Personalized Combination Therapy in Bladder Cancer: cAMP Modulators Synergize with 5-FU and Modulate Redox Programs

Simple Summary

Bladder cancer patients often show variable responses to chemotherapy, highlighting the need for more personalized treatment strategies. One promising approach is drug repurposing, which explores new anticancer uses for medications already approved for other diseases. In this study, we tested two cardiovascular drugs, milrinone and terbutaline, in combination with the commonly used chemotherapy drug 5-fluorouracil (5-FU). We found that these drugs significantly enhanced the anticancer effects of 5-FU in bladder cancer cells, but not in lung or prostate cancer models, indicating a tumor-specific benefit. Importantly, terbutaline also reduced harmful oxidative stress inside bladder cancer cells, which may help limit treatment resistance. These findings support the idea that selected heart and asthma medications could be repurposed to improve chemotherapy outcomes in bladder cancer when guided by appropriate biomarkers.

Abstract

Background/Objectives: Repurposed cAMP-elevating agents may personalize fluoropyrimidine therapy by exploiting pathway-specific vulnerabilities. Methods: We tested the PDE3 inhibitor milrinone and the β2-agonist terbutaline alone or combined with 10 μM 5-fluorouracil (5-FU) in UM-UC-5 (bladder), A549 (lung), and PC-3 (prostate) cells. Viability, migration, clonogenicity, and intracellular ROS (DCFDA) were measured; drug interactions used Chou–Talalay/CompuSyn. Results: In UM-UC-5, both agents reduced viability, migration, and clonogenicity and synergized with 5-FU (CI < 1 across Fa ≈ 0.42–0.57). 5-FU increased ROS, whereas terbutaline consistently lowered ROS below baseline and blunted 5-FU-induced oxidative signals; milrinone showed a dose-dependent redox profile without consistent ROS suppression. A549 combinations did not outperform 5-FU; PC-3 was largely unresponsive. Conclusions: cAMP modulators selectively potentiate 5-FU in bladder cancer cells and modulate redox programs (notably with terbutaline), supporting a biomarker-guided combination strategy (e.g., β2-AR/PDE3/PI3K–Akt features) for personalized therapy in bladder cancer; mechanistic and in vivo validation are warranted.

1. Introduction

Bladder cancer therapy remains challenged by heterogeneous responses and frequent relapses, underscoring the need for personalized combination strategies guided by molecular and functional biomarkers. Beyond standard fluoropyrimidine regimens, cyclic AMP (cAMP) signaling has emerged as a potential regulatory axis in urothelial carcinoma, with cell-type-specific expression of β₂-adrenergic receptors (β₂-AR) and phosphodiesterase 3 (PDE3) isoforms. These signaling nodes are known to influence proliferative capacity, migratory behavior, and redox homeostasis, processes that may condition tumor sensitivity to cytotoxic agents such as 5-fluorouracil (5-FU) in specific molecular contexts, including PI3K/Akt dependence and oxidative stress adaptation. Within this framework, pharmacological modulation of cAMP-linked pathways represents a testable strategy to support biomarker-driven personalization in bladder cancer.

Cancer continues to represent a major global health burden and a leading cause of death worldwide [1]. In 2020 alone, an estimated 19.3 million new cases and nearly 10 million cancer-related deaths were reported, with lung, prostate and bladder cancers accounting for a substantial proportion of the global incidence [2]. Despite continuous advances in oncology, current therapeutic strategies often face limitations such as resistance to chemotherapy, poor tumor selectivity, and significant side effects [3,4,5]. These challenges highlight the urgent need for more effective, safer, and economically viable treatment options. In this context, drug repurposing, the strategy of identifying new therapeutic indications for existing pharmacological agents, has gained increasing attention as a cost-effective and time-saving approach to drug development [6,7]. Repurposed drugs benefit from established safety profiles and manufacturing protocols, thereby accelerating clinical translation [8]. Indeed, several non-oncological agents, including antidiabetics, antihypertensives and anti-inflammatory drugs, have shown promise in preclinical and clinical oncology settings [9,10].

Among repurposing candidates, positive inotropic agents used in cardiovascular medicine have attracted attention due to their capacity to modulate intracellular signaling pathways relevant to cancer cell behavior [11,12]. In particular, agents that influence cyclic adenosine monophosphate (cAMP)-associated signaling have been reported to affect cellular proliferation, survival, migration, and oxidative stress responses, processes central to tumor progression and therapeutic resistance [13].

Milrinone, a phosphodiesterase 3 (PDE3) inhibitor, and terbutaline, a selective β2-adrenergic receptor agonist, represent two pharmacologically distinct approaches to elevating intracellular cAMP levels. Milrinone increases cAMP by preventing its degradation, whereas terbutaline stimulates cAMP production through β₂-AR–mediated Gs signaling [14,15]. Elevated cAMP levels can modulate key signaling cascades, including PKA and CREB, which in turn affect gene expression, cell survival and migration [16]. Although primarily indicated for acute heart failure and bronchospasm, respectively, accumulating evidence suggests that these agents can influence cancer-relevant phenotypes, either directly or by modulating cellular responses to cytotoxic stress [13]. Their combination with fluoropyrimidines such as 5-FU, which disrupt DNA and RNA synthesis, offers a rational framework to explore whether signaling modulation can sensitize tumor cells to chemotherapy-induced damage [17]. Notably, previous work from our group investigating the positive inotropic agent levosimendan in similar tumor models supports the feasibility of this repurposing strategy, motivating the evaluation of mechanistically distinct cAMP-modulating agents [18].

To interrogate tumor-context dependency and personalized therapeutic potential, we employed a panel of human solid tumor-derived cell lines representing distinct epithelial malignancies. UM-UC-5 bladder cancer cells were selected as the primary model given the emerging relevance of cAMP-associated signaling in urothelial tumor biology. PC-3 prostate and A549 lung adenocarcinoma cells were included as comparative models to assess whether the effects of cAMP-modulating agents reflect a lineage-specific vulnerability or a broader pan-cancer phenomenon. In this study, we evaluated the in vitro antitumor activity of milrinone and terbutaline, alone and in combination with 5-FU, by examining functional phenotypes including cell viability, morphology, migration, and long-term clonogenic capacity. While direct measurement of cAMP signaling was beyond the scope of this work, the observed phenotypic responses are discussed in the context of cAMP-linked regulatory programs that may govern stress adaptation and survival. Overall, this study aims to provide preclinical evidence supporting the repositioning of cardiotonic drugs as candidate adjuvants to standard chemotherapy and to inform future biomarker-guided strategies for tumor-selective deployment.

