Introduction

Cancer is one of the major health problems worldwide. In cancer, genetic and epigenetic alterations lead to the evasion of programmed cell death (apoptosis), resulting in the uncontrolled growth of abnormal cells, followed by their metastasis to other tissues and organs. This can lead to severe complications such as blood clots, infections, organ failure and ultimately death⁠. According to estimates from the International Agency for Research on Cancer, the total number of cancer cases in 2022 was 20.0 million, and it is projected to increase to 32.6 million in 20451. Among all types of cancer, the number of liver and intrahepatic bile ducts cancer cases is expected to rise from (2022) to 1.41 million (2045), and the number of breast cancer cases will increase from 2.3 million (2022) to 3.36 million (2045). These projections highlight an urgent need for more effective therapeutic strategies.

Despite advances in cancer therapy, resistance to conventional treatments and side effects continue to necessitate the development of new therapeutic agents that can selectively target cancer cells while sparing normal tissue2,3. Repurposing existing drugs for cancer therapy is a promising approach, enabling the faster development of effective treatments with established safety profiles4⁠.

Various types of cancer drugs cause serious side effects in patients, leading scientists to search for non-toxic alternatives worldwide5⁠. Recently, we observed anticancer properties of Antimycin-A, DAPG (2,4-diacetylphloroglucinol) and DPPG (2,4‑Dipropylphloroglucinol) against human cancer cell lines6,7. As a part of a series of research studies, we now focus on Eltrombopag olamine.

Eltrombopag (C29H36N6O6, Mr. 564.643) is a US Food and Drug Administration (FDA)-approved drug. Eltrombopag olamine, a thrombopoietin receptor agonist, is approved for treating chronic immune thrombocytopenia (ITP), hepatitis C virus (HCV)-associated thrombocytopenia, and aplastic anemia8. In addition to increasing platelet count and reducing the risks of bleeding, the molecule has iron-chelating properties and enhances the anti-neoplastic activity of cytarabine in pediatric acute myeloid leukemia cell lines9⁠.

Several studies have reported anticancer properties of Eltrombopag olamine in various cancer models. In murine models of breast cancer (4T1 cell line), Eltrombopag was found to reduce tumor burden and inhibit metastasis and angiogenesis through inhibition of HuR protein, a regulator of mRNA stability for several tumor-promoting genes such as Snail, Cox-2, and Vegf-c10[,11. It has been shown to inhibit proliferation of hepatocellular carcinoma (HepG2, Hep3B, Huh7) cells by altering intracellular iron levels and inducing G0/G1 cell cycle arrest12. Despite these promising findings, most of the previous studies focused on either the iron-chelating property or HuR inhibition, without a detailed analysis of how Eltrombopag modulates apoptotic pathways at the molecular level. The expression patterns of key apoptotic and survival-related genes and their contribution to cell death mechanisms remain insufficiently explored.

This study is the first to investigate the molecular mechanisms by which Eltrombopag olamine induces apoptosis in both human MCF-7 (breast adenocarcinoma) and SMMC-7721 (hepatocellular carcinoma) cancer cells. We aimed to evaluate its ability to inhibit cell proliferation, induce apoptosis, arrest the cell cycle, and modulate several apoptotic and pro-survival genes. Specifically, we analyzed the expression of FAS, TP53, BAX, TNFα, and cytochrome-C, alongside EGFR, ERK, MAPK, STAT3, NOTCH, and PARP1, which are associated with key cancer-related signaling pathways.

Furthermore, we conducted molecular docking simulations to evaluate interactions of Eltrombopag with EGFR, FAS, TP53, and TNFα proteins, adding structural support to our gene expression and functional findings. This study contributes to a deeper understanding of the molecular mechanisms involved in Eltrombopag-induced cell death and highlights its potential as a repurposed anticancer agent capable of targeting multiple pathways in both breast and liver cancer.

