Radiosensitization effect of iridium (III) complex on lung cancer cells via mitochondria apoptosis pathway.

{"full_text": " TYPE Original Research\n PUBLISHED 27 March 2025\n DOI 10.3389/fphar.2025.1562228\n\n\n\n\n Radiosensitization effect of\nOPEN ACCESS iridium (III) complex on lung\nEDITED BY\nMing Xu,\nShimonoseki City University, Japan\n cancer cells via mitochondria\nREVIEWED BY\nDapeng Chen,\n apoptosis pathway\nDalian Medical University, China\nKrzysztof Brzezinski,\nPolish Academy of Sciences, Poland\n Yuru Pang 1\u2020, Qiqi Meng 1\u2020, Yangchen Cui 1, Shiyi Liu 1, Huihui Jiang 1,\n*CORRESPONDENCE\n Chenlan Xu 1, Yan An 1, Yang Jiao 2,3, Qi Zhang 4* and Jihua Nie 1,4*\nJihua Nie, 1\n Department of Toxicology, School of Public Health, Medical College of Soochow University, Suzhou,\n niejihua@suda.edu.cn\n Jiangsu, China, 2State Key Laboratory of Radiation Medicine and Protection, School of Radiation\nQi Zhang,\n Medicine and Protection, Collaborative Innovation Center of Radiological Medicine of Jiangsu Higher\n qzhang2012@suda.edu.cn\n Education Institutions, Soochow University, Suzhou, China, 3Key Laboratory of Radiation Damage and\n\u2020 Treatment of Jiangsu Provincial Universities and Colleges, Collaborative Innovation Center of\n These authors have contributed equally to\nthis work Radiological Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, China,\n 4\n Key Laboratory of Radiation Medicine and Protection, Soochow University, Suzhou, China\nRECEIVED 17 January 2025\nACCEPTED 10 March 2025\nPUBLISHED 27 March 2025\n\nCITATION\nPang Y, Meng Q, Cui Y, Liu S, Jiang H, Xu C, An Y, Background: Lung cancer is the leading cause of cancer-related death in the\nJiao Y, Zhang Q and Nie J (2025) worldwide. Although cisplatin and other platinum-based drugs are widely used as\nRadiosensitization effect of iridium (III) complex\n radiosensitizers in radiotherapy and considered the \ufb01rst-line treatment for\non lung cancer cells via mitochondria\napoptosis pathway. advanced lung cancer, their clinical utility is often limited by drug resistance\nFront. Pharmacol. 16:1562228. and severe cytotoxic side effects. In recent years, iridium-based complexes and\ndoi: 10.3389/fphar.2025.1562228\n other transition metal cation complexes with similar structural properties have\nCOPYRIGHT garnered increasing research interest due to their potential anticancer properties.\n\u00a9 2025 Pang, Meng, Cui, Liu, Jiang, Xu, An, Jiao,\nZhang and Nie. This is an open-access article Methods: Recently, we synthesized a novel iridium (III) complex (Ir-1) and\ndistributed under the terms of the Creative\nCommons Attribution License (CC BY). The use,\n evaluated its safety and stability. The present study aimed to identify Ir-1 with\ndistribution or reproduction in other forums is potent anticancer activity by assessing its cytotoxic effects on lung cancer cells in\npermitted, provided the original author(s) and vitro. Additionally, it investigated Ir-1\u2019s radiosensitizing ef\ufb01cacy and the underlying\nthe copyright owner(s) are credited and that the\noriginal publication in this journal is cited, in\n mechanisms.\naccordance with accepted academic practice.\nNo use, distribution or reproduction is\n Results: The results demonstrated that Ir-1 exhibited signi\ufb01cant radiosensitizing\npermitted which does not comply with these effects on lung cancer cells. Ir-1 effectively reduced cell viability and colony\nterms. formation, arrested the cell cycle at the G2/M phase, inhibited cell migration and\n invasion, decreased mitochondrial membrane potential, and increased reactive\n oxygen species (ROS) generation in lung cancer cells. Importantly, these\n cytotoxic effects were selective, with minimal impact on normal cells.\n Mechanistic studies showed that Ir-1 enhanced radiation-induced cancer cell\n death by disrupting mitochondrial function and activating the mitochondrial\n apoptotic pathway. This was evidenced by upregulated expression levels of\n Bax, Cytochrome c (Cyt-C), and Caspase9 proteins, along with reduced level\n of Bcl-2 protein. Notably, the addition of a Cyt-C inhibitor signi\ufb01cantly reduced\n the expression of Cyt-C and Caspase9 proteins. Similarly, treatment with the\n Caspase9 inhibitor Z-LEHD-FMK also reduced Caspase9 protein level.\n\n\n\n\nFrontiers in Pharmacology 01 frontiersin.org\n\fPang et al. 10.3389/fphar.2025.1562228\n\n\n\n\n Conclusion: This study provides robust evidence that Ir-1 is a promising and safe\n radiosensitizer for lung cancer therapy. Its ability to enhance radiation-induced\n cytotoxicity through mitochondrial dysfunction and activation of apoptotic\n pathways highlights its potential for clinical application.