MMP9 and PCNA protein expression in tumor cells in the control and treatment groups Both the treatment group and the control group contained tumor cells that stained positively for MMP9 and PCNA. MMP9 protein expression was detected mainly in the cytoplasm of tumor cells while PCNA protein expression was seen in the nucleus. PCNA expression occurred in the nuclei of cells during the DNA synthesis phase of the cell cycle and provides an important marker indicating tumor proliferation. The tumor cells that positively stained for MMP9 were mainly distributed at the edge of normal tissue,

especially in the area between tumor tissue and skeletal muscle. In the center of the tumor mass, the percentage of positively stained cells was low. Immunohistochemical results showed statistically significant differences for mean percentage of MMP9 positively stained cells among the treatment groups CDK inhibitor (P = 0.00687, Figure 2B –a to -e). The CoCl2 + glibenclamide group had the lowest MMP9 expression. Results of immunohistochemical staining for PCNA showed that combined treatment with CoCl2 + glibenclamide inhibits tumor growth by decreasing tumor cell duplication, suggested by the mean percentage of positively stained cells that only reached 52.89% (Figure 2B –f to -j). The differences seen in the percentage of cells expressing PCNA among the treatment groups had statistical

significance Entospletinib concentration (P = 0.0348) (Table 1). The results of immnohistochemical staining show that combined treatment with CoCl2 + glibenclamide down-regulates MMP-9 and PCNA expression and inhibits tumor growth and invasiveness. Table 1 Comparison of the mean percentage of cells staining positive for MMP9 and PCNA among the treatment groups Group n MMP9   PCNA   DMSO 10 0.6312 ± 0.1527   0.9156 ± 0.1022   CoCl2 10 0.6028 ± 0.1337   0.8833 ± 0.1857   glibenclamide, 10 0.5711 ± 0.1637 F = 324.5 P = 0.00687 0.9017 ± 0.1772 F = 187.6 P = 0.0348 CoCl2 + glibenclamide 10 0.2856 ± 0.1234   0.5289 ± 0.1403   paclitaxel 10 0.3451 ± 0.1956   0.6574 ± 0.1945   MMP9 mRNA expression among the treatment groups After extracting total

mRNA from fresh tumor Baricitinib tissues taken from the control and treatment groups the concentrations were see more determined by UV spectrophotometer. Results of electrophoresis in 1% agarose gel showed that the mRNA had no obvious degradation. After performing real-time PCR the products were separated by 1% agarose gel electrophoresis. The MMP9 product was about 86 bp and the optimal annealing temperature was 64.2°C. Results of real time PCR demonstrated that the mRNA expression of MMP9 in the treatment groups was decreased compared with the control group. This trend follows what was seen with MMP9 protein expression. There was statistical significance for MMP9 (P = 0.021) mRNA levels among the groups (Table 2). Table 2 Comparison of the mRNA expression of MMP9 among the treatment groups Group n MMP9 mRNA   DMSO 10 1.320 ± 0.0524   CoCl2 10 0.881 ± 0.0723   glibenclamide 10 0.941 ± 0.