Materials and methods

Material

MTT, SYBR Green RT master mix, cDNA synthesis kit, and primers were purchased from Beijing TsingKe Biological Technology, China. RNA extraction kit from FAVORGEN (Taiwan), DMEM medium, Trypsin-EDTA, and FBS were obtained from Gibco (USA and Germany). FITC-annexin V/PI was purchased from Invitrogen (USA). Streptomycin and neomycin were from Amresco (USA). Propidium iodide (PI) and caspase inhibitors were obtained from Sigma (USA). HEPES and RNase A purchased from (Carl Roth, Germany).

Cell culture and treatment protocol

Three established cell lines were utilized in this study: MCF-7, SMMC-7721, and non-cancerous Human Embryonic Kidney (HEK) 293 T, all procured from ATCC. Cells were maintained in DMEM high-glucose medium containing L-glutamine, pyridoxine-HCl, and sodium pyruvate. Sodium bicarbonate and HEPES were added subsequently. The medium was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-neomycin. Cells were cultured under standard conditions (37 °C, 5% CO₂) and routinely passaged at 80% confluency to ensure optimal growth. For experimental assays, cells were seeded in 96-well plates at optimized densities: MCF-7 and HEK293T at 1.0 × 10⁴ cells/well, and SMMC-7721 at 2.0 × 10⁴ cells/well. Following a 24-hour stabilization period, cells were treated with varying concentrations of Eltrombopag olamine: MCF-7 (10–80 µg/ml), HEK293T (16–128 µg/ml), and SMMC-7721 (32–256 µg/ml) for 48 h. Then DMEM medium was carefully aspirated and replaced with MTT solution for viability assessment13,14. After 2 h of incubation, visible crystals formed, which were dissolved in the mixture of isopropanol and HCl following the removal of aliquots from each well. Then we recorded the absorbance of the magenta dye solution at 570 nm using a microplate reader (BioTek 800TS, USA). GraphPad Prism (8.0.2) and MS Excel (Microsoft Office 365) softwires were used to calculate cell growth inhibition and IC50 values, respectively.

Morphological examination of cells with a fluorescence microscope

Following the established protocol, cells were plated in 24-well plates at optimized densities (MCF-7: 4 × 10⁴ cells/well; SMMC-7721: 8 × 10⁴ cells/well) and exposed to cell-type-specific Eltrombopag olamine concentrations (MCF-7: 31 µg/mL; SMMC-7721: 154 µg/mL) for 24 h. Following aspiration of the culture medium, cells were rinsed with PBS and incubated with Hoechst 33342 (20 min, dark conditions). Cellular morphology was examined via fluorescence microscopy (Olympus IX71, Korea), with subsequent image acquisition.

Detection of early and late apoptosis by fluorescence microscope

MCF-7 cells and SMMC-7721 cells were treated with Eltrombopag olamine as described above. After 24 h, the cells were washed twice with PBS and annexin V binding buffer. The cells were then stained with FITC-annexin V/PI for 30 min in the dark at room temperature. Finally, an Olympus IX71 fluorescence microscope was used to detect early and late apoptotic cells.

Detection of apoptotic and necrotic cell death by flow cytometry

MCF-7 and SMMC-7721 cell lines were plated in 6-well culture dishes and exposed to Eltrombopag olamine for 12 h at concentrations of 80 µg/ml and 256 µg/ml, respectively. Following treatment, the cells were harvested and rinsed with annexin V binding buffer. Subsequently, they were stained with FITC-annexin V and PI for 20 min in the dark at room temperature. Cell apoptosis and necrosis were then analyzed using a CytoFLEX flow cytometer (Beckman Coulter, USA).

Cell cycle analysis by flow cytometry

MCF-7 cells were maintained in T-25 flasks and exposed to 15.5 µg/ml of Eltrombopag olamine for 12 h, following the previously described protocol. In parallel, SMMC-7721 cells cultured under the same conditions were treated with 154 µg/ml of Eltrombopag olamine for 12 h. After treatment, cells were detached by Trypsin-EDTA, rinsed with PBS, and resuspended in 1 ml of ice-cold PBS before being preserved at − 20 °C in 3 ml of 70% ethanol. Before experiment, ethanol was subsequently removed by PBS washing, and the cells were incubated with RNase A at 37 °C for 30 min. PI was then added for DNA staining, and the cell cycle distribution was assessed using a CytoFLEX flow cytometer.

