Article Figures & Data
Figures
- Figure 1.
Effects of calcitriol on tumor growth in vitro and in vivo. A, effect of calcitriol on the proliferation of CE81T and TE2 cancer cells in vitro as determined by viable cell counting. The y-axis represents the viable cell number. Data represent the means ± SEM. *, P < 0.05. B, effect of calcitriol treatment on ectopic tumor growth. Data represent the means ± SD of three independent experiments (9 animals per treated group in total). *, P < 0.05. Representative slides using Ki-67 nuclear staining in tumors 21 days after implantation was also shown with magnification ×400. C, effect of calcitriol treatment on cell death, as assessed by FACS with Annexin V–PI staining using cells under control condition or treated with calcitriol for 72 hours. The results of representative slides are shown. D, the changes of apoptosis and cell-cycle–associated proteins in vitro were evaluated by immunoblotting. E, immunoblotting analysis of VDR, p21, Bax, and Bcl-2 expressions using proteins extracted from tumor samples 21 days after implantation.
- Figure 2.
Effects of calcitriol on aggressive tumor behavior and EMT changes. A, effect of calcitriol on the invasiveness of esophageal cancer cells in vitro. Representative slides and quantitative data (y-axis shows the relative ratio, normalized to the number of invading cells under control conditions) are shown. *, P < 0.05. B, effect of calcitriol treatment on the invasive capacities was evaluated by murine orthotopic tumor implantation. The results are shown by representative slides and quantitative data. The y-axis represents the relative ratio of esophageal tumor invading into adjacent structure 2 weeks after implantation. *, P < 0.05 (white circle, excision location; black arrow, esophagus; red arrow, tumor). C, the changes of E-cadherin expression were evaluated by immunofluorescence in vitro, and the results of representative slides are shown. Scale bars, 25 μm. D, effect of calcitriol treatment on EMT-associated proteins. The changes in the levels of EMT-associated proteins were evaluated by immunoblotting in vitro. E, levels of VEGF and MMP-9 expression evaluated using immunohistochemical analysis in tumors 21 days after implantation. The results of representative slides are shown with magnification ×400.
- Figure 3.
Effects of calcitriol on IL6/STAT3 signaling in esophageal SCC. A, effect of calcitriol treatment on the expression of IL6 and activated STAT3 were evaluated by immunoblotting in vitro. B, the levels of IL6 in cellular supernatants were examined by ELISA in vitro. Columns represent the means ± SEM. *, P < 0.05. C, levels of IL6 and p-STAT3 evaluated using immunohistochemistry and immunoblotting analysis in ectopic tumors 21 days after implantation. The results of representative slides are shown with magnification ×400. Calcitriol treatment significantly decreased the expression of IL6. D, effect of calcitriol treatment on the expression of phosphorylated p38 was evaluated by immunoblotting. E, the expression levels of IL6 and p-STAT3 were evaluated by immunofluorescence using CE81T cells incubated in the presence of indicated dosage of calcitriol or p38 inhibitor for 48 hours and the results of representative slides are shown. Scale bars, 50 μm. F, effect of p38 MAPK inhibitor on the expression of IL6 and p-STAT3 was evaluated by immunoblotting.
- Figure 4.
Evaluation of esophageal tumor formation using histologic examination and animal micro-PET imaging and its relationship with IL6 levels and MDSC recruitment. A, images of the gross lesions and pathologic findings of tissue sections from animals treated with 4-NQO or vehicle for 16 weeks and then followed for 12 to 14 weeks. The representative slides are shown with magnification ×100 and ×400. B, representative PET images from mice treated with 4-NQO and diagnosed with invasive carcinoma, compared with benign lesions (hyperplasia/papilloma). The tumor-to-muscle SUVR was calculated from the micro-PET scans. Data points represent the means ± SEMs. C, flow cytometric analysis of CD11b+Gr1+ cells from mice exhibiting hyperplasia/papilloma or invasive carcinoma after histologic analysis. Representative images and quantitation are shown. Columns represent the means ± SEM. *, P < 0.05. D, IL6 levels in mice were measured using ELISA and quantitative data are shown. Columns represent the means ± SEM. *, P < 0.05.
- Figure 5.
Effect of calcitriol on tumor progression related to IL6 signaling in vivo. A, quantitative data of SUVR of esophageal lesion in 4-NQO–treated mice with or without calcitriol treatment or regulation of IL6. The tumor-to-muscle SUVR was calculated from the micro-PET scans. Columns represent the means ± SEM. B, representative images of gross lesions and the pathologic findings on sectioned tissue samples from 4-NQO mice with or without calcitriol or regulation of IL6. Quantitative data assessing the incidence of invasive esophageal tumors are shown. Columns represent the means ± SEM. *, P < 0.05. C, flow cytometric analysis of CD11b+Gr1+ cells from 4-NQO–treated mice with or without calcitriol or regulation of IL6. Representative images and quantitative data are shown. Columns represent the means ± SEM. D, effect of calcitriol treatment on the expression of IL6 in esophageal specimen from mice (control, C57 mice with vehicle only; 4NQO, 4-NQO–treated mice; 4-NQO + calcitriol, 4-NQO–treated mice with calcitriol injection; 4-NQO + IL6 KO, 4-NQO–treated IL6 KO mice) were evaluated by immunofluorescence and real-time RT-PCR. *, P < 0.05.
- Figure 6.
Effect of calcitriol treatments on the function and recruitment of MDSC in vivo. A, effect of calcitriol on MDSC recruitment in vivo (control, C57 mice without treatment; 4NQO, 4-NQO–treated mice; IL6, IL6-stimulated mice; IL6 + calcitriol, IL6-stimulated mice with calcitriol injection) were evaluated by FACS. B, immunofluorescence staining revealed that calcitriol attenuated the IL6-induced increase in the levels of activated STAT3 and ROS in MDSC from murine spleen. (top row, low power field with scale bars, 50 μm; bottom row, high power field with scale bars, 25 μm). Representative images and quantitative data are shown. C, the suppressing ability of MDSC for T-cell proliferation was evaluated by FACS. Representative images and quantitative data are shown (stimulation alone, CD8+ T cells with anti-CD3/CD28 stimulation beads without CD11b+ cells; +CD11b cells, T cells with stimulation and coculture with CD11b+ cells from control mice, or IL6-stimulated mice with or without calcitriol injection). *, P < 0.05.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary methods and legends - Supplementary methods and legends for supplementary Figures
- Supplementary Figure 1 - Supplementary Figure 1. Effects of Calcitriol on tumor cell proliferation in vitro
- Supplementary Figure 2 - Supplementary Figure 2. Effects of Calcitriol on migration capacity and EMT in vitro
- Supplementary Figure 3 - Supplementary Figure 3. Effects of blocking IL-6 on tumor growth and EMT in vitro
- Supplementary Figure 4 - Supplementary Figure 4. Effects of Calcitriol on IL-6/STAT3 signaling in esophageal SCC
- Supplementary Figure 5 - Supplementary Figure 5. IL-6 levels in 4-NQO-treated mice with or without Calcitriol or IL-6 injection were measured using ELISA
- Supplementary Figure 6 - Supplementary Figure 6. Effect of Calcitriol treatments on the function and recruitment of MDSC in vivo.
Volume 14, Issue 6
