Inhibition of Prostate Cancer DU-145 Cells Proliferation by Anthopleura anjunae Oligopeptide (YVPGP) via PI3K/AKT/mTOR Signaling Pathway

We investigated the antitumor mechanism of Anthopleura anjunae oligopeptide (AAP-H, YVPGP) in prostate cancer DU-145 cells in vitro and in vivo. Results indicated that AAP-H was nontoxic and exhibited antitumor activities. Cell cycle analysis indicated that AAP-H may arrest DU-145 cells in the S phase. The role of the phosphatidylinositol 3-kinase/protein kinase B/mammalian rapamycin target protein (PI3K/AKT/mTOR) signaling pathway in the antitumor mechanism of APP-H was investigated. Results showed that AAP-H treatment led to dose-dependent reduction in the levels of p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448), whereas t-AKT and t-PI3K levels remained unaltered compared to the untreated DU-145 cells. Inhibition of PI3K/AKT/mTOR signaling pathway in the DU-145 cells by employing inhibitor LY294002 (10 μM) or rapamycin (20 nM) effectively attenuated AAP-H-induced phosphorylation of AKT and mTOR. At the same time, inhibitor addition further elevated AAP-H-induced cleaved-caspase-3 levels. Furthermore, the effect of AAP-H on tumor growth and the role of the PI3K/AKT/mTOR signaling pathway in nude mouse model were also investigated. Immunohistochemical analysis showed that activated AKT, PI3K, and mTOR levels were reduced in DU-145 xenografts. Western blotting showed that AAP-H treatment resulted in dose-dependent reduction in p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448) levels, whereas t-AKT and t-PI3K levels remained unaltered. Similarly, Bcl-xL levels decreased, whereas that of Bax increased after AAP-H treatment. AAP-H also increased initiator (caspase 8 and 9) and executor caspase (caspase 3 and 7) levels. Therefore, the antitumor mechanism of APP-H on DU-145 cells may involve regulation of the PI3K/AKT/mTOR signaling pathway, which eventually promotes apoptosis via mitochondrial and death receptor pathways. Thus, the hydrophobic oligopeptide (YVPGP) can be developed as an adjuvant for the prevention or treatment of prostate cancer in the future.


Introduction
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Toxicity to Normal Cells
Cell proliferation is the foundation of an organism's growth, development, reproduction, and heredity. Perturbation in the balance between cell proliferation and apoptosis often leads to cancer [21]. Hence, inhibition of cell proliferation is an effective way of controlling cancer. In the previous study, we showed that AAP-H could inhibit the proliferation of prostate cancer DU-145 cells with a half-maximal inhibitory (IC 50 ) concentration of 9.605 mM, 7.910 mM, and 2.298 mM at 24 h, 48 h, and 72 h, respectively [20]. However, the toxicity of AAP-H to normal cells was not studied. In this study, the fibroblast NIH-3T3 cells were used as the normal cells [22], which were added to culture solution containing different concentrations of AAP-H (0, 1, 5, 10, 15, and 20 mM) and incubated for 24 h. As shown in Figure 1, AAP-H showed no inhibitory effect on the fibroblast NIH-3T3 cells.
We observed that the rate of inhibition of DU-145 cells increased in a dose-and time-dependent manner [20], whereas the cell viability of NIH-3T3 cells was not affected under the same concentration. Thus, these observations indicated that AAP-H was nontoxic and exhibited antitumor activities. Huang et al. [23] reported that the inhibitory rate of a tripeptide (QPK) extracted from Sepia ink on DU-145 cells is 74.62% at 15 mg/mL. Song et al. [6] reported that the antiproliferative peptide (YALPAH) from Setipinna taty inhibited the DU-145 cells with an IC 50 of 16.9 mM at 48 h. In comparison, the Anthopleura anjunae oligopeptides (YVPGP) showed lower IC 50  ink on DU-145 cells is 74.62% at 15 mg/mL. Song et al. [6] reported that the antiproliferative peptide (YALPAH) from Setipinna taty inhibited the DU-145 cells with an IC50 of 16.9 mM at 48 h. In comparison, the Anthopleura anjunae oligopeptides (YVPGP) showed lower IC50 values and better anticancer activity.