Gene expression by Real-time PCR

MCF-7 and SMMC-7721 cell lines were exposed to different concentrations of Eltrombopag olamine (31.0 µg/ml and 154 µg/ml, respectively) for 24 h. Total RNA extraction was performed using reagents obtained from FAVORGEN (Taiwan) according to the supplied protocol. Then cDNA was prepared using cDNA preparation kit (TsingKe, China). After that cDNA templates were mixed with 2× SYBR Green master mix (TsingKe, China) following the manufacturer’s specifications. The PCR conditions were set to 95 °C for 3 min followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s as supplied. Expression level of FAS, TP53, BAX, TNFα, Cytochrome-C, NOTCH2, EGFR, ERK, and WNT1 for MCF-7 and TNFα, TP53, BAX, STAT3, MAPK, NOTCH2, PARP1, EGFR, ERK, and WNT1 for SMMC-7721 cells were detected using a CFX96 Real-Time PCR detection system. 18 s and β-actin were used as housekeeping genes for MCF-7 and SMMC-7721 cells, respectively. The final Real-Time PCR data was analyzed using the double delta CT method according to Kabir et al.15. Primer’s list is shown in Table 1.

Table 1 Primer List.
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Involvement of caspases in the apoptosis process

The effects of caspases on the cytotoxicity of Eltrombopag olamine against MCF-7 and SMMC-7721 cells were assessed by Caspase-3/7 inhibitor (z-DEVD-fmk), caspase-8 inhibitor (z-IETD-fmk), and caspase-9 inhibitor (z-LEHD-fmk). Briefly, 1.0 × 104 MCF-7 cells and 2.0 × 104 SMMC-7721 cells were seeded in each well of a 96 well cultured plate and 24 h later cells were incubated in presence and absence of 2 µM of each Caspase-3/7, caspase-8, and caspase-9 inhibitors for 2 h at 37 °C with 5% CO2. Then MCF-7 and SMMC-7721 cells were incubated with 20.0 and 128.0 µg/ml of Eltrombopag olamine, respectively, for 48 h. Finally, MTT assay was used to detect cytotoxicity. Cells without Eltrombopag olamine and caspase inhibitors used as control.

In Silico study

The interactions of FAS, TP53, TNFα, and EGFR proteins with Eltrombopag olamine were verified by molecular docking simulations. The 3-dimensional structures of FAS, TP53, TNFα, and EGFR, proteins were downloaded from the Protein Data Bank (https://www.rcsb.org/) with the PDB codes 1DDF, 2OCJ, 1TNF, and 2GS2, respectively. To minimize energy, all water molecules and heteroatoms were removed from each protein using Biovia Discovery Studio 2021 to reduce the possibility of undesirable interactions with the receptors. During the docking process, PDB files were converted to pdbqt format. Eltrombopag (CID no. 135449332) was downloaded in 3D SDF format from the PubChem website (https://pubchem.ncbi.nlm.nih.gov/), minimized and converted to pdbqt format using PyRx tools. Protein-ligand binding results were obtained using PyRx Autodock Vina. We observed the responsible bonding interactions (hydrogen bonds, pi-alkali bonds, hydrophobic interactions) during protein-ligand interactions by using BIOVIA Discovery Studio 2021.

Statistical analysis

Quantitative data are expressed as mean values with their variability indicated by standard deviations (mean ± SD). For analytical processing, we employed a dual-software approach: Microsoft Excel facilitated preliminary data organization and basic calculations, while GraphPad Prism (v8.0.2) was utilized for advanced graphical representations and curve fitting. To determine intergroup variability, we implemented a one-way ANOVA framework with Duncan’s post hoc testing for multi-group comparisons using SPSS Statistics (v21.0). Duncan’s test was selected for its sensitivity in detecting treatment differences, although it is less conservative than methods such as Tukey or Bonferroni. No additional correction for multiple comparisons was applied. This analytical pipeline allowed comprehensive evaluation of treatment effects across experimental conditions. The threshold for statistical significance was maintained at α = 0.05, with p-values exceeding this threshold considered indicative of non-significant results.

