Figure 1.
Specific targeting of TMTP1-DKK peptides in vitro and in vivo.
A Fluorescent images of tumor cells treated with TMTP1-DKK were examined with Confocal laser scanning microscopy. (a) PC-3M-1E8 cells (b) PC-3M-2B4 cells (c) MKN-45sci cells (d) NIH/3T3 cells. FITC-TMTP1-DKK (green) and control peptide were examined in xenograft tumors including PC-3M-1E8 (e)TMTP1-DKK, (f)svTMTP1-DKK, MKN-45sci (g) TMTP1-DKK, (h) svTMTP1-DKK. Nuclei were co-stained with DAPI (blue). B Fluorescent images of nomal cells (normal mammary epithelial cell MCF-10A, normal liver cell LO2 and HEK293 cells) treated with TMTP1-DKK were examined with Confocal laser scanning microscopy. The morphological change of nomal cells was visualized using inverted microscope.
Figure 2.
Cytotoxicity of the TMTP1-DKK peptide in various cell lines. A, B, C
Cell survival rates were determined by MTT assays performed in triplicate (error bars, ±SD). The data presented represent the percentage of cells surviving compared to untreated cells. Representative results are shown. The differences of survival rates between 10 µM and 20 µM TMTP1-DKK are more significant in either PC-3M-1E8 or MKN-45sci cells (P<0.01). However, little or no effect was seen on murine fibroblast NIH/3T3 cell proliferation when they were treated with TMTP1-DKK. D Cells viability of MKN-45 and PC-3M-1E8 cancer cells treated different concentrations (0–20 µM) of svTMTP1-DKK for 24 hour was measured by MTT assay. E Morphological quantification of cellular apoptosis by inverted microscope in PC-3M-1E8 and MKN-45sci cells treated with 10 µM TMTP1-DKK. After treated with DKK, the cells showed cell shrinkage, membrane disintegration, and nuclear condensation/fragmentation. F Little Morphological change was observed by inverted microscope in PC-3M-1E8 and MKN-45sci treated with 10 µM svTMTP1-DKK for 24 hour.
Figure 3.
TMTP1-DKK induces apoptosis in cells. A
Cells were treated overnight with 10 µM TMTP1-DKK peptide and then analyzed by flow cytometry at 12 h, 24 h, and 48 h. Proliferating PC-3M-1E8 cells and MKN-45sci cells treated with TMTP1-DKK peptide (filled bars) showed a decrease in viability through apoptosis over time (P<0.01). However, murine fibroblast NIH/3T3 cells showed no significant changes. B Signaling molecules involved in the apoptotic effect of TMTP1-DKK peptide treated. Cultured cells treated with 10 µM TMTP1-DKK peptide for 24 h were harvested and lysed. Total cell proteins were resolved by 12% SDS-PAGE gel elctrophoresis and then transferred to a nitrocellulose membrane. After blocking with 5% nonfat milk, the membranes were incubated for 1 h with 1 µg/ml primary antibodies (caspase 9 (35 KD, 17 KD), caspase 8 (20 KD) and caspase 3(19 KD)) followed by horseradish peroxidase-conjugated anti-rabbit secondary antibodies. Blots were exposed to chemiluminescence substrate and developed with Hyperfilm MP. C PC-3M-1E8 cells and MKN-45sci cells were challenged with 10 µM TMTP1-DKK in the presence (+) of 40 µM Z-VAD-fmk for 24 h. After 24 hours, cell viability was analyzed. The broad range caspase inhibitor Z-VAD-fmk decreased the apoptosis rate of PC-3 M-1E8 and MKN-45sci cells.
Figure 4.
TMTP1-DKK inhibits cell migration. A
Cells were incubated with 2 µM TMTP1-DKK peptide for 12 h. Transwell migration assays of PC-3M-1E8 cells and MKN-45sci cells were performed. After 24 h incubation, cells from the upper side of the filter were removed and cells from the lower surface of the filter were fixed and stained. Data are the means ± SE of three independent experiments; each performed in triplicate. B Cellular migration was reduced by 52.38±3.3% in PC-3M-1E8 cells and 46.16±2.7% in MKN-45sci cells compared to the appropriate controls.
Figure 5.
TMTP1-DKK inhibits tumor growth and development by the mouse models of MKN-45sci orthotopic gastric cancer and PC-3M-1E8 prostate cancer.
A Mice treated with TMTP1-DKK peptide survived longer than control mice treated with an equimolar mixture of TMTP1 peptide or filter-sterile water, as shown by a Kaplan-Meier survival plot. B, C PC-3M-1E8 prostate cancer and MKN-45sci orthotopic gastric cancer treated with TMTP1-DKK peptide were smaller than those treated with control peptide. D Tumor growth in PC-3M-1E8 tumor-bearing mice undergoing TMTP1-DKK peptide therapy. Mice were treated over a period of 40 days (n = 9). During the treatment period, tumors were measured twice a week. Tumors volume in mice treated with TMTP1-DKK peptide was on average 18% the size of control. Tumor volumes were assessed on day 1 (○) and day 40 (•). P<0.05, t-test. E Animals were sacrificed six days after TMTP1-DKK treatment. The tumor was recovered and imaged immediately following analysis of apoptotic cell death by TUNEL assay. The number of apoptotic cells increased notably in TMTP1-DKK peptide–treated tumors. a, Magnification: ×100. b Magnification: ×200.
Figure 6.
TMTP1-DKK suppresses tumor metastasis and progression by MKN-45sci orthotopic xenografts in athymic mice. A, B
TMTP1-DKK peptide caused a dramatic and dose-dependent decrease in the number of metastatic tumors (yellow arrows) in MKN-45sci orthotopic gastric cancer. C Metastasis tissue samples were harvested and pathologically confirmed by H&E staining. D Orthotopic gastric tumors were recovered and imaged immediately following analysis of apoptotic cell death by TUNEL assay. Columns, average number of TUNEL-positive cells counted in three randomly selected fields in three tumor samples from each group; error bars, SD. *, P<0.001 by Student's t test compared with the control group.