AAP-H Induced Cycle Arrest of DU-145 Cells
The cell cycle is a tightly regulated and ordered event consisting of G1, S, G2, and M phases at the completion of which the newly replicated DNA is equally divided into daughter cells. The morphology and biochemistry of cells undergoing division change accordingly in these four stages [24,25]. The PI3K/AKT signaling pathways activate the cell-cycle-dependent protein kinase 2 (CDK-2) and CDK-4, which induces entry in S phase and DNA synthesis [26]. Evidence shows that AKT can actively and effectively regulate cell cycle progression from G2 to M phase and promote mitosis [27]. To investigate whether AAP-H affects cell cycle progression, DU-145 cells were treated with different concentrations of AAP-H (0, 5, 10, and 15 mM) for 24 h and assessed using flow cytometry. As shown in Figure 2, the number of cells in S phase increased in AAP-H treatment groups (p < 0.05) compared to the untreated controls. The percentages of cells in S phase after 24 h of AAP-H treatment (0, 5, 10, and 15 mM) were 7.3 ± 1.5%, 12.2 ± 1.4%, 17 ± 2.1%, and 20.9 ± 1.9%, respectively. To understand the mechanism underlying the AAP-H-induced S phase arrest, western blotting was used to analyze cell cycle regulatory proteins [28]. The levels of cyclin B1, cyclin D1, and CDK2 were dramatically reduced by varying degrees after treatment with high doses of APP-H (10-15 mM) ( Figure 2).
Flow cytometric and western blotting analysis showed that AAP-H regulated the cell cycle by altering the distribution of DU-145 cells in different phases of the cycle. Compared to the blank control group, the proportion of G2/M phase cells decreased, whereas that of S phase cells increased. This indicated that AAP-H may block the progression of cancer cells from S to G2/M phase, which eventually leads to apoptosis.

AAP-H Induced Cycle Arrest of DU-145 Cells
The cell cycle is a tightly regulated and ordered event consisting of G1, S, G2, and M phases at the completion of which the newly replicated DNA is equally divided into daughter cells. The morphology and biochemistry of cells undergoing division change accordingly in these four stages [24,25]. The PI3K/AKT signaling pathways activate the cell-cycle-dependent protein kinase 2 (CDK-2) and CDK-4, which induces entry in S phase and DNA synthesis [26]. Evidence shows that AKT can actively and effectively regulate cell cycle progression from G2 to M phase and promote mitosis [27]. To investigate whether AAP-H affects cell cycle progression, DU-145 cells were treated with different concentrations of AAP-H (0, 5, 10, and 15 mM) for 24 h and assessed using flow cytometry. As shown in Figure 2, the number of cells in S phase increased in AAP-H treatment groups (p < 0.05) compared to the untreated controls. The percentages of cells in S phase after 24 h of AAP-H treatment (0, 5, 10, and 15 mM) were 7.3 ± 1.5%, 12.2 ± 1.4%, 17 ± 2.1%, and 20.9 ± 1.9%, respectively. To understand the mechanism underlying the AAP-H-induced S phase arrest, western blotting was used to analyze cell cycle regulatory proteins [28]. The levels of cyclin B1, cyclin D1, and CDK2 were dramatically reduced by varying degrees after treatment with high doses of APP-H (10-15 mM) ( Figure 2).
Flow cytometric and western blotting analysis showed that AAP-H regulated the cell cycle by altering the distribution of DU-145 cells in different phases of the cycle. Compared to the blank control group, the proportion of G2/M phase cells decreased, whereas that of S phase cells increased. This indicated that AAP-H may block the progression of cancer cells from S to G2/M phase, which eventually leads to apoptosis. Mar. Drugs 2018, 16, x 4 of 16 The expression levels of cyclin B1, cyclin D1, and CDK2 in each group analyzed by densitometry normalized to β-actin. * p < 0.05 and ** p < 0.001 vs. the control group.

AAP-H Suppressed the PI3K/AKT/mTOR Signaling Pathway in DU-145 Cells
Recent studies have indicated that PCa cells can be inhibited via the PI3K/AKT/mTOR signaling pathway. Meng et al. [29] reported that ursolic acid, a pentacyclic triterpenoid, can inhibit cell growth and induce apoptosis by regulating the PI3K/AKT/mTOR pathway in human PCa cells. Kumar et al. [30] demonstrated that rottlerin (rott), an active molecule isolated from Mallotus philippensis, inhibited autophagy and apoptosis in PCa stem cells. Yasumizu et al. [31] observed that hypoxic microenvironments promoted PI3K/Akt/mTOR signaling pathways and morphological changes in human castration-resistant prostate cancer (CRPC) cells via the PI3K/AKT/mTOR signaling pathway.
Hence, the expression levels of p-PI3K (p85) [32], p-AKT (Ser473) [33], t-PI3K, t-AKT, and p-mTOR (Ser2448) [33] were investigated after 24 h treatment with AAP-H. As shown in Figure 3, AAP-H treatment led to a dose-dependent reduction in the levels of p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448), whereas t-AKT and t-PI3K levels remained unaltered compared to the nontreated group. Multiple signaling pathways operate and cross-talk inside cells; hence, when one pathway is stimulated, the levels of proteins related to that pathway, as well as other associated pathways, change concomitantly [34].

