1. Sinularin reduced cell viability of prostate cancer
The MTS assay was used to detect the effects of Sinularin (Fig. 1A) on cell viability. Prostate cancer cells were treated with Sinularin at indicated concentrations for 24 h and the results revealed that Sinularin dose dependently reduced the viability of tumor cells. However, Sinularin showed less effect on normal prostate epithelial cell (Fig. 1B). Furthermore, Sinularin exerts its anti-tumor effects at the concentration of 20 µM on DU145, 22Rv1 and LNCaP cells, and 40 µM on PC-3 cells. However, Sinularin showed less effect on normal cells at the same concentrations (20 or 40 µM). These results indicated that Sinularin selectively killed prostate cancer cells. The IC50 values of Sinularin on all prostate cancer cells were showed in Table 3. Among those prostate cancer cells, androgen-sensitive cell LNCaP and castration-resistant cell PC-3 were selected for the subsequent studies.
Table 3
The IC50 value of Sinularin on prostate cancer cells
Cells | IC50 (µM) |
DU145 | 55.4 |
LNCaP clone FGC | 67.8 |
22Rv1 | 78.3 |
PC-3 | 80.2 |
2. Sinularin exerted anti-tumor effects through apoptosis
To investigate how Sinularin reduces prostate cancer cell viability, we tested the effects of Sinularin on inducing cell apoptosis. As shown in Figure 2A and B, LNCaP and PC-3 cells were treated with Sinularin at the indicated concentrations for 24 h, and then cell apoptosis was determined by flow cytometry-based Annexin V-FITC/PI assay. The results showed that apoptotic cells increased significantly following the increasing Sinularin concentrations. However, Sinularin showed little effect on RWPE-1 cells at the same concentration (Fig 2C). Moreover, we examined the levels of apoptosis-related proteins Caspase-3 and poly (ADP-ribose) polymerase (PARP), the executor and marker of apoptosis respectively. Caspase-3 was activated by Sinularin and cleavage of PARP also increased only in tumor cells (Figure 2D). It is well known that highly conserved Caspase-family proteins play an important role in cell apoptosis [31, 32]. In this study we blocked the activity of Caspases by Z-VAD-FMK (an irreversible pan-caspase inhibitor) and found that cell apoptosis caused by Sinularin in both LNCaP and PC-3 cells was rescued dramatically (Figure 2E and F). These results indicated that Sinularin exerted anti-tumor effects via Caspases-dependent cell apoptosis on prostate cancer.
3. Sinularin induced intrinsic cell apoptosis in prostate cancer cells
Apoptosis is a form of programmed cell death that results in the orderly and efficient removal of damaged cells. Apoptosis can be triggered by signals from within the cells or by extrinsic signals [33-35]. In the study, we demonstrated that Sinularin activated caspase-9, which is one of crucial modulators of intrinsic apoptotic pathway (Figure 3A and B). Furthermore, we blocked the extrinsic and intrinsic apoptotic pathway by specific inhibitors respectively to explore the mechanism of Sinularin regulation on cell apoptosis. As shown in Figure 3C and D, the apoptotic level of prostate cancer cells was significantly reduced when the intrinsic apoptotic pathway was blocked by Z-LEHD-FMK (a selective and irreversible caspase-9 inhibitor). However, when the extrinsic apoptosis pathway was blocked by Z-IETD-FMK (a selective caspase-8 inhibitor), cell apoptosis induced by Sinularin did not change. In addition, we detected mitochondrial membrane potential via JC-1 fluorescence probe. The results showed that the mitochondrial membrane potential was markedly decreased in Sinularin-treated LNCaP and PC-3 cells (Figure 3E). Moreover, an increase of mitochondrial proteins Cyto c and Smac/DIABLO in the cytosol were detected followed by Sinularin treatment (Figure 3F and G). These results indicated that Sinularin exerted anti-tumor effects through inducing intrinsic cell apoptosis in prostate cancer cells.
4. Sinularin induced cell apoptosis via FOXO3
FOXO3 is a member of the FOXO subfamily of forkhead transcription factors that mediate a variety of cellular processes including apoptosis, proliferation, cell cycle progression, and oxidation stress [36, 37]. Emerging evidence indicates that FOXO3 might act as a tumor suppressor in cancers [38, 39]. In prostate cancer, the expression and activity of FOXO3 were inhibited [40-42]. Many small molecule drugs like Apigenin [43], Flavone [44], Vitexin [45, 46] and Epibrassinolide [47] have showed anti-tumor effects by activating FOXO3. In the study, we found that FOXO3 protein was increased dramatically in Sinularin treated cells (Figure 4A). In addition, we also found that Sinularin induced FOXO3 enrichment in nucleus (Figure 4B). According to these results, we hypothesised that Sinularin induced prostate cancer cell apoptosis may be via FOXO3. To verify this hypothesis, we suppressed FOXO3 through RNAi and determined Sinularin-induced cell apoptosis. As shown in Figure 4C-E, the apoptotic cells decreased significantly when FOXO3 was knocked-down in both Sinularin-treated LNCaP and PC-3 cells. In addition, the rescue of Sinularin-induced apoptosis was also achieved by cleavage of apoptosis-related proteins, such as Caspase-3 and PARP, which decreased when FOXO3 was knocked-down (Figure 4F and G). These results indicated that Sinularin induced cell apoptosis through FOXO3.
