engleski

Differential antiproliferative activity of adenovirally expressed Apoptin in vitro

Apoptin is a protein from chicken anemia virus that has the ability to kill human tumor cells. It enters the nucleus of tumor cells and redirects cell signaling toward apoptosis. Apoptin accumulates in the cytoplasm of normal cells, where it will be subsequently degraded by proteasomal activity. It has been found that Apoptin senses early stages of malignant transformation, such as alterations induced by the expression of the Simian virus 40 small t antigen. It is not clear what follows these early changes in cells that would be recognized by the Apoptin. In the study presented here, cytotoxic properties of Apoptin toward various tumor cell lines of different origin, and having various genetic backgrounds, were compared. An adenoviral vector that expresses Flag-tagged Apoptin gene and a control vector that expresses lacZ gene were used to transfect cells. Antiproliferative effect of Apoptin was measured using MTT and Trypan blue exclusion. The different modes of programmed cell death, apoptosis and autophagy, were evaluated using Annexin assay and Acridine Orange staining, respectively. The efficiency of transfection as well as localization of Apoptin within the cell was substantiated using immunofluorescence. Transformed primary mammary epithelial cells (HMLE), and mammary epithelial cells having stem cell properties (HMLE-shEcad), were introduced into the study because of their unique genetic background. Although Apoptin localised in the nucleus of these cells, it was not sufficient to induce apoptosis. Apoptin showed distinct levels of cytotoxicity towards various tumor cell lines, identifying NCI-H1299 (non-small cell lung cancer) as the most sensitive, and HCT 116 (colorectal carcinoma) as non-sensitive. In NCI-H1299 cells, Apoptin induced both apoptosis and autophagy, and localised primarily in the nucleus. As expected according to the literature, primary fibroblasts were resistant to Apoptin-specific killing, where Apoptin localised mainly in the cytoplasm. Our study confirmed Apoptin as a potent tumor-cell killer, although it didn’t affect cancer stem cells. The adenoviral system was the most efficient tool to transfect various cell lines, allowing us to compare its effects. We also showed for the first time that Apoptin affects autophagy, which will be further analysed and evaluated in more details.

Apoptin; adenoviral vectors; apoptosis

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