\n\n KEYWORDS\n\n iridium (III) complexes, lung cancer, mitochondria apoptosis pathway, radiation\n sensitization, ROS\n\n\n\n1 Introduction group that promotes cellular uptake of its iridium (III) complex\n when dissolved in DMSO (Cao et al., 2013; Liao et al., 2018). We\n Lung cancer is a malignant tumor with increasing morbidity and previously synthesized the iridium (III) complex Ir (MDQ)2 (acac)\nmortality worldwide (Xia et al., 2022). Clinically, the treatment and demonstrated its potential as a novel probe for tracking mouse\nstrategies for lung cancer are determined by factors such as disease neural stem cells (Xu et al., 2020), with excellent biocompatibility\nstage, histological type, and the patient\u2019s overall health status. Available and photostability (Li et al., 2019).\noptions include surgical resection, radiation therapy, chemotherapy, In this study, we con\ufb01rmed the inhibitory effects of iridium (III)\ntargeted therapy, and various combination therapies. Among these, complex on lung cancer cells, particularly its ability to enhance the\ntargeted therapy has introduced an innovative approach to cancer radiation-induced inhibition of lung cancer cells. Furthermore, we\ntreatment, offering the advantage of minimizing side effects investigated the potential mechanism by which iridium complexes\ncommonly associated with cytotoxic chemotherapy drugs (van den promote mitochondrial apoptosis through the activation of ROS\nBulk et al., 2018). However, it still has certain limitations (Zafar et al., signaling pathways.\n2021). Currently, for most patients with lung cancer, radiotherapy\ncombined with sensitizers remains a cornerstone treatment option.\nEspecially, chemoradiotherapy with platinum drugs is clinically 2 Materials and methods\nrecommended for the stage III-IIIA disease (Alexander et al., 2020).\nSince Rosenberg discovered cisplatin in the 1960s, platinum-based 2.1 Materials and chemicals\nchemotherapy has played a pivotal role in cancer treatment.\nHowever, its clinical effectiveness is often limited by the Ir-1 complex (Ir-1) [Ir (MDQ)2 (acac)] was synthesized and\ndevelopment of drug resistance (Okamoto et al., 2020) and dose- characterized by Dan Li from our research group (Li et al., 2019).\nlimiting toxicities, including neurotoxicity, nephrotoxicity, and Ir-2 [Ir (btpy)2 (acac)] and Ir-3 [Ir (btpy)3] were purchased from Jilin\nhepatotoxicity (Rosenberg et al., 1969; Santos et al., 2020). OLED Material Tech Co., Ltd. (Jilin, China, CAS numbers 343978-79-\nResearchers are committed to \ufb01nding new metal based anticancer 0 and 405289-74-9). Their chemical formulas were shown in Figure 1A.\nagents that are expected to replace platinum drugs with improved CDDP was used as a positive control drug purchased from Shanghai\nselectivity and safety. Transition metal cation complexes have emerged Aladdin Biochemical Technology Co., Ltd. (Shanghai, China, CAS\nas promising candidates in this context (Ni et al., 2022). Previous studies number 15663-27-1). All the chemicals and reagents were of\nhave demonstrated that certain metal complexes (such as iridium, analytical grade. Ir-1, known as Bis (2-methyldifenzo [f, h]\nruthenium, copper, and nickel) exhibit anticancer potential by quinoxaline) (acetylacetonate) Iridium (III), was reported to be\ninteracting with key organelles such as the nucleus, mitochondria, mostly used in two-photon imaging or OLED diodes and had\nand endoplasmic reticulum (Luo et al., 2021; Giorgi et al., 2022). anticancer activity (Li et al., 2019; Xu et al., 2020). Ir-2, known as\n Iridium belongs to the same family as platinum and is located in Bis [2-(2\u2032-benzothienyl) pyridinato-N, C3\u2019] (acetylacetonato) Iridium\nthe third row of transition metals, directly adjacent to platinum, (III), was reported to have certain anticancer activity (Zhang et al., 2020;\nresulting in many similar properties to those of platinum (Marloye Zhang et al., 2021; Yang et al., 2022). Ir-3, known as Tris [2-(benzo[b]\net al., 2016). Compared with platinum, which typically adopts a thiophen-2-yl) pyridine-C3, N] Iridium (III), could be used as an\nplanar structure, iridium shows the advantage of structural diversity, oxygen probe (Fischer et al., 2009). The antibodies for Bcl-2, Bax,\nenabling various ligand modi\ufb01cations through multiple Cyt-C, Caspase9 and Caspase3 were purchased from Abcam\ncoordination geometries (Konkankit et al., 2018). Liu et al. (Cambridge, MA, United States). The antibodies for \u03b2-tubulin and\nreported that Ir-Cpx, an organometallic iridium (III) complex GAPDH were purchased from Abbkine Biotechnology Co., Ltd.\nwith a semi sandwich structure, has cytotoxicity against (Wuhan, China). Cyt-C inhibitor was purchased from APExBIO\nA2780 cells (Liu et al., 2011). Similarly, Conesa et al. (Houston, TX, United States). Z-LEHD-FMK was purchased from\ndemonstrated that ACC25, a new iridium (III) complex with a MedChem Express (Monmouth Junction, NJ, United States).