Synergistic combination of the beta-3 antagonist SR59230A with common chemotherapeutic drugs and target therapies in cancer and endothelial cells

Synergistic combination of the beta-3 antagonist SR59230A with common chemotherapeutic drugs and target therapies in cancer and endothelial cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Synergistic combination of the beta-3 antagonist SR59230A with common chemotherapeutic drugs and target therapies in cancer and endothelial cells Arianna Bandini°, Letizia Biso°, Maria Cristina Viaggi, Maria Carla Pardini, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7147623/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Oct, 2025 Read the published version in Investigational New Drugs → Version 1 posted 7 You are reading this latest preprint version Abstract β3-adrenergic receptors (β3-ARs) are increasingly recognized as modulators of tumor progression and treatment resistance across multiple cancer types. SR59230A, a selective β3-AR antagonist, has shown preclinical antitumor activity through mechanisms involving mitochondrial reactivation, reactive oxygen species (ROS) production, and antiangiogenic effects. Based on this premise, this study aimed to investigate the in vitro synergistic effects of SR59230A combined with standard chemotherapeutics or targeted therapies in various human cancer cell lines (glioblastoma, melanoma, triple-negative breast cancer, anaplastic thyroid carcinoma) and endothelial cells (HUVECs). Cells were treated with SR59230A alone or in fixed-ratio combinations with temozolomide, paclitaxel, vemurafenib, lenvatinib, or sorafenib. Drug interactions were quantified using the Chou–Talalay method and validated with the Loewe additivity model. SR59230A exhibited dose-dependent antiproliferative activity, particularly in HUVECs and thyroid carcinoma cells. Synergistic effects were observed in all models, with the strongest synergy in A-2058 melanoma cells (SR59230A + vemurafenib), MDA-MB-231 breast cancer and 8505C thyroid carcinoma cells (SR59230A + paclitaxel), U-87 glioblastoma cells (SR59230A + temozolomide), and HUVECs (SR59230A + lenvatinib or sorafenib). Dose Reduction Index (DRI) values confirmed the potential to lower cytotoxic drug doses while preserving efficacy. These findings suggest that β3-AR antagonism via SR59230A may enhance the efficacy of conventional and targeted anticancer agents through multimodal mechanisms. The consistent synergistic effects across diverse tumor types support further investigation of β3-AR blockade as a promising strategy to overcome resistance and optimize cancer therapy. SR59230A temozolomide paclitaxel vemurafenib lenvatinib sorafenib Cancer therapy Combination treatment Synergism Figures Figure 1 Figure 2 Introduction In recent years, in cancer biology the involvement of the catecholaminergic system has gained increasing attention, with a particular interest in β-adrenergic receptors (β-ARs) ( 1 , 2 ). These receptors mediate the effects of catecholamines (i.e., norepinephrine and epinephrine), and β-adrenergic signalling may be implicated in different aspects of cancer progression, including tumour growth, angiogenesis, and metastasis ( 3 ). These hypotheses suggest that pharmacological agents targeting β-ARs have a therapeutic potential in oncology, particularly when used in conjunction with traditional chemotherapy, target therapies or immunotherapy ( 4 , 5 ). Commonly used β-adrenergic antagonists, such as β-blockers, which are widely used in hypertension treatment, have shown promising results as an additional treatment in various cancer types, including breast, prostate, brain, skin, and ovarian tumours, although the majority of clinical evidence comes from retrospective studies and still needs to be confirmed in more rigorous trials ( 6 – 12 ). Beyond frequently used β-blockers that are already on the market, other β-ARs compounds are under development and are currently being tested in preclinical settings. In particular, β3-AR has attracted attention due to its more restricted expression pattern compared to other β-AR subtypes, and because of its apparent involvement in specific conditions such as embryonic/fetal development and tumor growth. Both scenarios require cellular proliferation under highly hypoxic and theoretically inhospitable environments. In these contexts, β3-AR promotes cellular proliferation, hypoxia-induced angiogenesis, chemoresistance, a glycolytic metabolic shift (Warburg effect), and the induction of stemness ( 13 – 15 ). Among the β3-AR ligands, SR59230A is indicated as a β3-AR antagonist, although affinity for all β-ARs has been demonstrated depending from the animal species taken into consideration ( 16 ). In addition, in some cellular systems it can act as a partial agonist as well ( 17 ). SR59230A has shown preclinical efficacy in mouse B16F10 melanoma cells, by reducing cell proliferation and promoting cellular death, while also reducing tumour vascularisation by inducing endothelial cells apoptosis ( 18 ). One possible mechanism of this antitumour activity is an increase in mitochondrial reactive oxygen species (ROS) production, which cannot be neutralised in cancer cells, leading to reduced cell viability. The presence of β3-AR in the mitochondrial membranes could be responsible for the alterations in mitochondrial activity in human melanoma cells ( 19 ). Similarly, SR59230A increased apoptosis in a cellular myeloid leukaemia model, demonstrating its ability to increase doxorubicin resistance reversion ( 20 ). These preliminary findings indicate the possibility of using SR59230A in oncological settings, possibly as an adjunctive treatment to antineoplastic drugs. The aim of the present exploratory research is to demonstrate whether the β-AR antagonist SR59230A could exert a synergistic effect in vitro when combined to standard chemotherapeutic drugs and target therapies in human tumour and endothelial cell lines. This could represent a solid basis for the future use of this drug in a combination setting in various cancer types and in different lines of treatment. Materials and methods Cell Cultures, Reagents, and Drugs Five human cell lines were selected to represent a range of tumor types and biological contexts relevant to β-adrenergic signaling and cancer therapy. These included U-87 MG (glioblastoma), A-2058 (BRAF-mutated melanoma), MDA-MB-231 (triple-negative breast cancer), 8505C (anaplastic thyroid carcinoma), and HUVEC (human umbilical vein endothelial cells). The human glioblastoma U-87 MG (HTB-14™), the melanoma B-raf V600E mutated A-2058 (ATCC-CRL-11147), the human breast cancer MDA-MB-231 (ATCC CRM-HTB-26), and the human umbilical vascular endothelial cells HUVECs (ATCC PCS-100-010 ™) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA); the human anaplastic thyroid cancer (ATC) cell line 8505C (ACC 219) was purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Germany).The U-87 MG cells were cultured in Eagle’s minimum essential medium (MEM; Sigma Aldrich srl, Milan, Italy), supplemented with 10% heat inactivated fetal bovine serum (FBS; Sigma Aldrich srl), 1% L-glutamine, and 100 U/mL penicillin, 100 mg/mL streptomycin at 37°C in 5% CO 2 , 95% humidity; the A-2058 cell line was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma Aldrich srl); MDA-MB-231 cell line was maintained in RPMI 1640 medium (Sigma Aldrich srl) supplemented with 20% heat inactivated FBS, 1% Sodium Pyruvate, 1% L-glutamine, and 100 U/mL penicillin, 100 mg/mL streptomycin. HUVECs were cultivated in MCDB131 culture medium (Gibco, Gibco, Gauthersburg, MD) supplemented with 10% heat-inactivated FBS, L-glutamine (2 mM), heparin (10 U/ml), EGF (10 ng/ml), and basic fibroblast growth factor (5 ng/ml), whereas the cell line 8505C was maintained in RPMI 1640 medium supplemented with 15% FBS and L-glutamine (2 mM). Recombinant human vascular endothelial growth factor (rhVEGF) and recombinant human epidermal growth factor (rhEGF) were from PeproTech EC Ltd. (London, United Kingdom). Type A gelatin from porcine skin, above mentioned supplements, and all other chemicals not listed in this section were obtained from Sigma Aldrich srl, Milan, Italy. Plastics for cell culture were supplied by Sarstedt (Nümbrecht, Germany). Temozolomide, lenvatinib, sorafenib, vemurafenib and paclitaxel were obtained from SelleckChem (DBA Italia, Milan, Italy), and they were dissolved in dimethyl sulfoxide (DMSO) as well as SR59230A that was purchased from Merck Life Science (Milan, Italy). All drugs were dissolved in a stock solution of 10 mM for in vitro studies. DMSO concentration in the control’s media was the same used to make-up the highest concentration of the tested drug in growth media for the same experiment. Antiproliferative Assay In vitro chemosensitivity was tested on U-87MG, A-2058, MDA-MB-231, HUVECs and 8505C cell lines. Cells (2 × 10 4 ) were plated in 24-well sterile plastic plates and allowed to be attached overnight. Cells were treated with SR59230A, temozolomide, lenvatinib, sorafenib, vemurafenib and paclitaxel (0.001–100 µM) for 72 h or with vehicle as controls. At the end of the experiment, cells were harvested with trypsin/EDTA and the viable ones counted with a haemocytometer as previously described ( 21 ). Cell viability was assessed by trypan blue dye exclusion. The data are presented as the percentage of the vehicle-treated cells. All experiments were repeated, independently, three times with at least three samples for each concentration. The concentration of drug that reduced cell proliferation by 50% (IC 50 ) vs. controls were calculated by non-linear regression fit of the mean values of the data obtained in triplicate experiments (at least nine wells for each concentration). In Vitro Assessment of Synergism Between SR59230A and Temozolomide, Lenvatinib, Sorafenib, Vemurafenib and Paclitaxel on Cancer Cells and Endothelial Cells The effects of the combination between SR59230A and the above-mentioned drugs were investigated on all tumour cells and on endothelial cells with the concomitant treatment schedule at a fixed molar concentration ratio of 1:1 (SORAFENIB + SR59230; TMZ + SR59230), 1:10 (LENVATINIB + SR59230; VEMURAFENIB + SR59230) and 1:10000 (PACLITAXEL + SR59230) with the Chou’s method ( 22 ). The possible type of interaction (synergistic, additive, or antagonistic) between drugs was calculated using the multiple drug-effect equation and quantified by the