PDT treatment. Therefore, this work constructs a standard for further investigation and optimization of photosensitizer-HSA drug carrier system.

Topics: Caspase 3; Cell Line, Tumor; Chlorophyll; Humans; Jurkat Cells; Nanoparticles; Photochemotherapy; Photosensitizing Agents; Serum Albumin; Singlet Oxygen; Spectrometry, Fluorescence; Time Factors

Fibrillar aggregates of amyloid beta-peptides are major constituents of the plaques found in the brains of patients with Alzheimer's disease, and have been implicated in the neurotoxicity of Alzheimer's. We previously reported that the methanol extract of mulberry leaves inhibits the formation of amyloid beta-peptide (1-42)-fibrils in vitro, and protects hippocampal neurons from amyloid beta-peptide (1-42)-induced cell death. In this study, we identified antiamyloidogenic substances, pheophorbide a, kaempferol -3-O-glucoside, and kaempferol -3-O-(6-malonyl) glucoside, from the methanol extract of mulberry leaves. We also compared the antiamyloidogenic activity of pheophorbide a with that of other porphyrin-related compounds.

Topics: Amyloid beta-Peptides; Chlorophyll; Chromatography; Chromatography, High Pressure Liquid; Glucosides; Humans; Kaempferols; Mass Spectrometry; Methanol; Monosaccharides; Morus; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments; Plant Extracts; Plant Leaves; Porphyrins; Protein Multimerization

Multidrug resistance (MDR) is frequently observed after prolonged treatment in human hepatoma with conventional anti-tumor drugs, and photodynamic therapy (PDT) is a recently suggested alternative to overcome MDR. The therapeutic potential of PDT was evaluated in a multidrug resistance (MDR) human hepatoma cell line R-HepG2 with photosensitizer pheophorbide a (Pa).. Our results demonstrated that intracellular accumulation of Pa was not reduced by the overexpression of P-glycoprotein. Pa-based PDT (Pa-PDT) significantly inhibited the growth of R-HepG2 cells with an IC50 value of 0.6 microM. Mechanistic study demonstrated that genomic DNA fragmentation and phosphatidylserine externalization occurred where increase of intracellular singlet oxygen level triggers the phosphorylation of c-Jun N-terminal Kinase (JNK) and leads to activation of intrinsic apoptotic caspases cascade during the Pa-PDT treatment. The cytotoxicity of Pa-PDT, accumulation of sub-G1 population, and depolarization of mitochondrial membrane could be inhibited by JNK inhibitor in the Pa-PDT treated cells. Interestingly, the Pa-PDT induced JNK activation showed inhibitory effect on MDR by the down-regulation of P-glycoprotein in R-HepG2 cells in a dose-dependent manner. In addition, significant reduction of tumor size was obtained in Pa-PDT treated R-HepG2-bearing nude mice with no significant damages in liver and heart.. In summary, our findings provided the first evidence that PDT could inhibit the MDR activity by down-regulating the expression of P-glycoprotein via JNK activation using pheophorbide a as the photosensitizer, and our work proved that Pa-PDT inhibited the growth of MDR hepatoma cells by mitochondrial-mediated apoptosis induction.

Topics: Analysis of Variance; Animals; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Hepatocellular; Cell Line, Tumor; Chlorophyll; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Enzyme Activation; Flow Cytometry; Humans; JNK Mitogen-Activated Protein Kinases; Liver Neoplasms; Male; Membrane Potential, Mitochondrial; Mice; Photochemotherapy; Radiation-Sensitizing Agents; Xenograft Model Antitumor Assays

To evaluate the efficacy of photodynamic therapy (PDT) against methicillin resistant-Staphylococcus aureus (MRSA) by selecting different light sources for irradiation and combining them with the photosensitizer Na-Pheophorbide a (Na-Phde a).. The treatment of drug-resistant bacterial infection is a serious issue. Recently, as a new clinical approach against septic arthritis, an experimental in vivo and in vitro model for the inactivation of MRSA by PDT using the photosensitizer Na-Phde a has been developed.. Na-Phde a solution (280 micromol/L) was mixed with MRSA strain bacterial inoculum. After 60 minutes, light was irradiated for 30 minutes using the following light sources: GaA1p semiconductor laser (300 mW, 670 nm), halogen lamp (75 W), xenon lamp (300 W) and fluorescent lamp (27 W). Bacterial growth was evaluated after 24 hours incubation in a blood agar culture.. The semiconductor laser and halogen lamp groups showed perfect bactericidal effects after PDT. The xenon lamp and fluorescent lamp groups showed partial bactericidal effects.. The results of this experiment showed that PDT using the combination of Na-Phde a with a semiconductor laser or halogen lamp showed a better bactericidal performance than with xenon or fluorescent lamps. These findings indicated that PDT using Na-Phde a could be a useful treatment for septic arthritis and soft tissue infection.

Topics: Arthritis, Infectious; Chlorophyll; Methicillin-Resistant Staphylococcus aureus; Photochemotherapy; Radiation-Sensitizing Agents; Soft Tissue Infections; Staphylococcal Infections

Fatty acid beta-oxidation is essential for seedling establishment of oilseed plants, but little is known about its role in leaf metabolism of adult plants. Arabidopsis thaliana plants with loss-of-function mutations in the peroxisomal ABC-transporter1 (PXA1) or the core beta-oxidation enzyme keto-acyl-thiolase 2 (KAT2) have impaired peroxisomal beta-oxidation. pxa1 and kat2 plants developed severe leaf necrosis, bleached rapidly when returned to light, and died after extended dark treatment, whereas the wild type was unaffected. Dark-treated pxa1 plants showed a decrease in photosystem II efficiency early on and accumulation of free fatty acids, mostly alpha-linolenic acid [18:3(n-3)] and pheophorbide a, a phototoxic chlorophyll catabolite causing the rapid bleaching. Isolated wild-type and pxa1 chloroplasts challenged with comparable alpha-linolenic acid concentrations both showed an 80% reduction in photosynthetic electron transport, whereas intact pxa1 plants were more susceptible to the toxic effects of alpha-linolenic acid than the wild type. Furthermore, starch-free mutants with impaired PXA1 function showed the phenotype more quickly, indicating a link between energy metabolism and beta-oxidation. We conclude that the accumulation of free polyunsaturated fatty acids causes membrane damage in pxa1 and kat2 plants and propose a model in which fatty acid respiration via peroxisomal beta-oxidation plays a major role in dark-treated plants after depletion of starch reserves.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; alpha-Linolenic Acid; Arabidopsis; Arabidopsis Proteins; ATP-Binding Cassette Transporters; Chlorophyll; Cloning, Molecular; Darkness; DNA, Bacterial; Mutagenesis, Insertional; Mutation; Oxidation-Reduction; Peroxisomes; Photosynthesis; Photosystem II Protein Complex; Plant Leaves; Potassium Channels, Voltage-Gated; Starch

The digestive stability, efficiency of micellarization, and cellular accumulation of the chlorophyll pigments of different preparations of pea were investigated, using an in vitro digestion procedure coupled with human intestinal Caco-2 cells. Fresh pea (FP), cooked fresh pea (CFP), frozen pea (FZP), cooked frozen pea (CFZP), and canned pea (CP) were subjected to simulated digestion. Although after digestion the pigment profile was modified for all samples, except CP, allomerization reactions and greater destruction of chlorophylls were observed in only FP, which should be due to enzymes in FP that were denaturalized in the rest of the test foods. A pigment extract of CFZP was also subjected to in vitro digestion, showing a positive effect of the food matrix on the pigment digestive stability. The transfer of the chlorophyll pigments from the digesta to the micellar fraction was significantly more efficient in CFZP (57%, p < 0.0001), not significantly ( p > 0.05) different between CFP, FZP, and CP (28-35%), and lowest in FP (20%). Pheophorbide a stood out as the most-micellarized chlorophyll derivative in all of the samples, reaching levels of up to 98%. Incubation of Caco-2 cells with micellar fractions at the same concentration prepared from each test food showed that pigment absorption was considerably lower ( p < 0.006) in cells incubated with FP, whereas there were no differences among the rest of the preparations. Therefore, factors associated with the food matrix could inhibit or mediate the chlorophyll pigment absorption. These results demonstrated that the industrial preservation processes of peafreezing and canningas well as the cooking have a positive effect on the bioaccessibility and bioavailability of the chlorophyll pigments with respect to the FP sample, emphasizing CFZP with greater bioaccesibilty degree.

Topics: Biological Availability; Caco-2 Cells; Chlorophyll; Digestion; Drug Stability; Food Preservation; Freezing; Hot Temperature; Humans; Intestinal Mucosa; Micelles; Pisum sativum

Photodynamic therapy is a recently developed anticancer treatment that utilizes the generation of singlet oxygen and other reactive oxygen species in cancer tissue. Nrf2, an NF-E2-related transcription factor, plays a pivotal role in transcriptional upregulation of many target genes, including those for metabolizing enzymes and transporters essential for cellular defense in response to oxidative stress. In the present study, we examined the potential involvement of Nrf2 in the induction of human ABC transporter ABCG2 and heme oxygenase-1 (HO-1). When HepG2 cells were incubated with non-toxic concentrations of delta-aminolevulinic acid, protoporphyrin IX, or pheophorbide a and then exposed to visible light for 90 min, the mRNA level of HO-1 began increasing markedly, reaching the maximal level in 4 h. Following the transient induction of HO-1, the mRNA level of ABCG2 gradually increased in a time-dependent manner, whereas the ABCB6 mRNA level was little affected. Nrf2-specific siRNA treatments suppressed the induction of both ABCG2 and HO-1 after the photoactivation of porphyrins, suggesting that Nrf2 is a common regulator for transcriptional activation of the ABCG2 and HO-1 genes. On the other hand, the mRNA level of HO-1 was remarkably enhanced by Zn(2+)-protoporphyrin IX or hemin even in the absence of light. This induction may be attributed to inactivation of Bach1, a repressor for the HO-1 gene, by those compounds. Since patients have demonstrated individual defferences in their response to photodynamic therapy, transcriptional activation of the ABCG2 and HO-1 genes in cancer cells may affect patients' responses to photodynamic therapy.

Topics: Aminolevulinic Acid; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzothiazoles; Blotting, Western; Cell Line, Tumor; Chlorophyll; Chromatography, High Pressure Liquid; Diamines; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Heme Oxygenase-1; Humans; Neoplasm Proteins; Neoplasms; NF-E2-Related Factor 2; Organic Chemicals; Oxidative Stress; Photochemistry; Photochemotherapy; Photosensitizing Agents; Porphyrins; Protoporphyrins; Quinolines; RNA, Messenger; RNA, Small Interfering; Transfection

The inhibitory effects of four chlorophyll derivatives (chlorophyllide [Chlide] a and b and pheophorbide [Pho] a and b) on aflatoxin B1 (AFB1)-DNA adduct formation, and on the modulation of hepatic glutathione S-transferase (GST) were evaluated in murine hepatoma (Hepa-1) cells. Enzyme-linked immunosorbent assay showed that pretreatment with Chlide or Pho significantly reduced the formation of AFB1-DNA adducts, and that Pho was the most potent inhibitor. However, wash-out prior to adding AFB1 totally eliminated inhibition by Childe and partially eliminated inhibition by Pho, indicating that the inhibitory effect of Chlide, and to some extent Pho, was mediated through direct trapping of AFB1. Furthermore, spectrophotometric analysis showed that Pho treatment could increase GST activity in Hepa-1 cells. These observations indicate that the chlorophyll derivatives studied may attenuate AFB1-induced DNA damage in the Hepa-1 cell by direct trapping of AFB1. Pho provided additional protection not only by direct trapping, but also by increasing GST activity against hepatic AFB1 metabolites.

Topics: Aflatoxin B1; Animals; Anticarcinogenic Agents; Cell Line, Tumor; Chlorophyll; Chlorophyllides; DNA Adducts; Glutathione Transferase; Liver Neoplasms, Experimental; Mice

The unique ability of chlorophyll derivatives to accumulate in tumorous tissues and to cause a photodynamic effect under laser radiation has been successively used in photodynamic therapy. In this connection, enormous research on modifications of the peripheral substituents in chlorophyll has shown that the presence and position of the substituents in the chlorin derivatives make a remarkable difference in biological activity. As a result, we directed our attention to synthesize methyl pheophorbide-a derivatives, chlorophyll-a derivatives, possessing heterocyclic aromatic substituents or pyrazole moieties on the peripheral positions and to examine preliminary in vitro effects of these new derivatives on A549 cancer cells for PDT. From our experimental results, it was observed that the biological effect induced by photosensitizers on cancer cells may also depend upon the chemical structure of the photosensitizers.

Topics: Cell Survival; Chlorophyll; Chlorophyll A; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Lasers; Molecular Conformation; Pyrazoles; Stereoisomerism; Tumor Cells, Cultured

Disinfection of the tooth pulp-canal system is imperative to successful endodontic therapy. Yet, studies suggest that 30-50% of current endodontic treatments fail from residual bacterial infection. Photodynamic therapy using red-light chromophores (630 nm) to induce antimicrobial death mediated by generated reactive oxygen species (ROS) has been reported, but red-light also may thermally damage resident tissues. In the current study, we tested the hypothesis that several blue light chromophores (380-500 nm) generate intracellular reactive oxygen species but are not cytotoxic to mammalian cells.. THP1 monocytes were exposed to 10 microM of four chromophores (chlorin e6, pheophorbide-a, pheophorbide-a-PLL, and riboflavin) for 30 min before activation with blue light (27J/cm(2), 60s). After activation, intracellular ROS were measured using a dihydrofluorescein diacetate technique, and cytotoxicity was determined by measuring mitochondrial activity with the MTT method.. All photosensitizers produced intracellular ROS levels that were dependent on both the presence of the photosensitizer and blue light exposure. Riboflavin and pheophorbide-a-PLL produced the highest levels of ROS. Photosensitizers except riboflavin exhibited cytotoxicity above 10 microM, and all except pheophorbide-a-PLL were more cytotoxic after blue light irradiation.. The current study demonstrated the possible utility of blue light chromophores as producers of ROS that would be useful for endodontic disinfection.

Topics: Cell Line; Chlorophyll; Chlorophyllides; Coloring Agents; Fluoresceins; Fluorescent Dyes; Humans; Light; Mitochondria; Monocytes; Photosensitizing Agents; Polylysine; Porphyrins; Radiation-Sensitizing Agents; Reactive Oxygen Species; Riboflavin; Succinate Dehydrogenase; Tetrazolium Salts; Thiazoles

In this study, we examined the effect of photodynamic therapy (PDT) using Na-pheophorbide a (Na-Phde a) on normal joint tissue.. The treatment of methicillin-resistant Staphylococcus aureus (MRSA) infection is a serious issue. Recently, an experimental in vivo and in vitro model for the inactivation of MRSA by PDT using a photosensitizer, Na-Phde a, has been developed.. The knee joints of mice were injected with 560 or 280 micromol/L of Na-Phde a. Thirty minutes after injection, percutaneous laser irradiation was applied for 5 min using a semiconductor laser (power: 125 mW; wavelength: 664 nm; total energy: 12 J/cm2). The joint perimeter and body weight of the treated mice were monitored, and histological evaluation was also done.. Joint swelling was observed up to 3 wk after PDT (p < 0.05). On histology 1 wk post-PDT, the treated knees were found to have inflammatory changes, primarily in synovial tissue. Eight weeks after PDT, the synovitis was no longer present. No significant effects were observed on cartilage, bone marrow, or menisci.. The results of this experiment showed that PDT with Na-Phde a induced arthritis for a short time after treatment. However, this arthritis was reversible, and the PDT did not appear to induce osteoarthritic changes in normal joint tissue. These findings indicate that PDT using Na-Phde a caused minimal but reversible changes in joint tissue, suggesting that it would be a safe and useful treatment for bacterial septic arthritis.

