Publication: Caspase-dependent apoptotic mechanism of gallic acid and its derivatives isolated from quercus infectoria ethyl acetate extract against cervical cancer cells lines (hela)
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Date
2023-09
Authors
Ismail, Illyana
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Abstract
Cervical cancer is one of the most common cancers in women worldwide. In
2020, cervical cancer ranked the fourth most diagnosed cancer among Malaysian
women. The induction of apoptosis is one of the essential mechanisms to prevent the
process of carcinogenesis. The previous study indicated that natural products were
able to induce apoptosis and showed promising advantages in cancer treatment. The
Quercus infectoria galls (QI) have been reported to have antimicrobial, antioxidant,
anticancer and wound healing activities. However, the antiproliferative activity and
the underlying molecular mechanisms against human cancer cells have been poorly
elucidated. Hence, the present study was undertaken to examine the cell death
mechanisms of gallic acid and its derivatives isolated from Quercus infectoria ethyl
acetate extract (EAQI) against cervical cancer cells (HeLa). Gallic acid (GA) and its
derivative, methyl gallate (MG), were isolated by using a bioassay-guided isolation
technique. The antiproliferative effect that characterised by inhibitory concentration
at 50 % cell populations (IC50) of EAQI, GA and MG were determined by using the
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay at
various concentration ranging from 0.39 to 100 μg/ml at 72 hours of treatment in
HeLa cell lines and the control serving non-cancerous Vero cell lines. Cisplatin was
used as a positive control, while untreated HeLa and Vero cells served as the
negative control. Changes in cell morphology were measured by acridine orange/propidium iodide (AO/PI) staining for 24, 48 and 72 h. Viable, apoptotic and
necrotic cells were identified using a fluorescence microscope. Determination of
phosphatidylserine (PS) externalisation was performed using annexin-V Fluorescein
isothiocyanate (FITC) / propidium iodide (PI) dual staining assay. The cells were
treated for 3, 6 and 12 h and analysed by flow cytometry. Cellular deoxyribonucleic
acid (DNA) content was measured in HeLa cells using flow cytometry at 24, 48 and
72 h for cell cycle distribution. Apoptosis pathways were elucidated based on pro
and anti-apoptotic protein expressions (p53, Bax and Bcl-2) at 3 hours of treatment
and caspases activity (caspase-8 and -9) were analysed by flow cytometry technique
at 6 hours of treatment. The results showed that EAQI, MG and GA exhibited the
antiproliferative effect on HeLa cells with IC50 values of 11.50 ± 0.5 μg/ml, 11.00 ±
0.58 μg/ml and 10.00 ± 0.67 μg/ml, respectively. In the cell morphology analysis,
cells treated with IC50 value of EAQI, MG and GA displayed an increased apoptotic
cell population compared to untreated cells (p<0.05) at 72 hours of treatment. The
induction of apoptosis was confirmed by the externalisation of phosphatidylserine on
early apoptotic cells, which showed the treated cell population shifted from viable to
apoptotic quadrant. Based on the cell cycle distribution, the accumulation of cells at
the subG0 phase in treated cells indicated the discontinuity of deoxyribonucleic acid
DNA fragmentation and led to apoptosis. Furthermore, the results showed that p53
and Bax (pro-apoptotic proteins) were expressed in the treated cells, whereas Bcl-2
(anti-apoptotic protein) was not expressed at 3 hours of treatment. The caspase
analysis also revealed that EAQI, MG and GA had induced apoptosis by activating
caspase-8 and -9 at 6 hours of treatment. In conclusion, these findings suggested that
EAQI, MG and GA significantly induced apoptotic mechanisms via the regulation of intrinsic and extrinsic pathways, which should provide new insight into therapeutic
activity and anticancer agents of QI.