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.

Objective: The purpose of this study was to evaluate the anticancer effects of Ophiopogonin D′ (OPD′, a natural product extracted from a traditional Chinese medicine (Radix Ophiopogonis) against androgen-independent prostate cancer cells and to explore the underlying molecular mechanism(s) of action.Methods: The CCK-8 assay was used to assess the viability of prostate cancer cells. The cell morphology was examined by an ultrastructural analysis via transmission electron microscopy. Cells in apoptosis (early and late stages) were detected using an Annexin V-FITC/propidium iodide kit with a FACSCaliber flow cytometer. JC-1, a cationic lipophilic probe, was employed to measure the mitochondrial membrane potential (MMP) of PC3 cells. Changes in the protein expression of RIPK1, C-RIPK1, caspase 8, cleaved-caspase 8, Bim, Bid, caspase 10, and cleaved-caspase 10 were evaluated by Western blotting. The mRNA expression of Bim was examined by quantitative real-time reverse transcription polymerase chain reaction. Z-VAD-FMK (a caspase inhibitor) and necrostatin-1 (a specific inhibitor of RIPK1) were utilized to determine whether the cell death was mediated by RIPK1 or caspases. PC3 and DU145 xenograft models in BALB/c nude mice were used to evaluate the anticancer activity of OPD′ in vivo.Results: OPD′ was shown to exert potent anti-tumor activity against PC3 cells. It induced apoptosis via a RIPK1-related pathway, increased the protein expression levels of RIPK1 and Bim, and decreased the levels of cleaved-RIPK1, caspase 8, cleaved-caspase 8, Bid, caspase 10, and cleaved-caspase 10. OPD′ also increased the mRNA expression of Bim. The protein expression of Bim was decreased when cells were pre-treated with necrostatin-1. Treatment with OPD′ inhibited the growth of PC3 and DU145 xenograft tumors in BALB/c nude mice.Conclusion: OPD′ significantly inhibited the in vitro and in vivo growth of prostate cells via RIPK1, suggesting that OPD′ may be developed as a potential anti-prostate cancer agent.

Background: Voltage-gated sodium channels (VGSCs) are involved in several cellular processes related to cancer cell growth and metastasis, including adhesion, proliferation, apoptosis, migration, and invasion. We here in investigated the effects of S0154 and S0161, two novel synthetic sodium channel blockers (SCBs), on human prostate cancer cells (PC3, DU145, and LnCaP) and a prostate cancer xenograft model. Methods:The MTT assay was used to assess the anticancer effects of SCBs in PC3, DU145, and LnCaP cells. Sodium indicator and glucose uptake assays were used to determine the effects of S0154 and S0161 in PC3 cells. The impact of these SCBs on the proliferation, cell cycle, apoptosis, migration, and invasion of PC3 cells were determined using a CFDA-SE cell proliferation assay, cell cycle assay, annexin V-FITC apoptosis assay, transwell cell invasion assay, and wound-healing assay, respectively.The protein expression levels of Nav1.6, Nav1.7, CDK1, cyclin B1, MMP2, MMP9 in PC3 cells were analysis by Western blotting. The in vivo anticancer activity was evaluated using a PC3 xenograft model in nude mice.Results: S0154 and S0161 both showed anticancer and anti-metastatic effects against prostate cancer cells and significantly inhibited cell viability, with IC 50 values in the range of 10.51-26.60 μmol/L (S0154) and 5.07-11.92 μmol/L (S0161). Both compounds also increased the intracellular level of sodium, inhibited the protein expression of two α subunits of VGSCs (Nav1.6 and Nav1.7), and caused G2/M phase cell cycle arrest, with no or minor effects on cell apoptosis. Concentrations of 5 and 10 μmol/L of S0154 and S0161 significantly decreased the glucose uptake of PC3 cells. The compounds also inhibited the proliferation of PC3 cells and decreased their invasion in transwell assays. Furthermore, S0161 exerted antitumor activity in an in Jiajia Wang and Zongliang Lu contributed equally to this work. wileyonlinelibrary.com/journal/pros The Prostate. 2019;79:62-72.

Castration-resistant (androgen-independent) and PTEN-deficient prostate cancer is a challenge in clinical practice. Sorafenib has been recommended for the treatment of this type of cancer, but is associated with several adverse effects. Platycodin D (PD) is a triterpene saponin with demonstrated anti-cancer effects and a good safety profile. Previous studies have indicated that PC3 cells (PTEN -/-, AR -/-) are sensitive to PD, suggesting that it may also be a useful treatment for castration-resistance prostate cancer. We herein investigated the effects of combining PD with sorafenib to treat PTEN-deficient prostate cancer cells. Our data show that PD promotes sorafenib-induced apoptosis and cell cycle arrest in PC3 cells. Of interest, PD only promoted the anti-cancer effects of sorafenib in Akt-positive and PTEN-negative prostate cancer cells. Mechanistic studies revealed that PD promoted p-Akt ubiquitination by increasing the p-Akt level. PD also increased the protein and mRNA expression of FOXO3a, the downstream target of Akt. Meanwhile, PD promoted the activity of FOXO3a and increased the protein expression of Fasl, Bim and TRAIL. Interestingly, when FOXO3a expression was inhibited, the antitumor effects of both PD and sorafenib were individually inhibited, and the more potent effects of the combination treatment were inhibited. Thus, the combination of PD and sorafenib may exert potent anti-cancer effects specifically via FOXO3a. The use of Akt inhibitors or FOXO3a agonists, such as PD, may represent a promising approach for the treatment of androgen-independent and PTEN-deficient prostate cancer.