\nsemi sandwich structure protected by two chelating rings, exhibited\nanti-proliferative effects on MCF7 breast cancer cells, including\nmitochondria damage and compromising other organelles 2.2 Cell culture\n(Conesa et al., 2020). Several studies have highlighted that\niridium (III) complexes could exhibit different properties by Human non-small cell lung cancer (NSCLC) cells (H1299) and\nadjusting the ligands of iridium. Bis(2-methyldibenzo [f,h] human normal lung epithelial cells (BEAS-2B) were obtained from\nquinoxaline) (acetylacetonate) contains a lipophilic aromatic the Shanghai National Collection of Authenticated Cell Cultures\n\n\n\nFrontiers in Pharmacology 02 frontiersin.org\n\fPang et al. 10.3389/fphar.2025.1562228\n\n\n\n\n FIGURE 1\n Effects of iridium (III) complexes on cell viability. (A), Structures of three Iridium (III) complexes. (B, C), treatment with Ir-1, Ir-2, Ir-3 and positive\n control CDDP of at (0.1, 0.3, 1, 3, 10, and 30 \u03bcM) to lung cancer cells (B, A549 cell; C, H1299) and normal BEAS-2B cells (D) for 24 h. Data shown as mean \u00b1\n SD, n = 3. *p < 0.05 compared with 0 \u03bcM.\n\n\n\n\nFrontiers in Pharmacology 03 frontiersin.org\n\fPang et al. 10.3389/fphar.2025.1562228\n\n\n\n\n(Shanghai, China). Human NSCLC cells (A549) were obtained from cells were cultured into 6-well plates at a density of 1 \u00d7 106 cells per\nWuhan Procell Life Science & Technology Co., Ltd. (Wuhan, well and incubated overnight. After 24 h, the cells were divided into\nChina). All the cells were cultured in Dulbecco\u2019s Modi\ufb01ed Eagle Control group, Ir-1 group, 6 Gy group, and Ir-1+6 Gy group. Each\nMedium (DMEM) supplemented with 10% fetal bovine serum (FBS) group consisted of three replicate wells. Cells were pre-treated with\nand 1% penicillin/streptomycin. The cells were maintained under the respective drugs for 24 h, followed by 6 Gy irradiation. Samples\nstandard cell culture conditions at 37\u00b0C and 5% CO2 in a humid were collected and then centrifuged at 1,500 rpm for 3 min and \ufb01xed\nenvironment. Attached cells were harvested using trypsin and re- with 70% ethanol overnight in 4\u00b0C. The cells were washed with cold\nsuspended in a serum-containing medium before used in cell PBS to remove ethanol content and incubated at 37\u00b0C with RNase A.\ncryopreservation, passage and the assays, as described below. The cells were then stained with the propidium iodide (PI) for\n 30 min. Flow cytometry analysis was performed within 24 h to assess\n cell cycle distribution.\n2.3 Cell cytotoxicity\n\n The cell cytotoxicity was measured by CCK-8 assay. Cells were 2.7 Wound healing assay\nseeded into 96-well plates at a density of 1 \u00d7 104 cells per well and\ncultured in 200 \u03bcL of DMEM complete medium. Then, different To assess the impact of Ir-1 and radiation on lung cancer cells\nconcentrations (0 \u03bcM, 0.1 \u03bcM, 0.3 \u03bcM, 1 \u03bcM, 3 \u03bcM, 10 \u03bcM, and migration, a wound-healing assay was performed using A549 and\n30 \u03bcM) of Ir-1, Ir-2, Ir-3, and CDDP dissolved in DMSO were H1299 cells. Cells were divided into Control group, Ir-1 group, 6 Gy\ntreated on attached cells for 24 h, followed by addition of the mixture group, and Ir-1+6 Gy group. The cells were seeded in 6-well plates\nof CCK-8 and culture medium in a 1:9 ratio to each well and and allowed to reach a con\ufb02uent state, then a single scratch was\nincubation at 37\u00b0C for 1\u20132 h. The absorbance (OD value) of the cells made using a sterile 200 \u03bcL pipette tip. The cells were then washed to\nat 450 nm was measured using the ELISA reader. The data were remove debris and incubated with FBS-free culture medium. After\nanalyzed to calculate cell viability at different drug concentrations. scratch cleaning, images of the scratches were captured at 0 and 24 h\n with an inverted \ufb02uorescence microscope. The results were\n statistically analyzed by ImageJ. The effects of Ir-1 and 6 Gy\n2.4 Clonogenic assay irradiation on the migration ability of lung cancer cells\n were evaluated.\n A549, H1299 and BEAS-2B cells were seeded in 6-well plates at a\nrespective density of 200, 400, and 800 cells treated with 6 \u03bcM of Ir-1\nfor 24 h. The cells were exposed to 0, 2, 4, and 6Gy irradiation, and 2.8 Transwell assay\nthen were cultured in a 5% CO2 incubator at 37\u00b0C for 14 days. After\n\ufb01xed with 4% paraformaldehyde and stained with crystal violet, To assess the effect of Ir-1 and radiation on lung cancer cells\ncolonies containing \u226550 cells were counted under a microscope. The invasion ability, a Transwell assay was performed using 24-well\nexperiment was repeated three times to calculate the clone survival Matrigel invasion chambers. Each Transwell insert was \ufb01tted with a\nfraction and radiation sensitization ratio. polycarbonate membrane featuring 8 \u03bcm pores, precoated with\n 100 \u03bcL of Matrigel, and incubated at 37\u00b0C for 30 min to 1 h to\n facilitate gel solidi\ufb01cation. A549 cells and H1299 cells were divided\n2.5 Detection of apoptosis into Control group, Ir-1 group, 6 Gy group, and Ir-1+6 Gy\n group. The cells were pre-treated with Ir-1 for 24 h and 6 Gy\n Flow cytometry analysis was performed to assess the percentage irradiation. After 24 h post-irradiation, cells were harvested and\nof apoptotic cells following treatment with