AAP-H Suppressed the PI3K/AKT/mTOR Signaling Pathway in DU-145 Cells
Recent studies have indicated that PCa cells can be inhibited via the PI3K/AKT/mTOR signaling pathway. Meng et al. [29] reported that ursolic acid, a pentacyclic triterpenoid, can inhibit cell growth and induce apoptosis by regulating the PI3K/AKT/mTOR pathway in human PCa cells. Kumar et al. [30] demonstrated that rottlerin (rott), an active molecule isolated from Mallotus philippensis, inhibited autophagy and apoptosis in PCa stem cells. Yasumizu et al. [31] observed that hypoxic microenvironments promoted PI3K/Akt/mTOR signaling pathways and morphological changes in human castration-resistant prostate cancer (CRPC) cells via the PI3K/AKT/mTOR signaling pathway.
Hence, the expression levels of p-PI3K (p85) [32], p-AKT (Ser473) [33], t-PI3K, t-AKT, and p-mTOR (Ser2448) [33] were investigated after 24 h treatment with AAP-H. As shown in Figure 3, AAP-H treatment led to a dose-dependent reduction in the levels of p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448), whereas t-AKT and t-PI3K levels remained unaltered compared to the nontreated group. Multiple signaling pathways operate and cross-talk inside cells; hence, when one pathway is stimulated, the levels of proteins related to that pathway, as well as other associated pathways, change concomitantly [34].

PI3K/AKT/mTOR Signaling Involved in AAP-H-Induced Apoptosis
To further investigate whether AAP-H-induced apoptosis involves the PI3K/AKT/mTOR signaling pathway, the levels of p-AKT (Ser473), t-AKT, and cleaved-PARP were assessed using western blotting after DU-145 were treated with 10 mM AAP-H for 0, 2, 4, 8, 12, 18, and 24 h [35,36]. Results revealed that AAP-H reduced AKT phosphorylation in a time-dependent manner; however, AKT level remained unchanged. Cleaved-PARP level also increased in a time-dependent manner. Importantly, cleaved-PARP level started increasing concomitantly with decrease in AKT phosphorylation ( Figure 4). These results indicated that PI3K/AKT/mTOR inhibition was an early event that was initiated prior to cell death induction.
Furthermore, LY294002 [37] and rapamycin [38], were used to specifically inhibit the PI3K/AKT/mTOR signaling [39]. Figure 5 shows that the levels of phosphorylated AKT and mTOR were significantly reduced after treatment with LY294002 (10 μM) and rapamycin (20 nM), respectively. In addition, pretreatment with LY294002 and rapamycin effectively attenuated AAP-Hinduced phosphorylation of AKT and mTOR. At the same time, inhibitor addition further elevated AAP-H-induced cleaved-caspase-3 levels. These results implied that the PI3K/AKT/mTOR signaling pathway is one of the potential mechanisms via which APP-H induces DU-145 cell apoptosis.

PI3K/AKT/mTOR Signaling Involved in AAP-H-Induced Apoptosis
To further investigate whether AAP-H-induced apoptosis involves the PI3K/AKT/mTOR signaling pathway, the levels of p-AKT (Ser473), t-AKT, and cleaved-PARP were assessed using western blotting after DU-145 were treated with 10 mM AAP-H for 0, 2, 4, 8, 12, 18, and 24 h [35,36]. Results revealed that AAP-H reduced AKT phosphorylation in a time-dependent manner; however, AKT level remained unchanged. Cleaved-PARP level also increased in a time-dependent manner. Importantly, cleaved-PARP level started increasing concomitantly with decrease in AKT phosphorylation ( Figure 4). These results indicated that PI3K/AKT/mTOR inhibition was an early event that was initiated prior to cell death induction.
Furthermore, LY294002 [37] and rapamycin [38], were used to specifically inhibit the PI3K/AKT/mTOR signaling [39]. Figure 5 shows that the levels of phosphorylated AKT and mTOR were significantly reduced after treatment with LY294002 (10 µM) and rapamycin (20 nM), respectively. In addition, pretreatment with LY294002 and rapamycin effectively attenuated AAP-H-induced phosphorylation of AKT and mTOR. At the same time, inhibitor addition further elevated AAP-H-induced cleaved-caspase-3 levels. These results implied that the PI3K/AKT/mTOR signaling pathway is one of the potential mechanisms via which APP-H induces DU-145 cell apoptosis. Mar. Drugs 2018, 16, x 6 of 16

