Breast cancer is the most common malignant tumor among women worldwide, and its occurrence is closely associated with long-term exposure to environmental pollutants. Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants widely present in the living environment. Epidemiological studies indicate that exposure to PAHs increases the risk of breast cancer. PAH derivatives exhibit stronger toxicity or endocrine-disrupting activity than their parent compounds; however, research on their specific effects and mechanisms in breast cancer cells remains limited. For this purpose, this study selected 3-Hydroxybenz[a]anthracene, a PAH derivative with potential estrogenic activity, as the target compound. Using the estrogen receptor-positive breast cancer cell line MCF-7 as the model, we performed EdU staining, colony formation assays, scratch healing assays, Transwell invasion assays, and apoptosis assays and preliminarily examined changes in relevant signaling proteins via Western blot. Results indicate that 3-Hydroxybenz[a]anthracene promotes proliferation and migration in MCF-7 cells while inhibiting apoptosis under certain conditions, but it has no effect on cell invasion. Mechanistically, it upregulates key proteins including AKT, c-Myc, E-Cadherin, Vimentin, MMP2, MMP9 and Bcl-2 while downregulating p-AKT expression. This study confirms through in vitro experiments that 3-Hydroxybenz[a]anthracene exhibits estrogen-like effects and modulates malignant behavior in breast cancer cells by regulating relevant signaling pathways. These findings provide experimental evidence for further evaluating the potential role of this environmental contaminant in breast cancer initiation and progression.

3α-Hydroxysteroid dehydrogenase type 3 (3α-HSD3) is a key steroid-metabolizing enzyme involved in the regulation of intratumoral androgen and estrogen levels, predominantly in estrogen receptor-positive (ER⁺) MCF-7 breast cancer cells. Its dysregulation influences proliferation, survival, and resistance to hormone-based therapy. The main goal of this study is to identify potential natural inhibitors of 3α-HSD3 from Terminalia arjuna phytoconstituents to modulate MCF-7 breast cancer cell proliferation. A panel of nine phytochemicals were evaluated through molecular docking, ADMET profiling, HOMO-LUMO energy gap analysis, and prediction of activity spectra for substances (PASS) to assess their drug-likeness and preliminary activity. Subsequently, molecular dynamics (MD) simulations were performed on top candidates to explore the structural stability and interaction dynamics of protein-ligand complexes. The selected compounds exhibited favorable docking scores against 3α-HSD3, with Luteolin(CMP1), Leucocyanidin(CMP2), Gallic Acid(CMP3), and Ellagic Acid(CPM4) forming stable hydrogen bonds with key active site residues. This study showed that the top four compounds may serve as potential 3α-HSD3 inhibitors of breast cancer, warranting further in vivo validation.

Long-term recurrence in breast cancer is driven by reactivation of dormant disseminated tumor cells (DTCs) and remains a major clinical challenge, particularly in estrogen receptor-positive (ER+) tumors. This process is underpinned by regulation of the cell cycle machinery that controls quiescence maintenance and exit. HES1, a Notch pathway transcription factor, regulates key cell cycle genes and has been shown to demonstrate protein expression oscillations. Here, we sought to establish whether HES1 oscillations may regulate ER+ cancer cell quiescence and reactivation. To investigate this, we developed a fundamental in vitro model of cell cycle arrest and reentry based on reversible CDK4/6 inhibition (CDK4/6i) with palbociclib, compatible with quantitative single-cell live-imaging of a knock-in endogenous HES1 reporter. Consistent with earlier findings, HES1 exhibited ~24 h protein oscillations in cycling cells demonstrating a reproducible dip in protein expression prior to S-Phase. During CDK4/6i-mediated arrest, the ~24 h HES1 oscillation was lost, HES1 levels were maintained at a moderately higher level and HES1 exhibited smaller dips. Similar changes were observed in unperturbed, spontaneously quiescent cells. Following release from CDK4/6i and cell cycle reentry, these alterations were reversed and the characteristic G1/S HES1 dip was observed. Preventing this dip at the point of release, by inducibly sustaining HES1 with a Tet-On system, upregulated the cell cycle inhibitor p21, impeded cell cycle reentry and induced cell death. These findings suggest that manipulating HES1 dynamics could represent a promising therapeutic approach to prevent reactivation of dormant tumor cells.

