ER signaling plays a pivotal role in the proliferation and survival of ER+ breast cancers (36). In specific ER+ cell-line models, ATA showed stronger antiproliferative activity than tamoxifen under the tested experimental conditions (21).

In vitro studies demonstrate that ATA inhibits ER+ breast cancer cell growth in a dose-dependent manner over a concentration range of 1.56–25 µM, with selective suppression of ER+ cells observable at ~3 µM. The half-maximal inhibitory concentration (IC50) of ATA against ER+ breast cancer cell lines is ~1.4–1.5 µM, markedly lower than that of tamoxifen, while 6 µM has been established as a standard effective concentration for downstream mechanistic investigations. Mechanistically, the active ATA metabolite HTA directly binds to ERα and promotes its nuclear degradation via a ubiquitin-proteasome-dependent pathway, while simultaneously downregulating the transcription of estrogen receptor 1 (ESR1), the gene encoding ERα. This dual mechanism distinguishes ATA from classical selective ER degraders such as fulvestrant. In addition, ATA suppresses the transcription of ER-responsive genes, including growth regulating estrogen receptor binding 1, thereby effectively inhibiting ER-driven proliferation in breast cancer cells (21). Consistent with these findings, combination treatment with Tan IIA and fulvestrant produces synergistic antitumor effects in ER+ breast cancer models. In ER+ ZR-75-1 ×enograft tumors, combined therapy significantly inhibited tumor growth and elicited earlier therapeutic responses compared with either agent alone. Importantly, ¹⁸F-fluoroestradiol positron emission tomography/CT imaging enabled real-time monitoring of treatment response, with quantitative imaging signals closely correlating with intratumoral ERα expression, further validating the synergistic interaction between Tan IIA and fulvestrant in vivo (37). Thus, ERα degradation represents one of the more pharmacologically plausible and subtype-defined mechanisms, but it is primarily supported by ATA/HTA data in ER⁺ models rather than by evidence that native Tan IIA produces identical ERα target coverage in vivo.

Tan IIA has also shown antiproliferative effects in ER⁻ models, suggesting that ER-independent mechanisms may contribute under certain experimental conditions. Both in vitro and in vivo studies demonstrate that Tan IIA exhibits greater antitumor efficacy than tamoxifen in ER⁻ models, potentially through downregulation of p53 and Bcl-2 expression and suppression of genes involved in cell cycle progression, proliferation, apoptosis and DNA synthesis (38). Tan IIA displays an IC50 value of ~0.25 µg/ml in both ER+ and ER⁻ breast cancer cell lines, reflecting potent and relatively non-subtype-restricted antiproliferative activity. In vitro, Tan IIA inhibits cell growth in a dose-dependent manner over a concentration range of 0.0625–1.0 µg/ml, with 0.25 µg/ml commonly used for mechanistic studies. In vivo, subcutaneous administration of Tan IIA at 30 mg/kg significantly suppresses tumor growth and induces apoptosis, supporting its antitumor efficacy in animal models (38). Advances in nanomedicine have further expanded the therapeutic potential of Tan IIA by addressing its PK limitations and enhancing tumor targeting. Given that ER is overexpressed in various tumor cells, it has been explored as a target receptor for nanoparticle-based drug delivery. Molecular docking studies suggest that Tan IIA, acting as a phytoestrogen, possesses high ER-binding affinity and can be effectively incorporated into engineered nanocarriers. A modified derivative, Tan IIA-NH2, demonstrates favorable biocompatibility, enhanced tumor-targeting capability, robust antitumor efficacy and anti-metastatic potential. Furthermore, mesoporous silica nanoparticles co-loaded with Tan IIA and doxorubicin (Dox) (Tan-Dox-MSN) exhibit uniform particle size distribution, good dispersion and high drug-loading capacity. Both in vitro and in vivo experiments show that Tan-Dox-MSN achieves superior tumor accumulation and antitumor effects while significantly reducing systemic toxicity to normal tissues. These findings highlight the potential of leveraging high-ER-affinity phytoestrogens such as Tan IIA to improve nanodelivery systems for breast cancer therapy (22). However, ER-mediated targeting strategies should be evaluated carefully because ER expression is not restricted to tumor tissues, and systemic ER modulation may affect endocrine and inflammatory homeostasis in normal tissues (Fig. 1).

