The three drugs showed characteristic response patterns in HeLa cells. Etoposide induced apoptosis at high concentrations, but the effect was relatively mild, whereas staurosporine was a potent drug that rapidly induced apoptosis even at low concentrations (Fig. 5 (M, N, Bottom row)). Antimycin A caused the most significant mitochondrial dysfunction (Fig. 5 (O, Middle row)).Etoposide (100 µM) induced apoptosis, with Annexin V fluorescence intensity gradually increasing 12 hours after treatment (Fig. 5(M)). Staurosporine (10 µM) immediately caused the cells to shrink, and morphological changes were already observed at the start of imaging (T=0) (Fig. 5(C, Upper row)). Antimycin A (10 µM) caused an immediate decrease in MitoMP fluorescence intensity, with a reduction in mitochondrial membrane potential already observed at the start of imaging (Fig. 5(F, Upper row)). Furthermore, a loss of mitochondrial membrane potential (ΔΨm) was confirmed within 2 hours of treatment with Antimycin A (10 µM) (Fig. 5(O, Middle row)). Typically, mitochondrial dysfunction causes cell death or growth inhibition, but in this short-term observation (18 hours), the cell count increased by approximately 1.3 times. This is thought to be due to a metabolic shift involving the Warburg effect.

• Decrease in mitochondrial membrane potential: Antimycin A is a drug that inhibits mitochondrial electron transport chain complex III. By disrupting proton gradient formation, it rapidly reduces the mitochondrial membrane potential (ΔΨm). This is thought to stop ATP production via oxidative phosphorylation.
• Energy metabolism shift to glycolysis: HeLa cells, which are cancer cells, produce energy (ATP) primarily through glycolysis, independent of oxidative phosphorylation. This phenomenon is known as the “Warburg effect.”

Therefore, in this experiment, although Antimycin A treatment reduced the mitochondrial membrane potential, HeLa cells were able to continue supplying the energy required for cell proliferation over the short period of 18 hours by enhancing glycolysis. However, long-term Antimycin A treatment ultimately induces cell death (apoptosis or necrosis) and reduces cell numbers, as reported in numerous studies.

This experiment focused on early apoptosis, and data were acquired through 18-hour time-lapse imaging. Even during this 18-hour imaging period, the fluorescent probe MitoMP (MT-1) showed minimal fluorescence intensity decrease of only 10% in the control wells, demonstrating very low photobleaching and yielding highly accurate results. When analyzing later stages of cell death, such as late apoptosis or necrosis, co-staining with a dead cell detection fluorescent probe (PI: propidium iodide) and extending the observation time enables analysis. Furthermore, combining these with ROS (reactive oxygen species) detection probes enables a more detailed elucidation of the mechanisms underlying cell death pathways. Advances in fluorescent probes and microscopy technology are making significant contributions to the further development of medicine, pharmacology, and biology.