https://orcid.org/0000-0001-5761-2073
Figures
Abstract
Background
Cancer progression involves coordinated regulation of oncogenes and tumor suppressors. This study explores the interplay of ENOX2 (ecto-NADH oxidase disulfide-thiol exchanger 2), MMP2 (matrix metalloproteinase-2), and regulatory genes Ras Association Domain Family Member 1, Isoform A (RASSF1A), WAP Four-Disulfide Core Domain Protein 10A (WFDC10A), and Methyltransferase-Like Protein 7A (METTL7A) across multiple cancer cell lines, and evaluates the anticancer potential of repurposed mebendazole.
Methods
Eight human cell lines, including breast (MCF7 and MDAMB231), colorectal, pancreatic, lung, hepatocellular, leukemia, and endothelial models, were profiled by qRT-PCR and Western blotting. Expression was assessed under basal conditions and following mebendazole exposure (0.7 µM).
Results
Basal expression revealed elevated ENOX2 and MMP2 in aggressive cancers (MDA-MB-231, PANC1). Mebendazole significantly downregulated ENOX2 in HEPG2 (p < 0.01) and K562 (p < 0.05), and suppressed MMP2 in MDA-MB-231 (p < 0.05) and MCF7 (p < 0.01), indicating anti-invasive effects. Tumor suppressors were selectively induced: RASSF1A increased >200-fold in endothelial cells (p < 0.01) and was upregulated in HEPG2 and HT29 (p < 0.05), while WFDC10A was strongly elevated in MDA-MB-231 (>40-fold, p < 0.001). METTL7A displayed endothelial enrichment with heterogeneous tumor-specific regulation. Collectively, these findings reveal cell-type–specific modulation of oncogenic and suppressor pathways.
Conclusion
This multi-cancer investigation identifies ENOX2–MMP2 signaling as a functional driver of invasion and metastasis and demonstrates that mebendazole reprograms oncogenic–tumor suppressor networks. By integrating biomarker profiling with drug repurposing, our study highlights the translational potential of mebendazole as a cost-effective anticancer agent and supports the development of multi-gene biomarkers for diagnosis and therapy in aggressive malignancies.
Citation: Aqel RS, Ismail AS, El-Tanani M, Satyam SM (2026) Repurposing mebendazole to reprogram oncogenic and tumor-suppressor networks: Multi-cancer insights from ENOX2, MMP2, RASSF1A, WFDC10A and METTL7A. PLoS One 21(3): e0345701. https://doi.org/10.1371/journal.pone.0345701
Editor: Yusuf Ahmed Haggag, University of Michigan, EGYPT
Received: January 13, 2026; Accepted: March 9, 2026; Published: March 26, 2026
Copyright: © 2026 Aqel et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: ENOX2, ecto-NADH oxidase disulfide-thiol exchanger 2; MMP2, matrix metalloproteinase-2; RASSF1A, Ras association domain family member 1, Isoform A; WFDC10A, WAP four-disulfide core domain protein 10A; METTL7A, methyltransferase-like protein 7A; ATCC, American type culture collection; DMEM, Dulbecco’s modified eagle medium; RPMI, roswell park memorial institute medium; MEM, minimum essential medium; PBS, phosphate-buffered saline; RIPA, radioimmunoprecipitation assay; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; BSA, Bovine serum albumin; HRP, horseradish peroxidase; ECL, enhanced chemiluminescence; qRT-PCR, quantitative real-time polymerase chain reaction; cDNA, complementary DNA; RNA, ribonucleic acid; DNA, deoxyribonucleic acid; B2M, beta-2-microglobulin; EGCG, epigallocatechin gallate; DMSO, dimethyl sulfoxide; SD, standard deviation; ANOVA, analysis of variance.
Introduction
Cancer continues to pose one of the greatest challenges in modern medicine due to its multifactorial pathogenesis, heterogeneity, and resistance to current therapies. Cancer has emerged as one of the most critical health challenges worldwide, accounting for millions of deaths each year [1]. According to recent global estimates, over 19.3 million new cases were diagnosed in 2020, resulting in nearly 10 million deaths [2]. The persistently rising incidence and mortality burden highlight an urgent need for the discovery and development of more effective pharmacological interventions to manage and treat diverse cancer types. Breast cancer, lung cancer, colorectal cancer, liver cancer and pancreatic cancer are the most common types of cancer that cause many cancer deaths. The survival rates for numerous cancers remain poor because of insufficient progress in targeted therapy and immunotherapy and personalized medicine thus requiring immediate development of new biomarkers for early detection and treatment planning. Growing evidence indicates that tumor heterogeneity, therapeutic resistance, and disease relapse are strongly driven by cancer stem cell subpopulations and microenvironmental adaptation mechanisms, necessitating precision-guided biomarker discovery and individualized therapeutic strategies [3]. In addition, large-scale clinical analyses and chronobiology-based therapeutic optimization approaches emphasize that lifestyle modulation and time-adjusted pharmacological interventions can significantly influence long-term oncologic outcomes [4,5].
The biological process of cancer development results from multiple factors including genetic mutations and epigenetic changes and signaling pathway dysregulation and changes in tumor microenvironment composition [6]. The cancer hallmarks described by Hanahan and Weinberg serve as a theoretical model to study tumor development through their six key characteristics which include sustained cell growth signals and cell death resistance and tumor blood vessel formation and cancer cell invasion and metastasis and evasion of immune responses [7]. The framework identifies ENOX2 and MMP2 as essential molecular factors which promote cancer progression through their roles as tumor-associated NADH oxidase and matrix metalloproteinase-2 [8]. Beyond classical hallmarks, aberrant Wnt/β-catenin–Tcf-4 signaling and chromosomal condensation regulators contribute to tumor invasiveness, genomic instability, and metastatic competence, thereby expanding the molecular framework of cancer progression [9,10].