Results

In vitro and in vivo cell growth inhibition.

At concentrations of 10, 32, and 16 µg/ml of Eltrombopag olamine, 33.9%, 10.5%, and 2.5% of MCF-7, SMMC-7721, and HEK293T cell proliferation were inhibited, respectively. The inhibition of cell proliferation increased with increasing concentration and cell proliferation inhibition of 83.0%, 80.4%, and 49.8% were recorded at the highest concentrations of 80.0, 256.0, and 128 µg/ml of Eltrombopag olamine, respectively (Fig. 1A and B, HEK293T data was not shown). IC50 values of Eltrombopag olamine were calculated to be 31, 154, and 127 µg/ml for MCF-7, SMMC-7721, and HEK293T cells, respectively.

Fig. 1
figure 1

Antiproliferative activity of Eltrombopag olamine (EO) on MCF-7 and SMMC-7721 cells in vitro. (A) and (B) represent the antiproliferative activity of Eltrombopag olamine against MCF-7 and SMMC-7721 cells, respectively. (C) Detection of caspase activation in MCF-7 and SMMC-7721 cells. The antiproliferative activity of Eltrombopag olamine at concentrations of 20 and 128 µg/ml on MCF-7 and, SMMC-7721 cells in the presence and absence of caspase inhibitors. EO + Casp3i, EO + Casp8i, and EO + Casp9i represent Eltrombopag olamine mixed with Caspase-3/7, caspase-8 and caspase-9 inhibitors, respectively.

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Involvement of caspases in the cytotoxicity of Eltrombopag olamine

Approximately 44.0% of the proliferation of MCF-7 cells was inhibited by a concentration of 20 µg/ml of Eltrombopag olamine. Inhibition of cell proliferation was reduced by 28%, 24%, and 34.6% when MCF-7 cells were treated with Eltrombopag olamine in the presence of Caspase-3/7, −8, and − 9 inhibitors, respectively. On the other hand, 38% inhibition of SMMC-7721 cell proliferation was observed at 128 µg/ml Eltrombopag olamine, and inhibition of cell proliferation was reduced by 2%, 3%, and 16% in the presence of Caspase-3/7, −8, and − 9 inhibitors, respectively Fig. 1C.

Examination of morphological changes with Hoechst 33,342 stain

Fluorescence microscopy analysis demonstrated distinct apoptotic morphology in Eltrombopag olamine-treated cells (Fig. 2). Both MCF-7 and SMMC-7721 cell lines exhibited characteristic nuclear condensation and fragmentation (Fig. 2A and B). These morphological alterations are definitive markers of apoptotic progression, confirming the compound’s pro-apoptotic effects across different cancer cell types.

Fig. 2
figure 2

Detection of apoptotic cell death by Hoechst 33,342 staining. (A) and (B) represent Hoechst 33,342 stained MCF-7 and SMMC-7721 cells, respectively. Images were captured at 20× magnification. A white scale bar (50 μm) is shown in the control or treatment images.

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Detection of early and late apoptosis by FITC-annexin V/PI staining

The number of apoptotic MCF-7 and SMMC-7721 cells was very low before treatment with Eltrombopag olamine (Fig. 3A and B). Eltrombopag olamine treatment significantly increased the proportion of late apoptotic MCF-7 cells, while a remarkable increase in both early and late apoptotic SMMC-7721 cells was observed by fluorescence microscopy (Fig. 3A and B).

Fig. 3
figure 3

Detection of early and late apoptosis using FITC-annexin V/PI staining. (A) and (B) show the detection of early and late apoptosis in MCF-7 and SMMC-7721 cells, respectively, using fluorescence microscopy. Images were captured at 20× magnification. A white scale bar (50 μm) is shown in the control or treatment images. (C) and (D) show flow cytometry results for MCF-7 and SMMC-7721 cells, respectively. “co” represents control cells and “tr” represents Eltrombopag olamine treated cells. Flow cytometry quadrants Q1-LL, Q1-LR, Q1-UR, and Q1-UL indicate live cells, early apoptotic cells, late apoptotic cells, and necrotic cells, respectively.