Effect of AAP-H on DU-145 Xenografts
To investigate the anticancer effects of AAP-H on DU-145 xenografts, physiological saline, AAP-H, and DDP were injected in nude mice for 14 days. Mice body weight and tumor volume were determined every three days. As shown in the Figure 6A, the growth rate of tumor volume in the AAP-H and DDP treatment groups was slower than that of the control group. The inhibition rate was 36.93 ± 3.9% in the AAP-H low dose group, 62.22 ± 6.2% in the AAP-H high dose group, and 66.96 ± 5.7% in the DDP group. Although DDP had the best inhibitory effect ( Figure 6B), it elicited obvious toxic effects in nude mice. AAP-H not only reduced tumor weight but also slightly increased body weight and quality of life of nude mice during medication. Thus, we preliminarily concluded that AAP-H has no harmful effect on nude mice.

Effect of AAP-H on DU-145 Xenografts
To investigate the anticancer effects of AAP-H on DU-145 xenografts, physiological saline, AAP-H, and DDP were injected in nude mice for 14 days. Mice body weight and tumor volume were determined every three days. As shown in the Figure 6A, the growth rate of tumor volume in the AAP-H and DDP treatment groups was slower than that of the control group. The inhibition rate was 36.93 ± 3.9% in the AAP-H low dose group, 62.22 ± 6.2% in the AAP-H high dose group, and 66.96 ± 5.7% in the DDP group. Although DDP had the best inhibitory effect ( Figure 6B), it elicited obvious toxic effects in nude mice. AAP-H not only reduced tumor weight but also slightly increased body weight and quality of life of nude mice during medication. Thus, we preliminarily concluded that AAP-H has no harmful effect on nude mice.

Immunocytochemistry
The expression of members of the PI3K/AKT/mTOR signaling pathway (p-AKT (Ser473), p-PI3K (p85) and p-mTOR (Ser2448)) in tumor tissue from nude mice was examined using immunocytochemistry assay [33]. As shown in Figure 7, p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448) were detected in the cytoplasm and nucleus of the tumors; however, no expression was observed in the negative control group, whereas strong expression was observed in the positive control group. The positive expression started to decrease in the group treated with low dose of AAP-H (100 mg/kg). p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448) levels were negligible and cell morphology was poor when the concentration of AAP-H was increased to 150 mg/kg. The results of immunohistochemistry were further confirmed by western blotting analysis.
Recent studies have shown that mutations in PI3KCA of PI3K play crucial roles in the formation of human tumors [40]. Phosphorylated PI3K triggers the production of the second messenger inositol triphosphate, further activating AKT, which can phosphorylate a variety of downstream factors such as enzymes, kinases, and transcription factors to promote tumor production [41]. AKT is an evolutionarily conserved serine/threonine kinase with three subtypes that are expressed to varying degrees in most human tissues. p-AKT is a typical pro-cancer factor, which can promote proliferation of tumor cells via p21, p27, and p53, inhibiting apoptosis, and promoting invasion, metastasis, and angiogenesis of tumor cells. Reports show that p-AKT is expressed in various malignancies, such as oral cancer, breast cancer, and non-small cell lung cancer [42][43][44]. mTOR is a direct substrate of AKT kinase, and serine 2480 is the site of phosphorylation of the latter on mTOR; the deletion of the residue in the C terminal regulatory region of the protein increases mTOR activity [41]. Hence,

