Natural Coumarins Galbanic Acid and Auraptene Improved the Efficacy of Alkeran on Human Osteosarcoma Cells by Targeting ABC Transporters

ABSTRACT

Osteosarcoma is a severe bone malignancy, and current chemotherapeutic strategies often struggle to effectively halt disease progression. Galbanic acid (GBA) and auraptene (AUR) are natural sesquiterpene coumarins known for their diverse pharmacological activities. This study is the first to evaluate the ability of GBA and AUR to enhance Alkeran‐induced toxicity in osteosarcoma cells. GBA and AUR were isolated from Ferula szowitsiana, and the viability and apoptosis of osteosarcoma cells were assessed following treatments with GBA, AUR, and Alkeran—alone and in combination. An efflux assay was conducted to determine the functional interactions of AUR and GBA with ABC transporters, and molecular docking and dynamics simulations were performed to explore their potential interactions. Single treatment of cells with each agent did not induce significant toxicity: however, combination treatments of GBA or AUR with Alkeran significantly (p < 0.0001) reduced cell viability. Synergistic interaction was strong for both coumarins and Alkeran, supported by flow cytometry detection of apoptosis and ABC transporter activity. Molecular docking and dynamics simulations demonstrated favorable and stable interactions of coumarins with ABC transporters. In conclusion, this study provides strong support that GBA and AUR enhanced Alkeran efficacy in osteosarcoma cells by targeting ABC transporters.

1. Introduction

Bone sarcomas, including osteosarcoma, Ewing sarcoma, and chondrosarcoma, are aggressive tumors with high morbidity and mortality [1]. Osteosarcoma accounts for 20%–40% of pediatric bone cancers that primarily affect the metaphysis of long bones [2]. Despite the use of chemotherapy regimens, survival rates remain low, with a 5 year overall survival rate under 30%, especially for those with metastatic or recurrent disease [3]. Treatment efficacy is routinely hampered by the development of chemoresistance and the off‐target toxicity of conventional chemotherapeutics, which can reduce patient quality of life and limit therapy continuation [4]. This drawback emphasizes a critical need for innovative therapeutic strategies that can overcome resistance mechanisms and improve both survival and quality of life for osteosarcoma patients.

Drug resistance in osteosarcoma is commonly attributed to the elevated drug efflux. Multidrug resistance (MDR) is primarily caused by the overexpression of ATP‐binding cassette (ABC) family efflux transporters, including MDR1, also known as ABC subfamily B member 1 (ABCB1), and ABC subfamily G member 2 (ABCG2) [5, 6, 7]. There is direct evidence that elevated expression of ABC transporters, particularly ABCB1, contributes to chemoresistance and reduced responsiveness of human osteosarcoma cells to first‐line chemotherapy drugs [8, 9]. ABC transporters function through the interplay of nucleotide‐binding domains (NBDs) and transmembrane domains (TMDs). While most transporters, such as ABCB1, possess a full N‐TMD‐NBD‐TMD‐NBD‐C arrangement, ABCG2 functions as a homodimer containing only one NBD and one TMD per monomer. The transport cycle is driven by ATP binding at the NBDs, which induces domain rotation and dimerization to switch the transporter from an inward‐facing to an outward‐facing conformation, thereby effluxing substrates [10, 11, 12, 13, 14]. Consequently, inhibition strategies generally target either the ATPase activity at the NBDs or the substrate‐binding pocket within the TMDs. The evolution of ABC modulators has progressed from toxic first‐generation compounds (e.g., verapamil) and pharmacokinetically limited second‐generation inhibitors (e.g., VX‐710) to highly specific third‐generation agents such as tariquidar (TQR) and elacridar [15]. Notably, potent third‐generation inhibitors like TQR bind within the TMD central cavity, allosterically destabilizing NBD conformations and effectively trapping the transporter in a non‐functional state [16].