2. Materials and Methods

2.1. Compounds

5-Fluorouracil (5-FU) was obtained from Merck Life Science (Algés, Portugal), while Milrinone and Terbutaline were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). All compounds were used as received in the experiments.

2.2. Cell Culture

Human cancer cell lines UM-UC-5 (bladder), PC-3 (prostate) and A549 (lung adenocarcinoma) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco^®, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco^®) and 1% (v/v) penicillin-streptomycin (Sigma-Aldrich^®, Steinheim, Germany). Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO₂ and grown as adherent monolayers in T25 culture flasks (Thermo-Scientific^®, Waltham, MA, USA). Culture medium was replaced every 2–3 days, and cells were passaged at approximately 80% confluence using 0.25% trypsin-EDTA (Sigma-Aldrich^®, Steinheim, Germany).

2.3. Drug Exposure Protocol

The cytotoxic activity of 5-FU, Milrinone and Terbutaline was evaluated in UM-UC-5, PC-3, and A549 cell lines. Cells were treated with a range of concentrations (0.1 to 100 µM) for 48 h. Following treatment, cell viability was analyzed via the MTT assay.

2.4. Cell Viability Assay

For cell viability assessment, cells were seeded in 96-well plates at a density of 5000 cells per well in 200 µL of complete medium and allowed to adhere overnight. After 24 h, the medium was replaced with fresh medium containing the indicated drug concentrations. Following 48 h of treatment, cell viability was evaluated using the MTT assay. MTT reagent was added to each well and incubated to allow the reduction in MTT to insoluble formazan crystals by metabolically active cells. The resulting crystals were solubilized, and absorbance was measured at 570 nm using a Synergy HT spectrophotometer (BioTek Instruments Inc., Winooski, VT, USA). Absorbance values were used as an indicator of cell viability.

2.5. Morphological Assessment

Prior to conducting the viability assay, cells were imaged to assess morphological changes. Imaging was performed using a Leica DMI 6000B microscope coupled to a Leica DFC350 FX camera (Leica Microsystems, Wetzlar, Germany). Images were captured and analyzed using LAS X software (v3.7.4).

2.6. Wound Healing Assay

Cell migration was evaluated through a wound healing assay. Ibidi silicone inserts were placed in 12-well plates to create a cell-free area. Approximately 9 × 10⁵ cells were seeded in each chamber in 70 µL of medium and incubated for 24 h. Once inserts were removed, wells were gently washed with PBS and treated with drugs for 48 h. Migration into the wound area was documented at 0, 24, and 48 h using phase-contrast microscopy (100× magnification). The wound closure percentage was calculated using ImageJ software (version 1.53, FIJI, NIH, Bethesda, MD, USA), normalized to the initial wound area at 0 h.

2.7. Clonogenic Assay

To assess the long-term proliferation potential, clonogenic assays were performed. Cells were seeded at a low density (100 cells per well) in 6-well plates in triplicate. After overnight attachment, cells were treated with or without the indicated drugs for 48 h. Following treatment, cultures were maintained in drug-free medium for 14 days, with medium replacement every 2 days. Colonies were then fixed and stained with 0.5% crystal violet. Colonies larger than 0.5 mm and clearly separated were manually counted.

2.8. DCFDA Assay

Intracellular oxidative status was assessed using the 2′,7′-dichlorofluorescein diacetate (DCFDA) assay. After a 24 h cell adhesion period, cells were loaded with DCFDA (100 μM, diluted in PBS) at a volume of 100 μL per well and incubated for 30 min at 37 °C in the dark. Following dye loading, cells were gently washed, and fresh medium containing the indicated treatments was added. Cells were then exposed to the test compounds for 48 h at 37 °C.

At the end of each incubation period, intracellular fluorescence was measured using a fluorescence microplate reader (SpectraMax Gemini EM Microplate Reader, Molecular Devices, San Jose, CA, USA) with excitation and emission wavelengths set at 485 nm and 530 nm, respectively. Fluorescence intensity was used as an indirect measure of intracellular reactive oxygen species (ROS) levels. ROS were quantified with DCFDA (100 μM) optimized for signal-to-noise in this model; future work will incorporate compartment-specific redox readouts (e.g., GSH/GSSG, MitoSOX, NRF2 targets) to refine mechanistic attribution.

2.9. Synergistic Effect Analysis

To evaluate the interaction between 5-fluorouracil (5-FU) and each repurposed inotropic agent (milrinone or terbutaline), the Chou–Talalay method was applied using CompuSyn software (version 1.0; ComboSyn, Paramus, NJ, USA) to calculate the Combination Index (CI). CI values classify the interaction type: CI < 1 indicates synergism, CI = 1 denotes an additive effect, and CI > 1 indicates antagonism. A non-constant dose ratio design was used, maintaining a fixed concentration of 10 µM 5-FU while varying the concentrations of milrinone or terbutaline. Fraction affected (Fa) values were derived from cell viability data and used as input for the analysis.

2.10. Statistical Analysis

All experiments were performed in triplicate and independently repeated at least three times. Results are expressed as mean ± standard deviation (M ± SD). Statistical comparisons between untreated and treated groups were conducted using one-way ANOVA followed by Student’s t-test. Analyses included comparisons between untreated controls, individual drug treatments, and combinations, with specific evaluation of differences between 10 µM 5-FU and the corresponding combination groups. A p-value of ≤0.05 was considered statistically significant. Data analysis was performed using GraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA). Significance levels are denoted as follows: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 versus control; + p < 0.05; ++ p < 0.01; +++ p < 0.001; ++++ p < 0.0001 versus 10 µM 5-FU.

