br Sensitization to TRAIL mediated apoptosis by AC is
3.3. Sensitization to TRAIL-mediated apoptosis by AC is dependent on caspase proteins
Once TRAIL binds to either DR4 or DR5, the death signal propagates through the TRAIL-mediated signaling pathway, which leads to cas-pase-8-mediated activation of caspase-3, cleavage of PARP, and cas-pase-3 activation through caspase-9 (Khaider et al., 2012). To assess the molecular mechanism behind AC in TRAIL-mediated apoptosis, we analyzed several key proteins involved in TRAIL-induced apoptosis in primary prostate cancer cells. Treatment with AC or TRAIL alone slightly aﬀected expression levels of caspases, Bax, Bcl-2, and PARP in RC-58T/h/SA#4 Cell Counting Kit-8 (Fig. 3A). However, combined treatment with AC and TRAIL led to significant down-regulation of caspases-8, -9, -3 and
Fig. 4. Combined treatment with AC and TRAIL triggers caspase-independent apoptosis in RC-58T/h/SA#4 cells. (A) Cells were pretreated with 2 μM N-phenylmaleimide (N-PM), an AIF inhibitor, for 2 h and then incubated with AC and/or TRAIL for 24 h. Cell viability was determined by SRB assay. (B) After treatment with AC and/or TRAIL for 24 h, total cell lysates were subjected to detect expression levels of proteins. Expression levels of AIF, Endo G, and cytochrome c 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.
-10, and Bcl-2 as well as up-regulation of Bax and cleaved PARP in RC-58T/h/SA#4 cells. In addition, pre-treatment with 10 μM pan-caspase inhibitor (z-VAD-fmk) overcame apoptotic death by 62.41% upon co-treatment with AC and TRAIL (Fig. 3B). These results indicate that co-treatment with AC and TRAIL induced synergistic apoptosis via acti-vation of the caspase-dependent pathway.
3.4. Co-treatment with AC and TRAIL triggers caspase-independent cell death
To determine the role of caspase-independent apoptotic regulatory proteins in AC-induced TRAIL sensitization, RC-58T/h/SA#4 cells were cultured in the presence or absence of the AIF inhibitor N-phenylma-leimide (N-PM) (Fig. 4A). Pre-treatment of cells with N-PM significantly blocked apoptotic cell death induced by co-treatment with AC and TRAIL (Fig. 4A). Further, we investigated the eﬀect of co-treatment with AC and TRAIL on release of AIF, endonuclease G (Endo G), and cytochrome c proteins into the cytosol from mitochondria (Fig. 4B). Compared to single treatment with AC or TRAIL, expression levels of released AIF, Endo G, and cytochrome c into the cytosol were sig-nificantly elevated upon combined treatment with AC and TRAIL (Fig. 4B). These findings suggest that AC and TRAIL-induced synergistic
cell death utilized a caspase-independent signaling mechanism invol-ving AIF and Endo G.
3.5. AC sensitizes TRAIL-mediated apoptosis through up-regulation of DR5
To further explore the mechanism behind TRAIL-mediated apop-tosis sensitized by AC, expression levels of C/EBP homologous protein (CHOP), DR5, DR4, and p53 were evaluated by Western blot analysis. AC treatment elevated expression levels of CHOP, DR5, and p53 pro-teins in a dose-dependent manner, whereas expression of DR4 was not aﬀected (Fig. 5A). To clarify whether or not this increase in DR5 ex-pression could be responsible for AC and TRAIL-induced cell death, RC-58T/h/SA#4 cells were treated with 50–150 ng/mL of DR5 Fc chimera prior to combined treatment with AC and TRAIL. DR5 Fc chimeric proteins showing a high aﬃnity for DR5 will bind to DR5 instead of TRAIL and can be utilized in studies as DR5 inhibitors to identify re-ceptor bioactivity. Treatment with DR5 Fc chimera markedly reduced AC and TRAIL-induced cell death (Fig. 5B) as well as the apoptotic cell population (Fig. 5C). In addition, DR5 inhibitor pretreatment re-markably blocked expression of AC and TRAIL-induced pro-apoptotic proteins, such as CHOP, DR5, p53, cleaved PARP, cytosolic AIF, Bax, and caspases-9, -8, and -3, whereas anti-apoptotic Bcl-2 protein
Fig. 5. AC sensitizes TRAIL-mediated apoptotic cell death through DR5 up-regulation in RC-58T/h/SA#4 cells. (A) After treatment with AC for 24 h, total cell lysates were subjected to detect expression levels of proteins. Expression levels of CHOP, DR5, DR4 and p53 in RC-58T/h/SA#4 cells were analyzed by Western blotting. (B) Cells were pretreated with 0–150 ng/mL of DR5 FC chimera protein for 2 h and then incubated with AC and/or TRAIL for 24 h. Cell viability was determined by SRB assay. (C) Apoptotic cell population was quantified by Annexin V staining assay using Muse cell analyzer. Significant diﬀerences were compared with the control at *p < 0.05 and **p < 0.01 using one-way ANOVA.