RESUMEN

Photodynamic therapy (PDT) treats nonmelanoma skin cancer. PDT kills cells through reactive oxygen species (ROS), generated by interaction among cellular O2, photosensitizer and specific light. Protoporphyrin IX (PpIX) is a photosensitizer produced from methyl aminolevulinate (MAL) by heme group synthesis (HGS) pathway. In PDT-resistant cells, PDT efficacy has been improved by addition of epigallocatechin gallate (EGCG). Therefore, the aim of this work is to evaluate the effect of EGCG properties over MAL-TFD and PpIX production on A-431 cell line. EGCG's role over cell proliferation (flow cytometry and wound healing assay) and clonogenic capability (clonogenic assay) was evaluated in A-431 cell line, while the effect of EGCG over MAL-PDT was determined by cell viability assay (MTT), PpIX and ROS detection (flow cytometry), intracellular iron quantification and gene expression of HGS enzymes (RT-qPCR). Low concentrations of EGCG (<50 µM) did not have an antiproliferative effect over A-431 cells; however, EGCG inhibited clonogenic cell capability. Furthermore, EGCG (<50 µM) improved MAL-PDT cytotoxicity, increasing PpIX and ROS levels, exerting a positive influence on PpIX synthesis, decreasing intracellular iron concentration and modifying HGS enzyme gene expression such as PGB (upregulated) and FECH (downregulated). EGCG inhibits clonogenic capability and modulates PpIX synthesis, enhancing PDT efficacy in resistant cells.

Asunto(s)

Catequina , Proliferación Celular , Hemo , Fármacos Fotosensibilizantes , Protoporfirinas , Especies Reactivas de Oxígeno , Catequina/análogos & derivados , Catequina/farmacología , Protoporfirinas/farmacología , Protoporfirinas/metabolismo , Humanos , Hemo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Fármacos Fotosensibilizantes/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Fotoquimioterapia/métodos , Supervivencia Celular/efectos de los fármacos , Ácido Aminolevulínico/farmacología , Ácido Aminolevulínico/análogos & derivados

RESUMEN

Multi-charged nanoemulsions (NE) were designed to deliver Cannabidiol (CBD), Indocyanine green (ICG), and Protoporphyrin (PpIX) to treat glioblastoma (GBM) through Photodynamic Therapy (PDT). The phase-inversion temperature (PIT) method resulted in a highly stable NE that can be scaled easily, with a six-month shelf-life. We observed the quasi-spherical morphology of the nanoemulsions without any unencapsulated material and that 89% (± 5.5%) of the material was encapsulated. All physicochemical properties were within the expected range for a nanostructured drug delivery system, making these multi-charged nanoemulsions promising for further research and development. NE-PIC (NE-Protoporphyrin + Indocyanine + CBD) was easily internalized on GBM cells after three hours of incubation. Nanoemulsion (NE and NE-PIC) did not result in significant cytotoxicity, even for GBM or non-tumorigenic cell lines (NHF). Phototoxicity was significantly higher for the U87MG cell than the T98G cell when exposed to: visible (430 nm) and infrared (810 nm) laser light, with a difference of about 20%. From 50 mJ.cm-2, the viability of GBM cell lines decreases significantly, ranging from 65% to 85%. The NE-PIC was also effective for inhibiting cell proliferation into a 3D spheroidal GBM cell model, which is promising for mimicking the tumor cell environment. Irradiation at 810 nm was more effective in treating spheroid due to its deeper penetration in complex structures. NE-PIC has the potential as a drug delivery system for photoinactivation and photo diagnostic of GBM cell lines, taking advantage of the versatility of its active components.

Asunto(s)

Glioblastoma , Fotoquimioterapia , Humanos , Fotoquimioterapia/métodos , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Protoporfirinas/metabolismo , Línea Celular Tumoral

RESUMEN

As a tumor photodiagnostic agent, 5-aminolevulinic acid (ALA) is metabolized in the heme biosynthesis pathway to produce protoporphyrin IX (PpIX) with fluorescence. ALA-PpIX fluorescence was evaluated in human renal cell carcinoma (RCC) cell lines and non-tumor HK-2 cell lines. We found that extracellular PpIX level was correlated with ABCG2 activity, illustrating its importance as a PpIX efflux transporter. Extracellular PpIX was also related to the Km of ferrochelatase (FECH) that chelates PpIX with ferrous iron to form heme. The Vmax of FECH was higher in all RCC cell lines tested than in the HK-2 cell line. TCGA dataset analysis indicates a positive correlation between FECH expression and RCC patient survival. These findings suggest FECH as an important biomarker in RCC. Effects of iron chelator deferoxamine (DFO) on the enhancement of PpIX fluorescence were assessed. DFO increased intracellular PpIX in both tumor and non-tumor cells, resulting in no gain in tumor/non-tumor fluorescence ratios. DFO appeared to increase ALA-PpIX more at 1-h than at 4-h treatment. There was an inverse correlation between ALA-PpIX fluorescence and the enhancement effect of DFO. These results suggest that enhancement of ALA-PpIX by DFO may be limited by the availability of ferrous iron in mitochondria following ALA administration.

