Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, SI-1000 Ljubljana, Slovenia.
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, SI-1000 Ljubljana, Slovenia.
Int J Mol Sci. 2021 Mar 24;22(7):3305. doi: 10.3390/ijms22073305.
Identification of novel agents for bladder cancer treatment is highly desirable due to the high incidence of tumor recurrence and the risk of progression to muscle-invasive disease. The key feature of the cholesterol-dependent toxin listeriolysin O mutant (LLO Y406A) is its preferential activity at pH 5.7, which could be exploited either directly for selective targeting of cancer cells or the release of accumulated therapeutics from acidic endosomes. Therefore, our goal was to compare the cytotoxic effect of LLO Y406A on cancer cells (RT4) and normal urothelial cells (NPU), and to identify which cell membranes are the primary target of LLO Y406A by viability assays, life-cell imaging, fluorescence, and electron microscopy. LLO Y406A decreased viability, altered cell morphology, provoked membrane blebbing, and induced apoptosis in RT4 cells, while it did not affect NPU cells. LLO Y406A did not cause endosomal escape in RT4 cells, while the plasma membrane of RT4 cells was revealed as the primary target of LLO Y406A. It has been concluded that LLO Y406A has the ability to selectively eliminate cancer urothelial cells through pore-forming activity at the plasma membrane, without cytotoxic effects on normal urothelial cells. This promising selective activity merits further testing as an anti-cancer agent.
由于肿瘤复发率高且有进展为肌层浸润性疾病的风险，因此非常需要寻找新型膀胱癌治疗药物。胆固醇依赖性细胞毒素李斯特菌溶血素 O 突变体（LLO Y406A）的主要特点是在 pH5.7 时具有优先活性，这一特性可直接用于选择性靶向癌细胞，或使蓄积的治疗药物从酸性内涵体中释放。因此，我们的目标是比较 LLO Y406A 对癌细胞（RT4）和正常尿路上皮细胞（NPU）的细胞毒性作用，并通过细胞活力测定、活细胞成像、荧光和电子显微镜来确定 LLO Y406A 的主要靶细胞膜。LLO Y406A 降低了 RT4 细胞的活力，改变了细胞形态，引起细胞膜起泡，并诱导细胞凋亡，而对 NPU 细胞没有影响。LLO Y406A 未引起 RT4 细胞的内涵体逃逸，而 RT4 细胞的质膜则被揭示为 LLO Y406A 的主要靶标。研究结论为，LLO Y406A 能够通过质膜的成孔活性选择性地消除膀胱癌上皮细胞，而对正常尿路上皮细胞没有细胞毒性作用。这种有前景的选择性活性值得进一步作为抗癌药物进行测试。