combination index (CI), where CI 1 mean synergism, additive effect, and antagonism, respectively. Using the standard isobologram for mutually exclusive effects, the CI value was calculated according to the formula: CI = [(D) 1 /(DX) 1 ] + [(D) 2 /(DX) 2 ], where (D) 1 and (D) 2 are the concentrations at which the two drugs in combination cause a specific percentage of inhibition of cell proliferation. The dose-reduction index (DRI) indicates the degree of dose reduction that is possible in a combination for a given degree of effects as compared with the concentration of each drug alone: (DRI) 1 =(DX) 1 /(D) 1 and (DRI) 2 =(DX) 2 /(D) 2 . The CI and DRI indexes were calculated with the CalcuSyn v.2.0 software (Biosoft, Cambridge, UK). The synergistic, additive, and antagonistic effects of the combined drugs were also validated and graphically summarized using the Loewe additivity model, with the Combenefit software (v.2.021), an interactive platform for the analysis and visualization of drug combinations ( 23 ). Statistical analysis The data (mean ± SD or SEM) were analysed using ANOVA, followed by the Student–Newman–Keuls test, with significance set at P < 0.05 . Analyses were performed using GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego, CA). Results Antiproliferative effect of SR59230A on different cell lines After 72 hours of SR59230A treatment alone, antiproliferative activity was observed among the different cell lines, with IC 50 values in the micromolar range, demonstrating a concentration-dependent pharmacological activity. As shown in Fig. 1 , HUVEC endothelial cells were the most sensitive to the treatment (IC 50 = 6.45 µM), followed by BRAF mutated 8505C thyroid carcinoma cells (IC 50 = 13.09 µM) and U-87 glioblastoma cells (IC 50 = 18.21 µM). Meanwhile, in BRAF mutated A-2058 melanoma cells (IC 50 = 26.27 µM) and MDA-MB-231 triple-negative breast cancer cells (IC 50 = 34.83 µM), the drug showed minor antiproliferative activity. In addition, we evaluated the antiproliferative activity of conventional chemotherapeutic agents and target therapies after 72 h of treatment, observing IC 50 values in the nanomolar/micromolar range. Lenvatinib showed potent activity in HUVEC cells (IC 50 = 235 nM), while sorafenib showed a higher IC 50 (1,909 nM) in the same cell line. Vemurafenib showed an IC 50 of 1,242 nM in A-2058 melanoma cells. In glioblastoma U-87 cells, temozolomide had an IC 50 of 5,520 nM. Notably, paclitaxel showed strong cytotoxic activity in both 8505C thyroid carcinoma cells (IC 50 = 0.1499 nM) and MDA-MB-231 breast carcinoma cells (IC 50 = 1.046 nM). Synergistic effect of SR59230A with chemotherapeutic drugs The simultaneous combination of SR59230A with chemotherapeutic agents or target therapies resulted in a synergistic effect across all tested conditions, with varying degrees of synergy depending on the cell line and drug combination (Fig. 2 ). The strongest synergistic interaction was observed in A-2058 melanoma cells treated with vemurafenib and SR59230A (1:10 ratio), where the combination exhibited the lowest CI value (Fig. 2 A) and the highest dose reduction index (DRI), indicating the most substantial dose reduction potential among all tested conditions, as reported in Table 1 . Table 1 Dose Reduction Index (DRI) values for the drugs combination at 30%, 50%, 70% and 90% level of inhibition of HUVEC, 8505C, A-2058, U-87 MG and MDA-MB-231 cell growth after 72 hours of simultaneous combination treatment of SR59230A plus Temozolomide (TMZ), Paclitaxel (PTX), Vemurafenib, Lenfanib and Sorafenib respectively. DRI > 1 values indicate synergism. The DRI represents the theoretical magnitude of concentration reduction allowed for each drug when given in synergistic combination to achieve the same effect as that obtained with the concentration of each single agent. DrugS combination DRI values 30% 50% 70% 90% SR59230A Lenvatinib SR59230A Lenvatinib SR59230A Lenvatinib SR59230A Lenvatinib HUVEC cell line Lenvatinib + SR59230A 72 h (1:10) 76.8670 159.4460 10.0620 23.8910 1.3170 3.5800 0.0520 0.1740 SR59230A Sorafenib SR59230A Sorafenib SR59230A Sorafenib SR59230A Sorafenib HUVEC cell line Sorafenib + SR59230 72 h (1:1) 709.6830 13.3520 363.4610 11.5970 186.1450 10.0730 64.0990 8.0480 SR59230A TMZ SR59230A TMZ SR59230A TMZ SR59230A TMZ U-87 MG cell line Temozolomide (TMZ) + SR59230A 72 h (1:1) 312.4120 267.5840 164.4440 172.8920 86.5580 111.7100 31.1360 55.7040 SR59230A Vemurafenib SR59230A Vemurafenib SR59230A Vemurafenib SR59230A Vemurafenib A-2058 cell line Vemurafenib + SR59230A 72 h (1:10) 1892.7940 43.391000 1173.5360 39.838000 727.5940 36.576000 339.7240 31.922000 SR59230A PTX SR59230A PTX SR59230A PTX SR59230A PTX 8505C cell line Paclitaxel (PTX) + SR59230A 72 h (1:10000) 288.2480 359.4220 50.1020 88.8520 8.7080 21.9650 0.5360 2.3700 SR59230A PTX SR59230A PTX SR59230A PTX SR59230A PTX MDA-MB-231 cell line Paclitaxel (PTX) + SR59230A 72 h (1:10000) 53.122 3.865 43.9350 15.2960 36.3380 60.5300 26.8520 541.7160 In U-87 glioblastoma cells, the combination of temozolomide and SR59230A (1:1 ratio) also showed a robust synergistic effect (CI < 1), though to a lesser extent than vemurafenib + SR59230A (Fig. 2 B). Similarly, in HUVEC endothelial cells, the combination of sorafenib and SR59230A (1:1 ratio) demonstrated strong synergy, while lenvatinib and SR59230A (1:10 ratio) exhibited synergy from low affected fractions (Fa) up to approximately 60–70% (Fig. 2 C). Paclitaxel combined with SR59230A (1:10,000 ratio) in 8505C thyroid carcinoma cells showed a synergistic effect from small Fa values up to approximately 90%. In MDA-MB-231 triple-negative breast cancer cells, the same combination (PTX + SR59230A, 1:10,000) exhibited synergy from 10% affected fractions up to 100% (Fig. 2 D). To further validate the nature of these interactions, the Loewe additivity model was applied, confirming the degree of synergy observed across all cell lines and supporting the results obtained with the Chou-Talalay method. As shown in Fig. 2 E and F, the combination of SR59230A + Sorafenib and SR59230A + Temozolomide is synergistic, as indicated by the blue regions in the graphical representation, particularly at low-to-moderate levels of cell proliferation inhibition. Additionally, a wide area of additivity effect, represented in green, further supports the robustness of the observed interactions. Discussion The present exploratory study aimed to evaluate the pharmacological interactions between the β3-adrenergic receptor antagonist SR59230A and a panel of chemotherapeutic or targeted agents, including antiangiogenic drugs (Lenvatinib, Sorafenib), cytotoxics (Temozolomide, Paclitaxel), and targeted therapies (Vemurafenib), across multiple human cancer cell lines and endothelial cells. Drug combinations were tested at fixed molar ratios, and their interaction was quantified using the Chou-Talalay method, which allowed the calculation of CI and DRI ( 22 ), and validated with the Loewe additivity model. Our goal was to determine whether the use of SR59230A could improve the antiproliferative effects of standard therapies across different tumor and endothelial in vitro models. The selection of cell lines was guided by their clinical relevance and the documented expression and functional involvement of β-adrenergic receptor/signaling in these cell types. U-87 MG glioblastoma cells represent a highly aggressive and treatment-resistant tumor models where β-ARs expression has been implicated in cell survival and drug resistance via inhibition of the mTOR/p70S6K pathway ( 24 – 27 ). MDA-MB-231 were selected as a representative model of triple-negative breast cancer, a particularly aggressive subtype with limited therapeutic options ( 28 ). In this context, β3-adrenergic receptors are expressed and have been implicated in promoting tumor progression, stem-like traits, immune evasion, and resistance to chemotherapy ( 29 , 30 ). A-2058 melanoma cells, harboring the BRAF V600E mutation, were chosen to investigate potential synergy with vemurafenib ( 31 ). β3-ARs are particularly expressed in both tumor and stromal compartments of melanoma, where they promote angiogenesis, immune escape, and a metabolic shift toward glycolysis. This is achieved through the induction of mitochondrial dormancy mediated by uncoupling proteins (UCPs), which impair oxidative phosphorylation and enhance adaptation to hypoxic and nutrient-deprived conditions( 19 , 32 , 33 ). 8505C anaplastic thyroid carcinoma cells were included as a model of rare but aggressive thyroid cancer, where β3-AR expression has also been reported and associated with increased proliferation, invasiveness and metabolic reprogramming ( 34 – 36 ). Finally, HUVEC endothelial cells were included to assess potential antiangiogenic effects of β3-ARs antagonism ( 37 ). In endothelial cells, β3-ARs regulate nitric oxide (NO) production via eNOS activation and participate in angiogenic signaling, thus representing a relevant target for combination with anti-VEGFR agents ( 37 , 38 ). Based on these premises, we selected five anticancer agents with diverse mechanisms of action: temozolomide and paclitaxel (standard cytotoxics), vemurafenib (a targeted inhibitor of the MAPK pathway), and lenvatinib and sorafenib (antiangiogenic multikinase inhibitors). These drugs are commonly used in the clinical management of the selected tumor types ( 39 – 44 ). From a therapeutic perspective, combining SR59230A with conventional anticancer agents is expected to enhance efficacy through their complementary mechanisms of action. By antagonizing β3-adrenergic receptor, SR59230A may disrupt stress-adaptive pathways involved in tumor progression and resistance, thereby enhancing the overall efficacy of anticancer therapies. As a matter of fact, our results demonstrated synergistic interactions between SR59230A and all tested agents, although the extent of synergy varied depending on cell line and selected drug. The strongest synergism was observed in A-2058 melanoma cells treated with SR59230A + vemurafenib, U-87 MG glioblastoma cells treated with SR59230A + temozolomide, MDA-MB-231 and 8505C cells treated with SR59230A + paclitaxel, and HUVEC cells treated with SR5923A + sorafenib or lenvatinib. These findings support the hypothesis that β3-AR antagonism can enhance the activity of both cytotoxic and targeted therapies through complementary mechanisms of action, further supporting the rationale for