{"paper_id":"a284cd53-da12-45c5-ac6e-3ab426f22dda","body_text":"Selective targeting of genes \nregulated by zinc finger proteins in \nendometriosis and endometrioid \nadenocarcinoma by zinc niflumato \ncomplex with neocuproine\nIvana Špaková1, Lukáš Smolko1, Gabriela Sabolová1, Zuzana Badovská1, Katarína Kalinová2, \nCorina Madreiter-Sokolowski2, Wolfgang F. Graier2, Mária Mareková1, Janka Vašková1 & \nMiroslava Rabajdová1\nInadequate angiogenesis of endometriotic implants stimulated by the inflammatory microenvironment \nin the uterine region leads to the development of gynecological diseases, which significantly reduce \nthe fertility and vitality of young women. Angiogenic processes are controlled by factors whose \nactivities are regulated at the gene level by reactive oxygen species (ROS), hypoxia-induced factors \n(HIFs), and zinc-finger proteins (ZnFs) or posttranscriptionally via non-coding RNAs. The cooperation of \nthese factors is responsible for the manifestation of pathological stimuli in the form of endometriosis \nof the body of the uterus, ovaries, or peritoneum, from which endometrioid carcinoma can develop. \nMolecules that can control gene expression by their intercalation to target DNA sequence, such as \n[Zn(neo)(nif)2], could prevent the hyperactivation of pro-angiogenic pathways (decrease HIF-1α, \nVEGF-A, TGF-β1, COX2, and ANG2/ANG1), reduce the formation of ROS, and reduce the risk of uterine \nneoplasticity. The NSAID-metal complex [Zn(neo)(nif)2] shows an ability to intercalate into ZNF3-\n7 target DNA sequence at a higher rate, which could explain its effect on genes regulated by this \ntranscription factor. In addition, [Zn(neo)(nif)2] affects ROS production and Ca2+ level, possibly pointing \nto mitochondrial dysfunction as a potential cause for the described apoptosis.\nKeywords Endometriosis, Endometrioid adenocarcinoma, [Zn(neo)(nif)2], Angiogenesis, Ca2+, ROS\nPathologies in the pelvis primarily arise from the attachment and growth of endometriotic implants in a non-\nphysiological environment – outside the uterine cavity. This process is altered by uncontrolled angiogenesis, \nwhich bypasses the physiological immune response and elevates hypoxia-inflammatory conditions, driving the \ntransition of eutopic endometrium to ectopic endometriosis and its potential malignant transformation. The \ntransition of endometriosis to a malignant state is regulated by molecular mechanisms with key factors such as \nHIF-1α (hypoxia-inducible factor 1α), COX2 (cyclooxygenase 2), VEGF-A (vascular endothelial growth factor \nA), zinc-fingers (ZNFs)1, Nrf2-ARE (nuclear factor erythroid 2-related factor 2 – antioxidant response element)2 \nand microRNAs (miRs)3.\nAll these transcription factors (TFs) are necessary for the physiological regulation of uterine lining renewal \nduring the menstrual cycle and embryo implantation. For instance, hypoxia induced by a physiological decrease \nin progesterone (P4) levels increases the activity of the transcription factor HIF-1α together with COX2, which \nregulates the synthesis of prostaglandins (PGs)4. The rapid transition to severe hypoxia due to the interruption \nof blood supply in the uterine endothelium by vessel coiling5 activates apoptotic signals, allowing immune cells \nto remove epithelial cells during menstruation. Inadequate, prolonged local hypoxia significantly stimulates \npathological angiogenesis 6 by suppressing apoptosis while promoting an inflammatory microenvironment 7. \nHypoxia-accelerated implantation of viable endometriotic cells occurs through the production of pro-angiogenic \n1Department of Medical and Clinical Biochemistry, P . J. Šafárik University in Košice, Trieda SNP 1, 04011 \nKošice, Slovakia. 2Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging Molecular \nBiology and Biohemistry, Medical University of Graz, Neue Stiftingtalstrasse 6/4, T8010 Graz, Austria. email:  \nmiroslava.rabajdova@upjs.sk\nOPEN\nScientific Reports |        (2025) 15:10126 1| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports\n\n\nfactors (VEGF , PGF , TGF , Ang-1)8, which are released into the surrounding tissue and bind to capillaries and \narterioles, promoting the formation of new blood vessels9. This hypoxia-mediated angiogenesis is also targeted \nby miR-20610,11, -376a12, and let-7c13, which acts as an inhibitor of angiogenesis, whereas miR-133b or miR-23a \nserve as promoters of angiogenesis12.\nMicroRNAs can further regulate the expression of ZNFs, as they contain many seed-matched sequences \npredominantly localized to the ZNF regions coding the C2H2 domain 14. The absence of the specific ZNF3 \ndomain suppresses auto-ADP-ribosylation of PARP 15, which is involved in DNA repair, angiogenesis, \nand chemoresistance of gynecological pathologies 16. ZNF3 selectively inhibits PARP1 (poly(ADP-ribose) \npolymerase 1), which could serve as a potential therapeutic target for tumor treatment 17. In endometriosis, \nelevated reactive oxygen species (ROS) and mitochondrial dysfunction cause DNA strand breaks and activate \nDNA repair via PARP . Hyperactivation of PARP by ROS leads to depletion of NAD+ and ATP and can disrupt \ncalcium homeostasis (increasing intracellular Ca2+ levels), exacerbating cellular stress and ultimately leading to \ncell death18.\nThe Nrf2 plays a pivotal role in cellular defense against oxidative stress by regulating antioxidant response \nelements19 and has been linked to endoplasmic reticulum (ER) oxidative protein folding and calcium \nhomeostasis20. Impaired ER redox signaling can decrease Nrf2 nuclear translocation, resulting in ER calcium \noverload and increased calcium-dependent cell secretion20. In endometriosis, Nrf2 activity is often compromised, \nresulting in increased oxidative stress and mitochondrial dysfunction21.\nThe Nrf2-ARE pathway directly affects neoangiogenesis through the ANG2/ANG1 axis. Nrf2 activation \ninduces antioxidant enzymes (GPx, SOD) that lower reactive oxygen species (ROS) levels, thereby modulating \ninflammation and ensuring vascular stability 2. In both physiological and pathological conditions, such as \nendometriosis and endometrial carcinoma, the balance between angiopoietins ANG1 and ANG2 is essential for \nvascular homeostasis. ANG1 stabilizes blood vessels via the Tie2 receptor, while ANG2 antagonizes this effect, \npromoting vascular remodeling and increased permeability, particularly in the presence of VEGF22,23.\nOxidative stress and inflammatory cytokines can upset this balance by increasing ANG2 expression, leading \nto abnormal angiogenesis in gynecological disorders. The Nrf2-ARE pathway, through antioxidant responses, \nhelps counteract these effects by reducing ROS levels and restoring the ANG1/ANG2 ratio, promoting normal \nvascular function24.\nEvidence suggests that interactions between the Nrf2-ARE pathway and the ANG2/ANG1 axis influence \nendometrial lesion progression by regulating oxidative stress and inflammation. Prolonged hypoxia may trigger \npersistent Nrf2 activation, contributing to vascular dysfunction and increased permeability. Zinc-finger proteins \n(ZnFs) have been identified as potential regulators of the Nrf2-ARE pathway and angiopoietin expression, \noffering new therapeutic insights. ZnFs maintain redox balance and transcriptional regulation of genes related to \nendometriotic cell survival and apoptosis resistance25. In endometriosis and endometrial carcinoma, dysregulated \nZnFs may impair Nrf2 function, exacerbating oxidative stress and promoting pathological angiogenesis via the \nANG1/ANG2 axis26.\nFurthermore, ZNFs regulate the activity of tumor growth factor β (TGFβ), which contributes to TIEG \noverexpression and induces apoptosis27. Upregulation of ZNFs is associated with apoptosis resistance through \nregulating apoptotic genes such as BAX, Bcl-2, and Caspase-3 via ROS-induced oxidative damage28. Additionally, \nZNFs initiate an inflammatory response, support the implantation and survival of endometriotic lesions on the \nperitoneal surface, and contribute to the worsening course and development of endometriosis29.\nThe integrity of the newly formed vascular system is further regulated by ANG-1 (angiopoietin 1) and ANG-\n2 (angiopoietin 2), which may interact in the progression of endometriosis and represent potential therapeutic \ntargets for non-steroidal anti-inflammatory drugs (NSAIDs) that influence angiopoietin expression of 30,31. \nSuppression of the inflammatory mediator COX2 by NSAIDs poses a challenge in treating chronic inflammatory \ndiseases, as prolonged use of NSAIDs has been shown to increase oxidative stress and disrupt the sensitive \nantioxidant status of patients 32. Metal complexes with NSAIDs represent an innovative approach to treating \ninflammatory diseases, as their effect is not limited to COX2 inhibition but also involves interaction with nucleic \nacids and direct modulation of the enzyme activity, such as MMPs33.\nOur previous studies demonstrated the potential therapeutic effects of NSAID-biometal complex [Zn(neo)\n(nif)2] (neo = 2,9-dimethyl-1,10-phenathroline; nif = 2-[3-(trifluoromethyl)anilino]nicotinato), as it exhibited \nhigher cytotoxicity on cells with high inflammatory metabolism 33. Continuing our investigation of the \nmechanism of action of this complex, we analyzed the expression of targets involved in angiogenesis activated \nby hypoxia-inflammatory stimuli.\nResults\nDNA intercalation\nOur previous DNA binding studies performed on samples isolated from endometriotic 12Z and control HME1 \ncell lines indicated the binding specificity of [Zn(neo)(nif) 2]33. To further investigate the binding specificity \nof the studied complex, short double-stranded DNA (dsDNA) oligonucleotide sequences were selected for \nthe standard competitive fluorescence binding studies with ethidium bromide. The selected sequences were \nCCCTC-binding factor zinc-finger protein 3 (ZnF3-7) (5'- T A G C G C C C C C T G C T G G C-3'/3'- A T C G C G G G G G A \nC G A C C G-5’) and CCAAT/enhancer-binding proteins (C/EBP) (5'- A T T G C G C A A T-3'/3'- T A A C G C G T T A-5’). \nBoth sequences are located in the regulatory regions of their respective genes and are recognized by transcription \nfactors during transcription.