Nitrogen mustard‐based alkylating agents were among the first effective antitumor drugs developed and remain clinically significant across various cancer types. Alkeran, also known as Melphalan, is a phenylalanine derivative of nitrogen mustard that exploits the elevated amino acid uptake in malignant cells by utilizing high‐affinity L‐type amino acid transporters for intracellular entry [17]. As a bifunctional alkylator, Alkeran induces inter‐ and intra‐strand DNA cross‐links as well as DNA‐protein cross‐links, thereby disrupting replication, transcription, and DNA repair mechanisms, culminating in apoptosis of rapidly dividing tumor cells [18, 19]. It is effective against hematological malignancies, like multiple myeloma, and solid tumors such as osteosarcoma, melanoma, glioblastoma, and breast and ovarian carcinomas [20, 21, 22, 23, 24]. However, the clinical utility of Alkeran is significantly limited by dose‐dependent toxicities such as myelosuppression, worsened by renal impairment, along with gastrointestinal, cardiovascular, and pulmonary adverse effects [25]. Given these challenges, the development of combinatorial treatment strategies that preferably incorporate natural agents—known for their typically lower off‐target toxicity—is of significant interest. Such integrative approaches can potentiate the anticancer efficacy of Alkeran while enabling dose reduction to minimize adverse effects.

Coumarins are a prominent class of natural compounds predominantly found in the Apiaceae, Rutaceae, and Asteraceae families, which are native to specific geographical regions, including the Mediterranean and central Asia. Among these, galbanic acid (GBA), a sesquiterpene coumarin mainly isolated from Ferula species, displays a broad spectrum of pharmacological activities, including potent anticancer effects against lymphoma, melanoma, osteosarcoma, prostate, and breast cancers [26, 27, 28, 29, 30, 31]. Auraptene (AUR), a geranyloxycoumarin abundant in edible fruits and vegetables of the Ferula and Citrus genera, also exhibits diverse therapeutic properties, with documented anticancer activities across melanoma, glioblastoma, osteosarcoma, breast, colorectal, prostate, gastric, and esophageal carcinomas [32, 33, 34, 35, 36, 37, 38, 39]. Despite this broad anticancer profile, no studies have yet investigated whether GBA and AUR can potentiate the effects of Alkeran in cancer cells. The present study addresses this gap by pioneering the evaluation of these coumarins for their ability to enhance Alkeran‐induced toxicity in osteosarcoma cells and elucidating their underlying mechanism of action. To end this, GBA and AUR were first isolated from Ferula szowitsiana, and the viability and apoptosis of osteosarcoma cells were assessed following treatments with GBA, AUR, and Alkeran—alone and in combination. Additionally, a mitoxantrone‐based efflux assay was conducted to determine the functional interactions of AUR and GBA with ABC transporters. Finally, molecular docking and dynamics simulations were performed to explore the potential interactions of AUR with ABCB1 and ABCG2 (the experimental workflow is presented in Figure S1).

2. Methods

2.1. Extraction of GBA and AUR

To extract GBA (MW: 398.5 g/mol) and AUR (MW: 298.3 g/mol), F. szowitsiana was collected from the mountains of Golestan Forest, North of Iran. The plant was formally identified by a botanical expert at the Faculty of Pharmacy, Mashhad University of Medical Sciences (MUMS), to ensure the accuracy of the species. A voucher specimen of the roots (No. M1001) was deposited at the Department of Pharmacognosy and Biotechnology, Faculty of Pharmacy, MUMS, Iran. For extraction, the roots were air‐dried, powdered, and macerated in acetone (Merck). The acetone extract was concentrated under reduced pressure, yielding a thick residue. The fractionation process was carried out using thin‐layer chromatography (TLC) on silica gel plates (60 GF254, Merck), employing a petroleum ether and ethyl acetate solvent mixture. Fractions were identified under UV light (CAMAG spectrometer, 254 nm), eluted with acetone, and purified via preparative TLC to isolate GBA and AUR. The identity of compounds was confirmed by ^¹H‐NMR spectroscopy (Figure S2), and their high purity (>95%) was defined by HPLC (Figure S3). For cell treatment, various concentrations of GBA and AUR were prepared using dimethyl sulfoxide (DMSO) at 0.4% (v/v) as the solvent.