Asunto(s)

Carcinoma de Células Renales , Neoplasias Renales , Fotoquimioterapia , Humanos , Ácido Aminolevulínico/farmacología , Ácido Aminolevulínico/metabolismo , Deferoxamina/farmacología , Carcinoma de Células Renales/tratamiento farmacológico , Fluorescencia , Protoporfirinas/farmacología , Protoporfirinas/metabolismo , Hierro , Hemo , Neoplasias Renales/tratamiento farmacológico , Quelantes del Hierro/farmacología , Fármacos Fotosensibilizantes/farmacología , Línea Celular Tumoral , Fotoquimioterapia/métodos

RESUMEN

The aim of the present study was to investigate the effects of PDT using the photosensitizer 5-aminoulevulinic acid (5-ALA) in oral squamous cell carcinoma (OSCC) behavior, mainly regarding its role on the cancer stem cell (CSC) phenotypes and in maintenance of the stem cell properties. Two OSCC cell lines were used and divided in the groups: Control, 5-ALA, LED 6 J/cm2 and PDT. MTT and Neutral red assays were used to access cellular viability, cell migration was evaluated by the wound healing assay. The stem cell phenotype was analyzed by flow cytometry to evaluate the CD44high/ESAhigh, CD44high/ESAlow and CD44low populations, by the clonogenic and tumor sphere formation assays as well as by RT-qPCR. The presence of Protoporphyrin IX in each CSC fraction was evaluated by flow cytometry. The OSCC cell lines showed a significant decrease in cell viability and migration after PDT. The percentage of CD44high/ESAhigh cells decreased after PDT, which was associated with an increase in the CD44low cells and with a functional decrease in the colony and sphere formation capacity. CD44high/ESAhigh cells showed increased PpIX, which contributed for their greater sensitivity to PDT. INV gene increased significantly after PDT, indicating cellular differentiation. Altogether, our results demonstrate that 5-ALA mediated PDT decreases not only the fraction of oral CSC but also their functional capabilities, inducing their differentiation.

Asunto(s)

Carcinoma de Células Escamosas , Neoplasias de la Boca , Fotoquimioterapia , Ácido Aminolevulínico/farmacología , Ácido Aminolevulínico/uso terapéutico , Carcinoma de Células Escamosas/tratamiento farmacológico , Línea Celular Tumoral , Humanos , Neoplasias de la Boca/tratamiento farmacológico , Células Madre Neoplásicas/metabolismo , Rojo Neutro/uso terapéutico , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/uso terapéutico , Protoporfirinas/metabolismo

RESUMEN

(1) Background: coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been linked to hematological dysfunctions, but there are little experimental data that explain this. Spike (S) and Nucleoprotein (N) proteins have been putatively associated with these dysfunctions. In this work, we analyzed the recruitment of hemoglobin (Hb) and other metabolites (hemin and protoporphyrin IX-PpIX) by SARS-Cov2 proteins using different approaches. (2) Methods: shotgun proteomics (LC-MS/MS) after affinity column adsorption identified hemin-binding SARS-CoV-2 proteins. The parallel synthesis of the peptides technique was used to study the interaction of the receptor bind domain (RBD) and N-terminal domain (NTD) of the S protein with Hb and in silico analysis to identify the binding motifs of the N protein. The plaque assay was used to investigate the inhibitory effect of Hb and the metabolites hemin and PpIX on virus adsorption and replication in Vero cells. (3) Results: the proteomic analysis by LC-MS/MS identified the S, N, M, Nsp3, and Nsp7 as putative hemin-binding proteins. Six short sequences in the RBD and 11 in the NTD of the spike were identified by microarray of peptides to interact with Hb and tree motifs in the N protein by in silico analysis to bind with heme. An inhibitory effect in vitro of Hb, hemin, and PpIX at different levels was observed. Strikingly, free Hb at 1mM suppressed viral replication (99%), and its interaction with SARS-CoV-2 was localized into the RBD region of the spike protein. (4) Conclusions: in this study, we identified that (at least) five proteins (S, N, M, Nsp3, and Nsp7) of SARS-CoV-2 recruit Hb/metabolites. The motifs of the RDB of SARS-CoV-2 spike, which binds Hb, and the sites of the heme bind-N protein were disclosed. In addition, these compounds and PpIX block the virus's adsorption and replication. Furthermore, we also identified heme-binding motifs and interaction with hemin in N protein and other structural (S and M) and non-structural (Nsp3 and Nsp7) proteins.

