We observed 10 fields per section at 400× magnification, and the mean percentage of positively stained cells was used to determine the expression of the proteins in a section. All counts were performed blindly

for at least 3 randomly chosen sections from each mouse. Statistical analysis Statistical software SPSS 10.0 (Chicago, Illinois) was used in the Protein Tyrosine Kinase inhibitor analysis. A P value less than 0.05 was considered statistically significant. Differences among groups were assessed using the ANOVA test, and the LSD test was used to compare the differences in MMP-9 (protein and mRNA) and PCNA expression among the different groups. Results Combined CoCl2 and glibenclamide treatment influences tumor growth in TA2 mice inoculated with breast cancer cells The average growth rate of tumor in the mice that received combined treatment with CoCl2 + glibenclamide was obviously inhibited compared to the other groups according to the average tumor size that was measured every other day (Figure 1). All the mice were sacrificed 18 days after the initial inoculation and the tumors were removed. The average tumor volume in the CoCl2 + glibenclamide group was significantly reduced when compared with the other groups (Figure 1), and

the differences among these groups had statistical significance (F = 489.5 P = 0.0098). Figure 1 The growth curve of injected TA2 breast cancer cells in the control and treatment groups. Morphologic tumor changes in the treatment and control groups Immediately following sacrifice, breast cancer tissue samples were carefully collected. In the DMSO group, tumor cells invaded see more the surrounding normal tissue. As shown in Figure 2A, there were large areas of necrosis in tumor tissues from the paclitaxel and CoCl2 + glibenclamide groups, while a small amount of necrosis was observed in the DMSO (Figure 2A-a), CoCl2 (Black arrow heads, Figure 2A-b) and glibenclamide groups (Black arrow heads, Figure 2A-c). Moreover, numerous tumor cells in the CoCl2 + glibenclamide group displayed cell degeneration as suggested by the Progesterone presence of vacuoles within the cytoplasm (Black arrow heads, Figure 2A -d). Figure 2 The differences of morphology, MMP9 and

PCNA expression of TA2 breast cancer between the control and treatment groups. A. The morphologic characteristics of TA2 breast cancer in the control and treatment groups (HE staining, ×200). a. DMSO group. b. CoCl2 group. c. Glibenclamide group. d. CoCl2 + glibenclamide group. e. Paclitaxel group. B. Immunohistochemical staining for MMP9 and PCNA in the control and treatment groups (immunohistochemical staining, ×200). a. MMP9 staining of DMSO group. b. MMP9 staining of CoCl2 group. c. MMP9 staining of Glibenclamide group. d. MMP9 staining of CoCl2 + glibenclamide group. e. MMP9 staining of paclitaxel group. f. PCNA staining of DMSO group. g. PCNA staining of CoCl2 group. h. PCNA staining of Glibenclamide group. i. PCNA staining of CoCl2 + glibenclamide group. j.