ENOX2, encoded by the ENOX2 gene, is a cancer-specific cell surface protein belonging to the family of growth-related NADH oxidases. Unlike its constitutively expressed counterpart (CNOX), ENOX2 is expressed exclusively in transformed cells and is absent in normal healthy tissues. The enzyme shows periodic activity that affects the redox state of cells and controls both cell growth and proliferation. ENOX2 exists in serum and urine samples of cancer patients which makes it suitable for liquid biopsy-based non-invasive cancer diagnostic applications [11]. ENOX2 activity suppression leads to tumor cell growth reduction and programmed cell death activation [12]. The compounds capsaicin, green tea catechins (EGCG) and mebendazole show ENOX2 inhibitory activity which supports its role as a diagnostic tool and therapeutic target [13]. ENOX2 isoforms show particular expression patterns in cancer cells which makes them useful for identifying various cancer types [14].
Emerging mechanistic data suggest that redox-regulated oncogenic signaling may intersect with nucleocytoplasmic transport machinery and Myc-driven transcriptional amplification, thereby promoting tumor progression and chemoresistance [15,16]. Furthermore, targeted disruption of the Ran-RCC1 axis using nanoparticle-based inhibitory peptides has demonstrated significant anti-cancer efficacy in preclinical models [17].
ENOX2 has a parallel function to MMP2 which is also known as gelatinase A [18]. It is a zinc-dependent endopeptidase that is involved in the degradation of the extracellular matrix (ECM) and tumor invasion, angiogenesis and metastasis. MMP2 breaks down essential ECM components including type IV collagen which enables basement membrane destruction that is necessary for metastatic cell spread [19]. Research shows that MMP2 overexpression occurs in different solid tumors such as breast cancer and lung cancer and colorectal cancer and pancreatic cancer and these elevated levels are linked to worse patient outcomes and more aggressive tumor behaviour [20–22]. Genetic variations in the MMP2 gene have been linked to higher cancer risk for both lung and gastric cancer patients [23–25]. Multiple MMP inhibitors have undergone clinical testing, but their non-specific nature and harmful side effects restrict their medical use which requires new targeted strategies to control MMP2 activity or regulation. Regenerative and tissue-remodeling mechanisms observed in experimental wound-healing models further reinforce the biological interplay between extracellular matrix modulation, metabolic control, and proliferative signaling cascades relevant to tumor biology [26]. Extracellular matrix degradation and metastatic dissemination are closely coordinated with receptor tyrosine kinase signaling and cytoskeletal remodeling pathways, which can be therapeutically disrupted through combinatorial pharmacological strategies [27]. Advanced nanoformulation approaches have further demonstrated the potential to selectively target invasive tumor phenotypes in vivo [28].
ENOX2 and MMP2 have an unexamined relationship which plays a role in cancer biology. The complete regulatory functions of these molecules need further research to understand their individual roles in tumor growth promotion. ENOX2-generated redox changes could affect MMP2 expression or activity levels which would create a feedback mechanism that boosts tumor invasion and metastasis. Research into this connection has the potential to reveal new molecular pathways which scientists can use to develop innovative therapeutic approaches.
The current research investigates three more molecular factors RASSF1A WFDC10A and METTL7A which could potentially influence the ENOX2-MMP2 pathway. RASSF1A (Ras association domain-containing protein 1 isoform A) is a well-known tumor suppressor gene frequently silenced by promoter hypermethylation in a wide array of cancers. The protein functions as a cell cycle regulator and stabilizes microtubules and induces apoptosis while its decreased expression leads to more aggressive tumors and worse patient outcomes. WFDC10A (WAP four-disulfide core domain 10A), although less studied in the context of cancer, belongs to a family of proteins with potential roles in protease inhibition and immune regulation. WFDC family proteins show potential to affect tumor immune evasion and inflammatory pathways according to initial research findings yet WFDC10A's exact involvement in cancer development needs further investigation. The protein METTL7A (Methyltransferase-like protein 7A) in lipid metabolism exists as a dual-purpose protein which exhibits cancer-promoting and cancer-preventing effects according to various scientific studies. METTL7A has been implicated in cancer cell survival, chemoresistance, and modulation of the tumor microenvironment.
Metabolic dysregulation and cardiometabolic risk states are increasingly recognized as modulators of tumor suppressor signaling, lipid metabolism pathways, and inflammatory microenvironments that influence cancer progression [29]. Experimental studies further suggest that antioxidant-rich and micronutrient-based interventions can modulate oxidative stress, lipid profiles, and systemic inflammatory responses, thereby indirectly influencing proliferative and degenerative disease pathways [30–32]. The practice of drug repurposing has become more popular because it applies existing safe medications to develop new cancer treatments. Mebendazole, a benzimidazole anti-helminthic agent, exemplifies this strategy. The drug mebendazole received its first approval for treating parasitic worm infections yet researchers have found it shows anticancer effects in laboratory studies by blocking tubulin polymerization and stopping blood vessel formation and triggering cell death and blocking ENOX2 and other tumor-related enzymes [33]. The compound shows promise as a repurposed anticancer drug because it controls MMPs including MMP2. The drug's affordability and accessibility make mebendazole an attractive option for resource-limited areas which supports global health equity goals.
The expanding paradigm of drug repurposing and adjunctive therapy also includes agents that mitigate chemotherapy-induced organ toxicity and systemic adverse effects, thereby enhancing therapeutic tolerability and clinical feasibility [34–40]. Similarly, kinase-targeted approaches initially investigated in viral inflammatory conditions highlight the broader applicability of pathway-directed therapeutics across multiple disease systems, including oncology [41]. Recent advances in nanotechnology and biomaterial engineering have revolutionized targeted drug delivery systems, enabling enhanced tumor selectivity, improved pharmacokinetics, and reduced systemic toxicity [42,43]. In parallel, RNA-based therapeutic platforms with durable gene-silencing effects illustrate the translational feasibility of precision molecular modulation strategies that may be adaptable to oncologic applications [44].