Ir-1 and radiation, using reseeded into the upper chamber at a density of 1 \u00d7 105 cells/mL in\nthe Annexin V-PE/7-AAD apoptosis detection kit. Cells were 200 \u03bcL of serum-free RPMI-1640 medium. The lower chamber\ncultured into 6-well plates at a density of 1 \u00d7 105 cells per well in contained 800 \u03bcL of RPMI-1640 supplemented with 20% FBS. After\ncell incubator for 24 h. After the pre-treatment (6 \u03bcM Ir-1), cells incubation for 48 h, the medium was discarded. The cells that had\nwere exposed to 0, 2, 4, and 6 Gy irradiation to screen for optimal invaded the lower surface of the membrane were \ufb01xed in 4%\nradiation dose for cell apoptosis. Subsequently, the cells were paraformaldehyde for 20\u201330 min and stained with 0.1% crystal\nharvested, washed twice with phosphate-buffered saline (PBS), violet for 10\u201320 min. The cell membrane penetration was observed\nand re-suspended in 100 \u03bcL of buffer, which was then added under a \ufb02uorescence microscope and the number of invaded cells\nwith 5 \u03bcL of Annexin V-PE and 5 \u03bcL of 7-AAD, and then was quanti\ufb01ed for statistical analysis.\nincubated at room temperature in the dark for 15\u201320 min. Flow\ncytometry was then conducted to evaluate the apoptosis rates under\ndifferent treatment conditions. 2.9 Mitochondrial membrane potential assay\n\n To examine the effect of Ir-1 and radiation on lung cancer cells\n2.6 Cell cycle analysis mitochondrial membrane potential (\u0394\u03a8m), the mitochondrial\n membrane potential assay was carried out using the\n To determine the effect of Ir-1 and radiation treatment on the Mitochondrial Membrane Assay Kit and JC-1 (5,5\u2032,6,6\u2032-\ncell cycle regulation, cell cycle analysis was performed as follows: Tetrachloro-1,1\u2032,3,3\u2032-tetraethyl-imidacarbocyanine iodide) as\n\n\n\nFrontiers in Pharmacology 04 frontiersin.org\n\fPang et al. 10.3389/fphar.2025.1562228\n\n\n\n\n\ufb02uorescence probe. A549, H1299, and BEAS-2B cells were seeded TABLE 1 IC50 (\u03bcM) values of the iridium (III) complexes and CDDP.\ninto a 6-well plate at a density of 1 \u00d7105 cells per well and subjected\n IC50 (\u03bcM) A549 H1299 BEAS-2B\nto the same pretreatment conditions as described above. After 24 h\nincubation, cells were harvested and stained with JC-1 dye (green, Ir-1 11.16 \u00b1 0.51 53.89 \u00b1 5.63 >200\n\u03bbex = 490 nm, \u03bbem = 530 nm, red, \u03bbex = 525 nm, \u03bbem = 590 nm) in Ir-2 14.46 \u00b1 2.84 >200 >200\ndark for 20 min at 37\u00b0C. For the positive control, the cells were\n Ir-3 15.31 \u00b1 0.57 25.61 \u00b1 0.23 23.93 \u00b1 0.92\nexposed to carbonyl cyanide m-chlorophenylhydrazone (CCCP)\nfor 20 min. CDDP 12.94 \u00b1 0.22 6.52 \u00b1 0.39 28.13 \u00b1 0.58\n\n\n\n2.10 Intracellular ROS assay\n 19.0 for SNK-q test. The value of P < 0.05 was considered\n The intracellular reactive oxygen species (ROS) generation statistically signi\ufb01cant.\ninduced by Ir-1 and radiation was analyzed using the Reactive\nOxygen Species Assay Kit and \ufb02ow cytometry. A549, H1299 and\nBEAS-2B cells were seeded into a 6-well plate at a density of 1 \u00d7 3 Results\n105 cells per well, following the same pretreatment as described\nabove. The cells were then collected and re-suspended in a 3.1 Iridium (III) complexes reduced lung\nworking solution containing 10 \u03bcM of DCFH-DA (\u03bbex = cancer cells viability\n488 nm, \u03bbem = 525 nm) for 20 min at 37\u00b0C in the dark to\navoid light-induced ROS production. The samples were taken The effects of the Iridium (III) complexes on the viability of lung\nout every 4\u20135 min and mixed to ensure full contact between the cancer cells were evaluated using the CCK-8 assay and the\ncells and the probe. For the positive control, the cells were IC50 values were determined from the assay results. Results\nexposed to ROSUP for 20 min. The samples were then showed that Ir-1, Ir-2 and Ir-3 reduced cancer cell viability in a\nanalyzed by \ufb02ow cytometry and data analysis was conducted dose-dependent manner (Figures 1B\u2013D; Table 1). Speci\ufb01cally, Ir-1\nusing GraphPad Prism 8.0 software. reduced viability of A549 cells and H1299 cells, while showing no\n effect on normal epithelial MEAS-2B cells. Ir-2 selectively reduced\n the viability of A549 cells without affecting H1299 cells. In contrast,\n2.11 Western blot analysis Ir-3 reduced the viability of A549, H1299, and MEAS-2B cells.\n CDDP was used as a positive control in all experiments. Based on\n Cells were harvested as scheduled after treatment under these results, Ir-1 was chosen for the subsequent experiments. To\ndifferent conditions with Cyt-C inhibitor or Z-LEHD-FMK and investigate its potential to enhance the sensitivity of cancer cells to\nrinsed three times with PBS. The cells were then lysed on ice using radiotherapy, we selected the concentration of Ir-1 that achieved\na lysis buffer containing a protease inhibitor cocktail and cells viability at 75% (6 \u03bcM for A549 and 25 \u03bcM for H1299) for\nradioimmunoprecipitation assay (RIPA) buffer. After 20 min, subsequent experiments. A concentration of 6.5 \u03bcM CDDP was used\nthe cells were scraped and the lysate collected in an Eppendorf as a positive control in these experiments.