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International Journal of Molecular Sciences logoLink to International Journal of Molecular Sciences
. 2026 Mar 9;27(5):2504. doi: 10.3390/ijms27052504

Nitroxoline Enhances Radiosensitivity in Non-Small Cell Lung Cancer by Suppressing STAT3-AKT-MTOR Survival Signaling

Eun-Young Gong 1,†, Hye Won Lee 1,†, Hyunseung Oh 2, Jae-Ho Lee 3, Sang Jun Byun 4, Jeong-Woo Hwang 5, Sung Uk Bae 5,6, Hyowon Hong 7,*, Young Woo Do 8,*
Editor: Christos Mikropoulos
PMCID: PMC12985646  PMID: 41828719

Abstract

Radiotherapy is the central component in non-small cell lung cancer (NSCLC) treatment. Nonetheless, its therapeutic effectiveness is frequently compromised by adaptive engagement of prosurvival signaling pathways that foster radioresistance. STAT3 functions as the central signaling node that orchestrates cellular survival responses following radiation exposure. This study investigated whether nitroxoline, a clinically approved antimicrobial agent with STAT3-inhibitory activity, enhances radiosensitivity of NSCLC cells and how these effects are mechanistically regulated. We examined the combined effects of nitroxoline and radiation on cell viability and associated signaling pathways in NSCLC cells. Nitroxoline significantly enhanced radiation-induced cytotoxicity and suppressed clonogenic survival compared with radiation alone. Irradiation increased STAT3, AKT, and mTOR phosphorylation, whereas nitroxoline effectively suppressed the basal and radiation-induced activation of these pathways. The combination treatment markedly augmented radiation-induced apoptosis, as demonstrated by increased p53 expression and enhanced PARP and caspase-3 cleavage. Additionally, nitroxoline amplified radiation-induced DNA damage signaling, resulting in pronounced γ-H2AX and DNA-PKcs accumulation. Nitroxoline enhanced NSCLC cell radiosensitivity by suppressing STAT3–AKT–mTOR survival signaling, promoting apoptosis, and amplifying radiation-induced DNA damage, indicating the potential of repurposing nitroxoline as a radiosensitizer to improve radiotherapy outcomes in patients with NSCLC.

Keywords: nitroxoline, non-small cell lung cancer, radiosensitivity, radiosensitizer, STAT3, AKT, mTOR

1. Introduction

Non-small cell lung cancer (NSCLC) is the predominant histological subtype of lung cancer and a major contributor to cancer-associated mortality globally [1]. Although substantial developments have been made in surgical management, molecularly targeted agents, and immune-based therapies, radiotherapy (RT) continues to be critical in the management of patients with advanced-stage and disseminated diseases [2,3]. The effectiveness of RT is frequently undermined by the adaptive capacity of tumor cells to withstand radiation-induced stress through preexisting or treatment-emergent resistance programs, thereby allowing malignant cells to withstand irradiation-induced DNA damage and promoting local tumor persistence, disease recurrence, and unfavorable clinical outcomes [4,5]. Importantly, radioresistance in NSCLC is driven by both efficient DNA damage repair and adaptive activation of prosurvival signaling pathways that allow cancer cells to withstand radiation-induced cellular stress [5,6,7]. Consequently, overcoming these resistance mechanisms and enhancing tumor radiosensitivity remain critical unmet needs in NSCLC management.

Signal transducer and activator of transcription 3 (STAT3) is a multifunctional transcription factor that integrates signaling cues derived from cytokines and growth factors. Under physiological conditions, it regulates fundamental cellular functions, including cell survival, proliferation, and anti-apoptotic responses [8,9,10]. Nevertheless, aberrant or persistent STAT3 activation is frequently documented in colorectal, pancreatic, breast, and head and neck cancers, and is closely associated with tumor aggressiveness, therapeutic resistance, and poor clinical outcomes [11,12,13]. In NSCLC, elevated STAT3 expression and phosphorylation are associated with advanced disease stages, poor overall survival, and reduced targeted therapy response [14,15]. Moreover, ionizing radiation has been demonstrated to activate STAT3 signaling through phosphorylation, resulting in transcriptional programs that support DNA damage repair, suppress apoptotic responses, and promote epithelial–mesenchymal transition [12,16,17]. Therefore, STAT3 enables tumor cells to adapt to radiation-induced stress and serves as a key mediator of radioresistance. Thus, STAT3 signaling disruption via genetic or pharmacological approaches can increase cellular sensitivity to irradiation, supporting STAT3 as a key radioresistance mediator in NSCLC.

