RESUMO
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.
Assuntos
Clorofila/análogos & derivados , Lisossomos/efeitos dos fármacos , Radiossensibilizantes/toxicidade , Anticorpos Monoclonais , Carcinoma de Células de Transição , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Clorofila/toxicidade , Escuridão , Portadores de Fármacos , Humanos , Luz , Lipossomos , Células Tumorais Cultivadas , Neoplasias da Bexiga UrináriaRESUMO
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)
Assuntos
Clorofila/análogos & derivados , Fármacos Fotossensibilizantes/química , Porfirinas/química , Carcinoma/enzimologia , Clorofila/química , Clorofila/metabolismo , Clorofilídeos , Esterases/metabolismo , Esterificação , Ésteres/química , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/enzimologia , Solubilidade , Espectrometria de Fluorescência , Espectrofotometria , Neoplasias da Bexiga Urinária/enzimologiaRESUMO
We have isolated and characterized a cDNA clone containing DNA sequences coding for the noncollagenous carboxy-terminal domain of human pro alpha 2(IV) collagen. Using this cDNA clone in both Southern blot analysis of DNA isolated from human-mouse somatic-cell hybrids and in situ hybridization of normal human metaphase chromosomes, we have demonstrated that the gene coding for human pro alpha 2(IV) collagen is located at 13q33----34, in the same position on chromosome 13 as the pro alpha 1(IV) collagen gene.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 13 , Colágeno/genética , Pró-Colágeno/genética , Animais , Bandeamento Cromossômico , DNA/genética , Ligação Genética , Marcadores Genéticos , Humanos , Células Híbridas , Cariotipagem , Camundongos , Hibridização de Ácido NucleicoRESUMO
A model for focal bladder carcinoma in rats was developed with the use of an isograft technique. Bladder tumors developed by carcinogen induction with FANFT or MNU were grafted to bladders of syngeneic rats. Ninety-six per cent (56 our of 58) of the grafts were taken and 83 per cent (34 out of 41) of the grafted tumors remained neoplastic. Most of the grafts from FANFT-induced tumor remained localized at the original site whereas many from the MNU-induced tumor spread beyond the original graft area. The model system may be useful for the evaluation of chemotherapeutic agents as well as for the studies of basic mechanisms of tumor growth and spreading.
Assuntos
Carcinoma de Células de Transição/induzido quimicamente , FANFT , Metilnitrosoureia , Compostos de Nitrosoureia , Tiazóis , Neoplasias da Bexiga Urinária/induzido quimicamente , Animais , Carcinoma de Células de Transição/patologia , Transplante de Neoplasias , Neoplasias Experimentais/induzido quimicamente , Neoplasias Experimentais/patologia , Ratos , Ratos Endogâmicos , Transplante Isogênico , Neoplasias da Bexiga Urinária/patologiaRESUMO
Cultured cells of human transitional cell carcinoma line MGH-U1, in suspension, were assayed for galactosyl transferase by measurement of the transfer of [3H]galactose from uridine diphosphate:[3H]galactose to desialylated ovine submaxillary mucin. The assay was optimized with respect to time and to protein, uridine disphosphate:galactose, desialyated ovine submaxillary mucin, and Triton X-100 concentrations. This assay was then applied to fresh specimens of benign, inflamed, and neoplastic bladder epithelium from 33 patients who under went cold-cup biopsies at cytoscopy. Transitional cell carcinoma specimens gave values in the range of 24.7 to 184.8 cpm [3H]galactose transferred per microgram protein per hr [72.0 +/- 44.7 (S.D.); n = 25]; normal and inflamed specimens ranged from 0.8 to 46.1 cpm/microgram protein per hr [8.3 +/- 8.4 (S.D.); n = 35]. By using a known method of cell rupture, cell ghosts, representing cell-surface membranes, were isolated both from the cultured cell line and from two biopsy specimens of transitional cell carcinoma. Although a complete enzymatic and electron microscopic analysis was not undertaken, the coincidence of an enzyme marker with the cell ghost fraction containing the elevated galactosyl transferase made it appear probable that this enzyme is located in the cell surface.