3. Results

To investigate the potential repurposing of the positive inotropic agents milrinone and terbutaline in cancer therapy, we assessed their effects on viability, morphology, migration and clonogenic survival in UM-UC-5, PC-3 and A549 cell lines. The chemotherapeutic drug 5-fluorouracil (5-FU) was used as a reference for comparison, as it is widely employed in oncology. The concentration of 10 µM was selected based on previously published data [18,19], where it was shown to induce significant cellular effects across multiple cancer cell lines, including the ones used in this study. Although results with 5-FU alone have been previously published, they are included here to provide context and support comparative analyses. In addition to testing milrinone and terbutaline individually, we also evaluated their combination with 10 µM 5-FU to explore potential additive or synergistic effects.

3.1. Effect of Milrinone on Cell Viability and Morphology

To assess the potential cytotoxic effect of milrinone, UM-UC-5, PC-3, and A549 cancer cells were treated with increasing concentrations of the drug (0.1–100 µM) for 48 h. In parallel, the combination of each concentration of milrinone with a fixed dose of 10 µM 5-FU was tested. Cell viability was evaluated using the MTT assay and results are presented in Figure 1.

In UM-UC-5 cells (Figure 1A), milrinone alone exhibited a mild to moderate reduction in cell viability across concentrations. However, the combination with 5-FU led to a dramatic decrease in viability, with reductions ranging from 50% to 53% when compared to milrinone alone (

Table 1. Percentage change in cell viability following treatment with milrinone in combination with 10 µM 5-FU, compared to milrinone alone, in UM-UC-5, PC-3 and A549 cell lines. Values represent the difference between combination and single-agent treatments at matching concentrations of milrinone (0.1–100 µM), calculated using the formula: 100 − (B/A × 100), where A is the viability with milrinone alone and B is the viability with the combination. Negative values indicate decreased viability in the combination group.

Cell Line	Concentration	Δ (%)
UM-UC-5	0.1 µM MIL vs. 0.1 µM MIL + 10 µM 5-FU 1 µM MIL vs. 1 µM MIL + 10 µM 5-FU 10 µM MIL vs. 10 µM MIL + 10 µM 5-FU 25 µM MIL vs. 25 µM MIL + 10 µM 5-FU 50 µM MIL vs. 50 µM MIL + 10 µM 5-FU 100 µM MIL vs. 100 µM MIL + 10 µM 5-FU	−50 −51 −53 −52 −53 −52
PC-3	0.1 µM MIL vs. 0.1 µM MIL + 10 µM 5-FU 1 µM MIL vs. 1 µM MIL + 10 µM 5-FU 10 µM MIL vs. 10 µM MIL + 10 µM 5-FU 25 µM MIL vs. 25 µM MIL + 10 µM 5-FU 50 µM MIL vs. 50 µM MIL + 10 µM 5-FU 100 µM MIL vs. 100 µM MIL + 10 µM 5-FU	−18 −15 +1 −15 −14 −25
A549	0.1 µM MIL vs. 0.1 µM MIL + 10 µM 5-FU 1 µM MIL vs. 1 µM MIL + 10 µM 5-FU 10 µM MIL vs. 10 µM MIL + 10 µM 5-FU 25 µM MIL vs. 25 µM MIL + 10 µM 5-FU 50 µM MIL vs. 50 µM MIL + 10 µM 5-FU 100 µM MIL vs. 100 µM MIL + 10 µM 5-FU	−13 −16 −22 −27 −25 −36

). Crucially, statistical analysis demonstrated that the reductions observed with the combinations were also significant when compared directly to 10 µM 5-FU (p < 0.05 to p < 0.0001), reinforcing that milrinone enhances the cytotoxic effect of 5-FU at this standard dose. This suggests that combining both agents could allow therapeutic benefit while potentially reducing the required dose of 5-FU. In addition to viability, morphological analysis by phase-contrast microscopy was conducted to visually assess drug-induced cellular damage. UM-UC-5 cells treated with milrinone in combination with 5-FU showed marked changes in cell morphology, including reduced cell density, increased detachment, and signs of membrane retraction, when compared to either agent alone or to the untreated control. These qualitative alterations are consistent with the observed reductions in viability. Representative images are provided in Supplementary Figure S1A.

In contrast, PC-3 prostate cancer cells (Figure 1B) showed limited responsiveness to milrinone. The combination with 5-FU yielded variable outcomes, and statistical analysis revealed no significant improvements compared to 10 µM 5-FU alone. While a slight decrease (−18%) was observed at 0.1 µM, some concentrations (e.g., 10 µM) even resulted in increased viability (+1%) compared to milrinone alone, suggesting a lack of consistent synergism in this cell line and possibly even an antagonistic interaction at certain concentrations. In line with these findings, morphological analysis revealed no notable changes in PC-3 cells following treatment with milrinone, 5-FU, or their combination. Cell shape, confluence, and adhesion appeared similar to the control condition across all treatments, further supporting the limited sensitivity of this cell line. Representative phase-contrast images are provided in Supplementary Figure S1B.

In A549 lung cancer cells (Figure 1C), milrinone alone already induced a dose-dependent reduction in viability, particularly at concentrations ≥ 1 µM. Combination treatments resulted in further decreases when compared to milrinone alone (up to ~36%—Table 1), but statistical analysis showed no significant differences versus 10 µM 5-FU. This suggests that the cytotoxicity observed in A549 cells is largely attributable to 5-FU, with milrinone providing no additional measurable benefit. The greatest benefit from the combination was noted at 100 µM milrinone, reinforcing the relevance of dose selection. Morphological changes, including reduced confluence and cell shrinkage, were evident in cells treated with 10 µM 5-FU and became more pronounced when combined with milrinone, although not supported by significant statistical improvement (Supplementary Figure S1C).

Overall, these data indicate that milrinone exerts cell line-specific effects. A significant benefit of the combination with 5-FU was only detected in UM-UC-5 bladder cancer cells, while in PC-3 and A549 models, the cytotoxicity observed appears to be mainly mediated by 5-FU. These findings highlight the tumor-specific nature of the response and support further investigation of milrinone as a potential adjuvant specifically in bladder cancer.

3.2. Milrinone Inhibits Cell Migration and Clonogenic Potential

For wound healing and clonogenic assays, only 50 µM and 100 µM milrinone and terbutaline were tested. This choice was based on the concentrations previously used in our earlier studies with levosimendan [18,19], enabling a consistent comparison across structurally or mechanistically related positive inotropic agents.