Given the central role of STAT3 in promoting tumor progression and radioresistance, agents suppressing STAT3 signaling are of considerable therapeutic interest [12,18]. Nitroxoline, a long-standing antimicrobial drug widely used for the treatment of urinary tract infections, has recently gained recognition for its anticancer activities, including tumor proliferation inhibition, metastatic behavior suppression, and apoptotic cell death induction in multiple malignancies [19,20,21]. Furthermore, in several tumor models, nitroxoline can attenuate prosurvival signaling by reducing the expression of anti-apoptotic proteins, such as Bcl-2 and Mcl-1, while promoting caspase-dependent apoptotic pathways [22,23]. Nitroxoline can also inhibit STAT3 activation, suggesting its potential in targeting STAT3-driven oncogenic survival signaling [24]. Nonetheless, the ability of nitroxoline to modulate radiation responses in NSCLC, particularly through STAT3-dependent survival pathway regulation, remains unclear. Thus, nitroxoline may be a novel radiosensitizer that counteracts radiation-induced STAT3 activation and enhances therapeutic response in NSCLC.

Although nitroxoline has demonstrated promising anticancer activities and the ability to suppress STAT3-driven survival signaling, its mechanism of action in radiation responses in NSCLC remains unclear. To address this gap, we investigated whether nitroxoline enhanced radiosensitivity in NSCLC cells and evaluated the extent to which STAT3 inhibition contributed to this effect. Furthermore, we examined the downstream consequences of nitroxoline treatment, including apoptosis induction, survival pathway suppression, and DNA damage repair impairment, to define its functional role in modulating cellular responses to ionizing radiation. We aimed to establish nitroxoline as a potential radiosensitizer and to provide mechanistic insights into its capacity to counteract radiation-induced STAT3 activation in NSCLC.

2. Results

2.1. Nitroxoline Enhances Radiosensitivity and Suppresses Stemness-like Sphere-Forming Capacity in NSCLC Cells

To determine whether nitroxoline enhances radiation-induced cytotoxicity in NSCLC cells, we first assessed cell viability after 48 and 72 h treatment with nitroxoline and/or radiation. Nitroxoline alone reduced the viability of both A549 and H460 cells in a concentration-dependent manner, with H460 cells displaying a greater sensitivity to nitroxoline than A549 cells (Figure 1A). Similarly, radiation alone decreased cell viability in a dose-dependent manner, with H460 cells exhibiting a more pronounced response than A549 cells (Figure 1B). Combined treatment with nitroxoline and 3 Gy irradiation led to a significantly greater reduction in cell viability compared to radiation monotherapy in NSCLC cell lines (Figure 1C).

Figure 1.

Figure 1

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Nitroxoline enhances radiation-induced cytotoxicity and suppresses clonogenic survival in NSCLC cells. (A) A549 and H460 cells were treated with nitroxoline, and cell viability was assessed using MTT assay (n = 6/group). (B) A549 and H460 cells were exposed to RT and cell viability was measured using MTT assay (n = 6/group). (C) A549 and H460 cells were treated with nitroxoline and/or RT, and cell viability was determined using MTT assay (n = 6/group). (D,F) Representative images of clonogenic assays in NSCLC cells. (E,G) Quantification data of clonogenic assays shown in (D,F) (n = 3/group). *** p < 0.001. NSCLC, non-small cell lung cancer; RT, radiotherapy.

To evaluate long-term effects on cell growth, clonogenic assays were performed after treatment with nitroxoline, radiation, or a combination of both in A549 and H460 cells. The combination markedly suppressed the colony-forming ability of both A549 and H460 cells, resulting in substantially fewer colonies than either treatment alone (Figure 1D–G). Because clonogenic survival is associated with stemness-related properties in NSCLC, we next examined tumorsphere formation. Nitroxoline alone reduced the sphere-forming capacity, whereas radiation alone increased tumorsphere formation in both cell lines. Notably, nitroxoline attenuated the radiation-induced increase in sphere-forming capacity when used in combination with radiation (Figure 2A–C), suggesting suppression of radiation-associated stemness-like phenotypes. Collectively, these results indicate that nitroxoline enhances radiation-induced cytotoxicity and inhibits radiation-associated stemness-like sphere-forming capacity in NSCLC cells.

Figure 2.

Figure 2

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Nitroxoline attenuates radiation-induced tumorsphere formation in NSCLC cells. (A) Representative images of tumorsphere formation assay in NSCLC cells. (B,C) Quantification data of tumorsphere diameter of A549 and H460 cells (n = 5/group). * p < 0.05, ** p < 0.01, and *** p < 0.001. Scale bar = 100 µm. NSCLC, non-small cell lung cancer.

