Illuminating Superoxide Anion and pH Enhancements in Apoptosis of Breast Cancer Cells Induced by Mitochondrial Hyperfusion Using a New Two-Photon Fluorescence Probe

Mitochondrial morphology regulated by fusion and fission processes determines mitochondrial function and cell fate. Some studies showed hyperfused mitochondria could induce apoptosis in cancer cells, but the relevant molecular mechanisms remain elusive. Superoxide (O-2(center dot-)) and pH play vital roles in mitochondrial dysfunction and apoptosis. Therefore, it is worthwhile to explore if there is an intimate relationship between mitochondrial hyperfusion and simultaneous changes in O-2(center dot-) and pH levels, which will be helpful to uncover relevant detailed mechanism. For this purpose, we have developed a new reversible two-photon fluorescent probe (CFT) to simultaneously monitor O-2(center dot-) and pH in 4T1 cells and mice using dual-color imaging. With the assistance of probe, we found that inhibition of Dynamin-related protein 1 (Drp1) could transduce a signal through mitochondrial complexes I and II to enhance the O-2(center dot-) and pH levels and eventually induced mitohyperfusion and apoptosis in breast cancer cells. Together, these data indicate that CFT provides a robust tool for unveiling the roles of O-2(center dot-) and pH in signals associated with mitochondrial dysfunction in cells and in vivo.