the preclinical development of such combination strategies. Results from the combination studies demonstrated a cell line-specific profile of synergism, reflecting the heterogeneity of tumor biology and the variable relevance of β3-AR depending on its expression and functional role. In A-2058 melanoma cells, which harbor the BRAF V600E mutation, the combination of SR59230A with vemurafenib (at a 1:10 molar ratio) yielded the lowest CI values and the highest DRI in this study. These findings are in line with previous evidence indicating that β3-AR signaling contributes to resistance, immune evasion, and mitochondrial dormancy in melanoma. SR59230A has been shown to overcome these features by reactivating oxidative metabolism, inducing mitochondrial ROS production, and restoring sensitivity to BRAF inhibitors ( 19 , 32 ). In MDA-MB-231 triple-negative breast cancer cells, the combination of SR59230A with paclitaxel at a 1:10,000 ratio resulted in strong synergistic effects across a wide range of affected fractions, including Fa > 0.8. These data are particularly relevant given the aggressive nature and chemoresistance of triple negative breast cancer. Previous work demonstrated that β3-AR expression and blockade enhance sensitivity to anthracyclines and taxanes in triple negative models by reducing metastatic potential and modulating the tumor microenvironment ( 25 , 30 ). In U-87 MG glioblastoma cells, SR59230A combined with temozolomide (1:1 ratio) demonstrated moderate but consistent synergy. Nevertheless, previous studies suggest that β3-AR inhibition may sensitize glioma cells to DNA-damaging agents by promoting mitochondrial ROS accumulation and impairing mTOR signalling, thereby enhancing the cytotoxic activity of DNA-alkylating agents like temozolomide ( 24 , 26 ). In 8505C anaplastic thyroid carcinoma cells, synergy between SR59230A and paclitaxel (1:10,000 ratio) was again observed, suggesting that β3-AR antagonism may sensitize tumors to mitotic inhibitors. Although data on β3-AR in thyroid cancer remain limited, previous studies have reported its overexpression and link to proliferative and invasive behaviour ( 34 , 35 ). Finally, in HUVEC endothelial cells, strong synergistic effects were observed for SR59230A with lenvatinib (1:10) and sorafenib (1:1). β3-ARs are known to regulate nitric oxide production via eNOS and modulate angiogenic signaling; thus, their inhibition can synergize with VEGFR inhibitors by disrupting vascular support mechanisms ( 45 ). DRI analysis further supported the synergy, with DRI values consistently > 1 in all tested combinations. The highest values (> 10 at Fa > 0.7) were obtained in A-2058 cells (vemurafenib), MDA-MB-231 and 8505C cells (paclitaxel), and HUVEC cells (lenvatinib), indicating a substantial reduction in drug doses required to achieve equivalent effects, an aspect of particular clinical relevance to minimize toxicity. Our findings are in agreement with several preclinical studies demonstrating the therapeutic relevance of β-AR blockade in oncology ( 6 – 12 ). In triple-negative breast cancer, our observation of strong synergy between SR59230 and paclitaxel in MDA-MB-231 align with Chang et al., who demonstrated that β-blockade enhances anthracycline activity and reduced metastatic dissemination ( 7 ). The use of SR59230A, by selectively targeting β3-AR, may offer additional benefits over non-selective β-blockers like propranolol by avoiding cardiovascular side effects and directly disrupting tumor-specific signaling pathways. In melanoma, β3-ARs promote immune evasion and metabolic dormancy; their inhibition with SR59230A reduces tumor vascularization and stem-like features, supporting our findings in A-2058 cells. ( 19 , 32 ). In glioblastoma and neuroblastoma, SR59230A inhibits the mTOR/p70S6K pathway, and increase ROS production, sensitizing cells to genotoxic agents like temozolomide ( 26 , 27 ). These mechanisms are reflected in our glioblastoma model, where the combination demonstrated synergistic cytotoxicity. The antiangiogenic synergy observed in HUVEC cells treated with SR59230A + lenvatinib or sorafenib reinforces the hypothesis that β3-AR blockade disrupts endothelial signaling, as also reported in melanoma by Dal Monte et al. ( 45 ). Mechanistically, SR59230A is known to exert multimodal effects by disrupting key survival pathways in cancer and endothelial cells. Besides mitochondrial dysfunction, ( 19 , 26 ) another key mechanism is the inhibition of Uncoupling Protein 2 (UCP2), a mitochondrial regulator that buffers ROS and contributes to drug resistance. By downregulating UCP2, SR59230A promotes oxidative damage and increases the susceptibility of tumor cells to cytotoxic agents ( 46 ). Recent works in Ewing sarcoma has further revealed that SR59230A may promote ferroptosis, a regulated form of lipid-peroxidation–dependent cell death, especially under metabolic stress or nutrient depletion. These effects are amplified in combination with metabolic inhibitors like buformin and is associated with upregulation of ferroptosis markers and regulators like GPX ( 47 , 48 ). In glioblastoma and neuroblastoma, SR59230A has also been shown to suppress the mTOR/p70S6K pathway, a central signaling axis for cell growth, metabolism, and survival. This mechanism likely contributes to the synergy observed in our glioblastoma model when combined with temozolomide ( 26 , 27 ). Despite the promising results, this exploratory study presents some limitations. All experiments were conducted in vitro, which does not fully capture the complexity of the tumor microenvironment, including immune and stromal interactions. Moreover, the hypothesized mechanisms of synergy, such as ROS induction, ferroptosis, and mTOR inhibition, were not directly investigated and also the presence of β3-AR on cell lines was based on a solid scientific literature. In conclusion, this study provides strong preclinical evidence that β3-AR antagonism via SR59230A synergistically enhances the antiproliferative effects of a broad range of anticancer and antiangiogenic agents in vitro. Synergistic interactions were consistently observed across a panel of tumor and endothelial cell lines, particularly in aggressive cancer types such as TNBC, melanoma, glioblastoma, and anaplastic thyroid carcinoma. SR59230A not only enhanced antiproliferative activity but also enabled substantial dose reduction, potentially minimizing toxicity while improving efficacy. Further in vivo validation and mechanistic studies are needed to optimize these combination strategies and assess clinical feasibility. Declarations Funding The authors declare that no funds, grants, or other support were received during the preparation of this manuscript Author information Arianna Bandini e Letizia Biso contributed equally to this work and shared co-first authorship. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions Conceptualization: Marco Scarselli, Guido Bocci; Methodology: Arianna Bandini, Letizia Biso, Maria Cristina Viaggi, Maria Carla Pardini, Paola Orlandi, Marco Carli, Marta Banchi; Formal analysis and investigation: Arianna Bandini, Letizia Biso; Writing - original draft preparation: Arianna Bandini, Letizia Biso; Writing - review and editing: Marco Scarselli, Guido Bocci, Luca Filippi; Supervision: Marco Scarselli, Guido Bocci. All authors read and approved the final manuscript. Corresponding author Correspondence to Guido Bocci and Marco Scarselli. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics declarations Ethics approval Not applicable Consent to participate Not applicable. Consent for publication Not applicable. Competing interest The authors declare no competing interests. References Cole SW, Sood AK. Molecular Pathways: Beta-Adrenergic Signaling in Cancer. Clinical Cancer Research. 2012 Mar 1;18(5):1201–6. Schultze-Florey CR, Martínez-Maza O, Magpantay L, Breen EC, Irwin MR, Gündel H, et al. When grief makes you sick: Bereavement induced systemic inflammation is a question of genotype. Brain Behav Immun. 2012 Oct;26(7):1066–71. Coelho M, Soares-Silva C, Brandão D, Marino F, Cosentino M, Ribeiro L. β-Adrenergic modulation of cancer cell proliferation: available evidence and clinical perspectives. J Cancer Res Clin Oncol. 2017 Feb 5;143(2):275–91. Wang W, Cao X. 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Dal Monte M, Casini G, Filippi L, Nicchia GP, Svelto M, Bagnoli P. Functional involvement of β3-adrenergic receptors in melanoma growth and vascularization. J Mol Med. 2013 Dec 2;91(12):1407–19. Calvani M, Cavallini L, Tondo A, Spinelli V, Ricci L, Pasha A, et al. β3‐Adrenoreceptors Control Mitochondrial Dormancy in Melanoma and Embryonic Stem Cells. Oxid Med Cell Longev. 2018 Jan 13;2018(1). Calvani M, Dabraio A, Bruno G, De Gregorio V, Coronnello M, Bogani C, et al. β3-Adrenoreceptor Blockade Reduces Hypoxic Myeloid Leukemic Cells Survival and Chemoresistance. Int J Mol Sci. 2020 Jun 12;21(12):4210. Bandini A, Banchi M, Orlandi P, Vaglini F, Alì G, Fontanini G, et al. Melanocortin-4 Receptor Antagonism Inhibits Colorectal and Anaplastic Thyroid Cancer In Vitro and In Vivo. J Clin Med. 2025 Feb 11;14(4):1165. Chou TC. Drug Combination Studies and Their Synergy Quantification Using the Chou-Talalay Method. Cancer Res. 2010 Jan 15;70(2):440–6. Di Veroli GY, Fornari C, Wang D, Mollard S, Bramhall JL, Richards FM, et al. Combenefit: an interactive platform for the analysis and visualization of drug combinations. Bioinformatics. 2016 Sep 15;32(18):2866–8. Cole SW, Sood AK. Molecular Pathways: Beta-Adrenergic Signaling in Cancer. Clinical Cancer Research. 2012 Mar 1;18(5):1201–6. Sloan EK, Priceman SJ, Cox BF, Yu S, Pimentel MA, Tangkanangnukul V, et al. The Sympathetic Nervous System Induces a Metastatic Switch in Primary Breast Cancer. Cancer Res. 2010 Sep 15;70(18):7042–52. Deng J, Jiang P, Yang T, Huang M, Qi W, Zhou T, et al. Targeting β3-adrenergic receptor signaling inhibits neuroblastoma cell growth via suppressing the mTOR pathway. Biochem Biophys Res Commun. 2019 Jun;514(1):295–300. Boaretto A, Bruno G, Nastasi N, Favre C, Calvani M. Abstract 6721: β3-adrenergic receptor correlates with tumor growth and progression in neuroblastoma. Cancer Res. 2023 Apr 4;83(7_Supplement):6721–6721. Slotkin TA, Zhang J, Dancel R, Garcia SJ, Willis C, Seidler FJ. β-adrenoceptor signaling and its control of cell replication in MDA-MB-231 human breast cancer cells. Breast Cancer Res Treat. 