Origanum syriacum L. Attenuates the Malignant Phenotype of MDA-MB231 Breast Cancer Cells.

Breast cancer is the leading cause of cancer-related deaths among women. Among breast cancer types, triple negative breast cancer (TNBC) is the most aggressive, and is resistant to hormonal and chemotherapeutic treatments. As such, alternative approaches that may provide some benefit in fighting this debilitating pathology are critically needed; hence the utilization of herbal medicine. Origanum syriacum L., one of the most regularly consumed plants in the Mediterranean region, exhibits antiproliferative effect on several cancer cell lines. However, whether this herb modulates the malignant phenotype of TNBC remains poorly investigated. Here, we show that in MDA-MB-231, a TNBC cell line, Origanum syriacum L. aqueous extract (OSE) inhibited cellular viability, induced autophagy determined by the accumulation of lipidized LC3 II, and triggered apoptosis. We also show that OSE significantly promoted homotypic cell-cell adhesion while it decreased cellular migration, adhesion to fibronectin, and invasion of MDA-MB-231 cells. This was supported by decreased activity of focal adhesion kinase (FAK), reduced α2 integrin expression, and downregulation of secreted PgE2, MMP2 and MMP-9, in OSE-treated cells. Finally, we also show that OSE significantly inhibited angiogenesis and downregulated the level of nitric oxide (NO) production. Our findings demonstrate the ability of OSE to attenuate the malignant phenotype of the MDA-MB-231 cells, thus presenting Origanum syriacum L. as a promising potential source for therapeutic compounds for TNBC.

Production of isoform-specific knockdown/knockout Madin-Darby canine kidney epithelial cells using CRISPR/Cas9.

CRISPR-Cas9 gene editing has made it possible to specifically edit genes in a myriad of target cells. Here, a method for isoform-specific editing and clonal selection in Madin-Darby canine kidney (MDCK) epithelial cells is described in detail. This approach was used to address a long-standing question in virology of how adenovirus enters polarized epithelia from the apical surface. Our method relies on selecting two sgRNA sequences, cloning them into a suitable fluorescently labeled Cas9 vector system, and subsequently transfecting our MDCK epithelium and selecting isoform-specific Coxsackievirus and adenovirus receptor knockout clones. Utilization of this method is readily applicable to many other genetic targets in epithelial cells.•Simultaneous utilization of an sgRNA upstream and an sgRNA downstream of a target sequence allows for deletion of the intervening sequence, including whole exons.•Sorting of cells positive for fluorescent marker gene expression enhances the identification of partial and biallelic gene knockout.•PCR screening allows relatively fast and efficient determination of isoform-specific deletion.

Isoform specific editing of the coxsackievirus and adenovirus receptor.

The Coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and cell adhesion protein. CAR has two transmembrane isoforms that localize distinctly in polarized epithelial cells. Whereas the seven exon-encoded isoform (CAREx7) exhibits basolateral localization, the eight exon-encoded isoform (CAREx8) can localize to the apical epithelial surface where it can mediate luminal adenovirus infection. To further understand the distinct biological functions of these two isoforms, CRISPR/Cas9 genomic editing was used to specifically delete the eighth exon of the CXADR gene in a Madine Darby Canine Kidney (MDCK) cell line with a stably integrated lentiviral doxycycline-inducible CAREx8 cDNA. The gene-edited clone demonstrated a significant reduction in adenovirus susceptibility when both partially and fully polarized, and doxycycline-induction of CAREx8 restored sensitivity to adenovirus. These data reinforce the importance of CAREx8 in apical adenovirus infection and provide a new model cell line to probe isoform specific biological functions of CAR.