Despite the individual significance of ENOX2, MMP2, RASSF1A, WFDC10A, and METTL7A, an integrative analysis of their expression patterns across multiple cancer types and their modulation by mebendazole has not been comprehensively undertaken. Most research conducted to date has concentrated on individual genes and specific pathways in separate cancer models which restricts the development of universal applications. The research investigates how these molecular targets get regulated at both transcriptional and translational levels across eight human cancer cell lines which include MDA-MB-231 for triple-negative breast cancer and MCF7 for hormone-responsive breast cancer and HEPG2 for hepatocellular carcinoma and HT29 for colorectal adenocarcinoma and K562 for leukemia and A549 for lung adenocarcinoma and PANC1 for pancreatic carcinoma and EA.hy926 as control endothelial cells.
The main research question investigates how ENOX2 and MMP2 work together in cancer development through functional connections while RASSF1A WFDC10A and METTL7A affect their activity levels. The study investigates how mebendazole treatment affects ENOX2 and MMP2 expression to block tumor-promoting pathways. We used qRT-PCR and Western blotting and pharmacological treatment to study how these genes and proteins express themselves under normal conditions and when treated with drugs.
This study was aimed to achieve three main goals which include (1) determining how ENOX2 and MMP2 and RASSF1A and WFDC10A and METTL7A express in various human cancer cell lines (2) studying how mebendazole affects the gene expression and protein production of these markers and (3) determining the clinical value of these results for cancer detection and treatment planning.
This study introduces an original method which combines biomarker detection with drug repositioning and mechanism analysis through a single experimental framework. The research establishes links between ENOX2 and MMP2 and their associated regulatory factors to create a complete molecular image of cancer development and its treatment targets. The study demonstrates mebendazole's ability to affect these pathways which creates new opportunities for developing affordable cancer treatment options.
Materials and methods
This study was conducted exclusively using established, commercially available human cancer cell lines and did not involve human participants, identifiable data, or animal subjects; therefore, ethics approval and consent to participate were not applicable. All experimental procedures adhered to institutional research policies and standard laboratory guidelines governing the use of in vitro cell culture models, and consent for publication was not required because no human data or identifiable information were generated. The overall experimental design and analytical workflow used to evaluate the molecular effects of mebendazole across multiple cancer cell lines are summarized in S1 Fig.
Cell lines and culture conditions
Eight human cell lines were obtained from the American Type Culture Collection (ATCC): MDA-MB-231 (triple-negative breast cancer), MCF7 (hormone-responsive breast cancer), HEPG2 (hepatocellular carcinoma), HT29 (colorectal adenocarcinoma), K562 (chronic myeloid leukemia), A549 (lung adenocarcinoma), PANC1 (pancreatic carcinoma), and EA.hy926 (human umbilical vein endothelial cells, used as a non-malignant control). The MDA-MB-231, A549, and PANC1 cell lines were cultured in DMEM supplemented with 1% L-glutamine and sodium pyruvate (Euroclone, Italy). The K562, MCF7, and HT29 cell lines were maintained in RPMI-1640 medium with 1% L-glutamine (Euroclone, Italy), while EA.hy926 cells were grown in DMEM-F12 medium with 1% L-glutamine (Euroclone, Italy). The HEPG2 cell line was cultured in MEM medium with 1% L-glutamine (Euroclone, Italy). All media were further supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin to constitute complete growth medium. Cells were maintained at 37 °C in a humidified incubator with 5% CO₂, and media were refreshed every 2–3 days.
Experimental Design and Drug Treatment
Cell cultures were maintained in T-75 flasks and monitored under a microscope until they reached 60–80% confluence. Subculturing was performed under a laminar flow cabinet that had been sterilized with 70% ethanol and UV-irradiated for 30 min. All reagents were sterilized and pre-warmed in a 37 °C water bath prior to use. For adherent cell lines, monolayers were washed with Dulbecco’s Phosphate-Buffered Saline (PBS, 1X), followed by the addition of trypsin to facilitate cell detachment. Flasks were incubated with trypsin in a CO₂ incubator, tapped gently, and supplemented with complete medium. The cell suspension was transferred to a conical tube and centrifuged at 2000–2500 rpm for 10 min at 4 °C. The resulting pellet was resuspended in fresh medium [27]. For the K562 suspension cell line, trypsinization was not required; cells were washed with PBS, transferred directly to centrifuge tubes, and processed similarly.
Cell viability and density were assessed using Trypan Blue exclusion and an automated cell counter (Quad Count). Cultures with a viability greater than 4 × 10⁶ cells were seeded at a density of 1 × 10⁶ cells per T-25 flask under three conditions: untreated (medium only), vehicle-treated (DMSO), and drug-treated (mebendazole dissolved in DMSO). Cells were incubated at 37 °C in a humidified 5% CO₂ atmosphere for 24 h. The final concentration of mebendazole (0.7 µM) and the 24-hour exposure period were selected based on prior literature demonstrating effective modulation of oncogenic signaling pathways without inducing nonspecific cytotoxicity and were further validated in preliminary optimization experiments to ensure cellular viability and reproducibility of molecular responses [45–47].
