Abstract C83: The proliferation marker Ki-67 as novel molecular target in cancer therapy

Abstract

Abstract Introduction: For patients with advanced ovarian cancer there are no effective therapies. The need for alternative treatments with high specificity but with low systemic toxicity can be achieved by targeting key molecular markers associated with cancer cells. Here, we show an innovative proof-of-principle approach for efficient killing of proliferating ovarian cancer cells by inactivating a protein associated with cell proliferation namely, the nuclear Ki-67 protein (pKi-67), using nanotechnology-based photodynamic therapy (PDT). pKi-67 is highly expressed in all proliferating cells and antibodies against this protein are widely used as prognostic tools in tumor diagnosis. Materials and Methods: Anti pKi-67 antibodies were first conjugated to fluorescein isothiocyanate (FITC) and then encapsulated inside liposomes. The liposomes encapsulating antibodies were characterized by dynamic light scattering and transmission electron microscopy. We then evaluated the efficacy of Ki-67 targeting using two in vitro ovarian cancer models - monolayer cultures as well as 3D cultures established in our lab. Cell viability after irradiation with laserlight at 488 nm was assessed using MTT viability assay for monolayer cultures while a Live/Dead assay kit was used for analyzing 3D cultures. Results: After incubation of OVCAR-5 ovarian cancer cells with the liposomal constructs, we used confocal microscopy to image localization of the antibodies to the nucleoli of the cells. Irradiation of these cells with a 488 nm laser led to a significant loss of viability. The efficacy of this approach was further demonstrated in a 3D culture model of OVCAR-5 cells. Incubation of 3D cultures with the liposomal constructs followed by light irradiation led to destruction of their acinar structures over 72 h following treatment. Using two different anti pKi-67 antibodies, where one targets the “active” form of pKi-67 while the other binds to the “inactive” form of the protein; we show that cell killing is specific after inactivation of the “active” Ki-67 protein. Further, the specificity of this approach for pKi-67 positive cells is demonstrated in confluent human lung fibroblasts (MRC-5) where minimal cell death was observed. This is in agreement with the flow cytometry results which show that only small populations of confluent MRC-5 cells express pKi-67. Conclusions: Taken together, our findings suggest that pKi-67 is an attractive therapeutic target in cancer and this approach holds promise as an effective alternative cancer therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C83.