Dox and paclitaxel (taxol) remain frontline chemotherapeutic agents for breast cancer; however, their long-term use often results in drug resistance and severe dose-dependent toxicities, including cardiotoxicity, myelosuppression and weight loss (39,40). Because BCSCs contribute to drug resistance and relapse (41), this section focuses on chemosensitization in resistant models, whereas BCSC-specific mechanisms are discussed separately below. Accumulating evidence demonstrates that Tan IIA markedly enhances the sensitivity of breast cancer cells, including both parental MCF-7 cells and drug-resistant sublines such as MCF-7/Dox, to Dox and taxol (42–47). These chemosensitizing effects involve multiple complementary mechanisms, including inhibition of drug efflux transporters, modulation of oncogenic signaling pathways and enhancement of the overall efficacy of combination chemotherapy.

Mechanistically, Tan IIA downregulates key ATP-binding cassette transporters, including P-glycoprotein, breast cancer resistance protein and multidrug resistance-associated protein 1, thereby increasing intracellular accumulation of Dox and effectively eliminating both drug-sensitive and drug-resistant breast cancer cells (42,46). In parallel, Tan IIA suppresses activation of the PTEN/AKT pathway and inhibits β-catenin nuclear translocation, further restoring chemosensitivity in resistant cells (42,43). Consistent with these observations, Li et al (42) reported that Tan IIA, at concentrations described as non-toxic in the tested cell models (≤20 µg/ml), significantly potentiated the cytotoxicity of Dox, particularly in resistant breast cancer cells, by enhancing intracellular drug accumulation. However, these concentrations remain relatively high from a translational PK perspective and therefore require in vivo exposure confirmation. Combination treatment markedly reduced the IC50 of Dox, resulting in an ~3.9–4.4-fold increase in chemosensitivity compared with Dox alone (42).

Tan IIA also enhances the efficacy of taxol in resistant breast cancer models. In taxol-resistant MCF-7 cells, Tan IIA suppresses the expression of the microtubule-associated protein Tau, a known mediator of taxol resistance, thereby potentiating taxol-induced cytotoxicity (44). Notably, Tan IIA itself exhibits stronger antiproliferative activity than taxol in this model, with IC50 values of 8.4 µM for Tan IIA vs. 23.7 µM for taxol. Combination treatment within concentration ranges of 1–20 µM (Tan IIA) and 5–100 µM (taxol) predominantly produced additive effects, indicating that Tau suppression may contribute to improved taxol responsiveness in this resistant model. These data support an additive interaction in this model rather than a universally synergistic effect (44).

Beyond tumor-specific chemosensitization, Tan IIA exerts dual regulatory effects on ERK1/2 signaling that contribute to both efficacy enhancement and toxicity mitigation. In breast cancer cells, Tan IIA suppresses ERK1/2 activation to promote apoptosis, whereas in cardiomyocytes, it activates ERK1/2 signaling, thereby alleviating Dox-induced cardiotoxicity (45). Another study further revealed that Tan IIA can reverse hypoxia-induced Dox resistance and suppress epithelial-mesenchymal transition (EMT). Under hypoxic conditions, breast cancer cells such as MCF-7 and HCC1937 exhibit reduced E-cadherin expression and elevated vimentin levels, consistent with EMT and acquired chemoresistance. Treatment with Tan IIA restores these markers toward basal levels, significantly reduces cell viability and proliferation, and likely mediates these effects through downregulation of hypoxia-inducible factor 1α and TWIST family basic helix-loop-helix transcription factor-dependent signaling pathways. Notably, under hypoxic conditions, Tan IIA at 10 µM effectively reverses resistance to Dox (0.2 µg/ml), restoring drug sensitivity in breast cancer cells (47). The opposite regulation of ERK1/2 in tumor cells and cardiomyocytes also illustrates the context-dependent pharmacology of Tan IIA and supports the need to evaluate tissue-specific responses rather than assuming uniform pathway inhibition.

Collectively, available preclinical evidence suggests that Tan IIA can enhance the activity of Dox and taxol in selected breast cancer models, particularly resistant cell populations (42–47). However, the magnitude and nature of the interaction vary across models, ranging from additive to synergistic effects, and require confirmation at pharmacologically achievable exposures (Fig. 2).

