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

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. Supplementary Information The online version contains supplementary material available at 10.1007/s10142-024-01392-1.


Introduction
Cholangiocarcinoma (CCA) develops from the epithelial cells of the bile duct and is classified based on its anatomical location into intrahepatic cholangiocarcinoma (iCCA), periportal cholangiocarcinoma (pCCA), and distal cholangiocarcinoma (dCCA).Recent studies have reported an increasing incidence of and mortality rate due to CCA (Cho et al. 2022).Given the aggressive nature of CCA and its tendency to be first diagnosed in advanced stages, treatment options are often limited.Radical resection, in particular, is often constrained in these cases (Laschos et al. 2020), and may also lead to serious postoperative complications, including hepatorenal syndrome and progressive liver failure (Zhu and Wei 2020).Therefore, it is particularly important to develop effective methods to inhibit the proliferation and invasion of CCA cells.
Interstitial radiotherapy, a form of radiotherapy, is less active than conventional radiotherapy in terms of radiation Fuping Kang and Jing Wu contributed equally to this work.source activity, shorter treatment distance, and easier protection (Huang and O'Sullivan 2013).The radiation source can be directly implanted into tumor tissues, thus exerting a strong killing effect on tumor cells in the treatment target area and causing little damage to surrounding normal tissues (Zakeri et al. 2019).Iodine-125 seed ( 125 I seed), as the main radioactive particles used in clinical settings in China, are small in size and low in energy (Wu et al. 2018) and can release large doses of γ-rays into tumor tissues at close ranges, which can inhibit the mitosis of tumor cells and significantly reduce the re-proliferation of tumor tissues, causing no damage to normal tissues with fewer toxic side effects (Zhang et al. 2020).In the 1970s, 125 I seed brachytherapy was initially employed in prostate cancer (Zaorsky et al. 2017).At present, 125 I seed implantation is extensively utilized in the clinical treatment of malignant tumors in the chest region (Jiang et al. 2021), head and neck (Zhang et al. 2022), abdomen (Sugawara et al. 2011), and soft tissues (Chen et al. 2022a, b).After a long-term follow-up observation of patients receiving 125 I seed implantation treatment, the functions of the urinary system, reproductive system, and rectal system of patients were found to be well protected and maintained in a previous study (Buckstein et al. 2013).The 125 I seed can enhance the prognosis of patients who have experienced recurrence or failure of radiation therapy.Two recent clinical studies have demonstrated the therapeutic value of the 125 I seed in CCA (Luo et al. 2022;Chen et al. 2022a, b).However, unlike the widespread clinical development, research on the molecular mechanism underlying the anti-tumor effect of 125 I seed implantation is relatively lacking.Previous studies have identified inhibition of proliferation and apoptosis induction as potential antitumor mechanisms underlying 125 I seed brachytherapy (Zhang et al. 2016;Zhuang et al. 2009).However, comprehensive evidence on this topic, especially at the molecular level, is lacking.
Reactive oxygen species (ROS), including superoxide anions, hydrogen peroxide, and hydroxyl radicals, are byproducts of normal cellular metabolism (Cui et al. 2022), .Moderate ROS production is involved in regulating signal transduction and gene expression in normal cells, thus maintaining the cellular physiological balance.However, high ROS level can directly or indirectly cause apoptosis of cells.Directly, ROS can damage cellular nucleic acids, proteins, and lipids, eventually disrupting the biological molecular structure and function of cells.Indirectly, ROS can mediate multiple key apoptosis signaling pathways, such as regulating the release of cytochrome c, activating the cysteine protease family, and activating caspase, thereby accelerating the apoptosis of cells (Barbieri and Sestili 2012).Furthermore, compelling evidence suggests that the 125 I seed significantly elevates ROS level in tumor cells, in turn inducing apoptosis (Liu et al. 2018) but whether 125 I seed in CCA regulates proliferation and apoptosis through ROS signaling remains unclear.
p53 is induced by ROS and regulates the proliferation and apoptosis of tumor cells (Cao et al. 2020;Xing et al. 2022;Shi et al. 2020), and both p53 and ROS together inhibit the progression of CCA (Wandee et al. 2021;Ren et al. 2021).More importantly, p53 is induced by 125 I seed in non-small lung cancer and colorectal cancer (Ma et al. 2014;Zhang et al. 2021).However, whether the 125 I seed in CCA can promote p53 expression to play an inhibitory role in cancer through up-regulation of ROS production is unknown.
This study focused on the effects of 125 I seed treatment of different doses on CCA cells, and explored its possible regulatory mechanism, to provide experimental support for the development of new treatment options for CCA.

