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When used in combination with certain chemotherapies, curcumin has been shown to increase apoptosis in several cancer cell lines. Here, we report the combined effects of curcumin and cinobufacini on human cervical carcinoma cells. The aim of this study was to examine whether curcumin could enhance apoptosis induced by cinobufacini. 3-(4,5-Dimethylthiazol-2-y1)-2,5- diphenytetrazolium bromide (MTT) assays revealed that the growth and proliferation of HeLa cells could be inhibited by 75% after a combined treatment of 25 μg/mL cinobufacini and 8 μg/mL curcumin. The combined treatment is 3 times more effective than treatment with 25 μg/mL cinobufacini alone. Annexin V-FITC/PI staining, morphological changes and immunofluorescence verified a significant enhancement in cinobufacini-induced apoptosis when cells were also exposed to curcumin. The data showed that the proportion of early apoptotic cells significantly increased from 15.43% in cells treated only with 25 μg/mL cinobufacini to 49.2% in cells treated with 25 μg/mL cinobufacini and 8 μg/mL curcumin. Moreover, compared with treatment of only 25 μg/mL cinobufacini, ROS production increased 1.7-fold, the intracellular free Ca 2+ concentration increased 1.5-fold, and the mitochon- drial membrane potential decreased by 20% in the combined treatment. Simultaneously, the atomic force microscopy (AFM) re- sults suggest that cells treated with a combination of cinobufacini and curcumin varied significantly in shape and ultrastructure. Collapsed cells with leaking cytoplasm, blebbing pores and emerging apoptotic bodies were prevalent. The nanoparticle size increased from 70 nm when the cells were treated with 25 μg/mL cinobufacini to 190 nm when the cells were treated with 25 μg/mL cinobufacini and 8 μg/mL curcumin. The size increase resulted in the cell membrane becoming considerably rough. These results can improve our understanding of combination treatments. Specifically, the combination of cinobufacini and curcumin may potentially find use as a novel cervical carcinoma treatment. Additionally, AFM is a powerful tool that can be used to explore cellular morphologies and ultrastructures.

Cinobufacini-induced HeLa cell apoptosis enhanced by curcumin