Apoptosis and cell-cycle arrest are best interpreted as convergent downstream phenotypes rather than a single uniform mechanism, because the effective concentrations and upstream triggers vary considerably among derivatives and cell lines. Aberrant proliferation and dysregulated apoptosis in breast cancer cells are major contributors to tumor progression and therapeutic resistance (48,49). Tan IIA and its derivatives exert antitumor effects predominantly through the induction of apoptosis and cell cycle arrest across multiple breast cancer subtypes. Among these compounds, 1-hydroxy-Tan IIA has been identified as one of the most active derivatives. These relatively high concentrations suggest that this derivative may be useful as a mechanistic probe, but its direct pharmacological relevance requires further optimization. In MCF-7 and MDA-MB-231 breast cancer cells, 1-hydroxy-Tan IIA promotes DNA fragmentation, upregulates the pro-apoptotic protein Bax, downregulates the anti-apoptotic protein Bcl-2 and induces poly(ADP-ribose) polymerase cleavage, thereby triggering apoptosis (19). Consistent with these mechanistic findings, tanshinone derivatives isolated from Stachys parviflora exhibit measurable in vitro antitumor activity, with 1-hydroxy-Tan IIA showing the strongest cytotoxicity, characterized by an IC50 of ~22 µg/ml over a concentration range of 2.5–100 µg/ml (19). In addition to mitochondrial apoptosis, Tan IIA can induce cell death through ER stress-mediated pathways. In human breast cancer BT-20 cells, Tan IIA inhibits cell proliferation in both dose- and time-dependent manners, significantly increases the proportion of sub-G1 cells and activates ER stress-related apoptotic signaling, including caspase-12, growth arrest and DNA damage-inducible protein 153/C/EBP homologous protein, phosphorylated c-Jun N-terminal kinase (p-JNK) and phosphorylated p38 MAPK. Concurrently, Tan IIA suppresses the expression of anti-apoptotic Bcl-xL and phosphorylated ERK, collectively indicating the involvement of ER stress and MAPK pathway modulation in apoptosis induction (50). Quantitatively, Tan IIA exhibits time-dependent cytotoxicity in BT-20 cells, with IC50 values of 3.3 µg/ml at 24 h, 1.87 µg/ml at 48 h and 0.67 µg/ml at 72 h, within an effective concentration range of 0.25–8 µg/ml (50). In TNBC MDA-MB-231 cells, Tan IIA similarly suppresses proliferation in a dose- and time-dependent manner, markedly increases the sub-G1 population and activates apoptotic signaling by upregulating Bax and downregulating Bcl-2 (20).

Additionally, Tan IIA inhibits breast cancer cell proliferation and migration through modulation of the G protein-coupled estrogen receptor (GPER)/EGFR/ERK/c-Fos/c-Jun pathway. Flow cytometry and Transwell assays demonstrate that Tan IIA induces apoptosis and suppresses migration in MDA-MB-231 cells, accompanied by reduced expression of c-Fos, c-Jun and cell cycle-associated proteins. These effects are partially reversed by GPER inhibitors, implicating GPER as a key mediator. In vivo, oral administration of Tan IIA significantly reduced xenograft tumor volume and weight, with concomitant NF-κB downregulation and caspase-3 upregulation in tumor tissues (51). In MDA-MB-231 ×enograft models, long-term oral treatment with Tan IIA (20 or 60 mg/kg, 90 days) inhibited tumor growth, decreased NF-κB p65 and increased caspase-3 expression, underscoring apoptosis activation as a central mechanism (52). In the 4T1 breast cancer model, Tan IIA showed antiproliferative and pro-apoptotic effects associated with changes in key molecules, including p53, NF-κB, AKT, MYC and BCL-2, as well as enrichment of the p53, PI3K/AKT, MAPK and mTOR pathways. Molecular docking further suggested possible interactions between Tan IIA and proteins such as p53, Bcl-2 and NF-κB; however, these docking results should be considered hypothesis-generating unless validated by biochemical binding or in vivo target-engagement assays (53).