Topics: Animals; Chlorophyll; Female; Injections, Intra-Articular; Knee Joint; Lasers, Semiconductor; Mice; Mice, Inbred DBA; Photochemotherapy; Radiation-Sensitizing Agents

Many studies have been performed with the aim of developing effective resistance modulators to overcome the multidrug resistance (MDR) of human cancers. Potent MDR modulators are being investigated in clinical trials. Many current studies are focused on dietary herbs due to the fact that these have been used for centuries without producing any harmful side effects. In this study, the effect of tetrahydrocurcumin (THC) on three ABC drug transporter proteins, P-glycoprotein (P-gp or ABCB1), mitoxantrone resistance protein (MXR or ABCG2) and multidrug resistance protein 1 (MRP1 or ABCC1) was investigated, to assess whether an ultimate metabolite form of curcuminoids (THC) is able to modulate MDR in cancer cells. Two different types of cell lines were used for P-gp study, human cervical carcinoma KB-3-1 (wild type) and KB-V-1 and human breast cancer MCF-7 (wild type) and MCF-7 MDR, whereas, pcDNA3.1 and pcDNA3.1-MRP1 transfected HEK 293 and MXR overexpressing MCF7AdrVp3000 or MCF7FL1000 and its parental MCF-7 were used for MRP1 and MXR study, respectively. We report here for the first time that THC is able to inhibit the function of P-gp, MXR and MRP1. The results of flow cytometry assay indicated that THC is able to inhibit the function of P-gp and thereby significantly increase the accumulation of rhodamine and calcein AM in KB-V-1 cells. The result was confirmed by the effect of THC on [(3)H]-vinblastine accumulation and efflux in MCF-7 and MCF-7MDR. THC significantly increased the accumulation and inhibited the efflux of [(3)H]-vinblastine in MCF-7 MDR in a concentration-dependent manner. This effect was not found in wild type MCF-7 cell line. The interaction of THC with the P-gp molecule was clearly indicated by ATPase assay and photoaffinity labeling of P-gp with transport substrate. THC stimulated P-gp ATPase activity and inhibited the incorporation of [(125)I]-iodoarylazidoprazosin (IAAP) into P-gp in a concentration-dependent manner. The binding of [(125)I]-IAAP to MXR was also inhibited by THC suggesting that THC interacted with drug binding site of the transporter. THC dose dependently inhibited the efflux of mitoxantrone and pheophorbide A from MXR expressing cells (MCF7AdrVp3000 and MCF7FL1000). Similarly with MRP1, the efflux of a fluorescent substrate calcein AM was inhibited effectively by THC thereby the accumulation of calcein was increased in MRP1-HEK 293 and not its parental pcDNA3.1-HEK 293 cells. The MDR reversing properties of THC on P-

Topics: Adenosine Triphosphate; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Beryllium; Cell Line, Tumor; Chlorophyll; Curcumin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Etoposide; Fluoresceins; Fluorescent Dyes; Fluorides; Humans; Mitoxantrone; Molecular Structure; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Organic Anion Transporters; Rhodamine 123; Vinblastine

The two photosensitizers, chlorin e6 and pheophorbide a, were tested in an in vitro model of topical photodynamic therapy (PDT). Both dyes accumulate in HaCaT keratinocytes as verified by fluorescence measurement but pheophorbide a is enriched fivefold more strongly than chlorin e6 after 24 h. HaCaT cells are susceptible to PDT with both dyes. The phototoxicity measured by ATP bioluminescence is caused by necrosis and apoptosis depending on the photosensitizer used and the treatment modality. Chlorin e6 shows higher toxic potential because it elicits nearly 90% cell mortality 24 h after PDT comparable to pheophorbide a but with a fivefold lower rate of accumulation. These results implicate caution with topical PDT of oncologic diseases due to the risk of serious side effects on healthy skin in the course of topical photodynamic treatment. But the lack of dark toxicity and the time-dependent enrichment of both dyes in HaCaT cells are arguments for the application of these sensitizers in topical PDT of non-malign skin disorders. Further studies are necessary to discover appropriate lower doses and mechanisms of action of topical PDT with both compounds.

Topics: Apoptosis; Caspase 3; Cells, Cultured; Chlorophyll; Chlorophyllides; Humans; Keratinocytes; Photochemotherapy; Porphyrins; Structure-Activity Relationship

The safety and effectiveness of highly active antiretroviral therapy (HAART) is challenged by viral resistance to antiretrovirals and the frequent occurrence of drug interactions which may limit the access of these drugs to the target sites. In particular, drug distribution and elimination may be modified by active efflux transporters. While P-glycoprotein is well evaluated in this regard, the interaction of antiretrovirals with the ABC transporter BCRP (ABCG2) is far from being elucidated. The aim of this study was therefore to investigate the influence of all important anti-HIV drugs on BCRP activity in vitro in one assay to allow unrestricted comparison of the results.. BCRP inhibition was assessed by an increase in pheophorbide A accumulation in MDCKII-BCRP cells and compared with the corresponding parental cell line MDCKII lacking human BCRP.. According to the IC(50) estimation, the rank order for BCRP inhibition was lopinavir > nelfinavir > delavirdine > efavirenz > saquinavir > atazanavir > amprenavir > abacavir. Whereas nevirapine and zidovudine exerted weak inhibition, the inhibitory potency for ritonavir and tipranavir could not be estimated due to their low solubility and all other tested compounds (indinavir, didanosine, emtricitabine, lamivudine, stavudine, tenofovir and zalcitabine) were devoid of an effect.. Taken together, our study demonstrates significant inhibition of BCRP by many anti-HIV drugs. These results suggest that inhibition of BCRP might contribute to drug-drug interactions observed during HAART in vivo and possibly also the superior effectiveness of combination antiretroviral therapy.

Topics: Animals; Anti-HIV Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Blotting, Western; Cell Line; Cell Survival; Chlorophyll; Dogs; Drug Interactions; Epithelial Cells; Flow Cytometry; Humans; Neoplasm Proteins; Transfection

Pyrene mediated noncovalent attachment of a chlorophyll derivative, pyro-pheophorbide a, to a soluble single wall carbon nanotube is reported and the resultant CD, UV-Vis absorbance, fluorescence and 1H NMR spectra are discussed.

Topics: Chlorophyll; Nanotubes, Carbon; Spectrum Analysis

The function of ABCG2 (BCRP), a member of the ATP-binding cassette (ABC) superfamily of membrane-associated drug transporters, at the blood-brain barrier remains highly controversial. This project investigates the functional expression of endogenous ABCG2 in cultures of human and rodent brain cellular compartments.. RT-PCR, western blot and fluorescent immunocytochemical analyses were performed on ABCG2-overexpressing human breast cancer (MCF-MX100) cells, human and rat brain microvessel endothelial (HBEC and RBE4, respectively), and rat glial cells.. RT-PCR analysis detected ABCG2 mRNA in all the cell culture systems. Western blot analysis with anti-ABCG2 monoclonal BXP-21 antibody detected a robust band at approximately 72 kDa in the ABCG2-overexpressing MCF-MX100 cell line, whereas low expression was found in human and rat brain cell systems. Immunofluorescence microscopy detected predominant plasma membrane localization of ABCG2 in MCF-MX100 cells but weak signal in all brain cellular compartments. In the presence of ABCG2 inhibitors, the accumulation of (3)H-mitoxantrone and pheophorbide A, two established ABCG2 substrates, was significantly increased in MCF-MX100 cells but not in the human and rodent brain cell culture systems.. Our data show low endogenous ABCG2 protein expression, localization and activity in cultures of human and rat brain microvessel endothelial and glial cells.

Topics: Animals; Animals, Newborn; Astrocytes; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Blotting, Western; Brain; Breast Neoplasms; Cell Line, Tumor; Chlorophyll; Endothelial Cells; Female; Fluorescent Antibody Technique, Indirect; Humans; Indoles; Microcirculation; Microglia; Mitoxantrone; Neoplasm Proteins; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors; Transfection; Tritium

Scutellaria barbata, a Traditional Chinese Medicine native in southern China, has been widely used for treating liver diseases. In this study, the anti-proliferative effect of Pheophorbide a (Pa), an active component from S. barbata, was examined on a multi-drug resistant (MDR) human hepatoma cell line R-HepG2. Our study showed that Pa could significantly inhibit the growth of R-HepG2 cells with an IC50 value at 25.0 microM after 48 hours treatment. When compared with the parental HepG2 cells, Pa showed weak resistance to R-HepG2. Efflux of Pa out of R-HepG2 cells was not observed as its cellular uptake level showed no significant difference comparing with HepG2 cells. Interestingly, significant reduction of P-glycoprotein expression on Pa-treated R-HepG2 cells was found at both transcriptional and translational levels, leading to reduction of P-glycoprotein activity. In addition, mechanistic study elucidated that Pa induced cell cycle arrest at G2/M phase and inhibited the expressions of G2/M phase cell cycle regulatory proteins, cyclin-A1 and cdc2 in a dose-dependent manner.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Carcinoma, Hepatocellular; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chlorophyll; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Liver Neoplasms; Scutellaria

The stabilities of myoglobin, apo-myoglobin, and of two myoglobins with chlorophyllous chromophores (Zn-pheophorbide a and Zn-bacteriopheophorbide a), have been studied by thermal and chemical denaturation. With guanidinium chloride, the stability order is myoglobin>Zn-pheophorbide-myoglobin>Zn-bacteriopheophorbide-myoglobin approximately apo-myoglobin. The thermal behavior is more complex. The transition temperature of thermal unfolding of the apoprotein (62.4 degrees C) is increased by Zn-pheophorbide a (83.9 degrees C) and Zn-bacteriopheophorbide a (82.6 degrees C) to a similar degree as by the native chromophore, heme (83.5 degrees C). The recovery with Zn-pheophorbide (92-98%) is even higher than with heme (74-76%), while with Zn-bacteriopheophorbide (40%) it is as low as with the apoprotein (42%). Recovery also depends on the rates of heating, and in particular the time spent at high temperatures. It is concluded that irreversibility of unfolding is related to loss of the chromophores, which are required for proper re-folding.

Topics: Absorption; Animals; Bacteriochlorophylls; Chlorophyll; Circular Dichroism; Guanidine; Myoglobin; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Transition Temperature; Ultraviolet Rays

Breast cancer resistance protein (BCRP) is an efflux transporter that plays an important role in drug disposition. The goal of this study was to investigate the interactions of azole antifungal agents, ketoconazole, itraconazole, fluconazole, and voriconazole, with BCRP. First, the effect of the azoles on BCRP efflux activity in BCRP-overexpressing HEK cells was determined by measuring intracellular pheophorbide A (PhA) fluorescence using flow cytometry. We found that keotoconazole and itraconazole significantly inhibited BCRP-mediated efflux of PhA at low microM concentrations. However, fluconazole only mildly inhibited and voriconazole did not inhibit BCRP efflux activity at concentrations up to 100 microM. The IC(50) value of ketoconazole for inhibition of BCRP-mediated PhA efflux was 15.3 +/- 6.5 microM. Ketoconazole and itraconazole also effectively reversed BCRP-mediated resistance of HEK cells to topotecan. When direct efflux of [(3)H]ketoconazole was measured in BCRP-overexpressing HEK cells, we found that [(3)H]ketoconazole was not transported by BCRP. Consistent with this finding, BCRP did not confer resistance to ketoconazole and itraconazole in HEK cells. Taken together, ketoconazole and itraconazole are BCRP inhibitors, but fluconazole and voriconazole are not. These results suggest that BCRP could play a significant role in the pharmacokinetic interactions of ketoconazole or itraconazole with BCRP substrate drugs.

Topics: Antifungal Agents; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line; Cell Survival; Chlorophyll; Dose-Response Relationship, Drug; Drug Interactions; Drug Resistance, Neoplasm; Flow Cytometry; Fluconazole; Fluorescent Dyes; Humans; Inhibitory Concentration 50; Itraconazole; Ketoconazole; Neoplasm Proteins; Pyrimidines; Topotecan; Transfection; Triazoles; Voriconazole

Since porphyrins are regarded as endogenous substrates for the ATP-binding cassette (ABC) transporter ABCG2, it is hypothesized that functional impairment owing to genetic polymorphisms or inhibition of ABCG2 by drugs may result in a disruption of cellular porphyrin homeostasis. In the present study, we expressed ABCG2 genetic variants, i.e., V12M, Q141K, S441N, and F489L, as well as the wild type (WT) in Flp-In-293 cells to examine the hypothesis. Cells expressing S441N and F489L variants exhibited high levels of both cellularly accumulated pheophorbide a and photosensitivity, when those cells were incubated with pheophorbide a and irradiated with visible light. To further elucidate the significance of ABCG2 in cellular porphyrin homeostasis, we observed cellular accumulation and compartmentation of porphyrin and pheophorbide a by means of a new fluorescence microscopy technology, and found that accumulation of porphyrin and pheophorbide a in the cytoplasm compartment was maintained at low levels in Flp-In-293 cells expressing ABCG2 WT, V12M, or Q141K. When ABCG2 was inhibited by imatinib or novobiocin, however, those cells became sensitive to light. Based on these results, it is strongly suggested that certain genetic polymorphisms and/or inhibition of ABCG2 by drugs can enhance the potential risk of photosensitivity.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Benzamides; Cell Line; Cell Survival; Chlorophyll; Dose-Response Relationship, Drug; Drug Interactions; Homeostasis; Humans; Imatinib Mesylate; Inhibitory Concentration 50; Insecta; Light; Membrane Transport Modulators; Microscopy, Fluorescence; Neoplasm Proteins; Novobiocin; Photosensitivity Disorders; Photosensitizing Agents; Piperazines; Polymorphism, Single Nucleotide; Porphyrins; Pyrimidines; Quercetin; Risk Assessment; Transfection

Enzymatic removal of the methoxycarbonyl group of pheophorbide (Pheid) a in chlorophyll degradation was investigated in cotyledons of radish (Raphanus sativus). The enzyme pheophorbidase (PPD) catalyzes the conversion of Pheid a to a precursor of pyropheophorbide (PyroPheid), C-13(2)-carboxylPyroPheid a, by demethylation, and then the precursor is decarboxylated nonenzymatically to yield PyroPheid a. PPD activity sharply increased with the progression of senescence in radish, suggesting de novo synthesis of PPD. The enzyme activity was separated into two peaks in anion-exchange and hydrophobic chromatography; the terms type 1 and type 2 were applied according to the order of elution of these enzymes in anion-exchange chromatography. PPD types 1 and 2 were purified 9,999- and 6,476-fold, with a yield of 0.703% and 2.73%, respectively. Among 12 substrates tested, both enzymes were extremely specific for Pheids of the dihydroporphyrin and tetrahydroporphyrin types, indicating that they are responsible for the formation of these PyroPheids. Both PPDs had molecular masses of 113,000 kD on gel filtration and showed three bands of 16.8, 15.9, and 11.8 kD by SDS-PAGE. The partial N-terminal amino acid sequences for these bands of PPD (type 2) were determined. Based on their N-terminal amino acid sequences, a full-length cDNA of PPD was cloned. The molecular structure of PPD, particularly the molecular mass and subunit structure, is discussed in relation to the results of SDS-PAGE.

