Cytotoxic and Molecular Assessment of Hymenocrater longiflorus Plant in Human Carcinoma Cells

The present study was conducted to evaluate the effect of Hymenocrater ongiflorus methanolic extract on human osteosarcoma U2OS cancer cell line and colon RKO cancer cell line. Chemical analysis of the crude methanolic extract of the plant was done sing High Performance Liquid Chromatography – electrospray tandem mass spectrometry (HPLC-ESI /MS) analysis for the phenolic compounds flavonoids,
phenolic acids and their esters). Eleven compounds were detected in this extract, which were acacetin,  apigenin, apigenin-7-O-glucoside,caffeic acid, ferulic acid, arnosic acid, crismartin, genistein, sorhamantin, N-carboxybenzyloxy-cysteinyl ystein and rosmaric acid.The free radical scarvenging activity of H. longiflorus methanol extract was evaluated by using 2, 2,-diphenyl-1picrylhydrazyl (DPPH) assay. The results reveal that H. longiflorus has antioxidant activity, and this activity which is concentration dependent increases until reaching 86.226% antiradical activity (ARA) at concentration of 1000 µg ml -1 The in vitro study on cell viability reveals that the plant has an inhibitory effect on cancer cells (U2OS and RKO) using Dye exclusion assay and MTT assay which convert the yellow color to purple, the growth decreased and a significant effect was observed like decrease in the cell number as compared with non treated cells.Through assessment of cell cycle arrest using propidium iodine (PI) by uorescence
 Activated Cell Sorter (FACS) analysis using flow cytometry, indicate a mild effect was observed at G2 phase of U2OS and at G1 phase of  RKO cell line in comparison with non treated cells (control). Phosphorylation of H2AX was induced in response to DNA double strand breaks originated from diverge origins of γH2AX including external damage, replication fork collision, apoptosis and dysfunctional telomeres using immunoflourescence assay, by this assay accumulation of damage foci was observed as compared with non treated cells in both U2OS and RKO cell line.The study of the apoptosis using DAPI and Sulforhodamine stain, clarified decrease in cell size and shrinkage in both U2OS and RKO cell line, these results show the signs of apoptosis. Proteins and DNA damage using Western blot analysis was observed, different proteins were used (p21, p53, p-p53, γ H2AX and parp), Proteins increased in size with cleavage of parp protein into two bands, which is an indication of apoptosis in addition to increase in p53 and γ H2AX proteins for both U2OS and RKO cell line.From all obtained results, It is possible suggest that H. longiflorus methanol extract is a promising anticancer plant due to its effect on cell growth, cell cycle arrest DNA damage then lead to apoptosis, taking into consideration that RKO cell line was more sensitive than U2OS cell line.