Continuous and low-energy 125I seed irradiation changes DNA methyltransferases expression patterns and inhibits pancreatic cancer tumor growth

Abstract

Background: Iodine 125 (125I) seed irradiation is an effective treatment for unresectable pancreatic cancers. However, the radiobiological mechanisms underlying brachytherapy remain unclear. Therefore, we investigated the influence of continuous and low-energy 125I irradiation on apoptosis, expression of DNA methyltransferases (DNMTs) and cell growth in pancreatic cancers.

Materials and methods: For in vitro 125I seed irradiation, SW-1990 cells were divided into three groups: control (0 Gy), 2 Gy, and 4 Gy. To create an animal model of pancreatic cancer, the SW 1990 cells were surgically implanted into the mouse pancreas. At 10 d post-implantation, the 30 mice with pancreatic cancer underwent 125I seed implantation and were separated into three groups: 0 Gy, 2 Gy, and 4 Gy group. At 48 or 72 h after irradiation, apoptosis was detected by flow cytometry; changes in DNMTs mRNA and protein expression were assessed by real-time PCR and western blotting analysis, respectively. At 28 d after 125I seed implantation, in vivo apoptosis was evaluated with TUNEL staining, while DNMTs protein expression was detected with immunohistochemical staining. The tumor volume was measured 0 and 28 d after 125I seed implantation.

Results: 125I seed irradiation induced significant apoptosis, especially at 4 Gy. DNMT1 and DNMT3b mRNA and protein expression were substantially higher in the 2 Gy group than in the control group. Conversely, the 4 Gy cell group exhibited significantly decreased DNMT3b mRNA and protein expression relative to the control group. There were substantially more TUNEL positive in the 125I seed implantation treatment group than in the control group, especially at 4 Gy. The 4 Gy seed implantation group showed weaker staining for DNMT1 and DNMT3b protein relative to the control group. Consequently, 125I seed implantation inhibited cancer growth and reduced cancer volume.

Conclusion: 125I seed implantation kills pancreatic cancer cells, especially at 4 Gy. 125I-induced apoptosis and changes in DNMT1 and DNMT3b expression suggest potential mechanisms underlying effective brachytherapy.

Figure 1

¹²⁵I seed irradiation model developed in-house. In a 60-mm cell culture dish, eight ¹²⁵I seeds were embedded in the solidified paraffin evenly around the circumference of a 35-mm diameter, and one ¹²⁵I seed was placed at the center of dish. This arrangement produced a homogeneous dose distribution at the top of the cell culture dish, so that a 35-mm cell culture dish containing SW-1990 cells could be placed on it during the experiment.

Figure 2

Apoptosis of ¹²⁵I irradiated SW-1990 cells. The red region in the lower left quadrant represents apoptosis detected by flow cytometry in the 0 Gy (A), 2 Gy (B), and 4 Gy (C) groups. The quantitation is shown in D. *P < 0.05 compared with the 0 Gy (Control) group. ^#P < 0.05 compared with the 2 Gy group.

Figure 3

¹²⁵I irradiation induced expression changes of DNA methyltransferases mRNA in SW-1990 cells. DNMT1 (A), DNMT3a (B), and DNMT3b (C) mRNA expression in ¹²⁵I irradiated SW-1990 cells was detected as described in the Materials and Methods section. *P < 0.05 compared with the 0 Gy (Control) group. ^#P < 0.05 compared with the 2 Gy group. ^ΔP > 0.05 compared with the 0 Gy group.

Figure 4

¹²⁵I irradiation altered DNMTs protein expression in SW-1990 cells. Representative western blots of DNMT proteins are showed in the upper panel. DNMT1 (A), DNMT3a (B), and DNMT3b (C) protein expression in ¹²⁵I irradiated SW-1990 cells was detected as described in the Materials and Methods section. *P < 0.05 compared with the 0 Gy (Control) group. ^#P < 0.05 compared with the 2 Gy group. ^ΔP > 0.05 compared with the 0 Gy group.

Figure 5

¹²⁵I irradiation induced apoptosis in pancreatic cancer. The dark brown or brownish yellow spots represented the apoptotic cells detected by TUNEL staining in the 0 Gy (A), 2 Gy (B), and 4 Gy (C) groups. The average number of apoptotic cells per 200 objective fields were plotted (D). *P < 0.05 compared with the 0 Gy (Control) group. ^#P < 0.05 compared with the 2 Gy group.

Figure 6

Immunohistochemical staining for DNMTs in ¹²⁵I seed implanted pancreatic cancer. Representative staining sections for DNMT1 (upper), DNMT3b (middle) and DNMT3a (lower) were prepared as described in the Materials and Methods section. The brownish yellow spots represent positive staining. Scale bars represent 500 μm.

Figure 7

Pathology of ¹²⁵I implanted pancreatic cancer. Representative HE stained sections from the 0 Gy (A), 2 Gy (B), and 4 Gy (C) groups 28 d after ¹²⁵I seed implantation were prepared as described in the Materials and Methods section.

Figure 8

Tumor volume 0 and 28 d after ¹²⁵I seed implantation. The upper, middle, and lower panels show representative ultrasound images from 0 Gy (upper), 2 Gy (middle), and 4 Gy (lower) groups 0 and 28 d post ¹²⁵I seed implantation. *P < 0.05 compared with 0 d post-implantation; ^ΔP > 0.05 compared with 0 d post-implantation.