Evaluation of molecular mechanism(s) associated with the anti-proliferative and apoptosis-including properties of the crude methanolic and semi-purified extracts of dicerocaryum species in jurkat t cells

Abstract

Cancer is one of the largest single causes of death in both men and women worldwide. In the last decade, basic cancer research has produced remarkable advances in the understanding of cancer biology and genetics. Its control may benefit the lives of many individuals; therefore, there is a need to utilize different approaches in the prevention and cure of this killer disease. To date, the focus is on natural products that are implicated in cancer prevention and can also promote health without recognizable side-effects. Dicerocaryum species is an indigenous African plant used as a shampoo for hair due to its ability to remove dandruff from the scalp and its leaves are edible as a vegetable. The plant is also purported to possess some anti-inflammatory and antioxidant activities. The current study was performed to evaluate the anticancer and anti apoptosis-inducing properties of the crude methanolic and semi-purified extracts of Dicerocaryum species in Jurkat T cells. The leaves of Dicerocaryum species were collected from the grounds of University of Limpopo, dried and ground into a powder. Different solvents, namely: methanol, n-hexane, dichloromethane, n-butanol and water were used for the extraction of putative bioactive compounds with potential anticancer activities. The cells were routinely cultured in the presence of various concentrations of the extracts after which the cell proliferation and cytotoxicity effects were determined using 3-(4, 5-dimethylthiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue dye exclusion method, respectively. The results showed that the crude methanolic and dichloromethane/ethanol extracts exhibit significant growth inhibitory effects against Jurkat T cells in a dose- and time-dependent manner. Based on the IC50 values, dichloromethane/ethanol extract was found to be more potent than the crude methanolic, n-hexane, n-butanol and water extracts.In order to explore the mode of cell death elicidated by the crude methanolic and dichloromethane/ethanol extracts, cellular and nuclear morphological changes and the expression levels of major apoptotic genes and proteins xvi were investigated in the treated Jurkat T cells. Subsequently, nuclear shrinkage and chromatin condensation were observed in cells treated with crude methanolic and dichloromethane/ethanol extracts. The results obtained suggested that both the crude methanolic and dichloromethane/ethanol extracts induce cell death through the process of apoptosis as characterized by the afore mentioned features. In addition, the results obtained from the analysis of DNA fragmentation demonstrated a concentration-dependent DNA laddering in Jurkat T cells treated with the crude methanolic and dichloromethane/ethanol after 48 h. RT-PCR results indicated that the expression levels of major apoptotic genes such as bcl-2 were down-regulated whereas those of bax were up-regulated in a dose- and time-dependent manner, after treatment. The results obtained from the Western blot analyses were similar to that of RT-PCR whereby a dose-dependent decrease in Bcl-2 protein expression levels in Jurkat T cells treated with crude methanolic and dichloromethane/ethanol extracts was observed. Cells treated with 400 μg/ml and 600 μg/ml of crude methanolic extract showed a decrease in Bcl-2 protein expression after 24 and 48 h. Dichloromethane/ethanol extract also demonstrated down-regulation in expression levels of Bcl-2 protein after 24 and 48 h. No data were obtained from Bax protein expression in cells treated with both crude methanolic and dichloromethane/ethanol extracts after 12, 24 and 48 h, which suggested that Bax protein might have been a target of the ubiquitin/proteasome degradation pathway. Therefore, the results alluded to the fact that crude methanolic and dichloromethane/ethanol extracts of Dicerocaryum species exert their antiproliferative effects in Jurkat T cells through the dysregulation of apoptosis responsive genes. Thus, the effects that are elicited by the crude methanolic and dichloromethane/ethanol extracts could be due to up/down-regulation of the expression levels of major apoptotic genes and proteins. The findings also suggest that crude methanolic and dichloromethane/ethanol extracts from Dicerocaryum species are capable of reducing cell growth, cell survival and inducing apoptosis in Jurkat T cells. Thus, Dicerocaryum species could be xvii used as a valuable source for the isolation of novel and effective anticancer drugs.