Ugi-four component reaction of bile acids, formaldehyde, ethyl isocyanoacetate and 1,4-phenylenediamine produced dimeric steroid-glycylglycine conjugates in low yields. The structure of the new compounds consists of two steroid-dipeptide conjugate units linked by a 1,4-phenylene bridge. Detailed NMR characterization provided sufficient evidence on the obtained structures that were confirmed by single crystal X-ray diffraction. Interestingly X-ray crystallography revealed that the dimeric lithocholic acid-glycylglycine conjugate crystalizes in a conformation having the tertiary and secondary amides in the E and Z rotamers respectively.
The ER-α36 isoform is expressed in both ER-positive and ER-negative breast cancer cell lines. ER-α36 is implicated in promoting the proliferation, invasion, and metastasis of tumor cells. Runt-related transcription factor 2 (RUNX2) is widely recognized as a driver of bone-specific metastasis in breast cancer. The study aimed to examine the association between ER-α36 and RUNX2 in breast cancer and its relationship with cancer development. Immunohistochemical analysis of 25 cases revealed subcellular localization patterns of ER-α36 and RUNX2. ER-α36 showed nuclear positivity in 5 cases (20%), cytoplasmic/membranous positivity in 10 cases (40%), and combined nuclear/cytoplasmic positivity in 4 cases (16%). RUNX2 exhibited nuclear positivity in 5 cases (20%), cytoplasmic/membranous positivity in 9 cases (36%), and combined nuclear/cytoplasmic positivity in 6 cases (24%). Case-by-case analysis revealed that in specimens with nuclear ER-α36 positivity, RUNX2 was predominantly localized in the nucleus or nucleus/cytoplasm. Both proteins were predominantly expressed in grade 2 tumors. In vitro experiments, Knockdown of RUNX2 significantly reduced ER-α36 expression by 3.2-fold in MDA-MB-231 cells and by 8.9-fold in MCF-10ACE cells (**p < 0.01 and ***p < 0.001, respectively), whereas ER-α36 knockdown significantly decreased RUNX2 expression by 2.3-fold in MDA-MB-231 cells (*p < 0.05). The results of co-immunoprecipitation showed that the two proteins were significantly associated in MCF-10ACE and MDA-MB-231 cells. The luciferase reporter assay revealed that down-regulation of RUNX2 inhibited the binding of ER-α36 to estrogen response elements. These results suggest that RUNX2 interacts with ER-α36 and facilitates its transcriptional activation.
Organophosphate esters (OPEs) may disrupt hormonal balance, but their specific effects in pre- and post-menopausal women have been underexplored. This investigation delves into the menopause-specific associations of OPEs with sex hormones in the National Health and Nutrition Examination Survey, 2013-2016. We included 817 premenopausal and 657 postmenopausal women. Survey-weighted multivariable linear regression, restricted cubic splines (RCS), principal component analysis (PCA), and quantile-based g computation (qgcomp) models were conducted to investigate the associations of OPE metabolites and OPEs mixture with sex hormones in pre- and postmenopausal women. In postmenopausal women, bis(2-chloroethyl) phosphate (BCEP) and Bis(1,3-dichloro-2-propyl) phosphate (BDCPP) were negatively associated with testosterone (T) (BCEP: β = -0.06 [-0.10, -0.01]; BDCPP: β = -0.07 [-0.13, -0.01]) and free androgen index (FAI) (BCEP: β = -0.08 [-0.13, -0.02]; BDCPP: β = -0.06 [-0.12, -0.01]). Diphenyl phosphate (DPHP) was also negatively associated with FAI (β = -0.08 [-0.14, -0.02]). RCS analyses revealed non-linear exposure-response relationships between DPHP and T, between BDCPP and estrogen (E2), and between BDCPP and DPHP and the T/E2 ratio. PCA and qgcomp analyses consistently found negative associations between the OPEs mixture and T and FAI. Conversely, in premenopausal women, BCEP was linearly and positively associated with sex hormone-binding globulin (SHBG) (β = 0.05 [0.01,0.09]), whereas DPHP showed a nonlinear relationship with SHBG, with a curve rising then plateauing. Overall, OPEs, alone or as mixtures, might lower T and FAI levels in postmenopausal women. In contrast, BCEP and DPHP might increase SHBG levels in premenopausal women.
