br Western blot analysis br
2.5. Western blot analysis
Cells were harvested by trypsinization and pellets were lysed using radioimmunoprecipitation assay (RIPA) buﬀer (Pierce, USA). Then, the protein concentration was determined by Bio-Rad Bradford Protein assay (Bio-Rad, USA). A hundred micrograms of protein were loaded on a 12% SDS-PAGE gel and transferred to nitrocellulose membrane
(Millipore, Bedford, MA, USA). The membrane was probed with pri-mary Actinomycin D against topoisomerase II, STAT3, cyclin D1, p21 (Cell signaling Technology, MA, USA) and GAPDH (Calbiochem, Germany). Bound antibodies were detected by incubating the membrane with HRP-conjugated secondary antibody and visualized by a chemilumi-nescence detection using the SuperSignal™ West Dura Extended Duration substrate kit (Thermo Scientific, IL, USA) according to the protocol supplied with kit. Protein level was analyzed with ImageJ software.
2.6. Apoptosis assay and multi-caspase activation assay
To investigate the eﬀect of ( ± )-kusunokinin and ( ± )-bursehernin on apoptosis induction via multi-caspase activity, MCF-7 and KKU-M213 cell lines were seeded into 12-well plates at a density of 2 × 105 and 1 × 105 cells per well, respectively. The cells were treated with IC50 concentration for 96 h. Then, cells were harvested at 24, 48, 72, and 96 h, and resuspended in 1X PBS. The apoptotic cells were detected using the Muse® Annexin V Dead Cell Kit (Catalog No. MCH100105, Merck Millipore). Caspase activity was measured using the Muse® MultiCaspase Kit (Catalog No. MCH100109, Merck Millipore). Apoptosis and multi-caspase activity were performed following the protocol provided by the manufacturer and analyzed by MUSE® Cell Analyzer.
2.7. Statistical analysis
Data are presented as the mean ± standard deviation (SD) of at least two independent experiments. The data were statistically analyzed
Fig. 5. Eﬀect of ( ± )-kusunokinin and ( ± )-bursehernin on the induction of cell apoptosis. MCF-7 and KKU-M213 cells were treated with ( ± )-kusunokinin and ( ± )-bursehernin at IC50 concentration value 4.30 μM and 3.70 μM, respectively. Treated cells were incubated for 24, 48, 72, and 96 h. (A, B) After treatment, apoptotic cells were analyzed by the Muse® Annexin V Dead Cell Kit. The scatter plots are shown percentage of apoptotic cells for one experiment. (C, D) The graph is represented the summary mean percentages ± SD of apoptotic cells of three independent experiments. The statistical analysis of the data was carried out by Student t-test. p value less than 0.05 was considered to indicate a statistically significant diﬀerences compared to control group at 0 h.
using the Microsoft excel software, and the Student’s t-test was carried out. A p-value of less than 0.05 was considered to indicate a statistically significant diﬀerence between groups.
3.1. Cytotoxic eﬀects of ( ± )-kusunokinin and ( ± )-bursehernin on normal and cancer cells
To investigate the impact of ( ± )-kusunokinin and ( ± )-burse-hernin on the cell cycle, MCF-7 and KKU-M213 cell lines were treated with IC50 concentration of 4.30 μM of ( ± )-kusunokinin and 3.70 μM of ( ± )-bursehernin, respectively, for 24, 48 and 72 h. Cell cycle dis-tributions were assessed by flow cytometry. At 24 h after treatment, ( ± )-kusunokinin tended to cause cell cycle arrest at G2/M phase but not significantly increased whereas ( ± )-bursehernin significantly in-creased the cell population at G2/M-phase (p-value less than 0.05) (Fig. 3).
Topoisomerase II, STAT3, cyclin D1, and p21 are associated with
Fig. 6. Eﬀect of ( ± )-kusunokinin on multi-caspase in MCF-7 cells. (A, C) MCF-7 cells were treated with ( ± )-kusunokinin at IC50 concentration for 24, 48, 72, and 96 h. (B, D) MCF-7 cells were also treated with various concentrations of ( ± )-kusunokinin at 2.15, 4.30, and 8.60 μM for 48 h. Multi-caspases were determined using the Muse® MultiCaspase Kit. The plot is depicted the eﬀect of ( ± )-kusunokinin treatment on MCF-7 cells. (A, B) Each plot is a representative figure of the three replicates of each determination. (C, D) The graph is depicted the percentage of cell with multi-caspase activity. Values are mean ± SD (n = 3). The statistical analysis of the data was carried out by Student t-test. *p < 0.05 and **p < 0.01 were considered to indicate a statistically significant diﬀerences compared to control group at 0 h.
cell cycle and proliferation. The level of these proteins was evaluated on cancer cell lines treated with ( ± )-kusunokinin and ( ± )-bursehernin using Western blotting.
Topoisomerase II and STAT3 were significantly decreased after 4.30 μM ( ± )-kusunokinin treatment of MCF-7 cells at 72 h while cyclin D1 was significantly decreased at 48–72 h post treatment (Fig. 4A and C). Moreover, treatment of KKU-M213 cells with 3.7 μM ( ± )-burse-hernin significantly reduced cell cycle proteins topoisomerase II and cyclin D1 in a time dependent manner. STAT3 was significantly de-creased at 48 and 72 h after incubation (Fig. 4B and D). As shown in Fig. 4, p21 was significantly increased at 24 h after treatment with both compounds. These results suggest that ( ± )-kusunokinin and ( ± )-bursehernin have anti-proliferation eﬀects on breast cancer and cholangiocarcinoma cell lines.