\ntube was cleared by centrifugation at 12,000 rpm for 15 min at 4\u00b0C\nto remove debris. The protein concentration in the supernatant\nwas determined by the Bicinchoninic Acid Assay. For Western 3.2 Ir-1 increased radiation-induced\nblotting, equal amounts of total proteins were separated on 12.5% inhibition of colony formation in lung\nsodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- cancer cells\nPAGE) and transferred onto a polyvinylidene \ufb02uoride (PVDF)\nmembrane for 2 h. The membranes were blocked with 5% Next, we examined the synergistic effects of Ir-1 and\nskimmed milk at room temperature for 1 h, followed by radiation therapy in lung cancer cells. Firstly, the inhibitory\nwashing with PBST for 10 min. The membranes were incubated effect of Ir-1 in combination with different radiation doses (0,\nwith primary antibodies at 4\u00b0C for overnight. On the next day, after 2, 4, 6 Gy) on colony formation was evaluated in A549, H1299,\nwashed for 3 times, the membranes were incubated with HRP- and BEAS-2B cells. Figures 2A, B) illustrates that Ir-1 potentiated\nconjugated secondary antibody for 1 h at room temperature. the inhibitory effect of radiation on colony formation in\nImmunoreactive bands were visualized using an enhanced A549 cells. The results indicate that Ir-1 signi\ufb01cantly enhances\nchemiluminescence system. the inhibitory effect at a radiation dose of 6 Gy. A similar\n enhancement was observed in H1299 cells, as shown in\n Figures 2C, D, where Ir-1 signi\ufb01cantly increased the radiation-\n2.12 Statistical analysis induced inhibition of colony formation at 6 Gy. The sensitive\n enhancement ratio (SER) of Ir-1 in A549 cells (1.592) and\n Graphpad Prism 8.0 and ImageJ were used in statistical H1299 cells (1.191) were higher than compared to CDDP in\nanalysis. Numerical data were presented as mean \u00b1 standard the respective cell lines (1.099 and 1.138). Additionally, Ir-1 also\ndeviation (SD) from at least three experiments. The pairwise enhanced the radiation-induced inhibition of colony formation\ncomparison of different group results were made using SPSS in BEAS-2B cells (Figures 2E, F), the SER of Ir-1 in BEAS-2B cells\n\n\n\nFrontiers in Pharmacology 05 frontiersin.org\n\fPang et al. 10.3389/fphar.2025.1562228\n\n\n\n\n FIGURE 2\n Ir-1 increased radiation-induced inhibition of colony formation in lung cancer cells. The A549 (A, B), H1299 (C, D) and BEAS-2B (E, F) cells were\n treated with Ir-1 and CDDP for 24 h and exposure to 6 Gy radiation. Survival fraction was used to evaluate cell radiosensitivity. Data shown as mean \u00b1 SD,\n n = 3. **p < 0.01, ***p < 0.001, ****p < 0.0001.\n\n\n\nwas 1.003 which was lower than that in A549 and H1299 cells. both A549 cells and H1299 cells, the numbers of apoptotic cells in\nBased on these results, 6 Gy was selected as the optimal radiation the Ir-1 + 6 Gy radiation group were signi\ufb01cantly higher compared\ndose for subsequent experiments. to the 6 Gy radiation group (Figures 3A, B).\n\n\n3.3 Ir-1 increased radiation-induced 3.4 Ir-1 increased radiation-induced cell\napoptosis in lung cancer cells cycle arrested in lung cancer cells\n\n Following, we examined whether Ir-1 could enhance radiation- We then examined whether Ir-1 combined with radiation\ninduced lung cancer cells apoptosis. The results indicated that in in\ufb02uenced the cell cycle distribution of lung cancer cells. As\n\n\n\nFrontiers in Pharmacology 06 frontiersin.org\n\fPang et al. 10.3389/fphar.2025.1562228\n\n\n\n\n 3.5 Ir-1 enhanced radiation-induced\n inhibitory effect of migration and invasion in\n lung cancer cells\n\n The wound healing assay and Transwell assay were conducted to\n examine whether Ir-1 enhances the inhibitory effects of radiation on\n cancer cell migration and invasion. Figure 5A shows the wound healing\n assay results. The migration rate of A549 cells and H1299 cells in the Ir-\n 1+6Gy group was signi\ufb01cantly lower than that in the 6Gy\n group. Figure 5B shows Transwell assay results. The number of\n A549 cells and H1299 cells that penetrated the membrane in the Ir-\n 1+6Gy group was signi\ufb01cantly lower than that in the 6Gy group. These\n results indicate that Ir-1 complex enhances the radiation-induced\n inhibitory effect on lung cancer cell migration and invasion.