2.2. Treatment of Cells

Human osteosarcoma cells, MG‐63 cell line, were purchased from Pasteur Institute (Tehran, Iran). The cell line was authenticated by an expert before experimentation, and cells were tested and confirmed to be free from mycoplasma contamination prior to and during the experiments. Cells were cultured using dulbecco's modified eagle's medium (Capricorn) supplemented with 10% fetal bovine serum (Gibco). The cells were authenticated by an expert before experimentation, and they were tested and confirmed to be free from mycoplasma contamination prior to and during the experiments. Cells were maintained at 37°C with 5% CO₂ and passaged using 0.25% trypsin‐1 mM EDTA (Betacell).

The individual effects of GBA, AUR, and Alkeran were first assessed. MG‐63 cells were seeded in 96‐well plates and treated with 25, 50, and 100 µM of each agent for 24 and 48 h. After identifying sub‐lethal concentrations, combined treatments were performed by exposing cells to 50 and 100 µM of GBA or AUR + 100 µM Alkeran for 24 and 48 h. Controls included cells treated with 0.4% DMSO as a solvent control for single‐agent treatments, and cells treated with DMSO + Alkeran for combination treatments.

2.3. Viability Assay and Interaction Analysis

Cell viability following single and combined treatments was evaluated by resazurin assay. In brief, resazurin solution (0.1 mg/mL, Sigma) was added to cells at each specified time point and incubated at 37°C for 3 h. Subsequently, absorbance was measured at 600 nm using a microplate reader (Epoch). Cell viability (%) was calculated using the following equation: 100 × [100 − (absorbance of treated cells − absorbance of untreated cells)/(absorbance of blank control − absorbance of untreated cells)]. Viability assessment was conducted in four replicates and repeated at least three times.

The interaction between GBA and AUR + Alkeran was evaluated via the Chou–Talalay method using CompuSyn 1.0 software. This analysis calculated the combination index (CI) and dose reduction index (DRI). CI values interpret the interaction as follows: CI < 1 indicates synergy; CI = 1, additive effect; and CI > 1, antagonism. The DRI reflects how much the dose of each agent can be reduced in combination therapy compared to single treatments—a higher DRI indicates greater potential for lowering therapeutic doses, beneficial in minimizing toxicity during cancer treatment.

2.4. Cell Apoptosis Assay

To assess the apoptosis‐inducing effects of GBA, AUR, and Alkeran, MG‐63 cells were treated with 100 µM of each agent individually or in combination for 48 h. Following treatments, both adherent and floating cells from the treated and control groups were collected and washed with phosphate‐buffered saline (PBS). The cell pellets were then resuspended in a binding buffer containing FITC‐conjugated annexin V and propidium iodide (Sigma). Samples were analyzed by flow cytometry (BD FACSCalibur) using FL1‐H and FL2‐H channels. Ten thousand gated events were collected for all groups, and data analysis was performed with FlowJo V10 software to quantify the percentages of viable, necrotic, early apoptotic, and late apoptotic cells.

2.5. Efflux Assay for Detection of Mitoxantrone

To determine the effects of GBA and AUR on the activity of ABC transporters, an efflux assay was performed. Four experimental groups were included in this assay: untreated MG‐63 cells, cells treated with 0.4% DMSO, and cells treated with 100 µM GBA or AUR for 48 h. After the collection of cells, they were incubated in 10 µM mitoxantrone (Nanoalvand) at 37°C in the dark for 30 min (accumulation phase). Cells were then washed with ice‐cold PBS, resuspended in complete medium, and incubated at 37°C for 60 min (efflux phase). Cells were then washed and resuspended in ice‐cold PBS, and the intracellular mitoxantrone fluorescence was measured by flow cytometry (BD FACSCalibur) using the FL3 filter. Ten thousand gated events were collected for all groups, and data were analyzed by FlowJo V10 software.