Abstract Introduction The ESR1 gene encodes the estrogen receptor alpha (ERα), a transcription factor that regulates gene expression in response to estrogen. Mutations in ESR1 can lead to constitutive activation of ERα, allowing estrogen receptor-positive (ER+) breast cancer cells to proliferate even in the absence of estrogen; this proliferation contributes to resistance against standard hormone therapies such as treatment with aromatase inhibitors (AI), which function by reducing estrogen levels. ESR1 mutations are typically acquired during treatment and are most frequently observed in metastatic breast cancer. Detecting ESR1 mutations, often through circulating tumor DNA (ctDNA) in liquid biopsies, is critical for tracking disease progression and optimizing therapeutic strategies. As reported at the recent ASCO 2025 meeting, treatment switch before clinical progression based on the detection of ESR1 mutations in ctDNA can potentially improve outcomes in HR+/HER2- advanced breast cancer. Thus, identifying ESR1 mutations enables clinicians to make informed treatment decisions and supports the development of targeted approaches to overcome endocrine resistance. Molecular assays such as digital PCR (dPCR), real-time PCR (RT-qPCR), and next-generation sequencing (NGS) are commonly employed for detection of ESR1 mutations, with each platform offering distinct advantages in sensitivity and mutation coverage. However, a common limitation of these technologies is their sensitivity, which is compounded by the limited quantity of ctDNA that can be isolated from human specimens. To address these limitations, we developed a novel assay specifically designed for the detection of ESR1 mutations; the assay leverages the high specificity and sensitivity of the SuperRCA® technology developed by Rarity to enable accurate identification of low-frequency ESR1 variants. Methods The SuperRCA® uses 2 consecutive rolling circle amplification (RCA) reactions, which enable highly specific genotyping and accurate quantification of low-frequency variants. The sensitivity of the assay for ESR1 was assessed by testing a quantitative, serial dilution of synthetic controls of commonly found resistant variants and numerical positive signals detected using flow cytometry. Assay specificity was assessed by the number of positive signals from cfDNA extracted from normal individuals. Results The assay was capable of detecting rare ESR1 mutations at a sensitivity of 0.01% VAF down to 0.001% VAF. The number of positive signals was essentially zero for normal individuals , indicating high specificity. Conclusions These results demonstrate the SuperRCA® can be used to detect clinically relevant ESR1 variants, which may offer a powerful tool for improving the management of endocrine-resistant breast cancer.

