Methanolic extract of Anthocephalus cadamba induces apoptosis in Ehrlich ascites carcinoma cells in experimental mice

OBJECTIVE: Anthocephalus cadamba (Roxb.) Miq. (Family: Rubiaceae), a folk medicine commonly known as “Kadam” in Bengali, has been used for the treatment of tumor. The methanolic extract of A. cadamba (MEAC) showing antitumor activity on Ehrlich ascites carcinoma (EAC) cells treated mice was already reported. This study was designed to study the apoptosis-inducing property of MEAC and its mechanism in EAC cells in mice. MATERIALS AND METHODS: Apoptogenic morphology was determined by fluorescent DNA-binding double staining method using dyes acridine orange (AO)/ethidium bromide (EB). Comet assay was estimated to check the DNA damage. Flow cytometry (fluorescence-activated cell sorting [FACS]) was used to detect the apoptotic rate quantitatively by double labeling techniques using annexin V FITC/propidium iodide staining. Apoptotic protein expression was done using Western blotting assay method. STATISTICAL ANALYSIS: Results are expressed as mean ± standard deviation. Statistical analysis was performed using ANOVA followed by Dunnett's post hoc test of GraphPad Prism software. *P < 0.05, **P < 0.01 and ***P < 0.001 were considered statistically significant. RESULTS: Apoptosis-inducing effect of MEAC on EAC cells was confirmed from AO/EB staining and FACS analysis. MEAC treatment showed dose-dependent induction of DNA damage. Apoptosis was induced by increasing the expression of multiple downstream factors such as pro-apoptotic protein p53 and p21 in EAC. Bax was up-regulated and anti-apoptotic protein Bcl-2 was down-regulated resulting in decrease of the Bcl-2/Bax ratio by MEAC treatment. CONCLUSION: Experimental results revealed that MEAC induces apoptosis by modulating the expression of some pro-apoptotic and anti-apoptotic proteins in EAC and thus exerts its anti-tumor activity.