2000 Mar;60(2):153–66. Rains SL, Amaya CN, Bryan BA. Beta-adrenergic receptors are expressed across diverse cancers. Oncoscience. 2017 Aug 23;4(7–8):95–105. Mravec B, Horvathova L, Hunakova L. Neurobiology of Cancer: The Role of β-Adrenergic Receptor Signaling in Various Tumor Environments. Int J Mol Sci. 2020 Oct 26;21(21):7958. Orlandi P, Banchi M, Vaglini F, Carli M, Aringhieri S, Bandini A, et al. Melanocortin receptor 4 as a new target in melanoma therapy: Anticancer activity of the inhibitor ML00253764 alone and in association with B-raf inhibitor vemurafenib. Biochem Pharmacol. 2024 Jan;219:115952. Dal Monte M, Casini G, Filippi L, Nicchia GP, Svelto M, Bagnoli P. Functional involvement of β3-adrenergic receptors in melanoma growth and vascularization. J Mol Med. 2013 Dec 2;91(12):1407–19. Porcelli L, Garofoli M, Di Fonte R, Fucci L, Volpicella M, Strippoli S, et al. The β-adrenergic receptor antagonist propranolol offsets resistance mechanisms to chemotherapeutics in diverse sarcoma subtypes: a pilot study. Sci Rep. 2020 Jun 26;10(1):10465. Coelho M, Soares-Silva C, Brandão D, Marino F, Cosentino M, Ribeiro L. β-Adrenergic modulation of cancer cell proliferation: available evidence and clinical perspectives. J Cancer Res Clin Oncol. 2017 Feb 5;143(2):275–91. Chisholm KM, Chang KW, Truong MT, Kwok S, West RB, Heerema-Mckenney AE. β-Adrenergic receptor expression in vascular tumors. Modern Pathology. 2012 Nov;25(11):1446–51. Limaiem F, Kashyap S, Naing PT, Mathias PM, Giwa AO. Anaplastic Thyroid Cancer. 2024. Kou R, Michel T. Epinephrine Regulation of the Endothelial Nitric-oxide Synthase. Journal of Biological Chemistry. 2007 Nov;282(45):32719–29. Schena G, Caplan MJ. Everything You Always Wanted to Know about β3-AR * (* But Were Afraid to Ask). Cells. 2019 Apr 16;8(4):357. Strobel H, Baisch T, Fitzel R, Schilberg K, Siegelin MD, Karpel-Massler G, et al. Temozolomide and Other Alkylating Agents in Glioblastoma Therapy. Biomedicines. 2019 Sep 9;7(3):69. Onoda N, Sugino K, Higashiyama T, Kammori M, Toda K, Ito K ichi, et al. The Safety and Efficacy of Weekly Paclitaxel Administration for Anaplastic Thyroid Cancer Patients: A Nationwide Prospective Study. Thyroid. 2016 Sep;26(9):1293–9. Abu Samaan TM, Samec M, Liskova A, Kubatka P, Büsselberg D. Paclitaxel’s Mechanistic and Clinical Effects on Breast Cancer. Biomolecules. 2019 Nov 27;9(12):789. Cheng Y, Zhang G, Li G. Targeting MAPK pathway in melanoma therapy. Cancer and Metastasis Reviews. 2013 Dec 13;32(3–4):567–84. Une N, Takano-Kasuya M, Kitamura N, Ohta M, Inose T, Kato C, et al. The anti-angiogenic agent lenvatinib induces tumor vessel normalization and enhances radiosensitivity in hepatocellular tumors. Medical Oncology. 2021 Jun 21;38(6):60. Wilhelm S, Carter C, Lynch M, Lowinger T, Dumas J, Smith RA, et al. Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat Rev Drug Discov. 2006 Oct 1;5(10):835–44. Calvani M, Bruno G, Dabraio A, Subbiani A, Bianchini F, Fontani F, et al. β3-Adrenoreceptor Blockade Induces Stem Cells Differentiation in Melanoma Microenvironment. Int J Mol Sci. 2020 Feb 20;21(4):1420. Calvani M, Pelon F, Comito G, Taddei ML, Moretti S, Innocenti S, et al. Norepinephrine promotes tumor microenvironment reactivity through β3-adrenoreceptors during melanoma progression. Oncotarget. 2015 Mar 10;6(7):4615–32. Ascone M, Banella C, Amato R, Lotti M, Mattei G, Carrozzo F, et al. Abstract A048 SR59230A-induced ferroptosis sensitization of Ewing sarcoma cells via Beta-3 adrenergic receptor modulation: A novel therapeutic target. Cancer Res. 2024 Sep 5;84(17_Supplement):A048–A048. Banella C, Zocca L, Boaretto A, Mattei G, Mola M, Ballerini L, et al. Abstract A108: Metabolic oriented treatment: efficacy of sr59230a 𝜷3-adrenergic receptor antagonist, and sr plus buformin® in Ewing sarcoma. Mol Cancer Ther. 2023 Dec 1;22(12_Supplement):A108–A108. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 18 Oct, 2025 Read the published version in Investigational New Drugs → Version 1 posted Editorial decision: Revision requested 10 Aug, 2025 Reviews received at journal 10 Aug, 2025 Reviewers agreed at journal 20 Jul, 2025 Reviewers invited by journal 18 Jul, 2025 Editor assigned by journal 18 Jul, 2025 Submission checks completed at journal 18 Jul, 2025 First submitted to journal 17 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7147623","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":488522824,"identity":"4364f8a3-e274-443d-81b4-c956a22c75a8","order_by":0,"name":"Arianna Bandini°","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Arianna","middleName":"","lastName":"Bandini°","suffix":""},{"id":488522826,"identity":"fea6f481-97c6-4bcf-b449-284497123911","order_by":1,"name":"Letizia Biso°","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Letizia","middleName":"","lastName":"Biso°","suffix":""},{"id":488522828,"identity":"98e0c5bd-5428-49cb-97ea-a4c378e3f1c7","order_by":2,"name":"Maria Cristina Viaggi","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Cristina","lastName":"Viaggi","suffix":""},{"id":488522829,"identity":"f5e79bff-9dfc-4104-92c2-15114e4ecb96","order_by":3,"name":"Maria Carla Pardini","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"Carla","lastName":"Pardini","suffix":""},{"id":488522830,"identity":"664251c6-f5b9-4fc4-bcf8-2d89dc3e3757","order_by":4,"name":"Paola Orlandi","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Paola","middleName":"","lastName":"Orlandi","suffix":""},{"id":488522831,"identity":"218ed320-f1d9-49c1-a674-215635fa36d6","order_by":5,"name":"Marco Carli","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Marco","middleName":"","lastName":"Carli","suffix":""},{"id":488522835,"identity":"e5b305dc-a367-49f5-8260-45793e3f8477","order_by":6,"name":"Marta Banchi","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Marta","middleName":"","lastName":"Banchi","suffix":""},{"id":488522838,"identity":"1f09dd18-777a-4b1e-8180-f97b9602ac47","order_by":7,"name":"Luca Filippi","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Luca","middleName":"","lastName":"Filippi","suffix":""},{"id":488522839,"identity":"dc41dd1d-72a2-4980-96c4-dc536ce2a601","order_by":8,"name":"Guido Bocci","email":"","orcid":"","institution":"University of Pisa","correspondingAuthor":false,"prefix":"","firstName":"Guido","middleName":"","lastName":"Bocci","suffix":""},{"id":488522840,"identity":"8f92cb99-79d8-40d0-9f86-979ffc98d220","order_by":9,"name":"Marco Scarselli","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYNCDBwUScgwMjA0MCQwSRGpJMJAwhmkhUk+CAUNiA5SNU4tu+9mHHz4w1Mnzzz7+8EGCgUX6htvNbQ8eMFjU4dJidibdWHIGw2HDGedyjA2ADsvdcOdguwE+h5kdSGNj5mE4kMBwhodNAqzlRmKbBF4t55+BtNQlyJ9hf/4DqCXdgKCWG2BbmBMMzjCYgUIsgQgtz5glZxgcNtx4hscY5DDDmTcS20GekmzA6bA0xg8fKurk5c6wPwQz+G6kP3v4o6KOH5ctEGCAymXDECEI2EhUPwpGwSgYBcMcAACVM08L5A3/HAAAAABJRU5ErkJggg==","orcid":"","institution":"University of Pisa","correspondingAuthor":true,"prefix":"","firstName":"Marco","middleName":"","lastName":"Scarselli","suffix":""}],"badges":[],"createdAt":"2025-07-17 09:53:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7147623/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7147623/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10637-025-01587-8","type":"published","date":"2025-10-18T15:58:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":87275563,"identity":"ab4bab62-758b-4cf9-a674-8ede4d3e8078","added_by":"auto","created_at":"2025-07-22 09:01:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":65732,"visible":true,"origin":"","legend":"\u003cp\u003eDose–response curves of SR59230A in A-2058 (melanoma), MDA-MB-231 (triple-negative breast cancer), U-87 MG (glioblastoma), 8505C (anaplastic thyroid carcinoma), and HUVEC (endothelial cells). Cells were treated for 72 hours with increasing concentrations of SR59230A ranging from 0.01 µM to 100 µM. At the end of the treatment, viable cells were detached with trypsin/EDTA and manually counted using a haemocytometer. IC\u003csub\u003e50\u003c/sub\u003e values were calculated using nonlinear regression analysis using GraphPad Prism v.7.0 (GraphPad Software, San Diego, CA, USA). Data are expressed as mean ± SEM from at least three independent experiments performed in triplicate.\u003c/p\u003e","description":"","filename":"Bandinietal.Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7147623/v1/842c149b402db6637c38cc2a.jpg"},{"id":87277820,"identity":"d3acf079-edb3-443c-a514-c52cf6db5269","added_by":"auto","created_at":"2025-07-22 09:17:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":176392,"visible":true,"origin":"","legend":"\u003cp\u003eCombination index (CI)-fraction affected (Fa) plot of 72 h-concomitant combination of SR59230A + Vemurafenib (\u003cstrong\u003eA\u003c/strong\u003e) in A-2058 cells, SR59230A + Temozolomide (TMZ) in U-87 MG cells (\u003cstrong\u003eB\u003c/strong\u003e), SR59230 + Lenvatinib (\u003cstrong\u003eC\u003c/strong\u003e; red triangles) and SR59230+ Sorafenib (\u003cstrong\u003eC\u003c/strong\u003e; blue triangles) in HUVEC cells, SR59230 + Paclitaxel (PTX) in MDA-MB-231(\u003cstrong\u003eD\u003c/strong\u003e; red squares) and 8505C cells (\u003cstrong\u003eD\u003c/strong\u003e; blue rounds). CI\u0026lt;1, CI=1 (dotted line) and CI\u0026gt;1 indicate synergism, additive effect and antagonism, respectively. Three-dimensional landscape of the dose matrix of combination responses on the Loewe model, of 72 h SR59230A+ Sorafenib (\u003cstrong\u003eE\u003c/strong\u003e) and SR59230A + Temozolomide (TMZ) (\u003cstrong\u003eF\u003c/strong\u003e) concomitant combination cells. Blue and red colour reflect evidence of synergy and antagonism, respectively. The model supported synergy of the combinations in reducing cells viability\u003c/p\u003e","description":"","filename":"Bandinietal.Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7147623/v1/ac026fe0de9c08d12b1623dc.jpg"},{"id":93956040,"identity":"6d855d53-abe0-4688-b0bb-7a8ac33699ac","added_by":"auto","created_at":"2025-10-20 16:09:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1465848,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7147623/v1/f49b68b3-91d8-4178-87e4-8956d1f42523.