Abstract Background: PI3K/AKT pathway activation is implicated in resistance to endocrine therapy (ET) + CDK4/6i. Simultaneous inhibition of PI3K/AKT and CDK4/6 pathways may delay ET and/or CDK4/6i resistance, and/or re-sensitize tumors to ET plus CDK4/6i. Results from Phase 1b of the CAPItello-292 Phase 1b/3 trial (NCT04862663) have shown that triplet therapy with pan-AKT inhibitor capivasertib + CDK4/6i (palbociclib or ribociclib) + fulvestrant is tolerable in patients with HR+/HER2− ABC, with preliminary evidence of clinical activity. Most Phase 1b patients were heavily pre-treated and had received prior CDK4/6i treatment. This exploratory analysis from Phase 1b of CAPItello-292 examined matched pre- and post-treatment circulating tumor DNA (ctDNA) to identify potential genomic mechanisms associated with resistance to the triplet treatment. Methods: Baseline and end-of-treatment (EOT) ctDNA were tested using the Guardant360 Liquid assay on the Guardant Infinity platform. Somatic alterations of known/likely significance with variant allele fraction ≥0.3% (limit of quantification) were evaluated. Copy number changes, single nucleotide variants, fusions, and indels in EOT ctDNA were considered to be acquired only if absent in baseline ctDNA and archival tumor tissue. Separately, genome-wide CRISPR-Cas9 knockout screens were performed in estrogen receptor-positive (ER+) breast cancer cell lines (MCF7, CAMA-1, and T47D) to discover genomic modifiers of response/resistance following 3 weeks of treatment with combination treatments: capivasertib + fulvestrant and fulvestrant + CDK4/6i. Moreover, a stringent genome-wide CRISPR knockout screen (10 days treatment) was conducted in ER+ breast cancer cells to elucidate mechanisms of resistance to capivasertib + fulvestrant + palbociclib. Results: Across the CAPItello-292 Phase 1b population, paired ctDNA samples from 48 patients were analyzed; 42/48 patients discontinued treatment due to disease progression. 259 genetic alterations were detected at baseline and 304 alterations at the EOT. 30 patients had ≥1 newly detected alteration at EOT (30/48, 63%). Alterations in targets downstream of AKT were observed in 15% (7/48) at EOT, compared with 8% (4/48) at baseline, particularly loss-of-function mutations in TSC1, a tumor suppressor that controls mTORC1 signaling, and amplifications in RICTOR, which encodes the catalytic subunit of the mTORC2 complex. RAS pathway alterations were present in 25% (12/48) of patients at EOT versus 17% (8/48) at baseline. Cell cycle gene alterations were detected at EOT in 46% (22/48) versus 42% (20/48) at baseline. Newly detected PIK3CA/PTEN alterations were found at EOT in 6% (3/48) of patients. The specific PIK3CA/PTEN alterations identified at EOT included PIK3CA E545K, PIK3CA H1047R, and PTEN rearrangement, known to be sensitive to capivasertib. CRISPR resistance screen identified loss of TSC1, TSC2 and STK11 (AMPK pathway regulator) as key genes limiting response to capivasertib + fulvestrant. Resistance to the fulvestrant + palbociclib combination was associated with loss of cell cycle control in particular loss of RB. Identified mechanisms of resistance to capivasertib + fulvestrant + palbociclib combination further confirmed that loss of TSC1, TSC2 and STK11 attenuates the effectiveness of the triplet therapy. Conclusions: Consistent with AKT being a pivotal node in the PI3K/AKT signaling pathway, these data show that treatment with capivasertib + fulvestrant + CDK4/6i resulted in emergence of alterations associated with activation of alternate mechanisms, such as mTORC1, RAS, and AMPK. The CRISPR data provides additional support to clinical observations. Citation Format: D. R. Sudhan, C. Salinas-Souza, V. Cutano, I. De Toma, S. Dunn, J. Bradley, P. Neven, M. Beeram, E. Hamilton, B. Pistilli, A. Raskov Kodahl, P. Lau, V. F. Borges, M. Campone, T. Foukakis, E. Lim, I. Ługowska, P. Wysocki, I. Oshiomogho, V. Bhagawati Prasad, C. Gresty, U. McDermott, S. Barry, E. de Bruin, J. Armenia, H. S. Rugo. Mechanisms of resistance to capivasertib in combination with CDK4/6 inhibitor (CDK4/6i) plus fulvestrant in patients with hormone receptor-positive/HER2-negative (HR+/HER2−) advanced breast cancer (ABC): exploratory analysis from the Phase 1b CAPItello-292 study [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr RF4-04.

A novel class of hybrid compounds, such as thiadiazolyl-pyrazoles (14a-e) and benzothiazolyl-pyrazoles (15a-d), has been successfully synthesized via a simple condensation reaction. Newly synthesized compounds have been characterized by mass, 1H, and 13C NMR spectral analysis. The target compounds have been studied for their DFT docking, antibacterial, antioxidant, and cytotoxicity properties. Among the target compounds, heterocycle 14e showed significant binding with penicillin G acyl-Penicillin (1MWT) and Escherichia coli DNA gyrase B (6KZX) protein target, with binding scores -6.5 and -8.2 kcal/mol, respectively. In addition, the compounds 14c and 14e showed superior activity against both the microbes at different concentrations, whereas compounds 14a and 14e showed excellent antioxidant activity at 100 µg/mL concentration. Among the selected compounds, heterocycle 14e exhibited significant anticancer activity against both triple-negative breast cancer (MDA-MB-231) and oestrogen receptor-positive breast cancer (MCF-7) cell lines, with 68.49 and 57.38% of inhibition, respectively.