Immunocytochemistry
The expression of members of the PI3K/AKT/mTOR signaling pathway (p-AKT (Ser473), p-PI3K (p85) and p-mTOR (Ser2448)) in tumor tissue from nude mice was examined using immunocytochemistry assay [33]. As shown in Figure 7, p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448) were detected in the cytoplasm and nucleus of the tumors; however, no expression was observed in the negative control group, whereas strong expression was observed in the positive control group. The positive expression started to decrease in the group treated with low dose of AAP-H (100 mg/kg). p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448) levels were negligible and cell morphology was poor when the concentration of AAP-H was increased to 150 mg/kg. The results of immunohistochemistry were further confirmed by western blotting analysis.
Recent studies have shown that mutations in PI3KCA of PI3K play crucial roles in the formation of human tumors [40]. Phosphorylated PI3K triggers the production of the second messenger inositol triphosphate, further activating AKT, which can phosphorylate a variety of downstream factors such as enzymes, kinases, and transcription factors to promote tumor production [41]. AKT is an evolutionarily conserved serine/threonine kinase with three subtypes that are expressed to varying degrees in most human tissues. p-AKT is a typical pro-cancer factor, which can promote proliferation of tumor cells via p21, p27, and p53, inhibiting apoptosis, and promoting invasion, metastasis, and angiogenesis of tumor cells. Reports show that p-AKT is expressed in various malignancies, such as oral cancer, breast cancer, and non-small cell lung cancer [42][43][44]. mTOR is a direct substrate of AKT kinase, and serine 2480 is the site of phosphorylation of the latter on mTOR; the deletion of the residue in the C terminal regulatory region of the protein increases mTOR activity [41]. Hence, immunocytochemistry was used in this study to detect p-AKT in DU-145 cells. Our results showed that the expression of activated AKT, PI3K, and mTOR was increased in DU-145 xenografts, which resulted in continuous activation of the PI3K/AKT/mTOR pathway and rapid cell proliferation, thereby promoting tumor malignancy. immunocytochemistry was used in this study to detect p-AKT in DU-145 cells. Our results showed that the expression of activated AKT, PI3K, and mTOR was increased in DU-145 xenografts, which resulted in continuous activation of the PI3K/AKT/mTOR pathway and rapid cell proliferation, thereby promoting tumor malignancy.

AAP-H Suppressed the PI3K/AKT/mTOR Signaling Pathway in DU-145 Xenografts
The PI3K/AKT/mTOR signaling pathway plays a crucial role in growth, proliferation, metastasis, and malignant transformation in human cancer [45]. It can induce tumor formation via the following mechanisms: (1) It inhibits autophagy [46]. For example, mTOR is a key regulator of the initiation stage of autophagy, and its activation inhibits autophagy [47]. (2) Activated AKT inhibits caspases 3 and 9 by phosphorylating their Ser196 and ultimately inhibiting apoptosis [48,49]. Furthermore, it inhibits the Bcl-2 family of proteins. Bad promotes apoptosis with Bcl-2 or Bcl-xL, and activated AKT is a highly effective Bad kinase that can be blocked by phosphorylation of Ser136 to induce apoptosis. (3) Promotion of tumor metastasis, cell movement, and angiogenesis are related to PI3K/AKT signal transduction. The above observations suggest that the PI3K/AKT/mTOR pathway is closely related to the caspase and Bcl-2 signaling pathways. Changes in the levels of key proteins of the PI3K/AKT/mTOR signaling pathway (p-PI3K (p85), p-AKT (Ser473), t-PI3K, t-AKT, and p-mTOR (Ser2448)) in DU-145 xenografts treated with AAP-H for 14 days were analyzed using western blotting. Compared to the control group, AAP-H treatment resulted in dose-dependent reduction in the levels of p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448), whereas t-AKT and t-PI3K levels remained unaltered (Figure 8). These results were consistent with the in vitro results, indicating that AAP-H may inhibit the activity of DU-145 xenograft by regulating the PI3K/AKT/mTOR signal transduction pathway.

AAP-H Suppressed the PI3K/AKT/mTOR Signaling Pathway in DU-145 Xenografts
The PI3K/AKT/mTOR signaling pathway plays a crucial role in growth, proliferation, metastasis, and malignant transformation in human cancer [45]. It can induce tumor formation via the following mechanisms: (1) It inhibits autophagy [46]. For example, mTOR is a key regulator of the initiation stage of autophagy, and its activation inhibits autophagy [47]. (2) Activated AKT inhibits caspases 3 and 9 by phosphorylating their Ser196 and ultimately inhibiting apoptosis [48,49]. Furthermore, it inhibits the Bcl-2 family of proteins. Bad promotes apoptosis with Bcl-2 or Bcl-xL, and activated AKT is a highly effective Bad kinase that can be blocked by phosphorylation of Ser136 to induce apoptosis. (3) Promotion of tumor metastasis, cell movement, and angiogenesis are related to PI3K/AKT signal transduction. The above observations suggest that the PI3K/AKT/mTOR pathway is closely related to the caspase and Bcl-2 signaling pathways. Changes in the levels of key proteins of the PI3K/AKT/mTOR signaling pathway (p-PI3K (p85), p-AKT (Ser473), t-PI3K, t-AKT, and p-mTOR (Ser2448)) in DU-145 xenografts treated with AAP-H for 14 days were analyzed using western blotting. Compared to the control group, AAP-H treatment resulted in dose-dependent reduction in the levels of p-AKT (Ser473), p-PI3K (p85), and p-mTOR (Ser2448), whereas t-AKT and t-PI3K levels remained unaltered (Figure 8). These results were consistent with the in vitro results, indicating that AAP-H may inhibit the activity of DU-145 xenograft by regulating the PI3K/AKT/mTOR signal transduction pathway.