Asunto(s)

COVID-19/etiología , Hemoglobinas/metabolismo , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/metabolismo , Proteínas Estructurales Virales/metabolismo , COVID-19/sangre , Hemina/metabolismo , Hemoglobinas/ultraestructura , Humanos , Simulación del Acoplamiento Molecular , Unión Proteica , Dominios Proteicos , Proteómica , Protoporfirinas/metabolismo , SARS-CoV-2/patogenicidad , Proteínas no Estructurales Virales/ultraestructura , Proteínas Estructurales Virales/ultraestructura , Acoplamiento Viral , Replicación Viral

RESUMEN

PURPOSE: This study investigated the degree of tumor cell infiltration in the tumor cavity and ventricle wall based on fluorescent signals of 5-aminolevulinic acid (5-ALA) after removal of the magnetic resonance (MR)-enhancing area and analyzed its prognostic significance in glioblastoma. METHODS: Twenty-five newly developed isocitrate dehydrogenase (IDH)-wildtype glioblastomas with complete resection both of MR-enhancing lesions and strong purple fluorescence on resection cavity were retrospectively analyzed. The fluorescent signals of 5-ALA were divided into strong purple, vague pink, and blue colors. The pathologic findings were classified into massively infiltrating tumor cells, infiltrating tumor cells, suspicious single-cell infiltration, and normal-appearing cells. The pathological findings were analyzed according to the fluorescent signals in the resection cavity and ventricle wall. RESULTS: There was no correlation between fluorescent signals and infiltrating tumor cells in the resection cavity (p = 0.199) and ventricle wall (p = 0.704) after resection of the MR-enhancing lesion. The median progression-free survival (PFS) and median overall survival (OS) were 12.5 (± 2.1) and 21.1 (± 3.5) months, respectively. In univariate analysis, the presence of definitive infiltrating tumor cells in the resection cavity and ventricle wall was significantly related to the PFS (p = 0.002) and OS (p = 0.027). In multivariate analysis, the absence of definitive infiltrating tumor cells improved PFS (hazard ratio: 0.184; 95% CI: 0.049-0.690, p = 0.012) and OS (hazard ratio: 0.124; 95% CI: 0.015-0.998, p = 0.050). CONCLUSIONS: After resection both of the MR-enhancing lesions and strong purple fluorescence on resection cavity, there was no correlation between remnant fluorescent signals and infiltrating tumor cells. The remnant definitive infiltrating tumor cells in the resection cavity and ventricle wall significantly influenced the prognosis of patients with glioblastoma. Aggressive surgical removal of infiltrating tumor cells may improve their prognosis.

Asunto(s)

Ácido Aminolevulínico/metabolismo , Neoplasias Encefálicas/patología , Movimiento Celular , Glioblastoma/patología , Isocitrato Deshidrogenasa , Fármacos Fotosensibilizantes/metabolismo , Anciano , Ácido Aminolevulínico/administración & dosificación , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/cirugía , Ventrículos Cerebrales/metabolismo , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Femenino , Fluorescencia , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Glioblastoma/cirugía , Humanos , Estimación de Kaplan-Meier , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Fármacos Fotosensibilizantes/administración & dosificación , Pronóstico , Supervivencia sin Progresión , Protoporfirinas/metabolismo , Estudios Retrospectivos , Proteínas Supresoras de Tumor/genética

RESUMEN

Photodynamic therapy (PDT) has been used to treat certain types of non-melanoma skin cancer with promising results. However, some skin lesions have not fully responded to this treatment, suggesting a potential PDT-resistant phenotype. Therefore, novel therapeutic alternatives must be identified that improve PDT in resistant skin cancer. In this study, we analyzed the cell viability, intracellular protoporphyrin IX (PpIX) content and subcellular localization, proliferation profile, cell death, reactive oxygen species (ROS) detection and relative gene expression in PDT-resistant HSC-1 cells. PDT-resistant HSC-1 cells show a low quantity of protoporphyrin IX and low levels of ROS, and thus a low rate of death cell. Furthermore, the resistant phenotype showed a downregulation of HSPB1, SLC15A2, FECH, SOD2 and an upregulation of HMBS and BIRC5 genes. On the other hand, epigallocatechin gallate catechin enhanced the MAL-PDT effect, increasing levels of protoporphyrin IX and ROS, and killing 100% of resistant cells. The resistant MAL-PDT model of skin cancer squamous cells (HSC-1) is a reliable and useful tool to understand PDT cytotoxicity and cellular response. These resistant cells were successfully sensitized with epigallocatechin gallate catechin. The in vitro epigallocatechin gallate catechin effect as an enhancer of MAL-PDT in resistant cells is promising in the treatment of difficult skin cancer lesions.

