Función del mecanismo de doble diana en la regulación de proteínas periféricas de membrana

Abstract

OBJECTIVES: - Characterization of the hyperforin treatment of breast cancer cells in the presence and absence of PKCalpha expression. - Characterization of the salinomycin treatment of breast cancer cells in the presence and absence of PKCalpha expression. - Validation of the analysis of differential gene expression profiles of a model breast cancer cell line (MCF-7) in the presence and absence of PKCalpha. - Therapy design based on the molecular markers obtained in the analysis of differential gene expression. - Characterization the phosphorilation pathways affected under the control of PKCalpha on the breast cancer cell line MCF-7. - Characterization of the Rabphilin-3A and SNAP25 interaction on PC12 cells. MATERIALS AND METHODS: The different techniques required were: 1) Construction of expression plasmids, 2) Cell culture (MCF-7, MDA-MB-231, PC12), 3) Confocal microscopy and TEM, 4) Proliferation, Migration and Apoptosis measurements, 5) qPCR, 6) Proximity Ligation Assay (PLA), 7) Surface Plasmon Resonance, 8) siRNA interference, 9) Immunofluorescence, 10) Western-blot, 11) Image analysis (FIJI), 12) Human Phospho-Kinase Array. RESULTS We have determined the anti-proliferative and pro-apoptotic effect of hyperforin in MDA-MB-231 cells in the presence and absence of PKCalpha. Only the absence of PKCalpha reduced 20% the proliferation capacity of these cells. Hyperforin and the inhibition of PKCalpha together achieved a better effect with a lower concentration of hyperforin than in the presence of PKCalpha. We have studied the effect of salinomycin treatment with down-regulated PKCalpha on the proliferation capacity and on process like autophagy and apoptosis of two characteristics of malignant cancer cells, MCF-7 and MDA-MB-231 cells. The results obtained in proliferation assays determined a completed reduction of this capacity with 10 ?M of salinomycin in both breast cancer cell lines under the presence or the absence of PKCalpha expression. We validated these results by qPCR and designed a therapy that combines to inhibit PKCalpha expression and other of these PLCbeta4, PRKA, ERBB4 or PDGFR simultaneously. We studied this therapy through proliferation and migration capacity and the effect on apoptosis. The results obtained showed that BMS 599626 inhibitor, which is against ErbB proteins, might be a synergistic collaborator with PKCalpha to treat breast cancer. Another aspect studied was analyze the phosphorylation profile of kinases and their protein substrates and was essential for understanding how MCF-7 cells respond to changes in their environment like the inhibition of PKCalpha expression. We obtained a general reduction of the phosphorylation levels, STAT family and mTOR among others. Our group has resolved the crystal structures of Rph3A C2B-SNAP25 and C2B-PIP2 complexes in collaboration with the group of Dr. Verdaguer IBMB-CSIC, Barcelona. Biophysical and biochemical characterization performed in our group indicated that the bottom alpha-helix of C2B domain of rabphilin3A is crucial to interact with SNAP25, while the polybasic region is essential for the phosphoinositide-dependent interaction with the plasma membrane. The Raphilin3A and SNAP25 interactions were visualized at single-molecule resolution in an in situ proximity ligation assay (PLA) by a transient expression of the full-length HA-Rph3A and myc-SNAP25 in PC12 cells. Another aspect studied was the role in the membrane fusion of C2AB-Rph3A with small unilamellar vesicles (POPC/POPS/PIP2) and negative staining by transmission electron microscopy in a PIP2 and calcium dependent manner. CONCLUSIONS: 1. Hyperforin treatment shows an anti-proliferative and pro-apoptotic effect, and hyperforin also increases the synthesis of autophagic marker protein LC3B on MDA-MB-231 breast cancer cell line. 2. PKCalpha expression inhibition with hyperforin treatment achieves to completely reduce the proliferation rate of MDA-MB-231 cells. The hyperforin pro-apoptotic and pro-autophagic effect is independent of the presence and the absence of PKCalpha. 3. Salinomycin treatment inhibits the proliferation rate, to induce apoptosis and increase autophagic protein synthesis marker LC3B on MCF-7 and MDA-MB-231 cell lines. 4. PKCalpha down-regulation improves the salinomycin effect; this absence achieves better and faster results with a lower salinomycin dosis and this combination gives rise to a low toxicity treatment. 5. siRNA PKCalpha inhibition used for gen expression profile microarray of MCF-7 cells is validated by qPCR, and the down-regulation of EGFR and ITGB6 genes obtained in microarray analysis too. 6. The combination of PKCalpha down-regulation and specific inhibitors used against key up-regulated microarray genes (PLCbeta4, PKA, ErbB4 y PDGFR) obtained in the absence of PKCalpha, confirm our synergistic effect hypothesis on the proliferation rate, migration capacity and apoptosis in MCF-7 cells. 7. PKCalpha down-regulation reduce the relative levels of the phosphorilation protein families studied, mainly STAT protein family, FAK, mTOR, HSP60, PRAS4 and only two protein families are increased: ERK1/2 and p53. 8. We confirme residues implicated in the rabphilin3A and SNAP25 interaction. These residues belong to the alpha-helix C2B bottom face of rabphilin3A are K651A/K656A/K663A/H617A and E38A/D41A/R45A at N-terminal position of SNAP25. 9. The Rabphilin3A and SNAP25 interaction mechanism needs to bind PIP2 and this happen through C2AB of rabphilin3A. 10. We characterize in vitro rabphilin3A and SNAP25 interaction by surface plasmon resonance (SPR) measuring the dissociation constant of KD = 1 µM. 11. C2AB-rabphilin3A shows to be involved in the membrane phusion and curvature of the small unilamellar vesicules (SUV) in a PIP2/Ca2+-dependent manner by negative staining with transmission electron microscopy (TEM).