Cell lines and culture
The CCA cell lines, RBE and HCCC-9810, were acquired from the National Biomedical Experimental Cell Resource Bank of China located in Beijing.RBE and HCCC-9810 cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS).The cells were incubated at 37 ℃ in an incubator with 5% CO 2 .Specifically, 0.25% trypsin was used for passaging, and cells in the logarithmic growth phase were selected (after 48 h of culture) for experiments.In some experiments, cells were treated with 5 mmol/L NAC (Sigma) (Fan et al. 2020) or 100 nmol/L PFTα (Zhou et al. 2016).

Irradiation of CCA cells with the 125 I seed
125 I seed for medical use was obtained from HTA Co., Ltd., Beijing, China.Experiments were conducted using the 125 I seed at two different doses of 0.4 mCi and 0.8 mCi.RBE and HCCC-9810 cells were treated with various doses of the 125 I seed for 72 h.Control cell lines were not irradiated with the 125 I seed.We established an in vitro model of irradiation as follows: eight 125 I seeds were evenly buried in a 30 mm diameter circular groove, and one was positioned at the center.In the experiment, a 6 cm culture dish was positioned in the 125 I seed irradiation model within the chamber.RBE and HCCC-9810 cells (1 × 10 5 cells per well) were inoculated in dishes with 6 cm diameter.To ensure equal irradiation, all dishes were periodically rotated clockwise at specific time intervals, as described previously (Zhang et al. 2021;Ren et al. 2022).

Cell proliferation assay
Each group of cells was seeded into separate 96-well plates and incubated for different time intervals, i.e., 24, 48, and 72 h.Subsequently, each well of the 96-well plates was treated with 10 µL of the CCK-8 reagent (Dojindo Laboratories, Japan) and incubated at 37 °C for 2-3 h.The optical density values at 460 nm were measured using a Microplate Reader (Bio-Rad, USA) (Tian et al. 2015).

Terminal deoxynucleotidyl transferase (TdT)mediated dUTP nick end labeling (TUNEL) staining
Cells from each group were collected and adjusted to a final concentration of 1 × 10 5 cells/ml.Promega's Dead-End™ Colorimetric TUNEL System was used to perform TUNEL staining as described previously (Hiraoka et al. 2013).Apoptotic cells were assessed by optical microscopy (Olympus, C5060-ADU, Tokyo, Japan), and the proportion of TUNEL-stained cells was calculated by randomly counting the cells in three separate fields of view.

Flow Cytometry: apoptosis assay and cell cycle assay
In the cell apoptosis experiment, cells washed with PBS were collected and mixed with Annexin V and PI (5 mg/L) of 5 µL respectively.After incubation at 25 ℃ and dark for 15 min, 500 µL binding buffer was added and stood at 4 ℃ for 30 min.The apoptosis of cells was detected by flow cytometry (BD, Franklin Lakes, NJ, USA).
In a flow cytometry assay of the cell cycle, 1 × 10 6 cells were initially collected and then re-suspended in pre-cooled 70% ethanol, where they were stored overnight at 4 °C.The following day, the cells were centrifuged, stained with PI after re-suspension, and incubated at 37 °C for 30 min.Subsequently, the cell cycle distribution was analyzed using flow cytometry.

Reverse transcription-quantitative polymerase chain reaction (RTqPCR)
The TRIzol reagent (Invitrogen, Carlsbad, CA, United States) was used to extract total RNA from cultured cells.Reverse transcription was performed using a kit and realtime quantitative PCR analysis was conducted using the qPCR SYBR Green master mix dye method.The RNA was transcribed into cDNA before analysis.β-actin was the internal reference gene and was used for normalization.The relative expression level of the target gene was calculated using the 2 −△△Ct method (Pfaffl 2001).The primers used in the experiment are listed in Table 1.

Measurement of intracellular ROS level
To measure intracellular ROS level, flow cytometry with the DCFH-DA probe was employed as described in a previous study (Tian et al. 2015).Cells were treated with 15 µmol/L PEITC for 6, 9, and 12 h, followed by incubation with 10 µmol/L of DCFH-DA probe, away from light for 15 min.After incubation, digested cells were resuspended in PBS and the fluorescence intensity of DCF was measured by flow cytometry (BD, Franklin Lakes, NJ, USA).

