Abstract A115: Delivery of tumor-killing protein to cancer cells.
Authors: Zhao Muxun , Hu Biliang , Wang Pin , Tang Yi 
Published: June 2012

Virtually all human cancer cells have elaborate anti-apoptotic strategies to overcome apoptosis, including inactivation of the p53 pathway and hyperactivation of NF-κB. Therefore, the ability to rapidly resurrect apoptosis in tumor cells can be an effective option for cancer treatment. Although different strategies have been developed to counter the individual inactivation mechanisms, a more potent chemotherapy option is to directly arm the cancer cells with apoptosis-inducing proteins. From a therapeutic perspective, protein-based approaches are safer than gene therapy because no random or permanent genetic changes are involved, and only transient actions of proteins are needed for the desired results. However, compared to small molecule therapeutics, proteins suffer from serum instability and are inefficient or unable to penetrate the cell membrane. As a result, protein-based anti-tumor treatment remains elusive, despite its numerous therapeutic targets and vast potential. We have developed a simple and effective method to deliver proteins intracellularly. Using this method, Apoptin, a selective killer of various tumor cells, has been delivered to cancer cells both in vitro and in vivo.

Apoptin is a small protein isolated from chicken anemia virus that induces p53-independent apoptosis in a tumor-specific way. In normal cells, Apoptin stays in the cytoplasm, whereas in various tumor cell lines, it enters the nucleus and induces apoptosis. Due to its high selectivity and potency, Apoptin has become an attractive anti-tumor target for gene therapy.

To deliver Apoptin protein directly, we encapsulated recombinant maltose-binding-protein and Apoptin fusion protein (MBP-APO) in a degradable polymeric nanocapsule (NC). The NC was synthesized by first electrostatically depositing positively charged monomers on to the surface of the MBP-APO protein. Followed by the addition of a redox-sensitive crosslinker, radical polymerization was initiated to form a thin, positively charged polymeric layer around the protein. The particles were found to be positively charged and uniform in size. When incubated with tumor cell lines (MCF-7, HeLa and MDA-MB-231), the NC readily penetrated the cell membranes, disintegrated to release the MBP-APO and induced apoptosis. In contrast, no apoptotic effect was found on noncancerous cell lines such as HFF, highlighting the differential targeting on tumor cells only. Confocal microscopy of Rhodamine labeled MBP-APO NC showed that the protein entered the nuclei of cancer cells only, while remained in the cytosol of HFF. When injected intratumorally into mice with xenografted MCF-7 tumor, the NC inhibited the progression of tumor growth significantly, demonstrating the potential utility of this polymer-protein complex as a cancer treatment.

In conclusion, pro-apoptosis proteins, such as Apoptin, can be delivered into cancer cells in their active, functional forms using degradable protein NC. Our method is promising for the preparation and administration of other protein drugs for cancer treatment. For future studies, NC decorated with tumor-targeting moieties, as well as those containing tumor specific pro-apoptotic proteins, will be synthesized and delivered to achieve selective tumor killing.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A115.