Vitamin D deficiency is linked to cardiovascular disease (CVD), with oxidative stress (OS) playing a key role in the etiology of CVD. We hypothesized that vitamin D deficiency induces cardiac oxidative and endoplasmic reticulum stress (ERS), leading to apoptotic cell death. A rat model of vitamin D deficiency and insufficiency was employed for in vivo studies, while the rat cardiomyoblast cell line (H9C2) was used for in vitro experiments. (OS) parameters were assessed by spectrophotometry, gene and protein expression were determined using qPCR and western blotting. OS markers were elevated while antioxidant enzyme activities were decreased in the left ventricle of vitamin D deficient (VDD) rats compared to control rats. VDD downregulated nuclear factor-erythroid factor 2-related factor 2 (Nrf2) and its downstream proteins in the heart. Since elevated OS leads to ERS, we assessed ERS markers in the heart. Levels of ERS markers were elevated in both the VDD and insufficient rats. Pro-apoptotic and autophagy markers such as BAX, BIM and caspase 8 were upregulated in the heart of VDD rats. Experiments with the H9C2 cell line demonstrated that induction of mild OS using H2O2 upregulates ERS, autophagy and apoptotic markers. VD pretreatment of H9C2 cells led to increase in antioxidant enzyme activities, decrease in ERS and autophagy markers and apoptosis. 4PBA, an ERS inhibitor inhibited apoptosis indicative of a direct role for ERS. The findings suggest that vitamin D protects the heart from oxidative injury and cell death by antioxidant defence mechanism, upregulating Nrf2/HO-1 signaling while alleviating ER stress.
A synthesis of hybrids based on esters, amides of the ursane series, and 28-norursane ureas containing terminal alkyne or azide groups was performed via copper-catalyzed azide-alkyne cycloaddition (CuAAC). Derivatives of modified gallic acid, methyl azidobenzoates, 4-(azidomethyl)-3-methyl-1,2,5-oxadiazole-2-oxide, and dimethyl but-2-indiate were used as cyclization partners. The cytotoxicity of the resulting hybrids was evaluated on a panel of human cancer cell lines (MCF-7, HepG2, HeLa, U87MG, A549). Most of the ester and amide derivatives demonstrated weak antiproliferative activity and low selectivity. At the same time, urea conjugates 14a, 17a, 19, 19a showed pronounced cytotoxicity against MCF-7 and HeLa cells. The most active compound 19a effectively inhibited the growth of MCF-7 (IC₅₀ = 6.50 ± 0.49 μM) and HeLa (IC₅₀ = 7.87 ± 1.86 μM), while maintaining low toxicity for normal fibroblasts. The selectivity profile of 19a (SI = 10-12) significantly exceeds that of the reference drug doxorubicin (SI = 0.8-5.6). Hybrids based on 2-azidoethylurea were more active than propargylurea-derived congeners. The deprotection of OH groups of the polyphenol fragment increased cytotoxicity and selectivity. Molecular docking results indicate that urea hybrids with a 1,2,3-triazole linker may have a higher affinity for the ATP-binding site of Akt1 kinase compared to ester and amide analogs. This study substantiates a strategy for the development of selective antitumor agents based on triterpenoid ureas with polar substituents, which are promising for further preclinical optimization.
BACKGROUND:Hashimoto's thyroiditis is a common autoimmune endocrine disorder with a significant female predominance, suggesting that the estrogen/estrogen receptor axis may play a critical role in its pathogenesis. While classical nuclear receptors are well-studied, the role of G protein-coupled estrogen receptor 1 (GPER1) in Hashimoto's thyroiditis remains largely unexplored. This study aimed to evaluate serum GPER1 levels in patients with Hashimoto's thyroiditis and investigate its potential as a diagnostic biomarker. METHODS:This clinical study included 22 newly diagnosed, treatment-naive female patients with Hashimoto's thyroiditis and 40 healthy female controls. Serum GPER1 levels were measured using the ELISA method. Demographic data, thyroid function tests (TSH, FT4, FT3), and anti-thyroid peroxidase (anti-TPO) antibody levels were recorded for all participants. Statistical analyses, including ROC curve and Spearman's correlation, were performed to assess diagnostic efficiency and clinical relationships. RESULTS:Serum GPER1 levels were significantly higher in the Hashimoto's thyroiditis group compared to the control group. ROC curve analysis demonstrated that GPER1 had high diagnostic power, with an area under the curve (AUC) of 0.997 (95% CI: 0.990-1.000). Using a cut-off value of 0.400, GPER1 exhibited 95.5% sensitivity and 100% specificity for distinguishing Hashimoto's thyroiditis patients from healthy individuals. Furthermore, a strong positive correlation was identified between serum GPER1 and anti-TPO antibody levels. CONCLUSION:Our findings indicate that GPER1 levels are markedly elevated in patients with Hashimoto's thyroiditis and correlate strongly with thyroid autoimmunity. These results raise the possibility that GPER1 is related to inflammatory activity in Hashimoto's thyroiditis and could serve as a promising novel biomarker for its diagnosis. Further prospective studies are warranted to elucidate the exact mechanism of GPER1 in autoimmune thyroid disease.
