Ibrahim, Wan Suriyani Faliq Adeeba Wan
(2020)
Phytochemical characterization, induction of apoptosis and activation of natural killer (nk) cells by abrus precatorius leaves extract on human breast cancer cell line.
PhD thesis, Universiti Sains Malaysia.
Abstract
Cancer is still one of the global menace and poses a threat to the general world
population. The search for cancer cure is also still on the race. Although conventional
medicine remains the number one choice in cancer treatment, traditional approach is
also still one of the favourable choices made by cancer patients to deal with this
horrible disease. Traditional approaches, mainly by utilising medicinal plants are
widely sought after in many countries since centuries ago. The ability of medicinal
plants to exhibit their anti-proliferative activity, together with the ability to activate
immune responses would be the ideal strategy to beat the disease. Therefore,
understanding the mechanisms of medicinal plants displaying their anticancer
properties scientifically would fill the gap of unknown knowledge about them. A
medicinal plant known as Abrus precatorius or ‘saga’ were used in this study. This
plant has been utilised traditionally to cure various ailments including cancer. The
leaves of A. precatorius were selected to be extracted by different extraction
techniques which employed different types of solvent. GC-MS was employed to
provide the phytochemical analysis of the extracts. The ability of those extract to
inhibit proliferation in cancer cells were measured using MTT assay. The best extract
exhibiting the lowest inhibitory concentration (IC50) on the selected cancer cell, was
selected to determine the mechanisms of action in inducing the cell death. Cell cycle
arrest analysis, apoptosis staining with AnnexinV/PI and quantification of the
expression of p53, Bac, Bcl-2 and Caspase-3 proteins were used to determine themechanism of cell deaths. Finally, the ability of the extract to induce immune response
by activating NK cells was determined in a co-culture experiment of the NK cells with
the target cell, MDA-MB-231 cells. This was observed by the analysis of target cell
deaths and quantification of the secretion of cytokines, interleukin-2 (IL-2) and
interferon gamma (IFN-g); and the degranulation of the cytotoxic granules by
quantifying the perforin (PRF-1) and granzyme B (GzmB). The results showed that
the ethyl acetate and methanol extracts prepared using Soxhlet contained the highest
phenolic and terpenoid compounds comparing to the other extracts. The methanol
extract obtained by Soxhlet, APME (A. precatorius methanol extract), exhibited the
lowest IC50 value on MDA-MB-231 cells. Further analysis by flow cytometry revealed
APME induced cell death on MDA-MB-231 cells via apoptosis, through DNA arrest
at G0/G1 cycle, coupled with an increase of p53, Bax and Caspase-3 expression and
decrease of Bcl-2 expression. APME was also found to activate NK cells (from healthy
donor) by causing NK cell cytotoxic activity via apoptosis in the target cells. Increased
levels of INF-g and PRF-1 were also observed in this co-culture experiment. These
findings reflect the ability of A. precatorius leaves extract to exhibit the antiproliferative
effect on cancer cells and stimulatory effect on NK cells from the healthy
donors. This might be due to the presence of various phytochemical compounds in the
extract that might act synergistically.
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