OBJECTIVE:Hepatocellular carcinoma (HCC), the most common form of liver cancer, often develops in individuals with chronic liver diseases, especially cirrhosis. Cisplatin (Cisp), a chemotherapy agent commonly used in HCC treatment, is effective but is known to damage normal cells, including fibroblasts. Hesperetin (HST), a citrus flavanone found abundantly in citrus fruits, has demonstrated antioxidant, anti-inflammatory, and anticancer properties. This study aimed to investigate the synergistic cytotoxic effects and selective induction of senescence by HST in combination with Cisp in HepG2 cancer cells and NIH-3T3 fibroblast cells. METHODS:The cytotoxic effects of HST were assessed using the MTT assay to determine cell viability. The antiproliferative properties were evaluated using colony formation assays. Senescence was assessed using SA-β-gal staining, while flow cytometry was used to analyze cell cycle distribution and apoptosis. Protein expression related to proliferation and apoptosis was determined via Western blot analysis. RESULTS:MTT assay results indicated that both HST and Cisp reduced HepG2 cell viability in a dose-dependent manner, with IC50 values of 258 ± 2.47 µM and 5 ± 1.83 µM, respectively. Their combination (HST: 33-130 µM; Cisp: 0.6-2.5 µM) showed synergistic effects (combination index, CI < 1) co-treatment with HST (65 and 130 µM) significantly enhanced senescence in HepG2 cells. Clonogenic assays showed inhibition of colony formation, supported by reduced expression of p-ERK1/2 and Cyclin D1. Flow cytometry revealed increased apoptosis and G2/M phase arrest, with upregulation of Bax and caspase-3, and downregulation of Bcl-xL. In NIH-3T3 cells, HST showed minimal cytotoxicity (IC50 > 500 µM), and co-treatment with Cisp reduced senescence markers. CONCLUSION:These results suggest that HST and Cisp co-treatment synergistically reduces cancer cell viability while protecting normal fibroblasts from senescence, supporting its potential as a co-chemotherapeutic agent in HCC treatment, while also serving as a protective agent against senescence in healthy tissues.

Mesenchymal stem cells derived from Wharton’s Jelly (WJ MSCs) and their cell-free products are emerging candidates for cancer therapy but show context-dependent effects. This systematic review synthesized preclinical in vitro and in vivo evidence on the anticancer and protumorigenic roles of WJ MSCs, their secretome, and extracellular vesicles (EVs) across diverse malignancies. Searches of PubMed, Scopus, and Springer Nature Link (English, up to August 2025) followed PRISMA guidance. Experimental studies using WJ MSC-based interventions in cancer models were included, and two reviewers independently screened, extracted data, and assessed risk of bias using SYRCLE for animal studies and a QUIN-type tool for in vitro work. Twenty-six preclinical studies were eligible, predominantly in vitro with limited in vivo validation. Most reported antitumor effects—reduced proliferation, induction of apoptosis, inhibition of migration/epithelial–mesenchymal transition, and modulation of PI3K/AKT, NF κB, and STAT3 pathways—whereas a smaller subset described enhanced growth, EMT, or activation of HGF–AKT/ERK and β-catenin signaling. Overall methodological quality was moderate to high, although reporting of randomization and blinding was often incomplete. Current evidence supports WJ MSC-derived secretome and EVs as promising, potentially safer anticancer and drug-delivery platforms, but standardized production protocols, rigorous safety evaluation, and well-designed animal and clinical studies are required before clinical translation.

Background: Three-dimensional (3D) culture models have emerged as a promising preclinical platform for breast cancer research because they better mimic the tumor microenvironment (TME) of conventional two-dimensional cultures. With advances in tissue engineering, research related to 3D models has grown rapidly, necessitating a comprehensive evaluation of global research trends. Methods: A bibliometric analysis was performed using the Scopus database covering publications from 2007 to 2025, and only original research articles were included. Bibliometric indicators, including annual scientific output, leading countries, institutions, authors, journals, highly cited publications and collaboration patterns, were analyzed using Biblioshiny (R package), while keyword co-occurrence and temporal evolution were visualized using VOSviewer. Results: A total of 207 research articles published by 1,245 authors were analyzed. Scientific output showed a consistent annual growth rate of 13.32%, reflecting the increasing global interest in 3D breast cancer modeling. The United States was the leading contributor in publication productivity and scientific impact, while Biomaterials was the most productive journal. Highly cited publications predominantly focused on advanced tissue engineering technologies, including bioprinting, hydrogels, extracellular matrix engineering and microfluidics. Keyword co-occurrence analysis identified seven thematic clusters, while overlay visualization revealed a shift in research from conventional 3D cell culture toward more advanced tissue engineering approaches, particularly in biomaterials, tumor microenvironment reconstruction, drug screening and precision medicine. Conclusion: Research on 3D breast cancer models has evolved into a multidisciplinary field driven by tissue engineering innovations. This study provides a comprehensive knowledge map of the field, highlights emerging research directions and offers valuable guidance for future interdisciplinary collaborations and the development of physiologically relevant in vitro breast cancer models.