Canine distemper virus (CDV) induces central nervous system (CNS) demyelination, potentially influenced by local glucocorticoid metabolism via 11β-HSD enzymes and glucocorticoid receptors (GR). This study investigated the relationship between demyelination and local glucocorticoid synthesis in CDV infection. Cerebellar tissues from CDV-infected dogs (diseased group; n = 20) and clinically healthy dogs (control group; n = 10) were analyzed. Sections were immunohistochemically stained with anti-CDV, anti-11β-HSD1, anti-11β-HSD2, and anti-GR antibodies. In the diseased group, 11β-HSD1 immunopositivity was significantly higher than in controls in endothelial cells (p < 0.01), reactive astrocytes (p < 0.001), Purkinje neurons (p < 0.01), and molecular layer neurons (p < 0.001). 11β-HSD2 immunopositivity was significantly increased in reactive astrocytes (p < 0.01), white matter neurons (p < 0.001), and Purkinje neurons (p < 0.01). GR immunopositivity was elevated in reactive astrocytes (p < 0.01) and mossy fibers (p < 0.001). A strong positive correlation was observed between demyelination severity and 11β-HSD1 in reactive astrocytes (r = 0.915, p < 0.01), whereas a moderate negative correlation was found between demyelination severity and GR in reactive astrocytes (r = -0.461, p < 0.05). These findings indicate that local glucocorticoid metabolism is associated with CNS demyelination during CDV infection. This study provides novel insights into 11β-HSD enzymes and GR in CNS pathology, offering a translational model for understanding local glucocorticoid regulation in human demyelinating diseases such as multiple sclerosis.
Progesterone, the first sex hormone ever discovered, has a long history. Yet, it gained prominence in research during the 1930s. Currently, our understanding of progesterone goes beyond its role in the reproductive system, also encompassing the cardiovascular system. Studies have reported on the protective role played by this hormone in the cardiovascular system, which is primarily due to its actions on the vasculature. Progesterone stimulates vasoactive factors, modulates lipid profiles, reduces inflammatory responses, inhibits platelet aggregation and cell proliferation, and lowers blood pressure, thereby contributing to the reduction of cardiovascular disease risk. However, for a long time, hormone replacement therapy relied on the use of progestins, synthetic compounds that failed to replicate the beneficial effects of progesterone and often cause various adverse effects, which cannot be attributed to progesterone itself. In this context, this review compiles the most recent findings regarding the actions of progesterone in the vascular system, while also presenting a historical overview that contextualizes the research in both clinical and experimental areas related to progesterone. Thus, we seek to clarify the unique role played by progesterone in the cardiovascular system, dispelling misconceptions about its effects and discussing its therapeutic prospects.
BACKGROUND:MicroRNA-144 (miR-144) has been implicated in inflammation and glucocorticoid (GC) receptor regulation, but its role in GC sensitivity during acute respiratory distress syndrome (ARDS) is unclear. This study aimed to investigate the impact of miR-144 on GC therapeutic efficacy, elucidate its underlying molecular mechanisms in ARDS, and validate its clinical application value. METHODS:First, we established a sepsis-induced ARDS rat model with five groups (n = 6 each): control, model, GC, GC + miR-144 mimic, or GC + miR-144 inhibitor. Body weight, inflammatory markers, lung wet/dry ratio, histopathology, and miR-144/GRα/GRβ/NF-κB mRNA were assessed after the 5-day treatment. Second, we prospectively enrolled 63 ARDS patients receiving methylprednisolone and classified them as GC-sensitive (n = 43) or GC-resistant (n = 20) based on clinical outcomes. The correlations of baseline miR-144, GRα, GRβ, and NF-κB mRNA levels in patient blood were analyzed, and the predictive value of miR-144 for GC sensitivity was evaluated. RESULTS:In rats, miR-144 inhibitor enhanced GC efficacy (improved body weight recovery, reduced lung edema and inflammation), while mimic attenuated these effects. Mechanistically, in rat lung tissue, sepsis stimulation increased miR-144, GRβ, and NF-κB while reducing GRα expression; GC reversed these changes; miR-144 inhibitor further reduced GRβ/NF-κB, whereas mimic antagonized this without affecting GRα. In patients, GC-resistant individuals showed significantly higher miR-144, GRβ, and NF-κB levels than sensitive ones. Strong positive correlations were observed between miR-144 and both GRβ and NF-κB, and miR-144 effectively predicted GC resistance in ARDS. CONCLUSIONS:MiR-144 regulates GC sensitivity via the GRβ/NF-κB axis in ARDS. Baseline miR-144 level is a promising biomarker for predicting GC response, providing a basis for individualized treatment.
Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide, and current treatments are limited by low efficacy, toxicity, and resistance. Among phytosterol compounds, β-sitosterol (BST) was identified in abundance in plants and showed potential anticancer activity, as evidenced by several in vitro and in vivo studies. But the poor solubility of BST limits its efficacy toward anti-cancer therapeutics. Research showed that imidazolium-based ionic liquids (ILs) were found to be very effective for increasing the solubility as well as enhancing the bioavailability of anti-cancer drugs. To develop novel phytosterol-based drug candidates with improved anticancer potential, BST was chemically modified through the conjugation with imidazolium-based ionic liquid moieties to generate a series of BST ionic liquid derivatives (BST-ILs). The synthesized derivatives were characterized, and their physicochemical and pharmacokinetic properties were evaluated through in silico ADMET and density functional theory analyses. Notably, the ionic liquid derivatives displayed improved membrane permeability. The biological activities of BST and its BST-ILs derivatives were assessed in the human colorectal cancer (HCT116) cell line. The results demonstrated a concentration-dependent reduction in cell viability, with the ionic liquid conjugates exhibiting significantly enhanced cytotoxicity compared to the parent compound. Furthermore, the derivatives effectively inhibited cell migration and induced apoptosis, as confirmed by wound healing assay and gene expression analyses, including upregulated expression of apoptosis marker genes Bax and Caspase-3 and downregulation of Ki67 and Cyclin D1. Flow cytometric analysis in this study further demonstrated increased apoptotic cell populations following treatment with the derivatives. In addition, western blot analysis revealed altered pro-Caspase-3 protein expression, further supporting the modulation of apoptosis-associated signaling. Molecular docking and molecular dynamics simulations were performed to investigate interactions with key enzymes of the mevalonate pathway, namely farnesyl diphosphate synthase (FDFT1) and squalene synthase (SQS). These findings highlight BST-ILs conjugates as promising steroid-based candidates with the potential to target sterol biosynthesis pathways in colorectal cancer with significant anti-proliferative activities, highlighting the importance of these BST-ILs for further investigation for anti-cancer drug development.
Ten undescribed hydroxylated, acetylated, esterified and glycoside derivatives of lithocholic acid (LCA) were obtained through the biotransformation of Aspergillus minisclerotigenes, alongside four known compounds. Structural characterization employed integrated NMR spectroscopy, HR-ESI-MS, and X-ray crystallography. Biological evaluation identified compound 10 as a potent inhibitor of LPS-induced NO production in BV2 microglial cells (IC50 = 4.67 μM) and dose-dependently suppressed mRNA expression of IL-1β, TNF-α, iNOS, and COX-2. Furthermore, compound 10 promoted M2-polarization of LPS-activated microglia to attenuate neuroinflammation. Subsequent analysis demonstrated its induction of inflammatory autophagy in BV2 cells. These results suggested that biotransformation was an efficient strategy for structural diversification of LCA and discovery of potent anti-inflammatory leads.
