Morphological changes of ricin toxin-induced apoptosis in human cervical cancer cells

The morphological changes of ricin-induced apoptosis in a human cervical cancer cell line were studied. To shed light on the mechanism of action of ricin toxin (RT) at the cellular level, we examined cell growth, apoptosis, changes of mitochondrial membrane potential (MMP) and cytochrome C translocation in HeLa cells by exposing these cells to RT for indicated times. The effect of RT on cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), inner salt; MTS assay and apoptosis were measured using flow cytometry, fluorescence microscopy and electron microscopy. Changes in MMP were monitored using flow cytometry. Western blot analysis was used to evaluate the release of mitochondrial cytochrome C. RT noticeably inhibited the proliferation of HeLa cells, and the half maximal inhibitory concentration dose was about 100 ng/ml. HeLa cells treated with RT showed typical characteristics of apoptosis rather than necrosis, including phosphatidylserine exposed from the inner to the outer leaflet of the plasma membrane, abnormal cell morphology, chromatin condensation and nuclear fragmentation. In contrast, during the process of cellular apoptosis, the messenger RNA (mRNA) and protein expression of cytochrome C in treated and untreated Hela cells were not significantly changed (data not shown). However, when cells were treated with RT, the massive translocation of cytochrome C to the nucleus was evident. Our results indicate that RT-induced HeLa cell apoptosis, especially for cytochrome C translocation, may play an important role in apoptosis induced by RT.