br expression was elevated by AC and TRAIL co treatment
expression was elevated by AC and TRAIL co-treatment (Fig. 6). To-gether, these results indicate that AC-induced DR5 up-regulation is the initiating process of AC and TRAIL-induced synergistic apoptosis.
3.6. Co-treatment with AC and TRAIL suppresses phosphorylation of PI3K/ AKT/mTOR pathway
The PI3K/AKT/mTOR signaling pathway is a well-known regulator of cell survival, proliferation, and metastasis and is often targeted for chemopreventative agent-induced apoptosis (Duan et al., 2016). In-hibition of AKT and mTOR phosphorylation prevents uncontrolled cell proliferation, allowing these proteins to regulate various factors in-volved in apoptosis (Chang et al., 2003). To assess whether or not combined treatment with AC and TRAIL inhibits the PI3K/AKT/mTOR signaling pathway, protein expression was measured by Western blot analysis. Co-treatment with AC and TRAIL resulted in down-regulation of PI3K, phosphorylated AKT, and phosphorylated mTOR in RC-58T/h/ SA#4 Cell Counting Kit-8 (Fig. 7A). In addition, pretreatment with PI3K/AKT inhibitor (LY294002) followed by combined treatment of AC and TRAIL sig-nificantly facilitated cell death compared to non-LY294002 treated cells at 12 h and 24 h (Fig. 7B). These results demonstrate the partial
involvement of the PI3K/AKT/mTOR signaling pathway in RC-58T/h/ SA#4 cell growth inhibition induced by co-treatment with AC and TRAIL.
In the present study, AC isolated from F. philippinensis was screened by testing its synergistic apoptosis activity in combination with TRAIL in primary prostate cancer cells. AC was found to improve TRAIL-mediated apoptosis in TRAIL-resistant RC-58T/h/SA#4 primary pros-tate cancer cells. In addition, involvement of the DR5 apoptosis pathway in co-treatment with AC and TRAIL was investigated for the first time. Hence, the results of the current study, in which AC sensitized primary prostate cancer cells to TRAIL-induced apoptosis, can provide an eﬀective strategy for primary prostate cancer therapy using a food-derived compound.
Numerous studies have attempted to identify ideal therapeutics that selectively induce cancer cell death without significant cytotoxicity in normal cells. In our screening results, a high concentration of AC (7.5–10 μM) treatment significantly reduced RPWE-1 cell proliferation down to 75.49–43.18% compared to the control group, whereas an
Fig. 6. DR5 down-regulation suppresses apoptotic cell death induced by co-treatment with AC and TRAIL in RC-58T/h/SA#4 cells. Cells were pretreated with 100 ng/mL of DR5 FC chimera protein for 2 h and then incubated with AC and TRAIL for 24 h. After co-treatment with AC and TRAIL for 24 h, total cell lysates were subjected to detect expression levels of proteins. Expression levels of CHOP, DR5, p53, PARP, Bax, Bcl-2, and caspases-9, -8, and -3 in RC-58T/h/SA#4 cells were analyzed by Western blotting. Significant diﬀerences were compared with the control at *p < 0.05 and **p < 0.01 using one-way ANOVA.
equivalent concentration of AC did not aﬀect proliferation of RC-58T/ h/SA#4 primary prostate cancer cells. These data suggest that the primary cancer suppressive eﬀect of single AC treatment is accom-panied by undesirable cytotoxicity in prostate epithelial cells. Furthermore, some types of prostate cancer cells, including LNCaP, and RC-58T/h/SA#4 cells, are known to be resistant to apoptotic cell death induced by chemotherapeutics (Nesterov et al., 2001; Lee et al., 2014). In addressing tumor species showing resistance to chemotherapeutics, a combination of cancer therapeutics has shown promise for treating cancer (Tang et al., 2016; Jang et al., 2016; Shin and Park, 2013). In the case of advanced cancer, combination chemotherapy resulted in an 18% reduction of death risk, even though toxicity was significantly increased by treatment with poly-chemotherapeutics (Wagner et al., 2010). Furthermore, recent studies reported that a model for synergistic induction of apoptosis by TRAIL and other anti-cancer drugs can acti-vate additional cytotoxic mechanisms against normal human cells, such as hepatocytes, lymphocytes and osteoblasts (Newsom-Davis et al., 2009; Meurette et al., 2006). Interestingly, combined treatment with non-toxic doses of AC (5 μM) and TRAIL (100 ng/mL) significantly in-duced apoptotic cell death in TRAIL-resistant primary prostate cancer cells without normal cell cytotoxicity. Therefore, drug-sensitizing
natural compounds with low cytotoxicity in normal cells are believed to be promising candidates for eﬀectively overcoming various cancer species.
TRAIL is known to promote apoptosis in cancer cells by binding to two death receptors, DR4 and DR5, which are mainly expressed on the surface of cancer cells but not normal cells. However, there is also evidence that some cancer cells escape TRAIL-induced apoptosis via down-regulation of DR4 and/or DR5 on the cell surface (Zhang and Zhang, 2008). Thus, DR5 overexpression is expected to up-regulate apoptosis in cancer cells through eﬀective binding with TRAIL. In this study, AC moderately up-regulated DR5, CHOP, and p53 in RC-58T/h/ SA#4 cells, whereas expression of DR4 protein was not aﬀected by AC treatment. Furthermore, treatment with DR5-specific FC chimera pro-tein significantly inhibited AC and TRAIL-mediated apoptotic cell death. Similar to our results, ursolic acid was shown to up-regulate expression of p53, DR5, and CHOP, resulting in TRAIL-induced apop-tosis in metastatic prostate cancer cells (Shin and Park, 2013). In our previous study, we also reported that isoegomaketone sensitized TRAIL-resistant primary prostate cancer cells to apoptosis via up-regulation of p53, CHOP, and DR5 (Lee et al., 2014). Since CHOP and p53 have been reported to be involved in transcription of DR5 (Sessler et al., 2013; Lin