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Screening of various natural products in a search for novel inducers of apoptosis in human leukemia cells led us to identify the strong apoptosis-inducing activity in a fraction extracted with methanol from the roots of Sophora subprostrata Chun et T. Chen. We purified the compound that induced apoptosis in human leukemia cells and identified it as sophoranone. Sophoranone inhibited cell growth and induced apoptosis in various lines of cells from human solid tumors, with 50% inhibition of growth of human stomach cancer MKN7 cells at 1.2 +/- 0.3 microM. The growth-inhibitory and apoptosis-inducing activities of sophoranone for leukemia U937 cells were very much stronger than those of other flavonoids, such as daidzein, genistein and quercetin. At the early stages of treatment of U937 cells with sophoranone, reactive oxygen species were formed, mitochondrial permeability pores were opened and cytochrome c was released from mitochondria. Cytochrome c was also released upon treatment of isolated mitochondria with sophoranone. Inhibitors of complexes III and IV, but not complexes I and II, of the mitochondrial respiratory chain prevented the release of cytochrome c from isolated mitochondria by sophoranone, as well as the induction of apoptosis in U937 cells in response to sophoranone. Our results indicate that sophoranone might be a unique apoptosis-inducing anticancer agent that targets mitochondria.

The mechanism of the induction of apoptosis by arsenic trioxide (As2O3), which was demonstrated recently to be an effective inducer of apoptosis in patients with leukemia, was examined in detail in human leukemia U937 cells. Upon treatment of U937 cells with 50 microM of As2O3, complete inactivation of the kinases ERK1 and ERK2 was detected within 30 min. p38 was activated within 3 hr, and the maximum activity was detected at 6 hr, when DNA fragmentation remained undetectable. Experiments with transfected cells that expressed constitutively activated MEK1 and a specific inhibitor of p38 also suggested that inactivation of ERKs and activation of p38 might be associated with the induction of apoptosis by As2O3. In contrast to the inactivation of ERKs and the activation of p38, activation of JNK by As2O3 appeared to protect cells against the induction of apoptosis. Treatment of U937 cells with As2O3 also caused the Ca2+-dependent production of superoxide and intracellular acidification and a decrease in the mitochondrial membrane potential at the early stages of induction of apoptosis by As2O3. These changes preceded the release of cytochrome c from mitochondria and the activation of caspase-3. It should be possible to exploit the unusual characteristics of the mechanism of induction of apoptosis by As2O3 in U937 cells by making use of synergistic effects of this compound with other inducers of apoptosis.

In a previous study, we showed that geranylgeraniol (GGO) is a potent inducer of apoptosis in human leukemia cells, including HL60 promyelocytic leukemia cells. The present study describes the effects of activators of protein kinase C (PKC) on GGO-induced apoptosis in various lines of leukemia cells. Both 12-O-tetradecanoylphorbol-13-acetate (TPA) and diacylglycerol (DG) inhibited the GGO-induced morphological changes that are characteristic of apoptosis and the DNA fragmentation. Similar effects were observed with other lines of human and murine leukemia cells such as MLI, U937, MI and P388. Flow cytometric analysis also revealed that both TPA and DG prevented GGO-induced DNA degradation in a dose-dependent manner. These inhibitory effects of TPA and DG were antagonized by inhibitors of PKC such as H-7 and staurosporin, and by amiloride, an inhibitor of Na+/H+ antiporter. In contrast to the inhibitory effects of TPA and DG on GGO-induced apoptosis, 4α-TPA, which is unable to activate PKC, failed to prevent GGO-induced DNA fragmentation. However, the selective activator of PKC-β, 12-deoxyphorbol 13-phenylacetate 20-acetate, significantly inhibited GGO-induced DNA fragmentation. Our results suggest that PKC, and in particular the PKC-β isoenzyme, might be involved in the process of GGO-induced apoptosis. Int. J. Cancer 71:691-697, 1997. © 1997 Wiley-Liss, Inc.