OBJECTIVES: The study was aimed to purify obacunone and obacunone glucoside (OG) from seeds of Marsh White grapefruit and understand the mode of cytotoxicity of limonoids on colon cancer (SW480) cells. METHODOLOGY: Both limonoids were purified using chromatographic techniques. The structures and purity of limonoids were confirmed by NMR and HPLC analysis, respectively. RESULTS: Obacunone and OG inhibited SW480 cell proliferation with IC50 values of 97 and 109.7 μM respectively, at 24h. Sequence of events such as decreased ratio of bcl2/bax gene transcription, activation of caspase-3, fragmentation of DNA in cells treated with obacunone and OG demonstrated induction of apoptosis by limonoids. Additionally, higher induction of cytochrome-c in cytosol suggests the activation of intrinsic apoptosis by limonoids. Involvement of apoptosis was also confirmed through expression of bax, bcl2, pro-caspase-3 and caspase-9. Both the limonoids activated p21 and arrested cells at G1 and G2/M phase. Additive activity of proliferation inhibition and activation of caspase-3 by limonoids was observed when combined with camptothecin, demonstrating the induction of apoptosis. In conclusion, both limonoids induced apoptosis by activation of intrinsic apoptosis pathway and activation of p21 leading to arresting cells at G2/M phase of the cell cycle.
OBJECTIVES: The study was aimed to purify obacunone and obacunone glucoside (OG) from seeds of Marsh White grapefruit and understand the mode of cytotoxicity of limonoids on colon cancer (SW480) cells. METHODOLOGY: Both limonoids were purified using chromatographic techniques. The structures and purity of limonoids were confirmed by NMR and HPLC analysis, respectively. RESULTS:Obacunone and OG inhibited SW480 cell proliferation with IC50 values of 97 and 109.7 μM respectively, at 24h. Sequence of events such as decreased ratio of bcl2/bax gene transcription, activation of caspase-3, fragmentation of DNA in cells treated with obacunone and OG demonstrated induction of apoptosis by limonoids. Additionally, higher induction of cytochrome-c in cytosol suggests the activation of intrinsic apoptosis by limonoids. Involvement of apoptosis was also confirmed through expression of bax, bcl2, pro-caspase-3 and caspase-9. Both the limonoids activated p21 and arrested cells at G1 and G2/M phase. Additive activity of proliferation inhibition and activation of caspase-3 by limonoids was observed when combined with camptothecin, demonstrating the induction of apoptosis. In conclusion, both limonoids induced apoptosis by activation of intrinsic apoptosis pathway and activation of p21 leading to arresting cells at G2/M phase of the cell cycle.