Aberrant activation of Wnt signaling has been implicated in human osteosarcoma, which may provide a genetic vulnerability that can be targeted in osteosarcoma treatment. To test whether Wnt activation is necessary for osteosarcoma growth, colony formation, invasion, and metastasis, we treated human osteosarcoma cells with a small molecule inhibitor of Wnt/β-catenin, PRI-724, which suppresses Wnt/β-catenin-mediated transcription. We found increased protein levels of endogenous active-β-catenin in five human osteosarcoma cell lines. Treatment with PRI-724 was sufficient to inhibit human osteosarcoma 143B and SJSA-1 cell proliferation. Suppressed Wnt signaling was confirmed by decreased protein levels of the Wnt target Cyclin D1. Furthermore, we revealed significant inhibitory effects on cell migration, invasion, and colony formation in the human osteosarcoma cells. Using deposited data from next generation sequencing studies, we analyzed somatic mutations and gene expression of components in the Wnt/β-catenin pathway. We found somatic mutations and upregulated gene expression of many components in the Wnt/ β-catenin pathway, indicating activated Wnt signaling. Taken together, our results illustrate the critical role of Wnt/β-catenin signaling in human osteosarcoma pathogenesis and growth, as well as the therapeutic potential of Wnt inhibitors in the treatment of human osteosarcoma.

Key points This study demonstrates and evaluates the changes in rat vascular smooth muscle cell biomechanics following statin‐mediated cholesterol depletion. Evidence is presented to show correlated changes in migration and adhesion of vascular smooth muscle cells to extracellular matrix proteins fibronectin and collagen. Concurrently, integrin α5 expression was enhanced but not integrin α2. Atomic force microscopy analysis provides compelling evidence of coordinated reduction in vascular smooth muscle cell stiffness and actin cytoskeletal orientation in response to statin‐mediated cholesterol depletion. Proof is provided that statin‐mediated cholesterol depletion remodels total vascular smooth muscle cell cytoskeletal orientation that may additionally participate in altering ex vivo aortic vessel function. It is concluded that statin‐mediated cholesterol depletion may coordinate vascular smooth muscle cell migration and adhesion to different extracellular matrix proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell. Abstract Not only does cholesterol induce an inflammatory response and deposits in foam cells at the atherosclerotic plaque, it also regulates cellular mechanics, proliferation and migration in atherosclerosis progression. Statins are HMG‐CoA reductase inhibitors that are known to inhibit cellular cholesterol biosynthesis and are clinically prescribed to patients with hypercholesterolemia or related cardiovascular conditions. Nonetheless, the effect of statin‐mediated cholesterol management on cellular biomechanics is not fully understood. In this study, we aimed to assess the effect of fluvastatin‐mediated cholesterol management on primary rat vascular smooth muscle cell (VSMC) biomechanics. Real‐time measurement of cell adhesion, stiffness, and imaging were performed using atomic force microscopy (AFM). Cellular migration on extra cellular matrix (ECM) protein surfaces was studied by time‐lapse imaging. The effect of changes in VSMC biomechanics on aortic function was assessed using an ex vivo myograph system. Fluvastatin‐mediated cholesterol depletion (‐27.8%) lowered VSMC migration distance on a fibronectin (FN)‐coated surface (‐14.8%) but not on a type 1 collagen (COL1)‐coated surface. VSMC adhesion force to FN (+33%) and integrin α5 expression were enhanced but COL1 adhesion and integrin α2 expression were unchanged upon cholesterol depletion. In addition, VSMC stiffness (‐46.6%) and ex vivo aortic ring contraction force (‐40.1%) were lowered and VSMC actin cytoskeletal orientation was reduced (‐24.5%) following statin‐mediated cholesterol depletion. Altogether, it is concluded that statin‐mediated cholesterol depletion may coordinate VSMC migration and adhesion to different ECM proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell and aortic function.

Natural components continue to be an important source for the discovery and development of novel anticancer agents. Polymethoxyflavones are a class of flavonoids found in citrus fruits and medicinal plants used in traditional medicine. In the present study, the anticancer activity of the well-known nobiletin (5,6,7,8,3′,4′-hexamethoxyflavone) was compared against its less studied structural isomer 5,6,7,3′,4′,5′-hexamethoxyflavone. These compounds were evaluated on the Hs578T triple-negative breast cancer cell line and its more migratory subclone Hs578Ts(i)8. 5,6,7,3′,4′,5′-hexamethoxyflavone was found to be less toxic than nobiletin, while a similar growth inhibitory effect was observed after 72 h. Additionally, 5,6,7,3′,4′,5′-hexamethoxyflavone arrested the cell cycle in the G2/M phase, while no effect was observed on apoptosis or the migratory behavior of these cells. Furthermore, mechanistic studies revealed that the growth inhibition was concomitant with reduced phosphorylation levels of signaling molecules in the MAPK and Akt pathways as well as cell cycle regulators, involved in regulating cell proliferation, survival and cell cycle. In summary, the present study is the first to report on the anticancer activities of 5,6,7,3′,4′,5′-hexamethoxyflavone and to provide evidence that this flavone could have a greater potential than nobiletin for prevention or treatment of triple-negative breast cancer.