2.2. Nitroxoline Attenuates Radiation-Induced Survival Signaling in NSCLC Cells

To determine whether nitroxoline modulates survival pathways associated with radioresistance, STAT3, AKT, and mTOR expression and phosphorylation were evaluated in A549 and H460 cells. Western blotting was performed 48 h after radiation and/or nitroxoline treatment. Nitroxoline alone reduced total STAT3 and p-STAT3 levels in A549 and H460 cells, indicating inhibition of the STAT3 signaling axis under basal conditions. Conversely, 10 Gy radiation increased p-STAT3 expression without altering total STAT3 expression. After a combination treatment of nitroxoline and RT, p-STAT3 levels were lower than those after RT only, concomitant with a marked reduction in total STAT3 levels. Thus, the decrease in p-STAT3 appears to reflect, at least in part, reduced STAT3 abundance rather than a selective reversal of radiation-induced STAT3 phosphorylation (Figure 3A–D).

Figure 3.

Figure 3

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Nitroxoline suppresses basal and radiation-induced survival signaling in NSCLC cells. (A,C) Representative Western blot images displaying STAT3 and p-STAT3 expressions in NSCLC cells following treatment with nitroxoline and/or RT. β-Actin was used as a loading control for normalization. (B,D) Quantification data of Western blot from A and C (n = 3/group). (E,G) Representative Western blot images demonstrating the expression of mTOR, p-mTOR, AKT, and p-AKT in NSCLC cells following treatment with nitroxoline and/or RT. β-Actin was used as a loading control. (F,H) Quantification data of Western blot from (E,G) (n = 3/group). * p < 0.05, ** p < 0.01, and *** p < 0.001. NSCLC, non-small cell lung cancer; RT, radiotherapy.

Nitroxoline alone reduced basal AKT and mTOR phosphorylation in A549 and H460 cells, indicating suppression of intrinsic prosurvival signaling. Conversely, radiation markedly increased p-AKT and p-mTOR levels. Importantly, nitroxoline counteracted the radiation-induced increase in AKT and mTOR phosphorylation, restoring phosphorylation to basal levels (Figure 3E–H). This reversal was consistently observed in both cell lines, demonstrating that nitroxoline disrupts radiation-activated AKT/mTOR survival signaling. Therefore, nitroxoline appears to suppress basal and radiation-induced activation of STAT3 and AKT/mTOR survival pathways, thereby counteracting key prosurvival signals associated with radioresistance in NSCLC cells.

2.3. Nitroxoline Augments Radiation-Induced Activation of Apoptotic Markers in NSCLC Cells

To determine whether nitroxoline enhanced radiation-induced apoptotic signaling, we examined apoptosis-associated protein expression in A549 and H460 cells. Western blotting was performed 48 h after radiation and/or nitroxoline treatment. Nitroxoline alone modestly increased the expression of p53, cleaved PARP, and cleaved caspase-3 in both cell lines; conversely, radiation alone elevated these apoptotic markers (Figure 4A–D). Notably, combining nitroxoline and 3 Gy irradiation produced the most potent induction of all three markers, with substantially higher activation compared to that of either monotherapy in both A549 and H460 cells (Figure 3A–D). Thus, nitroxoline appears to potentiate radiation-induced apoptotic signaling in NSCLC cells.

Figure 4.

Figure 4

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Nitroxoline enhances radiation-induced apoptotic signaling in NSCLC cells. (A,C) Representative images displaying the expression of p53, cleaved PARP1, and cleaved caspase-3 in NSCLC cells following nitroxoline and/or RT treatment. β-Actin was used as a loading control for normalization. (B,D) Quantification data of Western blot analysis from (A,C) (n = 3/group). * p < 0.05, ** p < 0.01, and *** p < 0.001. NSCLC, non-small cell lung cancer; RT, radiotherapy.

2.4. Nitroxoline Enhances Radiation-Induced DNA Damage Signaling in NSCLC Cells

To determine whether nitroxoline influences radiation-induced DNA damage, we examined DNA-PKcs and γ-H2AX expression in A549 and H460 cells. Western blot and immunocytochemical analyses were performed 48 h following treatment with radiation and/or nitroxoline. Nitroxoline alone produced a modest increase in both DNA-PKcs and γ-H2AX relative to basal levels. Radiation moderately elevated the levels of these markers. Importantly, combining nitroxoline and irradiation resulted in the highest activation of both DNA-PKcs and γ-H2AX, with levels markedly exceeding those observed with either monotherapy (Figure 5A–D). Immunocytochemistry confirmed this pattern, with the most significant accumulation of γ-H2AX foci in the combination group (Figure 5E–H). Therefore, nitroxoline may amplify radiation-induced DNA damage signaling in NSCLC cells.

Figure 5.

