Induction of apoptosis through caspase-independent or caspase-9-dependent pathway in mouse and human osteosarcoma cells by a new nitroxyl spin-labeled derivative of podophyllotoxin

Previous study has found that a new nitroxyl spin-labeled derivative of podophyllotoxin, 4-4″-(2″,2″,6″,6″-tetramethyl-1″-piperidinyloxy)amino]-4′-demethyl-epipodophyllotoxin (GP7), can induce apoptosis in human leukemia cells. However, there have been no studies about the effects of GP7 on osteosarcoma (OS) cells. Here, we observed the anti-OS effects of GP7 in mouse and human OS cells with the comparison of etoposide. GP7 and etoposide inhibited the proliferation of a panel of mouse and human OS cells in a concentration- or time-dependent manner, and the inhibitory effect of GP7 on the proliferation of mouse LM8 or human U2OS cells was 1.28- or 1.35-fold higher than that of etoposide. GP7 or etoposide augmented the anti-OS effects of methotrexate, adriamycin, cisplatin, or their combination, and the combined inhibitory effects of GP7 with MTX on the proliferation of LM8 cells was higher than those of etoposide with MTX. GP7 arrested the cell cycle in S phase but etoposide in G₂/M phase. GP7 or etoposide induced sub-G₁ peak, apoptotic DNA fragmentation, activations of caspase-3, -8, -9, and DNA fragmentation factor, downregulation of Bcl-2 and Bcl-xL, upregulation of Bax and Bak, and cytochrome-c release from mitochondria in both mouse and human OS cells. GP7 or etoposide also induced endonuclease G translocation from mitochondria into cytosol in mouse cells. GP7- or etoposide-induced apoptotic DNA fragmentation of human OS cells was inhibited by the pan caspase inhibitor and caspase-9 inhibitor, not by caspase-8 inhibitor whereas it was not inhibited by the pan caspase inhibitor in mouse OS cells. Our findings indicate that GP7 is effective against mouse and human OS cells in vitro. The apoptotic DNA fragmentation in mouse OS cells may be mediated by caspase-independent pathway with the involvement of endonuclease G whereas in human OS cells by caspase-9-dependent pathway downstream of the cytochrome-c-initiated caspase cascade.