Background and Aims: Accurate endoscopic detection of premalignant lesions and earlycancers in the colon is essential for cure, since prognosis is closely related to lesion size andstage. Although it has great clinica...Background and Aims: Accurate endoscopic detection of premalignant lesions and earlycancers in the colon is essential for cure, since prognosis is closely related to lesion size andstage. Although it has great clinical potential, autofluorescence endoscopy has limited tumorto-normal tissue image contrast for detecting small preneoplastic lesions. We have developed amolecularly specific, near-infrared fluorescent monoclonal antibody (CC49) bioconjugate whichtargets tumor-associated glycoprotein 72 (TAG72), as a contrast agent to improve fluorescencebased endoscopy of colon cancer. Methods: The fluorescent anti-TAG72 conjugate was evaluated in vitro and in vivo in athymic nude mice bearing human colon adenocarcinoma (LS174T)subcutaneous tumors. Autofluorescence, a fluorescent but irrelevant antibody and the free fluorescent dye served as controls. Fluorescent agents were injected intravenously, and in vivowhole body fluorescence imaging was performed at various time points to determine pharmacokinetics, followed by ex vivo tissue analysis by confocal fluorescence microscopy and histology Results: Fluorescence microscopy and histology confirmed specific LS174T cell membrane targeting of labeled CC49 in vitro and ex vivo. In vivo fluorescence imaging demonstrated significant tumor-to-normal tissue contrast enhancement with labeled-CC49 at three hours postinjection, with maximum contrast after 48 h. Accumulation of tumor fluorescence demonstratedthat modification of CC49 antibodies did not alter their specific tumor-localizing properties, andwas antibody-dependent since controls did not produce detectable tumor fluorescence. Conclusions: These results show proof-of-principle that our near-infrared fluorescent-antibody probetargeting a tumor-associated mucin detects colonic tumors at the molecular level in real time,and offer a basis for future improvement of image contrast during clinical fluorescence endoscopy.展开更多
The phototoxicity in cancer cells of three series of octahedral Ir(III)complexes of the general formula[Ir(C^N)2(N^N)][PF6],where the N^N ligand is dipyrido[3,2-a:2′,3′-c]phenazine(dppz)and the C^N ligands are depro...The phototoxicity in cancer cells of three series of octahedral Ir(III)complexes of the general formula[Ir(C^N)2(N^N)][PF6],where the N^N ligand is dipyrido[3,2-a:2′,3′-c]phenazine(dppz)and the C^N ligands are deprotonated 2-phenyl-,2-(naphthalen-2-yl)-or 2-(thiophen-2-yl)-benzimidazole derivatives(series 1,2 and 3,respectively)with different substituents(H,methyl or 4-(trifluoromethyl)benzyl)on the imidazole units,has been studied.The 1-methyl benzimidazole compounds 2b and 3b were found to be the most photoactive in model human cervical cancer HeLa cells(IC50 about 20 nM under irradiation conditions,λexc=420 nm),inducing a substantial formation of apoptotic bodies as shown by flow cytometry,whereas the 4-(trifluoromethyl)benzyl derivatives 1c–3c showed the highest phototoxic indexes of the three series.Notably,the antiproliferative activity of the photoactivated Ir(Ⅲ)compounds was also significant under hypoxic conditions(2%O_(2)).Fluorescence confocal microscopy investigation for 1c,2c and 3c was undertaken,showing a significant subcellular localization of 3c in mitochondria,whereas 1c and 2c were located preferentially in the endoplasmic reticulum(ER).Further investigations on series 3 compounds using CellRox®(oxidative stress deep red reagent)have shown,after pretreatment with Tiron,that molecular superoxide radical(O_(2)^(·−))generation is mainly responsible for the oxidative stress induced by irradiation of the cells treated with the 2-(thiophen-2-yl)benzimidazole derivatives 3a–c.The photopotentiation of 3a and 3c also involves the formation of singlet oxygen(1O_(2)),but this does not occur with 3b,due to pretreatment with sodium azide.The ROS generation was also studied by standard droethidium(DHE)and singlet oxygen sensor green(SOSG)staining assays under both normoxic and hypoxic conditions.The results showed that in tumor HeLa cells,the generation of superoxide radicals competed with singlet oxygen production in cells in normoxia,but became dominant under hypoxia.展开更多