\n\n\n 3.6 Ir-1 enhanced radiation-induced\n reduction of the mitochondrial membrane\n potential in lung cancer cells\n\n To explore the underlying mechanism of Ir-1 action, we\n analyzed the changes in mitochondrial membrane potential\n levels using JC-1 dye as an indicator. As shown in Figure 6, in\n the Ir-1+6 Gy group of both A549 cells and H1299 cells, JC-1 dye\n exhibited brighter green \ufb02uorescence (monomers corresponding\n to low mitochondrial membrane potential) and weaker red\n \ufb02uorescence (aggregates at the high mitochondrial membrane\n potential) compared with other groups, especially the 6 Gy\n group. This suggests a signi\ufb01cant reduction in mitochondrial\n membrane potential when Ir-1 is combined with radiation. In\n contrast, BEAS-2B cells showed no signi\ufb01cant green \ufb02uorescence\n in the Ir-1 or Ir-1+6 Gy group, indicating that Ir-1 and radiation\n did not notably affect the mitochondrial membrane potential in\n normal lung epithelial cells. These \ufb01ndings suggest that the\n combination of Ir-1 and 6 Gy irradiation speci\ufb01cally reduces\n the mitochondrial membrane potential in lung cancer cells,\n which may trigger early apoptosis.\n\n FIGURE 3\n Ir-1 increased radiation-induced apoptosis in lung cancer cells.\n (A), Representative \ufb02ow cytometry results for A549, H1299 and BEAS-\n 3.7 Ir-1 enhanced radiation-induced the ROS\n 2B cells. (B), The data analysis from \ufb02ow cytometry results. Data generation in lung cancer cells\n shown as mean \u00b1 SD, n = 3. *P < 0.05.\n\n Mitochondrial membrane potential and ROS production have a\n strong positive correlation. Therefore, we next evaluated ROS\n generation in each treatment group. As shown in Figures 7A, B,\nshown in Figure 4, the cell cycle distribution of ROS were signi\ufb01cantly increased in the Ir-1 + 6 Gy group compared\nA549 cells and H1299 cells was signi\ufb01cantly altered in the Ir-1 with the 6 Gy group or Ir-1 group alone in both A549 and\n+ 6 Gy group, re\ufb02ecting that an increase at G2/M phase in Ir- H1299 cells. In contrast, no signi\ufb01cant change in ROS was\n1+6 Gy group compared with other groups. In the 6 Gy group, the observed in BEAS-2B cells (Figure 7C). These \ufb01ndings suggest\nproportions of A549 cells in the G0/G1, S, and G2/M phase were that the synergistic effect of Ir-1 and radiation in lung cancer\nrespectively 78.24% \u00b1 1.18%, 5.17% \u00b1 0.98%, and 16.59% \u00b1 cells may be mediated, at least in part, through the ROS pathway.\n0.28%, respectively. In the Ir-1 + 6 Gy group, these\nproportions changed to 71.48% \u00b1 1.25%, 4.37% \u00b1 0.24%, and\n23.79% \u00b1 1.36%, indicating a decrease of approximately 6.76% in 3.8 Ir-1 enhanced radiation-induced\nthe G0/G1 phase and an increase of approximately 7.2% in the apoptotic protein expression\nG2/M phase. The similar results were found in H1299 cells. In\nBEAS-2B cells, only slight changes in G0/G1, S and G2/M phase To further elucidate the mechanism by which Ir-1 enhances\nwere observed between 6Gy group and Ir-1 + 6 Gy group. radiation-induced apoptosis, the expression levels of apoptosis-\n\n\n\nFrontiers in Pharmacology 07 frontiersin.org\n\fPang et al. 10.3389/fphar.2025.1562228\n\n\n\n\n FIGURE 4\n Ir-1 increased radiation-induced cell cycle arrested in lung cancer cells. (A), Representative \ufb02ow cytometry results of cell cycle distribution in A549,\n H1299, and BEAS-2B cells following 24-hour treatment with Ir-1 or CDDP, with or without 6 Gy irradiation. (B), Cell cycle distribution analysis based on\n (A). Data shown as mean \u00b1 SD, n = 3.\n\n\n\n\nrelated proteins were analyzed in A549 cells. As shown in Figure 8A, Figure 8B, Cyt-C inhibitor pre-treatment reduced the expression\ncompared with other groups, the expression levels of pro-apoptotic levels of Cyt-C and cleaved-Caspase9 in the Ir-1 + 6 Gy\nproteins Bax, Cyt-C and cleaved- Caspase9 proteins were signi\ufb01cantly group. Similarly, Caspase9 inhibitor pre-treatment speci\ufb01cally\nincreased in Ir-1 + 6 Gy group, while anti-apoptotic protein Bcl-2 level decreased cleaved-Caspase9 expression levels in the Ir-1 + 6 Gy\nwas markedly decreased. To further con\ufb01rm that Ir-1 enhances the group (Figure 8C). Flow cytometry was used to detect the\ntherapeutic effect of radiation through the Cyt-C/Caspase9 signaling apoptosis cells, as shown in Figures 8D, E, the percentage of\npathway, A549 cells were pre-treated with Cyt-C or apoptosis cells was obviously decreased in the inhibitor groups\nCaspase9 inhibitors, followed by apoptosis assays. As shown in compared with the corresponding groups. These results strongly\n\n\n\n\nFrontiers in Pharmacology 08 frontiersin.org\n\fPang et al. 10.3389/fphar.2025.1562228\n\n\n\n\n FIGURE 5\n Ir-1 enhanced radiation-induced inhibition of migration and invasion in lung cancer cells. (A), Ir-1 increased radiation-induced inhibition of\n migration in A549 and H1299 cells. (B), Ir-1 increased radiation-induced inhibition of invasion in A549 and H1299 cells. Data shown as mean \u00b1 SD, n = 3.\n *P < 0.05.