5-bromo-2-deoxyuridine (BrdU) assay
Following the instructions specified in the BrdU labeling and detection kit (Roche, Mannheim, Germany) (Lee et al. 2016), when the cells grew to a confluence of approximately 50%, the culture was supplemented with BrdU-labeled media and incubated for 15-60 min.The culture medium was discarded and cells were fixed overnight with 70% ethanol.The cells were incubated with the mouse anti-BrdU analysis showed that 0.4 and 0.8 mCi 125 I seed treatment promoted the protein expression of Bax while inhibiting that of Bcl-2.The effect of 125 I seed was more obvious in the 0.8 mCi group (p < 0.01, Fig. 2F).Simultaneously, 125 I seed at both 0.4 and 0.8mCi doses promoted the mRNA expression of Bax, evidenced by RT-qPCR analysis, while inhibiting that of Bcl-2 in HCC-9810 cells (p < 0.001, Fig. 2G).Western blot showed in HCCC-9810 cells that with an increase in the 125 I seed dose, the enhancing effect of the 125 I seed on Bax and the inhibiting effect of Bcl-2 protein levels were more obvious (p < 0.001, Fig. 2H).Therefore, the 125 I seed induced apoptosis in CCA cells, and the higher the 125 I dose, the stronger the ability to induce apoptosis.

I seed activates ROS/p53 pathway in CCA cells
The 125 I seed induces increased ROS production (Wang et al. 2020) and p53 level (Xu et al. 2022).To further investigate whether the 125 I seed in CCA plays a role in cancer suppression by upregulating ROS generation and promoting p53 expression, we first treated RBE and HCCC-9810 cells with 0.4 and 0.8 mCi doses of 125 I seed for 72 h, respectively.Cells were subjected to DCFH-DA staining to detect ROS production.ROS level in RBE and HCCC-9810 cells of the 0 mCi group were low and increased significantly after treatment with the 125 I seed (0.4 and 0.8 mCi).ROS level tended to increase with higher doses of the 125 I seed (p < 0.001, Fig. 3A B).To determine whether 125 I seed induced p53 expression in CCA cells, we detected p53 level in RBE cells by RT-qPCR.p53 level increased remarkably after treatment with 0.4 and 0.8 mCi of the 125 I seed (p < 0.001, Fig. 3C).Western blot analysis showed that treatment with 0.4 and 0.8 mCi 125 I seed could promote the increase in p53 protein expression in RBE cells, and compared with the 0.4 mCi group, the induction of p53 expression was greater in the 0.8 mCi group (p < 0.001, Fig. 3D).Similar results were observed in HCCC-9810 cells (Fig. 3E F).Both mRNA and protein levels of p53 increased in a dose-dependent manner following treatment with 0.4 and 0.8 mCi 125 I seed in HCCC-9810 cells (p < 0.001).The above findings indicate that the ROS/p53 pathway regulates the proliferation and apoptosis of CCA cells following treatment with 125 I seed.

Inhibition of ROS generation blocks the 125 I seedinduced accumulation of ROS and upregulation of p53 in CCA cells
RBE cells were treated with 0.8 mCi of 125 I seed and NAC, a scavenger of ROS, to study the effect of ROS on 125 I seedinduced p53 upregulation.The DCFH-DA probe assay confirmed that the addition of NAC significantly reduced the increase in ROS level following treatment with 0.8 mCi primary antibody (BD Biosciences, San Jose, CA, USA).
Anti-mouse fluorescent antibody conjugated with FITC was the corresponding secondary antibody (Agilent Technologies, Santa Clara, California, USA).

Statistical analysis
All experiments were performed in a triplicate.Data were analyzed using IBM's SPSS 20.0 software.Measurement data are presented as mean ± standard deviation.One-way analysis of variance (ANOVA) was conducted to compare multiple groups.P < 0.05 indicated a statistically significant difference.

