Background: Carvacrol, a natural phenolic monoterpenoid, has been suggested to exert anticancer effects; however, its underlying mechanisms in breast cancer (BC) remain incompletely defined. Methods: MCF-7 (HR⁺) and MDA-MB-231 (TNBC) BC cell lines were treated with carvacrol at various concentrations. Cell viability was assessed using CVDK8 kit, while migration was evaluated by wound healing assays. Apoptosis was determined using Annexin V-FITC Kit, and ROS levels were measured by DCFH-DA assay. Flow cytometry was used for CD44/CD133 cancer stem cells markers analysis, and genes expression were quantified using qPCR. Results: Carvacrol significantly inhibited cell proliferation, and migration in both HR⁺ and TNBC cells. Additionally, carvacrol increased the BAX/BCL2 ratio, induced apoptosis, and decreased ROS levels, with greater antioxidant activity observed in MCF-7 cells. Moreover, carvacrol suppressed CD44⁺ levels, whereas CD133⁺ levels were not affected. Gene expression analysis revealed subtype-specific effects where ABCG2 was upregulated in MCF-7 cells but downregulated in MDA-MB-231 cells, while NF_KB1 expression increased in both lines. Conclusions: Carvacrol exerts multitargeted anticancer effects in BC by promoting apoptosis, reducing ROS, and suppressing CD44⁺, with distinct subtype-specific responses. These findings highlight carvacrol as a promising natural therapeutic compound for BC treatment; however, further in vivo studies and clinical investigations are required to validate its translational potential.

Melatonin, a pineal hormone with antioxidant and regulatory functions, has emerged as a key modulator of ovarian physiology. Its presence in follicular fluid suggests important roles in granulosa cell function, follicle development, and reproductive outcomes. However, its effects on granulosa cell tumour (GCT) biology and epigenetic regulation remain insufficiently defined. This study aimed to investigate the effects of melatonin on proliferation and global DNA methylation in human granulosa tumour cells (COV434) compared with healthy endothelial controls (HUVECs). COV434 and HUVEC cells were treated with melatonin at 1, 10, 100, and 1000 µM. Cell viability and proliferation were assessed using the MTT [3-(4.5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide] (Cambridge, UK) assay and xCELLigence RTCA system (Roche), while DNA methylation was quantified with a 5-mC ELISA kit (Epigentek Group Inc, USA). Experimental groups included negative, sham, melatonin-treated, and positive controls. Melatonin showed a cell type-dependent effect. In COV434 cells, proliferation was significantly inhibited, with an IC₅₀ of 10.55 µM, whereas HUVECs displayed increased proliferation at higher doses. DNA methylation levels decreased in both cell types in a dose-dependent manner, reaching the highest significance in COV434 cells at 1000 µM (p<0.001). In conclusion, melatonin demonstrated a dose-dependent inhibitory effect on COV434 cell proliferation while simultaneously reducing global DNA methylation levels.

Objectives: Noninvasive prenatal testing relies on the analysis of total cell-free DNA (cfDNA) in maternal plasma, where fetal-derived DNA constitutes only a minor fraction. This study aimed to preliminarily compare a modified TPY cfDNA extraction protocol with two commercial extraction kits for the downstream detection of Y-chromosome-specific markers in pregnancies carrying male fetuses. Methods: Plasma samples were obtained from 52 singleton pregnancies between 10 and 30 weeks of gestation with male fetal sex confirmed by ultrasonography. Total cfDNA was extracted from aliquots of the same maternal plasma samples using the modified TPY protocol, the QIAamp DSP Virus Kit, and the MagMAX™ Cell-Free DNA Isolation Kit. Quantitative real-time PCR was performed for the Y-chromosome-specific markers SRY and DYS14. At the same time, GLO was used as a reference marker to reflect the total cfDNA background. Extraction performance was assessed primarily using total cfDNA concentration and Ct values obtained from amplification of fetal-specific Y-chromosome markers. Results: Total cfDNA concentrations varied among the extraction methods, with the commercial kits yielding higher total cfDNA concentrations than the modified TPY protocol. In contrast, the TPY protocol yielded slightly lower mean Ct values for SRY and DYS14 than the commercial kits. SRY and DYS14 amplification was detected in 90.4% and 94.2% of samples, respectively. However, these Ct differences should be interpreted cautiously because fetal fraction, maternal DNA contamination, extraction recovery, and fragment size distribution were not directly measured. Conclusions: The modified TPY protocol showed preliminary technical feasibility for extracting total cfDNA from maternal plasma and enabling downstream amplification of Y-chromosome-specific markers in male pregnancies. Nevertheless, the observed lower Ct values do not establish selective fetal DNA enrichment, reduced maternal DNA contamination, or clinical superiority over commercial methods. Further analytical validation using standardized fetal fraction measurement, recovery efficiency testing, fragment size analysis, fetal-to-maternal DNA ratio assessment, and larger cohorts including both male and female pregnancies is required before broader clinical applicability can be determined.

Objective: Breast cancer (BC) is a heterogeneous malignancy driven by dysregulated molecular pathways that promote proliferation, invasion, and therapeutic resistance. Carvacrol, a natural monoterpenoid phenol, has demonstrated anticancer potential; however, its subtype-specific molecular mechanisms in BC remain unclear.Materials and Methods: This study combined bioinformatic and experimental approaches to evaluate the functional relationships and prognostic relevance of MKI67, E2F4, FOSL1, CD44, CDKN1A (P21), CASP9, ABCB1, ABCC1, and ABCC5. The STRING database was used to evaluate protein–protein interaction (PPI) networks. Overall survival (OS) and disease-free survival (DFS) analyses were conducted using GEPIA2 based on TCGA-BRCA data. MCF-7 and MDA-MB-231 BC cells were cultured and exposed to 400 µM carvacrol for 48 hours. Gene expression levels were evaluated by qPCR. Relative mRNA expression was calculated using the 2^−ΔΔCt method.Results: PPI analysis revealed significant functional connectivity among the studied genes (p=2.15 e−09). Survival analyses did not demonstrate consistent independent prognostic significance. Carvacrol induced subtype-dependent molecular alterations. In MCF-7 cells, CD44, FOSL1, and MKI67 were significantly downregulated (p