The impact of milrinone on cancer cell migration was assessed using a wound healing assay in UM-UC-5, PC-3 and A549 cells. Cells were treated for 48 h with milrinone alone (50 or 100 µM), 5-FU alone (10 µM), or their combination, and the percentage of wound closure was quantified at 0, 24, and 48 h (Figure 2).

In UM-UC-5 cells (Figure 2A), control cultures showed progressive wound closure over time, with nearly 70% closure at 48 h. Treatment with milrinone alone moderately impaired migration, resulting in only ~50% closure. A more pronounced effect was observed with 5-FU alone (~45% closure), while the combination treatment significantly reduced wound closure to below 30%, suggesting a strong additive or synergistic anti-migratory effect. Statistical analysis confirmed that, at both 24 h and 48 h, only the combination of 50 µM milrinone + 10 µM 5-FU produced a significantly greater reduction in closure rate compared to 10 µM 5-FU alone, whereas the 100 µM milrinone + 10 µM 5-FU combination did not reach significance versus 5-FU.Microscopic images taken during the wound healing assay show clear differences in wound closure between treated and control conditions, supporting the quantitative data. Representative images are shown in Supplementary Figure S3A.

In PC-3 cells (Figure 2B), milrinone and 5-FU alone had only a mild impact on migration, with wound closure at 48 h still exceeding 60% for both conditions. The combination treatment did not significantly enhance this effect, indicating that PC-3 cells are comparatively less responsive to both agents in terms of migration inhibition. Phase-contrast images captured during the assay confirmed the persistence of migration across conditions. Representative images are provided in Supplementary Figure S3B.

In contrast, A549 cells (Figure 2C) exhibited a moderate response to all treatments. Milrinone alone reduced migration to ~50% closure, and 5-FU produced a similar effect. The combination further decreased wound closure to approximately 35%, suggesting that A549 cells are moderately sensitive to cAMP-modulating agents in the context of migratory inhibition. However, statistical analysis showed that, at both 24 h and 48 h, only the combination of 50 µM milrinone + 10 µM 5-FU resulted in a significant reduction compared to 10 µM 5-FU alone, while the 100 µM milrinone + 10 µM 5-FU combination was not significantly different from 5-FU. These observations were visually supported by microscopic images obtained at 0, 24, and 48 h, as shown in Supplementary Figure S3C.

These data indicate that milrinone can impair cancer cell migration, particularly in UM-UC-5 and A549 lines, and that co-treatment with 5-FU enhances this effect, especially in the bladder cancer model. This suggests that the anti-migratory activity of milrinone may contribute to its overall antitumor potential, particularly when used in combination with established chemotherapeutics.

The long-term impact of milrinone on cancer cell survival and proliferation was evaluated using a clonogenic assay in UM-UC-5, PC-3, and A549 cells. Cells were exposed to 50 or 100 µM milrinone, 10 µM 5-FU, or their combination for 48 h, followed by a 14-day recovery period in drug-free medium to allow colony formation (Figure 3).

In UM-UC-5 cells (Figure 3A), treatment with milrinone alone significantly reduced clonogenicity in a dose-dependent manner (p < 0.05 for 50 µM and p < 0.01 for 100 µM vs. control). The combination of milrinone with 5-FU did not significantly surpass the effect of milrinone monotherapy, though a further reduction in colony formation was observed. Treatment with 5-FU alone also significantly reduced clonogenicity compared to control (p < 0.05), confirming the sensitivity of this cell line to both agents. When compared directly to 10 µM 5-FU, both combination treatments (50 µM milrinone + 5-FU and 100 µM milrinone + 5-FU) showed a significant additional reduction in clonogenic survival (+++ p < 0.001), indicating that milrinone can enhance the long-term inhibitory effect of 5-FU in UM-UC-5 cells. Representative images of colony formation under these treatment conditions are shown in Supplementary Figure S5A.

In contrast, PC-3 cells (Figure 3B) showed no statistically significant differences between any treatment groups. Neither milrinone alone nor in combination with 5-FU was able to meaningfully reduce colony formation, supporting the idea that PC-3 is the least responsive of the tested models, both in terms of viability and long-term proliferative capacity. Representative images are provided in Supplementary Figure S5B.

In A549 cells (Figure 3C), the combination treatment resulted in significantly reduced clonogenic survival compared to control (p < 0.05), but no statistically significant differences were detected when compared directly to 10 µM 5-FU, indicating that the inhibitory effect observed with the combinations is primarily attributable to 5-FU. Moreover, milrinone alone, at either 50 or 100 µM, did not significantly affect colony formation in this cell line, suggesting a lack of intrinsic long-term antiproliferative activity of the repurposed agent in A549 cells. Images corresponding to this condition are included in Supplementary Figure S5C.

These results indicate that milrinone enhanced the long-term inhibitory effect of 5-FU only in UM-UC-5 cells, while in A549, the effect of the combinations was comparable to 5-FU alone, and no impact was observed in PC-3 cells. The neutral findings in A549/PC-3 help delineate non-responsive contexts, reducing the risk of unnecessary combination therapy and supporting precision sparing.

3.3. Terbutaline Reduces Viability and Affects Cell Morphology

To evaluate the cytotoxic potential of terbutaline, UM-UC-5, PC-3, and A549 cells were treated with increasing concentrations of the drug (0.1–100 µM) for 48 h, either alone or in combination with 10 µM 5-FU. Cell viability was assessed by MTT assay, and the results are shown in Figure 4. Percentage differences in viability between terbutaline alone and the combination with 5-FU are detailed in

Table 2. Percentage change in cell viability following treatment with terbutaline in combination with 10 µM 5-FU, compared to terbutaline alone, in UM-UC-5, PC-3 and A549 cell lines. Values represent the difference between combination and single-agent treatments at matching concentrations of terbutaline (0.1–100 µM), calculated using the formula: 100 − (B/A × 100), where A is the viability with terbutaline alone and B is the viability with the combination. Negative values indicate decreased viability in the combination group.