\nExperimental results showed that the studied complex displaces ethidium bromide and binds to both \nsequences via intercalation, as indicated by quenching of fluorescence in the DNA-EB complex (Figure S1). \nA more thorough evaluation of the results revealed a higher affinity of the complex to the ZnF3-7 sequence \n(KSV = 2.17(2) × 105 M-1) in comparison with the EBP sequence (KSV = 1.10(3) × 105 M-1) (Fig. 1).\nScientific Reports |        (2025) 15:10126 2| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\nAngiogenic, inflammatory, antioxidant, and apoptotic gene expression\nWe determined the relative gene expression of angiogenic factors (VEGF-A, TGF-β1, ANG1, ANG2) (Table 1) \nas we hypothesize that the [Zn(neo)(nif)2] complex preferentially binds to a ZnF-like sequence, such as ZNF3-7 \nor other ZNFs. The intercalation of [Zn(neo)(nif)2] into DNA can potentially influence the activity of angiogenic \ntranscription factors. We proved this hypothesis by analyzing the expression of genes that regulate vascular \nformation in a spheroid model of HME1, 12Z, and A2780 cells.\nA non-significant increase in the ANG2/ANG1 gene expression ratio was observed in HME1 cells treated \nwith [Zn(neo)(nif) 2] (P = 0.9812), as well as in HME1 treated with cisPt (P = 0.7468). In the 12Z model, a \nsignificant elevation of ANG2/ANG1 ratio was found in the cells treated with cisPt (P < 0.0001), as well as in \nthose treated with [Zn(neo)(nif)2] (P < 0.0001), compared to untreated control 12Z 3D model cells (Fig. 2A). To \ncompare the efficiency of our compound with standard treatment (cisPt), the ANG2/ANG1 ratio showed a non-\nsignificant increase in A2780 cells with cisPt (P = 0.1243). In contrast, a significant increase in the ANG2/ANG1 \nratio was observed in A2780 cells treated with [Zn(neo)(nif)2] (P < 0.0001) (Fig. 2A).\nNext, we analyzed the VEGF-A/TGF-β1 ratio as an indicator of angiogenic activity in the samples. Our \nexperiments revealed a significant change in the spheroid cell model of HME1 under tested conditions. In the \nHME1 model treated with cisPt, a significant increase in the VEGF-A/TGF-β1 ratio was observed (P < 0.0001), as \nwell as in the HME1 model treated with [Zn(neo)(nif)2] (P < 0.0001). In 12Z cells treated with cisPt, a significant \nincrease in the VEGF-A/TGF-β1 ratio was found (P = 0.0019), whereas a non-significant change was observed \nin the 12Z model treated with [Zn(neo)(nif) 2]. In A2780 cells, treatment with both cisPt and [Zn(neo)(nif) 2] \nresulted in significant increases in the VEGF-A/TGF-β1 ratio (P = 0.0044 and P < 0.0001, respectively) (Fig. 2B).\nWe analyzed the relative gene expression of angiogenic factors (VEGF-A, TGF-b1, ANG1, ANG2), \npredicting that the studied complex preferentially binds to a ZnF-like sequence. Although significant changes \nHME1 VEGF-A (P value, significance) TGF-β1 (P value, significance) ANG1 (P value, significance) ANG2 (P value, significance)\ncontrol vs. cisPt 0.4763 (ns) ↓  < 0.0001 (***) ↓ 0.0002 (***) ↑ 0.1982 (ns) ↓\ncontrol vs. [Zn(neo)(nif)2] 0.0472 (*) ↓  < 0.0001 (***) ↓ 0.0028 (**) ↑ 0.1677 (ns) ↓\ncisPt vs. [Zn(neo)(nif)2] 0.4306 (ns) ↓ 0.9983 (ns) ↑ 0.3207 (ns) ↓ 0.0337 (*) ↓\n12Z\ncontrol vs. cisPt  < 0.0001 (***) ↑  < 0.0001 (***) ↑ 0.0056 (**) ↑ 0.3062 (ns) ↑\ncontrol vs. [Zn(neo)(nif)2] 0.9430 (ns) ↑ 0.9516 (ns) ↑ 0.0377 (*) ↑ 0.0235 (*) ↑\ncisPt vs. [Zn(neo)(nif)2]  < 0.0001 (***) ↓  < 0,0001 (***) ↓ 0.0088 (**) ↓ 0.8098 (ns) ↑\nA2780\ncontrol vs. cisPt 0.9940 (ns) ↑ 0.7830 (ns) ↑ 0.3315 (ns) ↓ 0.6487 (ns) ↑\ncontrol vs. [Zn(neo)(nif)2] 0.9858 (ns) ↑ 0.8925 (ns) ↑ 0.8926 (ns) ↑ 0.3668 (ns) ↓\ncisPt vs. [Zn(neo)(nif)2] 0.9982 (ns) ↓ 0.9754 (ns) ↓ 0.4056 (ns) ↑ 0.2783 (ns) ↓\nTable 1. Significance values of VEGF-A, TGF-β1, ANG1, and ANG2 (n = 6) under three tested conditions: \ncontrol (untreated spheroid cells), cisPt (spheroid cells treated with 10 μM cis-platin), [Zn(neo)(nif)2] \n(spheroid cells treated with 10 μM [Zn(neo)(nif)2]) across three experimental 3D models.\n \nFig. 1. Comparison of fitted Stern–Volmer plots from competitive binding studies with EB for ZnF3-7 (linear \nfit 95.8%) (green) and EBP (linear fit 98.4%) (red) dsDNA sequences.\n \nScientific Reports |        (2025) 15:10126 3| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\nin the individual expression levels of the calculated ratios were observed in specific groups (Table 1), their gene \nexpression did not reach significance across all conditions.\nTo evaluate the effects of our compound on inflammation and antioxidant activity, the Nrf2/COX2 gene \nexpression showed a non-significant increase in the HME1 model under both conditions: cisPt (P = 0.8172) \nand [Zn(neo)(nif) 2] (P = 0.4868). In 12Z cells treated with cisPt showed non-significant change (P = 0.6750), \nwhereas a significant elevation of Nrf2/COX2 ratio was observed in the 12Z model treated with [Zn(neo)(nif)2] \n(P < 0.0001) (Fig. 2C). In the A2780 model, the Nrf2/COX2 ratio significantly increased under both treated \nconditions (P < 0.0001).\nFurthermore, the COX2/HIF-1α ratio was analyzed to evaluate another aspect of the inflammatory impact \nof our compound. A significant increase in the COX2/HIF-1α gene expression ratio was observed in HME1 \ncells treated with both cisPt and [Zn(neo)(nif) 2] (P < 0.0001). The change in the COX2/HIF-1α ratio was also \nsignificant in 12Z cells treated with cisPt (P = 0.0001) and [Zn(neo)(nif)2] (P = 0.0044). In contrast, the change in \nthe COX2/HIF-1α ratio in A2780 cells treated with cisPt (P = 0.9844), as well as in those treated with [Zn(neo)\n(nif)2] (P = 0.9998), was non-significant (Fig. 2D).\nTo evaluate the effect of our test compound on apoptosis-associated gene expression, we selected CAS3 and \nBAX. A non-significant decrease in the CAS3/BAX gene expression ratio was observed in HME1 cells treated \nwith cisPt (P = 0.4299) and in the HME1 model treated with [Zn(neo)(nif) 2] (P = 0.8530) (Fig. 2E). Similarly, \na decrease in the CAS3/BAX ratio was noted in 12Z cells treated with cisPt (P = 0.8766), while a significant \nreduction was observed in the 12Z model treated with [Zn(neo)(nif) 2] (P < 0.0001). In the A2780 model, \nthe CAS3/BAX ratio remained non-significantly changed under both treatment conditions (cisPt P = 0.9997; \n[Zn(neo)(nif)2] P = 0.1528).\n \nScientific Reports |        (2025) 15:10126 4| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\nTable 2 summarizes the individual changes in the gene expression of the monitored inflammatory (COX2, \nHIF-1α), antioxidant (Nrf2), and apoptotic (CAS3, BAX) factors.\nBased on the detected changes in Nrf2 gene expression levels, we analyzed the gene expression of its two \nselected target gene products, GPx1 and SOD1 (Fig.  3). The relative gene expression of GPx1 significantly \nincreased in all studied cell lines under both tested conditions (P < 0.001). Different conclusions were drawn \nfor the relative gene expression of SOD1, where we observed a significant increase in expression in the HME1 \ncell model following treatment with [Zn(neo)(nif) 2] (P = 0.0188). In the 12Z cell model, a significant increase \nin expression was determined under the influence of both tested compounds (P < 0.0001), and in the A2780 cell \nmodel, we observed a significant increase in expression following treatment with cisPt (P < 0.0001).\nFig. 2. ( A): The gene expression ANG2/ANG1 ratio was analyzed under three tested conditions: control \n(untreated spheroid cells), cisPt (spheroid cells treated with 10 μM cis-platin), and [Zn(neo)(nif)2] (spheroid \ncells treated with 10 μM [Zn(neo)(nif) 2]) measured in six replicates (n = 6). Changes in the ANG2/ANG1 \nratio in the HME1 cell line were insignificant across all tested conditions (cisPt P = 0.9812; [Zn(neo)(nif)2] \nP = 0.7468; cisPt vs. [Zn(neo)(nif)2] P = 0.8665). Changes in the ANG2/ANG1 ratio in the 12Z cells, significant \nchanges were observed for control vs. cisPt (P < 0.0001, ***), control vs. [Zn(neo)(nif)2] (P < 0.0001, ***), and \ncisPt vs. [Zn(neo)(nif)2] (P < 0.0001, +  + +). In A2780 cells, the ANG2/ANG1 ratio changes were significant \nfor control vs. [Zn(neo)(nif)2] (P < 0.0001, ***) and cisPt vs. [Zn(neo)(nif)2] (P < 0.0001, +  + +), insignificant \nfor control vs. cisPt (P = 0.1243). (B): The VEGF-A/TGFβ1 ratio (n = 6) showed significant changes in the \nHME1 model under treatment with cisPt (P < 0.0001, ***), under treatment with [Zn(neo)(nif)2] (P < 0.0001, \n***), and between cisPt and [Zn(neo)(nif)2] treatments (P < 0.0001, +  + +). In the 12Z cells treated with \ncisPt, a significant change was observed (P = 0.0019, **), whereas treatment with [Zn(neo)(nif)2] showed an \ninsignificant change (P = 7334). The difference between 12Z cells treated with cisPt and [Zn(neo)(nif)2] was \nsignificant (P = 0.0109, +). In the A2780 cell line, significant changes were observed under treatment with \ncisPt (P = 0.0044, **) and treated with [Zn(neo)(nif)2] (P < 0.0001, ***), compared to the control. However, \nthe difference between A2780 cells treated with cisPt and [Zn(neo)(nif)2] was insignificant (P = 0.1622). \n(C): The gene expression ratio of Nrf2/COX2 (n = 6) showed insignificant changes in the HME1 cell line \nunder treatment with cisPt (P = 0.8172), [Zn(neo)(nif)2] (P = 0.4868), and between cisPt and [Zn(neo)\n(nif)2] treatments (P = 0.8468). In 12Z cells, significant changes were observed for control vs. [Zn(neo)(nif)2] \n(P < 0.0001, ***) and cisPt vs. [Zn(neo)(nif)2] (P < 0.0001, +  + +), while the change under treatment with \ncisPt was insignificant (P = 0.6750). In A2780 cells, significant changes were observed for control vs. cisPt \n(P < 0.0001, ***), control vs. [Zn(neo)(nif)2] (P < 0.0001, ***), and cisPt vs. [Zn(neo)(nif)2] (P < 0.0001, +  + +). \n(D): The gene expression COX2/HIF-1α ratio (n = 6) showed significant changes in the HME1 cell line \nfor control vs. cisPt (P = 0.0217, *), under treatment with [Zn(neo)(nif)2] (P < 0.0001, ***) compared to \ncontrol, and between cisPt vs. [Zn(neo)(nif)2] (P < 0.0001, +  + +). COX2/HIF-1α ratio changes of 12Z were \nsignificant under cisPt treatment (P = 0.0001, ***), with [Zn(neo)(nif)2] (P = 0.0044, **), and between tested \ncompounds cisPt and [Zn(neo)(nif)2] (P < 0.0001, +  + +). The COX2/HIF-1α ratio changes in A2780 cells were \ninsignificant for control vs. cisPt (P = 0.9844), control vs. [Zn(neo)(nif)2] (P = 0.9998), and cisPt vs. [Zn(neo)\n(nif)2] (P = 0.9806). E: The gene expression of CAS3/BAX ratio (n = 6) showed insignificant changes in the \nHME1 cell line under treatment with cisPt (P = 0.4299), [Zn(neo)(nif)2] (P = 0.8530), and also between cisPt \nvs. [Zn(neo)(nif)2] treatments (P = 0.7295). In 12Z cells, insignificant changes were found for control vs. cisPt \n(P = 0.8766), while significant changes were observed for control vs. [Zn(neo)(nif)2] (P < 0.0001), and cisPt vs. \n[Zn(neo)(nif)2] (P < 0.0001, +  + +). In A2780 cells, the CAS3/BAX ratio remained insignificant across all tested \nconditions: cisPt (P = 0.9997), [Zn(neo)(nif)2] (P = 0.1528), and between cisPt and [Zn(neo)(nif)2] (P = 0.1168).\n◂\nHME1 Nrf2 (P value, signif.) COX2 (P value, signif.) HIF-1α (P value, signif.) CAS3 (P value, signif.) BAX (P value, signif.)\ncontrol vs. cisPt 0.0071 (**) ↑ 0.0028 (**) ↓ 0.0223 (*) ↓ 0.2321 (ns) ↓ 0.0881 (ns) ↑\ncontrol vs. [Zn(neo)(nif)2] 0.0025 (**) ↑ 0.0096 (**) ↓ 0.1097 (ns) ↓ 0.0170 (*) ↓ 0.0782 (ns) ↑\ncisPt vs. [Zn(neo)(nif)2] 0.1114 ↑ 0.0015 (**) ↓ 0.4543 (ns) ↓ 0.0781 (ns) ↓ 0.0704 (ns) ↑\n12Z\ncontrol vs. cisPt 0.0065 (**) ↑ 0.0003 (***) ↓ 0.1602 (ns) ↓ 0.0013 (**) ↓ 0.0073 (**) ↑\ncontrol vs. [Zn(neo)(nif)2] 0.0191 (*) ↑ 0.0571 (ns) ↓ 0.5077 (ns) ↓ 0.4635 (ns) ↑ 0.4077 (ns) ↑\ncisPt vs. [Zn(neo)(nif)2] 0.0073 (**) ↑ 0.0076 (**) ↓ 0.1597 (ns) ↓ 0.0016 (**) ↑ 0.0180 (*) ↑\nA2780\ncontrol vs. cisPt 0.0004 (***) ↓ 0.5231 (ns) ↓ 0.0062 (**) ↑ 0.1597 (ns) ↑ 0.1452 (ns) ↑\ncontrol vs. [Zn(neo)(nif)2] 0.0004 (***) ↑ 0.1202 (ns) ↑ 0.0033 (**) ↑ 0.2337 (ns) ↑ 0.0464 (*) ↑\ncisPt vs. [Zn(neo)(nif)2] 0.9999 (ns) ↑ 0.5414 (ns) ↑ 0.0023 (**) ↑ 0.1557 (ns) ↑ 0.0162 (*) ↑\nTable 2. Significance values of Nrf2, COX2, HIF-1α, and CAS3, BAX (n = 6) under three tested conditions: \ncontrol (untreated spheroid cells), cisPt (spheroid cells treated with 10 μM cis-platin), [Zn(neo)(nif)2] \n(spheroid cells treated with 10 μM [Zn(neo)(nif)2]) across three experimental 3D models.\n \nScientific Reports |        (2025) 15:10126 5| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\nTable 3 summarizes the individual changes in the gene expression of the monitored antioxidant factors \n(GPx1 and SOD1).\nExpression of angiogenic and inflammatory microRNAs\nTo further support our findings on the potential effects of our compounds on inflammatory and angiogenic \npathways, we analyzed the delicate balance between pro-angiogenic (miR-23a, -133b, let-7c) and anti-angiogenic \n(miR-206, -376a) microRNA levels, which play a crucial role in the physiological regulation of vascular network \nformation and immune response.\nMicroRNAs are small yet highly significant molecules that regulate gene expression and control cellular \nmetabolism. Our focus was on determining the ratio of target miRNAs to selected angiogenic (VEGF-A, \nTGF-β1), antioxidant (Nrf2), and inflammatory factors (COX2, HIF-1α). The miRNA/mRNA ratio provides \ninsight into the extent of miRNA influence on the expression of the corresponding mRNA, as miRNAs can \npromote degradation and inhibit its translation into protein. This ratio can, therefore, help predict the direction \nof cellular metabolism.\nHME1 GPx1 (P value, signif.) SOD1 (P value, signif.)\ncontrol vs. cisPt  < 0.0001 (***) ↑ 0.0736(ns) ↑\ncontrol vs. [Zn(neo)(nif)2]  < 0.0001 (***) ↑ 0.0188 (*) ↑\ncisPt vs. [Zn(neo)(nif)2] 0.0024 (**) ↑ 0.7808 (ns) ↑\n12Z\ncontrol vs. cisPt  < 0.0001 (***) ↑  < 0.0001 (***) ↑\ncontrol vs. [Zn(neo)(nif)2]  < 0.0001 (***) ↑  < 0.0001 (***) ↑\ncisPt vs. [Zn(neo)(nif)2]  < 0.0001 (***) ↓ 0.3403 (ns) ↑\nA2780\ncontrol vs. cisPt  < 0.0001 (***) ↑  < 0.0001 (***) ↑\ncontrol vs. [Zn(neo)(nif)2]  < 0.0001 (***) ↑ 0.4126 (ns) ↑\ncisPt vs. [Zn(neo)(nif)2]  < 0.0001 (***) ↓  < 0.0001 (***) ↓\nTable 3. Significance values of GPx1 and SOD1 (n = 3) under three tested conditions: control (untreated \nspheroid cells), cisPt (spheroid cells treated with 10 μM cis-platin), [Zn(neo)(nif)2] (spheroid cells treated with \n10 μM [Zn(neo)(nif) 2]) across three experimental 3D model cells.\n \nFig. 3. ( A): The gene expression GPx1 (n = 3) under three tested conditions: control (untreated spheroid \ncells), cisPt (spheroid cells treated with 10 μM cis-platin), and [Zn(neo)(nif)2] (spheroid cells treated with \n10 μM [Zn(neo)(nif) 2]) showed a significant increase under treatment with cisPt (P < 0.0001, ***), [Zn(neo)\n(nif)2] (P < 0.0001, ***), and between cisPt and [Zn(neo)(nif)2] (P = 0.0024, + +). In 12Z cells, GPx1 expression \nwas significantly increased under both treatment conditions, cisPt and [Zn(neo)(nif)2] (P < 0.0001, ***), and \na significant difference was observed between cisPt vs. [Zn(neo)(nif)2] (P < 0.0001, +  + +). The A2780 model \nalso showed a significant increase under both treatment conditions (P < 0.0001, ***), as well as a significant \ndifference between cisPt and [Zn(neo)(nif)2] (P < 0.0001, +  + +). (B): The gene expression SOD1 (n = 3) under \nthe same three tested conditions: control (untreated spheroid cells), cisPt (spheroid cells treated with 10 μM \ncis-platin), and [Zn(neo)(nif)2] (spheroid cells treated with 10 μM [Zn(neo)(nif)2]) indicated an insignificant \nincrease in HME1 cells under treatment with cisPt (P = 0.0736), a significant increase under treatment with \n[Zn(neo)(nif)2] (P = 0.0188), and no significant change between cisPt and [Zn(neo)(nif)2] (P = 0.7808). In 12Z \ncells, a significant increase in SOD1 expression was observed under both cisPt and [Zn(neo)(nif)2] (P < 0.0001, \n***), while the difference between cisPt and [Zn(neo)(nif)2] was insignificant (P = 0.3403). In the A2780 model, \nSOD1 gene expression was significantly increased under treatment with cisPt (P < 0.0001, ***), increased \ninsignificantly under treatment with [Zn(neo)(nif)2] (P = 0.4126), and showed a significant difference between \ncisPt and [Zn(neo)(nif)2] treatments (P < 0.0001, +  + +).\n \nScientific Reports |        (2025) 15:10126 6| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\nWe determined the ratio of miR-206, -23a, -376a, -133b, and let-7c against VEGF-A, as it has previously \ndescribed that these miRs influence the expression of VEGF-A and angiogenesis. We observed a significant \nincrease in the ratio of miR-206/VEGF-A (Fig.  4A) in the HME1 model treated with cisPt (P < 0.0001). A \nsignificant decrease in miR-206/VEGF-A ratio was observed in 12Z cells treated with both cisPt (P < 0.00001) \nand [Zn(neo)(nif) 2] (P < 0.0001). In A2780 cells, we observed a significant decrease in mi-206/VEGF-A ratio \nonly in the group treated with [Zn(neo)(nif)2] (P = 0.0021).\nAn insignificant increase in the miR-23a/VEGF-A ratio (Fig.  4B) was observed in HME1 cells treated with \ncisPt (P < 0.0001). In the 12Z model, a significant decrease was observed under both treatment conditions, cisPt \n(P < 0.0001) and [Zn(neo)(nif)2] (P < 0.0001), while the A2780 cell model showed no significant changes.\nThe miR-133b/VEGF-A ratio (Fig. 4D) showed a significant increase in HME1 cells treated again with cisPt \n(P < 0.0001). In 12Z cells, a significant decrease in miR-376a/VEGF-A ratio was observed under both treatment \nconditions, cisPt (P < 0.0001) and [Zn(neo)(nif)2] (P < 0.0001). The A2780 cells did not show significant changes \nin the miR-376a/VEGF-A ratio.\nA significant increase in the miR-133b/VEGF-A ratio was observed in HME1 cells (P < 0.0001), while in \n12Z cells, a significant decrease was determined under both treatments (P < 0.0001). In the A2780 model, a \nsignificant decrease was observed with cisPt treatment (P = 0.0358) and [Zn(neo)(nif)2] (P = 0.0015).\nWe identified a significant increase in the let-7c/VEGF-A ratio in the HME1 model treated with cisPt \n(P < 0.0001). The 12Z cell model showed a significant decrease in the let-7c/VEGF-A ratio under both treatment \nconditions (P < 0.0001), as did the A2780 cells, which exhibited the same significant decrease (P < 0.0001) under \nboth tested conditions (Fig. 4E).\nThe expression of miR-133b and let-7c significantly impacts the expression of TGF-β1, as previously \ndescribed6,34. The levels of TGF-β1 and miR-133b, along with let-7c, influence the epithelial-mesenchymal \ntransition, which is characteristic of promoted endometriosis. The calculated ratio of miR-133b/TGF-β1 showed \na significant increase in the HME1 model under both treatment conditions: cisPt (0.0009) and [Zn(neo)(nif) 2] \n(P < 0.0001). The 12Z model showed a significant decrease under [Zn(neo)(nif)2] treatment (P = 0.0045), and the \nA2780 model exhibited a significant decrease with cisPt treatment (P = 0.0301) and [Zn(neo)(nif) 2] treatment \n(0.0100) as well (Fig. 5A).\nWe observed a significant increase in the let-7c/TGF-β1 ratio in HME1 cells treated with cisPt (P = 0.004) \nand [Zn(neo)(nif)2] (P = 0.0002). In the 12Z model, the let-7c/TGF-β1 ratio significantly decreased under both \ntreatment conditions (cisPt P = 0.0201; [Zn(neo)(nif)2] P = 0.0043). The A2780 model also showed a significant \ndecrease in the let-7c/TGF-β1 ratio under both treatment conditions (cisPt P = 0.0066; [Zn(neo)(nif) 2] \nP = 0.0041) (Fig. 5B).