Asunto(s)

Anticarcinógenos/farmacología , Carcinoma de Células Escamosas/tratamiento farmacológico , Catequina/análogos & derivados , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Terapia Combinada/métodos , Fotoquimioterapia/métodos , Neoplasias Cutáneas/tratamiento farmacológico , Ácido Aminolevulínico/análogos & derivados , Ácido Aminolevulínico/farmacología , Carcinoma de Células Escamosas/radioterapia , Catequina/farmacología , Muerte Celular/efectos de la radiación , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/genética , Hipoxia de la Célula/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Ferroquelatasa/genética , Ferroquelatasa/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Fármacos Fotosensibilizantes/metabolismo , Protoporfirinas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Neoplasias Cutáneas/radioterapia , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Estrés Fisiológico/efectos de la radiación , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Survivin/genética , Survivin/metabolismo , Simportadores/genética , Simportadores/metabolismo

RESUMEN

The ATP-Binding Cassette, subfamily G, member 2 (ABCG2) transporter is associated with the regulation of protoporphyrin IX transport and of other intermediates in heme biosynthesis. Because the hamster Harderian gland (HG) exhibits high concentrations of porphyrins and sexual dimorphism, we analyzed the hamster ABCG2. Cloned cDNA [2098-base pairs (bp)] contains an open-reading frame (ORF) of 1971-bp that encodes a 656 amino-acid protein with a molecular weight of 72844.56Da. The hamster ABCG2 sequence is conserved phylogenetically and shares a high percentage of identity with mouse (89%), rat (88%), and human (84%) transporters. Within its structure, a Walker A (G-X-X-G-X-G-K-S), a C signature motif characteristic of ABC transporters, and six putative transmembrane domains (TMDs) were identified. ABCG2 mRNA was detected in all hamster tissues, with higher amounts found in HG, brain, cerebellum, kidney, gut, ovary, and testis. Harderian ABCG2 expression exhibits a sexually dimorphic pattern where females display higher mRNA levels than males. Different patterns of transcriptional profiles of ABCG2 during the estrous cycle and after gonadectomy in both sexes were also observed. The differential expression between male and female HGs suggests that ABCG2 is under the regulation of gonadal steroids. The ABCG2 transporter is likely involved in the endogenous regulation of porphyrins in hamster HGs.

Asunto(s)

Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Hormonas Esteroides Gonadales/metabolismo , Glándula de Harder/metabolismo , Protoporfirinas/metabolismo , Animales , Cricetinae , ADN Complementario , Femenino , Humanos , Masculino , Mesocricetus , Ratones , Porfirinas/metabolismo , ARN Mensajero , Ratas , Caracteres Sexuales

RESUMEN

Protoporphyrin IX (PpIX) is a precursor of heme synthesis and is known to be an active photosensitizer and precursor of photosensitizers applied in photodynamic therapy (PDT) and photodynamic diagnostics (PDD). On irradiation with visible light, PpIX undergoes phototransformation, producing photoproducts which may also be phototoxic and increase its efficacy. The mechanism of PpIX phototransformation depends on environmental characteristics and can be different in vitro and in vivo. In this paper, we present a comparative study of the photoactivity of synthetic PpIX and PpIX extracted from the Harderian gland of ssp Rattus novergicus albinus rats, along with their photoproducts toward murine B16F-10 melanoma cells. It was observed that when irradiated with visible light the endogenous PpIX demonstrates photocytotoxicity ten times higher than the synthetic PpIX. The photoproduct of endogenous PpIX also possesses phototoxicity, though slightly lower than that of PpIX itself. The rate of cell internalization for both endogenous PpIX and its photoproduct was eightfold greater than that obtained for the synthetic porphyrin. This difference might result from a complexation of the native PpIX with some amphiphilic compounds during its synthesis within the Harderian glands, which facilitates the cell uptake of PpIX. Fluorescence microscopy images show that both endogenous and synthetic porphyrins are localized after uptake predominantly in the mitochondrial region of cells.