To overcome its poor water solubility and bioavailability, imidazole groups were introduced to generate a series of derivatives (TA01-TA12), which significantly inhibited proliferation, migration and invasion of MDA-MB-231 cells, with TA12 showing the most potent activity. In zebrafish xenograft models, TA12 effectively blocked cancer cell metastasis, potentially through S-phase arrest, ROS generation and DNA damage induction (54). Further in vitro and in vivo experiments demonstrate that Tan IIA inhibits breast cancer cell proliferation in a dose- and time-dependent manner, markedly reduces colony formation and BrdU incorporation, and regulates a broad genetic network. Gene expression profiling revealed that Tan IIA upregulated 41 and downregulated 24 genes associated with cell cycle regulation, proliferation, apoptosis, signal transduction, transcriptional regulation and cell adhesion. In a nude mouse xenograft model of invasive ductal carcinoma, intraperitoneal administration of Tan IIA (30 mg/kg) reduced tumor volume by 44.91%, with concomitant caspase-3 upregulation, indicating that its antitumor effects are mediated by multi-gene network regulation and apoptosis signaling activation. These findings collectively support the broad-spectrum antitumor activity of Tan IIA in both ER-positive and ER-negative breast cancers (55) (Fig. 3).

BCSCs are critical drivers of drug resistance and tumor relapse, with inflammatory cytokines and associated signaling pathways playing key roles in their maintenance (56). The IL-6/STAT3/NF-κB axis is therefore discussed here as a BCSC-maintenance pathway, while its contribution to drug resistance is cross-referenced in the chemosensitization section. Tan IIA, owing to its combined anticancer and anti-inflammatory activities, has been shown to markedly inhibit BCSC proliferation and mammosphere formation. In vitro, Tan IIA treatment significantly reduced BCSC proliferation and sphere-forming capacity, accompanied by decreased proportions of CD24⁻/CD44+ and aldehyde dehydrogenase 1+ subpopulations. Expression levels of inflammatory signaling proteins, including IL-6, STAT3, phosphorylated STAT3 (Tyr705), nuclear NF-κB p65 and cyclin D1, were also significantly suppressed. In vivo, Tan IIA administration inhibited tumor growth and reduced average tumor weight in xenograft models, confirming its capacity to effectively target and suppress BCSC-like populations (57). Quantitatively, Tan IIA exhibits stronger inhibitory activity against BCSCs than against parental breast cancer cells, with an IC50 of ~0.40 µg/ml for BCSCs compared with 0.65 µg/ml for bulk tumor cells, and an effective in vitro concentration range of 0.125–2.0 µg/ml (57). In the study by Li et al (58), Tan IIA effectively inhibited BCSC-associated traits within a concentration range of 2.5–20 µM (~0.74–5.92 µg/ml). Mechanistic analyses further demonstrated that Tan IIA attenuates BCSC stemness through modulation of the microRNA (miR)-125b/StAR-related lipid transfer domain protein 13 (STARD13) regulatory axis. Specifically, Tan IIA downregulates miR-125b while concomitantly upregulating its target gene STARD13, thereby suppressing miR-125b/STARD13 signaling, reducing stem-like properties and enhancing Dox sensitivity in breast cancer cells (58). Overexpression of miR-125b or knockdown of STARD13 reversed the inhibitory effects of Tan IIA on BCSC stemness, further validating the central role of this regulatory axis. Collectively, these findings indicate that Tan IIA suppresses BCSC properties and tumor-initiating capacity through inhibition of IL-6/STAT3/NF-κB signaling and modulation of the miR-125b/STARD13 axis, underscoring its therapeutic potential in overcoming drug resistance and preventing tumor recurrence (Fig. 4). These findings suggest that Tan IIA may suppress BCSC-like traits in selected models, but the durability of this effect and its contribution to relapse prevention remain to be validated in clinically relevant in vivo systems.

Ferroptosis is an iron-dependent form of programmed cell death that has emerged as a novel anticancer strategy (59,60). Tan IIA has recently been linked to ferroptosis regulation in breast cancer models. Mechanistic studies showed that Tan IIA downregulated lysine-specific histone demethylase 1A (KDM1A) expression, thereby reducing the transcriptional activity of protein inhibitor of activated STAT 4 (PIAS4) and inhibiting PIAS4-mediated SUMOylation of solute carrier family 7 member 11 (SLC7A11). Destabilization of SLC7A11 impaired cystine uptake and antioxidant defense, leading to lipid peroxide accumulation, increased labile iron and ferroptotic cell death in breast cancer cells (61).

