A Phytochemical Constituent, (E)-Methyl-Cinnamate Isolated from Alpinia katsumadai Hayata Suppresses Cell Survival, Migration, and Differentiation in Pre-Osteoblasts

Abstract

Background: (E)-methyl-cinnamate (EMC), a phytochemical constituent isolated from Alpinia katsumadai Hayata, is a natural flavor compound with anti-inflammatory properties, which is widely used in the food and commodity industry. However, the pharmacological effects of methyl-cinnamate on pre-osteoblasts remain unknown. This study aimed to investigate the pharmacological effects and mechanisms of EMC in pre-osteoblast MC3T3-E1 cells (pre-osteoblasts). Methods: Cell viability and apoptosis were evaluated using the MTT assay and TUNEL staining. Cell migration and osteoblast differentiation were examined using migration assays, as well as alkaline phosphatase activity and staining assays. Western blot analysis was used to examine intracellular signaling pathways and apoptotic proteins. Results: EMC decreased cell viability with morphological changes and increased apoptosis in pre-osteoblasts. EMC also induced the cleavage of Poly (ADP-ribose) polymerase (PARP) and caspase-3 and reduced the expression of anti-apoptotic proteins. In addition, EMC increased TUNEL-positive cells in pre-osteoblasts, decreased the activation of mitogen-activated protein kinases, and suppressed cell migration rate in pre-osteoblasts. Subsequently, EMC inhibited the osteoblast differentiation of pre-osteoblasts, as assessed by alkaline phosphatase staining and activity assays. Conclusion: These findings demonstrate that EMC has a pharmacological and biological role in cell survival, migration, and osteoblast differentiation. It suggests that EMC might be a potential phytomedicine for treating abnormalities of osteoblast function in bone diseases.

Effects of PIN on Osteoblast Differentiation and Matrix Mineralization through Runt-Related Transcription Factor

“…PIN (10 and 30 µM) was dissolved in 100% DMSO, diluted 1:1000 in final concentration, and 0.1% DMSO was used as the vehicle control. The medium was replaced every 2 days during the incubation period [ 46 ].…”

“…Cell migration was accessed using an in vitro wound healing assay [ 46 ]. Briefly, the cells were wound with a 200 μL pipette tip and cultured for 24 h at 37 °C in a humidified atmosphere of 5% CO 2 and 95% air.…”

“…Osteoblast differentiation was induced using OS containing 50 μg/mL L-AA and 10 mM β-GP with PIN (10 and 30 μM) for 7 days. The cell lysates were performed according to the manufacturer’s protocol using alkaline phosphatase activity colorimetric assay kit (Biovision, Milpitas, CA, USA) [ 46 ].…”

“…Cells were washed with 1 × PBS and then fixed in 10% formalin for 10 min at room temperature. After washing with distilled water, the cells were incubated with substrate solution for the reaction of ALP, followed according to the manufacturer’s protocol (Takara Bio Inc., Tokyo, Japan) as previously described [ 46 ].…”

Limonoid Triterpene, Obacunone Increases Runt-Related Transcription Factor 2 to Promote Osteoblast Differentiation and Function

“…Osteoblast differentiation was induced using osteogenic supplement medium (OS) containing 50 μg/mL L-ascorbic acid (L-AA) and 10 mM β-glycerophosphate (β-GP). The medium was replaced every 2 days during the incubation period as previously described [ 47 ].…”