caa a22 4500 445797177 CHVBK 20180317145129.0 cr unu---uuuuu 170323e20110201xx s 000 0 eng 10.1007/s10620-010-1294-2 doi (NATIONALLICENCE)springer-10.1007/s10620-010-1294-2 Targeting PI3K/Akt/HSP90 Signaling Sensitizes Gastric Cancer Cells to Deoxycholate-Induced Apoptosis [Elektronische Daten] [Maria Redlak, Thomas Miller] Background: The heat shock protein 90 (HSP90) plays a crucial role in the stability of several proteins that are essential for cell survival and for malignant transformation. The binding of HSP90 with pro-survival kinase Akt prevents proteosomal degradation of Akt and contributes to the functional stabilization of PI3K/Akt signaling and cell survival. Akt kinase and HSP90 are therefore highly over-expressed in a large panel of cancer cell lines and are present in multi-chaperoning complexes. In this paper, we investigated whether targeting both Akt and HSP90 would inhibit the survival pathway in AGS cells (human gastric mucosal cells), and how Akt/HSP90 inhibition modulates the deoxycholate (DC)-induced apoptosis. Methods: AGS cells in the presence of Akt inhibitors (LY294002 and wortmannin), or HSP90 inhibitor (geldanamycin, GA) for 30min or 18h, respectively, were treated with DC (50µM). Activation of PI3K/Akt signaling was evaluated by measuring the Akt and PTEN phosphorylation. HSP90, caspase-3 and caspase-9 were detected in whole lysates by Western blot analysis. AGS cells, transiently transfected with Akt siRNA, were treated with DC, and apoptosis was measured by caspase-3 activation. Apoptotic-positive cells were counted according to changes of cell morphology by Hoechst staining and fluorescence microscopy. Results: The intrinsic level of phospho-Akt (pAkt; active form), phospho-PTEN (pPTEN; inactive enzyme) and HSP90 were highly expressed in AGS cells indicating the active PI3K/Akt/HSP90 signaling. Although, deoxycholate at low concentration (50µM) slightly inhibited the expression of pAkt and cleaved HSP90 to 55KDa fragment, no significant effect on apoptosis induction, up to 4h (as assessed by caspase-3 activation) was observed. The higher concentrations of DC (100µM-300µM) resulted in progressive inhibition of pAkt, activation of PTEN, and specific cleavage of HSP90 to approximately 45 KDa fragments with significant induction of apoptosis. Although DC (50µM) had no profound effect on Akt/HSP90 and did not induce apoptosis, it became an inducer of apoptosis when cells were pretreated with LY294002, wortmannin, or geldanamycin. Consistent with these findings, significant activation of apoptosis in response to DC (50µM) was observed in cells with depleted Akt protein. Conclusions: These results demonstrate that down-regulation of PI3K/Akt pathway with specific cleavage of HSP90 to 45KDa modulates the pro-apoptotic effects of DC in gastric cells. They further indicate the importance of stable Akt/HSP90 complex in regulation of survival/death responses. Springer Science+Business Media, LLC, 2010 Cell signaling nationallicence Heat shock proteins nationallicence Apoptosis nationallicence AGS cell line nationallicence Redlak Maria Department of Surgery, Medical College of Virginia Commonwealth University, 23298, Richmond, VA, USA aut Miller Thomas Department of Surgery, Medical College of Virginia Commonwealth University, 23298, Richmond, VA, USA aut Digestive Diseases and Sciences Springer US; http://www.springer-ny.com 56/2(2011-02-01), 323-329 0163-2116 56:2<323 2011 56 10620 https://doi.org/10.1007/s10620-010-1294-2 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s10620-010-1294-2 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Redlak Maria Department of Surgery, Medical College of Virginia Commonwealth University, 23298, Richmond, VA, USA aut NATIONALLICENCE 700 1- Miller Thomas Department of Surgery, Medical College of Virginia Commonwealth University, 23298, Richmond, VA, USA aut NATIONALLICENCE 773 0- Digestive Diseases and Sciences Springer US; http://www.springer-ny.com 56/2(2011-02-01), 323-329 0163-2116 56:2<323 2011 56 10620 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer