Avocado extracts induce apoptosis via reactive oxygen in malignant human oral cells.

A99 Diets rich in fruits and vegetables has been associated with reduced risks for many types of cancers. Avocado (Persea americana) is a widely consumed fruit containing many cancer preventing nutrients, vitamins and phytochemicals. Additionally, the avocado is sodium, cholesterol and sugar free, and low in calories and fats making it a part of a healthy diet. In this study, aqueous and organic extracts were prepared from freshly peeled California Hass avocados and tested for growth inhibition and apoptosis in a malignant human oral cell line (CA). Among the five fractions tested, extract F001 (ethanol) exhibited the strongest growth inhibiting effect (IC50 = 14.1 μg/ml) after a three day treatment, whereas F003 (aqueous) extract was not effective up to 200 μg/ml. Further studies showed that the F001 extract induced apoptosis as indicated by flow cytometry, and Western blotting. Treatment of CA cells with 30 μg/ml of the F001 extract resulted in marked cleavage of PARP and activation of caspases 3, 8 and 9 after 12 hr incubation. Z-VAD-fmk, a pan-caspase inhibitor completely abolished F001 induced cleavage of PARP and activation of caspases. Over-expression of ΔFADD partially attenuated F001 induced apoptosis indicating the FAS/FADD pathway may participate in avocado extract F001 induced apoptosis. Flow cytometry revealed that the F001 extract induced a significant production of reactive oxygen species (ROS) within 3 hr of treatment. The addition of N-Acetyl-Cysteine (NAC) markedly reduced the levels of the ROS produced by the treatment of cells with the F001 extract. Coinciding with the reduced levels of ROS, Western blot analysis showed that NAC completely abolished F001 induced apoptosis. Our data demonstrates that avocado extracts have potent inhibitory effect on the proliferation and selective killing of human oral malignant cells. This inhibition appears to be mediated via ROS induced apoptosis. The further identification and characterization of the active components in avocados and their molecular targets in premalignant and malignant cells should help to better establish the nutritional components responsible for food-based chemopreventive activity.