Caspase-9 is traditionally considered the initiator caspase of the intrinsic apoptotic pathway. In the past decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial, and endosomal/lysosomal homeostasis. As previous studies revealed nonapoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts. Data-independent acquisition-parallel accumulation serial fragmentation (diaPASEF) proteomics was used to compare protein profiles of control and caspase-9 knockout cells. A total of 7669 protein groups were quantified, and 283 upregulated/141 downregulated protein groups were associated with the caspase-9 knockout phenotype. The deregulated proteins were mainly enriched for those associated with cell migration and motility and DNA replication/repair. Altered migration was confirmed in MC3T3-E1 cells with the genetic and pharmacological inhibition of caspase-9. ABHD2, an established regulator of cell migration, was identified as a possible substrate of caspase-9. We conclude that caspase-9 acts as a modulator of osteoblastic MC3T3-E1 cell migration and, therefore, may be involved in bone remodeling and fracture repair.

• Keywords
• ABHD2, Caspase 9, diaPASEF, migration, osteoblasts, proteomics,
• MeSH
• Cell Line MeSH
• Gene Knockout Techniques MeSH
• Caspase 9 * metabolism   genetics   MeSH
• Mice MeSH
• Osteoblasts * metabolism   cytology   MeSH
• Cell Movement * MeSH
• Proteomics * methods   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Casp9 protein, mouse MeSH Browser
• Caspase 9 * MeSH

Wedelolactone is a multi-target natural plant coumestan exhibiting cytotoxicity towards cancer cells. Although several molecular targets of wedelolactone have been recognized, the molecular mechanism of its cytotoxicity has not yet been elucidated. In this study, we show that wedelolactone acts as an inhibitor of chymotrypsin-like, trypsin-like, and caspase-like activities of proteasome in breast cancer cells. The proteasome inhibitory effect of wedelolactone was documented by (i) reduced cleavage of fluorogenic proteasome substrates; (ii) accumulation of polyubiquitinated proteins and proteins with rapid turnover in tumor cells; and (iii) molecular docking of wedelolactone into the active sites of proteasome catalytic subunits. Inhibition of proteasome by wedelolactone was independent on its ability to induce reactive oxygen species production by redox cycling with copper ions, suggesting that wedelolactone acts as copper-independent proteasome inhibitor. We conclude that the cytotoxicity of wedelolactone to breast cancer cells is partially mediated by targeting proteasomal protein degradation pathway. Understanding the structural basis for inhibitory mode of wedelolactone might help to open up new avenues for design of novel compounds efficiently inhibiting cancer cells.

• Keywords
• breast cancer, copper, proteasome, reactive oxygen species, wedelolactone,
• MeSH
• Proteasome Inhibitors chemistry   pharmacology   toxicity   MeSH
• Coumarins chemistry   pharmacology   toxicity   MeSH
• Humans MeSH
• Copper metabolism   MeSH
• Cell Line, Tumor MeSH
• Breast Neoplasms metabolism   MeSH
• Proteasome Endopeptidase Complex chemistry   metabolism   MeSH
• Proteolysis MeSH
• Reactive Oxygen Species metabolism   MeSH
• Molecular Docking Simulation MeSH
• Ubiquitination MeSH
• Protein Binding MeSH
• Cell Survival drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Proteasome Inhibitors MeSH
• Coumarins MeSH
• Copper MeSH
• Proteasome Endopeptidase Complex MeSH
• Reactive Oxygen Species MeSH
• wedelolactone MeSH Browser