Figure 1.
Western blotting analysis showed β-catenin in the membrane and cytoplasmic/nuclear cell fractions. α-tubulin was the loading control for the membrane fraction. Lamin A was the loading control for the cytoplasmic/nuclear fraction. Data are representative of at least 3 independent experiments. The graph shows the relative percentage of β-catenin in the corresponding location. Approximately 100 cells were evaluated, the odds ratio for cytoplasmic and or nuclear translocation of β-catenin in radioresistant cells versus their parents was 2.552 (95% CI: 1.443 to 4.512) and the Fisher's exact two-tailed was P = 0.0018.
Figure 2.
FIR-induced WISP-1 over-expression in esophageal cancer cells.
A. Western blotting analysis showed WISP-1 in the radioresistant cells was overexpressed, compared to their parents and normal esophageal epithelial cells. β-actin was the loading control. Data are representative of at least 3 independent experiments. The graph shows the mean band intensity (±SD). B. WISP-1 protein expression was determined in total protein lysates from KYSE-410 cells during FIR using Western blotting analysis. β-actin was the loading control. Data are representative of at least 3 independent experiments. 1, 2, 3, 4, 5, 6, 7, 8, 9f represented first, second, third, fourth, fifth, sixth, seventh, eight, and ninth fraction (2 Gy) of irradiation. The graph shows the mean values (±SD) of relative expression of WISP-1 versus β-actin. C. The increased expression of β-catenin and WISP-1 in radioresistant KYSE-410R cells was then further confirmed by immunofluorescence staining in vivo with tumor tissues from mice. D. WISP-1 protein expression was assessed using immunohistochemical staining on specimens from clinical patients before (upper panel, ×100) and after (bottom panel, ×100, ×400) radiotherapy.
Figure 3.
WISP-1 in medium promoted radioresistance of esophageal carcinoma cells.
A. ELISA assays (in triplicate) for WISP-1 levels in the conditioned medium (*P<0.05 when compared with non irradiated, **P<0.01 when compared with media alone or HET-1A, ***P<0.001 when compared with media alone or HET-1A or their parents). B. Recombinant WISP-1 stimulation elevated radioresistance in esophageal cancer cells. Clonogenic survival in recombinant WISP-1 protein (2 µg/ml) treated KYSE-410 and TE-1 cells after irradiation. The data points show mean survival fraction from 5 individual experiments (±SD).
Figure 4.
Depletion of extracellular WISP-1 protein attenuated the radioresistance of established radioresistant cancer cells.
Clonogenic survival in anti-WISP-1 antibody (4 µg/ml) treated radioresistant esophageal cancer cells after irradiation. The data points show mean survival fraction from 5 individual experiments (±SD).
Figure 5.
Depletion of extracellular WISP-1 protein promoted mitotic catastrophe in radioresistant cancer cells.
KYSE-410R cells were treated with anti-WISP-1 antibody (4 µg/ml), 4 Gy of radiation, or a combination. Immunofluorescence staining for nuclei (DAPI, blue), γ-H2AX (red) and β-tubulin (green). Scale bars, 10 µm. White arrows indicate γ-H2AX foci; red arrows indicate assembly of abnormal microtubules; blue arrows indicate atypical β-tubulin staining (green) co-stained withγ-H2AX (red). Yellow arrows indicate lagging chromosomal material. Data are representative of at least 3 independent experiments. Immunofluorescence was quantified by counting the mean number of nuclei per high-power field. The graph shows the quantification of nuclei positive for abnormal β-tubulin staining and β-tubulin and γ-H2AX co-staining. The graph shows the means ± SE of three independent experiments. For every experiment at least 200 nuclei were counted per treatment condition.
Figure 6.
WISP-1 antagonism specifically targeted FIR induced radioresistant cancer cells.
Graphs show the results of CCK-8 assays 48 hours (left panel) and 72 hours (right panel) after anti-WISP-1 treatment of KYSE-410 and KYSE-410R cells. Data are presented as means ± SD (n = 12). DMSO controls validated these results (data not shown).
Figure 7.
The role of WISP-1 mediated radioresistance in vivo.
NOD-SCID mice bearing KYSE-410 or KYSE-410R tumors were treated with radiotherapy, 2 µg/ml recombinant WISP-1 protein or 4 µg/ml anti-WISP-1 antibody, or a combination. The change in tumor volume was calculated as described in “Materials and Methods”. Data are presented as means ± SD (n = 6). BSA controls validated these results (data not shown).