In vitro, Tan IIA treatment suppressed breast cancer cell proliferation, colony formation and migration, while in vivo xenograft and lung metastasis models further supported its tumor-suppressive activity (61). However, ferroptosis-related effects have been reported over a broad concentration range, including high micromolar exposures. Therefore, ferroptosis should currently be regarded as a mechanistically interesting pathway but with PK-related uncertainty for native Tan IIA, particularly in light of its poor bioavailability, rapid metabolism and limited evidence of sustained tumor exposure.

Collectively, these findings identify the KDM1A/PIAS4/SLC7A11 axis as a potential mechanistic link between Tan IIA and ferroptotic vulnerability in breast cancer. Nevertheless, further PK/PD studies are required to determine whether this pathway can be engaged at achievable tumor concentrations and whether ferroptosis contributes substantially to the in vivo antitumor effects of Tan IIA or its optimized derivatives (Fig. 4).

HER2-overexpressing breast cancer cells frequently depend on HER2/EGFR signaling and downstream anabolic pathways, including lipid synthesis and protein biosynthesis (62,63). These metabolic pathways have thus emerged as attractive therapeutic targets. ATA, a derivative of Tan IIA, demonstrates superior antitumor activity compared with its parent compound in HER2-overexpressing breast cancer. In cell lines such as MDA-MB-453, SK-BR-3 and BT-474, ATA induced G1/S phase arrest and apoptosis, associated with marked downregulation of EGFR/HER2 receptor tyrosine kinases and inhibition of downstream prosurvival signaling. Furthermore, ATA triggered oxidative stress and endoplasmic reticulum stress, while activating the AMP-activated protein kinase (AMPK) signaling pathway, thereby inactivating key enzymes involved in lipid and protein biosynthesis and disrupting metabolic dependencies of tumor cells. In vivo, intraperitoneal administration of ATA significantly suppressed xenograft tumor growth in MDA-MB-453 models without inducing body weight loss or overt toxicity. Functional assays further demonstrated that ATA inhibited breast cancer cell migration, invasion and angiogenesis in vitro (64) (Fig. 4). Whether these findings are relevant to HER2-low or HER2-ultralow breast cancer remains unknown. Given the clinical expansion of HER2-directed ADCs, future studies should determine whether Tan IIA derivatives influence ADC sensitivity or resistance by altering HER2 expression, endocytosis, EMT oxidative stress or payload susceptibility.

Emerging evidence from non-breast cancer models suggests that Tan IIA may enhance anti-programmed cell death 1 (PD-1) efficacy by modulating tumor vasculature, endoplasmic reticulum stress, JNK signaling and immune-cell infiltration (28,29). However, direct evidence in breast cancer remains limited, and these findings should be regarded as hypothesis-generating. Future studies should evaluate whether Tan IIA-derived compounds can remodel the breast tumor microenvironment, enhance checkpoint blockade or influence PD-1/PD1 ligand 1 responsiveness in TNBC or other immunologically active breast cancer subtypes.

Because of their planar heterocyclic scaffold, certain Tan IIA derivatives have been investigated as G4 stabilizers. G4 DNA structures represent promising anticancer targets due to their regulatory roles in oncogene transcription and translation (65,66). A series of imidazole-modified Tan IIA derivatives (compounds 1–8), particularly compound 4, exhibited strong selectivity and binding affinity toward G4 DNA. These derivatives effectively stabilized multiple G4 structures, including those in c-Myc, K-ras and VEGF, leading to DNA damage and suppression of TNBC cell growth, metastasis and angiogenesis. Molecular docking and interaction studies further confirmed that these derivatives preferentially bind G4 DNA over duplex DNA, highlighting their specificity. These results provide a rationale for developing selected Tan IIA derivatives as G4-targeting leads, although their pharmacological relevance requires further PK/PD and target-engagement validation (67) (Fig. 4). Importantly, G4 stabilization is currently best supported for structurally optimized derivatives rather than for native Tan IIA itself. Therefore, this mechanism should be interpreted as a derivative-specific optimization strategy.