Topics: Amino Acid Sequence; Chlorophyll; Cloning, Molecular; Cotyledon; Esterases; Molecular Sequence Data; Plant Proteins; Raphanus; Sequence Alignment; Substrate Specificity

Scutellaria barbata has long been used as a Chinese medicine for the treatment of liver diseases such as hepatitis and hepatocellular carcinoma. In the present study, a bioassay-guided method was used to isolate the most active components from Scutellaria barbata. The anti-proliferative effects on human hepatoma HepG2 and Hep3B cells of each fraction at every stage of the purification were monitored. An active component, which is 97% pure by high performance liquid chromatographic analysis, was isolated. Based on nuclear magnetic resonance (NMR) and mass spectrophotometric (MS) analysis, this active component was identified to be pheophorbide a (C35H36N4O5). Mechanistic studies showed that pheophorbide a induced apoptosis in Hep3B cells, a viral-induced hepatoma cell line. However, it was found to be non-toxic in normal human liver cells WRL-68. DNA fragmentation, sub-G1 cell cycle arrest, as well as suppression of the anti-apoptotic protein Bcl-2, release of cytochrome c to the cytosol, and activation of pro-caspase 3 and pro-caspase 9 were observed when Hep3B cells were treated with 40 microg/mL (i. e., 67.5 microM) pheophorbide a for 48 hours. In conclusion, this is the first report describing the isolation of pheophorbide a from Scutellaria barbata using a bioassay-guided isolation method. The anti-proliferative activity and possible mechanisms of action of pheophorbide a on hepatoma Hep3B cells are also discussed.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chemical Fractionation; Chlorophyll; DNA Fragmentation; Flow Cytometry; Humans; Molecular Structure; Plant Extracts; Scutellaria

ABCG2 is a member of the adenosine triphosphate (ATP)-binding cassette family of multidrug transporters associated with resistance of tumor cells to many cytotoxic agents. Evaluation of modulators of ABCG2 activity has relied on methods such as drug sensitization, biochemical characterization, and transport studies. To search for novel inhibitors of ABCG2, a fluorescent cell-based assay was developed for application in high-throughput screening. Accumulation of pheophorbide a (PhA), an ABCG2-specific substrate, forms the basis for the assay in NCI-H460/MX20 cells overexpressing wild-type ABCG2. Treatment of these cells with 10 microM fumitremorgin C (FTC), a specific ABCG2 inhibitor, increased cell accumulation of PhA to 5.6 times control (Z' 0.5). Validation included confirmation with known ABCG2 inhibitors: FTC, novobiocin, tariquidar, and quercetin. Verapamil, reported to inhibit P-glycoprotein but not ABCG2, had insignificant activity. Screening of a library of 3523 natural products identified 11 compounds with high activity (> or = 50% of FTC, confirmed by reassay), including 3 flavonoids, members of a family of compounds that include ABCG2 inhibitors. One of the inhibitors detected, eupatin, was moderately potent (IC50 of 2.2 microM) and, like FTC, restored sensitivity of resistant cells to mitoxantrone. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel inhibitors of ABCG2 activity.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line, Tumor; Chlorophyll; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Drug Synergism; Humans; Indenes; Mitoxantrone; Neoplasm Proteins; Radiation-Sensitizing Agents

The ATP-binding cassette (ABC) transporter ABCG2 has been implicated to play a significant role in the response of patients to medication and/or the risk of diseases. To clarify the possible physiological or pathological relevance of ABCG2 polymorphisms, we have functionally validated single nucleotide polymorphisms (SNP) of ABCG2. In the present study, based on the currently available data on SNPs and acquired mutations, we have created a total of 18 variant forms of ABCG2 (V12M, G51C, Q126stop, Q141K, T153M, Q166E, I206L, F208S, S248P, E334stop, F431L, S441N, R482G, R482T, F489L, F571I, N590Y, and D620N) by site-directed mutagenesis and expressed them in insect cells. Because porphyrins are considered to be endogenous substrates for ABCG2, we have investigated the porphyrin transport activity of those variant forms in vitro. We herein provide evidence that the variants Q126stop, F208S, S248P, E334stop, and S441N are defective in porphyrin transport, whereas F489L exhibited impaired transport, approximately 10% of the activity observed for the wild type. Furthermore, Flp-In-293 cells expressing those variants were photosensitive. Thus, among those genetic polymorphisms of ABCG2, at least the hitherto validated alleles of Q126stop, S441N, and F489L are suggested to be of clinical importance related to the potential risk of porphyria.

Topics: Adenosine Triphosphate; Alleles; Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Transport; Camptothecin; Cell Line; Cell Survival; Chlorophyll; Daunorubicin; Doxorubicin; Drug Resistance; Hematoporphyrins; Humans; Irinotecan; Kinetics; Methotrexate; Mitoxantrone; Mutant Proteins; Mutation; Neoplasm Proteins; Polymorphism, Single Nucleotide; Porphyrins; Spodoptera; Transfection

The purpose of this study was to clarify the localization and function of the ATP-binding cassette transporter G2 (ABCG2; BCRP/MXR/ABCP) in retinal capillary endothelial cells, which form the inner blood-retinal barrier, as an efflux transport system.. The expression was determined by reverse transcriptase polymerase chain reaction and Western blotting. The localization was identified by immunostaining. The transport function of ABCG2 was measured by flow cytometry.. Western blotting indicated that ABCG2 was expressed as a glycosylated disulfide-linked complex in the mouse retina and in peripheral tissues, including liver, kidney, and small intestine. Double immunolabeling of ABCG2 and glucose transporter 1 suggested that ABCG2 was localized on the luminal membrane of mouse retinal capillary endothelial cells. ABCG2 mRNA and protein were found to be expressed in a conditionally immortalized rat retinal capillary endothelial cell line, TR-iBRB, and rat retina. Treatment with Ko143, an ABCG2 inhibitor, restored the accumulation of pheophorbide a and protoporphyrin IX in TR-iBRB cells.. ABCG2 is expressed on the luminal membrane of retinal capillary endothelial cells, where ABCG2 acts as the efflux transporter for photosensitive toxins such as pheophorbide a and protoporphyrin IX. ABCG2 could play an important role at the inner blood-retinal barrier in restricting the distribution of phototoxins and xenobiotics in retinal tissue.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line; Chlorophyll; Endothelial Cells; Glucose Transporter Type 1; Humans; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Photosensitizing Agents; Protoporphyrins; Rats; Retina; Retinal Vessels; RNA, Messenger; Transfection

The human ATP-binding cassette transporter, ABCG2, confers resistance to multiple chemotherapeutic agents and also affects the bioavailability of different drugs. [(125)I]Iodoarylazidoprazosin (IAAP) and [(3)H]azidopine were used for photoaffinity labeling of ABCG2 in this study. We show here for the first time that both of these photoaffinity analogues are transport substrates for ABCG2 and that [(3)H]azidopine can also be used to photolabel both wild-type R482-ABCG2 and mutant T482-ABCG2. We further used these assays to screen for potential substrates or modulators of ABCG2 and observed that 1,4-dihydropyridines such as nicardipine and nifedipine, which are clinically used as antihypertensive agents, inhibited the photolabeling of ABCG2 with [(125)I]IAAP and [(3)H]azidopine as well as the transport of these photoaffinity analogues by ABCG2. Furthermore, [(3)H]nitrendipine and bodipy-Fl-dihydropyridine accumulation assays showed that these compounds are transported by ABCG2. These dihydropyridines also inhibited the efflux of the known ABCG2 substrates, mitoxantrone and pheophorbide-a, from ABCG2-overexpressing cells, and nicardipine was more potent in inhibiting this transport. Both nicardipine and nifedipine stimulated the ATPase activity of ABCG2, and the nifedipine-stimulated activity was inhibited by fumitremorgin C, suggesting that these agents might interact at the same site on the transporter. In addition, nontoxic concentrations of dihydropyridines increased the sensitivity of ABCG2-expressing cells to mitoxantrone by 3-5-fold. In aggregate, results from the photoaffinity labeling and efflux assays using [(125)I]IAAP and [(3)H]azidopine demonstrate that 1,4-dihydropyridines are substrates of ABCG2 and that these photolabels can be used to screen new substrates and/or inhibitors of this transporter.

Topics: Adenosine Triphosphate; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Azides; Calcium Channel Blockers; Cell Line; Chlorophyll; Dihydropyridines; Drug Interactions; Humans; Mitoxantrone; Neoplasm Proteins; Nicardipine; Nifedipine; Photoaffinity Labels; Prazosin

Photodynamic therapy (PDT) is an effective treatment for cancer by inducing apoptosis or necrosis in the target cells. Pheophorbide a (Pa), a chlorophyll derivative, is a photosensitzier which can induce significant anti-proliferative effects in a number of human cancer cell lines. This study investigated the action mechanism of Pa-mediated photodynamic therapy (Pa-PDT) on the human hepatocellular carcinoma, Hep3B cells. Pa-PDT significantly inhibited the growth of Hep3B cells with an IC50 value of 1.5 microM. Intracellular ROS level was increased in Pa-PDT treated cells and the cytotoxic effect could be reversed when ascorbic acid was applied. Pa was found to be localized in the mitochondria and then induced the target cells to undergo apoptosis, which was confirmed by propidium iodide staining and DNA fragmentation assay. Pa-PDT treatment also led to the depolarization of mitochondrial membrane potential (Deltapim) and a release of cytochrome c from mitochondria to the cytosol. The caspase cascade was activated as shown by a significant decrease of procaspase-3 and -9 in Pa-PDT treated cells in a dose-dependent manner. Furthermore, in nude mice model, Pa-PDT treatment could reduce the tumor size by 57% after 14 days treatment.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Cell Growth Processes; Cell Line, Tumor; Chlorophyll; Humans; Liver Neoplasms; Male; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitochondria; Photochemotherapy; Radiation-Sensitizing Agents; Reactive Oxygen Species; Scutellaria; Xenograft Model Antitumor Assays

Curcumin (curcumin I), demethoxycurcumin (curcumin II), and bisdemethoxycurcumin (curcumin III) are the major forms of curcuminoids found in the turmeric powder, which exhibit anticancer, antioxidant, and anti-inflammatory activities. In this study, we evaluated the ability of purified curcuminoids to modulate the function of either the wild-type 482R or the mutant 482T ABCG2 transporter stably expressed in HEK293 cells and drug-selected MCF-7 FLV1000 and MCF-7 AdVp3000 cells. Curcuminoids inhibited the transport of mitoxantrone and pheophorbide a from ABCG2-expressing cells. However, both cytotoxicity and [(3)H]curcumin I accumulation assays showed that curcuminoids are not transported by ABCG2. Nontoxic concentration of curcumin I, II, and III sensitized the ABCG2-expressing cells to mitoxantrone, topotecan, SN-38, and doxorubicin. This reversal was not due to reduced expression because ABCG2 protein levels were unaltered by treatment with 10 mumol/L curcuminoids for 72 hours. Curcumin I, II, and III stimulated (2.4- to 3.3-fold) ABCG2-mediated ATP hydrolysis and the IC(50)s were in the range of 7.5 to 18 nmol/L, suggesting a high affinity of curcuminoids for ABCG2. Curcuminoids also inhibited the photolabeling of ABCG2 with [(125)I]iodoarylazidoprazosin and [(3)H]azidopine as well as the transport of these two substrates in ABCG2-expressing cells. Curcuminoids did not inhibit the binding of [alpha-(32)P]8-azidoATP to ABCG2, suggesting that they do not interact with the ATP-binding site of the transporter. Collectively, these data show that, among curcuminoids, curcumin I is the most potent modulator of ABCG2 and thus should be considered as a treatment to increase the efficacy of conventional chemotherapeutic drugs.

Topics: Adenosine Triphosphate; Anticarcinogenic Agents; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Azides; Biological Transport; Chlorophyll; Curcumin; Dihydropyridines; Drug Resistance, Neoplasm; Humans; Hydrolysis; Mitoxantrone; Mutation; Neoplasm Proteins; Prazosin; Toxicity Tests; Tumor Cells, Cultured

Hepatocellular carcinoma (HCC) ranks the sixth among the most common malignancies, with chronic HBV infection being the most common cause. HCC is more common in Africa, China and south-east Asia, but its incidence in the USA, Canada and Australia is rising. Current treatment modalities for HCC are not effective, and only a small percentage of patients are suitable for surgical resection and liver transplantation. Thus other treatment options and improvement of available modalities are badly in need. Photodynamic therapy (PDT) may have some therapeutic benefit for patients with HCC. The study by Tang et al. has implicated that coupled with Pheophorbide a (Pa), PDT may offer therapeutic benefit for patients with HCC. Inhibition of cell proliferation and induction of apoptosis by Pa may be mechanistically responsible for Pa-PDT. As Pa is an extract from a Chinese herbal medicine Scutellaria Barbata, which is widely available, less toxic and less expensive, such a combination may find a better clinical usage in the treatment of HCC patients. More studies are mandatory to fully elucidate the efficacy and mechanisms of Pa-mediated PDT.

Topics: Carcinoma, Hepatocellular; Chlorophyll; Humans; Liver Neoplasms; Photochemotherapy; Radiation-Sensitizing Agents; Scutellaria

The synthesis, physico-chemical properties, cellular localization and photocytotoxicity of estradiol-pheophorbide a conjugates in estrogen-dependent cancer and vascular endothelial cells are described with the aim of increasing the photodynamic activity by targeting the nucleus of both tumor and blood vessel cells.

Topics: Breast Neoplasms; Cell Line, Tumor; Chlorophyll; DNA Damage; Endothelium, Vascular; Estradiol; Humans; Photochemotherapy; Reactive Oxygen Species; Spectrometry, Fluorescence; Spectrum Analysis

The Qy absorption band of two chlorophyll derivatives, zinc chlorin e6 (ZnCe6) and zinc pheophorbide a (ZnPheida), in aqueous solution is bathochromically shifted on addition of quinones, e.g., 1,4-benzoquinone (BQ), with a corresponding shift of the fluorescence band. This is due to a complex formation of zinc chlorins induced by BQs and subsequent rearrangement. The time-resolved absorption spectra after laser pulse excitation show triplet quenching of the pigments by BQ and other quinones via electron transfer. The effects of electron transfer to noncovalently bound BQs were also studied with de novo synthesized peptides, into which ZnCe6 and ZnPheida were incorporated as model systems for the primary steps of photosynthetic reaction centers. Whereas the photophysical properties are similar to those of the unbound zinc chlorins, no BQ-mediated complex formation was observed.

Topics: Benzoquinones; Chlorophyll; Chlorophyllides; Kinetics; Organometallic Compounds; Porphyrins; Protein Conformation; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Zinc

Chlorophylls (Chl's) are the most abundant natural plant pigments. Four chlorophyll-related compounds (CRCs), including chlorophyllide a and b (Chlide a and b) and pheophorbide a and b (Pho a and b), were investigated for their antioxidative capacities to protect human lymphocyte DNA from hydrogen peroxide (H(2)O(2)) induced strand breaks and oxidative damage ex vivo. Lymphocytes exposed to H(2)O(2) at concentrations of 10 and 50 microM revealed an increased frequency of DNA single-strand breaks (ssb's; as measured by the comet assay) and also an increased level of oxidized nucleoside (as measured by 8-hydroxydeoxyguanosine, 8-OHdG). All Chl's reduced the level of DNA ssb's and 8-OHdG within human lymphocytes following exposure to 10 microM H(2)O(2). Only Pho a and b were able to decrease DNA ssb's and 8-OHdG following treatment of lymphocytes with 50 microM H(2)O(2), in a concentration-dependent fashion. It was demonstrated herein that Pho a and b were more antioxidative than others. We applied DPPH free-radical scavenge assays in vitro, and got similar results. Pho a and b had higher ability in scavenging capacities than others. We conclude that water-extract Chl's are able to enhance the ability of human lymphocytes to resist H(2)O(2)-induced oxidative damage, especially for Pho a and b.