\nMiR-206 recognizes the binding site of HIF-1α and can regulate the HIF transcription factor. It can inhibit \ncell proliferation and extracellular matrix accumulation by targeting HIF-1α. Based on the direct effect of miR-\n206 on HIF-1α, we performed an additional calculation of the miR-206/HIF-1α ratio, which showed a significant \nincrease in HME1 cells treated with cisPt (P < 0.0001) and [Zn(neo)(nif) 2] (P = 0.0039). In contrast, 12Z cells \nexhibited a significant decrease under treatment with [Zn(neo)(nif)2] (P = 0.0002), while A2780 cells treated \nwith cisPt showed a significant increase (P = 0.0023) (Fig. 5C).\nNrf2-dependent miR-206 plays an essential role in cell metabolism by targeting the pentose phosphate \npathway, leading to the inhibition of proliferation. We observed a significant decrease in the miR-206/Nrf2 \nratio in the 12Z model under cisPt treatment (P = 0.0013) and [Zn(neo)(nif)2] treatment (P = 0.0003). Similarly, \nA2780 cells exhibited a significant decrease under treatment with both compounds (cisPt P = 0.0047; [Zn(neo)\n(nif)2] P = 0.0021) (Fig. 5D). In contrast, HME1 cells showed a slight, non-significant reduction in miR-206/Nrf2 \nunder treatment of both tested conditions. This reduction may reflect elevated antioxidant activity, leading to \nincreased Nrf2 levels, which could, in turn, decrease miR-206 expression under studied conditions.\nIn the HME1 spheroids, we observed a significant elevation of the angiogenesis-promoting miR-133b (cisPt \nP = 0.0005; [Zn(neo)(nif)2] P = 0.0030) (Figure S2A), along with a considerable upregulation of the angiogenesis-\ninhibiting miR-206 (cisPt P = 0.0070; [Zn(neo)(nif)2] P = 0.0187) (Figure S2B). The levels of the other miRs did \nnot show considerable changes in either treatment group.\nIn the 3D model of the 12Z cell line (Figure S2C), a significant decrease was observed in the level of the \nangiogenesis-promoting miR-23a (cisPt P = 0.0471; [Zn(neo)(nif) 2] P = 0.0149) and let-7c (cisPt P < 0.0001; \n[Zn(neo)(nif)2] P = 0.0001) (Figure S2D). The expression levels of the remaining target miRs did not show \nsignificant changes in either treatment group.\nSpheroids of A2780 (Figure S2E) exhibited a significant downregulation of the angiogenesis-promoting \nmiR-133b ([Zn(neo)(nif)2] P = 0.0470), a decrease in the proangiogenic let-7c (cisPt P = 0.0019; [Zn(neo)(nif)2] \nP = 0.0002), and a significant upregulation of the angiogenesis-inhibiting miR-376 ([Zn(neo)(nif) 2] P = 0.0006) \n(Figure S2F). The levels of other miRs did not show considerable changes in either treatment group.\nAngiogenic, inflammatory, and antioxidant protein expression\nGene expression typically predicts the corresponding protein levels; however, these levels may be influenced by \npost-transcriptional and post-translational modifications, potentially leading to unexpected protein levels. We \nanalyzed the protein levels of angiogenic proteins VEGF-A and TGF-β1, the inflammatory marker COX2, and \nthe antioxidant marker Nrf2 (both in its total and phosphorylated (active) form) (Table 4).\nTo evaluate protein levels under tested conditions, we calculated the VEGF-A/TGF-β1 ratio, the Nrf2 active/\nCOX2 ratio, and the Nrf2 active/Nrf2 ratio. In the control model of HME1 cells, the VEGF-A/TGF-β1 ratio \nshowed a non-significant decrease (Fig. 6A). In the 12Z cell model, a significant increase was observed following \ntreatment with cisPt (P < 0.0001), while treatment with [Zn(neo)(nif) 2] resulted in a non-significant decrease \n(P = 0.1781). In A2780 cells, a significant increase was observed with cisPt (P = 0.0487), whereas a significant \ndecrease was noted with [Zn(neo)(nif)2] treatment (P = 0.0451).\nScientific Reports |        (2025) 15:10126 7| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\nThe Nrf2 active/COX2 ratio showed a significant increase only in the A2780 model treated with cisPt \n(P = 0.0045) (Fig. 6B), while no considerable changes were observed in the other tested models (HME1 and 12Z).\nThe regulatory action of the Nrf2 protein is exerted only in its phosphorylated form35. Therefore, we analyzed \nthe ratio of phosphorylated (active) Nrf2 to its total level in the tested groups (Fig.  6C). The results revealed a \nsignificant decrease in the Nrf2 active/Nrf2 ratio in A2780 cells treated with cisPt (P = 0.0366). No considerable \nchange was observed under any tested conditions, including treatment with cisPt or [Zn(neo)(nif)2].\nThe key regulatory factors COX2 and TGF-β1 cooperate in the development of inflammation. The COX2/\nTGF-β1 ratio showed a significant increase in 12Z cells treated with cisPt (P < 0.0001) (Fig. 6D) and in A2780 \ncells treated with [Zn(neo)(nif)2] (P = 0.0432).\nVEGF-A and Nrf2 are pivotal in regulating angiogenesis and cellular response to oxidative stress. Their \nreciprocal relationship is illustrated in Fig.  6E. In the HME1 model, the VEGF-A/Nrf2 ratio significantly \ndecreased under treatment with [Zn(neo)(nif)2] (P = 0.0254). In the 12Z model, this ratio significantly decreased \nunder treatment with both cisPt (P = 0.0017) and [Zn(neo)(nif)2] (P = 0.0123). In the A2780 model, a significant \ndecrease was observed under treatment with [Zn(neo)(nif)2] (P = 0.0225).\nThe final protein ratio analyzed was the TGF-β1/Nrf2 ratio (Fig.  6F), which reflects the regulation of \noxidative stress and inflammation at the cellular level. In the 12Z model, a significant decrease was observed \nunder treatment with [Zn(neo)(nif) 2] (P < 0.0001). Similarly, in the A2780 model, a significant decrease was \nobserved under treatment with cisPt (P = 0.0097).\nMitochondrial Ca2+, H2O2 levels, and cytosolic levels of Ca2+\nMitochondrial calcium overload, caused by Ca 2+ influx released from the endoplasmic reticulum under stress \nconditions, stimulates immune responses and ultimately leads to apoptosis. For live-imaging measurements, \nwe selected the epithelial cell lines HME1 and 12Z based on the gene expression results of angiogenic and \napoptotic factors. Excessive mitochondrial calcium accumulation can trigger the opening of the mitochondrial \n \nScientific Reports |        (2025) 15:10126 8| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\npermeability transition pore, resulting in the release of calcium from the mitochondria into the cytosol, serving \nas an indicator of apoptotic processes in the cell.\nWe quantified basal mitochondrial Ca2+ levels using the genetically encoded mitochondrial Ca 2+ biosensor \n4mtD3cpv. A significant increase in mitochondrial Ca2+ levels was observed in control HME1 cells in response \nto treatment with cisPt (P = 0.0069) and [Zn(neo)(nif) 2] (P = 0.0001) (Fig.  7A). Additionally, a significant \nincrease in the mitochondrial Ca 2+ level was detected in endometriotic 12Z cells treated with [Zn(neo)(nif) 2] \n(P < 0.0001), whereas no effect was observed with cisPt treatment (Fig. 7D).\nTo further investigate, cytosolic free Ca 2+ levels were measured using the Ca 2+ dye Fura-2. Significant \nchanges were observed in both tested epithelial cell lines (HME1, 12Z). In HME1 cells, basal cytosolic Ca2+ levels \nsignificantly changed in response to [Zn(neo)(nif)2] (P < 0.0001) (Fig. 7B). Similarly, in 12Z cells, cytosolic Ca2+ \nlevels significantly increased in response to cisPt (P < 0.0001) as well as [Zn(neo)(nif)2] (P < 0.0001) (Fig. 7E).\nMitochondrial metabolism is closely associated with Ca 2+ and ROS levels. To assess mitochondrial ROS \nlevels, we utilized the genetically encoded mitoHyPer7 biosensor. A significant increase in ROS was observed in \n12Z endometriotic cells following treatment with [Zn(neo)(nif) 2] (P = 0.0126), whereas the increase in HME1 \ncells was not statistically significant (Fig. 7C, F).\nDiscussion\nAngiogenesis is a physiological process that facilitates the formation of the primary vascular network necessary \nfor tissue growth and repair36. It regulates oocyte maturation, the development of functional corpora lutea, and \nuterine endometrial growth and decidualization 37. Disruption of this process due to the constant activation \nof angiogenic factors can lead to excessive vessel growth, contributing to the development and progression of \nendometriosis and its potential malignant transition38.\nThe complex interplay between the immune system, hormones, microelements, and genetic factors \nsignificantly influences the development and progression of endometriosis 39. Transcription factors such as \nZNFs and miRs play a dual role; they can reduce inflammation via immunosuppression, thereby promoting the \nspread and invasiveness of the condition40. Additionally, they can inhibit apoptotic cell death41 in endometriotic \ncells and wild-type tumors, such as endometrioid adenocarcinoma, ovarian cancer, or cervical squamous cell \ncarcinoma42.