RESUMO
BACKGROUND: The cellular temperatures of microorganisms are considered to be the same as those of their surroundings because the cellular volume is too small to maintain a cellular temperature that is different from the ambient temperature. However, by forming a colony or a biofilm, microorganisms may be able to maintain a cellular temperature that is different from the ambient temperature. In this study, we measured the temperatures of bacterial colonies isolated from soils using an infrared imager and investigated the thermogenesis by a bacterium that increases its colony temperature. RESULTS: The temperatures of some colonies were higher or lower than that of the surrounding medium. A bacterial isolate with the highest colony temperature was identified as Pseudomonas putida. This bacterial isolate had an increased colony temperature when it grew at a temperature suboptimal for its growth. Measurements of heat production using a microcalorimeter showed that the temperature of this extraordinary, microcalorimetrically determined thermogenesis corresponded with the thermographically observed increase in bacterial colony temperature. When investigating the effects of the energy source on this thermal behavior, we found that heat production by this bacterium increased without additional biomass production at a temperature suboptimal for its growth. CONCLUSIONS: We found that heat production by bacteria affected the bacterial colony temperature and that a bacterium identified as Pseudomonas putida could maintain a cellular temperature different from the ambient temperature, particularly at a sub-optimal growth temperature. The bacterial isolate P. putida KT1401 increased its colony temperature by an energy-spilling reaction when the incubation temperature limited its growth.
Assuntos
Temperatura Alta , Pseudomonas putida/isolamento & purificação , Pseudomonas putida/metabolismo , Microbiologia do Solo , Calorimetria , DNA Bacteriano/química , DNA Bacteriano/genética , Processamento de Imagem Assistida por Computador , Raios Infravermelhos , Dados de Sequência Molecular , Pseudomonas putida/classificação , Pseudomonas putida/genética , Análise de Sequência de DNA , TemperaturaRESUMO
Four platinum(II) and palladium(II) complexes with sugar-conjugated bipyridine-type triazole ligands, [Pt(II)Cl(2)(AcGlc-pyta)] (3), [Pd(II)Cl(2)(AcGlc-pyta)] (4), [Pt(II)Cl(2)(Glc-pyta)] (5), and [Pd(II)Cl(2)(Glc-pyta)] (6), were prepared and characterized by mass spectrometry, elemental analysis, (1)H- and (13)C-NMR, IR as well as UV/VIS spectroscopy, where AcGlc-pyta and Glc-pyta denote 2-[4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl]ethyl 2,3,4,6-tetra-O-acetyl-ß-D-glucopyranoside (1) and 2-[4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl]ethyl ß-D-glucopyranoside (2), respectively. The solid-state structure of complex 6 was determined by single-crystal X-ray-diffraction analysis. These complexes exhibited in vitro cytotoxicity against human cervix tumor cells (HeLa) though weaker than that of cisplatin.
Assuntos
Antineoplásicos/síntese química , Complexos de Coordenação/síntese química , Ligantes , Paládio/química , Platina/química , Triazóis/síntese química , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Carboidratos/química , Cisplatino/química , Cisplatino/uso terapêutico , Complexos de Coordenação/química , Complexos de Coordenação/uso terapêutico , Cristalografia por Raios X , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Neoplasias/tratamento farmacológico , Triazóis/química , Triazóis/uso terapêuticoRESUMO
The application of low-flux sunlight begins with the synthesis of effective antenna systems. This requires the development of dye integrates with optimized dye orientation for effective energy transfer. We here report a series of peptide-linked porphyrin arrays, denoted by Boc-(Por(Zn,S))(n)-OBu(t) (n = 2, 4, and 8), that change their dye orientation to increase fluorescence responsively to additive reagents. The B-band absorption (AB) regions of the arrays show blue shifts (dimer, 407.6 nm; tetramer, 408.2 nm; octamer, 407.8 nm) in organic solvents as compared to that of Boc-Por(Zn,S)-OBu(t) (monomer, 422.6 nm) and the fluorescence yield Phi' of the arrays decreases with increasing n, obeying the relationship Phi' = 0.03/n(1.5); however, the arrays are tuned up in fluorescence emission by the addition of 1,2-diaminoethane (en). The addition of a sufficient amount of en increases the fluorescence of the porphyrins in monomer, dimer, tetramer, and octamer by approximately 5, approximately 12, approximately 12, and >730 times, respectively, when compared with that observed in the absence of en. This also causes asymptotic red shifts in absorption (AB) bands (B-band lambda(max): 410 to 429-430 nm), as well as changes in circular dichroism (CD) spectra, and makes porphyrins approach new mutual asymmetric orientations. Our results show the potentiality of the tunable dye polymers that are a posteriori optimized in dye orientation and fluorescence emission by additive reagents for the development of effective light-harvesting materials.
