Iodine-125 seed inhibits proliferation and promotes apoptosis of cholangiocarcinoma cells by inducing the ROS/p53 axis

Abstract

With advances in radioactive particle implantation in clinical practice, Iodine-125 (¹²⁵I) seed brachytherapy has emerged as a promising treatment for cholangiocarcinoma (CCA), showing good prognosis; however, the underlying molecular mechanism of the therapeutic effect of ¹²⁵I seed is unclear. To study the effects of ¹²⁵I seed on the proliferation and apoptosis of CCA cells. CCA cell lines, RBE and HCCC-9810, were treated with reactive oxygen species (ROS) scavenger acetylcysteine (NAC) or the p53 functional inhibitor, pifithrin-α hydrobromide (PFTα). Cell counting kit-8 (CCK-8) assay, 5-bromo-2-deoxy-uridine (BrdU) staining, and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry assay were performed to test the radiation-sensitivity of ¹²⁵I seed toward CCA cells at different radiation doses (0.4 mCi and 0.8 mCi). 2,7-dichlorofluorescein diacetate (DCF-DA) assay, real-time quantitative polymerase chain reaction (RT-qPCR), and western blot analysis were performed to assess the effect of ¹²⁵I seed on the ROS/p53 axis. A dose-dependent inhibitory effect of ¹²⁵I seeds on the proliferation of CCA cells was observed. The ¹²⁵I seed promoted apoptosis of CCA cells and induced the activation of the ROS/p53 pathway in a dose-dependent manner. NAC or PFTα treatment effectively reversed the stimulatory effect of ¹²⁵I seed on the proliferation of CCA cells. NAC or PFTα suppressed apoptosis and p53 protein expression induced by the ¹²⁵I seed. ¹²⁵I seed can inhibit cell growth mainly through the apoptotic pathway. The mechanism may involve the activation of p53 and its downstream apoptotic pathway by up-regulating the level of ROS in cells.

Keywords: Apoptosis; Cholangiocarcinoma; Iodine-125 seed; Proliferation; ROS/p53 pathway.

Fig. 1

Irradiation with the¹²⁵I seed inhibits the proliferation of CCA cells. CCK-8 assay indicated that ¹²⁵I seed treatment gradually suppressed the viability of RBE (A) and HCCC-9810 cells (B). The proliferation of RBE (C) and HCCC-9810 cells (D) was gradually suppressed by ¹²⁵I seed treatment, as indicated by the BrdU assay. ***P < 0.001 vs. 0 mCi group; ###P < 0.001 vs. 0.4 mCi group

Fig. 2

The¹²⁵I seed induces apoptosis of CCA cells. TUNEL staining showed that ¹²⁵I seed promoted apoptosis of RBE (A) and HCCC-9810 cells. Flow cytometry analysis indicated that the treatment of ¹²⁵I seed for different doses induced apoptosis in RBE cells (C) and HCCC-9810 cells (D). RT-qPCR (E, G) and western blot analysis (F, H) showed that ¹²⁵I seed promoted Bax expression and inhibited Bcl-2 expression in a dose-dependent manner in RBE and HCC-9810 cells. **P < 0.01, ***P < 0.001 vs. 0 mCi group; ###P < 0.001 vs. 0.4 mCi group

Fig. 3

¹²⁵I seed activates ROS/p53 pathway in CCA cells. The intracellular ROS levels of RBE cells (A) and HCCC-9810 cells (B) were increased after ¹²⁵I seed treatment by flow cytometry. RT-qPCR (C, E) and western blot analysis (D, F) showed that ¹²⁵I seed promoted p53 expression in RBE and HCC-9810 cells in a dose-dependent manner. ***P < 0.001 vs. 0 mCi group; ###P < 0.001 vs. 0.4 mCi group

Fig. 4

Inhibition of ROS generation blocks the¹²⁵I seed-induced accumulation of ROS and upregulation of p53 in CCA cells. RBE cells were treated with 0.8 mCi of ¹²⁵I seed and ROS scavenger NAC. (A) Flow cytometry assay indicated that NAC reduced intracellular ROS levels in RBE cells compared with ¹²⁵I group. RT-qPCR (B) and western blot (C) analysis showed that NAC inhibited the mRNA and protein levels of p53 in RBE cells compared with ¹²⁵I group. ***P < 0.001 vs. Control group; ##P < 0.01, ###P < 0.001 vs. ¹²⁵I group

Fig. 5

Inhibition of ROS production blocks the inhibitory effect of the¹²⁵ I seed on the proliferation of CCA cells. RBE cells were treated with 0.8 mCi of ¹²⁵I seed and ROS scavenger NAC. (A) CCK-8 assay showed that NAC promoted the viability of RBE cells compared with ¹²⁵I group. (B) Flow cytometry showed that NAC blocked the inhibition of ¹²⁵I seed on CCA cell cycle. (C) BrdU assay showed that the proliferation of RBE cells was increased by NAC compared with ¹²⁵I group. RT-qPCR (D) and western blot analysis (E) showed that the mRNA and protein levels of p21 were significantly reduced in RBE cells after NAC treatment compared to the ¹²⁵I group. *P < 0.05, **P < 0.01,***P < 0.001 vs. Control group; #P < 0.05, ##P < 0.01 vs. ¹²⁵I group

Fig. 6

Inhibition of ROS production blocks the promoting effect of¹²⁵I seed on the apoptosis of CCA cells. RBE cells were treated with 0.8 mCi of ¹²⁵I seed and ROS scavenger NAC. (A) TUNEL staining showed that NAC inhibited RBE cell apoptosis compared with that in the ¹²⁵I group. (B) Flow cytometry showed that NAC inhibited RBE cell apoptosis induced by ¹²⁵I seed. RT-qPCR (C) and western blot analysis (D) showed that the mRNA and protein levels of Bcl-2 in RBE cells were promoted by NAC compared with ¹²⁵I seed group. RT-qPCR (E) and western blot analysis (F) showed that the mRNA and protein levels of Bax in RBE cells were inhibited by NAC compared with ¹²⁵I seed group. ***P < 0.001 vs. Control group; ##P < 0.01 vs. ¹²⁵I group

Fig. 7

Blockade of p53 attenuates the effect of the¹²⁵I seed on the proliferation and apoptosis of CCA cells. RBE cells were treated with 0.8 mCi of ¹²⁵I seed and PFTα, a functional inhibitor of p53. (A) CCK-8 assay indicated that PFTα could rescue the inhibitory effect of ¹²⁵I seeds on RBE cell proliferation. (B) Flow cytometry showed that PFTα blocked the inhibition of ¹²⁵I seed on CCA cell cycle. (C) BrdU assay showed that the proliferation of RBE cells was increased by PFTα compared with ¹²⁵I group. (D) TUNEL staining and Flow cytometry analysis (E) showed that the apoptosis of RBE cells was inhibited by PFTα compared to ¹²⁵I group. *P < 0.05, **P < 0.01,***P < 0.001 vs. Control group; #P < 0.05, ##P < 0.01 vs. ¹²⁵I group