Potential role of caspase-3 and-9 in arsenic trioxide-mediated apoptosis in PCI-1 head and neck cancer cells

Arsenic trioxide (As2O3) has been shown to inhibit the proliferation of hem atologic malignant cells. Previously, we reported that As2O3 had an antitum oral effect in head and neck cancer. Here, we investigated the induction of apoptosis and its mechanism in PCI-1 head and neck squamous carcinoma cell s, after treatment with As2O3. Treatment with 2 muM of As2O3 caused apoptos is in PCI-1 cells following 3 days of exposure, which was detected by the a nnexin V-PI and DAPI staining methods. The cell death population was marked ly increased, being 88% larger than the As2O3- untreated control cells. To address the mechanism of apoptosis, a Western blot assay was performed, sho wing that Bax was up-regulated without a change in Bcl-2. Activation of cas pase-9 during As2O3-induced apoptosis was substantiated by monitoring the p roteolysis of the caspase-9, which was associated with an increase of Apaf- 1 and cytochrome c protein. PCI-1 cells rapidly changed the mitochondria me mbrane potential (Delta psi (m)) after addition of As2O3. Furthermore, acti vation of caspase-3 was demonstrated by monitoring the proteolysis of the c aspase-3 and by measuring caspase-3 activity with a fluorogenic substrate, which was associated with the cleavage of poly(ADP-ribose) polymerase. To e xamine the in vivo effect of As2O3, C3H mouse inoculated with syngenic SCC7 cells was treated by intratumoral injection of As2O3 (300 mug) every day, demonstrating that tumor mass was dramatically reduced on day 4, and reveal ed induction of apoptosis by TUNEL assay. These results suggest that apopto sis of PCI-1 cells by As2O3 is induced by activation of caspase-3 via cytoc hrome c, caspase-9 and Apaf-1 complex.