Assuntos
Fluorescência , Porfirinas/química , Absorção , Dicroísmo Circular , Etilenodiaminas/química , Corantes Fluorescentes/química , Peptídeos/química , Polímeros/químicaRESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Heme Oxigenase-1/biossíntese , Fator 2 Relacionado a NF-E2/fisiologia , Proteínas de Neoplasias/biossíntese , Neoplasias/terapia , Fotoquimioterapia , Porfirinas/química , Porfirinas/efeitos da radiação , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Ácido Aminolevulínico/farmacologia , Benzotiazóis , Western Blotting , Linhagem Celular Tumoral , Clorofila/análogos & derivados , Clorofila/farmacologia , Cromatografia Líquida de Alta Pressão , Diaminas , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes , Heme Oxigenase-1/genética , Humanos , Fator 2 Relacionado a NF-E2/genética , Proteínas de Neoplasias/genética , Neoplasias/patologia , Compostos Orgânicos , Estresse Oxidativo , Fotoquímica , Fármacos Fotossensibilizantes/farmacologia , Protoporfirinas/farmacologia , Quinolinas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Interferente Pequeno/farmacologia , TransfecçãoRESUMO
Two water-soluble porphyrin self-assemblies consisting of bisacetylene- and monoacetylene-linked conjugated bis(imidazolylporphyrin) have been synthesized. Two-photon absorption (2PA) cross section values in water were determined as 7500 GM for bisacetylene- and 7900 GM for monoacetylene-linked bisporphyrin by femtosecond open-aperture Z-scan measurement. These values were almost the same as those for the similar structure reported previously in CHCl(3). Therefore, the structure is suggested to be similar in CHCl(3) and aqueous solutions. Both compounds were found to efficiently generate singlet oxygen upon one-photon irradiation in a manner similar to protoporphyrin, as demonstrated by the time-resolved luminescence measurement at the characteristic band of 1270 nm. Photocytotoxicities for HeLa cancer cells were examined and found to be as high as those of hematoporphyrin, demonstrating that these newly prepared compounds are potential candidates as 2PA-photodynamic therapy agents.
Assuntos
Imidazóis/síntese química , Fotoquimioterapia , Fármacos Fotossensibilizantes/síntese química , Porfirinas/síntese química , Acetileno/química , Células HeLa , Humanos , Imidazóis/química , Imidazóis/farmacologia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/química , Porfirinas/farmacologia , Oxigênio Singlete/metabolismo , Solubilidade , Relação Estrutura-Atividade , ÁguaRESUMO
The photosynthetic reaction center is an efficient molecular device for the conversion of light energy to chemical energy. In a previous study, we synthesized the hydrogenase/photosystem I (PSI) complex, in which Ralstonia hydrogenase was linked to the cytoplasmic side of Synechocystis PSI, to modify PSI so that it photoproduced molecular hydrogen (H2). In that study, hydrogenase was fused with a PSI subunit, PsaE, and the resulting hydrogenase-PsaE fusion protein was self-assembled with PsaE-free PSI to give the hydrogenase/PSI complex. Although the hydrogenase/PSI complex served as a direct light-to-H2 conversion system in vitro, the activity was totally suppressed by adding physiological PSI partners, ferredoxin (Fd) and ferredoxin-NADP+-reductase (FNR). In the present study, to establish an H2 photoproduction system in which the activity is not interrupted by Fd and FNR, position 40 of PsaE from Synechocystis sp. PCC6803, corresponding to the Fd-binding site on PSI, was selected and targeted for the cross-linking with cytochrome c3 (cytc3) from Desulfovibrio vulgaris. The covalent adduct of cytc3 and PsaE was stoichiometrically assembled with PsaE-free PSI to form the cytc3/PSI complex. The NADPH production by the cytc3/PSI complex coupled with Fd and FNR decreased to approximately 20% of the original activity, whereas the H2 production by the cytc3/PSI complex coupled with hydrogenase from Desulfovibrio vulgaris was enhanced 7-fold. Consequently, in the simultaneous presence of hydrogenase, Fd, and FNR, the light-driven H2 production by the hydrogenase/cytc3/PSI complex was observed (0.30 pmol Hz/mg chlorophyll/h). These results suggest that the cytc3/PSI complex may produce H2 in vivo.
