Induction of non-apoptotic cell death in human oral squamous cell carcinoma cell Lines by Rhinacanthus nasutus extract

Background: We recently reported that the MeOH extract of aerial parts and root of Rhinacanthus nasutus showed diverse biological activity, with most activity being concentrated into the EtOAc layer separated by sequential organic solvent extractions. In the present study, the EtOAc extracts were further separated by silica-gel column chromatography into five fractions (Frs. 1-5), and their cytotoxicity and apoptosis-inducing activity investigated. Materials and Methods: Cytotoxic activity was determined by 3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyltetrazolium bromide (MTT) method. The 50% cytotoxic concentration (CC 50) was determined from the dose-response curve. Tumor specificity (TS) was determined by the ratio of the mean CC 50 for normal cells to the one for tumor cell lines. DNA fragmentation was assayed by agarose gel electrophoresis. Caspase-31-7 activation was monitored by cleavage of substrates either spectrophotometrically or by western blot analysis. Results: Among five fractions of the EtOAc extract, Fr. 1, eluted with CHCl 3-MeOH (50:1), showed the highest tumor specificity (TS=3.3) as compared with other fractions eluted at higher concentrations of MeOH in CHCl 3 (TS=1.0-2.8). Fr. 1 did not induce internucleosomal DNA fragmentation or induced only marginal level of caspase-3 activity in either human promyelocytic leukemia HL-60 cells and human oral squamous cell carcinoma (OSCC) cell lines HSC-2. Conclusion: The present study suggests that hydrophobic substances of EtOAc extract show tumor specific cytotoxicity by inducing little or no apoptosis