Thyroid hormones (THs) regulate metabolism, proliferation, and genomic stability. Clinical studies have linked levothyroxine therapy with higher Oncotype DX Recurrence Scores in breast cancer (BC), suggesting a potential effect of thyroid hormone signaling on genomic risk. Here, we investigated the impact of triiodothyronine (T3) on DNA damage and repair pathways in estrogen receptor-positive T47D breast cancer and non-tumorigenic MCF10A cells. RNA sequencing revealed significant upregulation of RAD51 and enrichment of DNA repair pathways following 24 h T3 exposure. Consistently, T3 increased γH2AX and 53BP1 nuclear foci, indicating transient activation of the DNA damage response (DDR). These effects were transient, returning to baseline after 48 h, suggesting cellular adaptation. T3 also enhanced proliferation at 10 μM but inhibited growth at higher concentrations. Our findings indicate that acute exposure to T3 induces transient genomic stress, providing a potential mechanistic basis for the observed association between thyroid hormone therapy and increased BC recurrence risk.

Our environment, especially our diet, exposes us to numerous bioactive compounds that have the potential to interact with anticancer therapies. Although understanding these exposome-drug interactions is crucial for optimizing anticancer therapy, limited knowledge exists on the interference of contaminants, especially mycotoxins, with anticancer drugs. One of the most prevalent mycotoxins in food and feed is zearalenone (ZEN), which demonstrated estrogenic activity that is even stronger in its metabolite α-zearalenol (α-ZEL). Consequently, in this study, we investigated whether these mycoestrogens affect estrogen-dependent breast cancer therapy with doxorubicin, a standard treatment option. The effects of mycoestrogens on doxorubicin were investigated in MCF7 estrogen receptor-positive breast cancer cells. Cell viability assays revealed that both ZEN (between 1 and 10 µM) and α-ZEL (>0.01 µM) reduced the anticancer activity of doxorubicin. This decrease in doxorubicin activity by α-ZEL, and to a lesser degree ZEN, was due to a decrease in cell death (up to 30 % reduction of annexin V signal by both, 50 % more protein expression of the apoptosis-related proteins B-cell lymphoma 2 and 25 % less Bcl-2-associated X protein by 0.1 µM α-ZEL) and stimulation of the cell cycle progression (up to 6 % more cells in S phase and 5 % less in G2/M phase, and changes in protein expression of cell cycle-related proteins cyclin D1, p16, p21 and p53 by α-ZEL). Application of an estrogen receptor α inhibitor linked the ability of α-ZEL to interfere with cell viability and apoptosis to its activation of the receptor. Consequently, contamination of our diet with mycoestrogens might not only impact cancer growth but also impair the effectiveness of anticancer therapy in patients with estrogen-dependent cancer, warranting greater attention in its management.

