Ophiopogonin D' induces RIPK1‑dependent necroptosis in androgen‑dependent LNCaP prostate cancer cells

Abstract

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.

Figure 1

The chemical structure and biological activity of OPD'. (A) The chemical structures of OPD' and OPD. (B) The viability of LNCaP cells was examined by Cell Counting Kit-8 assay after treatment with OPD' or Sor (positive control) for 24 h. The concentrations that induced 50% growth inhibition (IC₅₀) were calculated (n=3). ^* P<0.05 vs. Sor. OPD, Ophiopogonin D; Sor, sorafenib.

Figure 2

OPD' induces necrosis in LNCaP cells. (A) Following LNCaP cell treatment with 2.5 or 5 µM OPD' for 24 h, the apoptotic rates were assessed by Annexin V-FITC/PI assay (n=3). ^*P<0.05 vs. vehicle. (B) The Cell Counting Kit-8 assay was used to analyze the effects of OPD' on cell survival following pre-treatment of LNCaP cells with Z-VAD-FMK (n=3). (C) Western blotting was used to examine the protein expression of MLKL and p-MLKL following 6-h treatment with OPD'. (D) Annexin V-FITC/PI staining was used to analyze the apoptotic rates of LNCaP cells following treatment with OPD' and NSA. (E) Treatment with 5 µM OPD' induced changes in cell morphology, as indicated by transmission electron microscopy. Arrows indicate leaked contents of the cell and cleavage of the cell nucleus. ^*P<0.05. OPD', Ophiopogonin D'; Sor, sorafenib; PI, propidium iodide; MLKL, mixed lineage kinase domain-like protein; NSA, necrosulfonamide; p, phosphorylated.

Figure 3

OPD' induces necroptosis in a RIPK1-dependentmanner. (A) Western blotting was used to examine the protein expression of RIPK1, cleaved-RIPK1, caspase 8 and cleaved-caspase 8 following treatment with OPD' for 6 h. (B) Annexin V-FITC/PI staining was used to analyze the LNCaP cell status after treatment with OPD' and/or Nec-1 (n=3). ^*P<0.05. OPD', Ophiopogonin D'; Nec-1, necrostatin-1; RIPK1, receptor-interacting serine/threonine-protein kinase 1; PI, propidium iodide; C, cleaved.

Figure 4

OPD' induces necroptosis by multiple pathways involving RIPK1 and RIPK3/MLKL. (A) Western blotting was used to examine the protein expression of RIPK3 following treatment with OPD' for 6 h. (B) LNCaP cells were exposed to 5 µM OPD' for 6 h, then the protein lysates were immunoprecipitated using anti-RIPK3 antibodies. The target proteins RIPK1, MLKL and RIPK3 were examined by western blotting. (C) The Cell Counting Kit-8 assay was used to analyze the survival of LNCaP cells in response to OPD' following pre-treatment with Nec-1 and/or NSA (n=3). (D) Annexin V-FITC/PI staining was used to analyze the apoptotic rates of LNCaP cells exposed to OPD' for 24 h following combined pre-treatment with Nec-1 and NSA for 2 h. ^*P<0.05. OPD', Ophiopogonin D'; Nec-1, necrostatin-1; NSA, necrosulfonamide; RIPK, receptor-interacting serine/threonine-protein kinase; MLKL, mixed lineage kinase domain-like protein; PI, propidium iodide.

Figure 5

OPD' regulates FasL, androgen receptor and prostate-specific antigen through RIPK1. (A) Western blotting was used to detect the effects of OPD' on target proteins FasL, Fas and Bim in LNCaP cells. (B) Western blotting was used to detect the effects of OPD' on the protein expression of FasL following treatment with Nec-1 and/or OPD'. (C) Western blotting was used to detect the effects of OPD' on target proteins AR and PSA in LNCaP cells. (D) Western blotting was used to detect the effects of OPD' on the protein expression of AR and PSA following treatment with Nec-1 and/or OPD'. OPD', Ophiopogonin D'; FasL, Fas ligand; Nec-1, necrostatin-1; Bim, Bcl-2-like protein 11; AR, androgen receptor; PSA, prostate-specific antigen.

Figure 6

OPD' determines the pattern of cell death mediated by FADD. (A) The baseline FADD protein expression in LNCaP, PC3 and DU145 cells was examined by western blotting. (B) LNCaP, PC3 and DU145 cells were treated with OPD' for 6 h, and FADD protein expression was examined by western blotting. (C) Annexin V-FITC/PI staining was used to analyze the apoptotic rates of LNCaP cells following treatment with OPD' and siRNA-FADD (n=3). (D) The Cell Counting Kit-8 assay was used to analyze the viability of LNCaP and PC3 cells following treatment with OPD' and NAC (n=3). ^*P<0.05. OPD', Ophiopogonin D'; FADD, Fas-associated death domain; siRNA, small interfering RNA; C, siRNA-control; siRNA-F1, siRNA-FADD#1; siRNA-F2, siRNA-FADD#2; PI, propidium iodide; NAC, N-acetylcysteine.

Figure 7

A schematic representation of the anti-cancer effects of OPD' in LNCaP and PC3 cells. RIPK, receptor-interacting serine/threonine-protein kinase; MLKL, mixed lineage kinase domain-like protein; FADD, Fas-associated death domain; FASL, Fas ligand; p, phosphorylated; siRNA, small interfering RNA.