Targeting PI3K/Akt/HSP90 signaling sensitizes gastric cancer cells to deoxycholate-induced apoptosis.
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 30 min or 18 h, respectively, were treated with DC (50 µM). 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 (50 µM) slightly inhibited the expression of pAkt and cleaved HSP90 to 55 KDa fragment, no significant effect on apoptosis induction, up to 4 h (as assessed by caspase-3 activation) was observed. The higher concentrations of DC (100 µM-300 µM) 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 (50 µM) 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 (50 µM) 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 45 KDa 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.
Redlak
Maria J
MJ
Department of Surgery, Medical College of Virginia Commonwealth University, Richmond, VA 23298, USA.
Miller
Thomas A
TA
D000730:Androstadienes / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D016227:Benzoquinones / Q000494:pharmacology; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002867:Chromones / Q000494:pharmacology; D003840:Deoxycholic Acid / Q000008:administration & dosage / Q000494:pharmacology*; D016503:Drug Delivery Systems; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D018841:HSP90 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047029:Lactams, Macrocyclic / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013274:Stomach Neoplasms; D000077191:Wortmannin
D000730:Androstadienes; D016227:Benzoquinones; D002867:Chromones; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D009025:Morpholines; D003840:Deoxycholic Acid; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D053148:Caspase 3; D053453:Caspase 9; D000077191:Wortmannin; C001277:geldanamycin