Potential anti-hepatocellular carcinoma properties and mechanisms of action of clerodane diterpenes isolated from Polyalthia longifolia seeds

Abstract

Diterpenes are secondary metabolites that have attracted much attention due to their potential biological activities including anti-cancer potential. The aim of the current study is to assess the anticancer potential of the six known clerodane diterpenes (1-6) isolated from Polyalthia longifolia seeds and their underlying molecular mechanisms. These compounds were evaluated for their cytotoxicity in vitro by using MTT assays. The "two-phase model" with NDEA and PB ad libitum was used for induction of HCC and sorafenib was used as the standard drug. Prophylactic studies were carried out for compounds 4/6 at both low (5 mg/kg b.w) and high (10 mg/kg b.w) doses. Based on the MTT assay results, the two best compounds, 4 and 6, were selected for in vivo studies. The results showed that treatment with compound 4/6 significantly restored the changes in biochemical parameters and liver morphology observed in (NDEA + PB)-induced HCC rats. Additionally, the docking studies showed that compound 4/6 interacted with several key proteins such as MDM2, TNF-α, FAK, thereby inhibiting these proteins and reversing the negative impacts of NDEA. In conclusion, our results suggested that compounds 4 and 6 are potential therapeutic agents for HCC, mostly due to their ability to control typical cancer pathways.

Figure 1

Chemical representation of clerodane diterpenes (1–6) from the Polyalthia longifolia (Sonn.) Thwaites seeds.

Figure 2

(a) Half-maximal inhibitory concentration (IC₅₀) values of clerodane diterpenes against HepG2 and Huh 7 cancer cell lines. Those with IC₅₀ values above 50 µg/mLwere considered as inactive and indicated with “x”. (b) The counteractive effect of 4 and 6 on body weight of (NDEA + PB)-induced hepatocellular carcinoma in Sprague–Dawley rats. Macroscopic features of livers from (c): Normal control; (d) Disease control (NDEA + PB); (e) Standard control (NDEA + PB + Sorafenib); and four tested groups: (f) NDEA + PB + 4-LD; (g) NDEA + PB + 4-HD; (h) NDEA + PB + 6-LD with a single nodule indicated by a red triangle; (i) NDEA + PB + 6-HD. NDEA: N-nitrosodiethylamine (200 mg/kg); PB: phenobarbital sodium (500 parts per million). Sorafenib: 30 mg/kg; 4-/6-LD: 5 mg/kg; 4-/6-HD: 10 mg/kg.

Figure 3

Effect of 4 and 6 on biochemical parameters of (NDEA + PB)-induced hepatocellular carcinoma in Sprague–Dawley rats. (a) Aspartate aminotransferase (AST) levels; (b) Alanine aminotransferase (ALT) levels; (c) Alkaline phosphatase (ALP) levels; (d) Gamma glutamyl transpeptidase (GGT) levels; (e) α-Fetoprotein; (f) CD4/CD8 ratio; (g) Vitamin D; (h) Tumor necrosis factor-α (TNF-α); (i) Tissue growth factor-β (TGF-β); (j) Interleukin-6 (IL-6); (k) Interleukin-10 (IL-10); (l) Interleukin-1 β (IL-1β); (m) Malondialdehyde (MDA); (n) Superoxide dismutase (SOD); (o) Catalase (CAT); (p) Glutathion reductase (GSH). All the values were expressed as mean ± standard error of the mean where n = 10 animals. The data were analyzed by one-way analysis of variances, followed by Tukey’s test, where *P < 0.05, **P < 0.01, and ***P < 0.001 statistically significant when compared with the disease control, and ^###P < 0.001 statistically significant when compared with normal control. The values in the parentheses indicate the percentage protection. NDEA: N-nitrosodiethylamine (200 mg/kg); PB: phenobarbital sodium (500 parts per million). Sorafenib: 30 mg/kg; 4-/6-LD: 5 mg/kg; 4-/6-HD: 10 mg/kg.

Figure 4

Effect of compounds 4 and 6 on histopathological properties of (NDEA + PB)-induced hepatocellular carcinoma in Sprague–Dawley rats at 100x. (a): Normal control; (b) Disease control (NDEA + PB); (c) Standard control (NDEA + PB + Sorafenib); and four tested groups: (d) NDEA + PB + 4-LD; (e) NDEA + PB + 4-HD; (f) NDEA + PB + 6-LD; (g) NDEA + PB + 6-HD. NDEA: N-nitrosodiethylamine (200 mg/kg); PB: phenobarbital sodium (500 parts per million). Sorafenib: 30 mg/kg; 4-/6-LD: 5 mg/kg; 4-/6-HD: 10 mg/kg. Yellow right arrow: Healthy hepatocytes; Blue isosceles triangle: central vein; Red teardrop: fibrosis and cytoplasmic fat infiltration; Red pentagon: regeneration of necrotic hepatocytes; Red chevron: tumor nodules.

Figure 5

Interaction between compounds 4 and 6 and the target proteins. The 3D interaction and hydrogen bonds between (a) 4-MDM2, (b) 4-TNF-α, (c) 4-IL-6, and (d) 4/6-FAK, (e) 6-MDM2, (f) 6-TNF-α, (g) 6-TGF-β. Hydrogen bond interaction between proteins and ligand shown in green color, and receptor active sites shown in red color.

Figure 6

General experimental procedure and the key protective effects of compounds 4 and 6 against (NDEA + PB)-induced HCC in rats. HCC: hepatocellular carcinoma; SCMC: sodium carboxymethyl cellulose; NDEA: N-nitrosodiethylamine; PB: phenobarbital sodium; Comp.: compound; b.w: body weight.