Cell Line	Concentration	Δ (%)
UM-UC-5	0.1 µM TERB vs. 0.1 µM TERB + 10 µM 5-FU 1 µM TERB vs. 1 µM TERB + 10 µM 5-FU 10 µM TERB vs. 10 µM TERB + 10 µM 5-FU 25 µM TERB vs. 25 µM TERB + 10 µM 5-FU 50 µM TERB vs. 50 µM TERB + 10 µM 5-FU 100 µM TERB vs. 100 µM TERB + 10 µM 5-FU	−56 −52 −44 −40 −46 −49
PC-3	0.1 µM TERB vs. 0.1 µM TERB + 10 µM 5-FU 1 µM TERB vs. 1 µM TERB + 10 µM 5-FU 10 µM TERB vs. 10 µM TERB + 10 µM 5-FU 25 µM TERB vs. 25 µM TERB + 10 µM 5-FU 50 µM TERB vs. 50 µM TERB + 10 µM 5-FU 100 µM TERB vs. 100 µM TERB + 10 µM 5-FU	−27 −27 −19 −23 −6 −12
A549	0.1 µM TERB vs. 0.1 µM TERB + 10 µM 5-FU 1 µM TERB vs. 1 µM TERB + 10 µM 5-FU 10 µM TERB vs. 10 µM TERB + 10 µM 5-FU 25 µM TERB vs. 25 µM TERB + 10 µM 5-FU 50 µM TERB vs. 50 µM TERB + 10 µM 5-FU 100 µM TERB vs. 100 µM TERB + 10 µM 5-FU	−7 −2 −13 −10 −13 −21

.

In UM-UC-5 cells (Figure 4A), terbutaline alone induced a clear dose-dependent decrease in viability, with reductions of approximately 40–50% at concentrations ≥ 25 µM. The combination with 5-FU significantly enhanced this cytotoxic effect across all doses tested, with viability reductions ranging from −40% to −56% compared to terbutaline alone (Table 2). Crucially, statistical analysis demonstrated that the decreases observed with the combinations were also significant when compared directly to 10 µM 5-FU (p < 0.05 to p < 0.0001), confirming that terbutaline potentiates the effect of 5-FU at this standard dose. Morphological evaluation supported these findings: terbutaline alone led to moderate changes in cell density and structure at higher concentrations, while the combination with 5-FU induced more pronounced alterations, such as cell shrinkage, loss of adhesion, and a clear reduction in confluence (Supplementary Figure S2A). These effects closely resembled those observed with milrinone + 5-FU, suggesting that both cAMP-modulating inotropes exert comparable cytotoxic profiles in bladder cancer cells.

In PC-3 cells (Figure 4B), terbutaline alone had only a modest impact on cell viability, with reductions not exceeding 15%. When combined with 5-FU, additional reductions were observed, most notably ~27% at both 0.1 and 1 µM. However, statistical comparisons revealed that these effects were not significantly different from 10 µM 5-FU alone. This indicates that the cytotoxic response in PC-3 cells is primarily mediated by 5-FU, with little or no contribution from terbutaline. Consistently, morphological analysis did not reveal any appreciable changes in PC-3 cells under any of the treatment conditions with terbutaline and/or 5-FU. Cell shape, confluence, and adhesion properties remained comparable to the untreated control, further reinforcing the resistance of this cell line to the tested inotropic agents (Supplementary Figure S2B).

In A549 cells (Figure 4C), terbutaline monotherapy produced moderate viability reductions at higher concentrations. Combination treatments led to slightly greater reductions (7–21% lower viability than terbutaline alone), with the most pronounced effect at 100 µM. However, statistical analysis showed no significant differences compared to 10 µM 5-FU, indicating that the observed cytotoxicity in A549 is largely attributable to 5-FU. Morphological evaluation revealed only slight changes with terbutaline alone, while the combination with 5-FU induced more evident alterations, including reduced confluence, cell shrinkage, and structural disorganization (Supplementary Figure S2C).

Overall, these findings demonstrate that terbutaline exerts cell line–dependent antitumor activity. The most striking and statistically significant sensitization to 5-FU was observed in UM-UC-5 bladder cancer cells, while in PC-3 and A549 the cytotoxic effect was mainly driven by 5-FU. This reinforces the notion that β2-adrenergic modulation may hold therapeutic potential in bladder cancer, whereas its role appears limited in prostate and lung models.

3.4. Terbutaline Suppresses Migration and Clonogenic Capacity

Cell migration was evaluated using the wound healing assay with Ibidi^® silicone inserts in UM-UC-5, PC-3 and A549 cells. After 24 h of seeding, inserts were removed to create a defined cell-free gap, and cells were treated for 48 h with 100 µM terbutaline, 10 µM 5-FU, or the combination. Wound closure was monitored at 0, 24 and 48 h using phase-contrast microscopy, and the percentage of closure was quantified using ImageJ software (Figure 5).

In UM-UC-5 cells (Figure 5A), the control group (no drug) showed the highest migratory capacity, reaching nearly complete wound closure by 48 h. Treatment with 100 µM terbutaline alone impaired migration, resulting in slower closure (~50%), while this concentration with 5-FU also reduced migration to a similar extent. The combination of 50 µM terbutaline and 5-FU produced the most pronounced inhibitory effect, with wound closure falling to ~30%, suggesting a potential additive or synergistic interaction in blocking migration in this bladder cancer model. Statistical analysis showed that, at 24 h, the combination of 50 µM terbutaline + 10 µM 5-FU was significantly different from 10 µM 5-FU alone (p < 0.05), and 100 µM terbutaline + 10 µM 5-FU also showed a significant difference (p < 0.01). At 48 h, the differences versus 10 µM 5-FU were even more pronounced, with +++ p < 0.001 for 100 µM terbutaline + 5-FU and ++++ p < 0.0001 for 50 µM terbutaline + 5-FU. Representative phase-contrast images illustrating these effects are shown in Supplementary Figure S4A.

In PC-3 cells (Figure 5B), all treatments, terbutaline, 5-FU, and their combination, resulted in wound closure percentages similar to the control, indicating that none of the conditions significantly impaired migration in this prostate cancer model. Microscopy images confirming the similarity in closure between treated and untreated groups are provided in Supplementary Figure S4B.