Irradiation with the 125 I seed inhibits the proliferation of CCA cells
We verified the effect of the 125 I seed on the proliferation of CCA cell lines (RBE and HCCC-9810 cells) by the CCK-8 assay after continuous irradiation with 0, 0.4, and 0.8 mCi for 24 h, 48 h, and 72 h.The 125 I seed significantly inhibited the viability of RBE and HCCC-9810 cells in a dosedependent manner (p < 0.001, Fig. 1A B).To determine further whether the 125 I seed inhibited the proliferation of CCA cells, BrdU assay was conducted and the results demonstrated that compared with the 0 mCi group, both 0.4 and 0.8 mCi 125 I seed dosages inhibited the proliferation of RBE and HCCC-9810 cells, with the effect being prominent in the 0.8 mCi-treatment group (p < 0.001, Fig. 1C D).Taken together, the 125 I seed negatively regulated the proliferation of CCA cells in a dose-dependent manner.
The 125 I seed induces apoptosis of CCA cells TUNEL staining after 72 h of 125 I seed irradiation at doses of 0, 0.4, and 0.8 mCi showed that the 125 I seed at doses of 0.4 and 0.8 mCi induced increased apoptosis in both RBE and HCCC-9810 cells, with the effect being more prominent in the 0.8 mCi group compared to 0.4 mCi group (p < 0.001, Fig. 2A B).The results of apoptosis measured by flow cytometry after 72 h of 125 I seed irradiation at doses of 0,0.4 and 0.8 mCi showed that 125 I seed at doses of 0.4 and 0.8 mCi induced increased apoptosis in both RBE and HCCC-9810 cells (p < 0.001, Fig. 2C D).Further, RT-qPCR analysis suggested that different doses of the 125 I seed could promote Bax expression and simultaneously inhibit Bcl-2 expression in RBE cells.The effect on the 0.8 mCi group was more significant compared to the 0.4 mCi group (p < 0.05, p < 0.001, Fig. 2E).In RBE cells, western blot

Blockade of p53 attenuates the effect of the 125 I seed on the proliferation and apoptosis of CCA cells
Finally, 125 I seed-induced RBE cells were treated with PFTα, a functional inhibitor of p53.Both CCK-8 and BrdU assays demonstrated that PFTα significantly rescued the inhibitory effect of 125 I seed on the viability and proliferation ability of RBE cells (p < 0.01, Fig. 7A C).To further elucidate the regulatory role of p53 in cell proliferation, we analyzed the cell cycle distribution of RBE cells using PI staining combined with flow cytometry.Flow cytometry showed that PFTα partially reversed 125 I-induced increase in the number of G0/G1 phase cells and decrease in the number of S phase cells compared with 125I group, but there was no significant change in G2/M phase cells in each group (p < 0.05, Fig. 7B).And results of the TUNEL staining and flow cytometry assay revealed that 125 I seed-induced apoptosis in RBE cells was reduced following treatment with PFTα (p < 0.01, Fig. 7D E).Thus, PFTα could significantly affect the regulatory effect of 125 I seed on the proliferation of CCA cells.

Discussion
Interventional chemoembolization or chemoinfusion were the main treatment options for inoperable progressive CCA (Bramlet et al. 1983), however, CCA is a tumor primarily without blood supply.Interventional chemoembolization or chemoinfusion thus fail to significantly improve the survival of these patients (Sahara et al. 2012). 125I seed implantation brachytherapy has been proven to be effective in various 125 I seed (p < 0.01, Fig. 4A).The mRNA level of p53, as detected by RT-qPCR, showed that NAC treatment could significantly inhibit the increase in p53 expression induced by the 125 I seed (p < 0.01, Fig. 4B).Western blot analysis confirmed that the addition of NAC significantly inhibited 125 I-induced increase in the protein level of p53 (p < 0.01, Fig. 4C).Collectively, inhibition of ROS generation effectively inhibited the accumulation of ROS and upregulation of p53 in RBE cells following 125 I seed treatment.