Figure 5

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Nitroxoline augments radiation-induced DNA damage signaling in NSCLC cells. (A,C) Representative Western blot images illustrating the expression of DNA-PKcs and γ-H2AX in NSCLC cells following treatment with nitroxoline and/or RT. β-Actin was used as a loading control for normalization. (B,D) Quantification data of Western blot from A and C (n = 3/group). (E,G) Representative immunocytochemistry images demonstrating γ-H2AX foci formation in A549 and H460 cells following nitroxoline and/or RT treatment. γ-H2AX foci are shown in red, and nuclei were counterstained with DAPI (blue). (F,H) Quantification data of γ-H2AX foci from (E,G) (F, n = 77–80/group; H, n = 73–78/group). * p < 0.05, ** p < 0.01, and *** p < 0.001. Scale bar = 20 µm (Merged) and 10 µm (Enlarge merged). NSCLC, non-small cell lung cancer; RT, radiotherapy.

2.5. Nitroxoline Suppresses Radiation-Induced Migratory and Invasive Phenotypes and Attenuates EMT-like Changes in NSCLC Cells

To determine whether nitroxoline modulates radiation-induced migratory phenotypes, we first performed a wound-healing assay in A549 and H460 cells. Wound images were obtained at 0, 36, and 72 h after treatment with radiation and/or nitroxoline. At 0 h, no significant differences in wound width were observed among the groups, indicating comparable initial conditions. Nitroxoline alone reduced the extent of wound closure compared with the control group at both 36 and 72 h, whereas radiation alone increased the extent of wound closure, consistent with enhanced migratory activity after irradiation (Figure 6A,B,D,E). Importantly, nitroxoline attenuated the radiation-induced increase in the extent of wound closure in both A549 and H460 cells, indicating suppression of radiation-enhanced cell motility.

Figure 6.

Figure 6

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Nitroxoline attenuates radiation-induced migratory activity in NSCLC cells. (A,D) Representative wound healing assay images of A549 and H460 cells treated with nitroxoline and/or RT. Images were acquired at 0, 36, and 72 h after scratching. (B,E) Quantification data of wound healing rate from A and D (n = 3/group). (C,F) Representative Western blot images of E-Cadherin, N-Cadherin, and Vimentin expressions in NSCLC cells following treatment with nitroxoline and/or RT. β-Actin was used as a loading control for normalization. * p < 0.05, ** p < 0.01, and *** p < 0.001. Scale bar = 200 µm. NSCLC, non-small cell lung cancer; RT, radiotherapy; EMT, epithelial–mesenchymal transition.

To examine whether these motility changes were accompanied by EMT-related phenotypic alterations, we analyzed the expression of E-cadherin, N-cadherin, and vimentin. Nitroxoline alone increased E-cadherin expression, whereas radiation alone decreased E-cadherin expression. In contrast, N-cadherin and vimentin expressions were reduced by nitroxoline but increased by radiation. Notably, combination treatment with nitroxoline and radiation restored E-cadherin expression and reduced N-cadherin and vimentin expressions relative to radiation alone (Figure 6C,F), indicating attenuation of radiation-associated EMT-like changes.

To functionally validate the migratory phenotype beyond the wound-healing assay, we additionally performed Transwell migration and Matrigel invasion assays. In both A549 and H460 cells, nitroxoline alone reduced migration and invasion, whereas radiation alone increased both phenotypes. Consistent with the wound-healing and EMT marker results, nitroxoline suppressed the radiation-induced increases in Transwell migration and Matrigel invasion in both cell lines (Figure 7A–D). Collectively, these findings indicate that nitroxoline inhibits radiation-enhanced migratory and invasive phenotypes and attenuates associated EMT-like changes in NSCLC cells.

Figure 7.

Figure 7

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Nitroxoline suppresses radiation-enhanced migration and invasion in NSCLC cells. (A,C) Representative migration and Matrigel invasion assays of A549 and H460 cells following treatment with nitroxoline and/or RT. (B,D) Quantification data of migration and Matrigel invasion assay from (A,C) (n = 7/group). *** p < 0.001. Scale bar = 100 µm. NSCLC, non-small cell lung cancer; RT, radiotherapy.

3. Discussion

In this study, we found that nitroxoline enhanced the radiosensitivity of NSCLC cells. Functionally, nitroxoline alone reduced cell viability and clonogenic survival, while its combination with radiation resulted in the greatest suppression of cell growth, indicating a clear radiosensitizing effect (Figure 1). In addition, nitroxoline reduced tumorsphere-forming capacity, and notably attenuated the radiation-associated increase in tumorsphere formation, indicating the suppression of stemness-like phenotypes linked to radioresistance (Figure 2). Beyond these functional outcomes, nitroxoline broadly counteracted radiation-induced prosurvival responses, enhanced apoptotic signaling, modulated DNA damage response (DDR)-associated DNA damage signaling (including γ-H2AX accumulation and DNA-PKcs expression), and suppressed radiation-driven increases in migratory capacity. Nitroxoline also reversed radiation-associated EMT-like marker changes, as reflected by restoration of E-cadherin and reduction in N-cadherin and vimentin expressions. Thus, nitroxoline appears to improve the cellular response to irradiation through coordinated modulation of survival signaling, cell death, DNA damage dynamics, and migratory behavior. To clarify the mechanisms underlying these effects, we examined the interaction of nitroxoline with radiation-activated signaling pathways.