Topics: Antioxidants; Chlorophyll; Chlorophyllides; DNA Damage; Humans; Hydrogen Peroxide; Lymphocytes

In an outbreak of food poisoning involving a dried purple laver product (called nori), four persons had allergic-like symptoms such as inflammation and red rash on their face, mouth and belly. The causative nori was extracted and smeared on the arm-skin of five volunteers. Three out of five volunteers had a slight allergic reaction after 5 to 30 min when they were exposed to sunlight. The levels of the chlorophyll derivatives, pheophorbide a and pyropheophorbide a, measured by HPLC were 851-906 and 5,460-5,624 microg/g, respectively, in the causative samples. Judging from the high contents of pyropheophorbide a and pheophorbide a and the symptoms of patients and volunteers, the causative agents were concluded to be the photosensitizers pyropheophorbide a and pheophorbide a.

Topics: Adult; Chlorophyll; Chromatography, High Pressure Liquid; Disease Outbreaks; Food Hypersensitivity; Foodborne Diseases; Humans; Male; Photosensitizing Agents; Seaweed

The binding reaction of methyl pheophorbide-a with bovine serum albumins (BSA) in aqueous solution was studied by fluorescence and UV-Vis absorption spectra. The results indicated that the combination reaction of them was a single static quenching process. In aqueous solution, methyl pheophorbide-a strongly bound BSA with the apparent molar ratio of 2 : 1 because of methyl pheophorbide-a polymerized by itself. The binding constant KB was 6.7 x 10(4) L x mol(-1). In mixture solvent of tetrahydrofuran and water, methyl pheophorbide-a existed as single molecule and bound BSA with a molar ratio of 1: 1. There is single position for combining methyl pheophorbide-a with BSA. The shortest binding distance (r = 3.50 nm) and energy transfer efficiencies (E = 0.39) between donor (BSA) and acceptor (methyl pheophorbide-a) were obtained by Förster's nonradiative energy transfer mechanism.

Topics: Algorithms; Animals; Cattle; Chlorophyll; Energy Transfer; Fluorescence; Furans; Kinetics; Models, Chemical; Molecular Structure; Porphyrins; Protein Binding; Serum Albumin, Bovine; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Water

In our screening program for new photosensitizers from the Malaysian biodiversity, we found five pheophorbide-related compounds from the leaves and stems of Aglaonema simplex. Detailed spectroscopic analyses showed that compounds 1-3 and 5 are pheophorbide and hydroxy pheophorbide derivatives of chlorophyll a and b. Compound 4, identified as 15(1)-hydroxypurpurin-7-lactone ethyl methyl diester, was isolated for the first time from the Araceae family. An MTT-based short-term survival assay showed that all five compounds exhibit moderate-to-strong photocytotoxic activities towards human leukemia (HL60) and two oral squamous carcinoma cell lines (HSC-2 and HSC-3). Compounds 4 and 5 showed the strongest photocytotoxicities, with IC(50) values of 0.30-0.41 muM (Table 2). Compounds 1-3 with Et chains at C(17(3)) were less photocytotoxic than the parent pheophorbide a (5).

Topics: Araceae; Chlorophyll; Dose-Response Relationship, Drug; HL-60 Cells; Humans; Photosensitizing Agents; Plant Extracts; Plant Leaves; Plant Stems

Cavendish banana (Musa Cavendishi, subgroup AAA) remains green upon ripening at tropical temperature (25-30 degrees C), due to incomplete degradation of chlorophyll (Chl). Earlier, evidence for the existence of two distinct degradative pathways--chlorophyllase and chlorophyll oxidase pathways in these bananas was provided. Here, an attempt has been made to understand further the mechanism of inhibition of Chl degradation at different stages of ripening and detecting various enzyme activities by partial purification. Soluble and Triton-solubilized protein fractions obtained from peel acetone powder from green-unripe, green-ripe and yellow-ripe bananas efficiently degraded Chl a. About 2-fold increase in Chl hydrolyzing/oxidizing and magnesium-dechelatase activities was observed in ripe, as compared to green-unripe bananas. The electrophoretic pattern of the soluble and detergent-solubilized proteins from the three stages of ripening revealed that the latter fraction contained only three slow moving proteins, which were found to be glycoproteins, as revealed in PAS staining. The soluble enzyme fraction contained all other bands along with the above three bands, as observed in the Native-PAGE of DEAE-Sepharose purified fractions. Only soluble fraction from 'green-ripe' bananas, catalyzed formation of an unknown intermediate (retention time 8.6 min), which was formed by the action of Triton-solubilized enzyme fractions, obtained from 'green-unripe' and 'yellow-ripe' bananas. The enzyme responsible for the formation of this intermediate might be involved in the stay-green character and could be a component of Chl oxidase pathway. Partial purification of soluble protein fraction by DEAE-Sepharose showed the presence of chlorophyllase, magnesium-dechelatase, pheophorbide a oxygenase, red fluorescent catabolite reductase and Chl oxidase. Native PAGE of pooled fractions showed separation of proteins in different bands. Pooled fractions IV and VI showed the presence of a single major band, resulting in almost a homogenous preparation in a single step. Fraction IV catalyzed dechelation of Mg by Mg-dechelatase, while fraction VI catalyzed the formation of 132-OH-Chl a by chlorophyll oxidase. Chlorophyll oxidase activity was stimulated by linolenic acid, indicating involvement of lipoxygenase in oxidative Chl degradation, thereby resulting in the formation of 13(2)-OH-Chl a as product. The results show the presence of various enzymes of chlorophyllase and chlorophyll

Topics: Biochemistry; Chlorophyll; Chromatography; Chromatography, High Pressure Liquid; Electrophoresis, Polyacrylamide Gel; Enzymes; Glycoproteins; Hydrolysis; Magnesium; Musa; Oxidoreductases; Oxygen; Powders; Solubility; Temperature; Time Factors

Pheophorbide a (PhA), a chlorophyll catabolite, was shown to be an ABCG2 substrate based on Abcg2(-/-) knockout mouse studies (J. W. Jonker et al., Proc. Natl. Acad. Sci. USA, 99: 15649-15654, 2002). We developed a functional assay for ABCG2 using PhA and the ABCG2 inhibitor fumitremorgin C. In selected cell lines expressing high levels of P-glycoprotein, multidrug resistance-associated protein 1, or ABCG2, PhA transport was observed only in cells expressing ABCG2. Fumitremorgin C-inhibitable PhA transport was found to correlate with cell surface ABCG2 expression as measured by the anti-ABCG2 antibody 5D3. We found that 100 micro M of the cyclin-dependent kinase inhibitor UCN-01 or 1 micro M of the P-glycoprotein inhibitor tariquidar inhibited ABCG2-mediated PhA transport. In 4-day cytotoxicity assays, ABCG2-mediated resistance to SN-38 and topotecan was abrogated in ABCG2-transfected HEK-293 cells treated with 1 micro M tariquidar, and ABCG2-transfected cells were 6-7-fold resistant to UCN-01. PhA is an ABCG2-specific substrate with potential value in measuring ABCG2 function and expression in clinical samples.

Topics: ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Breast Neoplasms; Camptothecin; Chlorophyll; Drug Resistance, Neoplasm; Humans; Irinotecan; Neoplasm Proteins; Quinolines; Staurosporine; Topotecan

Recent research on erythrocytes as model cells for photodynamic therapy showed differing behaviour of certain photosensitisers in erythrocytes compared to other cells. Differences of dye accumulation in the cell membrane were proposed to be the reason for the distinct photodynamic effects. Using pheophorbide a as an example, the combination of erythrocyte ghosts as models to follow the dye accumulation in the cell membrane and intact erythrocytes as model cells to show the photodynamic damage is provided. Evidence for the correctness of the combination of erythrocyte ghosts and intact erythrocytes as a functioning model system in photodynamic cell research is provided using the confocal laser scanning microscopy on intact, pheophorbide a loaded erythrocytes.

Topics: Chlorophyll; Erythrocytes; Humans; Microscopy, Confocal; Models, Biological; Photochemotherapy; Radiation-Sensitizing Agents; Research Design

In order to apply photodynamic therapy (PDT) to pigmented melanoma, the efficacy of PDT mediated by pheophorbide alpha from silkworm excreta (SPbalpha) and commercial Photofrin against B16F10 melanoma was comparatively studied from the in vivo assay using C57BL/6J mice. From in vitro PDT assay, the proliferation of B16F10 cells treated with SPbalpha (more than 0.5 microg/ml) and light illumination (1.2 J/cm2) were significantly inhibited with the necrotic response. This indicated that the photocytotoxicity of SPbalpha (665 nm) was not influenced by melanin from melanoma. From the assessment of the in vivo photosensitizing activity, the tumor growth was further delayed in groups treated with SPbalpha/PDT compared to that treated with Photofrin /PDT. The survival rate of tumor bearing mice treated with SPbalpha/PDT was closely associated with its photosensitizing effect. In addition, the photosensitizing effect of SPbalpha/PDT showed a dose dependent tendency in light illumination. These results demonstrated that B16F10 melanoma cells were significantly photosensitized by SPbalpha/PDT, regardless of the influence of melanin from melanoma, and SPbalpha/PDT at very low drug dose (1 mg/kg) and light dose (1.2 J/cm2) showed the photosensitizing efficacy surpassing Photofrin against B16F10 melanoma in mice system.

Topics: Animals; Bombyx; Cell Death; Cell Line, Tumor; Chlorophyll; Female; Light; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Photochemotherapy; Pigmentation; Survival Rate

We examined the damage to mitochondrial electron transport caused by photosensitization of a pheophorbide a derivative, DH-I-180-3, shown recently to induce necrosis of lung carcinoma cells with low dark toxicity. Confocal microscopy showed that DH-I-180-3 co-localized with dihydrorhodamine-123 suggesting that it mainly accumulates in mitochondria. The photosensitizer alone in the dark did not affect mitochondrial electron transport. Illumination of isolated mitochondria in the presence of DH-I-180-3 resulted in inhibition of both NADH- and succinate-dependent respiration. Measurement of the activity of each component of the electron transport chain revealed that Complex I and III were very susceptible to the treatment whereas Complex IV was resistant. We conclude that the photosensitizer is localized in mitochondria and, upon illumination, produces reactive oxygen species that inactivate Complexes I and III.

Topics: Animals; Benzoquinones; Cattle; Chlorophyll; Electron Transport; Electron Transport Complex I; Electron Transport Complex II; Electron Transport Complex III; Electron Transport Complex IV; Light; Mitochondria; NAD; Radiation-Sensitizing Agents; Succinic Acid

The mechanism of cell death by pheophorbide a (Pba) which has been established to be a potential photosensitizer was examined in experimental photodynamic therapy (PDT) on Jurkat cells, a human lymphoid tumor cell line. In 30-60 min after irradiation, Pba treated cells exhibited apoptotic features including membrane blebbing and DNA fragmentation. Pba/PDT caused a rapid release of cytochrome c from mitochondria into the cytosol. Sequentially, activation of caspase-3 and the cleavage of poly ADP-ribose polymerase (PARP) were followed. Meanwhile, no evidence of activation of caspase-8 was indicated in the cells. In experiments with caspase inhibitors, it was found that caspase-3 alone was sufficient initiator for the Pba-induced apoptosis of the cells. Pba specific emission spectra were confirmed in the mitochondrial fraction and the light irradiation caused a rapid change in its membrane potential. Thus, mitochondria were entailed as the crucial targets for Pba as well as a responsible component for the cytochrome c release to initiate apoptotic pathways. Taken together, it was concluded that the mode of Jurkat cell death by Pba/PDT is an apoptosis, which is initiated by mitochondrial cytochrome c release and caspase-3-pathways.

Topics: Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Chlorophyll; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Jurkat Cells; Light; Mitochondria; Photochemotherapy; Radiation-Sensitizing Agents

Previous research showed that berberine-containing Berberis species synthesise the substances 5'-methoxyhydnocarpin-D (5'-MHC-D) and pheophorbide a, which have no antimicrobial activity but inhibit the expression of multidrug resistant efflux pumps (MDRs) in Staphylococcus aureus and potentiate the action of berberine. The MDR pumps extrude synthetic and natural antimicrobials from bacterial cells. We searched for these compounds in Berberis aetnensis C. Presl. (Berberidaceae), an endemic plant of the volcano Mount Etna. This work confirms the presence of pheophorbide a and permits us to hypothesise the presence of 5'-MHC-D in leaf extracts. In fact, the activity of ciprofloxacin was improved when two chromatographic fractions isolated from leaf extracts were added. These results are indicative of the presence of MDR pump inhibitors. Moreover, crude extracts were tested on several micro-organisms and showed antimicrobial activity mainly against Gram-positive bacteria and yeasts.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Berberine; Berberis; Candida; Chlorophyll; Ciprofloxacin; Drug Interactions; Flavonoids; Gram-Negative Bacteria; Gram-Positive Bacteria; Molecular Structure; Multidrug Resistance-Associated Proteins; Plant Extracts; Staphylococcus aureus

In the course of our search for Acyl-CoA: cholesterol acyltransferase (ACAT) inhibitors from natural sources, a new type of ACAT inhibitor was isolated from a methanol extract of Diospyros kaki. On the basis of spectral and structural evidence, the compound was identified as pheophorbide A-methyl ester. Pheophorbide A-methyl ester inhibited ACAT activity in a dose dependent manner with an IC50 value of 1.85 microg/mL.

Topics: Acyl Coenzyme A; Animals; Chlorophyll; Chromatography, High Pressure Liquid; Diospyros; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Gas Chromatography-Mass Spectrometry; Korea; Methanol; Methyl Ethers; Microsomes, Liver; Molecular Structure; Plant Extracts; Plant Leaves; Rats; Sterol O-Acyltransferase

Chlorophyll (chl) breakdown during senescence is an integral part of plant development and leads to the accumulation of colorless catabolites. The loss of green pigment is due to an oxygenolytic opening of the porphyrin macrocycle of pheophorbide (pheide) a followed by a reduction to yield a fluorescent chl catabolite. This step is comprised of the interaction of two enzymes, pheide a oxygenase (PaO) and red chl catabolite reductase. PaO activity is found only during senescence, hence PaO seems to be a key regulator of chl catabolism. Whereas red chl catabolite reductase has been cloned, the nature of PaO has remained elusive. Here we report on the identification of the PaO gene of Arabidopsis thaliana (AtPaO). AtPaO is a Rieske-type iron-sulfur cluster-containing enzyme that is identical to Arabidopsis accelerated cell death 1 and homologous to lethal leaf spot 1 (LLS1) of maize. Biochemical properties of recombinant AtPaO were identical to PaO isolated from a natural source. Production of fluorescent chl catabolite-1 required ferredoxin as an electron source and both substrates, pheide a and molecular oxygen. By using a maize lls1 mutant, the in vivo function of PaO, i.e., degradation of pheide a during senescence, could be confirmed. Thus, lls1 leaves stayed green during dark incubation and accumulated pheide a that caused a light-dependent lesion mimic phenotype. Whereas proteins were degraded similarly in wild type and lls1, a chl-binding protein was selectively retained in the mutant. PaO expression correlated positively with senescence, but the enzyme appeared to be post-translationally regulated as well.

Topics: Apoptosis Regulatory Proteins; Arabidopsis; Arabidopsis Proteins; Cell Death; Cellular Senescence; Chlorophyll; DNA, Complementary; Dose-Response Relationship, Drug; Electron Transport Complex III; Electrons; Ferredoxins; Iron-Sulfur Proteins; Models, Chemical; Mutation; Oxidoreductases; Oxygen; Oxygenases; Phenotype; Plant Leaves; Protein Biosynthesis; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Zea mays

Oxygenation of pheophorbide a is a key step in chlorophyll breakdown. Several biochemical studies have implicated that this step was catalyzed by an iron-containing and ferredoxin-dependent monooxygenase, pheophorbide a oxygenase (PaO). It has been proposed that inhibition of its activity arrests the chlorophyll breakdown and leads to the "stay-green" phenotype. We searched the Arabidopsis genome for a possible PaO-encoding gene and hypothesized that it has homology to known iron-containing Rieske-type monooxygenase sequences. We identified three such open reading frames, Tic55, ACD1 and ACD1-like. We produced transgenic Arabidopsis plants which expressed antisense RNA as a method to inhibit the expression of these genes. The appearance of these antisense plants were indistinguishable from that of the wild type under illumination. However, after they were kept under darkness for 5 d and again illuminated, the leaves of the antisense ACD1 plants (AsACD1) were bleached. Leaves of AsACD1 accumulated 387 nmol (g FW)(-1) pheophorbide a which corresponded to 60% of chlorophyll a degraded. The rate of decrease in chlorophyll a was not influenced in senesced AsACD1 leaves. These results demonstrated that ACD1 is involved in PaO activity, and its inhibition led to photooxidative destruction of the cell instead of the "stay-green" phenotype.