In A549 cells (Figure 5C), the control group again showed efficient migration (~70% closure at 48 h). Terbutaline and 5-FU alone moderately reduced closure to ~55% and ~50%, respectively. The combination of 100 µM Terbutaline with 5-FU further decreased migration, with closure reduced to approximately 35%, suggesting enhanced anti-migratory activity when both agents are used together. Statistical analysis confirmed that, at 24 h, the combination of 100 µM terbutaline + 10 µM 5-FU was significantly different from 10 µM 5-FU alone (p < 0.0001), and at 48 h the same combination remained significantly different (p < 0.001). These observations are visually supported by phase-contrast images presented in Supplementary Figure S4C.

These results demonstrate that terbutaline can effectively inhibit cancer cell migration in a cell type-dependent manner, particularly in UM-UC-5 and, to a lesser extent, A549 cells, while PC-3 cells appear resistant to these effects. The findings support the idea that β2-adrenergic modulation may be a promising strategy to limit tumor cell migration in selected cancers, especially in combination with established chemotherapeutics.

To evaluate the long-term effects of terbutaline on the proliferative capacity of cancer cells, clonogenic assays were performed in UM-UC-5, PC-3, and A549 cell lines following 48 h of treatment with 50 or 100 µM terbutaline, 10 µM 5-FU, or the combination. After drug removal, cells were incubated in drug-free medium for 14 days to allow colony formation (Figure 6).

In UM-UC-5 cells (Figure 6A), treatment with 50 µM terbutaline alone led to a moderate reduction in colony formation relative to control. The combination of terbutaline and 5-FU further reduced the number of colonies, suggesting an additive effect on long-term proliferative suppression. Direct statistical comparison with 10 µM 5-FU showed that only the 100 µM terbutaline + 5-FU combination produced a significant additional reduction in clonogenic survival (p < 0.05). Representative images are shown in Supplementary Figure S6A.

In PC-3 cells (Figure 6B), clonogenic potential remained largely unaffected across all treatment conditions. Only the combination of 100 µM terbutaline + 10 µM 5-FU significantly reduced colony number compared to control (p < 0.05), but no statistically significant differences were found compared to 10 µM 5-FU alone, reinforcing the observation that PC-3 cells are relatively resistant to both inotropic agents, even over extended recovery periods. Images are provided in Supplementary Figure S6B.

In A549 cells (Figure 6C), treatment with 10 µM 5-FU significantly decreased colony formation compared to control (p < 0.05). The combination of 50 µM terbutaline + 5-FU also significantly reduced clonogenic survival compared to control (p < 0.01), but no statistically significant differences were found when either combination treatment was compared directly to 10 µM 5-FU, indicating that the observed reduction in colony formation is predominantly attributable to 5-FU. These results indicate that A549 cells are moderately sensitive to terbutaline in the context of combination treatment, but without evidence of a true additive effect over 5-FU alone. Representative images can be found in Supplementary Figure S6C.

Overall, these data support that terbutaline enhanced the long-term inhibitory effect of 5-FU only in UM-UC-5 cells, with no significant benefit in A549 or PC-3 when compared directly to 5-FU alone.

3.5. Impact of Terbutaline and Milrinone on Intracellular ROS Levels

To investigate the effects of the tested compounds on the redox status of UM-UC-5 cells, intracellular ROS levels were quantified using the DCFDA fluorescent probe (Figure 7). Hydrogen peroxide (H₂O₂, 350 μM) was used as a positive control and induced a marked increase in fluorescence compared to untreated cells, confirming the sensitivity of the assay.

Treatment with 10 μM 5-FU induced a slight but noticeable increase in ROS levels compared to the untreated control. In contrast, terbutaline (50 and 100 μM) effectively suppressed these levels below the baseline. When combined, terbutaline was able to counteract the pro-oxidant effect of the chemotherapy, with the 50 μM TERB + 10 μM 5-FU group showing the most significant reduction in oxidative stress. Regarding milrinone, a dose-specific response was observed: while the 50 μM MIL dose lowered ROS levels, the 100 μM dose, both alone and in combination with 5-FU, resulted in ROS levels that returned to or slightly exceeded the baseline.

3.6. Combination Index Analysis of 5-FU with Milrinone and Terbutaline in UM-UC-5 Cells

The interaction between 5-fluorouracil (5-FU) and the cAMP-elevating agents milrinone and terbutaline in UM-UC-5 bladder cancer cells was assessed using the Chou–Talalay method and CompuSyn software, following established protocols for quantifying drug synergy in preclinical oncology studies [20]. Combination index (CI) values were calculated for 10 µM 5-FU combined with increasing concentrations of milrinone or terbutaline and plotted against the fraction affected (Fa), representing the proportion of cells inhibited by treatment.

As illustrated in Figure 8 and detailed in

Table 3. CI values for UM-UC-5 cells treated with 10 µM 5-FU in combination with milrinone or terbutaline. CI in bold indicates drug pairs that are synergistic or additive.

Drug Combination	Fa	CI
0.1 µM MIL + 10 µM 5-FU	0.4217	0.2409
1 µM MIL + 10 µM 5-FU	0.4347	0.2347
10 µM MIL + 10 µM 5-FU	0.5073	0.2069
25 µM MIL + 10 µM 5-FU	0.5398	0.2051
50 µM MIL + 10 µM 5-FU	0.5623	0.2155
100 µM MIL + 10 µM 5-FU	0.5513	0.2637
0.1 µM TERB + 10 µM 5-FU	0.5146	0.1947
1 µM TERB + 10 µM 5-FU	0.4751	0.2193
10 µM TERB + 10 µM 5-FU	0.4620	0.2882
25 µM TERB + 10 µM 5-FU	0.4756	0.3796
50 µM TERB + 10 µM 5-FU	0.5066	0.5089
100 µM TERB + 10 µM 5-FU	0.5721	0.7033

, all tested combinations of 5-FU with either milrinone or terbutaline yielded CI values consistently below 1 (range: 0.205–0.264 for 10 µM 5-FU + Milrinone, 0.195–0.703 for 10 µM 5-FU + Terbutaline) across Fa values of ~0.42–0.57. This confirms a synergistic interaction, whereby the combined effect exceeds the expected additive activity of each agent alone.

The magnitude of synergy was particularly pronounced at lower to intermediate Fa values, suggesting that the combinations remain effective even when partial inhibition is achieved. This pattern is consistent with the viability and clonogenic assays (Section 3.2 and Section 3.4), where UM-UC-5 cells displayed the greatest sensitization to both inotropic agents. Importantly, no evidence of antagonism (CI > 1) was detected for either combination.

