Continuous low-dose irradiation by I-125 seeds induces apoptosis of gastric cancer cells regardless of histological origin

Figures & data

Figure 1. Cell viability of the three gastric cancer cell lines (MKN74, MKN45, and NUGC4) was assessed following incubation for 96 h with or without irradiation to maintain constant cell culture conditions. The viability of each of the 3 cell lines was significantly lower than that of the control group (*P < 0.05) (A). Apoptosis was determined by flow cytometry and the total apoptosis rate was calculated. The total apoptosis rate induced by irradiation was significantly increased in each of the three gastric cancer cell lines as compared with the control (*P < 0.05) (representative data was shown in [B]). Caspase-3 assay was performed and the activity of caspase-3 was increased significantly in all 3 irradiated gastric cancer cell lines (P < 0.05) (C). Experiments were performed at least 3 times.

Figure 2. Cell-cycle assay was performed on each of the three gastric cancer cell lines (MKN74, MKN45, and NUGC4) following incubation for 96 h with or without irradiation to maintain constant cell culture conditions (representative data was shown in [A]). Three cell-cycle segments are shown in order from left to right: G₁/G₀ phase, S phase, G₂/M phase, and the percentage of cells in each phase is shown in (B). Continuous low-dose-rate irradiation by I-125 seeds induced a lower percentage of G₀/G₁, and higher percentage of G₂/M phase cell-cycle arrest compared with the control for all 3 gastric cancer cell lines (P < 0.05). Experiments were performed at least 3 times.

Figure 3. Protocol for animal experiments using the MKN45 cell line (A). 2.5 × 10⁶ cells in 0.2 ml PBS were injected subcutaneously into the dorsa of each mouse. When tumors reached around 400 mm³ at about 3–4 weeks, I-125 seeds or cold seeds were implanted into each 5 mice per group via a needle. The untreated mice served as the no seed control group. The tumor’s macroscopic appearance was imaged on the day of implantation before the mouse was sacrificed (representative data was shown in [B]). Tumor size was measured once every 4 d. There were no significant changes in tumor volume during the first 2 weeks after seed implantation, but after that, I-125-irradiated tumors were much smaller than the others, and significant differences in tumor volumes were observed between the I-125 seed implanted group and the other 2 groups (P < 0.05) (C). The body weight of the animals was also measured every 4 d and mortality was monitored daily, but there were no significant differences between the 3 groups (D).

Figure 4. Histological experiments with MKN45 xenografts. Tumor sections were immunostained for Ki67 and a TUNEL assay was performed. Ki67 and TUNEL immunostaining are shown at 100× and 200× magnification (representative data was shown in [A]). Ki67 and TUNEL-positive cells were quantified in 20 randomly selected, high-power fields in each tissue section. The average number of Ki67-positive cells in the I-125 implanted group was clearly less than those in the control and cold seed implanted groups. In contrast to the proliferation rate, the average number of TUNEL-positive apoptotic cells in the I-125 implanted group was significantly increased over those in the control and cold seed implanted groups (*P < 0.05) (B). Tumor tissue samples were isolated and stained with annexin V-FITC and PI, then analyzed using a flow cytometer to clarify the induction of apoptosis. The apoptosis rate in the I-125 implanted group was significantly increased compared with those of the control and cold seed implanted groups (P < 0.05) (representative data was shown in [C]).