Figure 1.
Effect of TCS on the viability of different breast cancer cells.
A. The isolated TCS manifested a single band with a molecular mass of 27-kDa. B–D. MCF-7, BT-474, and MDA-MB-231 cells were cultured in the presence of different concentrations of TCS (ranging from 0.49 to 125 µM) for 24 h to 48 h. The remaining cell viability was measured by MTT assay. The results are shown as means ± S.D. of two independent experiments performed in triplicate.
Figure 2.
Effects of TCS on cell cycle distribution and proliferation of breast cancer cells.
A. TCS induced cell cycle arrest. MCF-7 and MDA-MB-231 cells were treated with different concentrations of TCS (as labeled in the figs.) for 24 h. Cells were harvested, fixed in ethanol, and stained with PI for flow cytometric DNA analysis. Data were analyzed by FCS Express 4 software. B. TCS inhibited cell proliferation. After treatment of MCF-7 and MDA-MB-231 cells for 24 h with 15 µM and 10 µM TCS respectively, cell counting was performed using a haemocytometer following staining with trypan blue solution.
Figure 3.
TCS-induced apoptosis in breast cancer cells.
A. Cells were treated with TCS at the indicated concentrations for 24 h. Cells were harvested and subsequently stained with Annexin V/PI solution for cell sorting under flow cytometry. Percentages of cells undergoing early apoptosis (right lower quadrant) and late apoptosis (right upper quadrant) were analyzed by FCS Express 4 software. B. Cells were treated with TCS at the indicated concentrations for 24 h, and cells with DNA fragmentation were detected by flow cytometry following TUNEL staining. C. Cells were treated with TCS at the indicated concentrations for 24 h, and nuclear morphological changes were examined by staining with Hoechst 33342 and fluorescence microscopy. Bars, 50 µm. In each panel of Fig. 3C, figures on the right represented fourfold magnification of the rectangular zones in figures on the left.
Figure 4.
The activation of caspase cascades was involved in TCS-induced cell apoptosis.
A. After treatment with TCS at the indicated concentrations, cells were harvested and protein levels of caspase-8, caspase-9, caspase-3, and PARP were determined by western blotting. Right panel, quantitative data from two independent experiments. Lower panel, The caspase-8 and caspase-9 activities were determined by ELISA kits. Asterisk, p<0.05 compared with control. B. TCS increased the number of tumor cells undergoing mitochondrial membrane depolarization. Cells were treated with TCS at the indicated concentrations for 24 h, and flow cytometry analysis of the mitochondrial membrane depolarization was carried out after JC-1 staining. C. Z-VAD-FMK pretreatment retarded TCS-induced cell apoptosis. After different treatments as indicated at the bottom of the histogram, cells were harvested, stained with Annexin V-PI, and processed for flow cytometry. Asterisk, p<0.05 compared with control.
Figure 5.
TCS inhibits MDA-MB-231 xenograft growth in nude mice.
A. Twelve female BALB/c nude mice received an injection of MDA-MB-231 cells and were divided into 2 groups. TCS was administered at a dose of 5.0 mg/kg every other day for a total of 7 injections. On day 16, mice were sacrificed and excised tumors are shown in the right panel. The dose applied had no detectable toxicological effects on nude mice, such as changes in body weight. B. A representative comparison between a TCS-treated mouse and a PBS-treated control mouse. C. TCS significantly reduced tumor volume from the 6th day of treatment onward. D. The mean tumor weight in TCS group was smaller than that of control. For asterisks in C and D, p<0.05.
Figure 6.
TCS-induced necrosis and apoptosis in MDA-MB-231 xenograft.
MDA-MB-231-bearing nude mice were treated with a TCS dose of 5.0 mg/kg every other day by i.p. injection for a total of 7 injections. On day 16 post-cell injection, tumors were excised carefully, and immunohistochemistry studies were performed as detailed in Materials and methods. Positive cells in each study were scored semi-quantitatively. A. HE staining. B. Tumor slides were examined for apoptosis by activated caspase-3. Representative images from both groups are shown, and positive cells were stained brown. C. Tumor slides were examined for apoptosis by cleaved PARP. Representative images from both groups are shown, and positive cells were stained dark brown. D. TUNEL assay was used for detecting DNA fragmentation. Representative images from both groups are shown with TUNEL-positive cells colored green. Microscopic magnification 200×.