Figure 1.
Decreased VLS in resveratrol-treated non-tumor mice.
Non-tumor bearing mice were given HDIL-2 and/or resveratrol. The vascular leak in the lungs and liver as well as TNF-α in the serum were measured. A. VLS in lung. B. VLS in liver. C. The TNF-α level in the serum was measured by sandwich ELISA.
Figure 2.
Decreased VLS and increased tumor regression in resveratrol-treated melanoma-bearing mice.
C57BL/6 mice were injected i.v. with B16F10 melanoma cells (5×105 cells/mouse) and adminsitered resveratrol as described in Fig. 1. On day 9, mice were treated with IL-2 as in Fig. 1. The mice were sacrificed for evaluation of VLS on day 12. The numbers of black metastatic nodules on the lung surfaces were counted under a microdissecting microscope. The area of each nodule was measured with software Image Pro. The values are shown as mean ±SEM. A. VLS in lung. B. VLS in liver. C. Enumeration of lung metastatic nodules. D. Area measurement of lung metastatic nodules. E. Histopathology of lung metastasis on the surface of one lobe.
Figure 3.
Resveratrol protects endothelial integrity.
The ultrastructural studies showed resveratrol protects endothelial cells. Panels 1 and 2 show PBS- or resveratrol-treated mice, respectively. The endothelial cell (En) lining the blood vessel is firmly anchored to the basal lamina (BL) with good integrity, thereby showing normal morphology. The cell has an intact nucleus (Nu) and is closely opposed to the intact basal lamina. Three RBCs lie within the blood vessel lumen. Panel 3 shows IL-2-treated mice with significant damage to the endothelial cells. Cell membrane is folding (F) and discontinuous (D). Vacuolation (V) is found under the folded cell membrane and within the cytoplasm. In addition, some endothelial cells have lost the normal morphology, with only extended cell membrane remnants remaining. Cellular debris from former endothelial cells can be found in the blood vessel lumen. An RBC fills the lumen. Panel 4 shows IL-2+resveratrol-treated mice with normal endothelial cell morphology. The cell membrane and cytoplasmic contents are well defined and closely adhere to the basal lamina. The intact nucleus is closely opposed to the intact basal lamina Three RBC fill the lumen. Original magnification, ×20,000.
Figure 4.
Histological studies of the lung and the liver for inflammatory cell infiltration.
Lungs and livers from VLS mice were harvested and preserved in 10% formalin solution. Sections were stained with hematoxylin and eosin. The level of perivascular infiltration was determined by counting the number of cells infiltrating around a venule. The data depicts the mean ±SEM of sections from four individual mice. A. The level of perivascular infiltration in lungs. B. The level of perivascular infiltration in livers. C. Statistic analysis of the infiltration depicts p>0.05 between IL-2 and IL-2+Resveratrol groups. Original magnification, ×200.
Figure 5.
Resveratrol protects endothelial cells from apoptosis.
Lungs from PBS, resveratrol, IL-2 or IL-2+resveratrol-treated mice were examined for apoptosis with TUNEL assay as described in Materials and Methods. Apoptotic cells are depicted by brown stained nuclei (arrow). The histogram shows the quantification of apoptotic cells. Original magnification, ×400.
Figure 6.
Resveratrol inhibits IL-2-induced expansion of Treg.
Splenocytes and infiltrating cells were stained with FITC-conjugated anti-CD4 mAb, fixed and permeabilized and incubated with PE-conjugated anti-FoxP3 mAb as described in Materials and Methods. The stained cells were analyzed by flow cytometry. The representative dot plots from spleen (A), lung (B), and liver (C) and the statistical analysis of the percentage mean ±SEM of Treg (D) are shown.
Figure 7.
Expansion of MDSC by IL-2 and/or resveratrol treatment.
Splenocytes and the infiltrating cells were stained with FITC-conjugated anti-CD11b and PE-conjugated anti-Gr-1 mAbs. The stained cells were analyzed by flow cytometry. The representative dot plots from spleen (A), lung (B), and liver (C) and the statistical analysis of the percentage mean ±SEM of MDSC (D) were shown.
Figure 8.
Analysis of the functional characteristics of MDSC.
Panel A: MDSC were isolated from IL-2 and IL-2+resveratrol treatment (labeled as IL-MDSC and R-MDSC respectively). LAK cells from IL-2 treated mice were isolated and incubated with MDSC for 2 h, and then used as effectors in the cytotoxicity assay against EC cell targets. Panel B: 3H-thymidine incorporation assay to measure the proliferation of the LAK cells in the panel A. Panel C: 1×106 MDSC were intravenously transferred to naive mice. VLS was then induced in the recipient mice. Panel D & E: Total RNA was extracted from the MDSC with the respective treatment. Expression of Arg1 (D) and AhR (E) were detected by QPCR. The relative expression was normalized to the endogenous 18S.
Figure 9.
Resveratrol enhances the expression of FoxO1 and the cytolytic susceptibility of melanoma.
Panel A and B: C57BL/6 mice were implanted s.c. with B16F10 melanoma cells (2×105 cells/mouse) and orally administered with resveratrol. On day 9, mice were treated with IL-2 as described in Fig. 1. The mice were sacrificed for evaluation of VLS on day 12. The primary tumors in the skin were resected and weighed. Total RNA was extracted. Expression of FoxO1 was detected by QPCR (A). The relative expression was normalized to the endogenous 18S. The mean ±SEM of weight is shown (B). Panel C: B16F10 cells were cultured with vehicle (Veh), IL-2 (IL) (1000 units/ml), or resveratrol at the indicated dose for 24 h. Total RNA was extracted. Expression of FoxO1 was detected by QPCR. Panel D: LAK cells were generated by culturing splenocytes from naïve mice with IL-2 (1000 units/ml) for 48 h. B16F10 cells were incubated with resveratrol (25 µM) for 2 h and labeled with 51Cr. The labeled B16F10 cells were then added to the LAK cells. After 4 h, the release of 51Cr was measured and percent cytotoxicity was calculated. Statistical analysis was performed at each effector to target ratio and showed p<0.001.