Persistent activation of the STAT3 and PI3K/AKT/mTOR signaling axes supports tumor cell survival and contributes to NSCLC resistance against anticancer therapies [25]. Excessive STAT3 phosphorylation is correlated with unfavorable clinical outcomes, increased tumor cell proliferation, and resistance to chemotherapy and RT in lung cancer [26]. AKT-mTOR signaling activation promotes radioresistance by enhancing the DNA repair capacity and suppressing apoptotic responses [27,28]. Consistent with these observations, irradiation increased STAT3, AKT, and mTOR phosphorylation in A549 and H460 cells, indicating activation of an adaptive prosurvival response. Relative to RT alone, nitroxoline suppressed basal STAT3/AKT/mTOR signaling and reduced total STAT3 abundance, resulting in a lower overall pathway activity in irradiated cells (Figure 3). Therefore, the effect of nitroxoline on p-STAT3 in the combination group should be interpreted as maintenance of suppressed STAT3 signaling (with reduced total STAT3), rather than a simple reversal of radiation-induced p-STAT3 elevation. This result is consistent with previous studies identifying nitroxoline as a functional STAT3 inhibitor in other tumor types, because nitroxoline suppresses STAT3-dependent prosurvival signaling. In addition to prosurvival signaling, cancer stem-like phenotypes are recognized as contributors to radioresistance, and radiation-survived A549 and H460 NSCLC tumor sphere cells display CSCs (cancer stem cells)/EMT-like features [29,30]. STAT3 signaling has been implicated in the maintenance of stemness and radioresistance, and PI3K/AKT/mTOR signaling has been linked to CSCs self-renewal and therapy resistance [31,32,33,34]. Consistent with this framework, we found that radiation increased tumorsphere formation, whereas nitroxoline reduced sphere-forming capacity and attenuated the radiation-induced increase in tumorsphere formation. Although direct pathway validation under sphere-forming conditions was not performed, these findings suggest that nitroxoline-mediated suppression of the STAT3–AKT–mTOR axis may contribute to limiting of the radiation-associated stemness-like phenotypes, thereby improving radiosensitivity.

Inhibition of these pathways translated to a pronounced proapoptotic response. Nitroxoline, particularly combined with irradiation, markedly increased p53 expression and enhanced cleaved PARP and cleaved caspase-3 activation compared with either treatment alone (Figure 4). These changes indicate that STAT3–AKT–mTOR signaling suppression lowers the apoptotic threshold and facilitates radiation-induced cell death. This interpretation is consistent with the results of studies demonstrating that STAT3 or AKT inhibition enhances apoptosis and radiosensitivity in lung cancer [35,36]. Additionally, nitroxoline induces apoptosis in small-cell lung cancer (SCLC) by inducing MDM2 degradation, stabilizing p53, and increasing PARP and caspase-3 cleavages [23]. Although SCLC and NSCLC differ biologically, these findings reinforce the notion that nitroxoline has an intrinsic proapoptotic activity for lung cancer cells and provides additional support for the apoptotic effects observed in our study. Overall, dual survival signaling suppression and apoptosis enhancement provide a coherent mechanistic basis for the nitroxoline radiosensitizing effect observed in functional assays.

Ionizing radiation generates DNA double-strand breaks (DSBs), leading to rapid H2AX phosphorylation (γ-H2AX) and DNA-PKcs activation, a core kinase in non-homologous end joining repair [37,38]. Increased γ-H2AX reflects DSB extension or persistence, whereas DNA-PKcs activation marks DDR engagement [39,40]. Survival pathways, including STAT3 and AKT/mTOR, have also been implicated in coordinating cellular tolerance to genotoxic stress, and pathway inhibition has been reported to promote sustained γ-H2AX accumulation in several tumor types [41,42]. Here, nitroxoline alone induced a modest increase in γ-H2AX and DNA-PKcs expressions, and irradiation produced a moderate elevation consistent with canonical DDR activation. Combined nitroxoline and irradiation treatment markedly increased γ-H2AX and DNA-PKcs levels and was accompanied by marked γ-H2AX foci accumulation. These changes indicate an increased burden of radiation-induced DNA damage and persistence of unresolved DSBs under suppressed survival signaling. Concurrent γ-H2AX and DNA-PKcs elevation in the combination group is consistent with amplified DNA damage signaling due to impaired DSB resolution, rather than enhanced repair capacity, in line with nitroxoline-mediated inhibition of STAT3–AKT–mTOR survival pathways (Figure 5). Collectively, these data indicate that nitroxoline enhanced radiation-associated DDR signaling and γ-H2AX accumulation, providing an additional mechanistic basis for the nitroxoline radiosensitizing effect in NSCLC cells.

