Table 1.
List of genes with their respective primers for GeXP multiplex analysis.
Fig 1.
Extraction and isolation of ethyl acetate extract of Dillenia suffruticosa.
Sequential solvent extraction using solvents with increasing polarity (hexane< dichloromethane<ethyl acetate) was carried out to obtain ethyl acetate fraction of D. suffruticosa. The extract obtained was subjected to isolation using column chromatography and thin layer chromatography by using different solvent systems.
Fig 2.
Involvement of oxidative stress in EADs-induced apoptosis in MCF-7 cells.
(A) and (B) represent the percentage of viability of MCF-7 cells pre-treated with vitamin C, α-tocopherol or EADs alone, at 24 and 48 hours, respectively. (C) Level of ROS in MCF-7 cells as determined using DCFH-DA assay. Data showed that pre-treatment of MCF-7 cells with α-tocopherol and ascorbic acid significantly reduced the cytotoxicity of EADs (P<0.05). On the other hand, EADs attenuated the intracellular ROS in MCF-7 cells in a concentration-dependent manner (P<0.05). The data are presented as mean±standard deviation of three replicates from at least three independent tests. An asterisk * indicates statistically significantly different from the untreated control (P<0.05).
Fig 3.
Involvement of caspase in EADs-induced apoptosis in MCF-7 cells.
General inhibitor Z-VAD-FMK did not inhibit the induction of apoptosis by EADs suggesting it is caspase-independent. (A) represents mean percentage of three independent experiments±SD. (B) Comparison of the percentage of apoptotic cells between caspase inhibitor negative group and caspase inhibitor positive group at different concentrations. The data are presented as mean±standard deviation of three replicates from three independent tests. An asterisk * indicates statistically significantly different from the untreated control (P<0.05).
Fig 4.
Mitochondria membrane potential of MCF-7 cells treated with EADs as determined by JC-1 fluorescent dye using flow cytometry analysis.
The increment of green fluorescence indicates the loss of ΔΨm in the mitochondria of EADs-treated MCF-7 cells. The data are presented as dot plots of JC-1 red fluorescence (Y-axis) against JC-1 green fluorescence (X-axis) of at least three independent tests.
Fig 5.
Expression level of the apoptotic-related genes in MCF-7 cells treated with EADs as determined by GeXP analysis.
EADs upregulated the expression of Bax and p21 and downregulated the expression of Bcl-2 and caspase-9. The expression of genes was normalized against beta actin and compared to the control. The data are represented as relative expression of genes in bars±SD of at least three replicates from three independent tests. An asterisk * indicates statistically significantly different from the untreated control (P<0.05).
Fig 6.
Expression level of the apoptotic-related proteins in MCF-7 cells treated with EADs at different time point as determined by Western blot analysis.
(A) Expression of p21, p53, Bax, Bcl-2, PARP and caspase-8 in MCF-7 cells treated with 25 and 50 μg/mL of EADs (B) Fold change of Bax to Bcl-2 ratio at 24 and 48 hours. (C) Expression of AKT-1, phosphor-AKT, JNK-1, phosphor-JNK, ERK-1 and phosphor-ERK1 in MCF-7 cells treated with 25 and 50 μg/mL of EADs. The expression of proteins was normalized against beta actin and compared to the control. The data are represented as mean ± SD of at least three replicates from three independent tests. An asterisk a indicates statistically significantly different from the untreated control (P<0.05).
Fig 7.
Chemical structure of the compounds isolated from EADs.
Structures of the isolated compounds were elucidated using 1H and 13C NMR spectroscopy. The isolated compounds were identified as kaempferide (1), kaempferol (2), protocatechuic acid (3), gallic acid (4), 3-epimaslinic acid (5) and β-sitosterol-3-O-β-D-glucopyranoside (6).
Table 2.
Cytotoxicity of the compounds isolated from EADs towards MCF-7 and MDA-MB-231 cells at 72 hours as reflected by IC50 value as determined by MTT assay.
Fig 8.
Proposed signalling pathway of EADs-induced apoptosis in MCF-7 cells.
It is postulated that EADs induces apoptosis in MCF-7 cells via the production of oxidative stress, p53- and p21-dependent cell cycle arrest, activation of JNK and NF-κB pathways and inactivation of AKT and ERK pathways. Regulation of these pathways eventually leads to the execution of mitochondrial-dependent and caspase-independent apoptosis.
Table 3.
Cytotoxicity of EADs towards the breast cancer and non-breast cancer cell line at different time point as reflected by IC50 value as determined using MTT assay.