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic combination of the beta-3 antagonist SR59230A with common chemotherapeutic drugs and target therapies in cancer and endothelial cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn recent years, in cancer biology the involvement of the catecholaminergic system has gained increasing attention, with a particular interest in β-adrenergic receptors (β-ARs) (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). These receptors mediate the effects of catecholamines (i.e., norepinephrine and epinephrine), and β-adrenergic signalling may be implicated in different aspects of cancer progression, including tumour growth, angiogenesis, and metastasis (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). These hypotheses suggest that pharmacological agents targeting β-ARs have a therapeutic potential in oncology, particularly when used in conjunction with traditional chemotherapy, target therapies or immunotherapy (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Commonly used β-adrenergic antagonists, such as β-blockers, which are widely used in hypertension treatment, have shown promising results as an additional treatment in various cancer types, including breast, prostate, brain, skin, and ovarian tumours, although the majority of clinical evidence comes from retrospective studies and still needs to be confirmed in more rigorous trials (\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Beyond frequently used β-blockers that are already on the market, other β-ARs compounds are under development and are currently being tested in preclinical settings.\u003c/p\u003e\u003cp\u003eIn particular, β3-AR has attracted attention due to its more restricted expression pattern compared to other β-AR subtypes, and because of its apparent involvement in specific conditions such as embryonic/fetal development and tumor growth. Both scenarios require cellular proliferation under highly hypoxic and theoretically inhospitable environments. In these contexts, β3-AR promotes cellular proliferation, hypoxia-induced angiogenesis, chemoresistance, a glycolytic metabolic shift (Warburg effect), and the induction of stemness (\u003cspan additionalcitationids=\"CR14\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the β3-AR ligands, SR59230A is indicated as a β3-AR antagonist, although affinity for all β-ARs has been demonstrated depending from the animal species taken into consideration (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). In addition, in some cellular systems it can act as a partial agonist as well (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). SR59230A has shown preclinical efficacy in mouse B16F10 melanoma cells, by reducing cell proliferation and promoting cellular death, while also reducing tumour vascularisation by inducing endothelial cells apoptosis (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). One possible mechanism of this antitumour activity is an increase in mitochondrial reactive oxygen species (ROS) production, which cannot be neutralised in cancer cells, leading to reduced cell viability. The presence of β3-AR in the mitochondrial membranes could be responsible for the alterations in mitochondrial activity in human melanoma cells (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Similarly, SR59230A increased apoptosis in a cellular myeloid leukaemia model, demonstrating its ability to increase doxorubicin resistance reversion (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). These preliminary findings indicate the possibility of using SR59230A in oncological settings, possibly as an adjunctive treatment to antineoplastic drugs.\u003c/p\u003e\u003cp\u003eThe aim of the present exploratory research is to demonstrate whether the β-AR antagonist SR59230A could exert a synergistic effect in vitro when combined to standard chemotherapeutic drugs and target therapies in human tumour and endothelial cell lines. This could represent a solid basis for the future use of this drug in a combination setting in various cancer types and in different lines of treatment.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003eCell Cultures, Reagents, and Drugs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFive human cell lines were selected to represent a range of tumor types and biological contexts relevant to β-adrenergic signaling and cancer therapy. These included U-87 MG (glioblastoma), A-2058 (BRAF-mutated melanoma), MDA-MB-231 (triple-negative breast cancer), 8505C (anaplastic thyroid carcinoma), and HUVEC (human umbilical vein endothelial cells). The human glioblastoma U-87 MG (HTB-14\u0026trade;), the melanoma B-raf V600E mutated A-2058 (ATCC-CRL-11147), the human breast cancer MDA-MB-231 (ATCC CRM-HTB-26), and the human umbilical vascular endothelial cells HUVECs (ATCC PCS-100-010 \u0026trade;) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA); the human anaplastic thyroid cancer (ATC) cell line 8505C (ACC 219) was purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Germany).The U-87 MG cells were cultured in Eagle\u0026rsquo;s minimum essential medium (MEM; Sigma Aldrich srl, Milan, Italy), supplemented with 10% heat inactivated fetal bovine serum (FBS; Sigma Aldrich srl), 1% L-glutamine, and 100 U/mL penicillin, 100 mg/mL streptomycin at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e, 95% humidity; the A-2058 cell line was cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM; Sigma Aldrich srl); MDA-MB-231 cell line was maintained in RPMI 1640 medium (Sigma Aldrich srl) supplemented with 20% heat inactivated FBS, 1% Sodium Pyruvate, 1% L-glutamine, and 100 U/mL penicillin, 100 mg/mL streptomycin. HUVECs were cultivated in MCDB131 culture medium (Gibco, Gibco, Gauthersburg, MD) supplemented with 10% heat-inactivated FBS, L-glutamine (2 mM), heparin (10 U/ml), EGF (10 ng/ml), and basic fibroblast growth factor (5 ng/ml), whereas the cell line 8505C was maintained in RPMI 1640 medium supplemented with 15% FBS and L-glutamine (2 mM). Recombinant human vascular endothelial growth factor (rhVEGF) and recombinant human epidermal growth factor (rhEGF) were from PeproTech EC Ltd. (London, United Kingdom). Type A gelatin from porcine skin, above mentioned supplements, and all other chemicals not listed in this section were obtained from Sigma Aldrich srl, Milan, Italy. Plastics for cell culture were supplied by Sarstedt (N\u0026uuml;mbrecht, Germany). Temozolomide, lenvatinib, sorafenib, vemurafenib and paclitaxel were obtained from SelleckChem (DBA Italia, Milan, Italy), and they were dissolved in dimethyl sulfoxide (DMSO) as well as SR59230A that was purchased from Merck Life Science (Milan, Italy). All drugs were dissolved in a stock solution of 10 mM for \u003cem\u003ein vitro\u003c/em\u003e studies. DMSO concentration in the control\u0026rsquo;s media was the same used to make-up the highest concentration of the tested drug in growth media for the same experiment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntiproliferative Assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn vitro chemosensitivity was tested on U-87MG, A-2058, MDA-MB-231, HUVECs and 8505C cell lines. Cells (2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) were plated in 24-well sterile plastic plates and allowed to be attached overnight. Cells were treated with SR59230A, temozolomide, lenvatinib, sorafenib, vemurafenib and paclitaxel (0.001\u0026ndash;100 \u0026micro;M) for 72 h or with vehicle as controls. At the end of the experiment, cells were harvested with trypsin/EDTA and the viable ones counted with a haemocytometer as previously described (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Cell viability was assessed by trypan blue dye exclusion. The data are presented as the percentage of the vehicle-treated cells. All experiments were repeated, independently, three times with at least three samples for each concentration. The concentration of drug that reduced cell proliferation by 50% (IC\u003csub\u003e50\u003c/sub\u003e) vs. controls were calculated by non-linear regression fit of the mean values of the data obtained in triplicate experiments (at least nine wells for each concentration).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn Vitro Assessment of Synergism Between SR59230A and Temozolomide, Lenvatinib, Sorafenib, Vemurafenib and Paclitaxel on Cancer Cells and Endothelial Cells\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe effects of the combination between SR59230A and the above-mentioned drugs were investigated on all tumour cells and on endothelial cells with the concomitant treatment schedule at a fixed molar concentration ratio of 1:1 (SORAFENIB\u0026thinsp;+\u0026thinsp;SR59230; TMZ\u0026thinsp;+\u0026thinsp;SR59230), 1:10 (LENVATINIB\u0026thinsp;+\u0026thinsp;SR59230; VEMURAFENIB\u0026thinsp;+\u0026thinsp;SR59230) and 1:10000 (PACLITAXEL\u0026thinsp;+\u0026thinsp;SR59230) with the Chou\u0026rsquo;s method (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). The possible type of interaction (synergistic, additive, or antagonistic) between drugs was calculated using the multiple drug-effect equation and quantified by the combination index (CI), where CI\u0026thinsp;\u0026lt;\u0026thinsp;1, CI\u0026thinsp;=\u0026thinsp;1, and CI\u0026thinsp;\u0026gt;\u0026thinsp;1 mean synergism, additive effect, and antagonism, respectively.\u003c/p\u003e\u003cp\u003eUsing the standard isobologram for mutually exclusive effects, the CI value was calculated according to the formula: CI = [(D)\u003csub\u003e1\u003c/sub\u003e/(DX)\u003csub\u003e1\u003c/sub\u003e] + [(D)\u003csub\u003e2\u003c/sub\u003e/(DX)\u003csub\u003e2\u003c/sub\u003e], where (D)\u003csub\u003e1\u003c/sub\u003e and (D)\u003csub\u003e2\u003c/sub\u003e are the concentrations at which the two drugs in combination cause a specific percentage of inhibition of cell proliferation.\u003c/p\u003e\u003cp\u003eThe dose-reduction index (DRI) indicates the degree of dose reduction that is possible in a combination for a given degree of effects as compared with the concentration of each drug alone: (DRI)\u003csub\u003e1\u003c/sub\u003e=(DX)\u003csub\u003e1\u003c/sub\u003e/(D)\u003csub\u003e1\u003c/sub\u003e and (DRI)\u003csub\u003e2\u003c/sub\u003e=(DX)\u003csub\u003e2\u003c/sub\u003e/(D)\u003csub\u003e2\u003c/sub\u003e. The CI and DRI indexes were calculated with the CalcuSyn v.2.0 software (Biosoft, Cambridge, UK). The synergistic, additive, and antagonistic effects of the combined drugs were also validated and graphically summarized using the Loewe additivity model, with the Combenefit software (v.2.021), an interactive platform for the analysis and visualization of drug combinations (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe data (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD or SEM) were analysed using ANOVA, followed by the Student\u0026ndash;Newman\u0026ndash;Keuls test, with significance set at \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e. Analyses were performed using GraphPad Prism 7.0 (GraphPad Software, Inc., San Diego, CA).