Effect of β-Eudesmol on NQO1 suppression-enhanced sensitivity of cholangiocarcinoma cells to chemotherapeutic agents

Figures

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  Fig. 1. Concentration responses of NQO1 suppression by β-eudesmol KKU-100 cells were seeded onto a 96-well microtiter plate overnight and cells were exposed to β-eudesmol (1–100 μM) and dicoumarol as a potent positive NQO1 inhibitor (1 μM) for 24 h. a The effect of β-eudesmol on NQO1 enzyme activity of KKU-100 cells analyzed by the enzymatic method, b The effect of β-eudesmol on NQO1 enzyme activity of cell lysates analyzed by the enzymatic method, and c The effect of β-eudesmol on the expression of the NQO1 protein by Western blot analysis using β-actin as an internal control for equal protein loading. The relative bar that was normalized with β-actin of each band is shown below. Each bar represents mean ± SD of three independent experiments, triplicate each. * indicates statistically significant difference with untreated control (paired t-test, p < 0.05)

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  Fig. 2. Potentiating effect of β-eudesmol on cytotoxic activities of 5-FU and DOX. KKU-100 cells were seeded onto a 96-well microtiter plate overnight and cells were treated with β-eudesmol (1, 10, 30 and 100 μM) in the absence and presence of 5-FU or DOX for 24 and 48 h. Cell death was determined using SRB assay. a KKU-100 cells were incubated with β-eudesmol (1, 10, 30 and 100 μM) for 24 and 48 h. The results are presented as the percentage of viable cells. b-e The cytotoxic effect of β-eudesmol in combination with chemotherapeutic agents was determined. b-c KKU-100 cells were treated with β-eudesmol (30 μM) in the absence and presence of 5-FU (3, 10, 30 and 100 μM) for 24 and 48 h. d-e KKU-100 cells were treated with β-eudesmol (30 μM) in the absence and presence of DOX (0.01, 0.1, 1 and 10 μM) for 24 and 48 h. Each bar represents mean ± SD of three independent experiments, triplicate each. * indicates statistically significant difference with each drug alone (t-test, p < 0.05). β-Eu; β-Eudesmol, 5-FU; 5-Fluorouracil, DOX; Doxorubicin

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  Fig. 3. Inhibitory effects of β-eudesmol in combination with 5-FU or DOX on CCA cells migration. Scratched wounds of monolayer KKU-100 cells were exposed to β-eudesmol (30 μM), 5-FU (30 μM), DOX (0.1 μM), and the combination of β-eudesmol (30 μM) with 5-FU (30 μM) or DOX (0.1 μM). Cell migration was monitored under phase-contrast microscopy (× 4 magnification). Representative images of wound healing were obtained at the time of the scratch and 48 h later (a). b The graph shows the level of cell migration into the wound scratch quantified as the percentage of wound closure at 48 h. Each bar represents mean ± SD of three independent experiments, triplicate each. * indicates statistically significant difference with each drug alone (paired t-test, p < 0.05). β-Eu = β-Eudesmol, 5-FU = 5-Fluorouracil, DOX = Doxorubicin

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  Fig. 4. Enhancing the effect of β-eudesmol on apoptotic activities of 5-FU and DOX on CCA cells. Flow cytometry analyses of KKU-100 cells using double staining with annexin V (annexin V, vertical line) and propidium iodide (PI, horizontal line). a control, b β-eudesmol (30 μM), c 5-FU (30 μM), d DOX (0.1 μM), e combination of β-eudesmol (30 μM) with 5-FU (30 μM), and f combination of β-eudesmol (30 μM) with DOX (0.1 μM) for 24 h. Flow cytometry apoptotic results are shown in four subpopulations which indicate: early apoptotic cells (upper left), late apoptotic cells (upper right), normal cells (lower left) and necrotic cells (lower right). g The levels of KKU-100 cell apoptosis expressed as the percentage of apoptotic cells are depicted in the graph (mean ± SD averaged from three independent experiments, triplicate each). *p < 0.05 vs untreated control. #p < 0.05 vs chemotherapeutic agents alone. * indicates statistically significant difference with each drug alone (paired t-test, p < 0.05). β-Eu = β-Eudesmol, 5-FU = 5-Fluorouracil, DOX = Doxorubicin

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  Fig. 5. Apoptotic bodies of KKU-100 CCA cells following treatment with β-eudesmol, 5-FU, and DOX, and the combination of β-eudesmol with 5-FU or DOX by DAPI staining. a control, b β-eudesmol (30 μM), c 5-FU (30 μM), d DOX (0.1 μM), e combination of β-eudesmol (30 μM) with 5-FU (30 μM), and f combination of β-eudesmol (30 μM) with DOX (0.1 μM) for 24 h. Apoptotic bodies were observed under inverted fluorescence microscopy (Scale bars represent 40 μm). β-Eu= β-Eudesmol, 5-FU= 5-Fluorouracil, DOX= Doxorubicin

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  Fig. 6. Caspase-3/7 activation in KKU-100 cells following treatment with β-eudesmol, 5-FU, and DOX, and the combination of β-eudesmol and 5-FU or DOX detected by fluorescence microscopy. a KKU-100 cells were treated with 30 μM of β-eudesmol, 30 μM of 5-FU, 0.1 μM of DOX and β-eudesmol (30 μM) combined with 5-FU (30 μM) or DOX (0.1 μM) for 24 h and labeled with CellEvent® Caspase 3/7 Green Detection Reagent and examined under the fluorescence microscope. Left panel, the grey image reveals KKU-100 CCA cells in the field of view. Middle and right panel show apoptotic caspase 3/7 positive cells green fluorescent in the same area of interest. Data are representative of at least 4–5 randomly selected fields’ images with similar results. Morphological changes were evaluated under a microscope. There were shrink and blabbing cells treated with β-eudesmol with or without 5-FU or DOX, indicating apoptosis induction. The scale bar corresponds to 40 μm. b The graph shows the level of caspase-3/7 activity as the percentage of caspase-3 /7 fluorescent cells expression at 24 h. Data are presented as mean ± SD of three independent experiments, triplicate each. * indicates statistically significant…

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  Fig. 7. Potentiating effect of β-eudesmol on suppression Bcl-2 and Bax protein expression by 5-FU and DOX on CCA cells. a-b KKU-100 cells were exposed to β-eudesmol (30 μM), 5-FU (30 μM), DOX (0.1 μM), and combination of β-eudesmol (30 μM) with 5-FU (30 μM) or DOX (0.1 μM) for 24 h. a Effect of β-eudesmol on potentiation of 5-FU and DOX sensitivity on NQO1 enzyme activity analyzed by enzymatic method. b Equal amounts of total protein were examined by Western blot analysis, with appropriate antibodies. β-actin was used as a loading control. Relative band intensities of NQO1, Bcl-2, and Bax were shown. Data are presented as mean ± SD of three independent experiments, triplicate each. * indicates statistically significant difference with each drug alone (paired t-test, p < 0.05). β-Eu= β-Eudesmol, 5-FU= 5-Fluorouracil, DOX= Doxorubicin