cytochrome-c-t and obacunone

Obacunone and obacunone glucoside (OG) are naturally occurring triterpenoids commonly found in citrus and other plants of the Rutaceae family. The current study reports the mechanism of cytotoxicity of citrus-derived obacunone and OG on human androgen-dependent prostate cancer LNCaP cells. Both limonoids exhibited time- and dose-dependent inhibition of cell proliferation, with more than 60% inhibition of cell viability at 100 μM, after 24 and 48 h. Analysis of fragmentation of DNA, activity of caspase-3, and cytosolic cytochrome-c in the cells treated with limonoids provided evidence for activation of programmed cell death by limonoids. Treatment of LNCaP cells with obacunone and OG resulted in dose-dependent changes in expression of proteins responsible for the induction of programmed cell death through the intrinsic pathway and down-regulation of Akt, a key molecule in cell signaling pathways. In addition, obacunone and OG also negatively regulated an inflammation-associated transcription factor, androgen receptor, and prostate-specific antigen, and activated proteins related to the cell cycle, confirming the ability of limonoids to induce cytotoxicity through multiple pathways. The results of this study provided, for the first time, an evidence of the cytotoxicity of obacunone and OG in androgen-dependent human prostate cancer cells.

Topics: Apoptosis; Benzoxepins; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Citrus paradisi; Cytochromes c; DNA Fragmentation; Dose-Response Relationship, Drug; Down-Regulation; Glucosides; Humans; Inflammation; Limonins; Male; Plant Extracts; Prostate-Specific Antigen; Prostatic Neoplasms; Proto-Oncogene Proteins c-akt; Receptors, Androgen; Signal Transduction; Triterpenes

Obacunone is one of the oxygenated triterpenoids found in rutaceae family. It has been demonstrated for various biological activities including inhibition of cancer cells proliferation and tumor. The current study is an attempt to understand the mode of cytotoxicity of obacunone using cultured pancreatic cancer (MDA Panc-28) cells. Obacunone exhibited dose and time dependant inhibition of Panc-28 cells proliferation . This was also associated with phosphatidylserine translocation from inner surface of plasma membrane to outer surface in the treated cells, suggesting the involvement of apoptosis. Analysis of cells treated with 50 and 100 μM of obacunone suggests activation of initiator caspase-9 and effector caspase-3, indicating involvement of caspases induced apoptosis. Obacunone upregulated expression of tumor suppressor protein p53, pro-apoptotic protein Bax and downregulated anti-apoptotic protein Bcl2. Furthermore, release of cytochrome-c into cytosol was observed in the cells treated with obacunone. It also resulted in down regulation of vital inflammatory mediators such as NFκB and Cox-2 suggesting the anti-inflammatory potential. For the first time, current study provides an evidence on activation of caspase dependant, cytochrome-c mediated intrinsic apoptosis and anti-inflammatory activity in Panc-28 cells by citrus obacunone. Induction of cytotoxicity and apoptosis was also confirmed by fluorescent tagged microscopic images of obacunone treated Panc-28 cells.

Topics: Anti-Inflammatory Agents; Apoptosis; Benzoxepins; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Proliferation; Citrus; Cytochromes c; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Inflammation Mediators; Limonins; Pancreatic Neoplasms; Time Factors

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.. Both limonoids were purified using chromatographic techniques. The structures and purity of limonoids were confirmed by NMR and HPLC analysis, respectively.. 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.

Topics: Amino Acid Sequence; Animals; Apoptosis; bcl-2-Associated X Protein; Benzoxepins; Camptothecin; Caspase 3; Caspase 8; Caspase 9; Cell Cycle; Cell Line, Tumor; Chromatography, High Pressure Liquid; Citrus paradisi; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; DNA Fragmentation; Down-Regulation; Glucosides; Humans; Limonins; Magnetic Resonance Spectroscopy; Mice; Molecular Sequence Data; NIH 3T3 Cells; Seeds; Triterpenes; Up-Regulation