2.6. Molecular Docking

To predict the preferred orientation of AUR within the binding site of ABCB1 and ABCG2, molecular docking was performed using AutoDock Vina 1.1.2. Three‐dimensional structures of the protein targets ABCB1 (PDB ID: 8Y6I, resolution 2.54 Å) and ABCG2 (PDB ID: 6HZM, resolution 3.09 Å) were retrieved from RCSB Protein Data Bank (https://www.rcsb.org/), and the three‐dimensional structure of AUR was obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/). Prior to docking, the protein structures were imported into Chimera 1.17.3 for refinement and energy minimization. This preparation included the removal of heteroatoms, water molecules, and residual ions, followed by the addition of hydrogen atoms, thereby optimizing the protein models for subsequent molecular docking analysis. For detailed visualization of ligand–protein interactions, BIOVIA Discovery Studio 2022 was employed to generate both two‐ and three‐dimensional interaction maps.

2.7. Molecular Dynamics Simulations

To evaluate the binding stability and flexibility of the AUR‐ABCG2 docking complex, which exhibited a more favorable binding energy, molecular dynamics simulations were performed using GROMACS 2024. The topology files for the ligand and protein were generated using the CGenFF server and the CHARMM36 force field. The TIP3P water system was used to solvate the complex in a 1.0 nm triclinic box, and Na⁺ and Cl⁻ ions were added to neutralize the system until the maximum force per atom was below 10.0 kJ/mol. The complex was simulated at a pressure of 1 bar and 310.15 K. By producing the root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (R _g), solvent accessible surface area (SASA), Lennard‐Jones short range (LJ‐SR) potential, and Coulombic SR (C‐SR) potential using the GROMACS tool, the generated trajectories were evaluated to demonstrate the stability and compactness of the structure.

2.8. Statistical Analysis

Data analysis was performed using GraphPad Prism 10. Statistical comparisons were conducted using one‐way analysis of variance (ANOVA) followed by Dunnett's post‐hoc test. All treatments were carried out in at least three independent experiments. Results are presented as mean ± standard deviation (SD). Statistical significance was defined at p‐values ranging from <0.05 to <0.0001.

3. Results

To evaluate whether GBA or AUR can enhance the effects of Alkeran in osteosarcoma cells, we first assessed the cytotoxicity of each agent individually. As illustrated in Figure 1, neither AUR nor GBA significantly affected cell viability at any concentration tested. Alkeran, however, exhibited concentration‐dependent cytotoxicity: while 25 and 50 µM had no significant effect, 100 µM reduced viability to 86.6% and 74.1% (p < 0.01) after 24 and 48 h, respectively. The calculated IC₅₀ values were >1000 µM for GBA at both time points, 447.1 and 597.8 µM for AUR at 24 and 48 h, respectively, and 495.2 and 181.3 µM for Alkeran at 24 and 48 h, respectively.

FIGURE 1.

Chemical structures and dose‐response curves of GBA, AUR, and Alkeran in osteosarcoma cells. Upon 24 and 48 h treatment of MG‐63 cells with GBA (A), AUR (B), and Alkeran (C), viability was calculated and compared with the relevant DMSO control. Viability assessment was conducted at least three times, and the results are presented as the mean ± SD. ALK: alkeran, AUR: auraptene, GBA: galbanic acid.

Combination treatments with GBA or AUR + Alkeran significantly reduced osteosarcoma cell viability compared to single‐agent treatments. As shown in Figure 2A, treatments with 100 µM GBA + 100 µM Alkeran produced the most pronounced effects, decreasing viability to 59.6% (p < 0.05) and 36.1% (p < 0.0001) after 24 and 48 h, respectively. Additionally, the combination of 100 µM AUR + 100 µM Alkeran reduced cell viability to 66.2% and 32.8% (p < 0.0001) after 24 and 48 h, respectively (Figures 2B). Morphological observations supported the viability assay results, as a reduced number of attached and viable MG‐63 cells were evident after 48 h treatment with 100 µM GBA or AUR + 100 µM Alkeran compared to controls (Figure 2C).

FIGURE 2.