RNA extraction and cDNA synthesis
The Quick-RNA MiniPrep Kit from Zymo Research enabled scientists to obtain total RNA from cultured cells through the execution of the provided protocol, as previously described in established RNA isolation methodologies [48]. The NanoDrop spectrophotometer measured RNA purity and concentration through spectrophotometric analysis (A260/A280 ratio between 1.8 and 2.0 was considered acceptable) [49]. The assessment of RNA integrity used agarose gel electrophoresis and Bioanalyzer RIN analysis to confirm that samples with RIN values above 7.5 were suitable for further analysis. The PrimeScript RT Master Mix from Takara Bio converted 1 μg of RNA into cDNA through reverse transcription while effectively eliminating genomic DNA contamination to produce high-fidelity results.
Quantitative Real-Time PCR (qRT-PCR)
qRT-PCR was performed using TB Green Premix Ex Taq II (Takara Bio) on an Applied Biosystems QuantStudio 5 real-time PCR system. The primers were constructed to extend across exon-exon junctions for preventing genomic DNA amplification. The specificity of primers was confirmed through melt-curve analysis and gel electrophoresis of the amplified products. The expression of ENOX2, MMP2, RASSF1A, WFDC10A, and METTL7A was quantified, and expression levels were normalized to the housekeeping gene B2M (beta-2-microglobulin). Relative gene expression was calculated using the 2-ΔΔCt method [50]. Technical duplicates were run for each sample to account for pipetting variability.
Protein extraction and western blotting
The cells underwent lysis through radioimmunoprecipitation assay (RIPA) buffer which contained protease and phosphatase inhibitors. Lysates were clarified by centrifugation at 14,000 × g for 15 minutes at 4°C. Protein concentrations were quantified using the SMART™ BCA Protein Assay Kit (iNtRON Biotechnology) according to the manufacturer’s instructions. For protein separation, 50 µg of total protein from each sample was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels, followed by transfer onto nitrocellulose membranes (Thermo Scientific™). Membranes were blocked with TBS-T containing 5% bovine serum albumin (BSA) for 1 h at room temperature and subsequently incubated with primary antibodies for 1 h at 37 °C. The following antibodies were used: monoclonal anti-MMP2 (1:2000, Abcam), polyclonal anti-ENOX2 (1:2000, Abcam), monoclonal anti-METTL7A (1:5000, Abcam), and polyclonal anti-β-actin (1:500, Abcam) as the loading control. After washing, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, and specific protein bands were detected using an enhanced chemiluminescence (ECL) system.
Statistical analysis
All data were analyzed using GraphPad Prism version 9.0. The Shapiro–Wilk test confirmed that the collected data followed a normal distribution. Parametric data were analyzed using one-way ANOVA followed by Tukey’s post-hoc test, while non-parametric data were analyzed using Kruskal–Wallis test followed by Dunn’s multiple comparison test. Student’s t-test provided the correct statistical approach for making individual pair comparisons. Results were expressed as mean ± standard deviation (SD). The research proved statistical significance through p-values which fell below 0.05. The study used three biological repeats together with three technical repeats to produce reliable results.
Results
The results of this study provide an integrated view of the transcriptional and translational regulation of ENOX2, MMP2, and associated genes (RASSF1A, WFDC10A, METTL7A) across eight human cancer cell lines under basal conditions and following treatment with mebendazole. The raw data underlying the gene and protein expression results are provided in S1 File to ensure transparency and reproducibility of the reported findings (see Figs 1–6).
(A–H) Quantitative RT-PCR showing relative ENOX2-4 expression after drug treatment in MDA-MB-231, MCF-7, HEP-G2, HT-29, A549, K562, PANC-1, and EA.hy926 cells. (Right panel) Basal ENOX2-4 levels across all tested cell lines, with EA.hy926 showing markedly higher expression than cancer cells. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns = not significant.
(A–H) Relative METTL7A mRNA expression following drug treatment in MDA-MB-231, MCF-7, HEP-G2, HT-29, A549, K562, PANC-1, and EA.hy926 cells, measured by quantitative RT–PCR and normalized to untreated controls. (Right panel) Basal METTL7A levels across the same cell lines, showing highest expression in EA.hy926 compared with cancer-derived lines. Data are mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.
(A–H) Quantitative RT-PCR analysis of RASSF1A expression in MDA-MB-231, MCF7, HEPG2, HT29, A549, K562, PANC1, and EA.hy926 cells following treatment compared to untreated/DMSO controls. (Right panel) Summary plot showing relative fold-change across all tested cell lines. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ns = not significant.
(A–E) Quantitative RT-PCR analysis of WFDC10A expression in MDA-MB-231, MCF7, HEPG2, HT29, and EA.hy926 cells following drug treatment compared with untreated/DMSO controls. (Right panel) Summary plot comparing WFDC10A fold-change across all tested cell lines. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns = not significant.
(A–D, G–H) Fold-change in ENOX2 mRNA expression in MDA-MB-231, MCF7, HEPG2, K562, HT29, and PANC1 cells under media, DMSO, and drug-treated conditions. (E, F, I) Immunoblot analysis showing ENOX2 protein expression (70 kDa) across the same cell lines with β-actin (42 kDa) used as loading control. Data indicate that ENOX2 is significantly downregulated in HEPG2 and K562 cells following drug treatment, while endothelial EA.hy926 cells display relatively stable ENOX2 expression.
Quantitative expression and protein validation of METTL7A and MMP2 in breast cancer cell lines (MDA-MB-231, MCF7) and endothelial cells (EA.hy926). In panel A–B, fold expression analysis shows that METTL7A was undetectable in MDA-MB-231 and MCF7 cells across media, DMSO, and drug-treated conditions. Panel C demonstrates by western blotting the absence of METTL7A in both breast cancer cell lines, whereas EA.hy926 endothelial cells exhibited a clear 28 kDa band, confirming endothelial-specific enrichment. In contrast, panel 2A–B shows that MMP2 expression was significantly downregulated by drug treatment in both MDA-MB-231 (p < 0.05) and MCF7 cells (p < 0.01) relative to control, while DMSO produced variable effects. Panel 2C validates these findings at the protein level, showing reduced MMP2 expression upon drug exposure with detectable bands at 28 kDa.