Pentagamavunon-1 (PGV-1) has potential as an anticancer drug for hepatocellular carcinoma (HCC) but has limitations in terms of solubility. This study aimed to enhance its solubility and cytotoxicity against liver cancer cells. Complexes were developed by combining bovine serum albumin (BSA) with lactose, aiming to improve the bioavailability of PGV-1 as an anticancer agent. These complexes were further modified through lactosylation, producing PGV-1-loaded lactosylated BSA (PGV-1 + BSA + Lac), to increase their cytotoxic effects on HCC cells. The glycation of BSA with d-lactose was conducted under dry-heat conditions at 60 degrees C for 0, 30, 60, 120, or 240 min. The extent of BSA glycation was monitored by assessing the availability of free peptides and examining the molecular weight profile. A straightforward formulation was proposed involving a complex of BSA lactosylated with PGV-1. The solubility of PGV-1 in aqueous solutions was assessed with varying amounts of BSA. Cytotoxicity was evaluated through an MTT assay using the HLF cancer cell line. The results revealed distinct variations in BSA conjugation that were dependent on the duration of heating. Modified BSA exhibited reduced peptide availability and slower migration in SDS-PAGE. Notably, PGV-1 demonstrated significantly higher solubility when combined with BSA + Lac compared with PGV-1 + BSA. The IC50 value for PGV-1 + BSA + Lac in HLF cells (0.008 +/- 0.002 & micro;M) was lower than that for PGV-1 (0.437 +/- 0.002 & micro;M) and PGV-1 + BSA (0.631 +/- 0.002 & micro;M), highlighting the potent inhibitory effect of PGV-1 + BSA + Lac on HLF cell proliferation. Thus, PGV-1 + BSA + Lac complexes may be promising candidates for targeted PGV-1 delivery in HCC therapy.

BACKGROUND:Lapatinib (LAP) significantly improves outcomes in HER2-positive (HER2+) breast cancer, including trastuzumab-resistant p95HER2-expressing tumors. However, primary and acquired resistance remain unmet clinical challenges. Multiple studies have highlighted the promising role of natural products in breast cancer therapy. Given their ability to modulate multiple oncogenic signaling pathways, these compounds also represent potential therapeutic agents for overcoming LAP resistance. PURPOSE:In this review, we integrate current evidence on the molecular mechanisms underlying LAP resistance with the therapeutic potential of natural products to overcome these resistance pathways. STUDY DESIGN:We analyzed studies published between 2006 and 2026 investigating natural products in breast cancer, with a particular focus on their modulation of LAP resistance-associated molecular networks. RESULTS:A total of 194 preclinical studies and 34 clinical trial reports were included. Apoptosis (n = 64) and PI3K/AKT/mTOR signaling (n = 59) were the most frequently targeted mechanisms, followed by RAS/RAF/MEK/MAPK (n = 32), autophagy (n = 17), cell plasticity (n = 13), HER and FOXO signaling (n = 11 each), with fewer studies targeting Src, MET, IGF-1R, FGFR, PTEN, PP2A, and BRK pathways. CONCLUSION:This review highlights the potential of natural products to target the complex molecular networks underlying LAP resistance, although further validation in LAP-resistant models and dedicated clinical trials is required to confirm their therapeutic value.

