ABSTRACT
Protosappanin-A (PrA) and oleanolic acid (OA), which are important effective ingredients isolated from Caesalpinia sappan L., exhibit therapeutic potential in multiple diseases. This study focused on exploring the mechanisms of PrA and OA function in podocyte injury. An in vitro model of podocyte injury was induced by the sC5b-9 complex and assays such as cell viability, apoptosis, immunofluorescence, quantitative real-time polymerase chain reaction, and western blot were performed to further investigate the effects and mechanisms of PrA and OA in podocyte injury. The models of podocyte injury were verified to be successful as seen through significantly decreased levels of nephrin, podocin, and CD2AP and increased level of desmin. The sC5b-9-induced podocyte apoptosis was inhibited in injured podocytes treated with PrA and OA, accompanied by increased protein levels of nephrin, podocin, CD2AP, and Bcl2 and decreased levels of desmin and Bax. The p-AKT/p-mTOR levels were also reduced by treatment of PrA and OA while AKT/mTOR was unaltered. Further, the effects of PrA and OA on injured podocytes were similar to that of LY294002 (a PI3K-AKT inhibitor). PrA and OA were also seen to inhibit podocyte apoptosis and p-AKT/p-mTOR levels induced by IGF-1 (a PI3K-AKT activator). Our data demonstrate that PrA and OA can protect podocytes from injury or apoptosis, which may occur through inhibition of the abnormal activation of AKT-mTOR signaling.
ABSTRACT
Secreted frizzledrelated protein 1 (sFRP1) is an inhibitor of canonical Wnt signaling; however, previous studies have determined a tumorpromoting function of sFRP1 in a number of different cancer types. A previous study demonstrated that sFRP1 overexpression was associated with an aggressive phenotype and the activation of transforming growth factor ß (TGFß) signaling. sFRP1 overexpression and sFRP1 knockdown cell models were established. Immunoblotting was conducted to examine the protein levels of the associated molecules. Immunofluorescence staining followed by confocal microscopy was performed to visualize the cytoskeleton alterations and subcellular localization of key proteins. sFRP1 overexpression restored glycogen synthase kinase 3ß (GSK3ß) activity, which activated Rac family small GTPase 1 (Rac1). GSK3ß and Rac1 mediated the effect of sFRP1 on the positive regulation of cell growth and migration/invasion. Inhibition of GSK3ß or Rac1 abolished the regulation of sFRP1 on TGFß/SMAD family member 3 (Smad3) signaling and the aggressive phenotype; however, GSK3ß or Rac1 overexpression increased cell migration/invasion and restrained Smad3 activity by preventing its nuclear translocation and limiting its transcriptional activity. The present study demonstrated a tumorpromoting function of sFRP1overexpression by selectively activating TGFß signaling in gastric cancer cells. GSK3ß and Rac1 serve an important function in mediating the sFRP1induced malignant alterations and signaling changes.
