The
camptothecin (
CPT) derivative
hydroxycamptothecin (
HCPT) containing 10-hydroxy represents one of the most potent
topoisomerase I inhibitors described. This
anticancer agent, currently undergoing clinical trials on gastric tumours, has been shown more active and less toxic than conventional camptothecins. To shed light on the mechanism of action of
HCPT at the cellular level, we examined cell growth, apoptosis, changes of mitochondrial membrane potential,
cytochrome c and AIF translocation in
cancer cells by exposing these cells to
HCPT for indicated time. The effect of
HCPT on cell proliferation was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl tetrazolium bromid) assay and apoptosis was measured using flow cytometry, fluorescence microscopy and electron microscopy. Changes of mitochondrial membrane potential were monitored by fluorescence microscope. Western blot analysis was used to evaluate the release of mitochondrial
cytochrome c and AIF; On the other hand, translocation of
cytochrome c and AIF from mitochondria to cytosol during apoptosis were confirmed by confocal microscopy.
HCPT could noticeably inhibit the proliferation of SMMC-7721cells and the IC(50) dose was about 0.22 microM; SMMC-7721 cells treated with
HCPT showed typical characteristics of apoptosis rather than necrotic including
phosphatidylserine (PS) exposed from the inner to the outer leaflet of the plasma membrane, abnormal cell morphology,
chromatin condensation and nuclear fragmentation; On the other hand, during process of cell apoptosis, mitochondrial transmembrane potential was reduced; Compared with the control group, the
mRNA and
protein expression of
cytochrome c and AIF in treated and untreated SMMC-7721 cells were not significantly changed (not shown). However, when cells were treated with
HCPT, the massive translocation of
cytochrome c and AIF to the nucleus was evident. Our results indicate that
HCPT can inhibit proliferation and induce apoptosis of human
hepatoma SMMC-7721 cells. Mitochondrial pathway of apoptosis, especially for
cytochrome c and AIF translocation, may play an important role in apoptosis induced by
HCPT.