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  • br Fig Combined treatment with


    Fig. 7. Combined treatment with AC and TRAIL inhibits the PI3K/AKT/mTOR pathway in RC-58T/h/SA#4 cells. (A) After treatment with AC and/or TRAIL for 12 h, total cell lysates were subjected to detect expression levels of proteins. Expression levels of PI3K, AKT, p-AKT, mTOR, and p-mTOR in RC-58T/h/SA#4 Cell Counting Kit-8 were analyzed by Western blotting. (B) Cells were pretreated with 10 μM LY294002, PI3K and AKT inhibitor, for 2 h and then incubated with AC and/or TRAIL for 24 h. Cell viability was evaluated by SRB assay. Significant differences were compared with the control at *p < 0.05 and **p < 0.01 using one-way ANOVA.
    et al., 2011; Yamaguchi and Wang, 2004), our results show that CHOP-and p53-mediated DR5 up-regulation contributed to the TRAIL-sensi-tizing effect of AC in TRAIL-resistant primary prostate cancer cells.
    Apoptosis, a type of programmed cell death, is initiated by two canonical pathways: (1) extrinsic and (2) intrinsic pathways (Elmore et al., 2007). Binding of cancer necrosis factor (TNF)-α, FAS ligand and TRAIL to their receptors triggers the extrinsic apoptosis pathway acti-vating adaptor molecules and caspases. On the other hand, the intrinsic pathway is usually initiated by response to DNA damage, hypoxia, and oncogenes and involves p53, PUMA, Bax, and p21 (Hofseth et al., 2004; Hengartner, 2000). In this study, we provide convincing evidence that AC significantly enhanced TRAIL-induced apoptosis through activation of extrinsic pathway proteins, including DR5, and caspases-8, -10 and −3. Our results also reveal that the apoptotic molecular mechanism induced by co-treatment with AC and TRAIL regulated intrinsic apop-totic pathway proteins such as AIF, Endo G, cytochrome c, Bax, Bcl-2, 
    cleaved-bid, and cleaved-PARP. Release of AIF, Endo G, and cyto-chrome c from mitochondria into the cytosol is involved in both the extrinsic and intrinsic apoptotic pathways (Elmore et al., 2007). In addition, natural compounds and TRAIL have been reported to sy-nergistically suppress TRAIL-resistant tumors through the extrinsic and intrinsic apoptosis signaling pathways (Zhang et al., 2016; Jang et al., 2016). Therefore, AC can increase sensitivity to TRAIL-induced apop-tosis via a synergistic combination of the extrinsic and intrinsic apop-tosis signaling pathways.
    The PI3K/AKT pathway is a prototypic survival signaling pathway that can phosphorylate many downstream substrates such as mamma-lian target of rapamycin (mTOR), NF-кB, Raf, and ASK1 related with proliferation, growth, survival, and metabolism (Hay, 2005). Once PI3K/AKT/mTOR are activated, they directly and/or indirectly regulate many other proteins, including Bax, MDM2, Bad, and FOXO, which are closely associated with drug resistance (Burris HA 3rd, 2013).
    Accumulative evidence has shown that several cancer cells are resistant to TRAIL due to high levels of constitutively active AKT and deficiency of death receptor expression (Nesterov et al., 2001; Xu et al., 2010). In our previous study, AC was reported to suppress the PI3K/AKT/mTOR signaling pathways in LNCaP prostate cancer cells (Cho et al., 2018a) and human umbilical vein endothelial cells (HUVECs) (Cho et al., 2018b) Consistent with these studies, co-treatment with AC and TRAIL also suppressed PI3K, phosphorylation of AKT, and phosphorylation of mTOR in RC-58T/h/SA#4, and pretreatment of LY294002 (PI3K and AKT inhibitor) significantly facilitate AC and TRAIL-induced cell death. These results suggest that combined treatment with AC and TRAIL may inhibit the PI3K/AKT/mTOR pathway to facilitate TRAIL-induced proliferation suppressive activity in primary prostate cancer cells.
    In conclusion, this study is the first to demonstrate that AC sensitizes primary prostate cancer cells to TRAIL by triggering up-regulation of DR5, leading to caspase-dependent and -independent TRAIL-mediated apoptosis. Cell Counting Kit-8 The present findings suggest that AC may activate the death ligand pathway to treat TRAIL resistance in primary prostate cancer cells without impairing the cancer selectivity of TRAIL.
    Conflicts of interest
    The authors declare that there are no conflicts of interest.
    This study was financially supported by the Dong-A University Research Fund.
    Transparency document
    Bertsch, U., Röder, C., Kalthoff, H., Trauzold, A., 2014. Compartmentalization of TNF-related apoptosis-inducing ligand (TRAIL) death receptor functions: emerging role of nuclear TRAIL-R2. Cell Death Dis. 5, e1390.
    Burris 3rd, H.A., 2013. Overcoming acquired resistance to anticancer therapy: focus on the PI3K/AKT/mTOR pathway. Cancer Chemother. Pharmacol. 71, 829–842.
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