These results reinforce the potential of cAMP pathway modulators to enhance the therapeutic efficacy of 5-FU in bladder carcinoma models. The strong synergy observed, particularly for milrinone, supports the concept of 5-FU dose reduction strategies that could maintain or improve anticancer efficacy while potentially mitigating treatment-related toxicity. The consistency of these findings with prior reports in related tumor types underscores their translational relevance and positions milrinone and terbutaline as promising adjuvant partners for chemotherapeutic regimens in bladder cancer.

4. Discussion

This study investigated the repurposing potential of two positive inotropic, cAMP-elevating agents, milrinone and terbutaline, alone and in combination with 5-FU across three solid tumor-derived cell lines, and after we reviewed the potential anticancer properties of these agents, highlighting molecular pathways such as cAMP/PKA signaling and PI3K/Akt interactions that could be exploited for therapeutic purposes [12]. The most consistent and clinically suggestive activity emerged in UM-UC-5 bladder cancer cells, where both agents reduced viability, migration, and clonogenic survival, and combinations produced statistically significant improvements over 5-FU alone in multiple assays. Importantly, Chou–Talalay analysis further supported a synergistic interaction (CI < 1) across the tested effect range, strengthening the rationale for combination strategies in this model. In contrast, A549 cells showed limited incremental benefit over 5-FU, and PC-3 cells remained largely unresponsive, underscoring the tumor-selective nature of the observed activity and the need for predictive markers to guide deployment. The selective benefit in UM-UC-5 underscores model-specific sensitivity, aligning with a biomarker-driven deployment strategy rather than a one-size-fits-all approach. While differential expression of PDE3 may contribute to the enhanced chemosensitizing effect of milrinone observed in UM-UC-5 cells, direct assessment of PDE3 levels was beyond the scope of this study. Accordingly, PDE3 expression should be regarded as a candidate predictive biomarker underlying tumor-context dependency rather than a validated molecular determinant.

A plausible unifying framework is that UM-UC-5 cells display heightened vulnerability to cAMP pathway modulation, where cAMP elevation can engage downstream effectors (e.g., PKA/CREB-centric signaling) that coordinate regulatory outputs relevant to cytoskeletal organization, stress adaptation, and survival. Although we did not directly profile transcriptional changes, the convergence of functional phenotypes (reduced migration and clonogenicity) together with consistent pharmacological synergy is compatible with engagement of cAMP-linked regulatory (gene) programs that may shape chemosensitivity. The redox readout provides an additional layer of functional specificity: 5-FU increased ROS, whereas terbutaline robustly lowered ROS below baseline and counteracted the 5-FU-associated oxidative signal, suggesting that its benefit may involve attenuation of oxidative cues that can support adaptive survival pathways. Milrinone showed a dose-dependent redox signature without consistently suppressing 5-FU-associated ROS, indicating that its synergistic contribution may rely more strongly on cAMP-dependent signaling interactions rather than direct redox quenching. Terbutaline’s ROS-lowering profile and milrinone’s synergy independent of consistent ROS suppression suggest complementary cAMP-linked mechanisms, broadening combination design space. It should be noted that the prolonged drug exposure used in vitro was designed to capture cumulative functional and phenotypic effects and was not intended to recapitulate clinical drug residence times, particularly in the context of intravesical therapy. Together, these observations support a biomarker-driven view of combination therapy, where pathway features such as β2-adrenergic receptor levels, PDE3 expression, and PI3K/Akt dependency may help identify responsive bladder cancer contexts.

These results suggest that UM-UC-5 cells possess a particular susceptibility to cAMP pathway modulation. Elevated intracellular cAMP, achieved via PDE3 inhibition (milrinone) or β2-adrenergic stimulation (terbutaline), can activate key effectors such as PKA and CREB, which regulate apoptosis, cytoskeletal organization and gene expression involved in stress responses [21]. The broad therapeutic relevance of PDE inhibitors further supports this rationale. In addition to their use in heart failure, PDE inhibitors are clinically employed to treat asthma, COPD, psoriasis and overactive bladder [22,23,24,25], and have been increasingly investigated for antitumor properties, including modulation of tumor signaling and microenvironment [26,27,28,29,30,31]. This expanding pharmacological profile reinforces the translational relevance of repositioning milrinone in oncology.

Molecular features of UM-UC-5 may further account for their distinct sensitivity. This cell line overexpresses EGFR and shows PI3K/Akt dependency, with prior studies demonstrating that PI3K inhibition induces apoptosis via Akt dephosphorylation [32]. As cAMP interacts with the PI3K/Akt pathway, it is plausible that these cells exhibit a critical vulnerability to cAMP elevation. Additionally, aberrant regulation of PRSS8 (prostasasin), a serine protease associated with epithelial integrity and tumor suppression, has been described in UM-UC-5, possibly contributing to their dysregulated survival control [33]. Another relevant aspect is the presence of functional TRAIL-mediated apoptotic signaling, which may enhance their sensitivity to cytotoxic and stress-inducing agents [34]. Altogether, these molecular attributes may converge to amplify the response of UM-UC-5 cells to cAMP-elevating agents and explain the strong additive effect observed when combined with 5-FU.