Viability and morphology of MG‐63 cells following combinatorial treatments. After 24 and 48 h treatment with GBA (A) or AUR (B) in combination with Alkeran, cell viability was calculated and compared with the relevant DMSO control. Phase contrast photomicrographs of cells after 48 h treatment with GBA or AUR, alone and in combination with Alkeran (C). The statistical significance of the results is represented as *p < 0.05, **p < 0.01, and ****p < 0.0001. Viability assessment was conducted in four replicates (N = 4) and repeated at least three times, and the results are presented as the mean ± SD. ALK: alkeran, AUR: auraptene, GBA: galbanic acid.

To characterize the nature of the pharmacological interactions between GBA or AUR and Alkeran, CI and DRI values were computed. As summarized in Table 1, the combination of 50 µM GBA + 100 µM Alkeran yielded a CI < 1 at 48 h, whereas 100 µM GBA combined with 100 µM Alkeran resulted in CI values < 1 at both 24 and 48 h, indicating synergism. The DRI, which quantifies the fold‐reduction in drug dose achievable in combination versus alone, was 3.1 and 2.27 for the 100 µM GBA combination at 24 and 48 h, respectively, exceeding values observed for 50 µM GBA. Similarly, for AUR, CI values remained < 1 across both concentrations (50 and 100 µM) at all time points. Consistent with GBA, the highest DRI values were observed for the 100 µM AUR combination (2.49 at 24 h and 2.45 at 48 h), surpassing those of 50 µM AUR. Collectively, these findings demonstrate that the synergistic potential of both GBA and AUR with Alkeran is dose‐dependent, being most pronounced at the highest tested concentration (100 µM) of these coumarins.

TABLE 1.

CI and DRI values for combinational treatment of MG‐63 cells with GBA and AUR combined with Alkeran during 24 and 48 h. *Fa: Fraction of affected cells.

	Alkeran
Coumarin	Dose (µM)	Time (h)	Fa*	CI	DRI
GBA	50	24	0.26	0.548	1.876
	100		0.404	0.329	3.106
	50	48	0.01	8.896	0.165
	100		0.639	0.484	2.279
AUR	50	24	0.03	5.689	0.290
	100		0.338	0.671	2.499
	50	48	0.16	1.851	0.734
	100		0.672	0.409	2.455

To further assess the effects of GBA or AUR in combination with Alkeran, cells were treated for 48 h, stained with annexin V‐FITC and PI, and analyzed by flow cytometry. As shown in Figure 3, cell viability after individual treatments with 100 µM GBA, 100 µM AUR, or 100 µM Alkeran was 87.6%, 88.7%, and 70.8%, respectively. Notably, the 100 µM GBA + 100 µM Alkeran treatment markedly altered cell populations: 48.8% were alive, 36.3% early apoptotic, 14.6% late apoptotic, and 0.5% necrotic. For 100 µM AUR + 100 µM Alkeran treatment, 47.2% of cells were alive, 45.3% early apoptotic, 8.4% late apoptotic, and 2.6% necrotic.

FIGURE 3.

Flow cytometry detection of apoptosis upon 48 h treatment of cells with GBA and AUR, alone and in combination with Alkeran. Representative quadrant dot plots indicating viable (Q4), early apoptotic (Q3), late apoptotic (Q2), and necrotic (Q1) cell populations (A). Quantification of total apoptosis presented as mean ± SD from three independent experiments (B). ALK: alkeran, AUR: auraptene, GBA: galbanic acid.

To investigate whether GBA and AUR modulate the efflux activity of ABCB1 and ABCG2 transporters, we measured intracellular mitoxantrone accumulation. Cells were divided into four experimental groups: untreated, vehicle control (0.4% DMSO), 100 µM GBA, and 100 µM AUR. Intracellular mitoxantrone levels were subsequently quantified using flow cytometry. As shown in Figure 4, cells treated with GBA or AUR exhibited notable differences in mitoxantrone fluorescence compared to the untreated and DMSO controls. These findings indicate that both coumarins functionally interact with ABC transporters.

