Effects of the amide alkaloid piperyline on apoptosis, autophagy, and differentiation of pre-osteoblasts.

Background: Amide alkaloidsare typical constituents in plants of the Piperaceae family. Most of the pharmacological properties of Piper nigrum L. are attributed to the major amide alkaloid, piperine. Piperyline (PIPE) is a further amide alkaloid that has been isolated from P. nigrum. Hypothesis/Purpose: This study was performed to examine the biological effects of PIPE on pre-osteoblasts and elucidate the underlying mechanisms. Study Design: We investigated the effects of PIPE in MC3T3E-1 cells, which are widely used for studying osteoblast behavior in in vitro cell systems. Results: We found that at concentrations ranging from 1 to 30 mu M, PIPE inhibited cell growth and induced apoptosis in pre-osteoblasts, which was accompanied by the upregulation of apoptotic proteins but downregulation of anti-apoptotic proteins. In contrast, PIPE had no appreciable effect on the autophagy pathway. Nevertheless, PIPE reduced cell adhesion and migration via the inactivation of non-receptor tyrosine kinase (Src)/focal adhesion kinase (FAK) and mitogen-activated protein kinases, and also promoted the downregulation of matrix metalloproteinase 2 and 9 levels. Furthermore, at concentrations of 10 and 30 mu M, PIPE suppressed osteoblast differentiation, as indicated by reductions in alkaline phosphatase staining and activity. In addition, PIPE reduced the protein levels of phospho-Smad1/5/8 and runt-related transcription factor 2, and the mRNA levels of osteopontin, alkaline phosphatase, and osteocalcin. Conclusion: The findings of this study indicate that PIPE has biological effects associated with cell adhesion, migration, proliferation, and osteoblast differentiation, and suggest a potential role for this alkaloid in the treatment of bone diseases.