Assuntos
Grupo dos Citocromos c/metabolismo , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Synechocystis/metabolismo , Synechocystis/efeitos da radiação , Proteínas de Bactérias/metabolismo , Sequência de Bases , Grupo dos Citocromos c/química , Grupo dos Citocromos c/efeitos da radiação , Primers do DNA , Transporte de Elétrons , Hidrogenase/química , Modelos Moleculares , Dados de Sequência Molecular , Fotoquímica , Complexo de Proteína do Fotossistema I/química , Complexo de Proteína do Fotossistema I/efeitos da radiação , Reação em Cadeia da Polimerase , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
In order to generate renewable and clean fuels, increasing efforts are focused on the exploitation of photosynthetic microorganisms for the production of molecular hydrogen from water and light. In this study we engineered a 'hard-wired' protein complex consisting of a hydrogenase and photosystem I (hydrogenase-PSI complex) as a direct light-to-hydrogen conversion system. The key component was an artificial fusion protein composed of the membrane-bound [NiFe] hydrogenase from the beta-proteobacterium Ralstonia eutropha H16 and the peripheral PSI subunit PsaE of the cyanobacterium Thermosynechococcus elongatus. The resulting hydrogenase-PsaE fusion protein associated with PsaE-free PSI spontaneously, thereby forming a hydrogenase-PSI complex as confirmed by sucrose-gradient ultracentrifuge and immunoblot analysis. The hydrogenase-PSI complex displayed light-driven hydrogen production at a rate of 0.58 mumol H(2).mg chlorophyll(-1).h(-1). The complex maintained its accessibility to the native electron acceptor ferredoxin. This study provides the first example of a light-driven enzymatic reaction by an artificial complex between a redox enzyme and photosystem I and represents an important step on the way to design a photosynthetic organism that efficiently converts solar energy and water into hydrogen.
Assuntos
Hidrogênio/metabolismo , Hidrogenase/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Cianobactérias/enzimologia , Cianobactérias/metabolismo , Fontes Geradoras de Energia , LuzRESUMO
The cellular uptake and photocytotoxity of poly-l-lysine (pL)-chlorin e6 (Ce6) conjugate were investigated. The cellularuptake of pL-Ce6 conjugate for HeLa cells was much higher than that of Ce6, and pL-Ce6 conjugate had high binding affinityfor HeLa cells. pL-Ce6 conjugate was accumulated in the nucleus of HeLa cells, and the effective photocytotoxity wasobserved by the irradiation.
