Fig 1.
HASPIN mRNA levels in breast cancer cell lines.
(A) Agarose electrophoresis confirming the specificity of the primer pair in RT (+) (top) and the absence of genomic DNA contamination in RT (−) (bottom). (B) Quantification of HASPIN mRNA levels in breast cancer cell lines and MCF10A cells. Data are presented as the mean ± SD of three independent experiments. (C) Doubling times ± SD (hours) of breast cancer cells and MCF10A cells (left panel). Data are presented as the mean ± SD of three independent experiments. Pearson’s correlation coefficient between HASPIN expression level and doubling time in breast cancer cells (r = −0.913, P < 0.05, right panel).
Fig 2.
CHR-6494 inhibits the proliferation of breast cancer cells in vitro.
(A) Immunoblotting analysis of pH3T3. The breast cancer cells were treated with DMSO or CHR-6494 (1000 nM) for 24 h. The effects of CHR-6494 on the viability of MDA-MB-231 (B), MCF7 (C), SKBR3 (D), and MCF10A (E) cells were determined by XTT assay. IC50 values are shown for each cell line. Data are presented as the mean ± SD of three independent experiments.
Fig 3.
CHR-6494 induces cell cycle arrest and apoptosis in vitro.
(A, B) Flow cytometry analysis of control and CHR-6494-treated breast cancer cells. DNA profiles (A) and phospho-MPM-2 profiles (B). Black boxes indicate the gated mitotic cell populations (B). (C) Dot plots showing the distributions of FITC-Annexin V/PI-stained control and CHR-6494-treated breast cancer cells. The cells in the lower left quadrant represent viable cells (FITC-Annexin V/PI-negative). The cells in the upper and lower right quadrants represent the late and early apoptotic cells, respectively. Data are presented as means ± SD of three independent experiments. *P < 0.05 (Student’s t test).
Fig 4.
Effects of CHR-6494 on the growth of MDA-MB-231 xenografts.
(A) Schematic representation of CHR-6494 treatment in MDA-MB-231 xenografted mice. Two weeks after MDA-MB-231 transplantation in immunodeficient nude mice, mice received four cycles of CHR-6494 (50 mg/kg) injected intraperitoneally (5 consecutive days over 35 days). (B) Tumor volume (mm3) was monitored in control and CHR-6494-treated mice. Data are presented as the means ± SD (n = 7 mice). (C) Tumor weight (g) was measured at the study endpoint. P = 0.71; Mann–Whitney U test. Data are presented as the mean ± SD (n = 7 mice). (D) Immunofluorescence of CD31-positive (top) and LYVE-1-positive (bottom) vessels in MDA-MB-231 tumors. Scale bars: 100m. (E) Vessel area of the tumors (LYVE-1-positive). P = 0.269; Student’s t test. Data are presented as the mean ± SD (n = 4 mice).