Cell line dependent regulation and basal enrichment of ENOX2–4 expression
Quantitative RT-PCR analysis revealed that ENOX2–4 expression was differentially regulated by drug treatment in a cell line–dependent manner (Fig 1.1). In MDA-MB-231, HT-29, and PANC-1 cells, drug exposure led to a significant downregulation of ENOX2–4 transcripts compared with untreated/DMSO controls (p < 0.05 to p < 0.01). By contrast, ENOX2–4 levels were significantly upregulated in A549, K562, and EA.hy926 cells (p < 0.05), while no significant change was observed in MCF-7 and HEP-G2 cells. These findings suggest a selective and heterogeneous regulatory effect of the drug on ENOX2–4, varying across tumor types.
Analysis of basal ENOX2–4 expression across the panel of cell lines further demonstrated marked variability (Fig 1.2). Endothelial-derived EA.hy926 cells exhibited the highest ENOX2–4 mRNA abundance (~22-fold higher than control), which was significantly greater than that observed in MDA-MB-231, MCF-7, HEP-G2, HT-29, A549, K562, and PANC-1 (p < 0.05 to p < 0.001). Among the cancer cell lines, MDA-MB-231 and A549 expressed the lowest ENOX2–4 levels (p < 0.001 vs. EA.hy926). Collectively, these results indicate that ENOX2–4 expression is intrinsically enriched in endothelial cells, while tumor cells maintain relatively low basal levels, and that pharmacological modulation of ENOX2–4 is strongly context-dependent.
Cell line specific drug modulation and endothelial enrichment of METTL7A
Quantitative RT–PCR demonstrated that METTL7A expression was significantly altered by drug treatment in a cell line–dependent manner (Fig 2.1). Upregulation was observed in MDA-MB-231 (p < 0.01), MCF-7 (p < 0.05), HEP-G2 (p < 0.05), K562 (p < 0.01), PANC-1 (p < 0.05), and EA.hy926 (p < 0.05) cells compared with untreated/DMSO controls. In contrast, a significant downregulation was detected in HT-29 (p < 0.001) and A549 (p < 0.01) cells.
Basal expression profiling (Fig 2.2) revealed that EA.hy926 cells displayed the highest METTL7A transcript levels, which were significantly greater than those in all tested cancer-derived cell lines (p < 0.01 to p < 0.0001). Among tumor lines, expression was generally reduced, with some approaching baseline. These results indicate that METTL7A is intrinsically enriched in endothelial cells, whereas tumor cells maintain comparatively low basal levels and display heterogeneous transcriptional responses to drug treatment.
Cell line specific drug modulation and endothelial upregulation of RASSF1A
As shown in Fig 3.1, quantitative RT-PCR analysis revealed that the effect of drug treatment on RASSF1A mRNA expression varied across different cell lines. In MDA-MB-231 (Fig 3.1A) and MCF7 (Fig 3.1B) breast cancer cells, no significant change in RASSF1A transcript levels was observed between drug-treated and untreated/DMSO groups (ns). In HEPG2 liver carcinoma (Fig 3.1C) and HT29 colorectal carcinoma cells (Fig 3.1D), drug treatment led to a significant increase in RASSF1A expression (p < 0.05). Conversely, A549 lung carcinoma (Fig 3.1E), K562 leukemia (Fig 3.1F), and PANC1 pancreatic cancer cells (Fig 3.1G) exhibited significant reductions in RASSF1A levels following treatment (p < 0.05). Interestingly, in EA.hy926 endothelial cells (Fig 3.1H), drug exposure resulted in a marked upregulation of RASSF1A expression (p < 0.01).
A consolidated representation across all tested cell lines is presented in Fig 3.2. This analysis confirmed that MDA-MB-231 and MCF7 cells remained unaffected by treatment (ns), whereas HEPG2 and HT29 cells showed significant increases in RASSF1A expression (p < 0.05). A549, K562, and PANC1 cells consistently demonstrated significant downregulation of RASSF1A upon treatment (p < 0.05). Strikingly, EA.hy926 cells exhibited a dramatic induction of RASSF1A mRNA, with more than a 200-fold increase relative to control conditions (p < 0.05), while this effect remained highly significant (p < 0.01) when analyzed across replicates. These findings indicate that the drug exerts a cell-type–specific regulatory effect on RASSF1A transcription, with particularly strong responsiveness in endothelial cells.
Cell line specific drug modulation and endothelial upregulation of WFDC10A
As shown in Fig 4.1, drug treatment resulted in a significant upregulation of WFDC10A mRNA across several cancer cell lines. In MDA-MB-231 breast cancer cells (Fig 4.1A), WFDC10A expression was dramatically induced by treatment, showing an increase of more than 40-fold compared to the untreated/DMSO control group (p < 0.001). In MCF7 breast carcinoma cells (Fig 4.1B), treatment significantly elevated WFDC10A levels by nearly threefold (p < 0.05). Similarly, in HEPG2 hepatocellular carcinoma (Fig 4.1C) and HT29 colorectal carcinoma cells (Fig 4.1D), WFDC10A transcripts were significantly increased upon drug exposure (p < 0.01 for both). In EA.hy926 endothelial cells (Fig 4.1E), drug treatment also produced a strong induction of WFDC10A expression, with nearly a twofold increase relative to untreated controls (p < 0.01).