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eAntiproliferative effect of SR59230A on different cell lines\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAfter 72 hours of SR59230A treatment alone, antiproliferative activity was observed among the different cell lines, with IC\u003csub\u003e50\u003c/sub\u003e values in the micromolar range, demonstrating a concentration-dependent pharmacological activity. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, HUVEC endothelial cells were the most sensitive to the treatment (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.45 \u0026micro;M), followed by \u003cem\u003eBRAF\u003c/em\u003e mutated 8505C thyroid carcinoma cells (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;13.09 \u0026micro;M) and U-87 glioblastoma cells (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;18.21 \u0026micro;M). Meanwhile, in \u003cem\u003eBRAF\u003c/em\u003e mutated A-2058 melanoma cells (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;26.27 \u0026micro;M) and MDA-MB-231 triple-negative breast cancer cells (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;34.83 \u0026micro;M), the drug showed minor antiproliferative activity.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn addition, we evaluated the antiproliferative activity of conventional chemotherapeutic agents and target therapies after 72 h of treatment, observing IC\u003csub\u003e50\u003c/sub\u003e values in the nanomolar/micromolar range. Lenvatinib showed potent activity in HUVEC cells (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;235 nM), while sorafenib showed a higher IC\u003csub\u003e50\u003c/sub\u003e (1,909 nM) in the same cell line. Vemurafenib showed an IC\u003csub\u003e50\u003c/sub\u003e of 1,242 nM in A-2058 melanoma cells. In glioblastoma U-87 cells, temozolomide had an IC\u003csub\u003e50\u003c/sub\u003e of 5,520 nM. Notably, paclitaxel showed strong cytotoxic activity in both 8505C thyroid carcinoma cells (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.1499 nM) and MDA-MB-231 breast carcinoma cells (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.046 nM).\u003c/p\u003e\u003cp\u003e\u003cb\u003eSynergistic effect of SR59230A with chemotherapeutic drugs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe simultaneous combination of SR59230A with chemotherapeutic agents or target therapies resulted in a synergistic effect across all tested conditions, with varying degrees of synergy depending on the cell line and drug combination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The strongest synergistic interaction was observed in A-2058 melanoma cells treated with vemurafenib and SR59230A (1:10 ratio), where the combination exhibited the lowest CI value (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and the highest dose reduction index (DRI), indicating the most substantial dose reduction potential among all tested conditions, as reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDose Reduction Index (DRI) values for the drugs combination at 30%, 50%, 70% and 90% level of inhibition of HUVEC, 8505C, A-2058, U-87 MG and MDA-MB-231 cell growth after 72 hours of simultaneous combination treatment of SR59230A plus Temozolomide (TMZ), Paclitaxel (PTX), Vemurafenib, Lenfanib and Sorafenib respectively. DRI\u0026thinsp;\u0026gt;\u0026thinsp;1 values indicate synergism. The DRI represents the theoretical magnitude of concentration reduction allowed for each drug when given in synergistic combination to achieve the same effect as that obtained with the concentration of each single agent.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eDrugS combination\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e\u003cp\u003eDRI values\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e30%\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e50%\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e70%\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e90%\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSR59230A\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eLenvatinib\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSR59230A\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLenvatinib\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eSR59230A\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eLenvatinib\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eSR59230A\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eLenvatinib\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHUVEC cell line\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eLenvatinib\u0026thinsp;+\u0026thinsp;SR59230A 72 h (1:10)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e76.8670\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e159.4460\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e10.0620\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e23.8910\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e1.3170\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e3.5800\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e0.0520\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e0.1740\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eSorafenib\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eSorafenib\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eSorafenib\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eSorafenib\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eHUVEC cell line\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eSorafenib\u0026thinsp;+\u0026thinsp;SR59230\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e72 h (1:1)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e709.6830\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e13.3520\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e363.4610\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e11.5970\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e186.1450\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e10.0730\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e64.0990\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e8.0480\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eTMZ\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eTMZ\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eTMZ\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eTMZ\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eU-87 MG cell line\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eTemozolomide (TMZ)\u0026thinsp;+\u0026thinsp;SR59230A 72 h (1:1)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e312.4120\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e267.5840\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e164.4440\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e172.8920\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e86.5580\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e111.7100\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e31.1360\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e55.7040\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003eVemurafenib\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003eVemurafenib\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003eVemurafenib\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003eVemurafenib\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eA-2058 cell line\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eVemurafenib\u0026thinsp;+\u0026thinsp;SR59230A 72 h (1:10)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e1892.7940\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e43.391000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e1173.5360\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e39.838000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e727.5940\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e36.576000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e339.7240\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e31.922000\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003ePTX\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003ePTX\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePTX\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003ePTX\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003e8505C cell line\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePaclitaxel (PTX)\u0026thinsp;+\u0026thinsp;SR59230A 72 h (1:10000)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e288.2480\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e359.4220\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e50.1020\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e88.8520\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e8.7080\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e21.9650\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e0.5360\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e2.3700\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003ePTX\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003ePTX\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003ePTX\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003eSR59230A\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003ePTX\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eMDA-MB-231 cell line\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePaclitaxel (PTX)\u0026thinsp;+\u0026thinsp;SR59230A 72 h (1:10000)\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cb\u003e53.122\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cb\u003e3.865\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cb\u003e43.9350\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e15.2960\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cb\u003e36.3380\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cb\u003e60.5300\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cb\u003e26.8520\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cb\u003e541.7160\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIn U-87 glioblastoma cells, the combination of temozolomide and SR59230A (1:1 ratio) also showed a robust synergistic effect (CI\u0026thinsp;\u0026lt;\u0026thinsp;1), though to a lesser extent than vemurafenib\u0026thinsp;+\u0026thinsp;SR59230A (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Similarly, in HUVEC endothelial cells, the combination of sorafenib and SR59230A (1:1 ratio) demonstrated strong synergy, while lenvatinib and SR59230A (1:10 ratio) exhibited synergy from low affected fractions (Fa) up to approximately 60\u0026ndash;70% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Paclitaxel combined with SR59230A (1:10,000 ratio) in 8505C thyroid carcinoma cells showed a synergistic effect from small Fa values up to approximately 90%. In MDA-MB-231 triple-negative breast cancer cells, the same combination (PTX\u0026thinsp;+\u0026thinsp;SR59230A, 1:10,000) exhibited synergy from 10% affected fractions up to 100% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eTo further validate the nature of these interactions, the Loewe additivity model was applied, confirming the degree of synergy observed across all cell lines and supporting the results obtained with the Chou-Talalay method. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE and F, the combination of SR59230A\u0026thinsp;+\u0026thinsp;Sorafenib and SR59230A\u0026thinsp;+\u0026thinsp;Temozolomide is synergistic, as indicated by the blue regions in the graphical representation, particularly at low-to-moderate levels of cell proliferation inhibition. Additionally, a wide area of additivity effect, represented in green, further supports the robustness of the observed interactions.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present exploratory study aimed to evaluate the pharmacological interactions between the β3-adrenergic receptor antagonist SR59230A and a panel of chemotherapeutic or targeted agents, including antiangiogenic drugs (Lenvatinib, Sorafenib), cytotoxics (Temozolomide, Paclitaxel), and targeted therapies (Vemurafenib), across multiple human cancer cell lines and endothelial cells. Drug combinations were tested at fixed molar ratios, and their interaction was quantified using the Chou-Talalay method, which allowed the calculation of CI and DRI (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e), and validated with the Loewe additivity model. Our goal was to determine whether the use of SR59230A could improve the antiproliferative effects of standard therapies across different tumor and endothelial in vitro models.\u003c/p\u003e\u003cp\u003eThe selection of cell lines was guided by their clinical relevance and the documented expression and functional involvement of β-adrenergic receptor/signaling in these cell types. U-87 MG glioblastoma cells represent a highly aggressive and treatment-resistant tumor models where β-ARs expression has been implicated in cell survival and drug resistance via inhibition of the mTOR/p70S6K pathway (\u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). MDA-MB-231 were selected as a representative model of triple-negative breast cancer, a particularly aggressive subtype with limited therapeutic options (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In this context, β3-adrenergic receptors are expressed and have been implicated in promoting tumor progression, stem-like traits, immune evasion, and resistance to chemotherapy (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). A-2058 melanoma cells, harboring the \u003cem\u003eBRAF V600E\u003c/em\u003e mutation, were chosen to investigate potential synergy with vemurafenib (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). β3-ARs are particularly expressed in both tumor and stromal compartments of melanoma, where they promote angiogenesis, immune escape, and a metabolic shift toward glycolysis. This is achieved through the induction of mitochondrial dormancy mediated by uncoupling proteins (UCPs), which impair oxidative phosphorylation and enhance adaptation to hypoxic and nutrient-deprived conditions(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). 8505C anaplastic thyroid carcinoma cells were included as a model of rare but aggressive thyroid cancer, where β3-AR expression has also been reported and associated with increased proliferation, invasiveness and metabolic reprogramming (\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Finally, HUVEC endothelial cells were included to assess potential antiangiogenic effects of β3-ARs antagonism (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In endothelial cells, β3-ARs regulate nitric oxide (NO) production via eNOS activation and participate in angiogenic signaling, thus representing a relevant target for combination with anti-VEGFR agents (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBased on these premises, we selected five anticancer agents with diverse mechanisms of action: temozolomide and paclitaxel (standard cytotoxics), vemurafenib (a targeted inhibitor of the MAPK pathway), and lenvatinib and sorafenib (antiangiogenic multikinase inhibitors). These drugs are commonly used in the clinical management of the selected tumor types (\u003cspan additionalcitationids=\"CR40 CR41 CR42 CR43\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). From a therapeutic perspective, combining SR59230A with conventional anticancer agents is expected to enhance efficacy through their complementary mechanisms of action. By antagonizing β3-adrenergic receptor, SR59230A may disrupt stress-adaptive pathways involved in tumor progression and resistance, thereby enhancing the overall efficacy of anticancer therapies.\u003c/p\u003e\u003cp\u003eAs a matter of fact, our results demonstrated synergistic interactions between SR59230A and all tested agents, although the extent of synergy varied depending on cell line and selected drug. The strongest synergism was observed in A-2058 melanoma cells treated with SR59230A\u0026thinsp;+\u0026thinsp;vemurafenib, U-87 MG glioblastoma cells treated with SR59230A\u0026thinsp;+\u0026thinsp;temozolomide, MDA-MB-231 and 8505C cells treated with SR59230A\u0026thinsp;+\u0026thinsp;paclitaxel, and HUVEC cells treated with SR5923A\u0026thinsp;+\u0026thinsp;sorafenib or lenvatinib. These findings support the hypothesis that β3-AR antagonism can enhance the activity of both cytotoxic and targeted therapies through complementary mechanisms of action, further supporting the rationale for the preclinical development of such combination strategies. Results from the combination studies demonstrated a cell line-specific profile of synergism, reflecting the heterogeneity of tumor biology and the variable relevance of β3-AR depending on its expression and functional role.\u003c/p\u003e\u003cp\u003eIn A-2058 melanoma cells, which harbor the \u003cem\u003eBRAF V600E\u003c/em\u003e mutation, the combination of SR59230A with vemurafenib (at a 1:10 molar ratio) yielded the lowest CI values and the highest DRI in this study. These findings are in line with previous evidence indicating that β3-AR signaling contributes to resistance, immune evasion, and mitochondrial dormancy in melanoma. SR59230A has been shown to overcome these features by reactivating oxidative metabolism, inducing mitochondrial ROS production, and restoring sensitivity to BRAF inhibitors (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In MDA-MB-231 triple-negative breast cancer cells, the combination of SR59230A with paclitaxel at a 1:10,000 ratio resulted in strong synergistic effects across a wide range of affected fractions, including Fa\u0026thinsp;\u0026gt;\u0026thinsp;0.8. These data are particularly relevant given the aggressive nature and chemoresistance of triple negative breast cancer. Previous work demonstrated that β3-AR expression and blockade enhance sensitivity to anthracyclines and taxanes in triple negative models by reducing metastatic potential and modulating the tumor microenvironment (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). In U-87 MG glioblastoma cells, SR59230A combined with temozolomide (1:1 ratio) demonstrated moderate but consistent synergy. Nevertheless, previous studies suggest that β3-AR inhibition may sensitize glioma cells to DNA-damaging agents by promoting mitochondrial ROS accumulation and impairing mTOR signalling, thereby enhancing the cytotoxic activity of DNA-alkylating agents like temozolomide (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). In 8505C anaplastic thyroid carcinoma cells, synergy between SR59230A and paclitaxel (1:10,000 ratio) was again observed, suggesting that β3-AR antagonism may sensitize tumors to mitotic inhibitors. Although data on β3-AR in thyroid cancer remain limited, previous studies have reported its overexpression and link to proliferative and invasive behaviour (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFinally, in HUVEC endothelial cells, strong synergistic effects were observed for SR59230A with lenvatinib (1:10) and sorafenib (1:1). β3-ARs are known to regulate nitric oxide production via eNOS and modulate angiogenic signaling; thus, their inhibition can synergize with VEGFR inhibitors by disrupting vascular support mechanisms (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). DRI analysis further supported the synergy, with DRI values consistently\u0026thinsp;\u0026gt;\u0026thinsp;1 in all tested combinations. The highest values (\u0026gt;\u0026thinsp;10 at Fa\u0026thinsp;\u0026gt;\u0026thinsp;0.7) were obtained in A-2058 cells (vemurafenib), MDA-MB-231 and 8505C cells (paclitaxel), and HUVEC cells (lenvatinib), indicating a substantial reduction in drug doses required to achieve equivalent effects, an aspect of particular clinical relevance to minimize toxicity.