\nThe regulatory gene sequences of angiogenic factors can vary depending on the specificity of the target ZNFs. \nFor example, ZNF471 has been shown to regulate the expression of EMT-related markers and transcription \nfactors involved in angiogenesis, cellular migration, and vasculogenic mimicry 43. Conversely, ZNF24 has been \nreported to repress VEGF transcription44. ZNF3 is known to be highly expressed in colorectal carcinoma cells45, \nFig. 4. ( A): The gene expression miR-206/VEGF-A ratio (n = 4) was analyzed under three tested conditions: \ncontrol (untreated spheroid cells), cisPt (spheroid cells treated with 10 μM cis-platin), and [Zn(neo)(nif)2] \n(spheroid cells treated with 10 μM [Zn(neo)(nif)2]). In HME1 cells, significant changes were found under \ntreatment with cisPt (P < 0.0001, ***), while changes under [Zn(neo)(nif)2] treatment were insignificant \n(P = 0.3425). A significant difference was observed between cisPt and [Zn(neo)(nif)2] (P < 0.0001, +  + +). In \n12Z cells, significant changes were found for control vs. cisPt (P < 0.0001, ***) and control vs. [Zn(neo)(nif)2] \n(P < 0.0001, ***), while the difference between cisPt. Vs. [Zn(neo)(nif)2] was insignificant (P = 0.0644). In \nA2780 cells, the changes were insignificant under cisPt treatment (P = 0.1882|, significant under [Zn(neo)\n(nif)2] treatment (P = 0.0021, **), and insignificant change cisPt vs. [Zn(neo)(nif)2] (P = 0.1341) (B): The \ngene expression miR-23a/VEGF-A ratio showed significant changes in HME1 under treatment with \ncisPt (P < 0.0001, ***), insignificant changes under [Zn(neo)(nif)2] treatment (P = 0.4761), and significant \ndifferences between cisPt and [Zn(neo)(nif)2] treatments (P < 0.0001, +  + +). In 12Z cells, significant changes \nwere observed under both cisPt and [Zn(neo)(nif)2] treatments (P < 0.0001, ***), as well as between cisPt \nand [Zn(neo)(nif)2] (P = 0.0152, +). In A24780 cells, the ratio showed insignificant changes across all tested \nconditions: cisPt (P = 0.2920), [Zn(neo)(nif)2] (P = 0.0524), and between cisPt and [Zn(neo)(nif)2] (P = 0.6294). \n(C): The gene expression miR-376a/VEGF-A ratio showed significant changes in HME1 under treatment \nwith cisPt (P < 0.0001, ***), insignificant changes under treatment with [Zn(neo)(nif)2] (P = 0.4752), and \nsignificantdifferences between cisPt and [Zn(neo)(nif)2] treatments (P < 0.0001, +  + +). In 12Z cells, significant \nchanges were observed under both cisPt and [Zn(neo)(nif)2] treatments (P < 0.0001, ***), as well as between \nand [Zn(neo)(nif)2] (P = 0.0066, + +). In the A2780 cells, no significant changes were observed across all tested \nconditions: cisPt (P = 0.9996), [Zn(neo)(nif)2] (P = 0.7412), and cisPt vs. [Zn(neo)(nif)2] (P = 0.7567). (D): \nThe gene expression miR-133b/VEGF-A ratio showed significant changes in HME1 cells under treatment \nwith cisPt (P < 0.0001, ***), insignificant changes under [Zn(neo)(nif)2] treatment (P = 0.3538), and significant \ndifference for cisPt vs. [Zn(neo)(nif)2] (P < 0.0001, +  + +). In 12Z cells, the ratio significantly changed under \nboth cisPt and [Zn(neo)(nif)2] treatment (P < 0.0001, ***), as well as between cisPt and [Zn(neo)(nif)2] \n(P = 0.0088, + +). In A2780 cells, significant changes were observed under cisPt treatment (P = 0.0358, *), \n[Zn(neo)(nif)2] (P = 0.0015, **), and insignificant changes between cisPt and [Zn(neo)(nif)2] (P = 0.4129). E: \nThe gene expression let-7c/VEGF-A ratio showed significant changes in HME1 for control vs. cisPt (P < 0.0001, \n***), insignificant changes for control vs. [Zn(neo)(nif)2] (P = 0.3580), and significant differences between \ncisPt and [Zn(neo)(nif)2] treatments (P < 0.0001, +  + +). In 12Z cells, significant changes were observed \nunder both cisPt and [Zn(neo)(nif)2] treatments (P < 0.0001, ***), as well as between cisPt and [Zn(neo)\n(nif)2] (P = 0.0147, +). In A2780 cells, significant changes were found for control vs. cis-Pt (P < 0.0001, ***) and \ncontrol vs. [Zn(neo)(nif)2] (P < 0.0001, ***), while changes between cisPt vs. [Zn(neo)(nif)2] were insignificant \n(P = 0.0913).\n◂\nScientific Reports |        (2025) 15:10126 9| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\nwhere it plays a role in cellular proliferation, migration, and invasion. If the [Zn(neo)(nif)2] intercalates into the \nZNF3 sequence, it could exert a suppressive effect on target genes, which aligns with the gene expression changes \nobserved in 12Z and A2780 cell lines.\nThe obtained data indicate that the [Zn(neo)(nif) 2] complex may influence gene regulation, as evidenced \nby its impact on expression of target gene associated with the promotion or suppression of angiogenesis (e.g., \nmRNA of ANG1, ANG2, TGF-β1, HIF-1α, COX2, Nrf2, BAX, and CAS3), on the expression of micro-RNA \n(e.g., miR-133b, miR-206, miR-376, miR-376, or let-7c), as well as on protein expression related to angiogenesis \n(e.g., COX2, VEGF-A, TGF-β1, and Nrf2). The molecular conformation of the complex suggests the possibility \nof intercalation, where the aromatic neocuproine ligand may intercalate between DNA base pairs, potentially \nstabilized by π-π stacking interactions, hydrogen bonding, van der Waals forces, and hydrophobic interactions46.\nFig. 5. ( A): The gene expression miR-133b/TGF-β1 ratio (n = 4) was analyzed under three tested conditions: \ncontrol (untreated spheroid cells), cisPt (spheroid cells treated with 10 μM cis-platin), and [Zn(neo)(nif)2] \n(spheroid cells treated with 10 μM [Zn(neo)(nif)2]). Significant changes in the miR-133b/TGF-β1 ratio \nwere observed in HME1 cells under treatment with cisPt (P = 0.0009, ***), with [Zn(neo)(nif)2] (P < 0.0001, \n***), and between cisPt and [Zn(neo)(nif)2] (P = 0.0008, ***). In 12Z cells, the ratio showed an insignificant \nchange under treatment with cisPt (P = 0.1162), a significant change under treatment with [Zn(neo)(nif)2] \n(P = 0.0045), a significant change between cisPt and [Zn(neo)(nif)2] (P = 0.0005, +  + +). In A24780 cells, a \nsignificant change was observed under treatment with cisPt (P = 0.0301, *), with [Zn(neo)(nif)2] (P = 0.0100, \n**), and between cisPt and [Zn(neo)(nif)2] (P = 0.0040, + +). (B): The gene expression let-7c/TGF-β1 ratio \n(n = 4) in HME1 cells showed significant changes for control vs. cisPt (P = 0.0004, ***), control vs. [Zn(neo)\n(nif)2] (P = 0.0002, ***), and cisPt vs. [Zn(neo)(nif)2] (P = 0.0008, +  + +). In 12Z cells, significant changes were \nobserved under treatment with cisPt (P = 0.0201), with [Zn(neo)(nif)2] (P = 0.0043, **), and between cisPt and \n[Zn(neo)(nif)2] (P = 0.0001, +  + +). In A2780 cells, significant changes were found under treatment with cisPt \n(P = 0.0066, **), with [Zn(neo)(nif)2] (P = 0.0041), and between the two treatments (P = 0.0044, + +). (C): The \ngene expression miR-206/HIF-1α ratio (n = 4) showed a significant elevation in HME1 cells under treatment \nwith cisPt (P < 0.0001, ***), with [Zn(neo)(nif)2] (P = 0.0039, **), and insignificant change between cisPt and \n[Zn(neo)(nif)2] (P = 0.1324). In 12Z cells, the ratio was insignificantly increased under treatment with cisPt \n(P = 0.5895) and significantly decreased under treatment with [Zn(neo)(nif)2] (P = 0.0002, ***). The difference \nbetween cisPt and [Zn(neo)(nif)2] treatment was insignificant (P = 0.0665). In A2780 cells, a significant \nincrease was observed for control vs. cisPt (P = 0.0023, **), an insignificant change for control and [Zn(neo)\n(nif)2] (P = 0.4254), and a significant difference between cisPt and [Zn(neo)(nif)2] treatments (P = 0.0012, + +). \n(D): The gene expression miR-206/Nrf2 ratio (n = 4) in HME1 model showed an insignificant decrease under \ntreatment with cisPt (P = 0.0692), an insignificant change under treatment with [Zn(neo)(nif)2] (P = 0.0659), \nand a significant decrease between cisPt vs. [Zn(neo)(nif)2] (P = 0.0233, +). In 12Z cells, the ratio significantly \ndecreased under treatment with cisPt (P = 0.0013, **), with [Zn(neo)(nif)2] (P = 0.0003, ***), and between cisPt \nand [Zn(neo)(nif)2] (P = 0.0123, +). In A2780 cells, a significant decrease was observed under treatment with \ncisPt (P = 0.0047, **), with [Zn(neo)(nif)2] (P = 0.0021, **), and a highly significant change between cisPt and \n[Zn(neo)(nif)2] (P < 0.0001, +  + +).\n \nScientific Reports |        (2025) 15:10126 10| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\nAlthough providing a definite explanation is challenging, the observed binding specificity towards the \nZnF3-7 sequence might involve the preference of specific base pair sequences (e.g., C-G), as suggested by recent \ncomputational studies on aromatic organic molecules 47. Since intercalation of the [Zn(neo)(nif) 2] complex \ninduces structural changes48 in the ZnF3-7 sequence, it may prevent ZnF3 from binding to the major groove49, \nthereby altering gene expression. Recognizing the ZnF3-7 sequence, where zinc-finger proteins bind, plays a \ncrucial role in regulating gene expression50, which might be a key aspect of the studied complex’s mechanism of \naction at the cellular level.\nSignificant changes in TGF-β1 expression were observed in the monolayer model of cell lines used in the \nexperiment (Table S3), with increased levels in endometriotic 12Z cells treated with cisPt and [Zn(neo)(nif) 2], \nand decreased levels in endometriotic adenoma A2748 cells treated with same compounds. It is well known that \nTGF-β1 acts as a potent immunosuppressor by regulating the proliferation and survival of immune system cells \nand inducing cell type-specific apoptosis 51. Additionally, TGF-β1 is a target of microRNA let-7c, which also \nregulates HIF-1α, estrogen receptor α, and several other genes involved in angiogenesis, cell cycle regulation, \nand signaling pathways34.\nLet-7c can also exhibit oncogenic effects, as it is highly expressed in ovarian cancers with poor prognosis and \ndecreased overall survival 52. Both VEGF-A and TGF-β1 play crucial roles in angiogenesis but have opposing \neffects on endothelial cells. We observed a decreasing trend in the let-7c/TGF-β1 ratio in the 12Z and A2780 \ncell models (Fig. 4B), which could be attributed to the apoptosis-inducing properties of TGF-β1 53. Conversely, \nan increased let-7c/TGF-β1 ratio was observed in HME1 cells. It is well known that let-7c has the ability to \ninhibit the TGF-β1 expression. A decreased level of let-7c may lead to TGF-β1-mediated induction of fibrosis \neffectors (e.g., collagen I), potentially predicting disease progression. On the other hand, studies demonstrated \nthat microRNAs of the let7-family can affect angiogenesis by modulating TGF-β1 signaling. This could reflect \na similar effect to that of let-7f., which has been linked to the activation of the anti-angiogenic TGF-β1/\nALK5 pathway54. Additionally, the observed elevation in the miR-133b/TGF-β1 ratio (Fig.  4A) in HME1 cells \nis significant, as miR-133b functions as an oncogene suppressor by regulating TGF-β1 receptor I and II 55. \nInterestingly, the miR-133b/TGF-β1 ratio decreased, which could be explained by the fact that TGF-β1 can act \nas both an oncogenic and a tumor-suppressive agent, depending on the tumor stage and type56.