Western Blotting Analysis of Bcl-2 Family Members
Apoptosis is triggered via the extrinsic (death receptor pathway) and intrinsic (mitochondrial pathway) pathways [50]. The Bcl-2 family of proteins, which regulates the release of cytochrome C from the mitochondria to the cytoplasm, is mainly composed of anti-apoptotic proteins (Bcl-2 and Bcl-xL) and apoptotic proteins (Bax, Bak). Anti-apoptotic and apoptotic proteins play opposite roles during the release of cytochrome C. For example, oligomeric Bax assists cytochrome C and other apoptotic proteins to pass through the mitochondrial membrane. However, Bcl-xL closes the voltage dependent anion channel (VDAC), which inhibits the release of cytochrome C from the Bax/VDAC channel [51]. To investigate whether apoptosis-related proteins participate in AAP-H-induced apoptosis, we analyzed the expression of Bcl-2 family members such as Bcl-xL and Bax. As shown in Figure 9, the level of the anti-apoptotic Bcl-xL was reduced, whereas that of the pro-apoptotic Bax was elevated post-AAP-H treatment. This indicated that AAP-H might affect apoptosis via the mitochondrial pathway.

Western Blotting Analysis of Bcl-2 Family Members
Apoptosis is triggered via the extrinsic (death receptor pathway) and intrinsic (mitochondrial pathway) pathways [50]. The Bcl-2 family of proteins, which regulates the release of cytochrome C from the mitochondria to the cytoplasm, is mainly composed of anti-apoptotic proteins (Bcl-2 and Bcl-xL) and apoptotic proteins (Bax, Bak). Anti-apoptotic and apoptotic proteins play opposite roles during the release of cytochrome C. For example, oligomeric Bax assists cytochrome C and other apoptotic proteins to pass through the mitochondrial membrane. However, Bcl-xL closes the voltage dependent anion channel (VDAC), which inhibits the release of cytochrome C from the Bax/VDAC channel [51]. To investigate whether apoptosis-related proteins participate in AAP-H-induced apoptosis, we analyzed the expression of Bcl-2 family members such as Bcl-xL and Bax. As shown in Figure 9, the level of the anti-apoptotic Bcl-xL was reduced, whereas that of the pro-apoptotic Bax was elevated post-AAP-H treatment. This indicated that AAP-H might affect apoptosis via the mitochondrial pathway.

Western Blotting of Caspases
The caspase family consists of cysteine-rich proteins, which are divided into initiator caspases (caspases 8 and 9) and executor caspases (caspases 3, 6, and 7). Caspases are activated by two major pathways. One involves the release of cytochrome C from the mitochondria to the cytoplasm, which eventually leads to apoptosis. The other pathway involves the death receptors [50].
Death receptor is a transmembrane protein on the cell membrane, which is responsible for transferring extracellular stimulating signal to the corresponding ligand and inducing apoptosis [52]. Caspase 8, one of the initiators of apoptosis, plays a key role in mediating caspase activation in the death receptor pathway [53] by activating the downstream executors, caspases 3, 6, and 7. However, caspase 8 exists mainly in an inactive state in human cancers. Recently, caspase 8 expression has been analyzed from various perspectives [54,55]. Here, we investigated the levels of initiator and executor caspases in treated and untreated tumors. As shown in Figure 10, initiator caspase 8 and 9 were activated and expressed to similar extents in the presence of 150 mg/kg AAP-H. This was paralleled by increase in the expression of the downstream executor caspase 3 and 7. These results indicated that AAP-H promoted apoptosis of DU-145 cells via both the mitochondrial and death receptor pathways.

Western Blotting of Caspases
The caspase family consists of cysteine-rich proteins, which are divided into initiator caspases (caspases 8 and 9) and executor caspases (caspases 3, 6, and 7). Caspases are activated by two major pathways. One involves the release of cytochrome C from the mitochondria to the cytoplasm, which eventually leads to apoptosis. The other pathway involves the death receptors [50].
Death receptor is a transmembrane protein on the cell membrane, which is responsible for transferring extracellular stimulating signal to the corresponding ligand and inducing apoptosis [52]. Caspase 8, one of the initiators of apoptosis, plays a key role in mediating caspase activation in the death receptor pathway [53] by activating the downstream executors, caspases 3, 6, and 7. However, caspase 8 exists mainly in an inactive state in human cancers. Recently, caspase 8 expression has been analyzed from various perspectives [54,55]. Here, we investigated the levels of initiator and executor caspases in treated and untreated tumors. As shown in Figure 10, initiator caspase 8 and 9 were activated and expressed to similar extents in the presence of 150 mg/kg AAP-H. This was paralleled by increase in the expression of the downstream executor caspase 3 and 7. These results indicated that AAP-H promoted apoptosis of DU-145 cells via both the mitochondrial and death receptor pathways.