Asunto(s)

Melanoma/patología , Fotoquimioterapia , Fármacos Fotosensibilizantes/farmacología , Protoporfirinas/farmacología , Animales , Transporte Biológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Oscuridad , Glándula de Harder/metabolismo , Espacio Intracelular/metabolismo , Masculino , Melanoma/tratamiento farmacológico , Ratones , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/aislamiento & purificación , Fármacos Fotosensibilizantes/metabolismo , Protoporfirinas/síntesis química , Protoporfirinas/aislamiento & purificación , Protoporfirinas/metabolismo , Ratas

RESUMEN

Some researchers have shown that carbon monoxide (CO) plays a role in emotional behavior modulation through intracellular 3'-5'-guanosine monophosphate mechanisms in the locus coeruleus (LC). In fact, the LC region has a high expression of the heme-oxygenase (HO) enzymes, which are responsible for the production of CO. However, the physiological mechanism by which the HO-CO pathway participates in the modulation of emotional responses in the LC still needs clarification. This study evaluates whether a systemic intraperitoneal treatment is able to alter behavioral responses (in the elevated plus-maze and the light-dark box test) and the expression of the HO-1 and HO-2 enzymes in the LC. The tested treatments are acute (3h before) or chronic (twice daily for 10days) and with a carbon monoxide releaser (tricarbonyldichlororuthenium [II] dimer, or CORM-2) or with a HO-1 inducer compound (cobalt protoporphyrin IX, CoPP). The results for the elevated plus-maze show that CO-for both acute or chronic administration of either drug-ncreased the number of entries into the open arms and the percentage of time spent in the open arms. Regarding the light-dark box test, chronic treatment with either drug increased the time spent in the light compartment. Additionally, treatment with CORM-2 or CoPP, either acutely or chronically, increased HO-1 enzyme expression in the LC cells. This study shows that systemic CO treatment can promote an anxiolytic-like effect and the expression of HO-1 enzymes in LC cells.

Asunto(s)

Hemo-Oxigenasa 1/biosíntesis , Locus Coeruleus/enzimología , Compuestos Organometálicos/farmacología , Protoporfirinas/farmacología , Animales , Ansiolíticos/metabolismo , Ansiedad/tratamiento farmacológico , Conducta Animal/fisiología , Monóxido de Carbono/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Masculino , Óxido Nítrico Sintasa de Tipo II/metabolismo , Compuestos Organometálicos/metabolismo , Protoporfirinas/metabolismo , Ratas , Ratas Wistar

RESUMEN

Photodynamic therapy (PDT) has been indicated for oral squamous cell carcinoma (OSCC) at early stages. Chemo and radio-resistance are frequently observed in OSCC, but it is unknown whether this tumor can develop resistance to PDT. It was investigated the process of acquiring resistance to multiple cycles of PDT by using OSCC cells. We also analyzed the expression of anti-apoptotic proteins and those related to Akt/mTOR pathway. Sub-lethal doses of PDT were applied, consisting of a constant concentration of 5-aminolevulinic acid (5-ALA) (1 mM, 4-h incubation) and increasing irradiation dose with LED (from 5.86 to 10.54 J/cm2 ). Cell viability, migration capacity, intracellular expression of protoporphyrin IX (PpIX), mitochondrial density, and pro-survival proteins were investigated in PDT-resistant cells. Six OSCC cell generations resistant to PDT were isolated. The resistant cells exhibited a survival phenotype characterized by a reduction in the expression of endogenous PpIX, increase in mitochondrial density, increase in migration capacity, and up-regulation of p-NFκB, p-survivin, iNOS, p-Akt Ser473 , cyclin D1, p-mTOR Ser2481 , and p-mTOR Ser2448 . OSCC cells are able to survive doses of 5-ALA-PDT by reducing PpIX synthesis and activating signaling pathways related to the inhibition of apoptosis and improvement of cell proliferation. Further studies are necessary to confirm whether this PDT-resistance phenotype can be clinically present, mainly in OSCC showing clinical recurrences after exposure to different PDT protocols.

Asunto(s)

Ácido Aminolevulínico/farmacología , Fotoquimioterapia/métodos , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Neoplasias de la Boca/metabolismo , Protoporfirinas/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo

RESUMEN

Many heme-containing proteins with a histidine in the distal E7 (HisE7) position can form sulfheme in the presence of hydrogen sulfide (H2S) and a reactive oxygen species such as hydrogen peroxide. For reasons unknown, sulfheme derivatives are formed specifically on solvent-excluded heme pyrrole B. Sulfhemes severely decrease the oxygen-binding affinity in hemoglobin (Hb) and myoglobin (Mb). Here, use of hybrid quantum mechanical/molecular mechanical methods has permitted characterization of the entire process of sulfheme formation in the HisE7 mutant of hemoglobin I (HbI) from Lucina pectinata. This process includes a mechanism for H2S to enter the solvent-excluded active site through a hydrophobic channel to ultimately form a hydrogen bond with H2O2 bound to Fe(III). Proton transfer from H2O2 to His64 to form compound (Cpd) 0, followed by hydrogen transfer from H2S to the Fe(III)-H2O2 complex, results in homolytic cleavage of the O-O and S-H bonds to form a reactive thiyl radical (HS(•)), ferryl heme Cpd II, and a water molecule. Subsequently, the addition of HS(•) to Cpd II, followed by three proton transfer reactions, results in the formation of a three-membered ring ferric sulfheme that avoids migration of the radical to the protein matrix, in contrast to that in other peroxidative reactions. The transformation of this three-membered episulfide ring structure to the five-membered thiochlorin ring structure occurs through a significant potential energy barrier, although both structures are nearly isoenergetic. Both three- and five-membered ring structures reveal longer NB-Fe(III) bonds compared with other pyrrole nitrogen-Fe(III) bonds, which would lead to decreased oxygen binding. Overall, these results are in agreement with a wide range of experimental data and provide fertile ground for further investigations of sulfheme formation in other heme proteins and additional effects of H2S on cell signaling and reactivity.