FIGURE 4.

Mitoxantrone‐based efflux assay to evaluate functional modulation of ABC transporters by GBA and AUR. MG‐63 cells were treated with 100 µM GBA (A) or AUR (B) for 48 h, followed by incubation with mitoxantrone. Intracellular mitoxantrone fluorescence was quantified by flow cytometry (10,000 events per sample). AUR: auraptene, GBA: galbanic acid.

To corroborate these functional observations and elucidate the structural basis of this interaction, particularly for AUR, molecular docking was conducted. As illustrated in Figure 5, AUR formed hydrogen bonds with Arg666 and Thr1078 within the ABCB1 binding pocket, stabilized by van der Waals interactions with Asp805, Gly1075, Ser1072, Cys1074, Lys1076, and Ser1077, resulting in a binding energy of −6.5 kcal/mol. Similarly, the interaction between AUR and ABCG2 involved a hydrogen bond with Lys76 and van der Waals forces with Ala52, Arg54, Ala73, and Phe75, exhibiting a favorable binding affinity of −7.3 kcal/mol.

FIGURE 5.

Molecular docking diagrams generated for predicting the interaction of AUR with the NBD domain of ABCB1 (A) and ABCG2 (B). 3D and 2D models were depicted using BIOVIA Discovery Studio 2022.

Molecular dynamics simulations were also carried out to investigate the dynamic behavior of the AUR‐ABCG2 complex (Figure 6). RMSD was examined to track the trajectories of amino acids, and results indicated that the AUR‐ABCG2 complex exhibited deviations below 0.5 nm in Cα atoms. To further assess protein stability and flexibility, RMSF analysis was conducted, and values of all Cα atoms of the AUR‐ABCG2 complex were predominantly less than 0.8 nm, which indicates their stability. Rg represents the mass‐weighted root‐mean‐square distance of atoms from their center of mass. Throughout the analysis, the AUR‐ABCG2 complex exhibited a consistent pattern in Rg values, falling below 3.4 nm. SASA was examined to analyze changes in the hydrophilic and hydrophobic, and the AUR‐ABCG2 complex showed a similar SASA value to ABCG2 alone, indicating accessibility throughout the simulation time. To quantify the strength of the interaction, LJ‐SR and C‐SR potentials were examined, and results indicated that AUR interacted with ABCG2 for the whole simulation time, further indicating their continuous interaction.

FIGURE 6.

Molecular dynamics simulations of the AUR‐ABCG2 complex. RMSD plot (A), RMSF plot (B), Radius of gyration plot (C), SASA per residue plot (D), LJ‐SR plot (E), and C‐SR plot (F) per 100 ns molecular dynamics simulations.

4. Discussion

A major challenge in managing osteosarcoma is that many patients are diagnosed at an advanced, high‐grade stage, at which point current chemotherapeutic treatments often fail to effectively control disease progression [40]. Although the standard approach combines surgery with chemotherapy, the emergence of chemoresistance and the harmful side effects of conventional drugs adversely affect patients’ quality of life and frequently lead to the discontinuation of treatment [4]. In response to these limitations, research is increasingly focused on incorporating natural compounds that can overcome chemoresistance by specifically targeting drug transporter mechanisms.

There is increasing interest in coumarins as safer and potentially more effective alternatives to traditional chemotherapy agents. Studies have demonstrated their promising anti‐tumor activity against osteosarcoma cells; for instance, Angedahurin A and Hesperidin have shown notable anticancer effects on MG‐63 cells by inducing cell cycle arrest and promoting apoptosis [41, 42]. In this study, we explored, for the first time, the ability of two specific coumarins, GBA and AUR, to enhance the therapeutic efficacy of Alkeran in human osteosarcoma cells. These agents have garnered attention due to their broad therapeutic potential, particularly their anticancer properties. Multiple mechanisms have been proposed to explain the anticancer effects of GBA and AUR, including downregulation of androgen and estrogen receptors, induction of mitochondrial‐mediated apoptosis through activation of caspase‐9 and caspase‐3 as well as PARP cleavage, inhibition of cyclin D1 and insulin‐like growth factor‐1, modulation of Bcl‐2 family proteins, suppression of angiogenesis, and reduction of metastatic potential [32, 43, 44, 45, 46, 47, 48, 49].