Western Blotting of Caspases
The caspase family consists of cysteine-rich proteins, which are divided into initiator caspases (caspases 8 and 9) and executor caspases (caspases 3, 6, and 7). Caspases are activated by two major pathways. One involves the release of cytochrome C from the mitochondria to the cytoplasm, which eventually leads to apoptosis. The other pathway involves the death receptors [50].
Death receptor is a transmembrane protein on the cell membrane, which is responsible for transferring extracellular stimulating signal to the corresponding ligand and inducing apoptosis [52]. Caspase 8, one of the initiators of apoptosis, plays a key role in mediating caspase activation in the death receptor pathway [53] by activating the downstream executors, caspases 3, 6, and 7. However, caspase 8 exists mainly in an inactive state in human cancers. Recently, caspase 8 expression has been analyzed from various perspectives [54,55]. Here, we investigated the levels of initiator and executor caspases in treated and untreated tumors. As shown in Figure 10

Materials and Reagents
The AAP-H (YVPGP) samples were purified from Anthopleura anjunae protein hydrolysates as mentioned previously [56] and stored at −20

Cell Toxicity Assay
The effect of AAP-H on the toxicity of NIH-3T3 cell viability was detected using the MTT assay according to the method described by Tang et al. [57], with slight modifications. In brief, the NIH-3T3 cells were first seeded in a 96-well (5 × 105 cell/mL) plate (Costar Corning, Rochester, NY, USA). After incubation for 24 h, 200 µL fresh culture medium containing different final concentrations of AAP-H (0, 1, 5, 10, 15, and 20 mM) was added to each well. After 24 h incubation, the cells were treated with 200 µL MTT reagents and incubated for another 4 h. Finally, the formazan crystals were dissolved in 100 µL dimethyl sulfoxide (DMSO), and the absorbance at 490 nm was recorded using an enzyme-linked immunosorbent assay (ELISA) reader (SpectraMa, Molecular Devices Co., San Jose, CA, USA). Different final concentrations of AAP-H (0, 1, 5, 10, 15, and 20 mM) were used for treatment prior to the MTT assay.

Determination of Cell Cycle Arrest
To investigate the cytotoxic effect of AAP-H on DU-145 cells, the relative sensitivities of the different phases of the cell cycle were observed using the cell cycle arrest test described by Chen et al. [58], with slight modifications. First, the cells were placed in a 6-well plate and different final concentrations (0, 5, 10, and 15 mM) of APP-H were added when the cells had adhered to the bottom of the plate. After 24 h incubation, the cells were collected by centrifugation, RNase A was added to the cell pellet, and incubated in a water bath at 37 • C for 30 min. Three hundred and fifty microliters of propidium iodide was added to the tubes in dark and incubated at 4 • C for another 40 min. Finally, the cells were sorted using a flow cytometer (Becton Dickinson, NJ, USA).

Western Blot Analysis
DU-145 cells were treated with culture solution containing different concentrations of AAP-H (0, 5, 10, and 15 mM). Then, the treated cells were incubated in a CO 2 incubator for 24 h. After washing thrice with phosphate buffered saline (PBS) (the tumor tissues were placed in a mortar, liquid nitrogen was added, and the tumor was rapidly ground and collected in a centrifuge tube), 200 µL radioimmunoprecipitation assay (RIPA) lysis solution containing 1% phosphatase inhibitor and phenylmethane sulfonyl fluoride (PMSF) was added to each group. The cracking liquid was transferred into a 1.5 mL centrifuge tube and sonicated for 30 min. Subsequently, the cell lysates were collected by centrifugation at 4 • C (12,000 rpm, 15 min). The total protein content of the supernatant was measured using the bicinchoninic acid (BCA) kit. The samples were then denatured by heating in a boiling water bath for 10 min, and the total protein (50 µg) was separated on preprepared 12% sodium dodecyl sulfate-polyacrylamide gels, followed by transfer to polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA). The membranes were stained with Ponceau S and then blocked with 5% nonfat milk for 1 h. After that, the membranes were incubated with the 1:1000 diluted primary antibodies against caspases 3, 7, 8, and 9, AKT, p-AKT (Ser473), PI3K, p-PI3K (p85), p-mTOR (Ser2448), Bax, Bcl-xL, PARP, CDK2, cyclin B1, and cyclin D1 in 5% w/v nonfat milk at 4 • C with gentle shaking for 16 h. Finally, the 1:1000 diluted secondary antibodies (goat anti-rabbit and goat anti-mouse) were added and incubated at room temperature for 1 h. The target bands were detected using enhanced chemiluminescence and quantified by densitometry as shown in DRAFT-alpha view using the Image J 1.38 software (NIH, Bethesda, MD, USA). β-actin was used as the loading control.

