Modifications of mitochondria in human tumor cells during anthracycline-induced apoptosis.

Adriamycin (ADR), one of the major antitumor agents used for the clinical treatment of a wide variety of human cancers and its glutathione(GSH)-conjugated adduct, ADRIGLU, induced apoptosis in K562 erythroleukemia and TVM-A12 clone 2 melanoma human cell lines. We have previously reported that ADR has nuclear localization and that ADRIGLU localizes exclusively in the cytoplasm. During ADR or ADRIGLU treatment, significant depletion of the cell energy state, demonstrated by a reduction in high-energy phosphates (ATP and GTP) and a decrease in energy charge potential (ECP), were recorded between 2 hours and 24 hours, by HPLC analysis. Transmission electron microscopy also revealed that between 2 hours and 24 hours of ADR or ADRIGLU treatment, mitochondria underwent evident morphological changes, from an initial "high amplitude swelling state" to a "shrinkage state" and finally, in early apoptotic cells, to an "abnormal shrinkage state", in which a marked accumulation of pycnotic mitochondria was also observed. Confocal microscopic analysis, using the potential-sensitive dye JC-1, showed that inhibition of cell energy metabolism was preceded by a rapid decrease in mitochondrial transmembrane potential (delta psi m). With the progression of exposure time, the early depolarization of the mitochondrial membrane was followed by a transient reversion to normal delta psi m until, in apoptotic cells, almost all mitochondrial subpopulations appeared to be hyperpolarized. Our results indicated that mitochondria are actively involved in anthracycline-induced programmed cell death, suggesting a novel mechanism that may be common to all forms of apoptosis.