Radiation has been reported to augment the migratory behavior of cancer cells by activating prosurvival and pro-motility pathways, contributing to a more aggressive phenotype following treatment [43,44]. Similarly here, irradiation increased wound closure in both NSCLC cell lines, whereas nitroxoline alone suppressed basal migration. Nonetheless, nitroxoline counteracted the radiation-induced increase in migratory activity. To further characterize the motility-related phenotype, we evaluated EMT-associated markers and observed that nitroxoline increased E-cadherin while reducing N-cadherin and vimentin, whereas irradiation produced the opposite result. Importantly, combined treatment restored E-cadherin and reduced N-cadherin/vimentin relative to RT alone, indicating attenuation of radiation-associated EMT-like changes (Figure 6). STAT3 activation promotes migration and invasion across multiple tumor types by regulating EMT-associated transcriptional programs, suggesting that suppressing STAT3 signaling can attenuate motility [45,46]. Although the direct STAT3 dependence of the migratory phenotype was not assessed here, the concurrent inhibition of STAT3 and the reduction in radiation-induced migration observed with nitroxoline are consistent with these reports. Importantly, because wound-healing assays alone provide a limited 2D readout of collective cell movement, we additionally performed Transwell migration and Matrigel invasion assays to strengthen the functional interpretation. These assays showed similar directional patterns in both A549 and H460 cells, with radiation increasing migration/invasion, nitroxoline reducing basal migration/invasion, and nitroxoline suppressing the radiation-enhanced migratory and invasive phenotypes (Figure 7). Thus, nitroxoline may mitigate radiation-induced promotility responses, providing additional functional benefits beyond its radiosensitizing effects.

Overall, our data revealed that nitroxoline enhanced the response of NSCLC cells to irradiation by suppressing STAT3–AKT–mTOR survival signaling, promoting apoptosis, enhancing radiation-associated DDR signaling/γ-H2AX accumulation, while also attenuating radiation-stimulated migratory activity. These coordinated effects suggest that nitroxoline interferes with the cellular adaptive programs that contribute to radioresistance. Because nitroxoline is well-tolerated and has been validated in clinical practice, its repurposing as a radiosensitizer may offer a practical strategy to improve RT outcomes in NSCLC. Nonetheless, the present study remains preliminary in translational terms because all experiments were performed in vitro. Although we strengthened the motility-related findings by adding Transwell migration and Matrigel invasion assays, these systems cannot fully recapitulate the in vivo tumor microenvironment [47]. In particular, stromal interactions, hypoxic conditions, and immune-mediated effects that can influence radiation response were not modeled in the present study [48,49]. Further in vivo validation and deeper mechanistic analyses are necessary to establish the translational relevance of nitroxoline as a radiosensitizer in NSCLC. Collectively, nitroxoline may enhance the therapeutic efficacy of RT in NSCLC.

4. Methods

4.1. Cell Lines and Cell Culture

A549 and H460 human NSCLC cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in RPMI-1640 medium (Invitrogen, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (both from Gibco, Waltham, MA, USA). Cultures were kept at 37 °C in a humidified incubator containing 5% CO2.

4.2. Drug Treatment and Irradiation

Radiation treatment was performed using a VitalBeam linear accelerator (Varian Medical Systems, Palo Alto, CA, USA) with 6-MV X-rays at a dose rate of 2 Gy/min, and the radiation dose was adjusted for each experimental condition. Nitroxoline (MedChemExpress, Monmouth Junction, NJ, USA) was dissolved in dimethyl sulfoxide (DMSO) and administered to cells at the indicated concentrations; DMSO served as the vehicle control. The cells were pretreated with nitroxoline for 2 h before irradiation.

4.3. MTT Assay

The MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma, St. Louis, MO, USA) was used to determine cell viability. For this analysis, A549 and H460 cells were seeded into 96-well plates at 4000 cells per well (six technical replicates per condition), and the membranes were incubated for 24 h. The cells were exposed to the specified concentrations of nitroxoline and incubated for an additional 2 h, followed by irradiation with 6-MV X-rays from a linear accelerator at a dose rate of 2 Gy/min. The treatments consisted of nitroxoline alone, radiation alone, or a combination of both. After 48 h or 72 h of incubation, the MTT solution (0.5 mg/mL) was added to each well and incubated for 2 h to allow formazan formation. The crystals were subsequently dissolved in DMSO, and absorbance was measured at 570 nm using an Infinite M200 Pro/F200 Pro microplate reader (TECAN, Männedorf, Switzerland).