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Detection of apoptotic and necrotic death cells by flow cytometry

Before treatment, MCF-7 cells showed 87.46% live, 3.01% early apoptotic, 3.14% late apoptotic, and 6.39% necrotic death cells. After treatment with Eltrombopag olamine, the number of late apoptotic cells increased significantly (59.23%) (Fig. 3C). On the other hand, after treatment of SMMC-7721 cells with Eltrombopag olamine, late apoptotic cells increased from 5.12% to 21.21% and necrotic cells rose from 9.74 to 29.29% (Fig. 3D).

Detection of alteration in gene expression

Eltrombopag olamine treatment led to the upregulation of several pro-apoptotic genes, including those involved in both intrinsic and extrinsic apoptotic pathways, in MCF-7 and SMMC-7721 cells. In MCF-7 cells, oncogenic signaling markers such as EGFR and ERK were downregulated, while NOTCH expression remained unchanged. In SMMC-7721 cells, pro-apoptotic gene expression also increased, whereas multiple oncogenic and survival-related genes, including STAT3, MAPK, NOTCH, PARP1, EGFR, and ERK, were markedly downregulated. WNT1 gene expression was completely abolished in both cell lines following treatment with Eltrombopag olamine (Fig. 4).

Fig. 4
figure 4

Effects of Eltrombopag olamine on the expression of several genes in MCF-7 and SMMC-7721 cells as detected by real-time PCR. (A) Relative mRNA expression of FAS, TP53, BAX, TNFα, Cytochrome-C (Cyt-C), NOTCH, EGFR and ERK genes in MCF-7 cells; (B) expression levels of TNFα, TP53, BAX, STAT3, MAPK, NOTCH, PARP1, EGFR and ERK genes in SMMC-7721 cells. The black dotted line indicates the 1.0 expression level.

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Cell cycle arrest

In untreated MCF-7 cells, the sub-G1, G0/G1, S and G2/M phases were 1.23%, 62.5%, 18.14%, and 17.75%, respectively. After treatment with Eltrombopag olamine, the sub-G1 and S phases increased to 2.58% and 21.61%, respectively, with a remarkable decrease to the G2/M phase (11.58%) and G0/G1 phase remained almost similar as untreated control (Fig. 5A). For SMMC-7721 cells, the sub-G1 phase increased from 1.23% to 8.56%, and the S phase increased from 7.56% to 11.54%. The G0/G1 phase and G2/M phase decreased from 78.19% to 72.84% and from 11.96% to 6.85%, respectively (Fig. 5B).

Fig. 5
figure 5

Detection of cell cycle arrest after Eltrombopag olamine treatment. (A) and (B), represent cell cycle phases in MCF-7 and SMMC-7721 cells, respectively. “Co” represents control cells and “Tr” represents cells treated with Eltrombopag olamine.

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In Silico study

Eltrombopag had the highest binding affinity for the EGFR protein with a binding energy of −9.3 kcal/mol. Eltrombopag formed four regular hydrogen bonds with residues Thr761, Val762, Gly833, and Glu734, as well as and Pi sigma interactions with residues Leu834 and Phe699 (Fig. 6A). Eltrombopag had a higher binding affinity for the FAS protein with a binding energy of −8.9 kcal/mol. In this case, two hydrogen bonds were formed with Asp301 and Thr219 and two Pi alkyl interactions were formed with residues Ile297 and Lys300 (Fig. 6B). The binding energy of Eltrombopag to TP53 was − 7.8 kcal/mol. Three hydrogen bonds were formed with Asn200, Ser227 and Thr231 along with different types of interactions with other amino acid residues (Fig. 6C). The binding affinity of Eltrombopag to TNFα was the lowest with a binding energy − 7.4 kcal/mol. In this case, five hydrogen bonds were formed with amino acid residues Ala33, Arg82, Ser86, Tyr87 and Gln125, and four different interactions were also observed (Fig. 6D).

Fig. 6
figure 6

Binding affinity between Eltrombopag and proteins. The 2D figures represent the binding affinity of Eltrombopag with EGFR (A), FAS (B), p53 (C), and TNFα (D), while the 3D figures represent the formation of hydrogen bonds.

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Discussion

Cell proliferation encompasses the series of events through which cells expand in size and undergo division to create additional cells. In healthy tissues, this process is tightly regulated to maintain normal cell function and balance. In cancer, however, these regulatory mechanisms are disrupted, leading to uncontrolled cell growth and tumor formation. As such, inhibiting abnormal proliferation is a key strategy in cancer therapy. Eltrombopag olamine exhibited dose-responsive inhibition of cell proliferation across MCF-7, SMMC-7721, and HEK293T cell line. This dose-response relationship supports its potential efficacy as an anticancer agent and helps inform optimal therapeutic dosing. The IC50 values revealed that MCF-7 cells were more sensitive to Eltrombopag olamine than SMMC-7721 and HEK293T cells, suggesting selective activity toward breast cancer cells while partially sparing normal cells. To explore the underlying mechanism of this antiproliferative effect, various analytical techniques were employed.

Hoechst 33,342 is a fluorescent dye that specifically binds to DNA, enabling visualization of nuclear changes under a fluorescence microscope. During apoptosis, chromatin condenses and fragments, resulting in bright nuclear staining that clearly distinguishes apoptotic cells from healthy ones. Apoptosis was observed following Hoechst 33342 staining in Eltrombopag olamine-treated MCF-7 and SMMC-7721 cells. Fluorescence microscopic analysis using FITC-annexin V/PI staining further confirmed the presence of late apoptosis in both cell lines.

Flow cytometry, a highly sensitive technique for measuring cellular characteristics (e.g., size, granularity, and marker expression), was employed to quantify apoptotic populations. By detecting FITC-annexin V binding (early apoptosis) and PI uptake (late apoptosis/necrosis), this analysis revealed distinct cell death patterns.

MCF-7 cells exhibited predominantly late apoptosis, while SMMC-7721 cells displayed both late apoptosis and necrosis. The cell-type-specific death responses, apoptotic in MCF-7 and apoptotic-necrotic in SMMC-7721, suggest that Eltrombopag olamine’s mechanism of action may be tissue-dependent, with potential implications for its therapeutic selectivity and toxicity profile in different malignancies.

Signaling pathways are key biological processes through which intracellular gene expression is regulated by extracellular signals16. These pathways often involve phosphorylation events, where enzymes called kinases transfer phosphate groups to specific proteins, activating or deactivating them. This cascade effect allows cells to respond rapidly and specifically to environmental cues such as stress, growth factors, or cytokines. Dysregulation of these pathways is frequently observed in cancer. The binding of a ligand to a membrane-bound receptor initiates a signal transduction process that triggers a cascade of intracellular signaling activities involving multiple kinases17. To investigate apoptotic signaling pathways in MCF-7 and SMMC-7721 cells, caspase enzyme expression and several gene levels were analyzed using caspase inhibitors and real-time PCR, respectively. The reduced antiproliferative activity of Eltrombopag olamine in the presence of Caspase-3/7, −8, and − 9 inhibitors in both cell lines indicated the involvement of these caspases in the apoptotic process.