Topics: Antisense Elements (Genetics); Apoptosis; Apoptosis Regulatory Proteins; Arabidopsis; Arabidopsis Proteins; Chlorophyll; Chloroplasts; Darkness; Light; Oxygen; Oxygenases; Phenotype; Phylogeny; Plants, Genetically Modified

In the course of our search for Acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors from natural sources, a new type of ACAT inhibitor was isolated from the methanol extract of Persicaria vulgaris. On the basis of spectral evidence, the structure of the active compound was identified as pheophorbide A. Pheophorbide A inhibited ACAT activity with an IC 50 value of 1.1 microg/ml in an enzyme assay using rat liver microsomes with a dose dependent fashion.

Topics: Animals; Chlorophyll; Dose-Response Relationship, Drug; Microsomes, Liver; Molecular Structure; Plant Extracts; Plant Leaves; Plant Stems; Polygonaceae; Porphyrins; Pyridines; Rats; Sesquiterpenes; Sterol O-Acyltransferase

The breast cancer resistance protein (BCRPABCG2) is a member of the ATP-binding cassette family of drug transporters and confers resistance to various anticancer drugs. We show here that mice lacking Bcrp1Abcg2 become extremely sensitive to the dietary chlorophyll-breakdown product pheophorbide a, resulting in severe, sometimes lethal phototoxic lesions on light-exposed skin. Pheophorbide a occurs in various plant-derived foods and food supplements. Bcrp1 transports pheophorbide a and is highly efficient in limiting its uptake from ingested food. Bcrp1(-/-) mice also displayed a previously unknown type of protoporphyria. Erythrocyte levels of the heme precursor and phototoxin protoporphyrin IX, which is structurally related to pheophorbide a, were increased 10-fold. Transplantation with wild-type bone marrow cured the protoporphyria and reduced the phototoxin sensitivity of Bcrp1(-/-) mice. These results indicate that humans or animals with low or absent BCRP activity may be at increased risk for developing protoporphyria and diet-dependent phototoxicity and provide a striking illustration of the importance of drug transporters in protection from toxicity of normal food constituents.

Topics: Administration, Oral; Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Bone Marrow Transplantation; Cell Line; Chlorophyll; Chromatography, High Pressure Liquid; Dermatitis, Phototoxic; Diet; Drug Resistance; Female; Fetus; Fibroblasts; Genetic Predisposition to Disease; Medicago sativa; Mice; Mice, Inbred Strains; Mice, Knockout; Molecular Structure; Neoplasm Proteins; Photosensitizing Agents; Porphyria, Hepatoerythropoietic; Porphyrins; Pregnancy; Protoporphyrins; Radiation Chimera; Topotecan

delta-Aminolevulinic acid (ALA), which is an intermediate in the biosynthesis of chlorophyll a, can be biosynthesized via the C5 pathway and the Shemin pathway in Euglena gracilis. Analysis of the (13)C-NMR spectrum of (13)C-labeled methyl pheophorbide a, derived from 13C-labeled chlorophyll a biosynthesized from d-[1-(13)C]glucose by E. gracilis, provided evidence suggesting that ALA incorporated in the (13)C-labeled chlorophyll a was synthesized via both the C5 pathway and the Shemin pathway in a ratio of between 1.5 and 1.7 to one. The methoxyl carbon of the methoxycarbonyl group at C-132 of chlorophyll a was labeled with (13)C. The phytyl moiety of chlorophyll a was labeled on C-P2, C-P3(1), C-P4, C-P6, C-P7(1), C-P8, C-P10, C-P11(1), C-P12, C-P14, C-P15(1) and C-P16.

Topics: Aminolevulinic Acid; Animals; Chlorophyll; Chlorophyll A; Euglena gracilis; Methionine

A methanolic crude extract of the plant Garuga pinnata Roxb. (Burseraceae) showed promising cytotoxic activity against a panel of human tumor cell lines in vitro, including KB and its drug-resistant sublines (Ferguson et al. Cancer Res. 1988, 48, 5956). Pheophorbide-a and-b methyl esters (3,4) were isolated as active principles with broad photo-dependent cytotoxic activities in the micromolar range. These findings prompted SAR studies of known and novel pheophorbide-a derivatives as photo-dependent and photo-independent cytotoxic agents. The results showed that zinc-protoporphyrin IX (10), zinc 13(R)-hydroxypheophorbide-a methyl ester (22), and zinc chlorin-e6 trimethyl ester (13) possessed photo-independent cytotoxic activity. Compounds 13 and 22 were the most active cytotoxic agents of the series (mean ED(50) 4.6 +/- 1.0 microM and 5.7 +/- 0.7 microM, respectively) against KB cells incubated in the dark.

Topics: Antineoplastic Agents; Cell Division; Chlorophyll; Chlorophyllides; DNA Damage; Drug Screening Assays, Antitumor; Humans; Light; Porphyrins; Protoporphyrins; Radiation-Sensitizing Agents; Structure-Activity Relationship; Tumor Cells, Cultured; Zinc

This study was designed to investigate the efficacy of photodynamic therapy (PDT) in treating colonic cancer in a preclinical study. Photofrin, a porphyrin mixture, and pheophorbide a (Ph a), a bacteriochlorin, were tested on HT29 human colonic tumor cells in culture and xenografted into athymic mice. Their pharmacokinetics were investigated in vitro, and the PDT efficacy at increasing concentrations was determined with proliferative, cytotoxic and apoptotic assessments. The in vivo distribution and pharmacokinetics of these dyes (30 mg/kg, intraperitoneal) were investigated on HT29 tumor-bearing nude mice. The inhibition of tumor growth after a single 100 J/cm2 PDT session was measured by the changes in tumor volume and by histological analysis of tumor necrosis. PDT inhibited HT29 cell growth in culture. The cell photodamage occurred since the time the concentrations of Ph a and Photofrin reached 5.10(-7) M (or 0.3 microg/mL) and 10 microg/mL, respectively. A photosensitizer dose-dependent DNA fragmentation was observed linked to a cleavage of poly(ADP-ribose) polymerase and associated with an increased expression of mutant-type p53 protein. PDT induced a 3-week delay in tumor growth in vivo. The tumor injury was corroborated by histological observation of necrosis 48 h after treatment, with a correlated loss of specific enzyme expression in most of the tumor cells. In conclusion, PDT has the ability to destroy human colonic tumor cells in vitro and in vivo. This tumoricidal effect is likely associated with a p53-independent apoptosis, as HT29 cells express only mutated p53. The current study suggests a preferential use of Photofrin in PDT of colonic cancer because it should be more effective in vivo than Ph a as a consequence of better tumor uptake.

Topics: Animals; Chlorophyll; Colonic Neoplasms; Dihematoporphyrin Ether; Humans; Male; Mice; Mice, Nude; Photochemotherapy; Photosensitizing Agents; Porphyrins; Transplantation, Heterologous; Tumor Cells, Cultured

Continuing our search for biogenic NF-kappa B inhibitors we investigated Solanum diflorum, used by the Istmo Sierra Zapotec Indians of Mexico in the treatment of inflammatory skin conditions. It became obvious very early that the active substance seems to be a degradation product of chlorophyll. Pheophorbide A was identified as one of the key compounds responsible for the NF-kappa B inhibitory activity. The compound interferes with NF-kappa B activation, was cytotoxic if exposed to light, but devoid of any cytotoxic activity in the dark.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Chlorophyll; Darkness; HeLa Cells; Humans; Mexican Americans; NF-kappa B; Plant Extracts; Plant Leaves; Plants, Medicinal; Skin Diseases; Solanaceae

Plasmid DNA is efficiently photocleaved by sodium pheophorbides (Na-Phdes) a and b in the absence of oxygen as well as in the presence of oxygen. Fluorescence microscopic observation shows a rapid incorporation of Na-Phde a into nuclei, mitochondria, and lysosome of human oral mucosa cells. In contrast Na-Phde b is incorporated only into the plasma membrane. The photodynamic activity of these pigments in living tissues is probably determined by the monomeric pigment molecules formed in hydrophobic cellular structures and involves two types of reactions: (i) direct electron transfer between DNA bases (especially guanine) and pheophorbide singlet excited state, and (ii) indirect reactions mediated by reactive oxygen species, including singlet oxygen whose production from molecular oxygen is sensitized by the Na-Phdes triplet state. A preliminary report has appeared in "Photodynamic Therapy of Cancer II," Proc. SPIE 2325, 416-424 (1994).

Topics: Chlorophyll; DNA; Humans; Microscopy, Fluorescence; Mouth Mucosa; Photochemotherapy; Radiation-Sensitizing Agents

Three pheophorbide-related compounds (1-3) were isolated from the leaves and stems of Clerodendrum calamitosum. The methyl ester of 3 (6) and the known (10S)-hydroxypheophytin a (7) also were isolated from leaves of the related plant Clerodendrum cyrtophyllum. Compounds 1 and 6 were isolated for the first time as naturally occurring products from a plant source. All structures were elucidated by detailed spectroscopic analysis. Biological evaluation showed that 1 and 2 exhibited strong cytotoxicity against human lung carcinoma (A549), ileocecal carcinoma (HCT-8), kidney carcinoma (CAKI-1), breast adenocarcinoma (MCF-7), malignant melanoma (SK-MEL-2), ovarian carcinoma (1A9), and epidermoid carcinoma of the nasopharynx (KB), and its etoposide- (KB-7d), vincristine- (KB-VCR), and camptothecin-resistant (KB-CPT) subclones. Compound 3 was less cytotoxic than 1 and 2. Compounds 4-6, the methyl esters of 1-3, showed strongly increased cytotoxicity compared with the parent acids. Interestingly, 6 was the most active derivative among these compounds. Compound 7 was inactive.

Topics: Adenocarcinoma; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Camptothecin; Cell Survival; Chlorophyll; Dose-Response Relationship, Drug; Drug Resistance; Etoposide; Female; Humans; Ileal Neoplasms; KB Cells; Kidney Neoplasms; Lung Neoplasms; Magnetic Resonance Spectroscopy; Melanoma; Molecular Structure; Ovarian Neoplasms; Plant Leaves; Plant Stems; Plants, Medicinal; Stereoisomerism; Structure-Activity Relationship; Taiwan; Tumor Cells, Cultured; Vincristine

The absorption and fluorescence properties of pheophorbide-a, Sodium salt of pheophorbide-a and its long chain (C20H39) ester (Pheophytine) were investigated in air-saturated micellar aqueous solutions before and after ultrasound irradiation (48 kHz, 10 min). The absorption spectra changed depending on the surfactant; cetyltrimethyl ammonium bromide (CTAB) or sodium dodecyl sulfate concentrations. The formation of different molecular species in various micellar solutions was estimated from the analysis of the absorption spectra. The absorption bands resulted from an aggregated form of the chromophore present in 50 mM phosphate buffer and in pre-micellar solutions. The specific bands of the aggregate disappeared with a simultaneous increase of the bands of monomer in normal micellar solution. The fluorescence spectra, the lifetimes and the fraction of each component (with a characteristic lifetime) of the chromophore in the micellar solutions changed significantly before and after ultrasound irradiation although the changes in absorption spectra were small. The fluorescence emission band at 710 nm due to the aggregate almost disappeared in the pre-micellar solution after ultrasound irradiation. The fraction of the short-lifetime component estimated for the aggregates decreased 55% in H2O or 85% in 2 mM CTAB, however the long-lifetime components increased after the ultrasound treatment. From these fluorescence properties, it was concluded that the aggregated molecules were converted to a stable monomeric form by ultrasound. Extrapolation of these data to in vivo situations suggests that pretreatment of certain photosensitizers with ultrasound in micellar solutions may lead to increased efficiency of photodynamic therapy since only the monomers are photodynamically active.

Topics: Cetrimonium; Cetrimonium Compounds; Chlorophyll; Micelles; Molecular Conformation; Pheophytins; Photosensitizing Agents; Radiation-Sensitizing Agents; Sodium Dodecyl Sulfate; Solutions; Spectrometry, Fluorescence; Spectrophotometry; Surface-Active Agents; Ultrasonics; Water

Oxygen consumption by cultured cells, through metabolism and photosensitization reactions, has been calculated theoretically. From this result, we have derived the partial oxygen pressure PO2 in the perfusion medium flowing across sensitized cultured cells during photodynamic experiments. The PO2 variations in the perfusate during light irradiation are related to the rate of oxygen consumption through photoreactions, and to the number of cells killed per mole of oxygen consumed through metabolic processes. After irradiation, the reduced metabolic oxygen consumption yields information on the cell death rate, and on the photodynamic cell killing efficiency. The aim of this paper is to present an experimental set-up and the corresponding theoretical model that allows us to control the photodynamic efficiency for a given cell-sensitizer pair, under well defined and controlled conditions of irradiation and oxygen supply. To demonstrate the usefulness of the methodology described, CHO cells cultured on microbeads were sensitized with pheophorbide a and irradiated with different light fluence rates. The results obtained, i.e. oxygen consumption of about 0.1 microM s(-1) m(-3) under a light fluence rate of 1 W m(-2), 10(5) cells killed per mole of oxygen consumed and a decay rate of about 1 h(-1) of living cells after irradiation, are in good agreement with the theoretical predictions and with previously published data.

Topics: Animals; Cell Death; Chlorophyll; CHO Cells; Cricetinae; Light; Microspheres; Models, Theoretical; Oxygen Consumption; Partial Pressure; Photochemotherapy; Photosensitizing Agents

This work reports on a monomer-dimer equilibrium state of pheophorbide a in solution. A methodology for controlling the equilibrium constant by use of temperature and solvent variation is described. The absorption spectrum of the dimer is calculated, using different prepared equilibria of monomer and dimer in solution. We propose that these aggregates provide a good model for understanding the dimerization process in tetrapyrroles.

Topics: Absorption; Chlorophyll; Dimerization; Fluorescence; Magnoliopsida; Mathematical Computing; Porphyrins; Radiation-Sensitizing Agents; Spectrophotometry

A series of modified chlorophylls (chlorophyll a, pyrochlorophyll a, Zn-pheophytin a and Zn-pheophorbide a) have been inserted into lamellar phases of sodium bis-(2-ethylhexyl)-sulfosuccinate (AOT). The role played by the different functional groups in affecting the bilayer formation and organisation has been investigated by means of the NMR quadrupolar splitting technique. Evidence is reported for the first time on the capacity of the phytyl chain of the chlorophylls to anchor the tetrapyrroles into the bilayer, favouring at the same time the regular formation of the lamellae.

Topics: Chlorophyll; Lipid Bilayers; Magnetic Resonance Spectroscopy; Pheophytins; Phytic Acid; Spinacia oleracea; Time Factors

The exposure of human lymphoid leukemia Molt 4B cells to pheophorbide a (PPB a), a moiety of chlorophyll a, led both to growth inhibition and induction of programmed cell death (apoptosis). The growth inhibition by PPB a was much stronger than that by chlorophyll a. Morphological change showing apoptotic bodies was observed in the Molt 4B cells treated with PPB a. The fragmentation by PPB a of DNA to oligonucleosomal-sized fragments, that are characteristics of apoptosis, was observed to be concentration- and time-dependent. These findings suggest that growth inhibition by PPB a of Molt 4B cells results from the induction of apoptosis and that PPB a, moiety of chlorophyll a, is essential for exertion of antitumor and apoptosis-inducing activity in these cells.