4.4. Clonogenic Assay

Long-term proliferative capacity was assessed using a clonogenic assay. A549 and H460 cells were seeded at 600 cells/well into 12-well plates and allowed to adhere for 24 h. Subsequently, the cells were treated with the indicated concentrations of nitroxoline and irradiated to assess the combined effects of drug treatment and radiation. The culture medium was changed every 2 days with the medium containing the corresponding concentration of nitroxoline. After a 14-day growth period, the cells were gently washed with phosphate-buffered saline (PBS), fixed in cold methanol for 20 min, and stained with 0.25% crystal violet for 30 min at room temperature. Colonies containing >50 cells were manually quantified using ImageJ software (version 1.54, NIH, Bethesda, MD, USA).

4.5. Tumorsphere Formation Assay

A549 and H460 cells were seeded at 1 × 105 cells/well into an ultra-low attachment 6-well plate (Corning Incorporated, Corning, NY, USA) and incubated with DMEM/F12 (Gibco, Waltham, MA, USA), 1× B27 (Gibco, Waltham, MA, USA), 20 ng/mL EGF (R&D Systems, Minneapolis, MN, USA), 20 ng/mL bFGF (R&D Systems, Minneapolis, MN, USA), and 1% penicillin-streptomycin. Cells were subsequently treated with nitroxoline and irradiated, then incubated for 10 days. The medium was refreshed every 2 days by replacing half of the culture volume with a fresh medium containing nitroxoline. Images were acquired using an eXcope K5 microscope (DIXI Science, Daejeon, Korea) at 200× magnification. Tumorsphere size was quantified by measuring the diameter (µm) of spheres on bright-field images using ImageJ software, and the mean diameter per well was used for statistical analysis.

4.6. Western Blot

To analyze protein expression, A549 and H460 cells were treated with nitroxoline, irradiation, or both. After 48 h, the cells were collected and lysed on ice for 20 min using a radioimmunoprecipitation assay buffer (Merck Millipore, Billerica, MA, USA) supplemented with a protease and phosphatase inhibitor cocktail (APExBIO, Houston, TX, USA). Lysates were centrifuged at 13,000 rpm for 15 min at 4 °C, after which the supernatants were harvested for analysis. Protein concentrations were quantified using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Equal protein amounts were resolved using 8–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Blocking was performed with 5% skim milk (BD, Franklin Lakes, NJ, USA) or bovine serum albumin (BSA, MP Biomedicals, Irvine, CA, USA) in Tris-Buffered Saline with Tween 20 for 1 h at room temperature, and incubated overnight at 4 °C with the primary antibodies. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Protein signals were detected using enhanced chemiluminescence detection reagents (Cytiva, Marlborough, MA, USA) and imaged using a Fusion Fx7 system (Vilber Lourmat, Marne-la-Vallée, France). Image analysis was performed using FusionCapt Advance software (Vilber Lourmat, Marne-la-Vallée, France). The antibodies used in this study are listed in Table 1.

Table 1.

List of antibodies used in this study.

Primary Antibodies
Immunogen Host species Dilution Manufacturer Catalog no. Application
p53 Rabbit 1:1000 Cell signaling (Danvers, MA, USA) #9282 WB
cleaved-PARP1 Rabbit 1:1000 Cell signaling (Danvers, MA, USA) #5625 WB
cleaved-caspase-3 Rabbit 1:1000 Cell signaling (Danvers, MA, USA) #9661 WB
p-STAT3Y705 Rabbit 1:1000 Cell signaling (Danvers, MA, USA) #9145 WB
STAT3 Mouse 1:1000 Cell signaling (Danvers, MA, USA) #9139 WB
p-mTOR Rabbit 1:1000 Cell signaling (Danvers, MA, USA) #2971 WB
mTOR Rabbit 1:1000 Cell signaling (Danvers, MA, USA) #2972 WB
p-AKT Rabbit 1:1000 Cell signaling (Danvers, MA, USA) #9271 WB
AKT Rabbit 1:1000 Cell signaling (Danvers, MA, USA) #9272 WB
DNA-PKcs Rabbit 1:1000 Cell signaling (Danvers, MA, USA) #4602 WB
γ--H2AX Rabbit 1:1000, 1:400 Cell signaling (Danvers, MA, USA) #2577 WB, ICC
E-Cadherin Rabbit 1:1000 Cell signaling (Danvers, MA, USA) #3195 WB
N-Cadherin Rabbit 1:1000 Cell signaling (Danvers, MA, USA) #13116 WB
Vimentin Rabbit 1:1000 Cell signaling (Danvers, MA, USA) #5741 WB
β-actin Mouse 1:5000 Santa Cruz (Dallas, TX, USA) sc-47778 WB
Secondary antibodies
Antibody Dilution Manufacturer Catalog no. Application
Goat anti-rabbit IgG, 555 1:200 Invitrogen A21428 ICC
Goat anti-rabbit IgG, HRP 1:5000 Enzo ADI-SAB-300-J WB
Goat anti-mouse IgG, HRP 1:5000 SantaCruz sc-2005 WB
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HRP, Horseradish peroxidase; ICC, Immunocytochemistry; IgG, Immunoglobulin.

