Induction of non-apoptotic cell death by Odontioda Marie Noel 'Velano' extracts in human oral squamous cell carcinoma cell lines

Abstract

Background: We recently reported that the MeOH extract from bulbs of Odontioda Marie Noel 'Velano' exhibited diverse biological activities but most of the activity was concentrated into the EtOAc layer separated by sequential organic solvent extractions. In the present study, the EtOAc layer was subjected to silica-gel column chromatography for further separation into five fractions, and the cytotoxicity and apoptosis-inducing activity of the fractions against human normal oral and tumor cells was further investigated.

Materials and methods: Cytotoxic activity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The 50% cytotoxic concentration (CC(50)) was determined by the dose-response curve. Tumor specificity (TS) was determined by the ratio of the mean CC(50) for normal cells to the one of tumor cell lines. DNA fragmentation was assayed by agarose gel electrophoresis, caspase-3/-7 activation was monitored by cleavage of substrates either spectrophotometrically or by western blot analysis.

Results: Among five fractions, the most hydrophobic fraction (Fr. 1) showed the highest cytotoxicity against all cell lines tested, followed by Fr. 2 >Fr. 3 >Fr. 4 >Fr. 5, in order of increasing polarity. Fr. 2 had the highest tumor-specificity, followed by Fr. 3, Fr. 4, Fr. 1 and Fr. 5. Fr. 1 induced caspase-3 activation more potently in promyelocytic leukemia HL-60 cells, than in oral squamous cell carcinoma (OSCC) HSC-2 cells, whereas it did not induce internucleosomal DNA fragmentation in either of these cell lines.

Conclusion: The present study suggests that hydrophobic substances in the EtOAc extract of Odontioda Marie Noel 'Velano' exhibit tumor-specific cytotoxicity without inducing apoptosis in the HSC-2 OSCC cell line.