Assuntos
Núcleo Celular/metabolismo , Fármacos Fotossensibilizantes/metabolismo , Polilisina/metabolismo , Núcleo Celular/química , Clorofilídeos , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Fármacos Fotossensibilizantes/análise , Polilisina/análise , Porfirinas/análise , Porfirinas/metabolismoRESUMO
Eight 5,10,15,20-tetrakis[3- or 4-(beta-D-glycopyranosyloxy)phenyl]chlorins were synthesized by means of the Whitlock method with diimide reduction and purified by reversed-phase thin layer chromatography (RP-TLC). All compounds were characterized by (1)H NMR spectroscopy, electron-spray ionization time-of-flight mass spectrometry (ESI-TOF MS), and UV-Vis spectroscopy. ESI-TOF MS could detect the 2H difference in molecular weight between a glycoconjugated chlorin and its corresponding porphyrin (i.e., 5,10,15,20-tetrakis[3- or 4-(beta-D-glycopyranosyloxy)phenyl]porphyrin). The cellular uptake of the eight chlorins was evaluated in HeLa cells. All glycoconjugated chlorins showed higher cellular uptake than tetraphenylporphyrin tetrasulfonic acid (TPPS), and 5,10,15,20-tetrakis[3-(beta-D-xylopyranosyloxy)phenyl]chlorin showed 50-fold higher uptake than TPPS. The photocytotoxicity of 5,10,15,20-tetrakis[3-(beta-D-glucopyranosyloxy)phenyl]chlorin, 5,10,15,20-tetrakis[3-(beta-D-xylopyranosyloxy)phenyl]chlorin and TPPS towards HeLa cells was examined at the concentration of 2x10(-7) M (mol/dm(3)). These photosensitizers had no cytotoxicity in the dark, but their photocytotoxicity decreased in the order of 5,10,15,20-tetrakis[3-(beta-D-glucopyranosyloxy)phenyl]chlorin>5,10,15,20-tetrakis[3-(beta-D-xylopyranosyloxy)phenyl]chlorin>TPPS. The results indicate that the photocytotoxicity is not related simply to cellular uptake.
Assuntos
Porfirinas/metabolismo , Porfirinas/toxicidade , Transporte Biológico , Glicosilação , Células HeLa , Humanos , Estrutura Molecular , Fotoquímica , Porfirinas/síntese química , Porfirinas/química , Análise EspectralRESUMO
Thirty-two glycoconjugated porphyrins were synthesized by a modification of Lindsey method in the presence of Zn(OAc)(2).2H(2)O as a template. The Zn(2+) ion template strategy improved the yield about three-fold in the case of meta-substituted tetraphenylporphyrins. In addition, free-base porphyrins were obtained almost quantitatively by demetalation with 4 M HCl. Sixteen deacetylated glycoconjugated porphyrins were tested as candidate photodynamic therapy (PDT) drugs using HeLa cells. Most of the deacetylated glycoconjugated porphyrins showed higher cellular uptake than tetraphenylporphyrin tetrasulfonic acid (TPPS), and 5,10,15,20-tetrakis[4-(beta-D-arabinopyranosyloxy)phenyl]porphyrin (p-5d) in particular showed 18.5-fold higher uptake than TPPS. The photocytotoxicity of 5,10,15,20-tetrakis[4-(beta-D-glucopyranosyloxy)phenyl]porphyrin (p-5a), p-5d and TPPS was examined with HeLa cells, using a light dose of 16 J/cm(2). These photosensitizers had no cytotoxicity in the dark, but their photocytotoxicity increased in the order of TPPS < p-5a < p-5d. These results suggest p-5d is a good candidate for a PDT drug.
Assuntos
Porfirinas/metabolismo , Porfirinas/toxicidade , Transporte Biológico , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Glicosilação , Células HeLa , Humanos , Estrutura Molecular , Fármacos Fotossensibilizantes/sangue , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/toxicidade , Porfirinas/sangue , Porfirinas/químicaRESUMO
BACKGROUND: Tumor biomarkers are commonly used for cancer screening and as indicators of treatment effects. We recently reported that urine porphyrin levels from tumor-bearing mice were elevated compared with those from normal mice after administration of 5-aminolevulinic acid (ALA). In the present study, we evaluated the use of urine samples from bladder cancer patients as tumor biomarkers. METHODS: ALA, 1.0 g, was orally administered to 66 bladder cancer patients and 20 healthy adults. The urine concentrations of uroporphyrin I (UPI), uroporphyrin III (UPIII), coproporphyrin I (CPI), coproporphyrin III (CPIII), and total porphyrins were measured using High Performance Liquid Chromatography (HPLC) system. RESULTS: Almost all of the urinary porphyrin concentrations from the patients with bladder cancer were higher than those from healthy adults. Moreover, 8h after ALA administration, urinary UPI and CPI showed high sensitivity (100 for UPI and CPI) and specificity (96.4 for UPI and 91.4 for CPI). CONCLUSION: These results indicate that the presence of urinary porphyrins after administration of ALA may function as tumor biomarkers. This method represents a possible new tumor screening method called photodynamic screening (PDS) using ALA-induced porphyrins.
