Article Figures & Data
Figures
- Figure 1.
As2O3 enhances radiation-induced endothelial cell and tumor cell death while protecting osteoblasts. Endothelial cells (HDMEC, 5 × 103), tumor cells (OSCC-3, 3 × 103), and osteoblasts (NHOst, 5 × 103) were plated in 96-well plates at 37°C and cultured overnight. A, cells were treated with 0.5, 1, 2.5, 5, 10, and 20 μmol/L doses of As2O3. B, cells were treated with As2O3 (AS, 5 μmol/L) followed by radiation treatment (IR, 7.5 Gy). After 72 h of culture, 10 μL of WST/ECS solution (BioVision) was added into each well and these cells were further incubated at 37°C for 2 to 4 h (2 h for OSCC-3 and 4 h for HDMEC and NHOst). The reaction was stopped by adding 1% SDS and plates were read on a microplate reader at a wavelength of 440 nm. The percentage of survival was calculated by adjusting the control group (no treatment, NT) to 100%. Each test group was run in eight wells and each assay was repeated at least thrice. *, P < 0.05, significant difference as compared with the control group. C, HDMEC, OSCC-3, and NHOst cells were cultured in 6 cm dishes. After 24 h, cells were treated with As2O3 (AS, 5 μmol/L) for 1 h and then exposed to a single dose of γ-irradiation (7.5 Gy) and cultured for an additional 72 h. Upon completion of incubation, cells were harvested and the percentage of apoptotic cells was evaluated by the TUNEL assay according to the manufacturer's instructions (Sigma). *, P < 0.05, significant difference as compared with the control group.
- Figure 2.
As2O3 enhances radiation-induced inhibition of endothelial cell tube formation. The Matrigel in vitro HDMEC tube formation assay was done in eight-well chamber slides. A and B, endothelial cells cultured on top of Matrigel were treated with VEGF and different doses of As2O3 (AS). A, photomicrographs of representative assays for no-treatment (NT), VEGF, VEGF + As2O3 (AS) 2.5 μmol/L, VEGF + AS 5 μmol/L, VEGF + AS 10 μmol/L, and VEGF + AS 20 μmol/L. B, quantitative data for HDMEC tube formation from three independent experiments (points, mean; bars, SE). C, HDMECs were cultured on top of Matrigel and were treated with VEGF alone or with radiation (7.5 Gy) and/or As2O3. *, P < 0.05, significant increase as compared with the no-treatment (NT) group; **, P < 0.05, significant decrease as compared with the VEGF group.
- Figure 3.
As2O3 enhances radiation-induced inhibition of tumor cell colony formation. The tumor cell colony formation assay was done in low–melting point agarose in 24-well plates. A and B, tumor cells (OSCC-3) were treated with different concentrations of As2O3 (2.5, 5, 10, and 20 μmol/L) and cultured for 14 d. C and D, tumor cells (OSCC-3 and UM-SCC-74A) were treated with As2O3 or radiation, or a combination of both and cultured for 14 d. The number of colonies formed in each test group was counted at low-power field (×50). The percentage of colonies formed was calculated by adjusting the control group (no treatment, NT) to 100%. *, P < 0.05, significant difference as compared with the no-treatment (NT) group; **, P < 0.05, significant difference as compared with As2O3 or radiation treatment alone.
- Figure 4.
As2O3 and radiation treatment significantly inhibit tumor growth in vivo. Gelfoam carrier (3 × 3 mm2) seeded with 2 × 106 BMSCs each were implanted into the flanks of 6-week-old SCID mice (eight animals/group). After 2 wk, tumor cells (OSCC-3 or UM-SCC-74A, 1 × 106) and HDMECs (1 × 106) were mixed with 100 μL of Matrigel and these cells were implanted into the flanks of SCID mice adjacent to the bone ossicles. Tumors were allowed to grow and vascularize for 8 d and then three doses of As2O3 (2 or 5 mg/kg, days 8, 12, and 15) and three fractionated doses of ionizing radiation (7.5 Gy × 3, days 9, 13, and 16) were administered. Tumor volume measurements began on day 1 (tumor inoculation) and continued twice a week until the end of the study. A, representative photomicrograph of tumor and bone ossicle growing side by side in a SCID mouse. B, cut tumors (OSCC-3) from no-treatment (NT), As2O3 5 mg/kg (AS 5 mg/kg), radiation treatment (IR), and As2O3 5 mg/kg + radiation treatment (AS 5 mg/kg + IR) groups. Dotted circle and arrow, areas of necrosis. C and D, tumor progression curves for OSCC-3 and UM-SCC-74A tumors treated with As2O3 (AS) or radiation treatment (IR) or As2O3 + radiation treatment (AS + IR). *, P < 0.05, significant difference as compared with the no-treatment group; **, P < 0.05, significant difference as compared with As2O3 or radiation treatment alone.
- Figure 5.
As2O3 and radiation treatment significantly inhibit tumor angiogenesis and tumor metastasis. A and B, paraffin-embedded tumor sections (OSCC-3, A; and UM-SCC-74A, B) were stained for tumor blood vessels using anti-human Von Willebrand factor antibodies. Representative photomicrographs of (1) no-treatment (NT), (2) As2O3 2 mg/kg (AS 2 mg/kg), (3) As2O3 5 mg/kg (AS 5 mg/kg), (4) radiation treatment (IR), (5) As2O3 2 mg/kg + radiation treatment (AS 2 mg/kg + IR), and (6) As2O3 5 mg/kg + radiation treatment (AS 5 mg/kg + IR). Microvessel density in the tumor samples was calculated by counting six random fields (×20). C and D, five paraffin-embedded (lungs) step sections of 5 μm with 100-μm spaces were cut and stained with H&E. The number of metastasis nodules present in these sections (OSCC-3, C; and UM-SCC-74A, D) was counted under the microscope. The treatment groups are the same as those described above (A and B). *, P < 0.05, significant difference as compared with the no-treatment group; **, P < 0.05, significant difference in AS + IR groups as compared with AS or IR alone.
- Figure 6.
As2O3 treatment protects bone from radiation-induced bone loss. The bone ossicles were fixed in aqueous buffered zinc formalin and analyzed by microcomputed tomography for the bone mineral density. A, representative microcomputed tomography photomicrographs of no-treatment (NT), As2O3 2 mg/kg (AS 2 mg/kg), As2O3 5 mg/kg (AS 5 mg/kg), radiation treatment (IR), As2O3 2 mg/kg + radiation treatment (AS 2 mg/kg + IR), and As2O3 5 mg/kg + radiation treatment (AS 5 mg/kg + IR). B, bone mineral density data are expressed as mg/cc. *, P < 0.05, significant difference as compared with the no-treatment group; **, P < 0.05, significant difference in the AS + IR group as compared with IR alone.
Volume 7, Issue 7
