CIr2 was synthesized by our group as previously described (Lin et al., 2024). The following reagents were procured from commercial suppliers: CCK-8, N-acetyl-L-cysteine (NAC), staurosporine, Z-VAD-FMK, necrostatin-1, SB202190, cisplatin, ATP Assay Kit (#S0026), Mito-Tracker Red (#C1049B), ROS Assay Kit (#S0033S), Apoptosis Detection Kit (#C1062S), tetramethylrhodamine ethyl ester (TMRE) were obtained from Beyotime Biotechnology (China). Ferrostatin-1 was acquired from MedChemExpress (USA). CB-5083 was purchased from TargetMol (USA). Sodium 4-Phenylbutyrate (4-PBA), Hydroxyurea (HU), Hydroxychloroquine sulfate (HCQ) were purchased from Macklin (China). The antibody against cleaved Caspase-9 (#9508), Caspase-3 (#14220), PARP (#5625), p-p38 (#4511), p38 (#8690), p-ERK 1/2 (#4370), ERK 1/2 (#4695), p-JNK (#4668), JNK (#9252), p-AKT (#4060), AKT (#4691), p-PDK1 (#3438), p-PTEN (#9551), Cyclin E1 (#20808), PCNA (#2586) were obtained from Cell Signaling Technology (USA). The antibody against p-Chk1 (#HA721189), Cyclin A2 (#ET1612-26) were purchased from HUABIO (China). The antibody against Bcl-2 (#12789-1-AP), Bax (#60267-1-Ig), CHOP (#15204-1-AP) were purchased from Proteintech (China). MCF-7, MDA-MB-231, MDA-MB-453, and MCF-10A cell lines were purchased from Procell (China) and cultured according to the supplier’s recommended protocols.

Cellular cytotoxicity was evaluated using the CCK-8 assay. 3,000–8,000 cells per well were plated in 96-well plates, and the cells were cultured overnight at 37 °C to allow full adhesion. Subsequently, culture medium with different concentrations of the drug was freshly prepared and added, followed by an additional 48-h incubation. Finally, CCK-8 reagent (10% of the total volume) was added to the medium, and the cells were incubated for 1–3 h before measuring the absorbance at 450 nm using a multimode microplate reader.

600 cells per well were plated in 6-well plates. After the preliminary formation of cell colonies (5-7 cells), various concentrations of drug were added. The culture medium containing freshly prepared drugs was replaced every 3 days. The cells were then cultured for 8–12 days until colonies containing more than 50 cells formed. Finally, the colonies were fixed in ice-cold methanol for 20 min and subsequently stained with 0.5% crystal violet.

3 × 10⁵ MDA-MB-231 cells per well were plated in a 6-well plate and cultured at 37 °C. A 200 μL pipette tip was used to create linear scratches once the cell monolayer reached about 95% confluency, and the cells were carefully rinsed with PBS to remove detached debris. To minimize the influence of cell proliferation, the culture medium was changed to L-15 medium supplemented with 1% FBS and CIr2 at final concentrations of 0, 100, or 200 μM. After 24 h of treatment, cell migration was quantified using the following formula: Migration area  % = empty area  0   h − empty area  24   h / empty area  0   h × 100 .

3 × 10⁴ MDA-MB-231 cells were seeded into the upper chambers of Transwell inserts (Corning, USA) in serum-free medium containing the indicated drugs. To serve as a chemoattractant, 400 μL of medium containing 20% FBS was filled in the lower chamber. Following 24 h culture, cells that had not migrated and remained on the upper surface of the insert were carefully removed. Subsequently, the cells that had traversed to the lower surface of the membrane were fixed and stained with 0.5% crystal violet. For quantification, five randomly selected fields (×100 magnification) were analyzed using phase-contrast microscopy.

2 × 10⁴ MDA-MB-231 and MCF-7 cells were plated in 4-well confocal dishes and cultured at 37 °C overnight. After exposure to 400 nM CIr2 for 12 h at 37 °C, the cells were washed with PBS, and afterwards stained with MitoTracker Red for 30 min under dark conditions. Finally, the samples were rinsed with PBS, images were obtained using an 810 Laser Scanning Confocal Microscopy System (Zeiss, Germany).

The fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to assess intracellular ROS accumulation. 3 × 10⁵ MDA-MB-231 cells per well were seeded in 6-well plates for 12 h at 37 °C, then cells were exposed to CIr2 for 24 h. Following PBS washing, cells were stained with DCFH-DA for 30 min at 37 °C under dark conditions. Then the cells were harvested and resuspended in PBS containing 0.5% FBS. Subsequently, the samples were examined using a Novo Sampler Q flow cytometer (Agilent Technologies, USA).

MMP alterations were evaluated using the MMP assay kit with TMRE. MDA-MB-231 cells were plated in 35 mm confocal dishes and cultured overnight at 37 °C. After treatment with various concentrations of CIr2 for 16 h, the cells were stained with TMRE for 30 min under dark conditions, then rinsed with incubation buffer, and promptly visualized using a confocal microscopy system.

ATP levels in cells were evaluated using a firefly luciferase-based assay kit. MDA-MB-231 cells were plated at 3 × 10⁵ cells per well in 6-well plates and cultured overnight at 37 °C. After exposure to various concentrations of CIr2 for 24 h, the cells were lysed in lysis buffer. A multimode microplate reader was used to measure the luminescence intensity.

Cell cycle assessment followed the manufacturer’s instructions. MDA-MB-231 cells were seeded in 6-well plates at 3 × 10⁵ cells per well and cultured overnight at 37 °C. After being exposed to different CIr2 concentrations for 24 h, the cells were collected and fixed for 6 h in 70% ice-cold ethanol. Following fixation, the cells were stained with a 20 μg/mL propidium iodide solution containing RNase under dark conditions for 30 min. The samples were then examined using flow cytometry.

To assess how CIr2 affects the induction of apoptosis, 3 × 10⁵ MDA-MB-231 cells per well were plated in 6-well plates and cultured at 37 °C overnight. Following exposure to various concentrations of CIr2 or cisplatin for 24 h, the cells were collected, rinsed with PBS, and stained with Annexin V-FITC solution under dark conditions for 30 min. The samples were then analyzed using flow cytometry.

MDA-MB-231 cells (3 × 10⁶ cells) were exposed to 500 nM CIr2 at 37 °C for 24 h. After treatment, cells were pre-fixed with 2.5% glutaraldehyde at 4 °C for 2 h, then washed with PBS, and post-fixed with 1% osmium tetroxide at 4 °C for 1 h. Following a wash with cold PBS, the cells underwent a dehydration process using a graded series of ethanol followed by acetone to ensure complete removal of water. They were then embedded in PON 812 resin (SPI Supplies, USA) at 65 °C for 72 h. The embedded samples were sectioned, stained with lead citrate, and subsequently analyzed using a transmission electron microscope (TEM) instrument (HT7700, Japan).

3 × 10⁵ MDA-MB-231 and MDA-MB-453 cells per well were plated in 6-well plates and cultured at 37 °C overnight. Following treatment with CIr2 and various inhibitors at the indicated concentrations and time points, for protein extraction, cells were lysed with RIPA lysis buffer containing protease inhibitors. Protein lysates were resolved by electrophoresis on 4%–20% SDS–PAGE gels (Beyotime, China) and subsequently transferred onto PVDF membranes. To block non-specific antibody binding, membranes were incubated with 5% skim milk for 1 h at room temperature. After blocking, they were exposed to primary antibodies at 4 °C overnight. Following several washes with TBST buffer, membranes were treated with HRP-linked secondary antibodies for 1 h. Protein bands were visualized using a ChemiDoc imaging system (Bio-Rad, USA).

3 × 10⁵ MDA-MB-231 cells per well were plated in 6-well plates and cultured at 37 °C overnight. Subsequently, cells were exposed to 500 nM CIr2 for another 24 h. Following ice-cold PBS washing, cells were lysed using Trizol reagent (Invitrogen, USA) to extract total RNA. The quality of RNA was evaluated by a NanoDrop spectrophotometer (Thermo Scientific, USA). RNA sequencing libraries were prepared and analyzed using an Illumina platform (Wuhan Metware Biotech Co., Ltd., China).