To investigate the carcinogenic mechanisms and potential molecular targets of the environmental endocrine disruptor Propylparaben in estrogen receptor-positive breast cancer. Based on a network toxicology strategy, this study first predicted and analyzed the physicochemical properties and in vivo/vitro toxicity of Propylparaben, screened its potential targets, and intersected them with estrogen receptor-positive breast cancer-related genes to obtain candidate toxicological targets. GO, KEGG, and PPI enrichment analyses were subsequently performed to identify key functional modules. Transcriptomic data from breast cancer were integrated, and core targets were screened using LASSO regression, SVM-RFE, and random forest algorithms. A multigene diagnostic model was then constructed, evaluated, and externally validated. Differential expression analysis, protein-level validation, and molecular docking were conducted to confirm expression patterns and binding capabilities of the core targets. Using MCF-7 cells as the in vitro model, dose-dependent Propylparaben intervention experiments were conducted to examine the transcriptional responses of the target genes. Drug sensitivity prediction, survival analysis, and GSEA-based functional annotation were further performed to evaluate the clinical potential and oncogenic mechanisms of the core targets. Propylparaben demonstrated strong estrogen receptor agonist activity, metabolic enzyme inhibition, and moderate carcinogenic risk, suggesting classical endocrine-disrupting properties. A total of 109 candidate toxicological targets associated with estrogen receptor-positive breast cancer were identified and found enriched in carcinogenic and tumor-suppressive pathways such as PI3K/AKT, mTOR, MAPK, and p53. Eight core targets were selected via machine learning, with SLC2A1 and KIF11 showing significant upregulation at both mRNA and protein levels, as well as stable binding affinities with Propylparaben. MCF-7 cell experiments confirmed their dose-dependent transcriptional upregulation upon Propylparaben treatment. Drug sensitivity analysis revealed that high expression of these targets correlated with increased sensitivity to Fulvestrant. KIF11 was predictive of neoadjuvant therapy response, while high SLC2A1 expression was significantly associated with poor survival outcomes. GSEA indicated that high expression of these genes significantly activated glycolysis, cell cycle, mTORC1, MYC, and E2F pathways, while suppressing antitumor mechanisms including p53 and complement cascades. Propylparaben may promote the occurrence and progression of estrogen receptor-positive breast cancer by upregulating SLC2A1 and KIF11, activating metabolic and proliferative pathways, and simultaneously suppressing tumor-suppressive signals.

Selective cytotoxicity of anhydroicaritin in ER-positive breast cancer via ESR1-mediated MAPK and apoptotic signaling.

This study reports the synthesis, characterization and pharmacological evaluation of novel series of 1,3,4-thiadiazole containing pyrazole Schiff base heterocycles. The structural elucidation of the synthesized compounds was performed using 1H and 13C NMR spectroscopy, mass spectrometry (MS) analysis, Fourier Transform Infrared spectroscopy (FT-IR) analysis. The antibacterial activity, antioxidant potential and cytotoxicity studies of newly synthesized compounds were assessed. The compound 8a exhibiting most potent inhibitory effect against S. aureus and E. coli with inhibition zones of 20 and 18 mm, respectively, at 50 µg/mL. The antioxidant potential of the compounds was evaluated using DPPH radical scavenging assay and FRAP assay which envisioned that the compounds 8a, 8c, and 8d showed highest potency. Cytotoxicity studies conducted on triple-negative breast cancer (MDA-MB-231) and estrogen receptor-positive breast cancer (MCF-7) cell lines showed that compound 8c exhibited the strongest anticancer activity with 65.29% inhibition against MDA-MB-231 and 54.36% of inhibition against MCF-7 cell line at 1000 µM concentration. These findings suggest that these hybrid heterocycles hold significant promise as potential candidates for antimicrobial, antioxidant and anticancer applications.

Obesity, a global health challenge, contributes to various cancers, including breast cancer. Complex metabolic dysregulation marks the development of both breast cancer and obesity. Here, the interplay between the obesity-derived adipokine leptin (LEP) and stearoyl-CoA desaturase 1 (SCD), a critical enzyme in fatty acid (FA) metabolism, was explored. While Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database analysis reported a significant protein-protein interaction between LEP and SCD, functional processing of the differentially expressed genes in LEP-treated breast cancer cells revealed a critical involvement of SCD in the interactome of deregulated proteins. Kaplan-Meier analyses linked LEP/SCD expression to poorer recurrence-free survival in patients with estrogen receptor α luminal A-like breast cancer. Functional studies demonstrated that LEP up-regulated SCD expression in luminal A-like breast cancer cells through LEP receptor-mediated signaling. Lipidomic profiling showed that SCD inhibition using MF-438 reduced LEP-induced FA desaturation, characterized by a shift from saturated to monounsaturated FAs. SCD inhibition also abolished the LEP-mediated mitochondrial respiration and ATP production. Additionally, LEP-induced oncogenic features, including enhanced growth and motility, were counteracted by pharmacologic/genetic SCD blockade, confirming SCD's role in leptin's protumorigenic effects. This study highlights the LEP-SCD axis as a driver of metabolic/functional alterations in estrogen receptor α-positive breast cancer, providing insights into the obesity-breast cancer link and identifying potential therapeutic targets (ie, SCD) to counter obesity-driven cancer progression.