Assuntos
Ácido Aminolevulínico , Biomarcadores Tumorais/urina , Detecção Precoce de Câncer/métodos , Porfirinas/urina , Neoplasias da Bexiga Urinária/diagnóstico , Neoplasias da Bexiga Urinária/urina , Adulto , Idoso , Cromatografia Líquida de Alta Pressão/métodos , Estudos de Viabilidade , Humanos , Pessoa de Meia-Idade , Fármacos Fotossensibilizantes , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Photodynamic diagnosis (PDD) using aminolevulinic acid (ALA) is widely used in clinical fields. In PDD, protoporphyrin IX (PpIX) is generated from ALA in tumors, allowing the detection of the tumors by PpIX fluorescence. However, it is well known that PpIX is bleached by light irradiation (photobleaching) resulting in reduced PDD efficiency. In this study, n-propyl gallate (NPG) was investigated as an enhancer of PDD efficiency. METHODS: Tumor cells were incubated with NPG after treatment with ALA, and reactive oxygen species and PpIX fluorescence were measured. RESULTS: The antioxidant NPG suppressed the production of reactive oxygen species from light-irradiated porphyrins and ameliorated photobleaching of PpIX generated from ALA in vitro and in vivo. CONCLUSION: Incubation with NPG decreased the production of reactive oxygen species from PpIX and suppressed PpIX photobleaching. These results indicate that the antioxidant NPG may significantly improve PDD efficiency.
Assuntos
Ácido Aminolevulínico , Aumento da Imagem/métodos , Microscopia de Fluorescência/métodos , Neoplasias Experimentais/patologia , Galato de Propila , Protoporfirinas , Linhagem Celular Tumoral , Combinação de Medicamentos , Sinergismo Farmacológico , Humanos , Fármacos Fotossensibilizantes , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: A detection method widely used of late in cancer surgery is 5-aminolevulinic acid-based photodynamic diagnosis (ALA-PDD), which relies on the tumor-specific accumulation of photosensitizing protoporphyrin IX (PpIX) after the administration of ALA. In this regard, we recently reported that peptide transporter PEPT1 and human ATP-binding cassette transporter ABCG2 are key players in regulating intracellular PpIX levels. In the present study, we re-evaluated in vivo the expression of genes involved in the porphyrin biosynthesis pathway. METHODS: Using quantitative real-time (qRT)-PCR, we measured the mRNA levels in a clinical specimen of bladder cancer from a patient who had been subjected to ALA-PDD. RESULTS: We confirmed that PEPT1 and ABCG2 are major contributors to the regulation of tumor-specific PpIX accumulation. qRT-PCR analysis revealed a predominantly high level of PEPT1 mRNA and a very low level of ABCG2 mRNA in the bladder cancer, corresponding to the roles of these genes in vitro. These findings were further confirmed by immunohistochemical studies with PEPT1- and ABCG2-specific antibodies. CONCLUSION: The induction of PEPT1 gene and the suppression of ABCG2 gene expression are among the key molecular mechanisms underlying tumor-specific PpIX accumulation after the administration of ALA in bladder cancer.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Ácido Aminolevulínico/administração & dosagem , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Protoporfirinas/farmacocinética , Simportadores/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Quimioterapia Combinada/métodos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Taxa de Depuração Metabólica/efeitos dos fármacos , Taxa de Depuração Metabólica/efeitos da radiação , Transportador 1 de Peptídeos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Células Tumorais CultivadasRESUMO
Photodynamic therapy (PDT) and photodynamic diagnosis of cancer are widely used in clinical fields. These are performed using photosensitizers. Many metalloporphyrin-related compounds have been developed as photosensitizers for use in PDT, and these tumor localization ability have been improved in recent research. Moreover, the precursor of porphyrin 5-aminolevulinic acid is used in fluorescence diagnosis using its tumor localization ability. In this review, these applications of photosensitizers in cancer therapy and diagnosis are summarized.