Apoptotic pathways are broadly categorized into intrinsic and extrinsic types. In the intrinsic apoptotic pathway, p53 activates BAX (a pro apoptotic protein), causing release of cytochrome-C (a soluble hemeprotein) from the mitochondria, forming a complex with Apaf-1 and caspase-9. This complex cleaves proCaspase-3/7, activating Caspase-3/7, which then cleaves PARP-1 (a DNA repair enzyme), thereby disrupting the DNA repair mechanism, ultimately triggering apoptosis18,19. In MCF-7 cells, elevated expression of BAX, p53, and cytochrome-C genes was detected, along with the presence of Caspase-3/7 and caspase-9 enzymes, indicating activation of the intrinsic apoptotic pathway. Similarly, in SMMC-7721 cells, the observed upregulation of BAX and p53, downregulation of PARP1, and the presence of Caspase-3/7, −8, and − 9 enzymes further supported the involvement of the intrinsic apoptotic pathway. Chen et al. reported that Eltrombopag could directly activate BAK (BCL2 antagonist/killer 1) protein expression and induce BAK-dependent apoptosis in mouse embryonic fibroblasts (MEFs) and Jurkat cells20. This finding supports the hypothesis that Eltrombopag may target multiple pro-apoptotic proteins, enhancing its ability to selectively induce apoptosis in various cancer cells. Such multi-targeted action is advantageous in overcoming resistance mechanisms often encountered in single-target therapies.

TNFα exhibits dual roles depending on the cellular context. While it can activate survival pathways such as NF-κB, its activation of caspase-8 via death receptor signaling typically leads to apoptosis. The balance between these opposing effects ultimately determines the cell’s fate. Consistent with this, TNFα gene expression was found to be increased, and caspase-8 enzyme detected in both MCF-7 and SMMC-7721 cells following treatment with Eltrombopag olamine. TNFα, a pro-inflammatory cytokine, can either promote cell survival or induce apoptosis by binding to TNFα receptor 1 or 2, with several pathway components involved—including caspase-821. Eltrombopag olamine may bind TNFα and potentially activate caspase-8 via FADD activation, suggesting involvement of death receptor-mediated apoptosis. Caspase-8 then initiates apoptosis by directly activating Caspase-3/7 or by inducing mitochondrial dysfunction in both cell lines. Moreover, the expression of the FAS gene was also significantly upregulated. Eltrombopag olamine activates FAS, which in turn triggers Caspase-3/7 activation through FAS protein and caspase-8 activity, leading to apoptosis in MCF-7 cells22.

The EGFR-RAS-RAF-MAPK-ERK pathway plays a critical role in normal cell proliferation, survival, and differentiation23. It is also a key contributor to oncogenesis and therapy resistance, as mutations or overexpression of EGFR or downstream components often result in persistent activation of survival signals. Consequently, targeting this signaling cascade is a major focus of current cancer therapies. The suppression of this pathway by Eltrombopag olamine underscores its potential as an inhibitor of oncogenic signaling. Upon activation, EGFR triggers MAPK via RAS and RAF proteins24, resulting in ERK activation. Phosphorylated ERK subsequently translocate to the nucleus, where it phosphorylates transcription factors to modulate gene expression23. Treatment with Eltrombopag olamine significantly decreased the gene expression of EGFR, MAPK, and ERK in SMMC-7721 cells, while only ERK expression was reduced in MCF-7 cells. These results suggest that Eltrombopag olamine induces apoptotic cell death in both cell lines, at least in part by inhibiting the EGFR-RAS-RAF-MAPK-ERK signaling pathway.

STAT3 regulates genes involved in cell cycle progression, anti-apoptotic signaling, and angiogenesis25. Constitutive activation of STAT3 is common in many cancers, leading to uncontrolled cell growth26. Therefore, inhibiting STAT3 via EGFR downregulation by Eltrombopag offers a promising approach to impair tumor survival and proliferation. In hepatocellular carcinoma, STAT3’s oncogenic role includes promoting proliferation, migration, invasion, stemness, anti-apoptosis, immune suppression, and angiogenesis by regulating the transcription of various oncogenic target genes27. EGFR activates STAT3 via tyrosine phosphorylation, promoting its dimerization and nuclear translocation to regulate gene transcription28. Additionally, reduced expression of both EGFR and STAT3 in SMMC-7721 cells may further drive apoptotic cell death.