Inhibition of ROS production blocks the inhibitory effect of the 125 I seed on the proliferation of CCA cells
Results of the CCK-8 assay demonstrated that cell viability decreased significantly after 125 I seed treatment compared with the control, while NAC could rescue the inhibitory effect of 125 I seed on the proliferation of RBE cells to some extent compared with the 125 I group (p < 0.01, Fig. 5A).
Next, flow cytometry analysis further demonstrated that 125 I seed inhibition of cell cycle progression was the reason for its anti-cell proliferation effect, and NAC could reduce this effect of 125 I seed.As shown in Fig. 5B, RBE cells showed different cell cycle distribution patterns under different treatments.Compared with the control group, 125 I seed induced G0/G1 and G2/M cycle arrest and a decrease in the number of S-phase cells, while, as expected, adding NAC to 125 I seed pretreated RBE cells resulted in a decrease in the number of G0/G1 phase cells and an increase in the number of S-phase cells, but no significant differences were seen in G2/M phase cells.And the results of the BrdU assay showed that NAC rescued the inhibitory effect of the 125 I seed on the proliferation of RBE cells to some extent (p < 0.01, Fig. 5C).p21, a key player in p53-mediated growth inhibition, is a tumor suppressor (Maheshwari et al. 2022).We next confirmed through RT-qPCR that the increase in p21 level induced by 125 I seed was reduced to a certain extent following the inhibition of ROS production (p < 0.01, Fig. 5D).Western blot analysis showed that the 125 I seed could significantly induce the expression of p21 protein, while NAC could partially rescue this effect (p < 0.01, Fig. 5E).Thus, our findings support that the inhibition of ROS rescues the inhibitory effect of 125 I seed on cell proliferation.
Fig. 2 The 125 I seed induces apoptosis of CCA cells.TUNEL staining showed that 125 I seed promoted apoptosis of RBE (A) and HCCC-9810 cells.Flow cytometry analysis indicated that the treatment of 125 I 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 125 I 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 that the effective inhibition dosage of 125 I seed ranged from 0.4 to 0.8 mCi (Ma et al. 2014).The 0.4 and 0.8 mCi doses of 125 I seed effectively inhibited the proliferation of CCA cells (Fig. 1), consistent with previously reported findings (Ma et al. 2014).However, Moon et al. concluded that CCA cells were radioresistant (Moon et al. 1997).The reason for cancers (Kobayashi et al. 2013;Moon et al. 2012;Reddi et al. 2012).125 I seed implantation is a low-dose, brachytherapy treatment.Radiotherapy, especially implanted particles in CCA cells, could partially improve prognosis and provide effective relief for tumor pain (Kuhn et al. 2002).Ma et al. verified  and western blot analysis (D, F) showed that 125 I 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 whereas tumor cells, by upregulating ROS level, cause organelle damage, ultimately inducing cell death (Lu et al. 2019).Accumulating evidence has shown that radiation therapy can elicit cancer cells to produce excess ROS (Tian et al. 2015;Hein et al. 2014;Leach et al. 2001).Hu et al. showed that 125 I seed irradiation caused mitochondrial damage, leading to a marked increase in intracellular ROS level in HCT116 cells (Hu et al. 2016).Therefore, in this study, the role of ROS in 125 I seed-induced apoptosis of CCA cells was determined.ROS level increased in a dose-dependent manner after 125 I seed treatment (Fig. 3A B).Further experiments on the effects of ROS showed that pre-treatment with a ROS scavenger (NAC) blocked the inhibition by 125 I seeds on the proliferation and the induction of apoptosis of CCA cells (Figs. 5 and 6), indicating that ROS is involved in the inhibition of growth and early apoptosis of CCA cells.
p53 is an important downstream regulator of ROSinduced apoptosis (Arnandis et al. 2018).Many studies have shown that ROS production can lead to DNA damage, in turn activating the ATM/ATR-p53 signaling pathway (Park et al. 2021).To confirm whether the 125 I seed treatment was dependent on the ROS-p53 pathway for induction of apoptosis, RT-qPCR and western blot showed that 125 I seed treatment could significantly induce protein expression of p53 compared to the control (Fig. 3C, D, E, F).Intracellular p53 protein level decreased significantly after pretreatment with NAC (Fig. 4), suggesting that ROS played a crucial role in inducing the upregulation of p53 expression.As an this difference may be different doses of radiation therapy used across studies.Accumulating evidence has shown that a mechanism by which 125 I seeds treat tumors is through releasing γ-rays, which damage the DNA double strands in tumor cells and induce apoptosis (Xie et al. 2015). 125I seed treatment can modulate the biological function of CCA cells, including apoptosis induction and cell cycle arrest (Zhou et al. 2022).In this study, cells were grown for 72 h post-125 I seed treatment to facilitate a meaningful comparison with previous findings (Ren et al. 2022).The 125 I seed exerted a dose-dependent effect on inhibiting proliferation and promoting apoptosis in RBE and HCCC-9810 cells (Figs. 1 and  2).However, we must acknowledge certain adverse effects of 125 I seed implantation brachytherapy exist (Almeida et al. 2011;Grewal et al. 2009).While the reported risk of persistent side effects due to 125 I seed therapy is relatively small, data suggest that extreme caution needs to be exercised when selecting patients to administer 125 I seed therapy (Ma et al. 2014).For patients with CCA with a moderate-to-high risk of recurrence, the smallest effective dose of 125 I seed should be administered to maximize potential benefits and minimize the risk of adverse events.Herein, both 0.4 and 0.8 mCi 125 I seed dosages inhibited the proliferation of CCA but given the side effects of the 125 I seed, 0.4 mCi dose may be preferred for subsequent in vivo experiments and clinical trials.
Mitochondria are key determinants of cell fate.Normal cells generally maintain low-to-moderate level of ROS, of Bax and Bcl-2.Therefore, we speculated that the proapoptotic mechanism of 125 I seed may be realized by upregulating intracellular ROS production, promoting p53 and Bax expression, and inhibiting Bcl-2 level.In addition, we also found that 125 I seed promoted the expression of p21 and induced RBE cells to increase in G0/G1 cells while decreasing in S phase.Therefore, we speculated that 125 I seed may important intracellular transcription factor, p53 can directly regulate the expression of its downstream apoptosis-related gene, Bax (Li et al. 2022).Bax level were significantly downregulated after NAC treatment, while those of Bcl-2 were significantly upregulated (Fig. 6C, D, E, F).The 125 I seed can induce apoptosis in CCA cells by antagonizing its anti-apoptotic effect through the formation of heterodimers also promote p21 through P53, thereby keeping cells in G1 phase and shortening S phase, thus inhibiting cell proliferation.However, it should be noted that normally, G1 phase could repair damaged DNA, but this study could not rely on blocking G1 phase to repair damaged DNA, because 125 I seed can also cause oxidative stress and other damage, thus inhibiting cell proliferation and inducing apoptosis (Fig. 5).The role of p53 expressional upregulation in mediating the effect of 125 I seed treatment on the biological functions of CCA cells is unclear.Therefore, PFTα, a functional inhibitor of p53, was used to suppress p53 level induced by 125 I seed treatment.We analyzed the proliferation and apoptosis of CCA cells in the PFTα pre-treatment group, which increased significantly after 125 I seed treatment, while apoptosis was significantly decreased (Fig. 7).Taken together, our findings suggest that the regulation of 125 I seed's effects on CCA cells depends on the p53 level.
However, it is worth noting that no studies have shown p53 mutations in the CCA cell lines used in this study (RBE and HCC-9810 cell lines), so this manuscript has certain limitations.Next, we will continue to explore the effects of 125 I seed on CCA cells carrying p53 mutations.

