Bostad Monica, Kausberg Marianne, Weyergang Anette, Olsen Cathrine Elisabeth, Berg Kristian, Høgset Anders, Selbo Pål Kristian
Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, and ‡Cancer Stem Cell Innovation Center (SFI-CAST), Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital , Ullernchausseen 70, 0310 Oslo, Norway.
Mol Pharm. 2014 Aug 4;11(8):2764-76. doi: 10.1021/mp500129t. Epub 2014 Jul 2.
We have used the site specific and light-depended drug delivery method photochemical internalization (PCI) to release an immunotoxin (IT), targeting the CD44 receptor, into the cytosol of target cells. The IT consisted of a pan CD44 mAb (clone IM7) bound to the ribosome inactivating protein (RIP) saporin by a biotin-streptavidin linker named IM7-saporin. PCI is based upon photosensitizing compounds localized in the membrane of endosomes and lysosomes causing membrane rupture upon illumination followed by release of the IT into the cytosol. In this in vitro study, we have used 7 different human cancer cell lines of various origins to investigate the cytotoxic effect of PCI-based targeting of the cancer stem cell (CSC) marker CD44. Epi-fluorescence microscopy shows both specific binding and uptake of the IM7-Alexa488, after 30 min and 18 h of incubation, and colocalization with the PCI-photosensitizer TPCS2a prior to light-triggered cytosolic release of the CD44-targeting IT. PCI of IM7-saporin resulted in efficient and specific cytotoxicity in CD44-expressing but not in CD44-negative cancer cells. A higher level of reactive oxygen species (ROS) was found in untreated and photodynamic therapy (PDT)-treated LNCaP (CD44(neg)) compared to that of DU145 (CD44(pos)) prostate cancer (PC) cells. This may explain the PDT-resistance observed in the DU145 cells. PCI-based targeting of CD44-expressing cancer cells gives very potent and specific cytotoxic effects and may represent a rational strategy for achieving site-selective elimination of CSCs in aggressive androgen-independent and treatment-resistant PC cells preventing cytotoxic effects on distant normal stem cells.
我们采用了位点特异性且依赖光的药物递送方法——光化学内化（PCI），将靶向CD44受体的免疫毒素（IT）释放到靶细胞的胞质溶胶中。该免疫毒素由一种泛CD44单克隆抗体（克隆IM7）组成，通过名为IM7 - 皂草素的生物素 - 链霉亲和素连接子与核糖体失活蛋白（RIP）皂草素结合。PCI基于定位在内体和溶酶体膜中的光敏化合物，光照后导致膜破裂，随后免疫毒素释放到胞质溶胶中。在这项体外研究中，我们使用了7种不同来源的人类癌细胞系，以研究基于PCI靶向癌症干细胞（CSC）标志物CD44的细胞毒性作用。落射荧光显微镜显示，孵育30分钟和18小时后，IM7 - Alexa488具有特异性结合和摄取，并在光触发靶向CD44的免疫毒素胞质释放之前，与PCI光敏剂TPCS2a共定位。IM7 - 皂草素的PCI在表达CD44的癌细胞中产生了高效且特异性的细胞毒性，而在CD44阴性的癌细胞中则没有。与DU145（CD44阳性）前列腺癌（PC）细胞相比，在未处理和光动力疗法（PDT）处理的LNCaP（CD44阴性）细胞中发现了更高水平的活性氧（ROS）。这可能解释了在DU145细胞中观察到的PDT抗性。基于PCI靶向表达CD44的癌细胞具有非常强大且特异性的细胞毒性作用，可能代表了一种合理的策略，用于在侵袭性雄激素非依赖性和治疗抗性PC细胞中实现对癌症干细胞的位点选择性消除，同时防止对远处正常干细胞产生细胞毒性作用。