All animal experiments were performed with the approval of the Ethics Committee of Guilin Medical University (protocol number GLMC-IACUC-20243159), following the institutional guidelines for animal care and use.

All cell culture experiments were performed with at least three independent biological replicates. Data are expressed as the mean ± standard error of the mean (SEM). The significance of differences between two groups was evaluated using Student’s t-test. P values <0.05 were considered statistically significant.

The chemical structure of CIr2 was shown in Supplementary Figure S1. The detailed syntheses and characterization process were described in our previous study (Lin et al., 2024).

Numerous iridium-based complexes have demonstrated notable cytotoxic effects against cancer cells. In the present study, we assessed the cytotoxicity of CIr2 across various breast cancer cell lines, including MCF-7 (Luminal A), MDA-MB-231 (TNBC), MDA-MB-453 (TNBC), and the normal breast epithelial cell line MCF 10A. Cisplatin served as the positive control. Table 1 presents the IC₅₀ values measured following 48 h of drug exposure.

As shown in Table 1, CIr2 exhibited potent anti-proliferative activity across all examined cell lines. Analysis of the IC₅₀ values revealed that CIr2 selectively inhibits TNBC cell lines, showing micromolar potency in MDA-MB-231 (0.15 ± 0.01 μM) and MDA-MB-453 (0.22 ± 0.01 μM) in contrast to the normal breast cell line MCF 10A (0.88 ± 0.06 μM). Notably, CIr2 exhibited markedly stronger cytotoxic effects toward TNBC cell lines compared to cisplatin, with cisplatin IC₅₀ values exceeding 50 μM in MDA-MB-231 and 26.50 ± 4.42 μM in MDA-MB-453, respectively. Since cisplatin primarily exerts its anticancer effects by inducing DNA damage and activating apoptotic signaling pathways, these findings indicate that CIr2 employs alternative mechanisms to suppress TNBC cell growth. MDA-MB-231 was chosen for mechanistic studies of CIr2 in TNBC due to its cytotoxic profile.

Additionally, we conducted colony formation assays in MDA-MB-231, MCF-7, and MCF 10A cells to further assess the inhibitory effects on cell proliferation of CIr2. The MDA-MB-453 cell line was excluded due to its semi-adherent nature, making it unsuitable for this assay. Exposure of MDA-MB-231 and MCF-7 cells to 50 nM CIr2 significantly reduced colony formation, and complete inhibition was observed at 100 nM. In contrast, CIr2 exhibited no significant cytotoxicity towards colony-forming ability of MCF 10A cells even at 100 nM (Figure 1A).

The highly aggressive migratory capacity of TNBC to distant organs makes metastasis a persistent challenge in its clinical treatment. Wound healing and Transwell assays were performed to evaluate the effects of CIr2 on the migration and invasion of MDA-MB-231 cells. As illustrated in Figures 1B–E, CIr2 dose-dependently inhibited the migratory and invasive abilities of the cells in contrast to the control. Notably, at lower concentrations (50–100 nM), CIr2 exhibited a suppressive trend on cell migration and invasion without significantly affecting cell viability after 24 h treatment, suggesting that the observed anti-migratory effects are not solely attributable to acute cytotoxicity (Supplementary Figures S2A-E). Taken together, all of the results indicate that CIr2 possesses promising antitumor activity, particularly against TNBC cells, and holds potential for further development as an anti-TNBC drug.