文摘Background and Aims: Accurate endoscopic detection of premalignant lesions and earlycancers in the colon is essential for cure, since prognosis is closely related to lesion size andstage. Although it has great clinical potential, autofluorescence endoscopy has limited tumorto-normal tissue image contrast for detecting small preneoplastic lesions. We have developed amolecularly specific, near-infrared fluorescent monoclonal antibody (CC49) bioconjugate whichtargets tumor-associated glycoprotein 72 (TAG72), as a contrast agent to improve fluorescencebased endoscopy of colon cancer. Methods: The fluorescent anti-TAG72 conjugate was evaluated in vitro and in vivo in athymic nude mice bearing human colon adenocarcinoma (LS174T)subcutaneous tumors. Autofluorescence, a fluorescent but irrelevant antibody and the free fluorescent dye served as controls. Fluorescent agents were injected intravenously, and in vivowhole body fluorescence imaging was performed at various time points to determine pharmacokinetics, followed by ex vivo tissue analysis by confocal fluorescence microscopy and histology Results: Fluorescence microscopy and histology confirmed specific LS174T cell membrane targeting of labeled CC49 in vitro and ex vivo. In vivo fluorescence imaging demonstrated significant tumor-to-normal tissue contrast enhancement with labeled-CC49 at three hours postinjection, with maximum contrast after 48 h. Accumulation of tumor fluorescence demonstratedthat modification of CC49 antibodies did not alter their specific tumor-localizing properties, andwas antibody-dependent since controls did not produce detectable tumor fluorescence. Conclusions: These results show proof-of-principle that our near-infrared fluorescent-antibody probetargeting a tumor-associated mucin detects colonic tumors at the molecular level in real time,and offer a basis for future improvement of image contrast during clinical fluorescence endoscopy.
基金supported by the Czech Science Foundation(Grant 17–05302S)Ministry of Education of the Czech Republic(Grant LTC17003)+1 种基金supported by the Spanish Ministry of Economy and Competitiveness and FEDER funds(Project CTQ2015-64319-R and RTI2018-096891-B-I00)the University of Murcia for a grant(R-1034/2016).
文摘The phototoxicity in cancer cells of three series of octahedral Ir(III)complexes of the general formula[Ir(C^N)2(N^N)][PF6],where the N^N ligand is dipyrido[3,2-a:2′,3′-c]phenazine(dppz)and the C^N ligands are deprotonated 2-phenyl-,2-(naphthalen-2-yl)-or 2-(thiophen-2-yl)-benzimidazole derivatives(series 1,2 and 3,respectively)with different substituents(H,methyl or 4-(trifluoromethyl)benzyl)on the imidazole units,has been studied.The 1-methyl benzimidazole compounds 2b and 3b were found to be the most photoactive in model human cervical cancer HeLa cells(IC50 about 20 nM under irradiation conditions,λexc=420 nm),inducing a substantial formation of apoptotic bodies as shown by flow cytometry,whereas the 4-(trifluoromethyl)benzyl derivatives 1c–3c showed the highest phototoxic indexes of the three series.Notably,the antiproliferative activity of the photoactivated Ir(Ⅲ)compounds was also significant under hypoxic conditions(2%O_(2)).Fluorescence confocal microscopy investigation for 1c,2c and 3c was undertaken,showing a significant subcellular localization of 3c in mitochondria,whereas 1c and 2c were located preferentially in the endoplasmic reticulum(ER).Further investigations on series 3 compounds using CellRox®(oxidative stress deep red reagent)have shown,after pretreatment with Tiron,that molecular superoxide radical(O_(2)^(·−))generation is mainly responsible for the oxidative stress induced by irradiation of the cells treated with the 2-(thiophen-2-yl)benzimidazole derivatives 3a–c.The photopotentiation of 3a and 3c also involves the formation of singlet oxygen(1O_(2)),but this does not occur with 3b,due to pretreatment with sodium azide.The ROS generation was also studied by standard droethidium(DHE)and singlet oxygen sensor green(SOSG)staining assays under both normoxic and hypoxic conditions.The results showed that in tumor HeLa cells,the generation of superoxide radicals competed with singlet oxygen production in cells in normoxia,but became dominant under hypoxia.