\nTGF-β1 can upregulate COX2 expression, leading to increased production of prostaglandin E2. This, in \nturn, influences the COX2 pathway and may induce invasiveness in cooperation with oncogenic signals 57. \nThis phenomenon could explain the increased COX2/TGF-β11 protein ratio (Fig.  6D). Further research \nhas demonstrated that TGF-β1 can elicit an Nrf2-mediated antioxidant response, contributing to its anti-\ninflammatory properties. For instance, the TGF-β1’s ability to induce Nrf2 activity has been associated with \nprotection against vascular wall rupture 58. On the other hand, Nrf2 has been shown to counteract TGF-β1-\nmediated growth inhibition, suggesting that Nrf2 may influence the pro-tumorigenic functions of TGF-β159. We \nanalyzed the decreased TGF-β1/Nrf2 ratio in 12Z and A2780 cells under treatment with cisPt (Fig.  6F), which \ncould represent its tumorigenic action in cooperation with COX2 and VEG-A levels.\nAnother significant target, VEGF-A, which protects endothelial cells from apoptosis 53, was unexpectedly \nelevated in the control HME1 cell line and showed an insignificant downregulation in the 12Z and A2780 cell \nlines (Table S3). Given that the simultaneous overexpression of VEGF-A and TGF-β1 are associated with poorer \ncancer prognoses43, we analyzed the gene expression ratio of those two markers. The VEGF-A/TGF-β1 ratio \n(Figs. 2B, 6A) decreased only in the 12Z cell line after treatment with [Zn(neo)(nif) 2], suggesting a potentially \nbetter prognosis. However, it has been reported that TGF-β1 suppresses VEGF-A-mediated angiogenesis in \ncolon cancer metastasis 61, despite the fact that aberrant TGF-β1 expression is critical in the development of \nendometriosis, which shares several parallels with tumorigenesis 62. We observed an increase in the VEGF-A/\nTGF-β1 ratio in both the A2780 and HME1 models, which may indicate the suppression of VEGF-A-mediated \nangiogenesis. The reciprocal interaction between VEGF-A and Nrf2 can drive a positive feedback loop that \nHME1 VEGF-A (P value, signif.) TGF-β1 (P value, signif.) COX2 (P value, signif.) Nrf2 (P value, signif.) Nrf2 active (P value, signif.)\ncontrol vs. cisPt 0.0528 (ns) ↓ 0.0159 (*) ↓ 0.3151 (ns) ↑ 0.1338 (ns) ↑ 0.7100 (ns) ↓\ncontrol vs. [Zn(neo)(nif)2] 0.0462 (*) ↓ 0.6167 (ns) ↑ 0.3151 (ns) ↑ 0.1131 (ns) ↑ 0.9862 (ns) ↑\ncisPt vs. [Zn(neo)(nif)2] 0.0445 (*) ↓ 0.0246 (*) ↑ 0.3151 (ns) ↓ 0.3792 (ns) ↓ 0.2154 (ns) ↑\n12Z\ncontrol vs. cisPt 0.0496 (*) ↓  < 0.0001 (***) ↓ 0.0524 (ns) ↑ 0.1121 (ns) ↓ 0.8652 (ns) ↓\ncontrol vs. [Zn(neo)(nif)2] 0.2219 (ns) ↓ 0.3118 (ns) ↑ 0.0049(**) ↑ 0.7900 (ns) ↑ 0.0547 (ns) ↑\ncisPt vs. [Zn(neo)(nif)2] 0.0228 (*) ↑ 0.0033 (**) ↑ 0.3151 (ns) ↑ 0.3380 (ns) ↑ 0.0789 (ns) ↑\nA2780\ncontrol vs. cisPt 0.0573 (ns) ↑ (ns) 0.3859 (ns) ↓ 0.8915 (ns) ↑ 0.0299 (*) ↑\ncontrol vs. [Zn(neo)(nif)2] 0.0342 (*) ↓ (ns) 0.1060 (ns) ↑ 0.1880 (ns) ↑ 0.8807 (ns) ↑\ncisPt vs. [Zn(neo)(nif)2] 0.0158 (*) ↓ (ns) 0.0203 (*) ↑ 0.2466 (ns) ↑ 0.0008 (***) ↑\nTable 4. Significance values of protein levels (VEGF-A, TGF-β1, Nrf2, phosphorylated Nrf2 = Nrf2 active) \n(n = 3) under three tested conditions: control (untreated spheroid cells), cisPt (spheroid cells treated with 10 \nμM cis-platin), [Zn(neo)(nif) 2] (spheroid cells treated with 10 μM [Zn(neo)(nif)2]) across three experimental \n3D model cells.\n \nScientific Reports |        (2025) 15:10126 11| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\nFig. 6. The protein levels in the cultivation media were analyzed in triplicates (n = 3) across three tested \nconditions: control (untreated spheroid cells), cisPt (spheroids treated with 10 μM cis-platin), [Zn(neo)(nif)2] \n(spheroids treated with 10 μM [Zn(neo)(nif)2]). (A): The VEGF-A/TGF-β1 protein expression ratio in HME1 \nshowed an insignificant decrease under treatment with cisPt (P = 0.6274) and [Zn(neo)(nif)2] (P = 0.0617), but \na significant change was observed between cisPt and [Zn(neo)(nif)2] treatments (P = 0.0391, +). In 12Z cells, a \nsignificant increase was detected for control vs. cisPt (P < 0.0001, ***), an insignificant decrease for control vs. \n[Zn(neo)(nif)2] (P = 0.1781), and a significant difference between cisPt and [Zn(neo)(nif)2] (P < 0.0001, +  + +). \nIn the A2780 model, a significant increase was observed under treatment with cisPt (P = 0.0487, *), [Zn(neo)\n(nif)2] (P = 0.0451, *), and between cisPt and [Zn(neo)(nif)2] treatments (P = 0.0097, + +). (B): The Nrf2 \nactive/COX2 protein expression ratio in HME1 showed an insignificant decrease under treatment with \ncisPt (P = 0.4140) and [Zn(neo)(nif)2] (P = 0.9889), with no significant change between cisPt and [Zn(neo)\n(nif)2] (P = 0.1881). In 12Z cells, an insignificant decrease was observed for control vs. cisPt (P = 0.5736), an \ninsignificant increase for control vs. [Zn(neo)(nif)2] (P = 0.1005), and an insignificant difference between cisPt \nand [Zn(neo)(nif)2] (P = 0.0664). In the A2780 model, a significant increase was observed under treatment \nwith cisPt (P = 0.0045, **), an insignificant decrease with [Zn(neo)(nif)2] (P = 0.2413), and a significant \ndifference between cisPt and [Zn(neo)(nif)2] (P = 0.0127, +). (C): The Nrf2 active/Nrf2 protein expression \nratio in HME1 showed an insignificant increase under treatment with cisPt (P = 0.5666) and [Zn(neo)(nif)2] \n(P = 0.9994), as well as between cisPt and [Zn(neo)(nif)2] treatments (P = 0.1857). In 12Z cells, an insignificant \ndecrease was observed for control vs. cisPt (P = 0.4890) and for control vs. [Zn(neo)(nif)2] (P = 0.9323), as \nwell as between both tested conditions cisPt and [Zn(neo)(nif)2] (P = 0.9543). The A2780 model showed \na significant decrease under treatment with cisPt (P = 0.0366, *), an insignificant increase with [Zn(neo)\n(nif)2] (P = 0.2785), and a significant difference between cisPt and [Zn(neo)(nif)2] (P = 0.0159, +). (D): The \nCOX2/TGF-β1 protein expression ratio in HME1 showed an insignificant increase under treatment with \ncisPt (P = 0.4886) and an insignificant decrease with [Zn(neo)(nif)2] (P = 0.9328), as well as between cisPt \nand [Zn(neo)(nif)2] treatments (P = 0.5281). In 12Z cells, a significant increase was observed for control vs. \ncisPt (P < 0.0001, ***), an insignificant decrease for control vs. [Zn(neo)(nif)2] (P = 0.5485), and a significant \ndifference between cisPt and [Zn(neo)(nif)2] (P < 0.0001, +  + +). The A2780 model showed an insignificant \nincrease under treatment with cisPt (P = 0.6724), a significant increase with [Zn(neo)(nif)2] (P = 0.0432, *), \nand significant difference between cisPt and [Zn(neo)(nif)2] (P = 0.0133, +). E: The VEGF-A/Nrf2 active \nprotein expression ratio in HME1 showed an insignificant decrease under treatment with cisPt (P = 0.8258), \na significant decrease with [Zn(neo)(nif)2] (P = 0.0254, *), and between cisPt and [Zn(neo)(nif)2] treatments \n(P = 0.0047, + +). In 12Z cells, a significant decrease was observed for control vs. cisPt (P = 0.0017, **), for \ncontrol vs. [Zn(neo)(nif)2] (P = 0.0123, *), and between cisPt and [Zn(neo)(nif)2] (P = 0.0022, + +). The A2780 \nmodel showed an insignificant decrease under treatment with cisPt (P = 0.3470), a significant decrease wit \n[Zn(neo)(nif)2] (P = 0.0225, *), and a significant difference between cisPt and [Zn(neo)(nif)2] (P = 0.0240, +). \nF: The TGF-β1/Nrf2 active protein expression ratio in HME1 showed an insignificant decrease under \ntreatment with cisPt (P = 0.9339) and [Zn(neo)(nif)2] (P = 0.8994), with no significant difference between \ncisPt and [Zn(neo)(nif)2] treatments (P = 0.9382). In 12Z cells, a significant decrease was observed for \ncontrol vs. cisPt (P < 0.0001, ***), an insignificant decrease for control vs. [Zn(neo)(nif)2] (P = 0.2798), and \na significant difference between cisPt and [Zn(neo)(nif)2] (P < 0.0001, +  + +). The A2780 model showed a \nsignificant decrease under treatment with cisPt (P = 0.0097, **), an insignificant decrease with [Zn(neo)(nif)2] \n(P = 0.3477), and significant difference between cisPt and [Zn(neo)(nif)2] (P = 0.0016, + +).\n \nScientific Reports |        (2025) 15:10126 12| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\npromotes angiogenesis63. The decreased VEGF-A/Nrf2 ratio (Fig.  6E) may indicate a potential reduction in \nangiogenic signals.\nThe role of ncRNA in cellular, tissue, and systemic metabolic processes is indisputable. MicroRNAs can \nexhibit both pro-angiogenic (miR-23a, -133b, let-7c) and anti-angiogenic (miR-206, -376a) effects. MicroRNAs \nknown to influence VEGF-A expression, such as miR-206, negatively regulate angiogenesis by directly targeting \nVEGF-A64. Similarly, miR-23a reduces VEGF-A levels 65 but also downregulates Nrf2 and CAT, potentially \naltering ROS levels66. MiR-133b, which plays an oncogenic role in the progression of cervical carcinoma 67 and \nbreast cancer68, did not show significant changes in its ratio to VEGF-A expression (Fig.  4). The HME1 cell \nmodel showed a significant increase under treatment with cisPt, while no significant change was observed with \n[Zn(neo)(nif)2] treatment. In contrast, the 12Z and A2780 models exhibited a decrease in ratios, which was \nmore pronounced and statistically significant in 12Z cells. Significant expression changes were observed in miR-\n206 in HME1 cells, miR-23a in 12Z cells, and miR-133b in HME1, 12Z, and A2780 cells (Figure S2). The anti-\nangiogenic miR-376a inhibits VEGF-A signaling by targeting SIRT1 or neuropilin 1 in various cancer cells69.