The role of ROS in cancer is complex, as they contribute to multiple stages of tumor progression, including initiation [35], growth [36], angiogenesis [37], invasion [38] and metastasis [39]. Beyond purely functional impairments, the modulation of the intracellular redox environment appears to be a critical determinant of the tumor-specific sensitivity observed in UM-UC-5 cells. As expected, H2O2 exposure significantly elevated DCFDA fluorescence, confirming the assay’s sensitivity. Furthermore, treatment with 10 μM 5-FU induced a significant accumulation of intracellular ROS, reinforcing the premise that conventional chemotherapeutic agents often leverage oxidative stress as a primary cytotoxic mechanism to induce DNA damage and apoptosis in bladder cancer cells [40]. In contrast, terbutaline demonstrated a robust capacity to suppress basal ROS levels at both concentrations. This antioxidant profile was notably preserved during co-treatment with 5-FU; specifically, the 50 μM terbutaline + 10 μM 5-FU combination yielded the lowest ROS levels across all experimental groups. By effectively neutralizing the pro-oxidant stimulus of 5-FU and reducing ROS below baseline, terbutaline may disrupt the “vicious cycle” where chemotherapy-induced oxidative stress inadvertently activates redox-sensitive survival pathways, such as NF-κB or MAPK signaling [41,42]. These results suggest that terbutaline’s synergistic antitumor effect does not rely on further ROS-mediated damage, but rather on the attenuation of oxidative signals that typically support tumor adaptation and chemoresistance. Milrinone exhibited a distinct, dose-dependent redox signature. The absence of a monotonic dose–response relationship and the emergence of biphasic effects at higher concentrations are consistent with the non-linear regulation of cAMP-associated signaling pathways, which are subject to threshold effects, feedback control, and context-dependent signaling outputs rather than linear pharmacological scaling. While the 50 μM dose moderately reduced ROS levels, the 100 μM concentration resulted in values that reached or slightly exceeded the baseline. This pattern persisted in combination with 5-FU, where milrinone failed to consistently mitigate the chemotherapy-induced oxidative surge. Unlike terbutaline, these findings suggest that milrinone’s contribution to the observed synergy in UM-UC-5 cells may be largely independent of direct redox quenching, potentially relying on alternative cAMP-mediated mechanisms or a more intricate balance between antioxidant defenses and pro-oxidant metabolic shifts at higher concentrations [12]. While modulation of oxygen tension could provide additional mechanistic insight into redox-dependent effects, systematic manipulation of oxygen levels introduces additional experimental variables that were beyond the scope of the present study. The current findings therefore establish redox modulation as a functional correlate of response under standard culture conditions, providing a foundation for future mechanistic refinement.

The A549 lung adenocarcinoma cell line exhibited an intermediate level of responsiveness. Milrinone or terbutaline alone induced moderate reductions in viability, migration, and clonogenic survival. In combination with 5-FU, further reductions were observed in some assays; however, direct statistical comparisons showed no significant differences between combinations and 5-FU alone, indicating that the observed effects were mainly driven by 5-FU. In clonogenic assays, neither milrinone nor terbutaline enhanced the long-term inhibitory effect of 5-FU, suggesting a lack of true additive or synergistic impact. Prior reports confirm that A549 cells express functional β2-adrenergic receptors and that cAMP signaling can influence proliferation, migration, and apoptosis [43,44], but in this model, cAMP-elevating agents did not confer additional benefit over standard chemotherapy.

PC-3 prostate cancer cells were largely unresponsive to both milrinone and terbutaline in all functional assays. In viability assays, no significant improvements over 5-FU alone were observed; in some cases, combinations with milrinone even slightly increased cell viability, suggesting possible antagonistic interactions. In migration and clonogenic assays, neither agent, alone or in combination, produced significant changes compared to control, except for a modest reduction in colony number with 100 µM terbutaline + 5-FU versus control, which was not significant versus 5-FU. This resistance may reflect low β2-adrenergic receptor expression, reduced cAMP sensitivity, or activation of compensatory pro-survival pathways [45,46,47]. These findings underscore the importance of tumor-specific strategies in drug repurposing and the necessity of careful preclinical evaluation to avoid unintended effects [48].

Beyond their cytostatic effects, milrinone and terbutaline impaired migration and clonogenic potential in a tumor-specific manner. The strongest and most consistent enhancements over 5-FU alone were observed in UM-UC-5 cells, where pharmacological synergy was confirmed. This pattern highlights the potential of cAMP pathway modulators to potentiate 5-FU efficacy in bladder cancer cells while preserving viability in non-malignant cells, as demonstrated in our normal cell toxicity assays. Such differential sensitivity supports the feasibility of dose-sparing strategies aimed at improving therapeutic tolerability. The absence of benefit in A549 and PC-3 cells underscores the need for predictive biomarkers, such as β2-adrenergic receptor levels, PDE3 expression, or PI3K/Akt pathway activation, to guide patient selection. In addition to molecular heterogeneity, biological sex has emerged as an important modifier of cancer incidence, progression and therapeutic response, including in bladder cancer [49,50,51]. Sex-specific differences in hormone signaling [52], adrenergic tone [53], immune regulation [54] and redox homeostasis [55] have been reported to influence treatment outcomes and drug sensitivity. While the present study was conducted in established tumor-derived cell lines and therefore does not model systemic or hormonal sex-related effects, it is plausible that cAMP-modulating agents such as milrinone and terbutaline may interact with sex-dependent signaling networks in vivo. Notably, β₂-adrenergic signaling and oxidative stress responses have been shown to differ between males and females in both physiological and pathological contexts [53,55]. These considerations support the need for future preclinical and clinical studies to explicitly address biological sex as a variable when evaluating cAMP-based combination strategies in bladder cancer.

These findings support the concept that these agents affect not only proliferation but also key functional properties linked to tumor progression and recurrence. Importantly, the lack of consistent benefit in non-bladder cancer models should not be interpreted as a limitation of the approach, but rather as evidence of tumor-context dependency. Such selectivity is a prerequisite for rational patient stratification and aligns with contemporary precision oncology paradigms, in which therapeutic efficacy depends on specific molecular and functional vulnerabilities rather than uniform pan-cancer responses. The consistency of these results with previous findings from levosimendan, another cAMP-elevating inotropic agent previously studied by our group, further strengthens the hypothesis that this class of drugs may offer broad repositioning potential in tumors characterized by defined signaling susceptibilities [18,19].

5. Conclusions

This study demonstrates that the positive inotropic agents milrinone and terbutaline exert tumor-selective antitumor activity in vitro and synergize with 5-fluorouracil in UM-UC-5 bladder cancer cells across viability, migration, and clonogenic endpoints. Notably, terbutaline consistently modulated intracellular redox balance by counteracting 5-FU–induced oxidative stress, highlighting functional divergence within cAMP-elevating agents beyond cytostatic effects. Together, these findings support the repositioning of selected cAMP-modulating cardiotonic drugs as candidate adjuvants to standard chemotherapy in biomarker-defined bladder cancer settings. The observed tumor-specific efficacy and preservation of non-malignant cell viability underscore the relevance of patient stratification strategies based on signaling vulnerabilities, including β2-adrenergic receptor expression, PDE3 levels, and PI3K/Akt pathway activation.