Briefly, tissue sections were baked, deparaffinized and microwaved at 98°C for 10 minutes in citrate buffer (0.01 M citric acid, pH6.0). After blocking the endogenous peroxidase by immersed the
sections in 3% H2O2, the sections were incubated with primary antibodies directing against human RhoA (sc-32039, 1:50; Santa Cruz) and RhoC (sc-12116, 1:50; Santa Cruz). Expression of RhoA or RhoC protein in tissue sections was detected with Anti-goat IgG/HRP Detection Kit(PV-6003; Zhongshan Biotechnology Limited Company, Beijing, China). The tissue sections were then counterstained with hematoxylin. Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End-labeling (TUNEL) Assay Assessment of cell death was performed by TUNEL GSK461364 in vivo method using an in situ cell death detection kit conjugated with horse-radish peroxidase (POD) (Roche Applied Science, Indianapolis, IN, USA), according to the manufacturer’s instructions. Five equal-sized fields in tissue sections were randomly chosen and analyzed under the Leica
DMI 4000B(Leica, Germany) light microscope. Density was evaluated in each positive staining field, yielding the density of dead cells (cell death index). Statistical Analysis All data were shown by mean Blebbistatin ± SD. Statistical analyses were performed using SPSS statistical software (SPSS Inc., Chicago, Illinois). Differences between two groups were assessed using a t test. A P value less than 0.05 was considered statistically significant. Results Ad-RhoA-RhoC-siRNA Inhibits Tumor Development in Nude Mice Tumors in the nude mice could be seen at 5th day from the implantation of HCT116 cells and Amylase all tumors had reached 5-7 mm in size at 9th day. The successful rate
of tumor implantation was 100%(Figure 1). After intratumorally injection, the growth speed of tumors in the three group was quite different. As shown in figure 2, the tumors in NS and Ad-HK group grew rapidly. In contrast, tumors in Ad-RhoA-RhoC group were significantly see more delayed. The dissected tumors in the NS and Ad-HK group had volumes of (699.62 ± 190.56)mm3 and (678.81 ± 155.39)mm3, which were 5.05 ± 0.48-fold and 4.58 ± 0.94-fold larger than the starting volume, whereas in the Ad-RhoA-RhoC group, the tumors had a volume of (441.38 ± 63.03)mm3, increased only 2.38 ± 0.56-fold (Figure 3). Tumor growth delay was statistically significant (P < 0.05). In addition, the mean tumor weight in NS, Ad-HK and Ad-RhoA-RhoC group was (0.75 ± 0.22) g, (0.78 ± 0.22) g and (0.36 ± 0.13) g, respectively. These data demonstrated that injection of Ad-RhoA-RhoC was able to slow down the growth of HCT116-derived xenografts. Figure 1 Tumor-bearing nude mice with 100% of tumor implantation rate. Figure 2 Growth curve of subcutaneous implanted tumors in nude mice treated with NS, Ad-HK, or Ad-RhoA-RhoC. Tumor volume is plotted against time elapsed. A significant delay in tumor growth is seen in the group treated with Ad-RhoA-RhoC.