Conclusion
In summary, 125 I seed inhibited cell growth through the apoptotic pathway.The mechanism may involve the activation of p53 and its downstream apoptotic processes by upregulating ROS production in cells.Region: Application of radioactive 125I particle stent in malignant biliary obstructive diseases, 2022BEG03152; Key R&D project of

Fig. 1
Fig. 1 Irradiation with the 125 I seed inhibits the proliferation of CCA cells.CCK-8 assay indicated that 125 I seed treatment gradually suppressed the viability of RBE (A) and HCCC-9810 cells (B).The

Fig. 3
Fig. 3 125 I seed activates ROS/p53 pathway in CCA cells.The intracellular ROS levels of RBE cells (A) and HCCC-9810 cells (B) were increased after 125 I seed treatment by flow cytometry.RT-qPCR (C, E)

Fig. 4 Fig. 5
Fig. 4 Inhibition of ROS generation blocks the 125 I seed-induced accumulation of ROS and upregulation of p53 in CCA cells.RBE cells were treated with 0.8 mCi of 125 I seed and ROS scavenger NAC.(A) Flow cytometry assay indicated that NAC reduced intracellular ROS

Fig. 6
Fig. 6 Inhibition of ROS production blocks the promoting effect of 125 I seed on the apoptosis of CCA cells.RBE cells were treated with 0.8 mCi of 125 I seed and ROS scavenger NAC.(A) TUNEL staining showed that NAC inhibited RBE cell apoptosis compared with that in the 125 I group.(B) Flow cytometry showed that NAC inhibited RBE cell apoptosis induced by 125 I seed.RT-qPCR (C) and western blot

Fig. 7
Fig. 7 Blockade of p53 attenuates the effect of the 125 I seed on the proliferation and apoptosis of CCA cells.RBE cells were treated with 0.8 mCi of 125 I seed and PFTα, a functional inhibitor of p53.(A) CCK-8 assay indicated that PFTα could rescue the inhibitory effect of 125 I seeds on RBE cell proliferation.(B) Flow cytometry showed that PFTα blocked the inhibition of 125 I seed on CCA cell cycle.(C) BrdU assay showed that the proliferation of RBE cells was increased by PFTα compared with 125 I group.(D) TUNEL staining and Flow cytometry analysis (E) showed that the apoptosis of RBE cells was inhibited by PFTα compared to 125 I group.*P < 0.05, **P < 0.01,***P < 0.001 vs.Control group; #P < 0.05, ##P < 0.01 vs. 125 I group

Table 1
Primer sequences used in this study