664 Molecular Mechanisms in Simvastatin-induced HCT116 Colorectal Cancer Cell Apoptosis

Abstract

Material and Methods: Mouse embryonic fibroblasts (MEFs) were isolated and cultured from E11-E13 embryos of control, Sos1-, Sos2or Sos1/2-null mice. To by-pass the lethality of embryos lacking Sos1, tamoxifen-induced Sos1-null mice were generated. Using our available Sos2-mutant mice, double knockout Sos1/Sos2 animals were also produced. Complete Sos1 depletion in MEFs was achieved after a nine day treatment of cultures with tamoxifen. To exclude tamoxifen off-target effects, control MEFs were also tamoxifen-treated. Cell signaling studies of our knockout MEFs were performed using PDGF or EGF as agonists. The ultrastructure of all experimental groups including control and Sos-deficient MEFs was examined by electron microscopy. Finally, flow cytometry analyses were employed to measure the level of oxidative stress in the MEFs. Results and Discussion: Light microscopy observations showed cytoplasmic swellings or inclusions in Sos1and double Sos1/Sos2-null MEFs. In contrast, tamoxifen-treated control MEFs did not exhibit these alterations. Ultrastructural analyses clearly demonstrated the occurrence of mitophagy in the cytoplasm of Sos1and Sos1/2-depleted MEFs, being more noticeable in the latest. Consistent with this, flow cytometry results seem to suggest an increase in the level of intracellular ROS (Reactive Oxidative Species) in mutant MEFs. Somewhat surprisingly, the absence of Sos proteins did not result in downregulation of Ras-MAPK signaling. In fact, quantitative measurements of RasMAPK activity in Sos-deficient MEFs showed unexpectedly enhanced levels in comparison to control fibroblasts. Conclusions: In conclusion, Sos proteins appear to be essential for normal homeostasis in MEFs. Additionally, the enhanced signal of Ras-MAPK pathway observed after depletion of Sos proteins suggests the occurrence of still undefined compensatory mechanisms. This work was supported by grants from FIS (PS09/01979), RTICC (RD06/0020/000), JCyL (SA044A08 and GR93), and Samuel SolorzanoBarruso Foundation.