For concentration-dependent inhibitory experiments
against the killing activity of PMN, c-Met inhibitor different concentrations of either parental A520C9 mAbs, or synthetic VHFR1C-10-VHCDR1-VHFR2-VLCDR3-VLFR4N-10 (South West University) were added with PMN (75 μg/ml) to incubate with MCF-7, Zr-75-30 or Raji cells, respectively (102-10-1nM), then living and dead cells were counted with 0.2% Trypan blue under an inverted microscope (IX-71, Olympus). The MCF-7 cells were grown and fixed as the above-mentioned procedure. Then original antibodies (OAbs) and the mimetic peptides were diluted to 100, 10, 1 and 0.1 μmol/L by PBS (pH7.45), respectively. The indirect enzyme-linked immunosorbent assays (ELISA) were introduced to analysis the relative affinity of the mimetics and OAbs to antigens. The value of absorbance at 490 nm wavelength was inspected by microplate reader (Bio-Rad), which was used to determine the concentration
of the OAbs and the mimetics when the saturation of Abs to antigens reached to one percent. The relative affinity was compared between OAbs and the mimetics at 50% saturation of Abs to antigens. In vivo activity and the biodistribution of PMN MCF-7 cells were Dabrafenib grown under the same condition as that of above described, and collected by centrifugation at 1,000 rpm. Cells were resuspended in FBS-free medium at a concentration of 108 cells/ml. Twenty-five 4–5-week-old female BALB/c athymic nude mice weighing 16–20 g were purchased from the Experimental Animal Center of West China Hospital. Before implanting tumor cells, mice were allowed to acclimatize for 3 days. A total of 6–7 × 107 MCF-7 cells were subcutaneously (s.c.) implanted into the left armpit of mice. Tumor growth was monitored daily until the average sizes of tumors reached 5 × 5 × 5 mm, then randomly separated those mice to the treatment group (PMN group; n = 5), wild type colicin Ia group (wt Ia group; n = 5), Fab-Ia group
(n = 5), Sc-Ia group (n = 5) and the PBS control group (PBS group; n = 5), and the treatment course began. The PMN group was treated with intraperitoneal (i.p.) injection of PMN at 1,200 μg/mouse/day (400 μg/8 hours, tid; n = 5). The wt Ia group, Fab-Ia group, Sc-Ia group and the PBS group were why injected with wt Ia protein, Fab-Ia protein, Sc-Ia protein (400 μg/8 hours, i.p. tid; n = 5) and PBS (450 μl/8 hours, i.p. tid; n = 5), respectively. Animals had free access to standard food and water throughout the treatment course. After 14 days, all mice were sacrificed to collect tumors and organs for weighing and for histopathological inspection. 150 μg PMN proteins labeled by FITC (EZ-labeled FITC protein labeling kit, pierce) were ip injected into BALB/c mice (n = 5), weighing 16–20 g, inoculated MCF-7 cells at armpit for 2 weeks. 2.5 hours later, the mice were fastened supinely on a black board under ether inhalation.