Background: The investigation of genetic modifications and epigenetic controls within the melanoma antigen gene family (MAGE), the cancer-testis antigen, in breast cancer still remains elusive. The present study aimed to detect genetic and epigenetic alterations in the MAGE family genes among breast cancer patients, specifically those with metastatic breast cancer and experienced resistance to chemotherapy. Method: In this bioinformatics study, MAGE family genes were retrieved from HUGO genes database and further analyzed for protein-protein interaction using STRING version 12.0, gene ontology using DAVID v2024q2, genetic alterations prognostic value using Kaplan-Meier Plotter, DNA methylation using MethSurv, and the correlation between immune cell infiltration using TIMER 2.0. P <0.05 was considered statistically significant. Results: Patients with metastatic breast cancer have experienced genetic abnormalities in four specific genes of MAGEF1, NSMCE3, MAGEL1, and NDN. The relapse-free survival indicated that NSMCE3 and NDN have an unfavorable prognosis, while MAGEF1 and MAGEL1 have a favorable prognosis in breast cancer patients. A moderate association between the mRNA levels of MAGEF1 and MAGEL1 and the efficacy of chemotherapy was observed. The DNA methylation analysis revealed two significant CpG sites within the MAGEL1 gene in which become a poor prognosis of patients with breast cancer. Conclusion: This work has the potential to pave the way for the creation of immunotherapy and improved treatment strategy for those struggling with metastatic breast cancer and chemoresistance. Further research is guaranteed to authenticate the outcomes of these bioinformatics discoveries.

Breast cancer stem cells (BCSCs) are one of the causes of drug resistance and disease recurrence due to their capacity for self-renewal and heterogeneity induction. A new BCSC-targeting agent has become a prospective approach to overcome resistance. Baicalein (5,6,7-trihydroxyflavone), a flavonoid extracted from Scutellaria baicalensis, has demonstrated anticancer activities in several models, including breast cancer. However, further elucidation of its effects on BCSCs is required. This study utilizes integrative bioinformatic approaches to identify the potential targets of baicalein in overcoming BCSC. In vitro experiments confirmed the top ten target genes recognized during a previous bioinformatic analysis of MCF-7 as a mammosphere for cytotoxicity and gene expression assays. We identified the potential baicalein target genes in BCSCs, which include CTNNB1, STAT3, BCL2, HIF1A, ESR1, TNF, CCND1, IL6, JUN, and MAPK3. Gene annotation and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis revealed a possible attenuation of the estrogen signaling pathway by baicalein and its involvement in cell cycle regulation. We successfully constructed a three-dimensional (3D) mammosphere culture and characterized it to possess higher levels of "stemness-associated" factors (OCT4 and SOX2) compared to two-dimensional (2D) cultures. Baicalein did not demonstrate any significant effects on cell viability in both 2D and 3D cultures, although a decline was observed in 2D cultures. qRT-PCR revealed that baicalein suppressed all hub genes. Furthermore, molecular docking confirmed the gene expression patterns and that baicalein had better binding affinity to CTNNB1, STAT3, TNF, JNK1, and mitogen-activated protein kinase (MAPK) than the respective native ligands. In addition, other proteins also interacted with baicalein, as reflected by the docking scores. Baicalein was identified to interact with ten potential targets through a bioinformatics study, although it did not exhibit cytotoxicity in 2D and 3D MCF-7 cultures. However, a downward trajectory was observed in the expression levels of hub genes related to kinase pathways, like Wnt/β catenin, PI3K/Akt, and MAPK, as well as inflammation-associated genes that correlate with BCSC survivability. One of the most prominent was the estrogen signaling pathway, which was supported by the molecular docking results. Future research directions included confirmation of baicalein’s efficacy and toxicity through in vivo approaches, as well as to understand its efficacy as a combination chemotherapeutic agent.

OBJECTIVE:Programmed cell death-1 (PD-1, encoded by PDCD1) regulatory network participates in glioblastoma multiforme development. However, such a network in trastuzumab-resistant human epidermal growth factor receptor 2-positive (HER2+) breast cancer remains to be determined. Accordingly, this study was aimed to explore the PD-1 regulatory network responsible for the resistance of breast cancer cells to trastuzumab through a bioinformatics approach. METHODS:The study used data mining tools like cBioportal and OMIM to identify genes involved in the programmed cell death-1-trastuzumab resistance regulatory network. The network was further examined using various tools like WebGestalt, DAVID, STRING, Cytoscape, CytoHubba, GEPIA, TNMPlot, and ROCPlot. RESULTS:The PDCD1 regulatory network in trastuzumab-resistant HER2+ breast cancer is linked to Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), interleukin (IL)-10, protein tyrosine phosphatase receptor type C (PTPRC), and FCGR2B. These factors have a significant prognostic power in pathological complete response in breast cancer patients treated with trastuzumab. Infiltration of B cells, CD8+ cells, CD4+ cells, neutrophils, dendritic cells, macrophages, and regulatory T cells is directly correlated with PTR expression. CONCLUSION:The study identified four genes (CTLA4, IL10, PTPRC, and FCGR2B) that are linked to the regulatory network of PD-1 in trastuzumab-resistant HER2+ breast cancer cells. Further research is needed to develop the therapeutic target against trastuzumab resistance in HER2+ breast cancer.