\n\n\n\n\nsuggest that Ir-1 enhances radiation-induced apoptosis by activating 5-year survival rate compared to other major cancers (Huang\nthe mitochondrial apoptosis pathway in lung cancer cells. et al., 2023). CDDP is widely used to be an adjuvant radiotherapy\n drug. However, platinum drugs have serious cytotoxic effects on\n normal tissue cells, limit their clinical application (Rosenberg\n4 Discussion et al., 1969; Santos et al., 2020). Therefore, a critical challenge is\n to develop novel anticancer agents such as cancer-speci\ufb01c\n Lung cancer remains one of the leading causes of cancer- radiosensitizers, that selectively enhance the cytotoxic effects\nrelated incidence and mortality worldwide, with a relatively low of radiation on tumor cells while minimizing damage to normal\n\n\n\n\nFrontiers in Pharmacology 09 frontiersin.org\n\fPang et al. 10.3389/fphar.2025.1562228\n\n\n\n\n FIGURE 6\n Ir-1 enhanced radiation-induced reduction the mitochondrial membrane potential of lung cancer cells.\n\n\n\n\ntissues (Kuo et al., 2015). In our previous study, we successfully potential of this iridium (III) complex in lung cancer cells.\nsynthesized a novel iridium (III) complex and demonstrated its Additionally, we investigated the underlying mechanisms by\nexcellent stability in mice (Li et al., 2019). Building on this which the iridium (III) complex potentiates the ef\ufb01cacy of\nfoundation, the current study investigated the radiosensitizing radiation therapy against lung cancer cells.\n\n\n\n\nFrontiers in Pharmacology 10 frontiersin.org\n\fPang et al. 10.3389/fphar.2025.1562228\n\n\n\n\n FIGURE 7\n Ir-1 enhanced radiation-induced the ROS generation in lung cancer cells. The A549 (A), H1299 (B) and BEAS-2B (C) cells were exposed to ROSUP\n (positive control), 6 \u00b5M of Ir-1 in the absence or presence of 6Gy irradiation. Data shown as mean \u00b1 SD, n = 3. *P < 0.05.\n\n\n\n Previous studies had con\ufb01rmed that some pyridinyl iridium targeted iridium (III) complex possessed enhancing ef\ufb01cacy to\n(III) complexes and half sandwich iridium (III) complexes had the restrain the cancer cell proliferation (Nam et al., 2016; Liu et al.,\nantitumor activity (Ma et al., 2019; Zhang et al., 2020; Zhang et al., 2017). Liu et al. synthesized a kind of polypyridine iridium (III)\n2021), primarily through ROS-mediated mitochondrial complex that induces apoptosis through targeting the lysosome\ndysfunction pathway (Wang et al., 2022). The intracellular and mitochondria (Du et al., 2019). Zhang et al. synthesized three\nROS could induce cell apoptosis, cycle arrest and senescence, iridium (III) complexes with a new ligand TFBIP entrapped in\nwhile also acting as signaling molecules in the intracellular liposomes, which targeted mitochondria, enhancing intracellular\nmitochondria apoptosis pathway. Compared with normal cells, ROS levels and inducing damage to inner and outer membrane\ncancer cells exhibit a severely disrupted redox balance, suggesting structures of mitochondria and release of Cyt-C (Zhang et al.,\nthat ROS regulation could be a potential target for cancer therapy 2022). Chen et al. synthesized two iridium (III) complexes having\n(Moloney and Cotter, 2018). Mitochondria serve as a major ligand THPIP, and con\ufb01rmed that they induced an accumulation\nsource of intracellular ROS and are related to apoptosis of toxic epoxidized lipid MDA and the ROS-mediated\ninduction. They function as key intracellular signaling hubs, mitochondrial dysfunction via activating the PI3K/AKT/mTOR\nemerging as important determinants in cancer development pathway (Chen et al., 2023). Additionally, a luminescent cyclic\nand progression (Guerra et al., 2017). The mitochondrial metallized iridium (III) diimide complex exhibited selective\nelectron transport chain maintains the stability of anticancer activity against various cancer cell lines (Lu et al.,\nmitochondrial membrane potential through redox reaction. 2015). Zhou Yi et al. reported that Ir (III)-BBIP promoted\nThe change of mitochondrial membrane potential is an apoptosis of A549 cells by regulating the apoptosis signaling\nimportant indicator to evaluate the physiological function of pathway (Zhou et al., 2021). Ma et al. comprehensively\nmitochondria (Sakamuru et al., 2022), and an early hallmark of reviewed a variety of iridium (III) complexes that can target\napoptosis. Apoptosis is the natural way of cell physiological death, apoptosis of cancer cells (Ma et al., 2019). Compared with\norderly regulated by apoptosis related signaling pathways, which previous reports, our study demonstrated a similar trend\nact as the anticancer therapies trigger apoptosis induction regarding the antitumor activity of iridium (III) complexes.\n(Mohammad et al., 2015). Radiation-induced DNA damage can Although the above studies showed the cytotoxicity of iridium\ntrigger G2/M phase arrest in the cell cycle, then affecting cell (III) complexes on cancer cells, its effect on apoptosis of normal\nproliferation (Pawlik and Keyomarsi, 2004). B-cell lymphoma-2 cells has been less involved. Our results address this gap by\n(Bcl-2) family proteins regulate programmed cell death by providing additional data on the effects of iridium (III)\ncontrolling intracellular signals of apoptosis and participate in complexes on both lung cancer cells and normal lung epithelial\nthe activation of the intracellular mitochondrial apoptosis cells, thereby offering a more comprehensive evaluation of their\npathway, including the anti-apoptotic protein Bcl-2 and the therapeutic potential and selectivity.