Asunto(s)

Hemo/análogos & derivados , Hemo/química , Peróxido de Hidrógeno/química , Sulfuro de Hidrógeno/química , Animales , Bivalvos/metabolismo , Dominio Catalítico , Hemoglobinas/química , Hemoglobinas/genética , Hemoglobinas/metabolismo , Enlace de Hidrógeno , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Protoporfirinas/química , Protoporfirinas/metabolismo , Teoría Cuántica

RESUMEN

In this work, we developed an experimental apparatus to directly measure transmittance and fluorescence in the stratum corneum (SC) ex vivo. The SC transmittance varied from ~6 to ~52 % in the wavelength range of 280-850 nm. For 260-300 nm excitation, the SC autofluorescence showed a strong emission band between 290 and 425 nm, which is associated with tryptophan, and another in the 600-670 nm range, which we attributed to a process involving resonance energy transfer to very hydrophobic keratin filaments. Weaker emission associated with less hydrophobic keratin filaments was also observed in the wavelength range of 350-480 nm. Protoporphyrin IX (PpIX) was incorporated into SC membranes, and its penetration was further increased by the addition of nerolidol to the treatment suspension. Both PpIX and the endogenous porphyrins showed fluorescence anisotropy consistent with their localization in SC membranes, and their molecular dynamics increased significantly in the presence of 1 % nerolidol. The emission and excitation spectra of PpIX and the endogenous SC porphyrins showed similar alterations during the photobleaching induced by 405-nm irradiation. This work also highlights the SC contribution to skin autofluorescence, which could be useful for fluorescence spectroscopy applications in the early diagnosis of skin diseases.

Asunto(s)

Epidermis/efectos de los fármacos , Epidermis/metabolismo , Fluorescencia , Fármacos Fotosensibilizantes/farmacología , Protoporfirinas/metabolismo , Sesquiterpenos/farmacología , Fenómenos Fisiológicos de la Piel/efectos de los fármacos , Animales , Animales Recién Nacidos , Uniones Intercelulares/efectos de los fármacos , Uniones Intercelulares/metabolismo , Membranas/efectos de los fármacos , Membranas/metabolismo , Fotoquimioterapia , Ratas , Ratas Wistar , Espectrometría de Fluorescencia

RESUMEN

Renal cell carcinoma (RCC) remains one of the greatest challenges of urological oncology and is the third leading cause of death in genitourinary cancers. Surgery may be curative when patients present with localized disease. Our previous results demonstrated the autofluorescence of blood PpIX in primary RCC mouse model and an increase in fluorescence intensity as a function of growth of the subcutaneous tumor mass. In another work, a nice correlation between the growth of the tumor mass and tissue fluorescence intensity was found. The aim of this study was to evaluate the expression profile of porphyrin biosynthesis pathway-related genes of human kidney cells. We used two kidney cell lines, one normal (HK2) and another malignant (Caki-1). Endogenous and 5-aminolevolinic acid (ALA) induced protoporphyrin IX (PpIX) HK2 and Caki-1 cells were analyzed by fluorescence spectroscopy. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to measure mRNA of those genes. Emission spectra were obtained by exciting the samples at 405 nm. For ALA untreated cells the maximum fluorescence intensity was detected at 635 nm. The mean peak area of emission spectra in both cells types increased linearly in function of cell number. Besides, basal levels of PpIX autofluorescence of each cell concentration of HK2 samples were significantly lower than those of Caki-1 samples. For ALA-treated cells the mean PpIX spectra shows PpIX emission peak at 635 nm with a shoulder at 700 nm. Analysis of PpIX fluorescence intensity ratio between tumor cells and HK2 cells showed that fluorescence intensity was, on average, 26 times greater in tumor cells than in healthy cells. qRT-PCR revealed that in Caki-1 ALA-treated cells, PEPT gene was significantly up-regulated and FECH and HO-1 genes were significantly down regulated in comparison with HK2 ALA-treated cells. In conclusion, our results demonstrate the preferential accumulation of ALA-induced PpIX in human RCC and also indicate that PEPT1, FECH and HO-1 genes are major contributors to this accumulation.