The WNT-β-catenin signaling pathway plays a crucial role in embryonic development and tissue homeostasis29. However, dysregulated WNT1 activation in cancer drives uncontrolled proliferation30,31, enhanced migration, and evasion of apoptosis, positioning this pathway as a key therapeutic target for novel anticancer strategies. Central to this pathway is WNT1 signaling, which is crucial for the growth and metastasis of various cancers and whose components are considered potential biomarkers and therapeutic targets32. When WNT1 binds to Frizzled and LRP receptors, it stabilizes β-catenin, allowing it to translocate to the nucleus and bind to TCF transcription factors, thereby converting them from repressors to activators of target gene expression33. Treatment with Eltrombopag olamine completely abolished WNT1 gene expression in both MCF-7 and SMMC-7721 cells, highlighting its potential to disrupt this oncogenic signaling pathway. Importantly, the simultaneous downregulation of both EGFR and WNT1 pathways by Eltrombopag olamine is particularly noteworthy. These two pathways are independently known to promote tumor proliferation, survival, and metastasis in various cancers30,34. Dual inhibition of EGFR and WNT1 is rarely observed with a single agent, especially with a repurposed drug not originally developed for oncology. This finding underscores the unique multi-targeted potential of Eltrombopag olamine and may provide a significant therapeutic advantage in overcoming resistance mechanisms associated with monotherapy targeting either pathway alone.

NOTCH signaling mediates cell-to-cell communication and plays a critical role in determining cell fate35,36. Depending on the cellular context and cancer type, it can function either as a tumor suppressor or as an oncogene35. In hepatic cancer models, the observed reduction in NOTCH expression in SMMC-7721 cells may reflect its involvement in promoting tumor progression. NOTCH signaling is essential for cell development and differentiation, and its dysregulation has been implicated in various cancers37. In this study, while NOTCH gene expression was not detected in MCF-7 cells following treatment with Eltrombopag olamine, a significant decrease was observed in SMMC-7721 cells. A graphical summary of these gene expression changes, including caspases, is presented in Fig. 7.

Fig. 7
figure 7

Overall results of gene expression and caspase detection shown graphically.

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The cell cycle progresses through four distinct stages—G1 phase, S phase, G2 phase and M phase. Dysregulation of this process can engage checkpoint mechanisms, prompting either DNA repair or activation of apoptotic pathways. In cancer therapy, inducing cell cycle arrest provides time for apoptotic mechanisms to fully activate, thereby enhancing treatment effectiveness. Arrest during the S phase is often linked to inhibited DNA synthesis and replication stress. Overall, cell cycle arrest halts cell division and growth, which can ultimately lead to apoptosis38. Eltrombopag olamine treatment induced S phase arrest in both MCF-7 and SMMC-7721 cell lines. Additionally, a significant increase in the sub-G1 phase was observed in SMMC-7721 cells, indicative of apoptosis caused by fragmented low molecular weight DNA released from these cells.

Molecular docking is a valuable computational method used in structural biology and drug design to predict how small molecules interact with biological macromolecules at the atomic level39. These in silico simulations help identify key binding sites, estimate interaction energies, and guide experimental design and drug optimization. Protein-ligand docking provides insights into active site recognition, ligand flexibility, and the molecular basis of binding. In this study, Eltrombopag demonstrated strong binding affinity to the 3D structures of EGFR, FAS, TP53, and TNFα proteins. These interactions suggest that Eltrombopag may activate or modulate these proteins, contributing to apoptosis through multiple signaling pathways.

While this investigation elucidates important anti-cancer properties of Eltrombopag olamine, several limitations should be noted. The study was confined to in vitro assessments using two cancer cell lines, without validation through in vivo models. Furthermore, potential synergistic effects with other chemotherapeutic agents remain unexplored, representing an important avenue for future research.

Conclusion

Eltrombopag olamine exhibits significant antiproliferative and pro-apoptotic activity in MCF-7 and SMMC-7721 cancer cell lines by targeting multiple molecular pathways. Through modulation of EGFR, FAS, TP53, TNFα, and downstream effectors, it engages both intrinsic and extrinsic apoptotic mechanisms. These findings support further investigation of Eltrombopag olamine as a potential repurposed therapeutic for breast and liver cancers.