Epitransciptomic marks, such as N6-methyladenosine (m6A) within RNA transcripts, have been implicated in multiple pro-tumorigenic activities. These modifications are controlled by writers, readers, and erasers, including the METTL3 m6A-methyltransferase. Recently, changes in expression or activity of epitranscriptomic enzymes have been shown to modulate metabolic pathways in multiple tumor types, including within endocrine-sensitive and -resistant estrogen receptor-positive (ERα+) breast cancer (ER+ BC) cells. Yet, a broad analysis of metabolic alterations, specifically with respect to METTL3 inhibition, has not been explored in these BC subtypes. Herein, we investigated the magnitude of pharmacological targeting of METTL3 (STM2457) on overall cellular metabolism in endocrine-sensitive (MCF-7 and ZR-75-1) and -resistant (LCC9 and ZR-75-1-4-OHT) ER+ BC cells. We found that STM2457 selectively decreased glycolytic activity in resistant cells and led to altered hexokinase 2 expression in LCC9 cells. STM2457 suppressed mitochondrial activity, while isotope tracing found diminished tricarboxylic acid (TCA) glucose oxidation in MCF-7 and LCC9 cell lines. This was accompanied by increased glutamine uptake and glutaminolysis, which was more pronounced in the endocrine-resistant LCC9 cells. We also observed differential expression of glutaminase 1 (GLS1) splice variants in the MCF-7 cells and an increase in the ASCT2 glutamine transporter. To determine combinatorial targeting potential, we co-treated cells with STM2457 and CB-839, which is a GLS1 inhibitor. CB-839 increased the potency of STM2457 only in the LCC9 and ZR-75-1-4-OHT endocrine-resistant cells. Our collective findings suggest that METTL3 inhibition leads to selective glycolytic and oxidative metabolic changes between these endocrine-sensitive and -resistant BC cells, which can be exploited for combinatorial therapy.

Background: Heat shock protein 90 (HSP90) is a molecular chaperone that stabilizes numerous oncogenic proteins and supports tumor survival. Small molecules targeting HSP90 offer a novel approach to overcome drug resistance and immune suppression in breast cancer. Methods: A novel thiazolyl benzodiazepine (TB) containing a hydrazone moiety was evaluated in breast cancer cell lines (ER+ MCF-7, TNBC MDA-MB-231, and HER2+ SK-BR-3). Cytotoxicity was assessed using the CCK-8 assay, followed by PCR sequencing, flow cytometry, RT-qPCR, protein profiling, and HSP90 binding assays. Results: TB showed the strongest activity in MCF-7 cells (IC50 = 7.21 µM) compared to MDA-MB-231 (IC50 = 28.07 µM) and SK-BR-3 (IC50 = 12.8 µM) cells. Mechanistic studies showed that TB binds to HSP90 (Kd = 3.10 µM), leading to disruption of the oncogenic signal. TB induced G2/M cell cycle arrest, promoted apoptosis via Bax and Caspase-3 activation, and suppressed cancer stem cell markers (NANOG, OCT4, SOX2). Additionally, TB activated immune-related pathways via ERK/MAPK signaling and upregulated genes such as SMAD2, SMAD3, and JUN.Conclusions: TB functions as an HSP90 inhibitor with dual anticancer and immunomodulatory properties in Estrogen Receptor-Positive (ER+) breast cancer cells. These findings suggest that TB represents a promising scaffold for the development of multi-targeted breast cancer therapies.