A-Kinase Anchoring Protein 3 (AKAP3), a Cancer-Testis Antigen (CTA), is involved in cell proliferation and is aberrantly expressed in triple-negative breast cancer (TNBC), making it a promising target for immunotherapy. This study aimed to design a novel multi-epitope vaccine targeting AKAP3 using immunoinformatics approaches. Predicted CTL, HTL, and B-cell epitopes were selected based on their immunogenicity, antigenicity, non-allergenicity, and non-toxicity. The final construct integrated three epitopes from each category, connected by appropriate linkers (EAAAK, AAY, GPGPG, KK) and adjuvanted with the 50S ribosomal protein L7/L12 to enhance the immune response. Population coverage analysis indicated high accessibility, with 99.28 % in Indonesia, 99.4 % in Southeast Asia, and 100 % globally. Structural modeling and validation confirmed the vaccine's stability and immunogenic profile. Molecular docking demonstrated strong binding affinity to TLR-4 (-839.8 kcal/mol), supported by stable interactions in molecular dynamics simulations. Immune simulations further predicted the robust activation of both cellular and humoral immunity. In silico cloning confirmed compatibility with the pET28a(+) expression system for potential recombinant production. Collectively, these findings support the designed vaccine as a promising candidate for TNBC immunotherapy, warranting further in vitro and in vivo evaluation.

Immunosuppressive regimen treatment in renal transplant recipients is necessary to prevent acute rejection from the body’s immune system. Mycophenolic acid (MPA), commonly prescribed for renal transplant recipients, exists in two formulations: mycophenolate mofetil (MMF) and enteric-coated mycophenolate sodium (EC-MPS). Both drugs act by inhibiting inosine-5’-monophosphate dehydrogenase (IMPDH), an enzyme that is responsible for the guanosine nucleotide synthesis pathway of T and B lymphocytes. IMPDH exists in two isoforms, IMPDH1 and IMPDH2, encoded by IMPDH1 and IMPDH2 gene, respectively. Polymorphisms in these genes may alter the enzyme activity, potentially influencing MPA pharmacodynamics and leading to variations in therapeutic responses among renal transplant patients taking MPA. A systematic literature search was performed using Scopus, PubMed, and Web of Science with the Boolean search strategy: “IMPDH AND Polymorphism* AND Mycophenol* AND ((Renal OR Kidney) Transplant* OR Graft*)”. The articles yielded from this literature search were screened, resulting in 15 articles that were included in this review. Some studies reported the association between the IMPDH1 or IMPDH2 polymorphism and acute rejection, while others found no significant correlation. Regarding toxicity, leukopenia was linked to IMPDH1 SNPs (rs2278293, rs2278294), although the results were inconsistent. Most of the studies found no significant association between IMPDH2 SNPs and leukopenia incidence.

Glycoproteins have emerged as promising agents in drug delivery due to their potential for targeted delivery. This study developed and evaluated complexes of Pentagamavunon-1 (PGV-1) with glycated bovine serum albumin to enhance cytotoxic effects against cancer cells. Bovine serum albumin nanoparticles (BSA NPs) and glycated BSA nanoparticles [BSA+Lactose (Lac) NPs] were synthesized and complexed with PGV-1. These complexes were characterized by particle size analysis, scanning electron microscopy, transmission electron microscopy, and Fourier transform infrared spectroscopy to confirm their successful synthesis and morphological properties. Cytotoxicity was assessed in HepG2, T47D, and Vero cell lines using MTT assays, while cellular senescence was evaluated by senescence-associated β-galactosidase staining. The results indicated that PGV-1+BSA+Lac NPs significantly increased cytotoxicity in HepG2 and T47D cancer cells compared to free PGV-1, while showing lower toxicity toward non-cancerous Vero cells and demonstrating enhanced selectivity. Furthermore, senescence analysis revealed reduced β-galactosidase activity in Vero cells treated with PGV-1+BSA+Lac NPs relative to free PGV-1, supporting the targeted action of these nanoparticles. In conclusion, PGV-1+BSA+Lac NPs not only improve the delivery efficiency and selectivity of PGV-1 but also present a promising strategy for cancer-specific drug delivery. This research advances the development of glycoprotein-based delivery systems, offering a potential platform for targeted cancer therapies.

