Figure 1.
The dose-dependent effects of CT
(A), T2A (B) and T1 (C) on the growth of human breast cancer cell lines (MCF-7, MDA-MB231, SKBR3 and MDA-MB453) and on normal mammary epithelial cells (HMEC) (D). Values were mean±SEM of at least three independent experiments, each in triplicates.
Figure 2.
Effects of T1 on Cell Cycle Progression and Protein Levels of Cell Cycle-Related Biomarkers (48h).
A and B: Effects of T1 on cell cycle arrests of estrogen-dependent MCF-7 (A) and estrogen-independent MDA-MB231 (B) cell lines. Data were from at least two independent experiments, each in duplicates; C: The representative Western blot images showing the effects of T1 on protein levels of cell cycle related biomarkers cyclinD, CDK4, cdc2, p-cdc2 and cyclinB; D and E: Quantitation of cyclinD, CDK4, cdc2, p-cdc2 and cyclinB protein levels in MCF-7 (D) and MDA-MB231 (E) by densitometry after normalization to β-actin. Values were mean±SEM of at least two independent experiments. Within the panel, the value with a letter was significantly different from that of the corresponding control, a, p<0.05; b, p<0.01; c, p<0.001.
Figure 3.
Effects of T1 on apoptosis of breast cancer cells and protein levels of apoptosis-related biomarkers (48h).
A: Effects of T1 on the proportion of DNA fragmentation (sub-G0), a marker of apoptosis, in MCF-7 and MDA-MB231 cell lines. Values were mean±SEM of at least two independent experiments, each in duplicates; B: The representative Western blot images showing the effects of T1 on protein levels of apoptosis related biomarkers PARP, c-PARP, bcl2 and bax; C: Quantitation of c-PARP protein levels in MCF-7 and MDA-MB231 by densitometry after normalization to β-actin. The images for quantitation were from at least two independent experiments. Within the panel, the value with a letter was significantly different from that of the corresponding control, a, p<0.05; b, p<0.01; c, p<0.001.
Figure 4.
Expressions of survivin and Aurora A in human breast tissues and breast cancer cells.
A and B: Expressions of survivin (A) and Aurora A (B) genes in normal breast tissues (n = 10), normal tissues adjacent to tumors (n = 12) and breast tumors (n = 14) by real-time RT-PCR; C and D: Expressions of survivin (C) and Aurora A (D) genes in human breast cancer cell lines (MCF-7, MDA-MB231, SKBR3 and MDA-MB453) and HMEC; E, Protein levels of survivin and Aurora A in HMEC and human breast cancer cell lines by western blot. Values were mean±SEM. Within the panel, the value with a letter was significantly different from that of the corresponding control, c, p<0.001.
Figure 5.
Effects of tanshinones on survivin and Aurora A protein levels in breast cancer cells (48 h).
A: Representative western blot images showing survivin and Aurora A protein levels in breast cancer cell lines (MCF-7, MDA-MB231, SKBR3, MDA-MB453) following tanshinone treatments with β-actin as the loading control; B and C: Quantitation of Aurora A (B) and survivin (C) protein levels by densitometry after normalization to β-actin. Values were mean±SEM of three independent experiments in duplicates. The images for quantitation were from at least two independent experiments. Within the panel, the value with a letter was significantly different from that of the corresponding control, a, p<0.05; b, p<0.01; c, p<0.001.
Figure 6.
Effects of Aurora A knockdown on the T1 activities in growth and apoptosis of MCF-7 breast cancer cells.
A: Western blot analysis showing knockdown of Aurora A protein level in MCF-7 cells by Aurora A siRNA; B: Effect of Aurora A knockdown on the growth-inhibition activity of T1; C: Effect of Aurora A knockdown on the apoptosis-induction activity of T1. Values were mean±SEM of three independent experiments in duplicates. Within the panel, the value with a letter was significantly different from that of the corresponding control (c, p<0.001), and the values with a “*” are significantly different (**, P<0.01; ***, P<0.001).
Figure 7.
Epigenetic modifications of Aurora A expression by T1 treatment in breast cancer cells.
A: Effect of the demethylating agent 5′-Azacytine (5-AZA) or the histone deacetylase inhibitor sodium butyrate (SB) treatment on Aurora A gene expression in MCF-7 cells; B: Identification of histone H3 acetylation level of DNA promoter areas in Aurora A gene that are associated with overexpression of Aurora A gene in MCF-7 cells by CHIP; C: Effects of T1 treatment (3 µM) on acetylation levels of histone 3 of Aurora A gene by CHIP; D: Effects of SB (1 mM) treatment on the activity of T1 in inhibiting the growth of MCF-7 cells; E: Scheme showing the CHIP primer locations for Aurora A gene. Values were mean±SEM of three independent experiments in triplicates. Within the panel, the value with a letter is significantly different from that of the corresponding control (a, p<0.05; b, p<0.01), and the values with a “*” are significantly different (*, P<0.05).