Topics: Apoptosis; Chlorophyll; DNA Fragmentation; DNA, Neoplasm; Humans; Leukemia, Lymphoid; Microscopy, Fluorescence; Tumor Cells, Cultured

A new method of bioactivity-directed fractionation, based on multidrug resistant pump (MDR) inhibition in Staphylococcus aureus, was demonstrated. This resulted in the isolation, from berberine-containing Berberis species, of two compounds that are themselves devoid of S. aureus antibacterial activity, but that form potent synergistic couples with a subinhibitory concentration of berberine. The bacterial MDR pump inhibitors were identified as the flavonolignan 2 and the porphyrin 3. Another natural flavonolignan, silybin (8) from Silybum marianum, was also shown to be a bacterial MDR pump inhibitor.

Topics: Berberine; Biological Assay; Chlorophyll; Chromatography, Gel; Chromatography, Thin Layer; Colorado; Drug Resistance, Microbial; Drug Resistance, Multiple; Flavonoids; Gas Chromatography-Mass Spectrometry; Magnetic Resonance Spectroscopy; Microbial Sensitivity Tests; Plant Leaves; Plants, Medicinal; Spectrometry, Mass, Fast Atom Bombardment; Spectrophotometry, Ultraviolet; Staphylococcus aureus

The aim of this study was to assess the efficiency of photodynamic therapy (PDT) on human pancreatic cancer cells in vitro and in an animal model.. Human pancreatic tumour cell lines were submitted to PDT with pheophorbide a (Ph a), a chlorophyll derivative, in culture and after grafting into athymic mice. Ph a was tested in culture (10-10-10-5 mol/l) with a 5-J/cm2 energy treatment and on tumour-bearing Nude mice (30 mg/kg intraperitoneally) with a 100-J/cm2 PDT session. The effect of PDT was assessed in vitro using proliferative, apoptotic and clonogenic tests and in vivo on tumour growth and on the induction of tumour necrosis.. PDT inhibited tumour cell growth in culture by affecting DNA integrity. This tumour cell photodamage started at low concentration (10-7 mol/l) as corroborated by clonogenic and tumour growth tests. A strong necrosis was achieved in vivo with a single PDT session.. PDT destroyed human pancreatic carcinoma after low photosensitizer supply and weak energy application. It exerted this tumoricidal effect via apoptosis induction with a gentle protocol, and apoptosis and/or necrosis with a stronger protocol.

Topics: Animals; Apoptosis; Cell Division; Chlorophyll; Electrophoresis; Humans; Male; Mice; Mice, Nude; Pancreatic Neoplasms; Photochemotherapy; Porphyrins; Radiation-Sensitizing Agents; Tumor Cells, Cultured

An assay method for determination of cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides (ChOOHs) in rat skin using high-performance liquid chromatography (HPLC) with a chemiluminescence detector has been developed. In the assay method, free form and free plus ester forms of ChOOHs could be separately determined by HPLC in combination with the treatment of a tissue extract by cholesterol esterase. Lower limits of quantitation for cholesterol 5alpha-, 7alpha-, and 7beta-hydroperoxides were 0.2, 0.1, and 0.5 nmol/g skin, respectively. This assay method showed that (i) good absolute recoveries of ChOOHs from rat skin (80-90% of radiolabeled ChOOHs added to rat skin); (ii) negligible autoxidation of cholesterol caused by the assay procedure (<9.4x10(-5)% of radiolabeled cholesterol added to rat skin); and (iii) good correlation between ChOOHs added to rat skin and ChOOHs determined, indicating this assay method is applicable to quantify ChOOHs in rat skin. By using this assay method, we observed that (i) cholesterol 5alpha-hydroperoxide was detected in skin of rats pretreated with oral doses of pheophorbide a and subsequent visible irradiation; (ii) concentrations of cholesterol 7-hydroperoxides in skin of rats in an ambient light room were not significantly different from those in a dark room for 12 weeks; and (iii) ultraviolet light B irradiation markedly enhanced the concentrations of cholesterol 7-hydroperoxides in the skin of rats.

Topics: Administration, Oral; Animals; Chlorophyll; Cholesterol; Isotope Labeling; Male; Molecular Structure; Oxidation-Reduction; Rats; Rats, Sprague-Dawley; Skin; Ultraviolet Rays

We identified singlet oxygen adduct of cholesterol, 3beta-hydroxy-5alpha-cholest-6-ene-5-hydroperoxide (5alpha-OOH), in skin of rats pretreated with oral doses of pheophorbide a and subsequent visible irradiation, that have been known to induce photosensitive diseases in animals and humans. In a living animal body, this is the first demonstration of presence of 5alpha-OOH, that is known to be formed exclusively by reaction in vitro between singlet oxygen and cholesterol. By the quantitative determination with high performance liquid chromatography equipped with a chemiluminescence detector, we observed time-dependent increase in concentrations of 5alpha-OOH in skin of rats pretreated with oral doses of pheophorbide a and subsequent visible irradiation, suggesting the occurrence of a labile activated oxygen species, singlet oxygen, in this system.

Topics: Animals; Chlorophyll; Cholesterol; Drug Stability; Linear Models; Lipid Peroxides; Male; Photochemistry; Photosensitizing Agents; Rats; Rats, Sprague-Dawley; Skin

The mechanism of caspase-3-dependent apoptosis induced by photodynamic therapy (PDT) of cultured Chinese hamster V79 cells with pheophorbide a (PPa) was investigated. The PPa-PDT induced rapid apoptosis within 30 min after irradiation of cells. This apoptosis was inhibited by the 1O2 quencher N3- and caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde, suggesting that 1O2 activated caspase-3 and then caused apoptosis. The intracellular calcium [Ca2+]i chelator (acetoxymethyl)-1,2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA-AM) and the cyclic adenosine monophosphate (cAMP)-increasing agent forskolin also inhibited not only the PPa-PDT-induced activation of caspase-3 but also apoptosis in V79 cells. Furthermore, PPa-PDT-induced cytochrome c release from mitochondria was found to be inhibited by the treatment with BAPTA-AM but not forskolin. These results indicated that [Ca2+]i and cAMP independently serve as regulators for PPa-PDT-induced apoptosis in the upstream of caspase-3.

Topics: Animals; Apoptosis; Caspase 3; Caspases; Cell Line; Chlorophyll; Colforsin; Cricetinae; Cytochrome c Group; Egtazic Acid; Photochemotherapy; Photosensitizing Agents

Chlorophyll breakdown in green plants is a long-standing biological enigma. Recent work has shown that pheophorbide a (Pheide a) derived from chlorophyll (Chl) is converted oxygenolytically into a primary fluorescent catabolite (pFCC-1) via a red Chl catabolite (RCC) intermediate. RCC, the product of the ring cleavage reaction catalyzed by Pheide a oxygenase, which is suggested to be the key enzyme in Chl breakdown in green plants, is converted into pFCC-1 by a reductase. In the present study, an in vitro assay comprising 18O2 Pheide a oxygenase and RCC reductase yielded labeled pFCC-1. Fast atom bombardment-mass spectrometric analysis of the purified pFCC-1 product revealed that only one of the two oxygen atoms newly introduced into Pheide a in the course of the cleavage reaction is derived from molecular oxygen. Analysis of the fragment ions located the oxygen atom derived from molecular oxygen on the formyl group of pyrrole B. This finding demonstrates that the cleavage of Pheide a in vascular plants is catalyzed by a monooxygenase. Chlorophyll breakdown is therefore indicated to be mechanistically related in higher plants and in the green alga Chlorella protothecoides.

Topics: Chlorella; Chlorophyll; Models, Chemical; Oxygenases; Plants; Porphyrins; Radiation-Sensitizing Agents; Spectrometry, Mass, Fast Atom Bombardment

The reduction of chlorophyllide b and its analogue zinc pheophorbide b in etioplasts of barley (Hordeum vulgare L.) was investigated in detail. In intact etioplasts, the reduction proceeds to chlorophyllide a and zinc pheophorbide a or, if incubated together with phytyldiphosphate, to chlorophyll a and zinc pheophytin a, respectively. In lysed etioplasts supplied with NADPH, the reduction stops at the intermediate step of 7(1)-OH-chlorophyll(ide) and Zn-7(1)-OH-pheophorbide or Zn-7(1)-OH-pheophytin. However, the final reduction is achieved when reduced ferredoxin is added to the lysed etioplasts, suggesting that ferredoxin is the natural cofactor for reduction of chlorophyll b to chlorophyll a. The reduction to chlorophyll a requires ATP in intact etioplasts but not in lysed etioplasts when reduced ferredoxin is supplied. The role of ATP and the significance of two cofactors for the two steps of reduction are discussed.

Topics: Adenosine Triphosphate; Alcohol Oxidoreductases; Chlorophyll; Chlorophyll A; Ferredoxin-NADP Reductase; Ferredoxins; Hordeum; Intracellular Membranes; Oxidation-Reduction; Pheophytins; Plastids; Subcellular Fractions; Zinc

Laser-induced fluorescence (LIF) of pheophorbide-a (Ph-a) was used for imaging of a rat pancreatic tumor. Using a dimensionless function (the ratio of Ph-a fluorescence by bluish autofluorescence), the fluorescence contrasts between excised tumors and their paired pancreas were investigated up to 48 h after a 9 mg kg-1 Ph-a intravenous administration. Among five tested excitation wavelengths, 355 and 610 nm excitations gave the best distinctive contrasts, both 48 h after dye injection. The LIF imaging of six intrapancreatic tumors and six healthy pancreas was carried out in vivo using two laser excitations: 355 nm (Nd:YAG + tripling) for bluish autofluorescence and 610 nm (rhodamine 6G dye) for reddish autofluorescence and dye emission. Images were recorded through bandpass filters at 470 and 640 nm (autofluorescence) and at 680 nm (dye + autofluorescence) with an intensified charged-coupled device camera. Autofluorescence as Ph-a fluorescence images did not allow accurate LIF diagnosis of pancreatic carcinoma. An image processing, including for each pixel a computed division of Ph-a fluorescence (after subtraction of reddish autofluorescence) by bluish autofluorescence intensity generated poorly contrasted tumor images in five of six and false tumor localization in one of three of the tumor-bearing pancreas. A fitting of the digital 640 nm autofluorescence up to the mean 680 nm fluorescence intensity in pancreas prior to subtraction allowed a safe diagnosis to be made with well-contrasted tumor images. To assess automation ability of the processing, a same fitting coefficient (mean of individual values) was applied. In this way, false-negative (one of six) and false-positive (two of six) images were present in tumor-bearing animals as false-positive in one-half of the controls. A successful standardized procedure was then applied with a normalization of 640 and 680 nm pancreas intensities to a same set threshold prior processing. In opposition to thin-layered hollow organs, such as bronchial tube or digestive tract, LIF imaging of carcinoma inserted in a compact organ is exhausting. The use of a dye excitable in the red wavelength range (610 nm for Ph-a) may partly solve this problem, rendering LIF imaging more accurate and potentially automated.

Topics: Animals; Chlorophyll; Fluorescence; Lasers; Pancreas; Pancreatic Neoplasms; Radiation-Sensitizing Agents; Rats; Spectrometry, Fluorescence

The sonodynamically induced antitumor effect of pheophorbide a (Ph-a) was investigated. Both in vitro and in vivo effects on sarcoma 180 were tested in combination with ultrasound at 2 MHz. The rate of ultrasonically induced cell damage in air-saturated suspension was enhanced by twice with 80 microM Ph-a. This enhancement was significantly inhibited by histidine, which may suggest it was mediated by ultrasonically induced oxidation. For mice, 5 mg/kg Ph-a was administered before the insonation, and ultrasound stopped the tumor growth at an intensity with which ultrasound alone showed only a slight antitumor effect.

Topics: Animals; Antineoplastic Agents; Cell Division; Chlorophyll; Histidine; Male; Mannitol; Mice; Mice, Inbred ICR; Radiation-Sensitizing Agents; Sarcoma 180; Superoxide Dismutase; Ultrasonic Therapy

Six chlorophyll-related compounds isolated from leaves of Neptunia oleracea (Leguminosae) inhibited the activation of tumor promoter-induced Epstein-Barr virus (EBV). Photo-irradiation of pheophorbide a, the major active constituent, did not enhance this inhibitory activity, and thus ruled out any photosensitizing effect playing the major role.

Topics: Antineoplastic Agents, Phytogenic; Chlorophyll; Fabaceae; Herpesvirus 4, Human; Molecular Structure; Plant Leaves; Plants, Medicinal; Tumor Cells, Cultured

Anti-tumor-promoting activity of pheophorbide a (PPBa) a chlorophyll-related compound, was examined in a two-stage carcinogenesis experiment in ICR mouse skin by 7,12-dimethylbenz[a] anthracene (DMBA, 0.19 mumol) and 12-O-tetradecanoylphorbol-13-acetate (TPA, 1.6 nmol). Topical application of PPBa (160 nmol) markedly reduced the average number of tumors per mouse and the ratio of tumor-bearing mice (inhibitory ratio: IR = 56%, P < 0.01 and 31%, P < 0.005, respectively). PPBa exhibited potent anti-inflammatory activity in ICR mouse ears and moderate inhibitory activity toward TPA-induced superoxide (O2-) generation in differentiated HL-60 cells. While CuPPBa, a synthetic copper complex of PPBa, exhibited higher anti-inflammatory activity than that of indomethacin, it showed little antioxidative effect against formation of lipid hydroperoxides (LOOHs) and malondialdehyde (MDA), suggesting that the antioxidative effect of PPBa might not be important for anti-inflammatory activity. These results imply that the active mechanism of PPBa for anti-tumor promotion might be partly involved in inhibition of TPA-induced inflammatory responses by suppressing leukocyte activation.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Anticarcinogenic Agents; Carcinogens; Chlorophyll; Drug Screening Assays, Antitumor; Female; Mice; Mice, Inbred ICR; Neoplasms, Experimental; Skin Neoplasms; Tetradecanoylphorbol Acetate

New water-soluble fullerene carboxylic acids (1 and 2) derived from C60 and C70 fullerenes, respectively, were examined for photocytotoxicity toward Raji cells (B lymphocyte). These compounds did not show any photocytotoxic effect even at 50 microM, while pheophorbide a showed significant photocytotoxicity at 0.5 microM. Therefore, fullerene derivatives derived from C60 and C70 would not be practical agents for photodynamic therapy.

Topics: B-Lymphocytes; Burkitt Lymphoma; Carbon; Carboxylic Acids; Chlorophyll; Fullerenes; Humans; Molecular Structure; Photochemotherapy; Radiation-Sensitizing Agents; Solubility; Tumor Cells, Cultured; Water

Laser-induced fluorescence of pheophorbide a (Ph-a) was used for in vitro photodynamic imaging (PDI) of a rat pancreatic acinar tumor. A 400 nm excitation induced a 470 nm autofluorescence and a 678 nm dye fluorescence in tumors and their surrounding pancreas 24 h after a 9 mg kg-1 body weight Ph-a intravenous administration. With lower intensities in these blood-rich tumors than in pancreas, Ph-a fluorescence signals are unable to provide tumor images. A dimensionless function (the ratio of Ph-a fluorescence by autofluorescence, called Rt for the tumor and Rp for the pancreas) was used for fluorescence contrast calculation (C = Rt/Rp) between six tumors and their paired pancreas. Among five available laser excitation wave-lengths, only the 355 nm excitation gave a distinctive contrast (C = 1.5). The PDI of six intrapancreatic tumors and their intraperitoneal metastasis and of two control normal pancreas was thus performed ex vivo using a 355 nm excitation source delivered by a tripled Nd:YAG laser and a charged-coupled device camera. Fluorescence images were recorded at 680 nm (dye), 640 nm (background) and 470 nm (autofluorescence) through three corresponding 10 nm width bandpass filters. Computed division for each pixel of Ph-a fluorescence values by autofluorescence generated false color image. In this way, contrasted tumor images were obtained. But in five out of six animals false-positive images were present due to an autofluorescence decrease in some normal pancreatic areas. A 470 nm autofluorescence imaging on the same tumors gave in all cases false-positive image and false-negative in half of the cases. These observations suggest that autofluorescence alone is unable to achieve accurate PDI of pancreatic carcinoma and that using Ph-a as a PDI dye needs strong improvements.