Asunto(s)

Carcinoma de Células Renales/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias Renales/patología , Protoporfirinas/biosíntesis , Animales , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Línea Celular Tumoral , Humanos , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Ratones , Protoporfirinas/metabolismo

RESUMEN

Aminolevulinic acid (ALA)-mediated protoporphyrin IX (PpIX) production is being explored for tumor fluorescence imaging and photodynamic therapy (PDT). As a prodrug, ALA is converted in heme biosynthesis pathway to PpIX with fluorescent and photosensitizing properties. To better understand the role of heme biosynthesis enzymes in ALA-mediated PpIX fluorescence and PDT efficacy, we used lentiviral shRNA to silence the expression of porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD) and ferrochelatase (FECH) in SkBr3 human breast cancer cells. PBGS and PBGD are the first two cytosolic enzymes involved in PpIX biosynthesis, and FECH is the enzyme responsible for converting PpIX to heme. PpIX fluorescence was examined by flow cytometry and confocal fluorescence microscopy. Cytotoxicity was assessed after ALA-mediated PDT. Silencing PBGS or PBGD significantly reduced ALA-stimulated PpIX fluorescence, whereas silencing FECH elevated basal and ALA-stimulated PpIX fluorescence. However, compared with vector control cells, the ratio of ALA-stimulated fluorescence to basal fluorescence without ALA was significantly reduced in all knockdown cell lines. PBGS or PBGD knockdown cells exhibited significant resistance to ALA-PDT, while increased sensitivity to ALA-PDT was found in FECH knockdown cells. These results demonstrate the importance of PBGS, PBGD and FECH in ALA-mediated PpIX fluorescence and PDT efficacy.

Asunto(s)

Ácido Aminolevulínico/metabolismo , Silenciador del Gen , Hemo/biosíntesis , Fotoquimioterapia , Protoporfirinas/metabolismo , Línea Celular Tumoral , Humanos

RESUMEN

Photodynamic therapy (PDT) is used for skin treatments of premalignant and cancer lesions and recognized as a non-invasive technique that combines tissue photosensitization and subsequent exposure to light to induce cell death. However, it is limited to the treatment of superficial lesions, mainly due to the low cream penetration. Therefore, the improvement of transdermal distribution of aminolevulinic acid (ALA) is needed. In this study, the kinetics and homogeneity of production of ALA-induced PpIX after the skin pre-treatment with microneedles rollers of 0.5, 1.0 and 1.5 mm length were investigated. An improvement in homogeneity and production of PpIX was shown in a porcine model. Widefield fluorescence imaging three hours after the topical application of ALA-cream in the combined treatment with microeedles rollers.

Asunto(s)

Ácido Aminolevulínico/farmacología , Agujas , Protoporfirinas/metabolismo , Piel/efectos de los fármacos , Piel/metabolismo , Ácido Aminolevulínico/metabolismo , Animales , Cinética , Masculino , Profármacos/metabolismo , Profármacos/farmacología , Sus scrofa

RESUMEN

This paper describes the elimination of porphyrins by feces. It was demonstrated that porphyrin accumulates substantially more in tumors than in normal tissues, and consequently more PPIX reaches the blood of patients and animals with tumors, and then, it needs to be eliminated. The fluorescence of feces revealed that there are large amounts of PPIX in the excreta of animals with cancer comparing with healthy animals. The autofluorescence of feces porphyrin extracted with acetone was analyzed using fluorescence spectroscopy of animals inoculated with DU145 cells into the prostate and healthy animals to monitor the PPIX concentration. Emission spectra were obtained by exciting the samples at 405 nm. Significant differences were observed in autofluorescence intensities measured in the 575-725 nm spectral regions for the studied groups. The results showed a noninvasive, simple, rapid and sensitive method to detect cancer by feces analysis.

Asunto(s)

Técnicas y Procedimientos Diagnósticos , Heces/química , Neoplasias de la Próstata/patología , Protoporfirinas/metabolismo , Animales , Línea Celular Tumoral , Humanos , Masculino , Ratones , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/metabolismo , Protoporfirinas/biosíntesis , Espectrometría de Fluorescencia