\npro-apoptotic protein Bax. Bcl-2 and Bax proteins could In conclusion, the novel iridium (III) complex Ir-1 [Ir (MDQ)2\nregulate the release of Cyt-C and then activate the caspase (acac)] demonstrated to exert potent anticancer effects and enhance\nfamily including Caspase9 and Caspase3, showing caspase radiosensitivity in lung cancer cells. Our \ufb01ndings revealed that Ir-1\ncascade reaction and triggering the response of apoptosis signi\ufb01cantly augmented the inhibitory effects of radiation by\nprocess (Guo et al., 2021). Thus, inhibiting cancer cell inducing apoptosis, causing G2/M phase cell cycle arrest,\nproliferation and promotion of apoptosis are fundamental inhibiting cell migration and invasion, promoting intracellular\nstrategies for the treatment of cancer. Liao C et al. reported ROS production and activating mitochondria apoptosis pathway\nthat an iridium (III) complex liposome delivery system in lung cancer cells. The \ufb01ndings provided compelling evidence that\nincreased ROS levels, causing cellular oxidative damage, Ir-1 represents a promising candidate as a selective and effective\nmitochondrial dysfunction, and inhibiting proliferation (Liao radiosensitizer for lung cancer treatment, warranting further\net al., 2018). Huang et al. reported a type of mitochondria- investigations.\n\n\n\nFrontiers in Pharmacology 11 frontiersin.org\n\fPang et al. 10.3389/fphar.2025.1562228\n\n\n\n\n FIGURE 8\n Ir-1 enhanced radiation-induced apoptotic protein expression. (A), Ir-1 and radiation impacted on Bax, Bcl-2, Cyt-C and cleaved-Caspase9\n expression. (B, C), the Cyt-C and cleaved-Caspase9 expression level after treated with Cyt-C inhibitor and Caspase9 inhibitor. (D), Representative \ufb02ow\n cytometry results of A549 cell apoptosis after indicated treatment. (E), Data analysis from (D). Data shown as mean \u00b1 SD, n = 3. *P < 0.05.\n\n\n\n\nData availability statement curation, Investigation, Supervision, Writing\u2013original draft,\n Writing\u2013review and editing. YC: Data curation, Investigation,\n The raw data supporting the conclusions of this article will be Methodology, Software, Validation, Writing\u2013original draft. SL:\nmade available by the authors, without undue reservation. Investigation, Supervision, Validation, Writing\u2013original draft. HJ:\n Investigation, Methodology, Validation, Writing\u2013review and\n editing. CX: Investigation, Methodology, Validation,\nAuthor contributions Writing\u2013review and editing. YA: Investigation, Methodology,\n Validation, Writing\u2013review and editing. YJ: Investigation,\n YP: Data curation, Formal Analysis, Investigation, Validation, Methodology, Validation, Writing\u2013review and editing. QZ:\nWriting\u2013original draft, Writing\u2013review and editing. QM: Data Funding acquisition, Investigation, Project administration,\n\n\n\nFrontiers in Pharmacology 12 frontiersin.org\n\fPang et al. 10.3389/fphar.2025.1562228\n\n\n\n\nSupervision, Writing\u2013original draft, Writing\u2013review and editing. Generative AI statement\nJN: Funding acquisition, Investigation, Project administration,\nSupervision, Writing\u2013original draft, Writing\u2013review and editing. The author(s) declare that no Generative AI was used in the\n creation of this manuscript.\n\nFunding\n Publisher\u2019s note\n The author(s) declare that \ufb01nancial support was received for the\nresearch and/or publication of this article. This work was supported All claims expressed in this article are solely those of the authors\nby the National Health Commission (NHC) Key Laboratory of and do not necessarily represent those of their af\ufb01liated\nNuclear Technology Medical Transformation (Mianyang Central organizations, or those of the publisher, the editors and the\nHospital, 2023HYX005) and Undergraduate Training Program for reviewers. Any product that may be evaluated in this article, or\nInnovation and Entrepreneurship, Soochow University claim that may be made by its manufacturer, is not guaranteed or\n(202310285067Z). endorsed by the publisher.\n\n\nCon\ufb02ict of interest Supplementary material\n The authors declare that the research was conducted in the The Supplementary Material for this article can be found online\nabsence of any commercial or \ufb01nancial relationships that could be at: https://www.frontiersin.org/articles/10.3389/fphar.2025.1562228/\nconstrued as a potential con\ufb02ict of interest. full#supplementary-material\n\n\nReferences\n Alexander, M., Kim, S. Y., and Cheng, H. (2020). Update 2020: management of non- Liao, C., Xu, D., Liu, X., Fang, Y., Yi, J., Li, X., et al. (2018). Iridium (III) complex-\nsmall cell lung cancer. Lung 198 (6), 897\u2013907. doi:10.1007/s00408-020-00407-5 loaded liposomes as a drug delivery system for lung cancer through mitochondrial\n dysfunction. Int. J. 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