Although an increasing amount of data indicate that DATS can suppress the growth of cultured cancer cells by causing cell cycle arrest at G2/M phase and generation of apoptosis [26–29, 35–37], little is known about the effects of this compound on the growth of human leukemia cells. In the present study, we demonstrated that DATS-induced anti-proliferative effects in four leukemia cell lines (U937, THP-1, HL60 and K562) were related to induction of apoptosis, as confirmed by measurement of chromatin condensation of nuclei, DNA fragmentation, and induction of sub-G1 phase. Our data also indicated that DATS induced apoptosis of U937 cells through generation of ROS and mitochondrial dysfunction, suggesting that ROS act as upstream signaling molecules for initiation of cell death.
Mounting evidence suggests that damaged mitochondria stimulate increased ROS production, subsequently resulting in activation of the signaling pathways that control cancer cell growth. However, loss of MMP as a result of mitochondrial depolarization in association with apoptosis appears to be more common. The mechanisms by which ROS cause or regulate apoptosis typically include caspase activation and modulation of Bcl-2 family protein expression [38, 39]. Caspases, a family of cystein-containing aspartate-specific proteases, are known to play key roles during apoptosis and to lead to initiation and execution of apoptosis. Activation of initiator caspases, such as caspase-8 and −9, resulted in downstream activation of effector caspases, such as caspase-3 and −7 [40, 41]. The decrease in MMP causes disruption of the outer mitochondrial membrane and contributes to release of cytochrome c. Release of cytochrome c has been reported to contribute to activation of caspase-9, which in turn causes activation of caspase-3. In particular, activation of capase-3 is responsible for proteolytic degradation of many key proteins, including PARP and β-catenin, finally leading to apoptosis [42, 43]. Modulation of anti- and pro-apoptotic proteins of the Bcl-2 family also controls mitochondrial function. In mammals, members of the Bcl-2 family can be divided into two subfamilies; the anti-apoptotic protein family, including Bcl-2, and the pro-apoptotic protein family, including Bax. Balance between anti-apoptotic and pro-apoptotic members also determines the fate of the cell through mitochondrial dysfunction [44, 45]. In addition, activation of caspase-8 by apoptotic stimuli converts Bid to truncated Bid (tBid), leading to conformational changes in Bax, mitochondrial depolarization, and release of cytochrome c from mitochondria. This leads finally to activation of caspase-3 and induction of apoptosis via a complex of apoptotic protease activating factor-1 (Apaf-1), procaspase-9, and cytochrome c after translocation of tBid to the mitochondria [41, 44–46]. Furthermore, members of the IAP family, which includes XIAP, cIAP-1, and cIAP-2, have been reported to exert their anti-apoptotic effects due to their function as direct inhibitors of activated caspases. Therefore, down-regulation of IAPs relieves the triggering block of proapoptotic signaling and execution of caspases, thus activating cell death [47, 48].
In this study, our data indicated an association of DATS-induced apoptosis of U937 cells with increased generation of ROS and enzymatic activity of both the extrinsic and intrinsic caspase cascades, including caspase-8 and −9. Although the truncated form of Bid fwas not detected, the levels of intact Bid proteins were gradually down-regulated by DATS in a concentration-dependent manner. DATS also caused a significant reduction in MMP values, which was connected with activation of caspase-3 and concomitant degradation of PARP and β-catenin. In addition, down-regulation of anti-apoptotic Bcl-2 and IAP family proteins, including Bcl-2, XIAP, and cIAP-1, was observed in U937 cells exposed to DATS, as compared with control cells. Thus, the results indicated that caspase-8 activation by DATS appeared to trigger mitochondrial apoptotic events by inducing conformational changes in apoptotic proteins.
ROS-mediated caspase activation and mitochondrial dysfunction have been suggested as critical for DATS-induced apoptosis in several cancer cell lines [26–29]; however, the current role of mitochondrial functional changes associated with ROS generation in the response of human leukemia cells to DATS has not yet been explored. Therefore, we next investigated the question of whether these observations and apoptosis by DATS in U937 cells were associated with generation of ROS. The results revealed that activation of caspase-9 and −3, degradation of PARP, and inhibition of Bcl-2 in DATS-treated cells were ROS-dependent and that co-culture with NAC, a commonly used ROS scavenger, effectively blocked DATS-induced apoptosis in U937 cells. Findings from the present study also indicated that activation of caspase-8 in DATS-treated cells is ROS-dependent, which suggests that ROS may act upstream of caspase-8 activation in U937 cells. Therefore, it is reasonable to assume that the initial signal for activation of caspase-8 after treatment with DATS is also derived from ROS. In addition, blocking of ROS generation prevented DATS-induced down-regulation of XIAP and cIAP-1. Because IAP family proteins are also substrates of caspase-3 [49, 50], the observed decrease in XIAP and cIAP-1 expression may be due to caspase-3-mediated processing following DATS treatment. Thus, our data indicate that ROS production and mitochondrial dysfunction are possible contributing factors to DATS toxicity.