\nIn conclusion, we analyzed the let-7c/VEGF-A ratio (Fig. 4E) to further investigate the microRNA effect on \nVEGF-A expression. This pro-angiogenic microRNA showed a decreased ratio in all tested conditions in 12Z \nand A2780 cells. Chrishev et al. reported elevated expression of let-7c in ovarian tissue compared to endometrial \ntissue, suggesting that let-7c may have oncogenic effects with poor prognosis and lower overall survival34, which \naligns with our observations. The decreased let-7c expression observed in our tested conditions may indicate a \nbetter prognosis.\nSince Ang1 stabilizes blood vessels while Ang2 induces angiogenesis, the elevated Ang2/Ang1 ratio (favoring \nAng2) (Fig. 2A) likely reflects an active angiogenesis phase 70. We hypothesize the observed increase in ANG2 \nexpression alongside a simultaneous decrease in ANG1 under tested conditions may serve as an independent \npredictor of cell death, similar to findings reported by Ong et al. 71. The ANG2/ANG1 ratio may be a valuable \nprognostic biomarker of endothelial activation in endometriosis or endometrioid adenocarcinoma, particularly \nin combination with altered expression of VEGF-A and TGF-β172.\nBased on gene expression changes, we hypothesize that alterations in the Nrf2/COX2 ratio may reflect shifts \nin the regulatory roles of HIF-1α and COX2 in the Nrf2-mediated inflammatory response. The decreased CAS3/\nBAX ratio (Fig.  2E, 6B) suggests enhanced pro-apoptotic stimuli resulting from mitochondrial dysfunction \nclosely linked to endoplasmic reticulum stress73, potentially influenced by studied compounds in HME1 cells.\nFig. 7. ( A, D): Mitochondrial Ca2+ levels under three tested conditions: control (untreated cells: HME1 \nn = 4; 12Z n = 14), cisPt (cells treated with 10 μM cis-platin: HME1 n = 6; 12Z n = 14), [Zn(neo)(nif)2] (cells \ntreated with 10 μM [Zn(neo)(nif) 2]: HME1 n = 5; 12Z n = 14). (B, E): Cytosolic Ca2+ levels under three tested \nconditions: control (untreated cells: HME1 n = 46; 12Z n = 102), cisPt (cells treated with 10 μM cis-platin: \nHME1 n = 43; 12Z n = 88), [Zn(neo)(nif)2] (cells treated with 10 μM [Zn(neo)(nif) 2]: HME1 n = 50; 12Z \nn = 99). C, F: Mitochondrial H2O2 levels under two tested conditions: control (untreated cells: HME1 n = 3; 12Z \nn = 3), [Zn(neo)(nif)2] (cells treated with 10 μM [Zn(neo)(nif) 2]: HME1 n = 3; 12Z n = 4).\n \nScientific Reports |        (2025) 15:10126 13| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\nThe observed elevation in the CAS3/BAX ratio in 12Z and A2780 under treatment with [Zn(neo)(nif)2] may \nindicate the activation of CAS3 in programmed cell death processes74.\nMiR-206 has been reported to influence HIF-1α and Nrf2 expression in relation to ROS production and \naccumulation75, as it inhibits cell growth even under high glucose metabolism conditions typical for cancer \ncells. We observed a decrease in the miR-206/HIF-1α ratio across all tested cell lines following cisPt treatment. \nIn HME1 cells, treatment with [Zn(neo)(nif) 2] resulted in a significant increase in the miR-206/HIF-1α ratio, \nwhereas a decrease was observed in 12Z and A2780 cells. This decrease may indicate increased resistance to \napoptosis and could be indicative of disease progression76.\nThe miR-206/Nrf2 ratio suggests upregulation of Nrf2 across all tested conditions, which may enhance \nantioxidant defense, cytoprotection, and resistance to oxidative stress-induced apoptosis. Conversely, a decrease \nin the miR-206/Nrf2 ratio may indicate the promotion of tumor progression, as Nrf2 can support cancer cell \nsurvival under stressful conditions. On the other hand, in oxidative disorders, a lower miR-206/Nrf2 ratio may \nprotect by reducing oxidative damage77 (Fig. 5C).\nNrf2 is a crucial regulator of endothelial miR-206-attenuated expression and can drive tumorigenesis through \ndysregulation of the Krebs cycle or pentose phosphate pathway78. The NRF2 pathway exhibits dual roles; it can \nact as a tumor suppressor by reducing ROS levels through its antioxidant function 79, yet it can also promote \ntumorigenesis by inducing ROS production and enhancing tumor growth 80. The precise role of Nrf2 in the \nstudied epithelial cell lines treated with tested compounds requires further investigation.\nThe findings suggest that the studied [Zn(neo)(if) 2] complex may contribute to mitochondrial calcium \noverload, resulting in increased ROS production. This mitochondrial Ca 2+ accumulation could be associated \nwith the activation of apoptotic genes (BAX, CAS3) and potential involvement of the mitochondrial permeability \ntransition pore (mPTP). Furthermore, Ca2+ transfer through the mPTP may lead to elevated Ca2+ levels, which, \ntogether with increased ROS levels, could play a role in the induction of apoptosis or apoptosis-like cell death81,82.\nMaterial and methods\nDNA binding studies\nThe double-stranded oligonucleotides ZnF3-7 (5'- T A G C G C C C C C T G C T G G C-3'/3'- A T C G C G G G G G A C G A C \nC G-5’) and EBP (5'- A T T G C G C A A T-3'/3'- T A A C G C G T T A-5’) were prepared by annealing forward and reverse \nsingle-stranded oligonucleotide sequences, which were obtained from commercial suppliers (Sigma Aldrich).\nCompetitive fluorescence binding studies were conducted following a conventional procedure. Ethidium \nbromide (2,5 μM) was added to the respective oligonucleotides to form the DNA-EB complex. The studied \ncompound was gradually added to this mixture at 0 to 5 μM concentrations.\nThe fluorescence emission spectra (λ EX = 520 nm) were recorded after each addition of the complex, and \nthe maximum emission intensity values were used to calculate the binding constants (K SV) using the standard \nStern–Volmer equation: F0\nF =1+ KSV[Q].\nCell lines and cultivation protocol\nWe conducted experiments on three epithelial cell lines: HME1, 12Z, and A2780. The HME1 cell line (ExPASy \nhtTERT-HME1) is an hTERT-immortalized cell line with epithelial morphology, derived from the breasts of \na 53-year-old female patient undergoing reduction mammoplasty with no history of breast cancer. HME1 \ncells were used as a model of physiological angiogenesis. The 12Z cell line (a kind donation from Prof. Anna \nStarzinski-Powitz, Goethe-Universität Frankfurt) is an SV40 virus-immortalized cell line obtained from a \n37-year-old female patient undergoing laparoscopy. This cell line exhibits expression of markers characteristic \nof endometriotic lesions observed in vivo. The A2780 cell line (a kind donation from Dr. Martina Šemeláková \nPhD., Pavol Jozef Šafárik University in Košice) is a human ovarian cancer cell line, originally established from \nan endometrioid adenocarcinoma of an untreated patient. The cell cultures were maintained to cell-specific \nprotocols using appropriate culture media: Roswell Park Memorial Institute (RPMI) 1640 Medium for A2780 \ncells, Dulbecco’s Modified Eagle’s Medium (DMEM) for 12Z cells, Human Mammary Epithelial Cell Growth \nMedium (MEBM) mixed with Nutrient mixture medium F-12 Ham (1:1) for HME1 cells. All culture media were \nsupplemented with 10% Fetal Bovine Serum (FBS) and 1% Penicillin/Streptomycin. The cells were incubated at \n37°C in a humidified atmosphere containing 5% CO2.\nCell transfection and treatment with tested compounds\nAll microscopic experiments were performed on 30 mm glass coverslips plated with cells in 6-well plates. \nCells were transfected at 50–60% confluency with organelle-targeted biosensors: mitoHyPer7 (1.5 μg/well), \nmtD1GoCam (1.5 μg/well), the FRET-based Ca 2+ biosensor 4mtD3cpv (1.5 μg/well), and cytosolic Ca 2+ \nindicator Fura-2 acetoxy-methyl-ester (Fura-2AM) (1.5 μg/well), using 3 μL of TransFast transfection reagent \n(Promega, Madison, WI, USA) in 1 mL of serum and antibiotic-free medium for 8–12 h. Following transfection, \nthe medium was replaced with 2 mL of experimental EH-loading buffer (Table S4), and measurements were \nconducted for 2–3 h at room temperature.\nThe tested compounds, cis-platin (cisPt) and [Zn(neo)(nif) 2], were used at a final concentration of 10 μM \n(based on IC 50 – Table S5) in the appropriate complete cultivation medium. The compounds were applied to \nadherent cells (at the confluence 50–60%) or spheroid cells for 8 h, based on the results of the Cell Viability \nAssay (Figure S3).\n3D tissue models\nOur experiments utilized 3D tissue spheroids to study mRNA/miRNA expression, providing a more reliable \ntissue model for angiogenesis compared to the conventional 2D monolayer in vitro experiments. To form the \nspheroids, we used U-bottom 96 well-plates, with their surface coated with 0.8% LE agarose to create a thin \nScientific Reports |        (2025) 15:10126 14| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\nfilm non-adhesive film. Cells were seeded as a single-cell suspension (5—120 × 104 cells/mL, depending on the \ndoubling time of each experimental tissue culture) in 200 μL of complete medium per well in the microtitration \nplates. The morphology of the spheroids for all experimental cell lines and in the tested conditions is shown in \nFigure S4.\nTotal RNA was extracted from the cell suspension using the RNeasy Mini Kit (Qiagen; Hilden, Germany) with \na modified manufacturer’s protocol. The isolated nucleic acid was transcribed into cDNA using the ProtoScript \nFirst Strand cDNA Synthesis Kit (New England Biolabs; Ipswich, MA, United States) and a thermocycler (Techne \nTC-3000X). qRT-PCR amplification was performed using SensiMIX II (Bioline Meridian Bioscience; London, \nEngland) on the Rotor-Gene Q system (Qiagen; Hilden, Germany) to detect the target mRNA expression.