The invasion areas were measured with ImageJ. CCK8 assay Cells were plated in 96 well plates at a density of 5×103 cells per well. due to its ability to affect regulators of EMT such as Twist1. This result may facilitate the development of strategies that target Twist1 to treat metastatic breast cancer, as Twist1 is usually expressed at a high LCK (phospho-Ser59) antibody level in metastatic breast cancer cells but not in normal cells. Introduction In 2020, an estimated 276,480 new cases of invasive breast cancer are expected to be diagnosed in women in the U.S., along with 48,530 new cases of non-invasive (in situ) breast cancer . The majority of deaths from breast cancer are not due to the primary tumor itself, but are the result of metastasis to other parts of the body . Metastasis is a process comprised of a series of sequential steps, starting with local invasion of surrounding tissues by cells originating from the primary tumor and continuing until tumor cells invade and intravasate into blood or lymphatic vessels [3, 4]. At the DPA-714 initial stage of metastasis, invasive tumor cells first alter cell-to-cell adhesion and cell adhesion to the extracellular matrix (ECM). Proteins in the DPA-714 Cadherin family are important in mediating cell-to-cell adhesion and play a predominant role in breast cancer metastasis . E-Cadherin maintains cell-cell junctions and its down-regulation correlates with the development of metastatic breast cancer cells . On the other hand, N-Cadherin is closely associated with mesenchymal cells and related to epithelial-to-mesenchymal transition (EMT) during the gastrulation stage . Increasing evidence shows that EMT is associated with cancer progression [8, 9] by assisting invasion and intravasation into the bloodstream and inducing proteases involved in the degradation of the ECM . During EMT, cells undergo changes from an epithelial phenotype to a mesenchymal-like phenotype . EMT starts with the disintegration of cell-cell adhesion by loss of epithelial markers, such as E-Cadherin, and the expression of mesenchymal markers, such as Vimentin and N-Cadherin. Accordingly, the expression DPA-714 of transcriptional repressors of E-Cadherin, including zinc finger E-box-binding homeobox 1 (ZEB1), zinc finger E-box-binding homeobox 2 (ZEB2), twist-related protein (Twist), zinc finger protein, Snail, DPA-714 and Slug, is usually associated with poor prognosis in breast carcinoma . The Twist family of basic helix-loop-helix transcription factors, which includes Paraxis, Scleraxis, Hand1, Hand2, Twist1, and Twist2 , is usually involved in EMT . Both Twist1 and Twist2 function in the transcriptional regulation of developmental processes, but Twist1 is better studied and is a known activator of EMT in cancer cells . It promotes EMT by activating several target genes that promote cellular de-differentiation and cell mobility. In addition, Twist1 is also reported to promote the cancer stem cell phenotype, inhibit apoptosis, and contribute to chemotherapy resistance . Overexpression of Twist1 is usually common in metastatic carcinomas including in aggressive and metastatic forms of breast cancer [16C18]. As a grasp regulator of EMT in breast epithelial cells, Twist1 is usually a promising target for metastatic breast cancer therapy. What makes Twist1 a particularly attractive target is usually that it is rarely expressed in normal adult tissues . This makes it relatively safe to target Twist1 in the treatment of cancers such as metastatic breast cancer. Successful inactivation of Twist1 in cancer cells by siRNA or chemotherapeutic approaches has been reported [20C23], and inhibitors targeting either the upstream regulator or downstream effector of Twist1 signaling have also been identified for cancer therapy . Harmine is usually a beta-carboline alkaloid found in a variety of plants, such as the Middle Eastern herb harmal or Syrian rue (Peganum harmala) and the South American vine. It is reported to have cytotoxic activity against human tumor cell lines . In MDA-MB-231 breast cancer cells that overexpress breast cancer resistance protein (BCRP), harmine inhibits BCRP . Harmine is also reported to antagonize transcriptional coactivator with PDZ-binding motif (TAZ), suppress breast cancer cell proliferation and migration, and promote cancer cell apoptosis in vitro . Recently, in an unbiased screen, harmine was identified as the first pharmacologic inhibitor of Twist1 with significant anti-tumor activity in oncogene-driven lung cancer . Treatment with harmine causes Twist1 degradation and induces senescence or apoptosis in NSCLC cells . Given the importance of Twist1 in promoting EMT in breast carcinoma cells, in.