The loss of therapeutic efficacy in breast cancer treatment remains a significant challenge, with increasing evidence that the tumor microenvironment (TME) plays a crucial role in driving resistance. Understanding how stromal cells remodel the TME is critical for advancing cancer treatment strategies and drug development. Among the key cellular components of the TME, fibroblasts are recognized for their pivotal role in cancer progression through extracellular matrix remodeling. To enable in vitro interrogation of tumor-stromal interactions relevant to endocrine response, a microfluidic droplet-based 3D coculture platform was developed to demonstrate the feasibility of uniform breast cancer-fibroblast coculture models. As proof of concept, this system was used to investigate the impact of stromal remodeling by coculturing an estrogen receptor-positive (ER+) breast cancer cell line with either primary fibroblasts or immortalized lung fibroblasts. Collagen I and Ki67 expressions were evaluated following exposure to endocrine therapy. The 3D coculture of ER+ breast cancer cells with primary fibroblasts resulted in two distinct spatial distributions of collagen I, termed "hotspots". One cohort exhibited a single hotspot with reduced collagen I expression, while the other displayed multiple hotspots with increased collagen I expression. Notably, the stratification of these populations was independent of the spheroid size. Coculture with immortalized fibroblasts produced similar hotspot patterns but with higher overall collagen I levels. Notably, during endocrine response studies, an increase in Ki67 and collagen I expression was found in the coculture spheroids, suggesting that fibroblasts contribute to proliferation and modulate endocrine response. Collectively, this study establishes a versatile 3D microfluidic model for probing tumor-stromal interactions and provides a foundation for future mechanistic studies of ECM-mediated remodeling in breast cancer.

Background/Objectives: Breast cancer remains one of the leading causes of cancer-related mortality in women worldwide, highlighting the urgent need for effective and less toxic therapeutic strategies. Thymoquinone (TQ), a bioactive phytochemical derived from Nigella sativa, possesses antioxidant and anticancer activities. Methotrexate (MTX), a widely used folate antagonist, is an established chemotherapeutic agent but is limited by toxicity and resistance. This study aimed to investigate the potential synergistic effects of TQ and MTX in estrogen receptor-positive MCF-7 breast cancer cells. Methods: MCF-7 cells were exposed to TQ (0–100 μM), MTX (0–10 μM), and their combinations for 24–72 h. Cell viability was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and drug interactions were evaluated using the Chou–Talalay method. Apoptosis was quantified by Annexin V/Propidium Iodide (PI) flow cytometry, and cell cycle distribution was analyzed by PI staining. Intracellular reactive oxygen species (ROS) generation was measured using a 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) assay, while antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT)) activities were quantified spectrophotometrically. Gene expression of Bax, Bcl-2, NF-κB, MMP-2, and MMP-9 was determined by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Results: TQ and MTX each reduced cell viability in a dose- and time-dependent manner, while combination treatment significantly enhanced cytotoxicity compared with single agents (p < 0.01). Combination Index (CI) values < 1 confirmed a synergistic interaction, particularly at 50 μM TQ + 5 μM MTX and 100 μM TQ + 10 μM MTX. Combination therapy increased total apoptosis up to 83.6%, markedly elevated the Bax/Bcl-2 ratio, and enhanced caspase-3 activation. Cell cycle analysis revealed pronounced G2/M arrest. ROS levels increased approximately six-fold, accompanied by significant suppression of SOD and CAT activities. qRT-PCR results demonstrated upregulation of pro-apoptotic Bax and downregulation of anti-apoptotic B-cell lymphoma 2 (Bcl-2), nuclear factor kappa B (NF-κB), matrix metalloproteinase (MMP)-2, and MMP-9. Conclusions: TQ potentiates the anticancer activity of MTX in MCF-7 breast cancer cells by synergistically inducing apoptosis, oxidative stress, and cell cycle arrest while suppressing metastasis-related genes. This combination may represent a promising therapeutic strategy for breast cancer, warranting further validation in in vivo and clinical studies.