BACKGROUND:Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder, often associated with insulin resistance and obesity. As a heterogeneous condition, diverse mechanisms could be involved in its pathogenesis. Data on PCOS among vegetarian populations, particularly from Indian subcontinent, remain limited. This study aimed to assess the association between biochemical, hormones, and metabolic parameters in lean and obese women with PCOS. METHODS:A multicentre, observational case-control study conducted among 40 women diagnosed with PCOS and 43 matched by BMI healthy controls classified as either lean (BMI < 25) and obese (BMI ≥ 25). Groups were compared using analysis of covariance (ANCOVA) with age as a covariate based on biochemical, metabolic, hormonal and hematological parameters. Age and BMI adjusted partial spearman correlation analysis was performed to examine the association of HOMA-IR and testosterone with study variables. Univariate and multivariate regression analyses, adjusted for age and BMI, were employed to identify independent predictor of HOMAIR and testosterone, with multiple testing correction applied. RESULT:Significant differences were found in TG, VLDL, HDL, SGPT, ALP, FSH, Testosterone, HOMA-IR, AMH and WBC. HOMA-IR was elevated in both lean and obese PCOS groups compared to control groups. In age and BMI adjusted multivariate regression, BMI (p = 0.002) and SGPT (p = 0.0032) were independent predictor of HOMA-IR, while WBC showed a borderline association (p = 0.068). AMH was the only independent predictor of testosterone (p = 0.005). CONCLUSION:Although our vegetarian women with PCOS, had normal value of liver enzymes, they were higher in comparison to control groups. SGPT was independently association with insulin resistance (IR), suggesting disturbance of liver enzyme in POCS and inflammatory marker (WBC) were mainly influenced by BMI. The association between AMH and testosterone suggests that hyperandrogenism and ovarian dysfunction in PCOS and may occur independently of adiposity.
We have synthesized a trifluoromethyl phenyldiazirine derivative, namely N-(4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl)-20-azapregn-5-en-3β-ol (DAMDz1). In vitro assays using Saccharomyces cerevisiae demonstrated that DAMDz1 undergoes 3-O-acetylation by yeast alcohol acetyltransferase Atf2p, indicating its ability to cross the plasma membrane. A bacterial 3beta-hydroxysteroid dehydrogenase was found to convert DAMDz1 into its 3-keto derivative. In silico computations have indicated phospholipid membrane permeability of DAMDz1 and the possibility of its localization within the active sites of aforementioned enzymes and some other proteins that interact with oxysterols and aminosterols.
BACKGROUND AND AIMS:Inhibition of Δ24-dehydrocholesterol reductase (DHCR24) leads to accumulation of the cholesterol biosynthesis intermediate desmosterol, which is the endogenous ligand for liver X receptors (LXRs). Activation of LXRs is considered to be atheroprotective as it suppresses inflammation and stimulates cholesterol efflux in macrophages via upregulation of ATP-binding cassette transporters A1 and G1. In the current study we aimed to investigate the effect of the selective DHCR24 inhibitor SH42 on regression of atherosclerotic plaques in APOE*3-Leiden.CETP mice, a well-established model for human-like lipid metabolism. METHODS:Mice were fed a Western-type diet (16% fat, 0.15% cholesterol) for 20 weeks to promote plaque development. Subsequently, treatment was started with vehicle or SH42 for 8 weeks while the animals were kept on a regular chow diet. RESULTS:Treatment with SH42 significantly upregulated desmosterol levels in liver and plasma, and led to a small reduction in plasma triglyceride, nonHDL-cholesterol and HDL-cholesterol levels on top of the lipid-lowering effect caused by the dietary switch. While SH42 treatment did not affect immune cell numbers in blood, bone marrow, or spleen, SH42 strongly reduced the number of macrophages found in the peritoneum after thioglycolate injection. CONCLUSIONS:While the dietary switch halted atherosclerotic lesion progression, SH42 treatment did not accelerate atherosclerotic lesion regression further.