The potent anticancer activity of CIr2 led us to explore its fundamental mechanisms. In our previous study, CIr2 was demonstrated to trigger ceramide redistribution and induce apoptosis in pancreatic cancer cells (Lin et al., 2024). To investigate the apoptotic effect of CIr2 on MDA-MB-231 cells, we performed Annexin V labeling assays to detect phosphatidylserine externalization, an early marker of apoptosis (Balasubramanian et al., 2007). Surprisingly, CIr2 treatment caused only a minimal increase in Annexin V-positive cells compared to the cisplatin- and heat-treated positive controls (Figure 3A), indicating that the cell membrane integrity remained largely intact, even at concentrations exceeding twice the IC₅₀. Additionally, since caspase activation is a hallmark of apoptosis (Riedl and Shi, 2004), Western blot analysis was employed to examine the expression of apoptosis-related proteins, including cleaved PARP, Caspase-9, Caspase-3, Bcl-2, and Bax, in MDA-MB-231 cells following CIr2 treatment. As shown in Figure 3B, CIr2 did not activate Caspase-9, Caspase-3 or PARP, nor did it significantly affect the levels of Bcl-2 and Bax. A similar trend was found in the MDA-MB-453 TNBC cells. Conversely, cells exposed to staurosporine, an established apoptosis inducer, showed clear PARP cleavage (Supplementary Figure S4). Furthermore, treatment with Z-VAD-FMK, a pan-caspase inhibitor, failed to reduce the cytotoxicity of CIr2 (Figure 3C). The absence of caspase activation and the ineffectiveness of Z-VAD-FMK indicate that CIr2 acts independently of the canonical apoptotic machinery. This distinguishes CIr2 from conventional metal-based chemotherapeutics (Wu et al., 2024; Guo et al., 2025; Pang et al., 2025), suggesting that the compound engages unique signaling networks to bypass apoptosis resistance frequently observed in TNBC cells.

Given the potent in vitro activity of CIr2, its anticancer activity was assessed in vivo in nude mice bearing MDA-MB-231 tumors. As shown in Figures 6A–E, following 25 days of treatment, tumor volume in the control group grew sharply, while tumor growth in the CIr@F127-treated group was markedly suppressed, with a tumor inhibition rate of 75.4%. Importantly, treatment with CIr@F127 was well tolerated, with no mortality or notable changes in body weight observed over the course of the study. Furthermore, our previous study demonstrated that CIr@F127 treatment at the same dose (8 mg/kg) did not significantly affect the major organs of mice (Lin et al., 2024), indicating minimal severe side effects under these conditions. In addition, immunohistochemical analysis was conducted to evaluate the antitumor effects of CIr@F127 treatment. HE staining demonstrated that tumors in the control group exhibited a high density of cell nuclei, closely packed intercellular spaces, and relatively preserved cellular morphology (Figure 6F). Conversely, the CIr@F127-treated group displayed a decreased nuclear density along with enlarged and irregular intercellular spaces, suggesting that CIr2 disrupts tumor tissue architecture and exerts anticancer effects. However, as shown in Figure 6G, TUNEL staining indicated minimal difference in the red fluorescence marking apoptotic cells between the control and CIr@F127-treated groups, with only a few cells showing TUNEL positivity. It should be noted that, unlike apoptosis, paraptosis currently lacks validated molecular markers for direct identification in vivo and is primarily defined by distinctive morphology features, which are most reliably assessed by transmission electron microscopy. Consequently, most studies characterize paraptosis based on comprehensive in vitro morphological and mechanistic evidence, while in vivo experiments mainly focus on antitumor efficacy and exclusion of apoptosis. In this context, our in vivo analyses were designed to evaluate therapeutic efficacy and biosafety, whereas the paraptotic nature of CIr2-induced cell death was rigorously established in vitro. Future studies incorporating ultrastructural analyses of tumor tissues will be important to further elucidate paraptosis under physiological conditions. Collectively, these in vivo results reinforce the translational promise of paraptosis-inducing iridium complexes, consistent with previous reports that IrM2 effectively suppressed tumor growth in animal models without overt toxicity (He et al., 2018).

Collectively, our findings delineate a non-apoptotic mechanism by which CIr2 induces paraptosis in TNBC cells (Figure 7). CIr2 accumulation in mitochondria causes mitochondrial dysfunction, oxidative stress, and ATP depletion, which in turn activate ER stress and p38 MAPK signaling. The resulting mitochondrial–ER crosstalk leads to extensive cytoplasmic vacuolation and caspase-independent paraptotic cell death. These results suggest that CIr2 holds therapeutic potential as a paraptosis-inducing agent for triple-negative breast cancer.