5. PUMA, a target molecule by FOXO3, was up-regulated by Sinularin in prostate cancer cells
Intrinsic pathway is a important manner of cell apoptosis, and mitochondria plays a key role in this process [35]. In the study, we found that Sinularin induced mitochondrial membrane potential collapses and release of mitochondrial proteins in prostate cancer cells (Figure 3). To study the molecular mechanisms by which Sinularin triggered intrinsic cell apoptosis, we investigated the expression of the Bcl-2 family of proteins, the important intrinsic cell apoptosis mediators. The results showed that anti-apoptosis protein Bcl-2 decreased and pro-apoptosis protein Bax and PUMA increased in Sinularin treated tumor cells (Figure 5A).
PUMA is a member of the Bcl-2 family of proteins and belongs to the BH3-only pro-apoptotic subclass. PUMA binds to anti-apoptotic Bcl-2 family members to induce mitochondrial outer membrane permeabilization and apoptosis [48, 49]. Furthermore, PUMA was reported as a target of FOXO3 [50]. Herein, we investigated that FOXO3 regulation on PUMA expression at both mRNA and protein levels in prostate cancer cells (Figure 5B and C). In addition, PUMA up-regulation by Sinularin could be rescued via FOXO3 knockdown (Figure D). These results indicated that PUMA could be up-reguleted by Sinularin via FOXO3 in prostate cancer cells.
6. PUMA participated in Sinularin-induced cell apoptosis in prostate cancer cells
We then sought to determine whether PUMA was involved in Sinularin-mediated apoptosis in prostate cancer cells. We suppressed PUMA expression through RNAi and detected Sinularin-induced cell apoptosis. As shown in Figure 6A and B, the rescue of Sinularin-induced apoptosis was detected on apoptosis-related proteins, such as cleavage of Caspase-3 and PARP. They both decreased when PUMA was knocked down. In addition, the apoptotic cells decreased significantly when PUMA was knockdown in both Sinularin-treated LNCaP and PC-3 cells (Figure 6C and D). Furthermore, the mitochondrial membrane potential was also detected and revealed that Sinularin-induced mitochondrial membrane potential collapses was markedly rescued when PUMA was knocked down (Figure 6E and F). These results indicated that Sinularin induced cell apoptosis through PUMA.
7. Sinularin stabilized cellular FOXO3 protein level post-translationally
To investigate how Sinularin up-regulate FOXO3 expression, we detected its mRNA levels in LNCaP and PC-3 cells. Interestingly, the FOXO3 mRNA showed no significant changes after Sinularin treatment (Figure 7A and B). Emerging evidences indicate that as a crucial tumor suppressor, FOXO3 is commonly down-regulated by post-transcriptional suppression, especially by ubiquitin-proteasome degradation pathway [51, 52]. According to these reports and our results, we speculated that Sinularin up-regulated FOXO3 may be via suppressing the ubiquitin-proteasome degradation pathway. As shown in Figure 7C, ubiquitinated FOXO3 dramatically decreased in Sinularin treated cells when compared to negative control (NC). FOXO3 ubiquitination is mediated by 14-3-3. 14-3-3 could bind to FOXO3 and lead to FOXO3 translocation from nucleus to cytoplasm and ubiquitination [51, 53-55]. In the study, we also found that FOXO3 interacted with 14-3-3 in prostate cancer cells (Figure 7D, E).
8. Sinularin stabilized FOXO3 via inhibiting AKT and ERK/MAPK
In tumor cells, the primary mechanism of FOXO3 expression and activity regulation is by controlling the translocation of FOXO3 between nucleus and cytoplasm, which could be achieved by phosphorylation by a series of kinases. The protein kinases such as protein kinase B (PKB/AKT), extracellular signal-regulated kinase (ERK), serum/glucocorticoid regulated kinase (SGK), and IκB kinase isoform β (IKKβ), promote FOXO3 phosphorylation on specific amino acid followed by nuclear export and ubiquitin-proteasome degradation [53, 54, 56, 57]. In this study, we demonstrated that Sinularin significantly inhibited AKT and ERK1/2 phosphorylation (Figure 8A). Moreover, we observed that phosphorylated FOXO3 at Ser253 and Ser294 also decreased in Sinularin treated cells (Figure 8B). FOXO3 phosphorylation at Ser253 and Ser294 are mediated by AKT and ERK1/2 respectively, and phosphorylation at these sites lead to FOXO3 binding with 14-3-3 protein. The binding of 14-3-3 protein with phosphorylated FOXO3 helps FOXO3 translocate from nucleus to cytoplasm and subsequent ubiquitin-proteasome degradation [51, 53-55]. According to these reports, we considered that Sinularin up-regulated FOXO3 via stabilizing FOXO3 protein achieved through inhibition of AKT- and ERK1/2-mediated ubiquitin-proteasome degradation. To verify this hypothesis, small molecule activators were utilized. Western blot assay demonstrated that re-activated AKT by SC79 (a unique specific AKT activator) in Sinularin treated cells led to FOXO3 down-regulation (Figure 8C). Meanwhile, re-activated ERK1/2 by TBHQ (ERK1/2 activator) also led to FOXO3 down-regulation (Figure 8D).