br Acknowledgment This study was supported by

Acknowledgment This study was supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (KAKENHI) [grant number 15K15031].
Introduction Glioma is the leading malignancy of astrocyte origin in the brain. The most aggressive, invasive, and destructive glioma is called glioblastoma multiforme. Their highly invasive and diffuse nature precludes curative surgical therapy due to the resistance to radiotherapy, chemotherapy, immunotherapy, and others [12], [19], [42]. Besides, the blood-brain barrier further blocks the passage of most antineoplastic drugs into the brain, restricting their therapeutic effects. Despite the advances in clinical care, current treatment options for glioma are still limited and the prognosis with malignant glioma remains poor. To meet clinical demands, a deeper understanding of antineoplastic actions and novel therapeutic strategies are crucial to improve the treatment outcome of glioma patients. The molecular events in the development of malignancies are multifactorial. Increasing evidence has pointed to the importance of cyclooxygenase-2 (COX-2). For example, the level of COX-2 7ACC1 is well correlated with tumor malignancy and aggressiveness. A number of epidemiological, clinical, and experimental studies suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) have promising antineoplastic effects on cancers including glioma [12], [15], [21], [28], [43], [46]. Among the NSAIDs, non-selective COX inhibitor indomethacin has a profound antineoplastic effect on a variety of malignancy. Specifically, the induction of apoptosis via an off-target action could underlie its antineoplastic mechanism through mitogen-activated protein kinase (MAPK), Akt, β-catenin, CHOP, AMP-activated protein kinase (AMPK), or Aurora B kinase [11], [32], [33], [35], [45], [48]. Indomethacin suppresses glioma through the inhibition of cell growth and differentiation, and in particular the induction of apoptosis [3], [4], [8], [27], [33], [38]. The antineoplastic effects against glioma can be further improved by structural modification and formulation, both in vivo and in vitro models [5], [6], [7]. The proapoptotic nature of indomethacin in glioma however remains unclear. Therefore, this study was conducted to determine the molecular mechanisms of indomethacin in the apoptosis it induced in glioma cells.
Materials and methods
Results
Discussion Beyond their inhibitory effects on eicosanoid production, NSAIDs are increasingly implicated in the prevention and treatment of cancers as chemotherapeutics [12], [15], [21], [28], [43], [46]. A growing body of evidence suggests an emerging utility of indomethacin as chemotherapeutics and highlights its antineoplastic outcomes against glioma [3], [4], [5], [6], [7], [8], [27], [33], [38]. To extend the scope of relevant studies, current results of our study showed that the antineoplastic effect of indomethacin involved apoptosis. Indomethacin is known to trigger both mitochondria- and death receptor-mediated apoptotic programs [31], [32]. The ceramide/PP2A/Akt axis is a crucial inducer of glioma apoptosis and may be a possible COX-independent target for indomethacin. Apoptosis inhibitory proteins such as Mcl-1 and FLIP are potential downstream effectors of this axis and their downregulation turns on apoptotic program in indomethacin-treated glioma cells. Apoptosis is a type I programmed cell death controlled by a network of genes. In particular mitochondria are critical organelles to the induction of apoptosis by releasing proapoptotic mediators through pore channels formed by Bax. Establishing the formation of Bax pore channel and mitochondrial permeability is counterbalanced by the actions of the Bcl-2 family proteins with either antiapoptotic or proapoptotic potential. Other than mitochondrial Bax oligomerization, the cluster of particular membrane receptors activated by engaging with extracellular ligands is also important in initiating caspase cascades and apoptosis. Besides Bcl-2 family proteins and death receptors, caspases could be alternative targets for the negative control of apoptosis through inhibitor of apoptosis protein family (IAP) and FLIP [2], [16], [26], [30]. In this study, we found that indomethacin-induced apoptosis was accompanied by elevated caspase 3 activity and the same event was attenuated by caspase inhibitor. We found no changes in the protein levels of Bax, Bad, Bid, and Bcl-2 in the glioma cells under indomethacin treatment; instead, the protein and mRNA levels of Mcl-1 and FLIP dropped and Bax mitochondrial distribution expanded. Mcl-1, a member of the Bcl-2 family protein, prevents Bax pore channel formation and mitochondria-mediated apoptosis by antagonizing the actions of proapoptotic Bcl-2 family proteins [2], [16]. FLIP is a component of the death-inducing signaling complex (DISC) and its presence prevents proteolytic activation of caspase 8 and consequently death receptor-mediated apoptosis [26], [30]. Therefore, reduced levels of Mcl-1 and FLIP render their ineffectiveness in antagonizing Bax oligomerization and DISC activation leading to apoptosis. Several regulatory molecules are involved in the activation of mitochondria- and death receptor-mediated apoptotic pathways. A number of reports have shown that increased Bax mitochondrial distribution, upregulation of both proapoptotic Bcl-2 family proteins and death receptors, and downregulation of antiapoptotic Bcl-2 family proteins as well as IAPs are essential for the indomethacin-induced apoptosis [11], [31], [35], [45]. Our results further suggested that the downregulation of Mcl-1 and FLIP is one underlying mechanism of indomethacin-induced apoptosis in glioma cells.