Our previous studies have demonstrated that AUR and GBA enhanced the cytotoxic effects of multiple chemotherapeutic agents—including cisplatin, paclitaxel, 5‐fluorouracil, doxorubicin, vincristine, and arsenic trioxide—as well as radiotherapy and thermal therapy across various cancer types such as adult T‐cell leukemia/lymphoma, gastric, esophageal, colon, and prostate cancers [35, 36, 39, 50, 51, 52, 53, 54, 55]. The current findings further support the therapeutic potential of natural coumarins in combination with cancer therapy. Specifically, our results show that combining GBA or AUR with Alkeran significantly increased cytotoxicity and induced apoptosis in osteosarcoma cells. To note, dose‐dependence of interaction metrics was observed for both coumarins. The lower dose of AUR and GBA (50 µM) exhibited antagonism at 24 h, while the higher dose (100 µM) showed synergism at both time points. Accordingly, the combination of these natural coumarins with Alkeran was beneficial as the administered dose was increased. These outcomes align with prior research where pretreatment with natural reversal agents such as resveratrol enhanced the efficacy of Alkeran in breast carcinoma cells [56]. Similarly, co‐treatment with curcumin and Alkeran promoted apoptosis in breast cancer cells, and metformin was shown to amplify Alkeran‐induced DNA damage in multiple myeloma cells [19]. To further elucidate the mechanism underlying the synergistic effects of GBA and AUR, an efflux assay was performed, and results indicated notable differences in the level of mitoxantrone fluorescence between GBA‐ or AUR‐treated cells compared to controls. These findings emphasize the promising role of GBA and AUR as adjuvants to improve conventional chemotherapy efficacy in osteosarcoma by targeting ABC transporters.

To further validate the functional data and characterize the molecular interactions underlying coumarin binding to target ABC transporters, we conducted molecular docking and molecular dynamics simulations. These analyses demonstrated that AUR forms stable, energetically favorable complexes with ABCB1 and ABCG2, specifically targeting the NBD. While conventional inhibitors often occupy the TMD to lock transporters in an inward‐facing state and prevent efflux [13, 14], our findings indicated that AUR interacts directly with the NBD. This interaction disrupts the ATP binding and hydrolysis essential for substrate translocation [10, 11, 12]. This mechanism aligns with our previous demonstration that the structurally related coumarin GBA reverses ABCB1‐mediated efflux [50].

The current study has a certain limitation that warrants further investigation. Although efflux assay and molecular simulations support transporter involvement, mostly consistent with ABCG2, they do not yet conclusively demonstrate ABCB1 and ABCG2 targeting. Conducting further assessments using transporter‐specific controls (e.g., Ko143 for ABCG2 and/or verapamil for ABCB1) would provide deeper insights into the observed effects.

In conclusion, this study presents compelling support that GBA and AUR enhanced the efficacy of Alkeran chemotherapy in osteosarcoma cells, partially through targeting the ABC transporters ABCB1 and ABCG2. Consequently, combining these coumarins with chemotherapy holds significant promise for improving therapeutic outcomes in osteosarcoma. These findings lay a strong foundation for further preclinical and clinical investigations into the use of GBA and AUR as natural reversal agents.

Author Contributions

Parastoo Azadbeigi: experiments and data analysis. Sara Seyedshazileh: experiments and data analysis. Ahmadreza Gharaiean‐Morshed: revision. Mehrdad Iranshahi: supplied GBA and AUR. Fatemeh B. Rassouli: project conceptualization, supervision, and manuscript revision.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the present work.

Supporting information

Supporting File 1: cbdv71235‐sup‐0001‐FigureS1.png

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Data Availability Statement

The data supporting the findings of this study will be available upon request from the corresponding author.