A consolidated comparison across all tested cell lines is presented in Fig 4.2. This analysis revealed striking variability in WFDC10A induction, with MDA-MB-231 cells showing the highest expression levels among all cell types (p < 0.05 when compared with most other lines). EA.hy926 cells also demonstrated significantly elevated expression compared to control conditions (p < 0.01), whereas MCF7, HEPG2, and HT29 cells exhibited moderate but significant increases in WFDC10A following treatment (p < 0.05). In contrast, A549 lung carcinoma, K562 leukemia, and PANC1 pancreatic carcinoma cells did not show statistically significant changes (ns). These findings indicate that WFDC10A is differentially regulated in a cell line–specific manner, with particularly robust induction in breast carcinoma cells and a notable upregulation in endothelial cells.
Cell line specific drug modulation and endothelial regulation of ENOX2
As shown in Fig 5.1, analysis of ENOX2 expression at both transcript and protein levels revealed variable responses to drug treatment across different cancer and endothelial cell lines. In MDA-MB-231 breast cancer cells (Fig 5.1A), ENOX2 mRNA was undetectable under all conditions, and this was consistent with the protein blot in Fig 5.1E showing no clear ENOX2 signal. In MCF7 cells (Fig 5.1B), ENOX2 expression was observed in all groups, but treatment with the drug did not lead to a marked reduction compared with media and DMSO controls, which was further supported by the corresponding immunoblot (Fig 5.1E). In HEPG2 hepatocellular carcinoma cells (Fig 5.1C), ENOX2 mRNA was strongly downregulated following treatment compared with untreated and DMSO controls, which was corroborated by a marked reduction in ENOX2 protein levels as seen in Fig 5.1F. Similarly, in K562 leukemia cells (Fig 5.1D), ENOX2 transcript levels were significantly decreased in the drug-treated group, with the Western blot (Fig 5.1F) confirming reduced protein expression relative to controls.
In HT29 colorectal carcinoma cells (Fig 5.1G), ENOX2 expression was moderately reduced in drug-treated cells compared with controls, a finding mirrored by the immunoblot in Fig 5.1I. In PANC1 pancreatic carcinoma cells (Fig 5.1H), transcript levels also showed a slight decrease following drug treatment, and this was in line with a visible reduction at the protein level (Fig 5.1I). Interestingly, EA.hy926 endothelial cells exhibited detectable ENOX2 protein across all conditions (Fig 5.1F and 5.1I), but treatment did not produce a pronounced reduction, indicating a relatively stable ENOX2 regulation in endothelial cells.
Overall, these results demonstrate that drug treatment leads to a significant downregulation of ENOX2 in certain cancer cell types, particularly HEPG2 and K562, while expression in endothelial cells appears comparatively less affected.
Endothelial-specific enrichment of METTL7A and drug-mediated suppression of MMP2 in breast cancer cells
To determine the expression pattern of METTL7A in breast cancer cell lines, we first examined its transcript levels in MDA-MB-231 and MCF7 cells. As shown in Fig 6.1A and 6.1B, METTL7A mRNA expression was undetectable in both cell lines under basal conditions and remained unchanged upon vehicle (DMSO) or drug treatment. This absence of detectable expression was confirmed at the protein level, where immunoblotting revealed no METTL7A band in MDA-MB-231 or MCF7 cells, regardless of treatment. In contrast, a distinct band at the expected molecular weight of 28 kDa was observed in EA.hy926 endothelial cells (Fig 6.1C), indicating selective enrichment of METTL7A in endothelial lineage cells. Together, these results demonstrate that METTL7A is not expressed in breast cancer cells but is prominently detectable in endothelial cells, supporting a cell type–specific role.
We next assessed the effect of drug exposure on MMP2 expression as an invasion-associated marker. In MDA-MB-231 cells, MMP2 expression was significantly downregulated in the presence of the drug compared with media control (p < 0.05), with vehicle treatment also showing partial suppression (Fig 6.2A). Similarly, in MCF7 cells, drug treatment caused a marked reduction in MMP2 transcript levels relative to control (p < 0.01), whereas DMSO produced a variable effect (Fig 6.2B). These transcriptional changes were supported by immunoblotting, which revealed a reduction in MMP2 protein expression upon drug treatment in both MDA-MB-231 and MCF7 cells, with the expected 28 kDa band detected across conditions (Fig 6.2C). Collectively, these data highlight that while METTL7A is absent in breast cancer cells but enriched in endothelial cells, MMP2 is significantly suppressed by drug exposure in both MDA-MB-231 and MCF7, underscoring the anti-invasive potential of the treatment.
Discussion
This study investigated how ENOX2 and MMP2 and RASSF1A and WFDC10A and METTL7A genes are regulated at the transcriptional and translational levels in multiple human cancer cell lines and endothelial cells under normal conditions and after mebendazole treatment. The research design allowed scientists to study both natural gene expression patterns and drug effects on these patterns which revealed important information about tumors and their blood vessel networks and drug treatment possibilities. The research evaluates gene regulation in eight different cell lines which include solid tumors and blood cancers to create detailed models for understanding how cancer-related and tumor-controlling pathways develop in specific cell types and how these pathways can be targeted by drugs.
This study shows ENOX2–4 expression changes differently between cell lines when treated with mebendazole because MDA-MB-231 and HT-29 and PANC-1 cells show decreased expression but A549 and K562 and EA.hy926 cells show increased expression. ENOX2 modulation produces different effects on cells because MCF7 and HEPG2 cells show minimal response to the treatment [51,52]. ENOX2 functions as a member of the ECTO-NOX family to support cancer cell growth through its enzymatic functions while maintaining redox regulation [53]. The observed decrease in ENOX2–4 expression in specific cancer cells indicates mebendazole disrupts redox equilibrium and cell growth in particular types of tumors. The drug causes lung adenocarcinoma and leukemia cells to express more genes through two mechanisms: compensatory responses and activation of alternative signaling pathways. ENOX2 targeting strategies need to be evaluated through frameworks that consider the unique characteristics of each tumor type. ENOX2–4 basal enrichment in EA.hy926 endothelial cells confirms previous studies that ENOX family members function in normal vascular biology as well as cancer cells [54]. The endothelial bias indicates that ENOX2 should not be considered as a single tumor biomarker because its expression depends on specific cellular contexts which might need individualized therapeutic approaches [55].