Topics: Animals; Chlorophyll; Evaluation Studies as Topic; Fluorescence; Image Processing, Computer-Assisted; Lasers; Pancreatic Neoplasms; Photosensitizing Agents; Rats; Rats, Inbred Lew

The inhibition of cytokine and monoclonal antibody binding cell surfaces caused by an extract of Psychotria acuminata, a medicinal plant used in the traditional medicine of the people of Belize (Central Africa), was attributed to the presence of pheophorbide a and pyropheophorbide a. Since the binding of tumor necrosis factor-alpha, interleukin-8, complement factor 5a as well as epidermal growth factor to target cells was dramatically reduced, the inhibition was not receptor or cytokine specific. In addition, the respective binding of radiolabeled monoclonal antibodies CL203 and R15.7 to the cell surface antigens intracellular cell adhesion molecule-1 and lymphocyte function-associated antigen-1 beta-chain was decreased by pretreatment of cells with pheophorbide a as well. In all cases, the inhibition by pheophorbides was dependent on the simultaneous presence of light, indicating causative involvement of a photodynamic process. These observations are not unique to pheophorbides and can be extended to porphyrins as well as to other photodynamic agents. Cytotoxicity resulting from photodynamic therapy (PDT) has been documented by many studies. Our investigations suggest that the inactivation of cell surface receptors contributes not only to an antitumor effect of PDT but also to the systemic immunosuppression, a serious side effect of PDT.

Topics: Belize; Chlorophyll; Complement C5a; Humans; Interleukin-8; Photosensitizing Agents; Plants, Medicinal; Porphyrins; Receptors, Cell Surface; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Pheophorbide a prepared from the algae Spirulina was derivatized at the C(7)-carboxylic group by linking amino alkyls of various lengths and terminal functional groups. The compounds were purified by thin-layer chromatography (TLC) and by high-pressure liquid chromatography (HPLC). Solubilization of compounds by serum lipoproteins, the kinetics of compound uptake into mammalian cells, and photosensitizing effectiveness when activated by 673 nm laser light have been studied. Optimal photosensitizer uptake into cells and the greatest photosensitizing activity were observed with compounds having side-chain lengths of 4-6 carbon atoms which terminated in -OH and -CH3 groups. The most effective compounds were 3 orders of magnitude more potent than Photofrin in the degree of photoinactivation of cultured EMT-6 tumor cells. HDL and LDL significantly promoted the efflux of these photosensitizing drugs from cells, suggesting that their long-term retention in normal tissues in vivo would be minimal and produce little phototoxicity.

Topics: Animals; Chlorophyll; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Eukaryota; Humans; Lipoproteins, HDL; Lipoproteins, LDL; Mice; Photic Stimulation; Photosensitizing Agents; Tumor Cells, Cultured

Many crude drugs were screened for their capacity to inhibit the binding of endothelin-1 (ET-1) to ET receptors; several crude drugs showed significant binding inhibitory activity. Pheophorbide a (1), a potent non-peptide ET receptor antagonist, was isolated from Altemisiae capillaris Flos ("Inchinko" in Japanese), which has been utilized as a remedy for hepatitis in Oriental medicine. In receptor binding experiments, compound 1 inhibited ET-1 binding specifically to both the ETA receptor (ETAR) and ETB receptor (ETBR), with IC50 values of 8.0 x 10(-8) and 2.1 x 10(-7) M, respectively. Thus, compound 1 is an ET-1 binding inhibitor; however, it exhibited no affinity for the other receptors of angiotensin II and atrial natriuretic peptide. We also evaluated the inhibitory activity of porphyrin compounds, and found that some exhibited moderate activity.

Topics: Animals; Brain; Chlorophyll; Endothelin Receptor Antagonists; Japan; Medicine, East Asian Traditional; Muscle, Smooth, Vascular; Plant Extracts; Rats; Structure-Activity Relationship; Swine

Photodynamic therapy (PDT) is a new modality to treat cancer. In clinical use, the procedure is limited by the poor penetration depth of the curative light, which ranges between 3 and 6 mm. The cause of this situation is not clear. Two hypotheses are discussed: shielding effect or hypoxia. To test both hypotheses, we designed an in vitro model. Suspensions and sediments of OAT-75 cells were incubated at 37 degrees C with pheophorbide a or 13(2)-hydroxy-bacteriopheophorbide a methyl ester, both second-generation sensitizers. Irradiation was done with laser light of suitable wavelength. In all experiments, cell death was seen on the surface of the sediment, i.e., near the border of the oxygen-containing atmosphere and opposite the site of the irradiation beam. Therefore, we should accept the hypoxia thesis.

Topics: Absorption; Carcinoma, Small Cell; Cell Hypoxia; Chlorophyll; Humans; Laser Therapy; Lung Neoplasms; Photochemotherapy; Radiation-Sensitizing Agents; Tumor Cells, Cultured

Selective histological necrosis of experimental pancreatic carcinoma by photodynamic therapy (PDT) has been successful with haematoporphyrin derivatives and phthalocyanine as photosensitizers. This report describes the feasibility of PDT with pheophorbide A as the photosensitizer to treat azaserine-induced pancreatic rat carcinoma and analyses survival of the animals. An organ distribution study 24 h after pheophorbide A administration (9 mg/kg intravenously) gave a selectivity ratio of 13.5:1 between tumour and surrounding tissue. Light of 660 nm and 100 J/cm2 induced selective necrosis of the tumour. Six of nine rats were cured in 120 days whereas all 36 control animals died within 35 days (P < 0.01). The pancrease and hepatic pedicle were relatively unaffected by PDT, but the duodenum was injured.

Topics: Adenocarcinoma; Animals; Chlorophyll; Duodenum; Necrosis; Pancreas; Pancreatic Neoplasms; Photochemotherapy; Radiation-Sensitizing Agents; Rats; Rats, Inbred Lew; Survival Analysis; Time Factors

Pheophorbide a is a photocytotoxic agent. To develop a tissue-specific, intracellularly targeted photoactive system, pheophorbide a was incorporated into immunoliposomes coated with a monoclonal antibody (T-43) directed against the T-24 bladder tumor cell line. The efficacy of this system was studied in vitro using the human bladder tumor cell line MGH-U1. Uptake and localization were determined by the fluorescence of the immunoliposome markers within biochemically resolved subcellular components. The results demonstrate localization of the immunoliposome markers within the lysosomes of the tumor cells. Specific monoclonal antibody enhancement of the immunoliposomes uptake by MGH-U1 cells was demonstrated by the use of soluble T-43 monoclonal antibody as a competitive inhibitor. Pheophorbide-a-loaded immunoliposomes were shown to be photocytotoxic towards MGH-U1 cells at concentrations equivalent to photosensitizer at 500 ng ml-1. Treated cells, when protected from light, showed no cytotoxicity. These results demonstrate that uptake of pheophorbide-a-containing immunoliposomes by target cells and subsequent delivery to the lysosomes cause photoactivated killing of tumor cells. The utilization of immunoliposomes for intracellular lysosomal targeting of photoactive drugs to tumor cells constitutes a potentially valuable approach to photodynamic therapeutics.

Topics: Antibodies, Monoclonal; Carcinoma, Transitional Cell; Cell Line; Cell Survival; Chlorophyll; Darkness; Drug Carriers; Humans; Light; Liposomes; Lysosomes; Radiation-Sensitizing Agents; Tumor Cells, Cultured; Urinary Bladder Neoplasms

We propose the use of acetoxymethyl esters of pH-sensitive amphipathic photosensitizers (PS) for photodynamic therapy (PDT). These compounds may be applicable for PDT involving endocytosis of lipophilic carriers leading to lysosomal uptake of the esterified PS by target cells. Partial and/or total enzymatic de-esterification may result in the extralysosomal distribution of the photoactive agents, possibly culminating in a multisite photochemical response. We report here the synthesis and properties of chlorin e6 triacetoxymethyl ester (CAME) and pheophorbide a acetoxymethyl ester (PAME). Chlorin e6 and pheophorbide a are photocytotoxic chlorins that possess free carboxylate groups and exhibit optimum wavelengths of excitation substantially red shifted relative to hematoporphyrin derivative. Acetoxymethyl esterification of chlorin e6 and pheophorbide a was accomplished with bromomethyl acetate. High-performance liquid chromatography allowed for the purification of PAME, in 87% purity, and CAME, in 63% yield and 94% purity, as well as the detection of the presumed mono- and diesters of chlorin e6 as transient intermediates in the synthesis of CAME. The ultraviolet-visible absorption, fluorescence excitation and emission, NMR and mass spectra of the chlorin e6 triester are consistent with those expected for CAME. The pH-sensitive amphipathicity of pheophorbide a and chlorin e6 but not CAME was demonstrated using a water/1-octanol partition assay. The production of pheophorbide a from PAME and the sequential formation of the di- and monoesters and free chlorin e6 from CAME, by the action of lysosomal esterases obtained from cancer cells, demonstrate the potential of cellular enzymes to convert the lipophilic esters to pH-sensitive amphipathic PS.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Carcinoma; Chlorophyll; Chlorophyllides; Esterases; Esterification; Esters; Humans; Hydrogen-Ion Concentration; Lysosomes; Photosensitizing Agents; Porphyrins; Solubility; Spectrometry, Fluorescence; Spectrophotometry; Urinary Bladder Neoplasms

The photophysical properties of pheophorbide a (Pheo a) under two-step laser activation were investigated. For the first time quantum yields of higher excited state formation were calculated. It was shown that the quantum yields of the formation of these states depend strongly on the pulse duration of the excitation source. The nonlinear properties of Pheo a are quite different in dependence on the excitation wavelength but the quantum yields of the higher excited state formation in both cases (lambda exc = 337 nm and lambda exc = 667 nm) rise up increasing the photon flux density of the laser light up to 10(26) phot cm-2 s-1. A further increase of the photon flux density has no effect on the quantum yields.

Topics: Chlorophyll; Lasers; Mathematics; Models, Theoretical; Photochemistry; Quantum Theory; Radiation-Sensitizing Agents; Spectrometry, Fluorescence; Spectrophotometry

The in vivo administration and distribution of a potent new photosensitizer, pheophorbide A (PH-A), was investigated in rats. The spectral characteristics were determined. This hydrophobic compound was solubilized by an ethanol/phosphate-buffered saline (PBS) mixture (v/v) and sonicated immediately before i.v. administration. Tissue distribution and the affinity of PH-A for an acinar pancreatic tumor were determined in Lewis rats for up to 48 h after a single i.v. administration of 3 mg kg-1 body wt. Methanol-extracted PH-A was quantitatively determined by fluorescence spectrophotometry at 665.6 nm. The PH-A uptake pattern showed that the reticulo-endothelial system is the major target of PH-A, followed by the gut and then the lung and pancreas. PH-A concentrations in skin were very low. The presence of an enterohepatic cycle was suggested by the PH-A biliary output, intestinal uptake and blood concentrations. Tumor PH-A retention was longer than pancreatic retention. The ratio of tumoral to peri-tumoral pancreas PH-A was 6.7:1, 24 h after i.v. administration. With its similar tissue pattern, better absorption spectrum and lower skin toxicity, PH-A could be a more potent photosensitizer than hematoporphyrin derivatives.

Topics: Animals; Chlorophyll; Molecular Structure; Pancreas; Pancreatic Neoplasms; Photochemotherapy; Photosensitizing Agents; Porphyrins; Rats; Rats, Inbred Lew; Spectrometry, Fluorescence; Tissue Distribution

Pheophorbide a-induced photo-oxidation, in vitro, of cytochrome c oxidase and cytochrome c results in irreversible modifications to both protein components. Photo-oxidation of cytochrome c, as exhibited by change in its heme oxidation state, displays exponential kinetics and is detected with a lag period. Both the photo-induced inactivation of the enzyme, and destruction of the substrate ability of cytochrome c occur as complex multi-process events. Under similar experimental conditions, the loss of the substrate capability of cytochrome c develops approximately three times faster than inactivation of the enzyme. The slight lag in the photo-oxidation of cytochrome c is due to pheophorbide a-induced superoxide production. However, the relative amount of photo-oxidant produced is considerably more effective than the cytochrome c reducing capacity of the superoxide. Neither hydroxyl radical nor hydrogen peroxide are involved in the photo-oxidation of the heme function. The possibilities of heme oxidation by a singlet oxygen mediated pathway or direct electron abstraction involving the heme or apoprotein are not excluded. It is proposed that a multi-site oxidation of numerous reduced energy cofactors within cells may augment collateral enzyme inactivation in maximizing photosensitizer-induced cytotoxicity. Accordingly, amphipathic photosensitizers, capable of accessing both lipid and aqueous compartments containing reduced cofactors, may be more effective agents for photodynamic therapy than those which exhibit a high specificity of subcellular localization.

Topics: Chlorophyll; Cytochrome c Group; In Vitro Techniques; Oxidation-Reduction; Photochemistry; Photochemotherapy; Radiation-Sensitizing Agents; Superoxides

The role of the reactive oxygen species (ROS) in the induction of tissue injury has been well documented. Regarding glomerular injury, the focus of past studies has been on superoxide, hydrogen peroxide, hydroxyl radical and C10-. However, whether singlet oxygen, an exceptionally reactive oxygen metabolite, may cause glomerular lesions has not been investigated. In the present study we documented the effects of locally generated singlet oxygen in the kidney. To generate singlet oxygen, kidneys were perfused with the pigment pheophorbide a, which was followed by exposure to light. One hour after irradiation, severe morphological damage of the glomerular cell had developed. Extensive deposits of fibrin and accumulation of platelets were seen in the glomerular capillary lumina. By contrast, kidneys not exposed to light showed no, or only minimal, abnormalities. Glomerular injury was not inhibited by pretreatment with superoxide dismutase. These results suggest that singlet oxygen produced by pheophorbide a by photodynamic reaction leads to tissue injury in vivo.

Topics: Animals; Chemotherapy, Cancer, Regional Perfusion; Chlorophyll; Female; Glomerulonephritis; Kidney; Light; Oxygen; Radiation-Sensitizing Agents; Rats; Rats, Inbred Strains; Singlet Oxygen; Superoxide Dismutase

The authors describe a new photodynamic therapy (PDT) method for malignant brain tumors. Pheophorbide a (Ph-a), the photosensitizer, has low toxicity, causes no skin sensitization and is activated with an acoustic Q switched neodymium yttrium-argon-garnet (Nd:YAG) laser which achieves deep tissue penetration. The Ph-a distribution in Fisher 344 (F344) rats bearing rat T9 glioma at 24 hours after intravenous injection was very low in the normal brain tissue, but significantly higher in the T9 glioma giving a tumor to normal brain tissue concentration ratio of 7.5:1. The in vitro survival rate of T9 glioma cells pretreated with Ph-a was 68.8 +/- 5.4% after laser irradiation for 20 minutes, significantly lower than in the control groups. This indicates that Ph-a was activated with the acoustic Q switched Nd:YAG laser causing the photodynamic effect. The survival rate after Ph-a pretreatment and laser irradiation in a waterbath at 44.0 degrees C was further reduced to 15.8 +/- 3.3%. In vivo PDT studies using T9 glioma cells inoculated into the dorsal region of F344 rats showed tumor eradication in four of six rats. The combination of PDT and laser hyperthermia produced tumor eradication in all six rats. The combination of PDT and hyperthermia is a promising method for tumor treatment.