Effect of AAP-H on DU-145 Xenografts
Male BALB/c nude mice (nu/nu) were obtained from the Zhejiang Laboratory Animal Center (Hangzhou, China) and housed in specific pathogen-free conditions. DU-145 cells were cultured in Ham F12 medium (10% FBS) at 37 • C in a 5% CO 2 incubator. A suspension of DU-145 cells (in the logarithmic growth stage) were then prepared (0.2 mL, with 1 × 10 6 cells) and injected into the right armpit of nude mice in sterile environment. The tumor nodes appeared after 7 days, which confirmed the successful establishment of the DU-145 tumor-bearing nude mouse model. The body weight was measured after every three days, and the tumor volume was calculated according to the following formula: volume (mm 3 ) = 1/6 × π × width 2 × length. When the average tumor volume reached 60 mm 3 , the mice were randomly divided into four groups with five mice in each group (negative control, positive control treated with 4 mg/kg cisplatin (DDP), and two treatment groups with 100 mg/kg and 150 mg/kg AAP-H). All mice were injected intraperitoneally for 14 days. The experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee of the Zhejiang Ocean University (SYXK, 2014-0013) and adhered to the code of the World Medical Association (Declaration of Helsinki).

Immunocytochemistry (IHC)
The animal experiments were divided into four groups, and each group contained five nude mice. All animals were euthanized, and the three transplanted tumors were selected randomly and cut into 1-cm 3 pieces for IHC. IHC was performed as follows: First, the tumors were placed in 4% paraformaldehyde for 24 h and washed with flowing water overnight. Second, the tumors were dehydrated using an ethanol gradient (75%, 95%, and 100%) for 5 min and transparentized with xylene for 40 min. Then, the tumors were put in paraffin to maintain constant temperature (60 • C) for 1 h. Finally, the tumors were put into a square box, fluid wax was poured into these boxes, and the boxes were refrigerated at 4 • C. Subsequently, 4-µm slices were prepared using a Leica slicer (RM2135, Leica Instruments GmbH, Wetzlar, Germany). The adhered slices were peeled off using a spreading machine (III1210, Leica Instruments GmbH, Wetzlar, Germany) and processed using a baking machine (II1220, Leica Instruments GmbH, Wetzlar, Germany). The paraffin section was used for IHC.

Conclusions
AAP-H exhibited anticancer activity on DU-145 prostate cancer cells by targeting the PI3K/AKT/mTOR signaling pathway and was not toxic for normal fibroblast cells. In addition, AAP-H showed good antiproliferative effect on DU-145 cells. The antitumor mechanism of APP-H on DU-145 cells may involve regulation of the activity of the PI3K/AKT/mTOR signaling pathway, which eventually promotes apoptosis via both the mitochondrial and death receptor pathways ( Figure 11). Therefore, the hydrophobic oligopeptide (YVPGP) will be further studied for use as a functional food or adjuvant for prevention or treatment of prostate cancer.
Mar. Drugs 2018, 16, x 13 of 16 dehydrated, transparentized with xylene, and sealed with a resin. The primary antibodies were replaced with PBS in the negative control. Antigen expression was analyzed using light microscopy (Olympus, Tokyo, Japan).

Conclusions
AAP-H exhibited anticancer activity on DU-145 prostate cancer cells by targeting the PI3K/AKT/mTOR signaling pathway and was not toxic for normal fibroblast cells. In addition, AAP-H showed good antiproliferative effect on DU-145 cells. The antitumor mechanism of APP-H on DU-145 cells may involve regulation of the activity of the PI3K/AKT/mTOR signaling pathway, which eventually promotes apoptosis via both the mitochondrial and death receptor pathways ( Figure 11). Therefore, the hydrophobic oligopeptide (YVPGP) will be further studied for use as a functional food or adjuvant for prevention or treatment of prostate cancer.