This study demonstrates METTL7A shows significant enrichment in endothelial cells although its functions in cancer and vascular biology remain poorly understood. The highest basal METTL7A expression levels were found in EA.hy926 cells but tumor cell lines expressed lower levels of METTL7A which depended on drug treatment. The MDA-MB-231, MCF7, HEPG2, K562, PANC-1 and EA.hy926 cell lines showed upregulation but HT-29 and A549 showed significant downregulation. METTL7A shows context-dependent behavior which suggests its role in distinct signaling pathways that function between different cell types and potentially link to methyltransferase activities and epigenetic modifications. The endothelial specificity of METTL7A corresponds to the growing scientific evidence that endothelial cells function as key players in tumor angiogenesis and drug resistance mechanisms [56,57]. The therapeutic perspective shows that METTL7A expression levels rise in breast and liver cancer cells to help tumors survive under stress but its reduced expression in colorectal and lung cancer cells makes it a promising therapeutic candidate.
The tumor suppressor RASSF1A showed an identical range of regulatory patterns. The treatment did not affect MDA-MB-231 and MCF7 cells but HEPG2 and HT-29 cells showed strong RASSF1A induction while A549 and K562 and PANC-1 cells showed decreased levels. Endothelial cells showed high sensitivity to mebendazole treatment because the compound caused RASSF1A expression to increase by more than 200 times. The observation shows how pharmacological agents can bring back or boost tumor suppressor gene function especially in endothelial cells. Given that RASSF1A is frequently silenced by promoter hypermethylation in cancers, its pharmacological induction offers a promising therapeutic strategy [58,59]. The endothelial-specific robustness of this induction makes it suitable for tumor vasculature normalization which could improve anti-angiogenic treatment effectiveness. The observed downregulation in lung, pancreatic and hematologic malignancies shows that mebendazole does not universally restore RASSF1A activity but instead produces different epigenetic and transcriptional effects based on the cell type.
WFDC10A functions as a tumor suppressor gene which also exhibits protease inhibitory properties and mebendazole treatment led to its strong induction in different tumor cell lines with MDA-MB-231 breast cancer cells showing the greatest increase at more than 40-fold. The cells MCF7, HEPG2, HT-29 and EA.hy926 showed moderate to strong induction. The three cell lines A549, K562 and PANC-1 showed no change. WFDC10A stands out as a highly drug-inducible gene according to these results because it shows the strongest drug response in breast carcinoma cells which depend on protease activity for their invasive and metastatic behavior. WFDC10A induction through its protease-inhibitory function would reduce extracellular matrix breakdown and tumor cell spread which provides a possible explanation for mebendazole's anti-invasive effects.
The research into ENOX2 at both mRNA and protein levels reveals its complex regulatory systems. The results of HEPG2 and K562 cell experiments showed that mebendazole effectively reduces ENOX2-driven redox metabolism in these cell types but HT-29 and PANC-1 cells show only slight sensitivity to the drug. The ENOX2 expression of endothelial cells remained constant after drug treatment which demonstrated their resistance to drug effects compared to tumor cells. ENOX2 shows therapeutic potential because cancer cells express high levels of this protein, but endothelial cells express it at normal levels yet further in vivo research must confirm this specificity.
The study provides its most significant mechanistic finding through its demonstration of how MMP2 invasion and metastasis activity becomes suppressed by mebendazole in breast cancer cells. The two cell lines MDA-MB-231 and MCF7 showed a strong anti-invasive effect through their downregulation of MMP2 at both mRNA and protein levels. The fact that MMP2 was unaffected in endothelial cells further underscores the tumor selectivity of this effect. The study shows that mebendazole stops cancer cell invasion because METTL7A exists in endothelial cells but not in breast cancer cells. The dual mechanism of action provides strong evidence for using mebendazole as an anti-metastatic drug.
The research combines multiple tumor suppressors and oncogenes into a single pharmacological system which makes this study unique according to these results. The research presents a different approach than previous studies because it examines how ENOX2, MMP2, RASSF1A, WFDC10A and METTL7A function as a network that shows different patterns of regulation between various tumor types and endothelial cells. The discovery of METTL7A enrichment in endothelial cells together with RASSF1A pharmacological induction through treatment provides new insights for biomarker research. Importantly, the cell-type–specific modulation of ENOX2, MMP2, RASSF1A, WFDC10A, and METTL7A identified in this study highlights their potential utility as clinically testable molecular signatures for patient stratification, pharmacodynamic monitoring, and prediction of therapeutic response in aggressive cancers, particularly in the context of drug-repurposing strategies. The suppression of MMP2 in breast cancer cells helps achieve clinical goals to prevent cancer cell invasion and metastasis which indicates its potential for human medical applications.
This study benefits from its solid methodology and its well-organized research structure. The research uses eight cell lines which cover both blood cancers and solid tumors to demonstrate wide applicability and show how different cancer types affect experimental results. The combination of qRT-PCR with Western blotting allows researchers to confirm transcriptional findings at the protein level which helps prevent errors that can occur when transcription and translation are not properly linked. The inclusion of endothelial cells serves as a non-cancerous reference point which establishes physiological relevance for the study results to distinguish tumor-related effects from endothelial cell responses. The experimental design with untreated, vehicle-treated, and drug-treated groups allows scientists to differentiate between drug-induced effects and any effects that could result from the vehicle. The reliability of conclusions receives additional support from multiple replicates and suitable parametric and non-parametric analysis methods.
