Figure 5

HNK treatment inhibits CNI-induced renal tumor growth in vivo; and it is associated with decreased tumor vasculature and increased tumor cell apoptosis. Nude mice were subcutaneously, xenografted with 786-O cells (1 × 10⁶), and when palpable tumor appeared around 10 days later, mice (n = 5 each group) were treated intraperitonealy with different combinations of CsA (10 mg/Kg/day) and HNK (2 mg/Kg/day); and the mice in control group were treated with vehicle alone. Treatments were continued for up to 20 days (i.e. 30 days after tumor injection). (A) Tumor volumes were calculated (as described in “Materials and Methods” section) for the four treatment groups. The mean change in tumor volume is shown in the tumor growth curve, (*CsA compared to vehicle treatment, p < 0.0001; ⁺(CsA + HNK) treatment compared to CsA alone, p < 0.0001; and #, HNK alone compared to (CsA + HNK) treatment, p < 0.0001). The data reflect representative of two independent experiments. (B) Tumors were excised after euthanizing mice at the end of study, and two representative tumors from each group is shown to depict tumor size. (C) IHC stains showing the expression of CD31 in harvested tumor tissues of mice from all treatment groups. Representative photomicrographs (magnification, x400) of tissue sections from each treatment group are shown. Side panel, bar diagram depicts mean vessel density calculated by standard grid counting of CD31-positive vessels. The data reflect ±S.D. of three different tissues from each group, in which three to four non-overlapping fields of each specimen were analyzed in a blinded manner. (D) Total tissue lysates, prepared from the excised tumors, were utilized to measure the expression level of cleaved caspase-3 and β-actin by Western blot. The bands from adjacent lanes were cropped from the same blot (full-length blots are included in a supplementary file). Representative blots of three different tumor tissue lysates from each group are shown.