Endometriosis is a chronic inflammatory disease characterised by ectopic endometrial tissue, progressive fibrosis and chronic pain, with a pathogenesis that goes beyond oestrogen dependence. The central question this review seeks to answer is: are steroidal alkaloids - nitrogenous plant-derived steroids with a unique structure - a mechanistically sensible, non-hormonal alternative that can simultaneously target the inflammatory, fibrotic and epigenetic drivers of endometriotic lesion persistence, while preserving fertility and avoiding systemic hormonal suppression? A growing body of evidence points to non-hormonal molecular networks including immune dysregulation, angiogenesis, and resistance to apoptosis as drivers of lesion persistence. These molecular disturbances are associated with treatment resistance and limit the long-term efficacy of hormone-based therapies. This review synthesises recent mechanistic advances that describe the non-hormonal signalling pathways that maintain endometriotic lesions, with a specific emphasis on NF-κB-mediated sterile inflammation, inflammasome activation, PI3K/Akt/mTOR-dependent survival signalling, Hedgehog-driven fibrosis and epigenetic repression of progesterone receptor expression. Steroidal alkaloids are critically reviewed as multitarget modulators suppressing inflammatory signalling, inhibiting invasive and fibrotic remodelling, and restoring apoptotic sensitivity in this pathophysiological context, without direct systemic oestrogen deprivation. Structural determinants of steroidal alkaloid activity, including glycosylation status, nitrogen topology and configuration of the steroidal scaffold, are discussed in the context of pathway selectivity, pharmacokinetics and toxicity. Preclinical in vitro and in vivo evidence is critically reviewed in the context of key translational limitations such as bioavailability constraints, narrow therapeutic index and teratogenic risk from developmental pathway inhibition. This review highlights steroidal alkaloids as a mechanistically rational, non-hormonal approach to target lesion persistence and fibrosis in endometriosis, integrating molecular, pharmacological and structural insights, and outlines the major hurdles that must be overcome for clinical translation. The main objective of this review is to address a specific unanswered question: can steroidal alkaloids with their multitarget pharmacology offer a biologically coherent, non-hormonal strategy for the treatment of endometriosis that goes beyond oestrogen deprivation to target the molecular machinery of lesion survival, fibrosis and immune dysregulation. This question is addressed by reviewing the nonhormonal signalling networks that support ectopic lesions, the structural and pharmacokinetic properties of steroidal alkaloids that are relevant to these pathways, and the translational challenges that need to be addressed for clinical translation.
Cyclopamine, a jervatrum-type steroidal alkaloid from Veratrum species, is one of the earliest natural inhibitors of the Hedgehog (Hh) signaling pathway and was instrumental in identifying Smoothened (SMO) as a therapeutic target in oncology. Initially recognised as a potent teratogen, its activity was later attributed to direct SMO inhibition, suppressing GLI-mediated transcription and revealing Hh signaling as a driver of diverse malignancies, including basal cell carcinoma, medulloblastoma, pancreatic ductal adenocarcinoma, and glioblastoma. Despite strong pathway specificity, clinical translation of cyclopamine is hindered by poor solubility, acid lability, low bioavailability, and teratogenic risk. Medicinal chemistry optimization, prodrug strategies, and advanced nanocarrier systems, such as micelles, lipid nanoparticles, albumin carriers, and polymer-drug conjugates, have been explored to overcome these limitations and enable tumor-targeted delivery. Moreover, cyclopamine analogues have inspired the development of clinically approved SMO inhibitors, validating the pathway as a therapeutic target. Collectively, cyclopamine's trajectory from developmental toxicant to molecular probe and lead scaffold underscores its enduring impact on Hh-directed drug discovery and highlights the ongoing relevance of formulation-driven strategies in oncology.
Our research group has previously constructed a series of 3-biotin-B-norcholesteryl benzimidazole derivatives; nevertheless, their underlying biological mechanisms against breast cancer remain poorly defined. In the present work, three representative analogs were assessed in MCF-7 human breast cancer cells to explore whether the optimal lead candidate restrains tumor cell proliferation and migration via modulating apoptosis and cell-cycle progression. MTT and colony formation assays identified compound 1 as the most potent antiproliferative agent, with an IC50 of 6.53 μM in MCF-7 cells, which outperformed tamoxifen under identical experimental conditions. By contrast, compound 1 exhibited a markedly elevated IC50 of 15.19 μM in non-tumorigenic MCF10A human mammary epithelial cells, yielding a selectivity index of 2.33 toward MCF-7 cells. Furthermore, compound 1 suppressed colony formation and scratch wound closure in a concentration-dependent fashion. Morphological examination combined with Hoechst 33258 staining revealed hallmark apoptotic phenotypes, including cellular shrinkage, apoptotic body generation, as well as nuclear condensation and fragmentation. Annexin V-FITC/PI double-staining flow cytometry validated concentration-dependent apoptosis predominantly featured by early apoptotic populations, while PI cell cycle staining demonstrated prominent G1-phase cell cycle arrest. RT-qPCR quantification revealed that compound 1 treatment markedly upregulated the transcript levels of p53 and c-Myc yet downregulated MDM2, whereas the expression of ERα remained statistically unaltered. Collectively, these data demonstrate that compound 1 elicits antiproliferative and antimigratory activities in MCF-7 cells by triggering robust early apoptosis and G1-phase arrest, an effect mediated at least partially through regulation of the MDM2-p53 signaling axis. Taken together, these observations establish compound 1 as a promising antitumor lead scaffold for subsequent structural modification. Further investigations regarding its selectivity profile, systemic toxicity, pharmacokinetic properties, and in vivo therapeutic efficacy are therefore warranted.