\u003c/p\u003e\u003cp\u003eOur findings are in agreement with several preclinical studies demonstrating the therapeutic relevance of β-AR blockade in oncology (\u003cspan additionalcitationids=\"CR7 CR8 CR9 CR10 CR11\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In triple-negative breast cancer, our observation of strong synergy between SR59230 and paclitaxel in MDA-MB-231 align with Chang et al., who demonstrated that β-blockade enhances anthracycline activity and reduced metastatic dissemination (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The use of SR59230A, by selectively targeting β3-AR, may offer additional benefits over non-selective β-blockers like propranolol by avoiding cardiovascular side effects and directly disrupting tumor-specific signaling pathways. In melanoma, β3-ARs promote immune evasion and metabolic dormancy; their inhibition with SR59230A reduces tumor vascularization and stem-like features, supporting our findings in A-2058 cells. (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In glioblastoma and neuroblastoma, SR59230A inhibits the mTOR/p70S6K pathway, and increase ROS production, sensitizing cells to genotoxic agents like temozolomide (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). These mechanisms are reflected in our glioblastoma model, where the combination demonstrated synergistic cytotoxicity. The antiangiogenic synergy observed in HUVEC cells treated with SR59230A\u0026thinsp;+\u0026thinsp;lenvatinib or sorafenib reinforces the hypothesis that β3-AR blockade disrupts endothelial signaling, as also reported in melanoma by Dal Monte et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMechanistically, SR59230A is known to exert multimodal effects by disrupting key survival pathways in cancer and endothelial cells. Besides mitochondrial dysfunction, (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) another key mechanism is the inhibition of Uncoupling Protein 2 (UCP2), a mitochondrial regulator that buffers ROS and contributes to drug resistance. By downregulating UCP2, SR59230A promotes oxidative damage and increases the susceptibility of tumor cells to cytotoxic agents (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Recent works in Ewing sarcoma has further revealed that SR59230A may promote ferroptosis, a regulated form of lipid-peroxidation\u0026ndash;dependent cell death, especially under metabolic stress or nutrient depletion. These effects are amplified in combination with metabolic inhibitors like buformin and is associated with upregulation of ferroptosis markers and regulators like GPX (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). In glioblastoma and neuroblastoma, SR59230A has also been shown to suppress the mTOR/p70S6K pathway, a central signaling axis for cell growth, metabolism, and survival. This mechanism likely contributes to the synergy observed in our glioblastoma model when combined with temozolomide (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Despite the promising results, this exploratory study presents some limitations. All experiments were conducted in vitro, which does not fully capture the complexity of the tumor microenvironment, including immune and stromal interactions. Moreover, the hypothesized mechanisms of synergy, such as ROS induction, ferroptosis, and mTOR inhibition, were not directly investigated and also the presence of β3-AR on cell lines was based on a solid scientific literature.\u003c/p\u003e\u003cp\u003eIn conclusion, this study provides strong preclinical evidence that β3-AR antagonism \u003cem\u003evia\u003c/em\u003e SR59230A synergistically enhances the antiproliferative effects of a broad range of anticancer and antiangiogenic agents in vitro. Synergistic interactions were consistently observed across a panel of tumor and endothelial cell lines, particularly in aggressive cancer types such as TNBC, melanoma, glioblastoma, and anaplastic thyroid carcinoma. SR59230A not only enhanced antiproliferative activity but also enabled substantial dose reduction, potentially minimizing toxicity while improving efficacy. Further in vivo validation and mechanistic studies are needed to optimize these combination strategies and assess clinical feasibility.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eArianna Bandini e Letizia Biso\u0026nbsp;contributed equally to this work and shared co-first authorship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Marco Scarselli, Guido Bocci; Methodology:\u0026nbsp;Arianna Bandini, Letizia Biso, Maria Cristina Viaggi, Maria Carla Pardini, Paola Orlandi, Marco Carli, Marta Banchi; Formal analysis and investigation: Arianna Bandini, Letizia Biso; Writing - original draft preparation: Arianna Bandini, Letizia Biso; Writing - review and editing: Marco Scarselli, Guido Bocci, Luca Filippi; Supervision: Marco Scarselli, Guido Bocci.\u003cbr\u003eAll authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Guido Bocci and Marco Scarselli.\u0026nbsp;\u003c/p\u003e\n\u003ch4\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/h4\u003e\n\u003cp\u003e\u003cem\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCole SW, Sood AK. 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Medical Oncology. 2021 Jun 21;38(6):60. \u003c/li\u003e\n\u003cli\u003eWilhelm S, Carter C, Lynch M, Lowinger T, Dumas J, Smith RA, et al. Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat Rev Drug Discov. 2006 Oct 1;5(10):835\u0026ndash;44. \u003c/li\u003e\n\u003cli\u003eCalvani M, Bruno G, Dabraio A, Subbiani A, Bianchini F, Fontani F, et al. \u0026beta;3-Adrenoreceptor Blockade Induces Stem Cells Differentiation in Melanoma Microenvironment. Int J Mol Sci. 2020 Feb 20;21(4):1420. \u003c/li\u003e\n\u003cli\u003eCalvani M, Pelon F, Comito G, Taddei ML, Moretti S, Innocenti S, et al. Norepinephrine promotes tumor microenvironment reactivity through \u0026beta;3-adrenoreceptors during melanoma progression. Oncotarget. 2015 Mar 10;6(7):4615\u0026ndash;32. \u003c/li\u003e\n\u003cli\u003eAscone M, Banella C, Amato R, Lotti M, Mattei G, Carrozzo F, et al. Abstract A048 SR59230A-induced ferroptosis sensitization of Ewing sarcoma cells via Beta-3 adrenergic receptor modulation: A novel therapeutic target. Cancer Res. 2024 Sep 5;84(17_Supplement):A048\u0026ndash;A048. \u003c/li\u003e\n\u003cli\u003eBanella C, Zocca L, Boaretto A, Mattei G, Mola M, Ballerini L, et al. Abstract A108: Metabolic oriented treatment: efficacy of sr59230a \u0026amp;#120631;3-adrenergic receptor antagonist, and sr plus buformin\u0026reg; in Ewing sarcoma. Mol Cancer Ther. 2023 Dec 1;22(12_Supplement):A108\u0026ndash;A108. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"investigational-new-drugs","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"drug","sideBox":"Learn more about [Investigational New Drugs](https://www.springer.com/journal/10637)","snPcode":"10637","submissionUrl":"https://submission.nature.com/new-submission/10637/3","title":"Investigational New Drugs","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"SR59230A, temozolomide, paclitaxel, vemurafenib, lenvatinib, sorafenib, Cancer therapy, Combination treatment, Synergism","lastPublishedDoi":"10.21203/rs.3.rs-7147623/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7147623/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eβ3-adrenergic receptors (β3-ARs) are increasingly recognized as modulators of tumor progression and treatment resistance across multiple cancer types. SR59230A, a selective β3-AR antagonist, has shown preclinical antitumor activity through mechanisms involving mitochondrial reactivation, reactive oxygen species (ROS) production, and antiangiogenic effects. Based on this premise, this study aimed to investigate the in vitro synergistic effects of SR59230A combined with standard chemotherapeutics or targeted therapies in various human cancer cell lines (glioblastoma, melanoma, triple-negative breast cancer, anaplastic thyroid carcinoma) and endothelial cells (HUVECs). Cells were treated with SR59230A alone or in fixed-ratio combinations with temozolomide, paclitaxel, vemurafenib, lenvatinib, or sorafenib. Drug interactions were quantified using the Chou\u0026ndash;Talalay method and validated with the Loewe additivity model. SR59230A exhibited dose-dependent antiproliferative activity, particularly in HUVECs and thyroid carcinoma cells. Synergistic effects were observed in all models, with the strongest synergy in A-2058 melanoma cells (SR59230A\u0026thinsp;+\u0026thinsp;vemurafenib), MDA-MB-231 breast cancer and 8505C thyroid carcinoma cells (SR59230A\u0026thinsp;+\u0026thinsp;paclitaxel), U-87 glioblastoma cells (SR59230A\u0026thinsp;+\u0026thinsp;temozolomide), and HUVECs (SR59230A\u0026thinsp;+\u0026thinsp;lenvatinib or sorafenib). Dose Reduction Index (DRI) values confirmed the potential to lower cytotoxic drug doses while preserving efficacy. These findings suggest that β3-AR antagonism via SR59230A may enhance the efficacy of conventional and targeted anticancer agents through multimodal mechanisms. The consistent synergistic effects across diverse tumor types support further investigation of β3-AR blockade as a promising strategy to overcome resistance and optimize cancer therapy.\u003c/p\u003e","manuscriptTitle":"Synergistic combination of the beta-3 antagonist SR59230A with common chemotherapeutic drugs and target therapies in cancer and endothelial cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-22 09:01:26","doi":"10.21203/rs.3.rs-7147623/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-10T23:02:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-10T22:42:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"150302998785620442340245992664059675502","date":"2025-07-20T12:29:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-18T07:13:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-18T06:18:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-18T06:16:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Investigational New Drugs","date":"2025-07-17T09:51:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"investigational-new-drugs","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"drug","sideBox":"Learn more about [Investigational New Drugs](https://www.springer.com/journal/10637)","snPcode":"10637","submissionUrl":"https://submission.nature.com/new-submission/10637/3","title":"Investigational New Drugs","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"eda34934-f901-44a2-a5de-2eeb9a705fe2","owner":[],"postedDate":"July 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-20T16:03:15+00:00","versionOfRecord":{"articleIdentity":"rs-7147623","link":"https://doi.org/10.1007/s10637-025-01587-8","journal":{"identity":"investigational-new-drugs","isVorOnly":false,"title":"Investigational New Drugs"},"publishedOn":"2025-10-18 15:58:08","publishedOnDateReadable":"October 18th, 2025"},"versionCreatedAt":"2025-07-22 09:01:26","video":"","vorDoi":"10.1007/s10637-025-01587-8","vorDoiUrl":"https://doi.org/10.1007/s10637-025-01587-8","workflowStages":[]},"version":"v1","identity":"rs-7147623","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7147623","identity":"rs-7147623","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}