IMMUNOHISTOCHEMICAL DETECTION OF CASPASE 8 EXPRESSION AND APOPTOTIC INDEX ACTIVITIES OF CALVATIA CRANIIFORMIS CRUDE EXTRACT IN BALB/C MICE INOCULATED WITH H22 CELLS

Objectives: To identify Immunomodulatory, apoptosis induction and anticancer activities of aqueous and alcoholic extracts of Calvatia craniiformis regarding the size of tumor mass and immunohistochemical detection of caspase 8 expressions   and apoptotic index in mice inoculated   with H22 mouse hepatocellular carcinoma cells as therapeutic experimental system for human hepatocellular carcinoma. Materials and methods: Forty-eight Balb/C albino mice were injected in legs with H22 cells. Tumor size was measured twice a week.  Immunohistochemistry used for evaluation of Caspase-8 protein expression and apoptotic index was determined. Results: Administration of aqueous extract of C. craniiformis in (0.3, 0.6, 1.2) mg/kg and alcoholic extract in (0.25, 0.5, 1) mg/kg to H22 tumor-bearing mice show significant difference (P≤0.01) in tumor size compared with control group.  Inhibitory activity of aqueous and alcoholic extracts was dose and duration dependent. Effective doses   in reduction of tumor size were 1.2mg/kg   for aqueous and 1mg/kg for alcoholic extract in which H22 tumor mass reduced in size for up to 87.9 %. H22 bearing mice treated with C. craniiformis aqueous and alcoholic extracts shows significant increase (P ≤ 0.05) in apoptotic index % compared with untreated control group. Conclusions: Extracts of C. craniiformis were highly efficient in H22 tumor growth inhibition, causing reduction in the tumor size clinically and increase the expression of caspase 8 gene product in tumor tissue. This effect causing increase in apoptotic index of H22 tumor cells taken from legs of inoculated animals, causing protection of H22 inoculated legs from losing compared with untreated control group which loss their legs due to necrosis and destruction in the bones. Antitumor activity of C. craniiformis aqueous and alcoholic extract was dose and duration dependent. These finding indicate the usefulness of C. craniiformis extracts as a novel antitumor agent for hepatocellular carcinoma, with its proved apoptosis induction through caspase 8 activation pathway.