Mohammad A Khasawneh, Adrian Koch, Hanan M Elwy, Alaaeldin A Hamza and Regine Schneider- Stock
Leptadenia pyrotechnica has been commonly used traditionally for the treatment of tumors in Gulf countries. However, the molecular mechanism of the anti-cancer activity of Leptadenia pyrotechnica remains unclear. In the present study, 80% ethanolic extract of Leptadenia pyrotechnica and its fractions (n-hexane, EA, n-butanol and aqueous) were evaluated for in vitro cytotoxicity against colon cancer cell lines (HCT 116 wild type and HCT 116 p53−/−). The hexane fraction of Leptadenia pyrotechnica (LHE) is identified as the major anti-cancer fraction in Leptadenia pyrotechnica solvent extracts. Cells were incubated with different concentrations of LHE. Cell viability was quantified by crystal violet and MTT assays. Apoptotic cells were determined using Annexin V-propidium iodide (Annexin V/PI) staining and γH2AX assay for damaged DNA. Bax and Bcl-2, caspase-8, 9, 3 protein expression was analyzed by western blotting. LHE decreased cell viability in colon cancer cells in a dose- and time-dependent manner with an IC50 of 100 μg/ml after 24 h. Further experiments on colon cancer cells treated with LHE at 100 μg/ml for 24 h and 48 h revealed that it induces a p53-dependent apoptosis through intrinsic as well as extrinsic pathways. Analysis of DNA fragmentation by flow cytometry and H2AX assay showed a higher apoptotic cell death in p53 wildtype colon cancer cells treated with LHE in comparison to p53-/- cells. Data of western blots further demonstrated that LHE treatment caused activation of caspases, up regulation of Bax and down regulation of Bcl-2 in a time-dependent manner in HCT 116 cell lines. GC-MS analysis of LHE revealed the presence of derivatives of linoleic acid (octadecadienoic), diterpenes (phytol), triterpenes (squalene), and lupeol as the major constituents. These findings collectively suggest that LHE induces apoptosis in HCT 116 cells possibly mediated by ROS generation and involved multiple pathways including extrinsic, intrinsic and caspase-mediated pathways. Our results suggest that LHE may be applied as a potential anti-cancer drug against human colon cancer having intact p53 function.