Black seed (Nigella sativa L.) is well known for its pharmacological properties, particularly its anticancer activity, with previous studies demonstrating its cytotoxic effects on several cell lines, such as A‐549, DLD‐1, MDA‐MB231, or HCT. This study aims to investigate the effects of black seed oil (BSO) on the migratory activity of 4T1 triple‐negative breast cancer (TNBC) cells, focusing on its bioactive properties. BSO was extracted via hydro‐distillation and analyzed for its phytochemical composition using gas chromatography–mass spectrometry (GC‐MS). The cytotoxicity of BSO and doxorubicin (Dox) was assessed using the MTT assay. The effects of BSO and Dox on cell migration and matrix metalloproteinase‐9 (MMP‐9) expression were evaluated using a scratch wound‐healing assay and gelatin zymography method respectively. Additionally, intracellular reactive oxygen species (ROS) levels were measured using 2’,7’‐dichlorofluorescin diacetate (DCFDA) staining. GC‐MS analysis identified p‐cymene as a major component of BSO, along with various other bioactive compounds. BSO exhibited low toxicity toward 4T1 cells, while its combination with Dox reduced cell viability in a dose‐dependent manner. Furthermore, BSO in combination with Dox inhibited cell migration and suppressed MMP‐9 expressions in 4T1 cells. BSO treatment also led to an increase in ROS levels. In conclusion, BSO exhibits potential anticancer properties by inhibiting cell migration and downregulating MMP‐9 expression, highlighting its possible therapeutic role in TNBC treatment.

Natural biodegradable polymers have been extensively studied as scaffolds for three-dimensional (3D) cancer cell culture in high-throughput screening (HTS) for anticancer drug discovery. This study fabricated a chitosan-based scaffold combined with pectin at different ratios: 10:90, 40:60, 60:40, and 90:10. Collagen I, the most abundant component of breast cancer extracellular matrix (ECM), was added to the scaffold formula. The composite scaffold displayed an interconnected, open-pore structure with tunable porosity, swelling, and degradable characteristics at different chitosan-to-pectin ratios. A high ratio of chitosan to pectin (60:40 and 90:10) exhibited the ideal properties for a 3D scaffold suitable for cell culture. These scaffolds supported the attachment and growth of the T47D breast cancer cell line. Additionally, this 3D cell culture demonstrated doxorubicin and tamoxifen resistance when compared to 2D culture. Therefore, it is a feasible and promising tool for more reliable anticancer drug screening.

Context: The development of curcumin analog compounds containing boron as anticancer agents is ongoing. Diboronocurcumin analogs are promising for development as selective and targeted anti-breast cancer agents. We designed five novel diboronocurcumin analogs (CCB-4 to CCB-8) featuring heterocyclic ketones to address current limitations in breast cancer treatment, including resistance and toxicity. Aims: To identify potential targets and molecular pathways of diboronocurcumin analogs in breast cancer, utilizing a bioinformatics technique and structurebased in silico research. Methods: Target prediction was performed using databases including TargetNet, GeneCards, OMIM, and NCBI. Functional network analysis tools, such as STRING, Cytoscape, GO, and KEGG, were utilized to identify key signaling pathways in breast cancer. Molecular docking was employed to evaluate the binding interactions between diboronocurcumin analogs and the predicted target proteins. Results: ESR1, RELA, HDAC1, and MMP9 were four targets of diboronocurcumin analogs implicated in the processes of breast cancer cell proliferation, metastasis, and death, according to bioinformatics approaches. Molecular docking studies revealed that diboronocurcumin analogs had a high affinity for the four targets. CCB-4 to CCB-8 demonstrated stronger binding to ESR1 (-10.18, -10.49, -9.19, -10.24, and -10.85 kcal/mol, respectively) than tamoxifen (-9.52 kcal/mol). In addition, CCB-5 and CCB-8 exhibited greater binding to IKK (upstream pathway kinase of RELA), compared to doxorubicin, tamoxifen, and lapatinib. Conclusions: This study provides insights into the multi-target potential of diboronocurcumin analogs in suppressing breast cancer progression. Further in vitro and in vivo validation is supported by encouraging in silico results, particularly for CCB-5 and CCB-8.