Topics: Animals; Brain Neoplasms; Chlorophyll; Glioma; Laser Therapy; Photochemotherapy; Radiation-Sensitizing Agents; Rats; Rats, Inbred F344; Survival Analysis; Tumor Cells, Cultured

To evaluate whether administration of pheophorbide-a, a new photosensitizer, followed by use of Q-switched Nd:YAG laser produces a photodynamic reaction, we administered pheophorbide-a to female nude mice (BALB/c-nu) that had been implanted with human hepatocellular carcinoma. Intra-tumoral concentrations of pheophorbide-a were measured by high-performance liquid chromatography. 3 hours after peroral administration of 1 mg/kg body weight, the intra-tumoral concentration was too low to reveal photodynamic effects. Peroral administration of 250 mg/kg body weight, intra-peritoneal administration of 5 mg/kg body weight, and intra-tumoral injection of 200 micrograms yielded 0.24 micrograms/g, 0.83 micrograms/g and 3.68-108 micrograms/g tumor concentrations, respectively. All tumors were irradiated interstitially using a Q-switched Nd:YAG laser at 1064 nm. Only tumors that had been intra-tumorally injected had areas of necrosis larger than those in control tumors. The results suggest that the injection of pheophorbide-a followed by interstitial irradiation using a Q-switched Nd:YAG laser does not induce sufficient photodynamic reaction if the intra-tumoral pheophorbide-a concentration is less than 0.83 micrograms/g tumor tissue, and that photodynamic therapy may be useful if the pheophorbide-a tumor concentration is within the range of 0.83-108 micrograms/g.

Topics: Animals; Chlorophyll; Female; Humans; Lasers; Liver; Liver Neoplasms, Experimental; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Neoplasm Transplantation; Photochemotherapy; Radiation-Sensitizing Agents

Remarkably effective photocleavage of plasmid DNA with pheophorbide a, as a visible light sensitizer, has been found for the first time. DNA is photocleaved even in the absence of oxygen. The novel oxygen-independent scission is ascribed to specific photodynamic function of the ring V in pheophorbide a.

Topics: Chlorophyll; DNA; Molecular Structure; Oxygen; Photochemistry; Plasmids; Radiation-Sensitizing Agents

This is the first report on the use of photodynamic therapy (PDT) for rat pituitary tumor in vivo. Rat pituitary tumor (GH3) cells were cultured, GH3 tumor was subcutaneously implanted in nude mice, and pheophorbide-a (Ph-a) and white light were prepared. Photocytotoxicity proportional to Ph-a concentration, intensity of irradiation, and incubation time was observed in vitro. Despite the delay in the disappearance of Ph-a from the tumor, Ph-a in the pituitary gland rapidly decreased after intravenous administration in vivo. Through PDT, the tumor grossly disappeared, the plasma levels of rat growth hormone secreted from the tumor also remarkably decreased, and the development of giantism was inhibited. These results indicate that PDT is effective against rat pituitary tumor.

Topics: Animals; Chlorophyll; Chromatography, High Pressure Liquid; Female; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Photochemotherapy; Pituitary Neoplasms; Radiation-Sensitizing Agents; Spectrometry, Fluorescence; Tumor Cells, Cultured

The triplet states of pheophorbide a and pheophytin a were studied in several environments by direct measurement of the phosphorescence of the pigments and photosensitized singlet oxygen (1O2) luminescence. The spectra, lifetimes and quantum yields of phosphorescence and the quantum yields of 1O2 generation were determined. These parameters are similar for monomeric molecules of both pigments in all the environments studied. Aggregation of the pigment molecules leads to a strong decrease in the phosphorescence and 1O2 luminescence intensities, which is probably due to a large decrease in the triplet lifetime and triplet quantum yield in the aggregates. The results obtained for pheophorbide a and pheophytin a are compared with those previously reported for chlorophyll alpha. The data suggest that the photodynamic activity of the pigments in living tissues is probably determined by the monomeric pigment molecules formed in hydrophobic cellular structures. Aggregated molecules seem to have a much lower activity.

Topics: Chlorophyll; Chlorophyll A; Kinetics; Luminescent Measurements; Oxygen; Pheophytins; Photochemistry; Pigments, Biological; Singlet Oxygen; Solvents; Spectrophotometry

Topics: Chlorophyll; Light; Radiation-Sensitizing Agents; Structure-Activity Relationship

The effect on photodynamic therapy (PDT) of using pheophorbide-a as a photosensitizer and Nd: YAG laser (Q-switch) was evaluated. Two hundred micrograms (0.1 ml) of pheophorbide-a was injected into tumors that had been subcutaneously implanted into the backs of nude mice (BALB/c-nu). Interstitial Nd: YAG laser irradiation (Q-switch; mean power 0.5 W, duration 10 min.) was performed 72 hours after pheophorbide-a injection. Forty-eight hours after laser irradiation, the areas of tumor necrosis were measured; these were larger in the group with the injection of pheophorbide-a than in those without. The areas of tumor necrosis after Nd: YAG laser irradiation at a higher mean power (Q-switch; mean power 2 W, control temperature 43-43.5 degrees C, duration 10 min.) were also measured. Again, the areas were larger in the group injected with pheophorbide-a. These results show that the injection of pheophorbide-a and interstitial irradiation using Nd: YAG laser induce a photodynamic reaction, and that this combination is useful in the treatment of deep-seated tumors.

Topics: Animals; Carcinoma, Hepatocellular; Chlorophyll; Liver Neoplasms; Mice; Photochemotherapy; Rabbits

Intra-tumoral concentrations of pheophorbide-a, a new photosensitizer, were measured by high-performance liquid chromatography after administration of pheophorbide-a to nude mice (BALB/c-nu) that had been implanted with human hepatocellular carcinoma. The results were as follows: 1) 0.009 +/- 0.007 microgram/g tissue in 1 mg/kg body p.o. administration group, 2) 0.22 +/- 0.06 microgram/g tissue in 250 mg/kg body p.o. administration group, 3) 0.85 +/- 0.14 microgram/g tissue in 5 mg/kg body intra-peritoneal administration group, 4) 3.42 +/- 2.84 microgram/g tissue in not-injected side, and 116 +/- 24 microgram/g tissue in injected side of 200 micrograms intra-tumoral administration group. Tumors in each group were irradiated using an Nd:YAG laser (Q-switch; mean power 0.5 W, duration 10 min.). Areas of tumor necrosis were larger than in the control group only in the intra-tumoral administration group. These results suggest that no photodynamic reaction occurs if the intra-tumoral pheophorbide-a concentration is less than 0.85 microgram/g tissue.

Topics: Animals; Carcinoma, Hepatocellular; Chemical Phenomena; Chemistry; Chlorophyll; Female; Humans; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Photochemotherapy; Tissue Distribution

Topics: Animals; Chlorophyll; Electron Spin Resonance Spectroscopy; Erythrocytes; Goats; Hemolysis; In Vitro Techniques; Photolysis; Radiation-Sensitizing Agents

A new sensitizing method of photodynamic therapy for malignant tumors and its effects was studied. Prepared for the study were pheophorbide a (Phd), dissolved in an oily contrast medium, Lipiodol (LPD-Phd), and water-soluble pheophorbide a (W-S Phd) as sensitizers, and VX-2 tumor in rabbit livers. The Phd distribution was compared after intraarterial (i.a.) administration of LPD-Phd or W-S Phd and intravenous (i.v.) administration of W-S Phd. Phd was extracted with methanol at 24 h after injection, and the supernatant absorbance was measured at 670 nm by spectrophotometry. The tumor showed higher values of Phd than did the liver with LPD-Phd i.a. and W-S Phd i.a. (P less than .01). Conversely, the tumor accumulated less Phd than did the liver with W-S Phd i.v. (P less than .05). We subsequently produced severe photo-destruction in a Walker tumor in a Sprague-Dawley rat liver with slight damage to adjacent liver tissue using LPD-Phd i.a. and Nd-YAG dye laser irradiation at 670 nm. The intraarterial administration of a photosensitizer may make it possible to treat liver tumors by photodynamic therapy.

Topics: Animals; Chlorophyll; Iodized Oil; Lasers; Liver; Liver Neoplasms, Experimental; Male; Photochemotherapy; Rabbits; Rats; Rats, Inbred Strains

An investigation of the production of radical species by photoexcitation pheophorbide alpha, haematoporphyrin and protoporphyrin was performed. In an aqueous solution containing different amounts of ethanol, the superoxide radical was detected by the spin trapping technique. In addition, secondary radicals were observed. The generation of oxygen radicals was found to dominate in solutions with a low ethanol content.

Topics: Chlorophyll; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Ethanol; Free Radicals; Hematoporphyrins; Hydrogen-Ion Concentration; Light; Photochemistry; Porphyrins; Protoporphyrins; Time Factors

As models for chlorophyll a (Chl a), methyl ester ClFe(III)pheophorbides (1, pheophorbide a; 2, mesopheophorbide a; and 3, mesopyropheophorbide a) were examined by Fourier transform infrared (FTIR) absorption and resonance Raman (RR) spectroscopy. The infrared (IR) chlorin band above 1600 cm-1, assigned as a Ca-Cm mode (Andersson et al. (1987) J. Am. Chem. Soc. 109, 2908-2916) is shown to be metal-sensitive and responsive to spin state and coordination number for dihydroporphyrins, as well as being diagnostic for the chlorin vs. porphyrin or bacteriochlorin macrocycle. Frequency variations for this metallochlorin IR band thus parallel those of the v10 RR mode of porphyrins in their predictive utility. Qy excitation SERRS spectra of Chl a were compared with Qy excitation RR spectra of 1 and methyl Ni(II)pyropheophorbide a. The data demonstrate that 5-coordinate ClFe(III)pheophorbides are better models for chlorophylls than are ruffled 4-coordinate Ni(II)pheophorbides. Major spectral differences between the three chlorophyll models are associated with the C-9 keto and/or C-10 carbomethoxy vibrational modes. The approx. 1700 cm-1 IR band was formerly assigned solely to v(C = O) of the C-9 keto group. However, this IR feature shifts down to approx. 1685 cm-1 and nearly doubles in intensity when the C-10 carbomethoxy is removed, as for 3. Similar frequency downshifts coupled with intensity increases in the IR are found in the literature on chlorophylls. RR spectra of pheophorbides having the C-10 carbomethoxy group (1 and 2) have bands at both approx. 1700 and approx. 1735 cm-1. However, the C-9 keto v(C = O) mode of pyrophorbins also downshifts to approx. 1685 cm-1, as in the IR spectra. The approx. 1735 cm-1 ester RR mode disappears in the case of pyrophorbins, and is never RR active for nonconjugated esters of porphyrins or chlorins. These data demonstrate an interaction between the C-10 and C-9 carbonyls of phorbins. They also indicate that phorbins tend toward conjugation of the C-10 ester. Biological examples of such conjugation effects have recently been reported, e.g., for the Chl a pi-cation radical (Heald et al. (1988) J. Phys. Chem. 92, 4820-4824). Because the phorbin E ring is the major structural feature distinguishing chlorophylls from non-photosynthetic systems, the participation of the C-10 ester in ring conjugation is suggestive of its biological importance.

Topics: Chlorophyll; Fourier Analysis; Porphyrins; Spectrophotometry; Spectrophotometry, Infrared; Spectrum Analysis; Spectrum Analysis, Raman

Normal mouse skin has a prominent fluorescence peak at 674 nm. Fluorescence emission and fluorescence excitation spectroscopy, carried out both in vitro and in vivo, led to the conclusion that the chromophore(s) responsible for this naturally occurring fluorescence is/are pheophorbide a and/or pheophytin a, degradation products of chlorophyll a that are derived from the mouse food.

Topics: Animals; Chlorophyll; Chlorophyll A; Fluorescence; Male; Mice; Mice, Inbred BALB C; Skin; Spectrometry, Fluorescence

Topics: Animals; Chlorophyll; Cholesterol; Free Radicals; Humans; Light; Neoplasms; Oxygen; Photochemistry; Photosensitivity Disorders

Topics: Animals; Chlorophyll; Male; Photosensitivity Disorders; Rats

The fluorescence decay I(t) and time-resolved spectra I(lambda, t) of some porphyrins and chlorins in ethanol and phosphate-buffered aqueous solution were investigated with a time-correlated single-photon-counting apparatus with a mode-locked Ar+ laser (514.5 nm) as the excitation source. The fluorescence of hematoporphyrin, mesoporphyrin and pheophorbide aa is considerably influenced by the conditions of aggregation (these compounds undergo aggregation in phosphate-buffered solution but not in ethanolic solution). The fluorescence decay of chlorin e6 which remains monomeric in both solvents is single exponential in all cases. The fluorescence spectra of hematoporphyrin, mesoporphyrin and pheophorbide a in phosphate-buffered solution are shifted with respect to the spectra obtained in ethanol; moreover, a new emission band (X band) appears, whose intensity increases on increasing the amount of equilibrium aggregates and shows a fast fluorescence decay. For hematoporphyrin and mesoporphyrin the appearance of the X band emission appears to be correlated with irreversible photoprocesses leading to fluorescent photoproducts. Analysis of the reported fluorescence spectra of cancer cells after incubation with hematoporphyrin derivative suggests that the fluorescent photoproducts might be formed also in vivo.

Topics: Chlorophyll; Ethanol; Hematoporphyrins; Kinetics; Mesoporphyrins; Porphyrins; Solutions; Spectrometry, Fluorescence; Structure-Activity Relationship; Time Factors; Water

Topics: Chlorella; Chlorophyll; Chromatography, High Pressure Liquid; Drug Stability; Tablets

Swelling followed by erythematopurpuric lesions on sun-exposed areas of the body developed in five patients. All patients were found to have ingested chlorella. The histopathologic changes consisted of swelling of endothelial cells and thrombosis of small blood vessels in the dermis and the subcutaneous fatty tissue. The photosensitizing agent contained in chlorella tablets was proved to be pheophorbide -a and its ester.

Topics: Adult; Aged; Chlorella; Chlorophyll; Diet Fads; Female; Food Hypersensitivity; Humans; Male; Middle Aged; Photosensitivity Disorders

Topics: Chlorella; Chlorophyll; Diterpenes; Gas Chromatography-Mass Spectrometry; Hydrolysis; Phytol; Vegetables

Topics: Chlorophyll; Chlorophyllides; Chromatography, High Pressure Liquid

The preventive effects of L-ascorbic acid (V.C) and pantothenic acid (PaA) against photosensitized hemolysis in the presence of pheophorbide a or hematoporphyrin were investigated in vitro. The development of hematoporphyrin-induced photosensitivity and the preventive effects of V.C and PaA were also investigated in male mice. V.C and PaA exerted preventive effects against photosensitized hemolysis in the presence of pheophorbide a or hematoporphyrin. In such cases, V.C and PaA had not only an additive effect but also a synergistic one. The time required for the photosensitized acute shock-like death (i.e., survival time), was significantly extended by simultaneous administration of V.C and PaA. This supports the findings of in vitro experiments.

Topics: Animals; Ascorbic Acid; Chlorophyll; Drug Synergism; Hematoporphyrins; Hemolysis; Light; Male; Mice; Pantothenic Acid; Photosensitivity Disorders; Rats