4.7. Immunocytochemistry

A549 and H460 cells were cultured on glass coverslips and treated with nitroxoline and/or irradiation. After treatment, cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and blocked for 1 h in PBS containing 3% BSA and 0.1% Triton X-100. Cells were then incubated overnight at 4 °C with primary antibodies diluted in PBS containing 1% BSA and 0.1% Triton X-100. After washing, Alexa Fluor 555–conjugated goat anti-rabbit secondary antibodies were applied for 1 h at room temperature. Coverslips were mounted on glass slides using a DAPI-containing mounting medium (Vector Laboratories, Burlingame, CA, USA). Fluorescence images were acquired using a Leica Stellaris 5 confocal microscope (Leica Microsystems, Wetzlar, Germany) and analyzed using ImageJ software (NIH, Bethesda, MD, USA). A complete list of antibodies used for immunocytochemistry is shown in Table 1.

4.8. Wound Healing Assay

The cell migratory capacity was determined using a wound-healing assay. We seeded A549 and H460 cells at 5 × 105 cells/well into 12-well plates and cultured them until approximately 90% confluence was reached. The cells were then treated with nitroxoline and subsequently exposed to irradiation. After treatment, a uniform scratch was generated across the cell monolayer using a sterile 200 µL pipette tip. Wound images were captured at 0, 36, and 72 h under a light microscope at ×10 magnification. Migratory activity was quantified by measuring wound closure relative to the initial wound area using the ImageJ software.

4.9. Migration and Matrigel Invasion Assay

Transwell migration and Matrigel invasion assays were performed using 6.5 mm Transwell inserts with 8-µm pore polycarbonate membranes (Corning Incorporated, Corning, NY, USA). For the migration assay, A549 and H460 cells were seeded at 5 × 104 cells/well in the upper chamber in 200 µL of a medium containing 1% fetal bovine serum (FBS), and the lower chamber was filled with 500 µL of a medium containing 10% FBS as a chemoattractant. For the Matrigel invasion assay, the upper chamber was coated with Matrigel (Corning Incorporated, Corning, NY, USA) and incubated for 2 h before cell seeding. After 24 h, cells were treated with nitroxoline and irradiated, and then incubated for an additional 48 h. Non-migrated/non-invaded cells on the upper surface were gently removed using a cotton swab. Cells on the lower surface were fixed with cold methanol and stained with 0.5% crystal violet. Images were acquired by light microscopy, and stained cells were quantified using the ImageJ software.

4.10. Statistical Analysis

All experiments were performed independently at least three times. Quantitative data are presented as mean ± standard deviation. Statistical comparisons among multiple groups in the in vitro assays were conducted using one-way analysis of variance. Data processing, statistical analysis, and graphical presentation were performed using GraphPad Prism software (version 8.0; GraphPad Software, San Diego, CA, USA). Statistical significance was denoted by * p < 0.05, ** p < 0.01, and *** p < 0.001. To maintain figure clarity, statistical significance was annotated in the figures, only for the pre-specified comparisons assessing the additional effect of combination treatment (CFI-400945 vs. CFI-400945+RT and RT vs. CFI-400945+RT). Complete statistical results for all group comparisons are provided in Supplementary Table S1.

Acknowledgments

The authors thank the members of the laboratory for their support and constructive discussions.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms27052504/s1.

ijms-27-02504-s001.zip (144.1KB, zip)

Author Contributions

E.-Y.G. and H.W.L. contributed equally to this work. Y.W.D. and H.H. co-supervised the study.; Conceptualization, H.O. and H.W.L.; Methodology and validation, H.H. and Y.W.D.; Investigation and writing—original draft, E.-Y.G. and J.-W.H.; Resources and validation, S.J.B.; Writing—review and editing, H.W.L. and S.U.B.; Supervision and resources, J.-H.L. and H.H.; Funding acquisition, Y.W.D. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data that support the findings of this study are available within the article or from the corresponding authors on reasonable request.

Conflicts of Interest

The authors have no conflicts of interest to declare.

Funding Statement

This work was supported by Biomedical Research Institute grant, Kyungpook National University Hospital (2018).

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ijms-27-02504-s001.zip (144.1KB, zip)

Data Availability Statement

All data that support the findings of this study are available within the article or from the corresponding authors on reasonable request.

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