Immunomodulatory, Apoptosis Induction and Antitumor Activities of Aqueous and Methanolic Extract of Calvatia Craniiformis in Mice Transfected with Murine Hepatocellular Carcinoma Cells

Abstract

OBJECTIVES: To evaluate the Immunomodulatory, apoptosis induction and antitumor effects of aqueous and methanolic extracts of Calvatia craniiformis regarding the size of tumour mass, caspase-8 expression and apoptotic index (AI%) in mice transfected with murine hepatocellular carcinoma cell line (H22) as an experimental therapeutic system for human hepatocellular carcinoma.

MATERIAL AND METHODS: Forty-eight Balb/C albino mice were transfected in legs with H22 cells. Tumour size was measured twice a week. Caspase-8 protein expression and apoptotic index determination evaluated by Immunohistochemistry.

RESULTS: Tumor size significantly differed between the two groups of mice transfected with H22 cells; the first was treated with C. craniiformis aqueous extract (0.3, 0.6, 1.2) mg/kg and the second group was treated with C. craniiformis methanolic extract (0.25, 0.5, 1.0) mg/kg compared with control group. The inhibitory activity of aqueous and methanolic extracts was dose and duration dependent. The size of the tumour mass was reduced up to 87.9% when treated with 1.2 mg/kg aqueous extract and 1 mg/kg for methanolic extract. Caspase-8 expression was increased in a dose-dependent manner among H22 bearing mice treated with C. craniiformis aqueous extract (0.3, 0.6, 1.2) mg/kg. At 0.3 mg/kg, the intensity of expression was strong in (33.33%) and very strong in (66.67%). While at 0.6 mg/kg and 1.2 mg/kg the intensity of expression was strong in (33.33%) and very strong in (100%) with a significant difference (P ≤ 0.001). H22 bearing mice treated with (0.25, 0.5, 1.0) mg/kg C. craniiformis methanolic extract shows increased caspase-8 expression in a dose-dependent manner. At 0.25 mg/kg, the intensity of expression was strong in (33.33%) and very strong in (66.67%). While at 0.5 mg/kg, the intensity of expression was strong in (33.33%) and very strong in (100%). At 1.0 mg/kg, the intensity of expression was strong in (16.67%) and very strong in (83.33%) with significant difference (P ≤ 0.001). AI% of H22 bearing mice treated with C. craniiformis aqueous and methanolic extracts were significantly increased (P ≤ 0.05) compared with the untreated control group. No significant difference was reported in AI% between aqueous and methanolic extracts treated groups.

CONCLUSIONS: Extracts of C. craniiformis were highly efficient in tumour growth inhibition, causing a reduction in the tumour size clinically and increase the expression of caspase-8 gene product in tumour tissue, causing increase apoptotic index of H22 cells taken from the legs of inoculated mice leading to loss of legs due to bone necrosis. Antitumor activity of C. craniiformis aqueous, and the methanolic extract was dose and duration dependent.