Cancer remains a significant global challenge, affecting millions, but progress has been made in understanding its development and advancement. The discovery of cancer drugs focuses on identifying "new dimension" hallmarks of cancer, such as phenotypic plasticity, senescence, polymorphic microbiota, and non-mutational epigenetic reprogramming. These elements are crucial in tumor development and treatment. Recent small molecule anticancer drugs target these characteristics and are currently undergoing preclinical testing, clinical trials, and approval. This review aids in developing strategies for cancer treatment. This review examines pharmacological agents aimed at the new dimension hallmarks of cancer, specifically those that address phenotypic plasticity, such as dedifferentiation, blocked differentiation, and trans-differentiation. Drugs that eliminate senescent cells are categorized as senolytic and senomorphic agents. This review analyzes microbial metabolites that could affect the efficacy of chemotherapeutic agents, specifically those derived from Fusobacterium nucleatum, Helicobacter pylori, Escherichia coli, Bacteroides fragilis, Streptococcus gallolyticus, Porphyromonas gingivalis, Mycoplasma hyorhinis, and Enterococcus faecalis. Drugs targeting non-mutational epigenetic reprogramming encompass DNA methylation inhibitors, histone modification modulators, BET inhibitors, and chromatin remodeling complexes. This review contributes to the development and exploration of strategies aimed at overcoming challenges in cancer treatment. This will lead to improved outcomes for patients and a more optimistic future in cancer treatment.

The PI3K/Akt pathway plays a crucial role in regulating a broad network of proteins involved in the proliferation of HER2-positive breast cancer. The ethyl acetate fraction of Vernonia amygdalina, which contains cardiac glycosides, has been shown to reduce the expression of PI3K and mTOR. However, the specific cardiac glycoside compounds with significant potential as PI3K inhibitors have yet to be clearly identified. This study employs machine learning to perform virtual screening of cardiac glycosides from V. amygdalina against the p110 subunit of PI3K. Initially, Lipinski’s Rule of Five was used to filter the PIK3CA inhibitor database via KNIME software. Subsequently, QSAR modeling was conducted using KNIME’s machine learning platform, employing six different algorithms. Cardiac glycosides from V. amygdalina were then evaluated using the best-performing QSAR model. The top three compounds identified underwent molecular docking and molecular dynamics simulations. The random forest algorithm was selected as the primary predictive model, which identified Vernoamyosides A (VG-1), Vernoniamyosides D (VG-8), and Vernoniosides A4 (VG-10) as the compounds with the highest confidence levels. Molecular docking results indicated that these three compounds exhibited stronger and more stable interactions with the PIK3CA receptor compared to alpelisib, a known PIK3CA inhibitor. Furthermore, molecular dynamics simulations revealed that VG-10 had the lowest binding free energy, as determined by MM-GBSA analysis. The findings of this study provide a foundational basis for preclinical and clinical investigations aimed at developing PI3K inhibitors derived from cardiac glycosides of V. amygdalina for the treatment of HER2+ breast cancer.

Background Trastuzumab (TRZ) is the first drug used to treat HER2-positive breast cancer, but some patients become resistant to it because of the PI3K/Akt pathway and other pathways that counteract it. TRZ, in conjunction with other therapeutic agents, is needed to overcome resistance. α-chaconine (CHA), a glycoalkaloid from the Solanaceae family, can suppress lung cancer cell proliferation in vitro by inhibiting PI3K/Akt signaling, one of the key regulatory pathways in TRZ resistance. Methods This study used integrative bioinformatics analysis to screen for possible targets of CHA that can help fight breast cancer that is resistant to TRZ. In vitro experiments were used to confirm the target genes using TRZ-resistant HCC-1954 (HCC-TRZ) cells for cytotoxicity, gene expression studies, and enzymatic assay. Results We identified several potential target genes of CHA, including EGFR, VEGF, ACHE, and ADORA. We generated HCC1954-TRZ cells, which showed an increase in cell viability after sequential treatment of the parental HCC1954 cells with TRZ. Further experiments showed the high sensitivity of HCC-TRZ toward TRZ when TRZ was combined with CHA. The combination of CHA and TRZ significantly increased the mRNA expression levels of various genes compared to a single TRZ treatment. Additionally, CHA alone and combined with CHA-TRZ inhibited acetylcholinesterase (AChE) activity in HCC-TRZ cells. Conclusion CHA increased the sensitivity of HCC-TRZ cells to TRZ by targeting several potential target genes and AChE activity. This study highlights the potential of using CHA in combination with TRZ to overcome TRZ resistance in HER2+ breast cancer cells.

