Platycodin D (PD), a major saponin derived from Platycodin grandiflorum, exerted cytotoxicity against prostate cancer cell lines (PC3, DU145 and LNCaP cells) with IC50 values in the range of 11.17 to 26.13μmol/L, whereas RWPE-1cells (a non-malignant human prostate epithelial cell line) were not significantly affected. A further study in these cell lines showed that PD could potently affect cell proliferation (indicated by the bromodeoxyuridine assay), induce cell apoptosis (determined by Annexin V-FITC flow cytometry) and cause cell cycle arrest (indicated by PI staining). After being treated with PD for 48 hours, DU145 and LNCaP cells were arrested in the G0 /G1 phase, and PC3 cells were arrested in the G2/M phase. A Western blotting analysis indicated that PD increased the expression of the FOXO3a transcription factor, decreased the expression of p-FOXO3a and MDM2 and increased the expression of FOXO-responsive genes, p21 and p27. MDM2 silencing (transiently by siRNA-MDM2) increased the PD-induced FOXO3a protein expression, while MDM2 overexpression (in cells transiently transfected with a pcDNA3-MDM2 plasmid) decreased the PD-induced expression of the FOXO3a protein. Moreover, PD dose-dependently inhibited the growth of PC3 xenograft tumors in BALB/c nude mice. A Western blotting analysis of the excised xenograft tumors indicated that similar changes in protein expression also occurred in vivo. These results suggest that PD exhibits significant activity against prostate cancer in vitro and in vivo. The FOXO3a transcription factor appears to be involved in the activity of PD. Together, all of these findings provide a basis for the future development of this agent for human prostate cancer therapy.

Forkhead box O3 (FOXO3a) is a transcription factor with tumor suppressor functions that plays an important role in prostate cancer. Daidzein, one of the soy isoflavones present in soy-based foods, has been shown to exert anti-tumor effects in vitro and in vivo. We herein investigated the inhibitory effects of S-equol, an isoflavandiol metabolized from daidzein by bacterial flora in the intestines, on the LnCaP, DU145 and PC3 human prostate cancer cell lines. Our results showed that S-equol and R-equol inhibited the growth of all three cell lines. Additional studies revealed that S-equol caused cell cycle arrest in the G2/M phase in PC3 cells by downregulating Cyclin B1 and CDK1 and upregulating CDK inhibitors (p21 and p27), as well as inducing apoptosis by upregulating Fas ligand (FasL) and the expression of proapoptotic Bim. Additionally, S-equol increased the expression of FOXO3a, decreased the expression of p-FOXO3a and enhanced the nuclear stability of FOXO3a. S-equol also decreased the expression of MDM2, which serves as an E3 ubiquitin ligase for p-FOXO3a, thus preventing p-FOXO3a degradation by the proteasome. Mechanistic studies showed that S-equol targeted the Akt/FOXO3a pathway, which is important for prostate cancer cell survival, cell cycle progression and apoptosis. Moreover, treatment with S-equol inhibited the growth of PC3 xenograft tumors in BALB/c nude mice. Overall, the data from the present study demonstrate that S-equol has significant anti-prostate cancer activities in vitro and in vivo, and indicate that its anticancer effects were likely associated with the activation of FOXO3a via an Akt-specific pathway and inhibitory effects on MDM2 expression. The results not only provide a better understanding of the molecular mechanisms of this unique secondary metabolite of a natural anti-cancer compound, but also provide a basis for the development of daidzein and its analogs as novel anticancer agents.

Ophiopogonin D' (OPD') is a natural compound extracted from Ophiopogon japonicus, which is a plant used in traditional Chinese medicine. Our previous study has indicated that OPD' exhibits antitumor activity against androgen-independent prostate cancer (PCa), but the effects and the underlying molecular mechanism of action of OPD' in androgen-dependent PCa were unclear. In the present study, OPD' induced significant necroptosis in androgen-dependent LNCaP cancer cells by activating receptor-interacting serine/threonine-protein kinase 1 (RIPK1). Exposure to OPD' also increased Fas ligand (FasL)-dependent RIPK1 protein expression. The OPD'-induced necroptosis was inhibited by a RIPK1 inhibitor necrostatin-1, further supporting a role for RIPK1 in the effects of OPD´. The antitumor effects of OPD' were also inhibited by a mixed lineage kinase domain-like protein (MLKL) inhibitor necrosulfonamide. Following treatment with inhibitors of RIPK1 and MLKL, the effects of OPD' on LNCaP cells were inhibited in an additive manner. In addition, co-immunoprecipitation assays demonstrated that OPD' induced RIPK3 upregulation, leading to the assembly of a RIPK3-MLKL complex, which was independent of RIPK1. Furthermore, OPD' increased the expression of Fas-associated death domain, which is required to induce necroptosis in LNCaP cells. OPD' also regulated the expression levels of FasL, androgen receptor and prostate-specific antigen in a RIPK1-dependent manner. These results suggested that OPD' may exhibit potential as an anti-PCa agent by inducing RIPK1-and MLKL-dependent necroptosis.

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BackgroundThe extract of Platycodon grandiflorum has been reported to have effective spermicidal activity. This study was designed to evaluate the spermicidal and contraceptive activity, as well as the safety, of Platycodin D (PD), a major saponin in Platycodon grandiflorum.MethodsUsing the computer-aided sperm analysis (CASA) test criteria, the sperm-immobilizing activity of PD was studied using highly motile human sperm. The sperm viability was assessed by fluorescent staining using SYBR-14 (living sperm) and propidium iodide (dead sperm). The sperm membrane integrity was assessed by evaluating the hypo-osmotic swelling (HOS) and examinations by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The in vivo contraceptive efficacy was evaluated in rats using post-intrauterine PD application. The comet assay was employed to determine whether PD caused DNA damage in the sperm. Vaginal biopsies were also performed to determine whether the PD gel induced vaginal inflammation.ResultsA dose-dependent effect of PD on the sperm motility and viability was observed. The maximum spermicidal effect was observed with a 0.25 mM concentration of PD. More than 70% of the PD-treated sperm lost their HOS responsiveness at a concentration of 0.20 mM PD, indicating that PD caused injury to the sperm plasma membrane. TEM and SEM revealed significant damage to both the head and tail membranes of the sperm. PD decreased the fertility to zero in rats, was non-DNA damaging and was not harmful to the vaginal tissue in the rats.ConclusionPD has significant spermicidal activity that should be explored in further studies.