cytochrome-c-t and amooranin

25-Hydroxy-3-oxoolean-12-en-28-oic acid (Amooranin-AMR) is a triterpene acid isolated from the stem bark of a tropical tree (Amoora rohituka) grown wild in India. A herbal preparation used for the treatment of cancer by the Ayurvedic system of medicine contains the stem bark of Amoora rohituka as one of the ingredients. In this paper, we show that AMR displays a strong inhibitory effect on survival of human breast carcinoma MDA-468, breast adenocarcinoma MCF-7 cells compared to breast epithelial MCF-10A control cells. A 50% decrease in cells (IC50) ranged from 1.8 to 14.6 microM and cell growth was suppressed by arresting cell cycle at G2 + M phase. AMR effectively induces apoptosis and triggered a series of effects associated with apoptosis including cleavage of caspase-8, -9, -3, Bid and ER stress in MDA-468 cells and caspase- 8, -9, -6 and Bid in MCF-7 cells, release of cytochrome c from the mitochondria, cleavage of poly (ADP-ribose) polymerase (PARP) and DNA fragmentation with a concomitant upregulation of p53, Bax and down-regulation of Bcl-2 in MDA-468 cells, but Bax unchanged in MCF-7 cells. The use of caspase blocking peptides and acridine orange staining confirmed the involvement of primarily caspase-9 and -3 in MDA-468 cells with mutated p53 and primarily caspase-8, -9 and -6 in MCF-7 cells expressing wt p53. We also observed in MCF-7/p53siRNA cells AMR treatment caused reduced expression of Bcl-2 without affecting levels of Bax similar to MCF-7 cells treated with AMR and proteolytic activation of Bax in MDA-468 cells. These results suggest that AMR induces apoptosis in human breast carcinoma cells via caspase activation pathway and likely it is a p53-independent apoptosis.

Topics: Apoptosis; Blotting, Western; Breast Neoplasms; Caspase 3; Caspase 7; Caspase 9; Caspase Inhibitors; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Female; Flow Cytometry; Humans; Molecular Structure; RNA, Small Interfering; Signal Transduction; Time Factors; Triterpenes; Tumor Suppressor Protein p53

Amooranin (AMR), a natural triterpenoid drug isolated and characterized from Amoora rohituka stem bark, is cytotoxic to SW620 human colon carcinoma cell line with an IC(50) value of 2.9 microg/ml. This novel compound caused depolarization of mitochondrial membrane and decrease of membrane potential, indicating initial signal of apoptosis induction. The percentage of cells with decreased mitochondrial potential ranged from 7.4% at 1 microg/ml to 60.5% at 100 microg/ml AMR. Flow cytometric analysis of apoptosis using Annexin-V-FITC staining showed that the percentage of apoptotic cells ranged from 7.5% at 1 microg/ml to 59.2% at 100 microg/ml AMR. AMR-induced apoptosis was accompanied by redistribution of cytochrome c from mitochondria to cytosol as well as down regulation of Bcl-2 and Bcl-X(L) proteins in a dose-dependent manner. SW620 human colon carcinoma xenograft mice treated with AMR showed significant reduction in tumor growth rates compared to saline- and doxorubicin-treated groups. The reduction in tumor growth rate was better in xenografts treated with 2 mg/kg AMR than 5 and 10 mg/kg treated mice. The analysis of global gene expression changes induced by AMR in xenograft tumors by microarray hybridization revealed that several genes involved in energy pathways, transport, apoptosis, immune response, nucleic acid metabolism, protein metabolism, cell growth and/or maintenance, signal transduction and cell communication, were affected by this natural cancer drug. These results suggest that the anticancer properties of AMR in SW620 human colon carcinoma cell line are mediated through its effects on functional genomics, targeting the apoptotic process.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-X Protein; Carcinoma; Cell Line, Tumor; Colonic Neoplasms; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Down-Regulation; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; Transplantation, Heterologous; Triterpenes