Breast cancer is a potentially fatal illness that affects millions of women worldwide. Methotrexate (MTX) may be beneficial for treating breast cancer; however, high doses and prolonged use can cause drug resistance. Although certain citrus flavonoids—nobiletin, sinensetin, tangeretin, hesperidin, hesperetin, and naringenin—may overcome resistance to chemotherapy, no study has investigated MTX resistance. This study investigated the potential of natural chemicals, specifically nobiletin and sinensetin, to overcome MTX resistance in breast cancer cells using MTX-resistant MCF-7 (MCF-7/MTX) and MCF-7 cells. Protein targets of citrus flavonoids were identified from multiple databases and were collected using Venny 2.1. Microarray data of MCF-7 and MCF-7/MTX cells were acquired from the Gene Expression Omnibus. Subsequently, we constructed a protein–protein interaction network and selected the hub proteins. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, drug– and disease–gene enrichment analyses, genetic alteration examination, receiver operating characteristic curve analysis, mRNA levels analysis, prognostic value analysis, and molecular docking analysis were performed along with in vitro experiments. Cytotoxicity of citrus flavonoids (individually and combined) was assessed in MCF-7/MTX cells. Nobiletin and sinensetin significantly enhanced the cytotoxicity of MTX in MCF-7/MTX cells. BCL2L1, CDK1, EGFR, PTGS2, PLK1, MMP2, ACHE, ABCG2, and KIT genes were enriched in cholinesterase activity, cell cycle regulation, and the PI3K/Akt signaling pathway. Nobiletin and sinensetin impeded PLK1, CDK1, and ACHE activities based on molecular docking. Nobiletin and sinensetin in combination with MTX may overcome breast cancer cell resistance to MTX.

Epigenetic alterations in regulatory genes, genetic factors, and genomic instability, which cause breast cancer, can also contribute to disease resistance. HORMAD , which encode proteins containing HORMA domains and are involved in homologous recombination, have important roles in cancer emergence and progression. In this study, we uncovered putative breast cancer therapeutic targets by examining HORMAD1 and HORMAD2 genetic and epigenetic alterations. mRNA levels of HORMAD1 and HORMAD2 in breast cancer samples and normal breast tissues, as well as mRNA levels in normal, breast cancer, and metastatic breast cancer samples, were analyzed using TNMplot. Prognostic value, genetic alterations, epigenetic alterations, genetic variations, ROC plots, functional prediction, and immune infiltration of HORMAD1 and HORMAD2 were conducted with KMPlotter, cBioportal, methsurv, ClinVar, ROC Plotter, PredictSNP, PANTHER, and TIMER 2.0, respectively. Both HORMAD1 and HORMAD2 mRNA levels were lower in breast cancer samples, and lower in metastatic breast cancer samples. Patients expressing higher HORMAD1 and HORMAD2 levels had favorable overall survival (OS) rates than the opposite groups. HORMAD1 and HORMAD2 gene amplifications and deletions were also observed. Pathway enrichment analyses showed that Wnt signaling alterations contributed to cell proliferation. Increased DNA methylation levels were identified in HORMAD2 when compared with HORMAD1 in patients. Two 1021C>T (Q334) and 430A>G (T144A) variants of HORMAD1 were shown to have clinical significance in patients. Also, functional prediction mutant analysis of HORMAD1 confirmed that S287F exerted a deleterious effect on amino acid impact, however, further investigations are warranted. Receiver operating characteristic (ROC) plot data indicated a significant correlation between HORMAD2 levels and anti-human epidermal growth factor receptor 2 (HER2) sensitivity. Genetic and epigenetic changes in HORMAD1 and HORMAD2 genes may be used as indicators and targets for overcoming breast cancer resistance and limiting metastasis in breast cancer cells via Wnt targeting. Further research is required to verify our findings.