\nFor micro-RNA analysis, the isolated miRNA was processed using the TaqMan™ MicroRNA Reverse \nTranscription Kit (Applied Biosystems™) with the Techne TC-3000X thermocycler, followed by qRT-PCR \namplification using the TaqMan™ Universal Master Mix II no UNG (Applied Biosystems™) on the thermocycler \nRotor-Gene Q System (Qiagen; Hilden, Germany) to detect the target miRNA expression. The primer sequences \nand TaqMan probes used are listed in supplementary data (Table S6).\nThe obtained data were analyzed using Rotor-Gene Q 2.5.3 Software (Qiagen; Hilden, Germany), with relative \nmRNA expression normalized to the housekeeping gene β-Actin, and relative miRNA expression normalized to \nCt40, as described by Gevaert et al.83.\nELISA\nThe VEGF-A protein level was analyzed using the Human VEGF ELISA Kit (AB100662), while the TGF-β1 \nprotein level was determined with the Human TGF beta 1 ELISA Kit (AB100647). The COX2 protein level was \nassessed using the Human COX2 ELISA Kit (AB267646), and the Nrf2 transcription factor was analyzed using \nthe Human Nrf2 ELISA Kit (AB277397). The phosphorylated Nrf2 transcription factor was determined with the \nNrf2 Transcription Factor Assay Kit (AB207223). All analyses were conducted on cell suspensions and followed \nthe manufacturer’s instructions (Abcam, Cambridge, UK).\nELISA plates were read at 450 nm using the SYNERGY HTX multi-mode reader (BioTek Instruments, \nWinooski, Vermont, USA), and data analysis was performed using Gen5 3.10 Software (BioTek Instruments). \nQuantification of the prepared samples was carried out using standard curve analysis.\nLive-Cell Imaging\nWe conducted live-cell imaging experiments using the following equipment:\n – A Zeiss array confocal laser scanning microscope (Axio Observer.Z1 from Zeiss, Gottingen, Germany) \nequipped with a 100 × objective lens (Plan-Fluor × 100/1.45 Oil, Zeiss, Germany), a motorized filter wheel \n(CSUX1FW , Y okogawa Electric Corporation, Tokyo, Japan) on the emission side, and an AOTF-based laser \nmerge module for the 405, 445, 473, 488, 514, and 561 nm laser lines (Visitron Systems). The system included \na Nipkow-based confocal scanning unit (CSU-X1, Y okogawa Electric Corporation). Data acquisition and \nfluorescence microscope control were performed using Visiview 4.2.01 (Visitron, Puchheim, Germany)84.\n – An inverted wide-field microscope Anglerfish (Observer.A1, Carl Zeiss GmbH, Vienna, Austria) with a \n40 × oil immersion objective (Plan Apochromat 1,3 NA Oil DIC (UV) VISIR, Carl Zeiss GmbH, Vienna, \nAustria) and a standard CFP/YFP filter cube. Emission collection was facilitated by a 505dcxr beam-splitter, \ndirecting light to both sides of the camera (CCD camera, Coolsnap Dyno, Photometrics, Tucson, AZ, USA). \nVisualization was carried out using the NGFI AnglerFish C-Y7G imager for emission collected with Angler-\nfish.\nA constant buffer perfusion flow was maintained using the NGFI perfusion system (PS9D, NGFI, Graz, Austria).\nMitochondrial H2O2 measurements\nWe measured mitochondrial H 2O2 levels using the genetically encoded H 2O2 sensors mitoHyper7. The \nmitoHyPer7 signals were imaged by alternately exciting the cells with a motorized dual filter system equipped \nwith LED 480nm (excitation filter 480nm/17nm) and LED 430nm (excitation filter 433nm/24nm) beam splitters. \nEmissions were alternately collected using78 a 535/22 BrightLine HC emission filter, as previously described by \nTawfik et al.85.\nCells were initially perfused with HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) to record \nH2O2 production for the first 2 min. Subsequently, [Zn(neo)(nif)2] (10 µM) was added for the following 6 min, \nand finally, cells were perfused again with HEPES for an additional 2 min.\nThe acquired data were saved as image files during the experiments and analyzed using Fiji software (ImageJ2). \nBackground and photobleaching corrections were performed in Excel, and data were analyzed further using \nGraphPad Prism 8.01.\nCytosolic Ca2+ measurements\nWe measured cytosolic Ca 2+ concentrations in cells incubated with the fluorescent cytosolic Ca 2+ indicator \nFura-2 acetoxy-methyl-ester (Fura-2AM) (TEFLabs, Austin, TX) for 30 min in EH-loading buffer. Cells stained \nwith Fura-2AM were illuminated at 340 nm and 380 nm, with emission captured at 515 nm, as previously \ndescribed86. The measurements were recorded as the F380/F340 ratio using live-acquisition software v2.0.0.12 \n(Till Photonics) and analyzed using GraphPad Prism 8.01. Background subtraction was performed using a \ndesignated background region of interest (ROI), and bleaching correction was applied using an exponential \ndecay fit of the basal fluorescence extrapolated across the entire measurement. The results represent the maximal \n(Δmax) in cytosolic Ca 2+ levels in response to ATP or histamine (100 µmol/L) stimulation of the cells.\nScientific Reports |        (2025) 15:10126 15| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\nMitochondrial Ca2+ measurements\nMitochondrial Ca2+ measurements were conducted using the genetically encoded biosensor 4mtD3cpv. The \nexcitation wavelength for 4mtD3cpv was set at 440 nm (440AF21, Omega Optical, Brattleboro, VT, USA), and \nemissions were captured at 480 and 535 nm (480AF30 and 535AF26, Omega Optical, Brattleboro, VT, USA) \nas previously described 86. Data acquisition was performed using NIS-Elements AR software (Nikon, Vienna, \nAustria) and analyzed using GraphPad Prism 8.01. Measurements were corrected by a background region of \ninterest (ROI), and photobleaching correction was applied using an exponential decay fit. The results represent \nthe maximal change (Δmax) in mitochondrial Ca 2+ levels in response to ATP or histamine (100 µmol/L) \nstimulation of the cells.\nStatistical analysis\nThe experimental qRT-PCR mRNA data were analyzed using GraphPad Prism 8.01 (GraphPad Software, San \nDiego, CA, USA) and are represented as mean values ± SD of three independent measurements provided in \nduplicate (one independent measurement was provided in duplicate for miRNA determination and one \nindependent measurement was provided in triplicate for GPx1 and SOD1, respectively).\nCytosolic calcium measurement data were evaluated using GraphPad Prism 8.01 and are expressed as mean \nvalues ± SD of three independent measurements for untreated cells: HME1 (n = 46) and 12Z (n = 102); cells \ntreated with 10 μM cis-platin: HME1 (n = 43) and 12Z (n = 88); and cells treated with 10 μM [Zn(neo)(nif) 2]: \nHME1 (n = 50) and 12Z (n = 99).\nMitochondrial calcium measurement data were analyzed using GraphPad Prism 8.01 and are represented as \nmean values ± SD of three independent measurements for untreated cells: HME1 (n = 4) and 12Z (n = 14); cells \ntreated with 10 μM cis-platin: HME1 (n = 6) and 12Z (n = 14); and cells treated with 10 μM [Zn(neo)(nif) 2]) \nHME1 (n = 5) and 12Z (n = 14).\nMitochondrial H2O2 data were analyzed using GraphPad Prism 8.01 and are represented as mean values ± SD \nof three independent measurements for untreated cells: HME1 (n = 3) and 12Z (n = 3); and cells treated with 10 \nμM [Zn(neo)(nif) 2]: HME1 (n = 3) and 12Z (n = 4).\nStatistical analysis was performed using the Student’s t-test and nonparametric analysis of variance (ANOV A), \nfollowed by Tukey’s post hoc test and Dunnett ‘s Multiple Comparison test. Statistically significant results were \nfound as follows: P-value < 0.05 (*, significant), P-value < 0.01 (**, highly significant), and P-value < 0.001 (***, \nstrongly significant).\nData availability\nThe datasets used and/or analyzed during the current study are available from the corresponding author upon \nreasonable request.\nReceived: 11 June 2024; Accepted: 12 March 2025\nReferences\n 1. Huang, M., Chen, Y ., Han, D., Lei, Z. & Chu, X. Role of the zinc finger and SCAN domain-containing transcription factors in \ncancer. Am. J. Cancer Res. 9, 816–836 (2019).\n 2. Nguyen, T., Nioi, P . & Pickett, C. B. The Nrf2-Antioxidant Response Element Signaling Pathway and Its Activation by Oxidative \nStress. J. Biol. Chem. 284, 13291–13295 (2009).\n 3. Tang, Y . et al. MicroRNAs and angiogenesis: a new era for the management of colorectal cancer. Cancer Cell Int. 21, 221 (2021).\n 4. Zhao, Y ., Xing, C., Deng, Y ., Y e, C. & Peng, H. 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Biol. 9, 614668 (2021).\nAuthor contributions\nConceptualization, I.Š. and M.R.; methodology, I.Š., and L.S.; validation, Z.B., K.K., and C.M.-S.; formal analysis, \nI.Š., L.S., Z.B., K.K.; investigation, I.Š.; resources, J.V .; data curation, I.Š. and L.S.; writing—original draft prepa-\nration, G.S., L.S., and I.Š.; writing—review and editing, L.S., J.V ., C.M.-S., and M.R.; visualization, I.Š., and L.S.; \nsupervision, M.R., W .F .G., and J.V .; project administration, M.R., M.M., and J.V .; funding acquisition, M.R. and \nW .F .G. All authors reviewed the manuscript.\nFunding\nThis research was funded by Slovak Grant Agency VEGA 1/0435/23 and the Austrian Science Funds (FWF) \n[DOI: 10.55776/W1226].\nDeclarations\nCompeting interests\nThe authors declare no competing interests.\nAdditional information\nSupplementary Information The online version contains supplementary material available at  h t t p s : / / d o i . o r g / 1 \n0 . 1 0 3 8 / s 4 1 5 9 8 - 0 2 5 - 9 4 2 4 9 - x     .  \nCorrespondence and requests for materials should be addressed to M.R.\nReprints and permissions information is available at www.nature.com/reprints.\nPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and \ninstitutional affiliations.\nScientific Reports |        (2025) 15:10126 18| https://doi.org/10.1038/s41598-025-94249-x\nwww.nature.com/scientificreports/\n\nOpen Access  This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives \n4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in \nany medium or format, as long as you give appropriate credit to the original author(s) and the source, provide \na link to the Creative Commons licence, and indicate if you modified the licensed material. 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