Evaluating anticancer activity of emodin by enhancing antioxidant activities and affecting PKC/ADAMTS4 pathway in thioacetamide-induced hepatocellular carcinoma in rats

Abstract

Emodin is a naturally occurring anthraquinone derivative with a wide range of pharmacological activities, including neuroprotective and anti-inflammatory activities. We aim to assess the anticancer activity of emodin against hepatocellular carcinoma (HCC) in rat models using the proliferation, invasion, and angiogenesis biomarkers. After induction of HCC, assessment of the liver impairment and the histopathology of liver sections were investigated. Hepatic expression of both mRNA and protein of the oxidative stress biomarkers, HO-1, Nrf2; the mitogenic activation biomarkers, ERK5, PKCδ; the tissue destruction biomarker, ADAMTS4; the tissue homeostasis biomarker, aggregan; the cellular fibrinolytic biomarker, MMP3; and of the cellular angiogenesis biomarker, VEGF were measured. Emodin increased the survival percentage and reduced the number of hepatic nodules compared to the HCC group. Besides, emodin reduced the elevated expression of both mRNA and proteins of all PKC, ERK5, ADAMTS4, MMP3, and VEGF compared with the HCC group. On the other hand, emodin increased the expression of mRNA and proteins of Nrf2, HO-1, and aggrecan compared with the HCC group. Therefore, emodin is a promising anticancer agent against HCC preventing the cancer prognosis and infiltration. It works through many mechanisms of action, such as blocking oxidative stress, proliferation, invasion, and angiogenesis.

Keywords: ADAMTS4; Aggrecan; Angiogenesis; ERK5; HO-1; MMP3; Nrf2; PKCδ; VEGF; emodin; hepatocellular carcinoma.

Figure 1.

Timeline of the animal treatment in the study.

Figure 2.

Effect of oral administration of emodin (40 mg/kg, P.O) on both mRNA and protein levels of the antioxidant biomarkers; Nrf2 and HO-1. (a) The relative expression of the hepatic Nrf2 mRNA, (b) the relative hepatic NRF2 protein levels, (c) the relative expression of the hepatic HO-1 mRNA and (d) the relative hepatic HO-1 protein. * Significant difference as compared with the control groups at p < 0.05. # Significant difference as compared with the HCC group at p < 0.05. C, control; HCC, hepatocellular carcinoma; HO-1; Heme oxigenase variant 1; Nrf2, nuclear factor erythroid 2-related factor 2.

Figure 3.

Effect of oral administration of emodin (40 mg/kg, P.O) on oxidative status of the hepatic cells. (a) The relative hepatic tissues of MDA. (b) The relative hepatic tissues of reduced glutathione. * Significant difference as compared with the control groups at p < 0.05. # Significant difference as compared with the HCC group at p < 0.05. C, control; HCC, hepatocellular carcinoma; MDA; malondialdehyde.

Figure 4.

Effect of oral administration of emodin (40 mg/kg, P.O) on the percent survival and hepatic nodules. (a) The percent of the survival of the four groups of the experiment over sixteen weeks. (b) Representative images of livers separated from different treated groups. (c) Relative serum cancer market AFP in tested groups. (d) The average number of hepatic nodules in groups. * Significant difference as compared with the control groups at p < 0.05. # Significant difference as compared with the HCC group at p < 0.05. C, control; HCC, hepatocellular carcinoma; AFP, alpha Fetoprotein.

Figure 5.

Effect of oral administration of emodin (40 mg/kg, P.O) on liver sections stained with Haematoxylin/eosin. (a) Representatives of microscopic pictures of liver sections stained with haematoxylin/eosin showing normally arranged hepatic cords around central veins. Liver section from HCC group show well-differentiated HCC. Cells of HCC are polygonal with distinct cell membranes, an eosinophilic granular cytoplasm and prominent nucleoli (black arrows). Sections from HCC group orally treated with 40 mg/kg emodin show improvement of the structure of hepatocytes. Scale bare represented 50 µm. (b) Mitotic score calculated using high field powers in 10 different areas of each rat. The results of the mitotic figure are presented as the mean ± SE. * Significant difference as compared with the control groups at p < 0.05. # Significant difference as compared with the HCC group at p < 0.05. C, control; HCC, hepatocellular carcinoma.

Figure 6.

Effect of oral administration of emodin (40 mg/kg, P.O) on both mRNA and protein levels of the mitogenic activation biomarkers; PKCδ and ERK5. (a) The relative expression of the hepatic PKCδ mRNA, (b) the relative hepatic PKCδ protein, (c) the relative expression of the hepatic ERK5 mRNA and (d) the relative hepatic ERK5 protein. * Significant difference as compared with the control groups at p < 0.05. # Significant difference as compared with the HCC group at p < 0.05. C, control; ERK5, extracellular-signal-regulated kinase 5; HCC, hepatocellular carcinoma; PKCδ; Protein kinase C delta type.

Figure 7.

Effect of oral administration of emodin (40 mg/kg, P.O) on liver sections stained with anti-ADAMTS4 antibodies. (a) Representative images of hepatic tissues stained with anti-ADAMTS-4 antibodies in different treated groups showing increased staining in the HCC group, which was reduced by treatment with emodin. (b) The positive staining score was determined through immunohistochemistry in ten different fields. Black arrows indicated the areas of positive immune staining. Scale bar represents 100 μm.

Figure 8.

Effect of oral administration of emodin (40 mg/kg, P.O) on mRNA expression of hepatic ADAMTS4, aggrecans, MMP3 and VEGF. (a) The relative expression of the hepatic ADAMTS-4 mRNA and (b) the relative expression of the hepatic aggrecans mRNA. (c) the relative expression of the hepatic MMP3 mRNA. (d) the relative expression of the hepatic VEGF mRNA. * Significant difference as compared with the control groups at p < 0.05. # Significant difference as compared with the HCC group at p < 0.05. ADAMTS4, A disintegrin and metalloproteinase with thrombospondin motifs 4; C, control; HCC, hepatocellular carcinoma; MMP3, matrix metallopeptidase 3; VEGF, vascular endothelial growth factor.

Figure 9.

Effect of oral administration of emodin (40 mg/kg, P.O) on protein expression of hepatic ADAMTS4, aggrecans, MMP3 and VEGF. (a) Representative images of Western blot analysis of hepatic ADAMTS4, aggrecans MMP3 and VEGV. (b) ROD of Western blot analysis of ADAMTS4 in relation to control. (c) ROD of Western blot analysis of aggrecans in relation to control. (d) ROD of Western blot analysis of MMP3 in relation to control. (e) ROD of Western blot analysis of VEGF in relation to control. * Significant difference as compared with the control groups at p < 0.05. # Significant difference as compared with the HCC group at p < 0.05. ADAMTS4, A disintegrin and metalloproteinase with thrombospondin motifs 4; C, control; HCC, hepatocellular carcinoma; MMP3, matrix metallopeptidase 3; VEGF, vascular endothelial growth factor.