Zearalenone at environmental levels promoted ER- positive breast cancer cell lines through the hedgehog pathway.

In this study, a smart hybrid liposome system was achieved combining a self-assembling biopolymer conjugate (human serum albumin-hyaluronic acid) with targeting and redox-responsive activity with dioleoyl phosphatidylethanolamine, a fusogenic phospholipid. The obtained hybrid liposomal structures were found to possess suitable physicochemical properties for cancer therapy application, with mean size of 65 nm, negative surface charge (-27 mV) and the ability to efficiently encapsulate Doxorubicin in the inner liposomal core with high efficiency. The drug loaded hybrid liposomes (DOX@HBLs) were able to trigger the drug release under simulated acidic and redox conditions of the tumor environment, whereas the biological characterization demonstrated the safety and the selectivity of the formulation, able to target the cancer cells (toxicity similar to that of the free drug) while sparing the healthy cells (viability > 90 % in all cases). Furthermore, compared to free drug, DOX@HBLs were able to reduce cell motility, impair metabolic pathways essential for cancer progression, and effectively inhibit spheroid formation (by almost 50 % after 20 days incubation) in both Estrogen Receptor-positive and Triple Negative Breast Cancer cells, demonstrating their high potential as unconventional drug delivery vectors in cancer therapy.

The effects of a methanol extract of Nymphaea odorata (MeNO) rhizomes, its fractions and the active compound (methyl gallate, MeG) were investigated in estrogen receptor-positive (ER+) breast cancer cell lines MCF-7 and T47-D:A18, as well as ER-negative line SKBr3. Cell viability and cytotoxicity were determined using CellTiter-Glo® 2.0 assays at concentrations ranging from 1 to 100 μg/mL. Caspase activity and apoptosis were determined using Caspase-Glo® 3/7, Caspase-Glo® 8, and ApoTox-Glo™ triplex assays, as well as qPCR. Total RNA was isolated from MCF-7 cells treated with MeG. RNA-seq libraries were prepared using a Universal Plus mRNASeq kit, and sequencing was performed on a NovaSeq 6000. MeNO inhibited the growth of MCF-7 cells with an IC50 of 14.1 μg/mL, as well as T47-D:A18 (IC50 of 25.6 μg/mL) and SKBr3 cells (IC50 of 35.5 μg/mL). Bioassay-guided fractionation of MeNO in MCF-7 cells identified the active fraction containing one compound, namely methyl gallate (MeG). MeG had an IC50 of 8.6 μg/mL in MCF-7 cells. Transcriptomic analysis of MeG-treated MCF-7 cells showed differential expression of 10,634 genes, with 5643 upregulated and 4991 downregulated (FDR < 0.05). Ingenuity pathway analysis revealed the involvement of 43 canonical pathways, with the top upregulated pathways including apoptosis, autophagy, and the unfolded protein response pathways.

Estrogen-responsive breast cancer has been treated with tamoxifen since 1998, yet challenges such as limited selectivity and emerging resistance remain significant hurdles to improving therapeutic outcomes. In recent years, the incorporation of fluorine atoms in the structure of potential drugs has gained importance due to their unique properties. Perfluoroalkyl chains, known for their chemical inertness and ability to target estrogen, offer promising modifications to improve treatment efficacy. In this study, we evaluated the biological activity of 21 perfluoroalkylated tamoxifen derivatives, synthesized under mild conditions with high stereoselectivity. Seven of these compounds exhibited superior cytotoxic and selectivity activity against estrogen receptor-positive breast cancer cells (MCF-7), with IC50 values of 10.68-18.18nM compared to 29.41nM for 4-hydroxytamoxifen, which is used in standard therapy. Preliminary mechanism-of-action studies, supported by siRNA knockdown of ESR1 (the estrogen receptor gene), revealed that the compounds act through a similar mechanism to tamoxifen, further confirming their potential as next-generation therapeutic agents for estrogen receptor-positive breast cancer.