Expression level of the apoptotic-related genes in MCF-7 cells treated with EADs as determined by GeXP analysis.

<p>EADs upregulated the expression of <i>Bax</i> and <i>p21</i> and downregulated the expression of <i>Bcl-2</i> and <i>caspase-9</i>. 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).</p

Extraction and isolation of ethyl acetate extract of <i>Dillenia suffruticosa</i>.

<p>Sequential solvent extraction using solvents with increasing polarity (hexane< dichloromethaneD. <i>suffruticosa</i>. The extract obtained was subjected to isolation using column chromatography and thin layer chromatography by using different solvent systems.</p

List of genes with their respective primers for GeXP multiplex analysis.

<p>Forward universal primer sequence (5’-AGGTGACACTATAGAATA-3’); Reverse universal primer sequence (3’-GTACGACTCACTATAGGG-5’).</p><p>List of genes with their respective primers for GeXP multiplex analysis.</p

Expression level of the apoptotic-related proteins in MCF-7 cells treated with EADs at different time point as determined by Western blot analysis.

<p>(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).</p

Involvement of caspase in EADs-induced apoptosis in MCF-7 cells.

<p>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).</p

Proposed signalling pathway of EADs-induced apoptosis in MCF-7 cells.

<p>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.</p

Chemical structure of the compounds isolated from EADs.

<p>Structures of the isolated compounds were elucidated using 1^H and 13^C NMR spectroscopy. The isolated compounds were identified as kaempferide (<b>1</b>), kaempferol (<b>2</b>), protocatechuic acid (<b>3</b>), gallic acid (<b>4</b>), 3-epimaslinic acid (<b>5</b>) and β-sitosterol-3-O-β-D-glucopyranoside (<b>6</b>).</p

Involvement of oxidative stress in EADs-induced apoptosis in MCF-7 cells.

<p>(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).</p

Mitochondria membrane potential of MCF-7 cells treated with EADs as determined by JC-1 fluorescent dye using flow cytometry analysis.

<p>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.</p