Figure 1.
Irradiation models of 125I seeds.
(A) In vitro model, eight 125I seeds were evenly taped around a 30-mm diameter circumference, with one 125I seed placed in the center. (B) In vivo model, a transverse CT scanning was performed on mice, and the dose distribution was calculated by TPS and the GTV (the red circle) should be kept inside the 90% isodose curve (blue one) in every plan. 8 Seeds were implanted into different position by the needle (the three yellow vertical lines) according to TPS.
Figure 2.
125I seed and X-ray irradiation inhibit CNE2 cell proliferation.
(A) Representative pictures of colony formation of cells exposed to 125I seed and X-ray at various doses for 14 days. (B) The survival fraction of colony formation assay was fitted by single-hit multitarget theory formula. (C) Cell viability of CNE2 cells treated for irradiation was examined by MTT assay. (D) Cell proliferation of CNE2 cells treated for irradiation was examined by EdU assay. Significant difference between 125I seed and X-ray groups under the same dose is indicated by *P<0.05 and **P<0.01.
Figure 3.
125I seed irradiation induces apoptosis of CNE2 cells.
Apoptosis was examined by Annexin V–PI co-staining flow cytometric analysis (A, B), caspase-3 activity assay (C) and TUNEL assay (D). Cells exposed to irradiation were harvested 24 hours after irradiation. Then, apoptosis was measured. Significant difference between 125I seed and X-ray groups under the same dose is indicated by *P<0.05 and **P<0.01.
Figure 4.
Effects of 125I seed irradiation on cells migration and invasion.
Cell suspensions were obtained 24 hours after irradiation at a total dose of 4 Gy, and then they were plated in 60-mm culture plate. (A) Wound healing assay was performed 12 hours after plating. The total distance migrated by wounded cells was expressed as percentage of initial distance. (B) The inhibition of cell invasion was measured by transwell and Boyden chamber assay. The number of cells was counted to calculate the average number of migrated cells. Data are presented as mean ± SD (n = 3). *P<0.05, **P<0.01 versus the control group.
Figure 5.
Induction of G2/M arrest and ROS generation by 125I seed irradiation.
The cells were exposed to 125I seed and X-ray irradiation at various doses. 24 hours after irradiation, the effects of 125I seed on the cell cycle distribution of CNE2 cells was examined by flow cytometric analysis (A). Quantification of the percentage of G2/M phase (B) and apoptosis reflected by Sub G1(C). (D, E) Effects of 125I seed on the expression levels of apoptosis and cell cycle arrest-associated proteins was analyzed by western blotting. (F) The level of ROS was measured by flow cytometry. Data are presented as mean ± SD (n = 3). Significant difference between 125I seed and X-ray groups under the same dose is indicated by *P<0.05 and **P<0.01.
Figure 6.
Inactivation VEGF-A/ERK signaling pathway by radioactive 125I seeds.
(A) Suppression of VEGF-A expression by 125I seed irradiation as measured by immunofluorescent assay. (B) Western blotting analysis of the expression levels of VEGF-A/ERK in cells exposed to 125I seeds. (C) The extracellular levels of VEGF-A was measured by ELISA. (D, E) VEGF-A (20 ng/ml) significantly blocks the 125I seed irradiation-induced inhibition on cell migration by recovering p-ERK protein levels.
Figure 7.
125I seed irradiation exhibits greater
in vivo anticancer activity than X-ray. (A) Representative images of tumors treated with 125I seed and X-ray irradiation. (B, C) Effects of 125I seed and X-ray irradiation on tumor and body weights. Data are presented as mean ± SD (n = 4). Significant difference between 125I seed and X-ray groups under the same dose is indicated by *P<0.05 and **P<0.01. (D, E) Expression of VEGF-A, p-ERK, Cleaved-PARP and Cdc2/cyclinB1 in xenograft tumors detected by IHC or western blotting.
Figure 8.
Proposed signal pathways of apoptosis and cell cycle arrest induced by 125I seeds.
125I seeds caused DNA damage to activate the sensory ATM/ATR kinases, finally results in cell apoptosis and G2/M arrest. At the same time, 125I seeds inhibit cells migration by inactivation VEGF-A/ERK pathway. VEGF-A which can increase p-ERK levels was inhibited by 125I seeds to regulate cellular proliferation, survival and migration.