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Abstract Background Medicinal plants represent alternative means for the treatment of several chronic diseases, including inflammation. The genus Ranunculus, a representative of the Ranunculaceae family, has been reported to possess anti-inflammatory, analgesic, antiviral, antibacterial, antiparasitic and antifungal activities, possibly due to the presence of anemonin and other. Different studies have shown the occurrence of unusual fatty acids (FAs) in Ranunculaceae; however, their therapeutic role has not been investigated. The purpose of this study is to characterize potential anti-inflammatory bioactivities in Ranunculus constantinopolitanus D'Urv., traditionally used in Eastern Mediterranean folk medicine. Methods The aerial part of R. constantinopolitanus was subjected to methanol (MeOH) extraction and solvent fractionation. The bioactive fraction (I.2) was further fractionated using column chromatography, and the biologically active subfraction (Y2+3) was identified using infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). The effects of I.2 and Y2+3 on cell viability were studied in mouse mammary epithelial SCp2 cells using trypan blue exclusion method. To study the anti-inflammatory activities of I.2 and Y2+3, their ability to reduce interleukin (IL)-6 levels was assessed in endotoxin (ET)-stimulated SCp2 cells using enzyme-linked immunosorbent assay (ELISA). In addition, the ability of Y2+3 to reduce cyclooxygenase (COX)-2 expression was studied in IL-1-treated mouse intestinal epithelial Mode-K cells via western blotting. Data were analyzed by one-way analysis of variance (ANOVA), Student-Newman-Keuls (SNK), Tukey HSD, two-sample t-test and Dunnett t-tests for multiple comparisons. Results The chloroform fraction (I.2) derived from crude MeOH extract of the plant, in addition to Y2+3, a FA mix isolated from this fraction and containing palmitic acid, C18:2 and C18:1 isomers and stearic acid (1:5:8:1 ratio), reduced ET-induced IL-6 levels in SCp2 cells without affecting cell viability or morphology. When compared to fish oil, conjugated linoleic acid (CLA) and to individual FAs as palmitic, linoleic, oleic and stearic acid or to a mix of these FAs (1:5:8:1 ratio), Y2+3 exhibited higher potency in reducing ET-induced IL-6 levels within a shorter period of time. Y2+3 also reduced COX-2 expression in IL-1-treated Mode-K cells. Conclusion Our studies demonstrate the existence of potential anti-inflammatory bioactivities in R. constantinopolitanus and attribute them to a FA mix in this plant.

microRNAs (miRNAs) serve as novel noninvasive cancer biomarkers. In an HMT-3522 S1 (S1) breast epithelial risk-progression three-dimensional (3D) culture model, non-neoplastic S1 cells form a fully polarized epithelium. When silenced for the gap junction and tumor suppressor Cx43, Cx43-KO-S1 cells recapitulate pre-neoplastic phenotypes observed in tissues at risk for breast cancer in vivo. To delineate the role of miRNAs in breast tumorigenesis and identify key miRNA players in breast epithelial polarity, the miRNA profile specific to Cx43 loss in Cx43-KO-S1 compared to S1 cells was sequenced, revealing 65 differentially expressed miRNAs. A comparative analysis was conducted between these miRNAs and tumor-associated miRNAs from a young Lebanese patient validation cohort. miR-183-5p, downstream of Cx43 loss, was commonly upregulated in the patient cohort and the 3D culture model. miR-492, not attributed to Cx43 loss, was only specifically up-regulated in the young Lebanese patients. Ectopic expression of either miR-183-5p or miR-492 in S1 cells, through pLenti-III-miR-GPF vectors, resulted in the formation of larger multi-layered acini devoid of lumen, with disrupted epithelial polarity, as shown by an altered localization of Cx43, ß-catenin and Scrib, and decreased nuclear circularity in 3D cultures. Enhanced proliferation and invasion capacity were also observed. Over-expression of miR-183-5p or miR-492, therefore, induces pre-neoplastic phenotypes similar to those reported upon Cx43 loss, and may act as oncomiRs and possible biomarkers of increased breast cancer risk., (© 2022. The Author(s).)

(1) Background: The expression of connexin 43 (Cx43) is disrupted in breast cancer, and re-expression of this protein in human breast cancer cell lines leads to decreased proliferation and invasiveness, suggesting a tumor suppressive role. This study aims to investigate the role of Cx43 in proliferation and invasion starting from non-neoplastic breast epithelium. (2) Methods: Nontumorigenic human mammary epithelial HMT-3522 S1 cells and Cx43 shRNA-transfected counterparts were cultured under 2-dimensional (2-D) and 3-D conditions. (3) Results: Silencing Cx43 induced mislocalization of β-catenin and Scrib from apicolateral membrane domains in glandular structures or acini formed in 3-D culture, suggesting the loss of apical polarity. Cell cycle entry and proliferation were enhanced, concomitantly with c-Myc and cyclin D1 upregulation, while no detectable activation of Wnt/β-catenin signaling was observed. Motility and invasion were also triggered and were associated with altered acinar morphology and activation of ERK1/2 and Rho GTPase signaling, which acts downstream of the noncanonical Wnt pathway. The invasion of Cx43-shRNA S1 cells was observed only under permissive stiffness of the extracellular matrix (ECM). (4) Conclusion: Our results suggest that Cx43 controls proliferation and invasion in the normal mammary epithelium in part by regulating noncanonical Wnt signaling., Competing Interests: The authors declare no conflict of interest.

Despite recent advances in chemotherapy, aggressive and metastatic breast cancers remain refractory to targeted therapy and the development of novel drugs is urgently needed. Retinoids are crucial regulators of cellular proliferation, differentiation, and cell death, and have shown potent chemotherapeutic and chemopreventive properties. The major drawback of the use of all-trans retinoic acid (ATRA) in cancer therapy is disease relapse. Therefore, synthetic retinoids, specifically ST1926, have emerged as potent anticancer agents. Given the importance of the microenvironment in modulating the response of cancer cells to chemotherapeutic drugs, we investigated the antitumor activities of ST1926 in two-dimensional (2D) and different three-dimensional (3D) human breast cancer models and compared them with ATRA. We have shown that in 2D cell culture models, ATRA-resistant MCF-7 and MDA-MB-231 cells were sensitive to ST1926 at submicromolar concentrations that spared the 'normal-like' breast epithelial cells. ST1926 induced apoptosis and S-phase arrest, caused DNA damage, and downregulated the Wnt/β-catenin pathway in breast cancer cells in 2D and 3D cell culture models. ST1926-mediated growth inhibition was independent of the retinoid receptor-signaling pathway. Long-term treatments with low submicromolar ST1926 concentrations reduced the anchorage-independent growth and decreased the sphere-forming ability of breast cancer progenitor cells in the sphere formation assay. Furthermore, ST1926 potently induced cell death of breast cancer cells under 3D conditions and spared the lumen-forming ability of normal-like breast epithelial cells. In tested 3D models, ATRA had minimal effects on the growth of breast cancer cells compared with ST1926. In summary, our results highlight the therapeutic potential of ST1926 in breast cancer and warrant its further clinical development.