Fujii Tomohiko, Kamiya Mako, Urano Yasuteru
Graduate School of Medicine and ‡Graduate School of Pharmaceutical Sciences, The University of Tokyo , 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Bioconjug Chem. 2014 Oct 15;25(10):1838-46. doi: 10.1021/bc5003289. Epub 2014 Sep 19.
It is difficult to completely remove carcinomas in unguided ablative surgery because they cannot be distinguished with the unaided human eye. Therefore, in order to precisely visualize tiny tumors and the borders between cancerous lesions and normal tissues, we have been developing fluorescence probes activatable only in cancer cells. We previously reported the hydroxymethylrhodamine green (HMRG)-based fluorescence probe gGlu-HMRG for γ-glutamyltransferase (GGT), which is overexpressed in a variety of cancer cells, and we showed that it enables in vivo rapid detection of human ovarian cancer SHIN-3 nodules with a high tumor-to-background (T/B) fluorescence ratio in model mice. However, cancer cell lines with low GGT expression could hardly be detected with gGlu-HMRG. Here we developed two new HMRG-based fluorescence probes for the cathepsin family of cysteine proteases, including cathepsin B (CatB) and cathepsin L (CatL), which show increased expression and/or activity, secretion, and altered localization in many kinds of cancer cells. The developed probes, Z-Phe-Arg-HMRG and Z-Arg-Arg-HMRG, are colorless and nonfluorescent at the physiological pH of 7.4, but are hydrolyzed to HMRG upon reaction with purified cathepsins, resulting in a more than 200-fold fluorescence increase. These probes could visualize human ovarian cancer cell lines SHIN-3, SK-OV-3, and OVCAR-3, of which the latter two were hardly detectable with gGlu-HMRG. Z-Phe-Arg-HMRG showed higher applicability than Z-Arg-Arg-HMRG for in vivo imaging, and we confirmed that 0.5-mm-sized SK-OV-3 tumor nodules disseminated on the mesentery in a mouse model could be rapidly visualized by Z-Phe-Arg-HMRG, with a T/B fluorescence ratio of 4.2. Further, intraperitoneally disseminated tumor could be visualized in real time in vivo by fluorescence endoscopy after spraying Z-Phe-Arg-HMRG, with a T/B ratio of 3. In conclusion, our HMRG-based activatable probes targeted to cathepsins have expanded the detectable range of cancers, and appear to be suitable for clinical application.
在非引导性消融手术中，很难完全切除癌组织，因为肉眼无法将其区分。因此，为了精确可视化微小肿瘤以及癌性病变与正常组织之间的边界，我们一直在研发仅在癌细胞中可激活的荧光探针。我们之前报道了基于羟甲基罗丹明绿（HMRG）的γ-谷氨酰转移酶（GGT）荧光探针gGlu-HMRG，GGT在多种癌细胞中过表达，并且我们表明它能够在模型小鼠体内以高肿瘤与背景（T/B）荧光比快速检测人卵巢癌SHIN-3结节。然而，gGlu-HMRG几乎无法检测到GGT表达低的癌细胞系。在此，我们研发了两种基于HMRG的新型荧光探针，用于半胱氨酸蛋白酶组织蛋白酶家族，包括组织蛋白酶B（CatB）和组织蛋白酶L（CatL），它们在多种癌细胞中表达和/或活性增加、分泌增加且定位改变。所研发的探针Z-Phe-Arg-HMRG和Z-Arg-Arg-HMRG在生理pH值7.4时无色且无荧光，但与纯化的组织蛋白酶反应后会水解为HMRG，导致荧光增加超过200倍。这些探针能够可视化人卵巢癌细胞系SHIN-3、SK-OV-3和OVCAR-3，其中后两种细胞系用gGlu-HMRG几乎无法检测到。Z-Phe-Arg-HMRG在体内成像方面比Z-Arg-Arg-HMRG具有更高的适用性，并且我们证实，在小鼠模型中，Z-Phe-Arg-HMRG能够快速可视化肠系膜上0.5毫米大小的SK-OV-3肿瘤结节，T/B荧光比为4.2。此外，在喷洒Z-Phe-Arg-HMRG后，通过荧光内镜可在体内实时可视化腹膜内播散的肿瘤，T/B比为3。总之，我们基于HMRG的靶向组织蛋白酶的可激活探针扩大了癌症的可检测范围，似乎适用于临床应用。