RESUMEN

OBJECTIVE: To investigate red fluorescence found in the digestive tract of Wistar rats submitted to stress produced by a liquid diet of 5% glucose and maintenance in darkness. BACKGROUND DATA: Protoporphyrin IX (PpIX) is produced by the Harderian gland, located in the inner corner of the eyes of rats. Under stressful conditions this gland increases the production of PpIX, which can be detected in different regions of the body, in a manner reminiscent of a porphyria. MATERIAL AND METHODS: Sixty-five Wistar rats were used in this study. The fluorescence spectra were registered with optical resolution better than 1.7 nm. The rats were fed a 5% glucose diet, exclusively, up to 120 h. The animals were evaluated throughout the diet period, which included two sequential experiments: considering the red fluorescence of their intestinal tract and the fluorescence that appeared in some external parts of their bodies (paw, tail, nose, and scrotum). The normal diet was reintroduced and new spectra were obtained after 24 and 48 h. RESULTS: Experiment I showed a marked, time-dependent increase in the intestinal content of porphyrin in rats fed the glucose diet. The fluorescence spectrum of the material identified it as PpIX. The spectra collected in Experiment II showed an increase in fluorescence in the four external areas associated with the duration of the diet. This fluorescence disappeared after reintroduction of the regular diet. CONCLUSION: The feeding of a restricted diet (5% glucose) to Wistar rats resulted in reversible porphyria. Measurement of the fluorescence intensity may be a reliable method for monitoring the porphyrin content of tissues.

Asunto(s)

Fluorescencia , Glucosa/administración & dosificación , Mucosa Intestinal/metabolismo , Protoporfirinas/metabolismo , Animales , Dieta , Masculino , Distribución Aleatoria , Ratas , Espectrometría de Fluorescencia/métodos , Factores de Tiempo

RESUMEN

Hematophagy is a feeding habit that involves the ingestion of huge amounts of heme. The hematophagous hemipteran Rhodnius prolixus evolved many genetic resources to protect cells against heme toxicity. The primary barrier against the deleterious effects of heme is the aggregation of heme into hemozoin in the midgut lumen. Hemozoin formation is followed by the enzymatic degradation of heme by means of a unique pathway whose end product is dicysteinyl-biliverdin IX-γ (Rhodnius prolixus biliverdin, RpBv). These mechanisms are complemented by a heme-binding protein (RHBP) in the hemolymph that attenuates the pro-oxidant effects of heme. In this work, we show that when insects are fed with blood enriched with a heme analog, Sn-protoporphyrin (SnPP-IX), both hemozoin synthesis and RpBv production are inhibited in a dose-dependent manner. These effects are accompanied by increased oxidative damage to the midgut epithelium and inhibition of oviposition, indicating that hemozoin formation and heme degradation are protective mechanisms that work together and contributed to the adaptation of this insect to successfully feed on vertebrate blood.

Asunto(s)

Hemo/metabolismo , Hemoproteínas/metabolismo , Metaloporfirinas/metabolismo , Protoporfirinas/metabolismo , Rhodnius/fisiología , Animales , Sangre , Femenino , Tracto Gastrointestinal/metabolismo , Oviposición , Conejos

RESUMEN

The cellular traffic of haem during the development of the human malaria parasite Plasmodium falciparum, through the stages R (ring), T (trophozoite) and S (schizonts), was investigated within RBC (red blood cells). When Plasmodium cultures were incubated with a fluorescent haem analogue, ZnPPIX (Zn protoporphyrin IX) the probe was seen at the cytoplasm (R stage), and the vesicle-like structure distribution pattern was more evident at T and S stages. The temporal sequence of ZnPPIX uptake by P. falciparum-infected erythrocytes shows that at R and S stages, a time-increase acquisition of the porphyrin reaches the maximum fluorescence distribution after 60 min; in contrast, at the T stage, the maximum occurs after 120 min of ZnPPIX uptake. The difference in time-increase acquisition of the porphyrin is in agreement with a maximum activity of haem uptake at the T stage. To gain insights into haem metabolism, recombinant PfHO (P. falciparum haem oxygenase) was expressed, and the conversion of haem into BV (biliverdin) was detected. These findings point out that, in addition to haemozoin formation, the malaria parasite P. falciparum has evolved two distinct mechanisms for dealing with haem toxicity, namely, the uptake of haem into a cellular compartment where haemozoin is formed and HO activity. However, the low Plasmodium HO activity detected reveals that the enzyme appears to be a very inefficient way to scavenge the haem compared with the Plasmodium ability to uptake the haem analogue ZnPPIX and delivering it to the food vacuole.

Asunto(s)

Eritrocitos/parasitología , Plasmodium falciparum/metabolismo , Protoporfirinas/metabolismo , Biliverdina/metabolismo , Eritrocitos/metabolismo , Hemo Oxigenasa (Desciclizante)/clasificación , Hemo Oxigenasa (Desciclizante)/genética , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemoproteínas/metabolismo , Hemina/metabolismo , Humanos , Malaria Falciparum/parasitología , Plasmodium falciparum/crecimiento & desarrollo , Unión Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factores de Tiempo