The induction of apoptosis in human cancer cells by terpenoid and solvent extracts of anacardiaceae plant species

Abstract

The Anacardiaceae or Cashew family is the source of bioactive phytochemical extracts with antimicrobial, antioxidant, anti-inflammatory and cytotoxic properties. These extracts include essential oils, solvent extracts and resins. The essential oil (EO), which is extracted from the plant by steam or hydrodistillation, consists ofmonoterpenoid and sesquiterpenoid secondary metabolites. The alcoholic extract constituents include a number of larger molecule metabolites, such as triterpenoids and flavonoids, extracted by maceration. The resin, which is obtained from the inner bark after wounding and allowed to ooze out and harden, is also predominantly made from triterpenoids. This study involved obtaining EO, resin, solvent and aqueous extracts of leaves, twigs, fruit and flowers from a number of Anacardiaceae species in four genera, namely: Pistacia lentiscus, Pistacia terebinthus and its hybrids, Pistacia chinensis, Schinus molle, Schinus terebinthifolius, Schinus lentiscifolius, Rhus lancea and Lithraea molleoides. Samples were taken every season over a one-year period. The growth inhibitory and cytotoxic potential of each of the extracts on cancer cell lines was analysed in vitro, as was the allelopathic effect on seed germination. A number of cancer cell lines including leukaemia cells HL60 and K562, human glioblastoma-astrocytoma cells ofneuroectodermal origin U373 and prostate cancer cells PC3 were treated with different concentrations of the extracts for three exposure intervals. The MIT assay was used to assess the cytotoxic potential of each extract. Morphological studies were used to confirm the mode of cell death taking place and determine the extent of apoptosis in the treated cancer cells using Haematoxylin and Eosin staining and fluorescent staining by Quinacrine Dihydrochloride. Germination inhibition tests were also carried out to determine potential allelopathic activity exerted on monocotyledonous and dicotyledonous seeds by the aqueous extracts of the Anacardiaceae species. Results show that Anacardiaceae spring leaves, in particular the Schinus species, provide the highest EO yields, when compared to Pistacia, Lithraea and Rhus species. However, during the fruiting season, the fruit EO yield exceeds that of the leaves. Polar solvents gave higher yields of organic phytochemicals from spring leaves of Pistacia lentiscus and Schinus terebinthifolius. Higher yields of extracted phytochemicals may possibly coincide with the formation of new leaves and fruits to enhance the plant's defenses against herbivory and pathogen infection. The spring leaves EOs, resins and polar solvent extracts exhibited the highest cytotoxic potential against the four cancer cell lines tested. Cancer cell cytotoxicity was dependent on extract concentration and exposure duration, with highest proliferation inhibition provided by the highest concentrations (50-100 ppm) and longest exposure duration (72 hours) in most cases. The K562 cancer cells were more prone to cytotoxicity by the Anacardiaceae extracts, often reaching growth inhibition of 50% of the cell population (GI50) at low extract concentrations. Treated U373, PC3 and HL60 cancer cells provided higher GI50 values in most cases. Staining of treated cancer cells confirmed that the mode of cell death showed hallmarks of apoptosis and that the number of apoptotic cells increased with concentration and exposure duration. Germination inhibition tests confirmed the allelopathic effect of Anacardiaceae aqueous extracts, which was more effective on monocotyledonous than on dicotyledonous seeds.