However, there are certain limitations of this study. The study takes place in a laboratory setting which restricts the ability to predict how gene regulation would behave in the intricate tumor microenvironment that contains immune cells and stromal and vascular elements. Immortalized endothelial cells lack the natural behavior of primary endothelial cells and the various features of tumor-associated blood vessels. The study design limited drug exposure to a single concentration at a single time point which made it impossible to study dose–response effects and drug kinetics. The study lacks functional assays for invasion and apoptosis and angiogenesis which prevents direct demonstration of gene modulation effects. ENOX2 and MMP2 protein changes were validated but not all transcriptional results were confirmed at the protein level which creates doubt about their biological effects.
The research needs to validate these findings through in vivo experiments using xenograft and patient-derived organoid models which duplicate the natural microenvironment. The process of determining pharmacological thresholds and drug dynamics needs dose–response and time-course research studies. The evaluation of tumor phenotypes requires functional assays to determine how gene modifications affect cell invasion and proliferation and apoptosis. The drug response analysis begins by using multi-omics methods to detect first determinants through the combination of epigenomic and transcriptomic profiling. The evaluation of METTL7A, RASSF1A and WFDC10A as treatment response biomarkers and ENOX2 and MMP2 suppression as pharmacodynamic efficacy indicators should be included in clinical studies. The study needs complete therapeutic potential realization through the combination of molecular and functional and translational endpoints.
The research makes significant progress in the field by showing that mebendazole produces different yet important effects on cancer-related genes across multiple types of cancer. The study demonstrates how drug responses depend on specific conditions and it establishes mebendazole as a potential cancer treatment through its anti-invasive and anti-angiogenic effects. The research provides two important outcomes by showing how genes work in detail and creating a complete method to study tumor and endothelial cell behavior.
Conclusion
This study provides a comprehensive analysis of ENOX2 and MMP2 together with RASSF1A and WFDC10A and METTL7A in various human cancer cell lines to understand their roles in cancer development and invasion. The highest levels of ENOX2 and MMP2 were found in aggressive subtypes MDA-MB-231 and PANC1 cells which showed post-transcriptional regulation according to protein analysis. Mebendazole demonstrated its ability to block essential cancer-causing mechanisms while affecting WFDC10A and METTL7A based on specific conditions which makes it suitable as a cost-effective cancer treatment candidate.
ENOX2 activates MMP2 through a functional pathway according to the research which also identifies drug-sensitive molecular targets for biomarker-based diagnostics and personalized treatments. The research provides important findings for developing non-invasive cancer biomarkers and low-cost treatments for aggressive tumors that do not respond to therapy even though it has limitations in laboratory settings.
Supporting information
S1 Fig. Experimental workflow for evaluating the molecular effects of mebendazole in human cancer cell lines.
This schematic illustrates the experimental workflow used to investigate the molecular effects of mebendazole treatment in multiple human cancer cell lines. Human cancer cell lines (MDA-MB-231, MCF-7, HEPG2, HT29, K562, A549, and PANC1) and a non-malignant endothelial control cell line (EA.hy926) were cultured under standard conditions. Cells were assigned to untreated, vehicle-treated (DMSO), or drug-treated groups (0.7 µM mebendazole for 24 h). Total RNA was extracted using a Quick-RNA MiniPrep kit, and RNA purity was assessed by NanoDrop spectrophotometry (A260/A280 ratio 1.8–2.0). Complementary DNA (cDNA) was synthesized using PrimeScript RT Master Mix. Gene expression analysis was performed by quantitative real-time PCR (qRT-PCR) using the QuantStudio 5 system, targeting ENOX2, MMP2, RASSF1A, WFDC10A, and METTL7A, with B2M as the housekeeping gene. Relative gene expression levels were calculated using the 2 − ΔΔCt method. Protein expression was further validated by Western blot analysis following protein extraction with RIPA buffer containing protease and phosphatase inhibitors, quantification using the SMART™ BCA assay, separation by SDS-PAGE (50 μg protein per lane), and transfer to nitrocellulose membranes. Primary antibodies against MMP2, ENOX2, and METTL7A were used with β-actin as a loading control, followed by HRP-conjugated secondary antibodies and chemiluminescent detection (ECL).
https://doi.org/10.1371/journal.pone.0345701.s001
(TIF)
S1 File. Raw Data of Gene and Protein Expression Analyses.
This file contains the complete raw data supporting the results presented in Figures 1–6 of the manuscript. It includes quantitative RT-PCR data showing relative fold changes of ENOX2–4, METTL7A, RASSF1A, and WFDC10A in multiple human cancer cell lines (MDA-MB-231, MCF7, HEPG2, HT29, K562, A549, PANC1) and a non-malignant control (EA.hy926) under untreated, vehicle-treated (DMSO), and drug-treated conditions. Statistical significance (P-values) and the direction of gene expression (up- or down-regulated) are indicated. The file also includes original uncropped Western blot images for ENOX2, METTL7A, MMP2, and β-actin across selected cell lines, with molecular weights noted and band intensities measured using Image J software. All data correspond to the experimental findings reported in the main manuscript and support the conclusions regarding drug-induced modulation of gene and protein expression.
https://doi.org/10.1371/journal.pone.0345701.s002
(PDF)
Acknowledgments
The authors sincerely acknowledge the esteemed administrations of RAK Medical and Health Sciences University and Al-Ahliyya Amman University for providing the research facilities essential for this study and gratefully thank RAK Medical and Health Sciences University for supporting the article processing charges for open-access publication.
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