Assuntos
Neoplasias/terapia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/química , Humanos , Metaloporfirinas/uso terapêutico , Neoplasias/diagnóstico , Especificidade de ÓrgãosRESUMO
BACKGROUND: Recently, 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is being widely used in cancer therapy owing to the tumor-specific accumulation of photosensitizing protoporphyrin IX (PpIX) after the administration of ALA. In the present study, by focusing on genes involved in the porphyrin biosynthesis pathway, we aimed to explore biomarkers that are predictive for the efficacy of ALA-PDT. METHODS: We used five lines of human gastric cancer cells to measure the ALA-based photocytotoxicity. ALA-induced production of PpIX in cancer cells was quantified by fluorescence spectrophotometry. To examine the potential involvement of PEPT1 and ABCG2 in the ALA-PDT sensitivity, stable cell lines overexpressing PEPT1 were established and ABCG2-specific siRNA used. RESULTS: We observed that three cell lines were photosensitive, whereas the other two cell lines were resistant to ALA-based photocytotoxicity. The ALA-based photocytotoxicity was found to be well correlated with intracellular PpIX levels, which suggests that certain enzymes and/or transporters involved in ALA-induced PpIX production are critical determinants. We found that high expression of the peptide transporter PEPT1 (ALA influx transporter) and low expression of the ATP-binding cassette transporter ABCG2 (porphyrin efflux transporter) determined ALA-induced PpIX production and cellular photosensitivity in vitro. CONCLUSION: PEPT1 and ABCG2 are key players in regulating intracellular PpIX levels and determining the efficacy of ALA-based photocytotoxicity against gastric cancer cells in vitro. Evaluation of the expression levels of PEPT1 and ABCG2 genes could be useful to predict the efficacy of ALA-PDT. Primers specific to those target genes are practical and useful biomarkers for predicting the photo-sensitivity to ALA-PDT.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Ácido Aminolevulínico/administração & dosagem , Proteínas de Neoplasias/metabolismo , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Simportadores/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Transportador 1 de Peptídeos , Neoplasias GástricasRESUMO
BACKGROUND: Tumor markers are commonly used for cancer screening and as indicators of therapeutic effects. Certain types of tumor have been known to produce a variety of porphyrins after 5-aminolevulinic acid (ALA) administration. In this study, porphyrins in tumor-bearing mouse urine were analyzed after oral administration of ALA in order to identify new tumor markers excreted in the urine. METHODS: Porphyrin concentrations in the urine of tumor-bearing mice were measured after administration of 1.0mg of ALA (approximately 50mgkg(-1)). RESULTS: Porphyrin concentrations in the urine of tumor-bearing mice increased after administration of ALA. HPLC analysis of the urine revealed the existence of uroporphyrin (UP) and coproporphyrin (CP) in the urine of ALA-treated tumor-bearing mice. Furthermore, at 3h after ALA administration, UP concentrations in the urine of tumor-bearing mice significantly increased compared to those in the urine of normal mice. CONCLUSION: These results suggest that UP as a precursor of heme detected in the urine of tumor-bearing mice after ALA administration is a potential marker of tumor development.
Assuntos
Ácido Aminolevulínico/administração & dosagem , Biomarcadores Tumorais/urina , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/urina , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Porfirinas/urina , Animais , Carcinoma Hepatocelular/diagnóstico , Linhagem Celular Tumoral , Camundongos , Camundongos Endogâmicos C3H , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Resultado do TratamentoRESUMO
BACKGROUND: 5-Aminolevulinic acid (ALA) is a precursor of heme that is fundamentally important in aerobic energy metabolism. Among the enzymes involved in aerobic energy metabolism, cytochrome c oxidase (COX) is crucial. In this study, the effect of ALA on cytochrome c oxidase activity was measured. FINDINGS: c57BL/6N species of mice were administered ALA orally for 15 weeks. After ALA administration, mice were sacrificed and livers were obtained. COX activity in mitochondria from ALA-administered mouse livers was 1.5-fold higher than that in mitochondria from PBS-administered mouse livers (P < 0.05). Furthermore, ATP levels in ALA-administered mouse livers were much higher than those in PBS-administered mouse livers. These data suggest that oral administration of ALA promotes aerobic energy metabolism, especially COX activity. CONCLUSIONS: This is the first report of a drug that functions in aerobic energy metabolism directly. Since COX activity is decreased in various diseases and aging, the pharmacological effects of ALA will be expanding.
RESUMO
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.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Proteínas de Neoplasias/fisiologia , Polimorfismo de Nucleotídeo Único/genética , Porfirinas/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/farmacologia , Alelos , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Clorofila/análogos & derivados , Clorofila/farmacologia , Daunorrubicina/farmacologia , Doxorrubicina/farmacologia , Resistência a Medicamentos/genética , Hematoporfirinas/metabolismo , Humanos , Irinotecano , Cinética , Metotrexato/metabolismo , Mitoxantrona/farmacologia , Proteínas Mutantes/genética , Proteínas Mutantes/fisiologia , Mutação/genética , Proteínas de Neoplasias/genética , Spodoptera , TransfecçãoRESUMO
Heme (Fe-protoporphyrin IX), an endogenous porphyrin derivative, is an essential molecule in living aerobic organisms and plays a role in a variety of physiological processes such as oxygen transport, respiration, and signal transduction. For the biosynthesis of heme or the mitochondrial heme proteins, heme or its biosynthetic precursor porphyrin must be transported into mitochondria from cytosol. The mechanism of porphyrin accumulation in the mitochondrial inner membrane is unclear. In the present study, we analyzed the mechanism of mitochondrial translocation of porphyrin derivatives. We showed that palladium meso-tetra(4-carboxyphenyl)porphyrin (PdTCPP), a phosphorescent porphyrin derivative, accumulated in the mitochondria of several cell lines. Using affinity latex beads, we showed that 2-oxoglutarate carrier (OGC), the mitochondrial transporter of 2-oxoglutarate, bound to PdTCPP, and in vitro PdTCPP inhibited 2-oxoglutarate uptake into mitochondria in a competitive manner (Ki = 15 microM). Interestingly, all types of porphyrin derivatives examined in this study competitively inhibited 2-oxoglutarate uptake into mitochondria, including protoporphyrin IX, coproporphyrin III, and hemin. Furthermore, mitochondrial accumulation of porphyrins was inhibited by 2-oxoglutarate or OGC inhibitor. These results suggested that porphyrin accumulation in mitochondria is mediated by OGC and that porphyrins are able to competitively inhibit 2-oxoglutarate uptake into mitochondria. This is the first report of a putative mechanism for accumulation of porphyrins in the mitochondrial inner membrane.
Assuntos
Ácidos Cetoglutáricos/metabolismo , Mitocôndrias/metabolismo , Porfirinas/metabolismo , Sequência de Aminoácidos , Animais , Células HeLa , Heme/química , Hemina/química , Humanos , Ácidos Cetoglutáricos/química , Dados de Sequência Molecular , Porfirinas/química , Ratos , Ratos Wistar , Transdução de Sinais , Succinatos/químicaRESUMO
Cytochrome c(3) from Desulfovibrio vulgaris has four hemes per molecule, and a redox change at the hemes alters the conformation of the protein, leading to a redox-dependent change in the interaction of cytochrome c(3) with redox partners (an electron acceptor or an electron donor). The redox-dependent change in this interaction was directly monitored by the high-performance electrochemical quartz crystal microbalance (EQCM) technique that has been improved to give high sensitivity in solution. In this method, cytochrome c(3) molecules in solution associate electrostatically with a viologen-immobilized quartz crystal electrode as a monolayer, and redox of the associating cytochrome c(3) is controlled by the immobilized viologen. This technique makes it possible to measure the access of cytochrome c(3) to the electrode or repulsion from the electrode, and hence interconversion between an electrostatic complex and an electron transfer complex on the cytochrome c(3) and the viologen as a mass change accompanying a potential sweep is monitored. In addition, simultaneous measurement of a mass change and a potential step reveals that the cytochrome c(3) stores electrons when the four hemes are reduced (an electron pool effect), that is, the oxidized cytochrome c(3) facilitates acceptance of electrons from the immobilized viologen molecule, but the reduced cytochrome c(3) donates the accepted electrons to the viologen with difficulty.