BACKGROUND: The differential sensitivity of tumor cells to TRAIL-induced apoptosis may be mediated by different intracellular inhibitors of apoptosis, and only a few reports have described the pathway(s) that are activated in response to TRAIL in prostate cells. METHODS: LNCaP was transfected with a dominant-negative form of FADD (FADD-DN) and cells were selected in the presence of hygromycin. Cell viability was estimated by calcein assay. Apoptosis was estimated by caspase activation using both fluorogenic substrates and Western blot analysis of activated caspases. To detect cytochrome c release, mitochondria-free cytosol was prepared and Western blot analysis was performed. RESULTS: LNCaP is resistant to TRAIL but TRAIL transiently induces DEVDase activity and activation of caspase-8; caspase-2, -3, -7, and -9 were not activated. Wortmannin, an inhibitor of the PI3K/Akt pathway, converted the phenotype of LNCaP from TRAIL-resistant to -sensitive. In the presence of wortmannin TRAIL induced activation of caspase-2, -3, -7, -8, and -9, as well as dissipation of mitochondrial transmembrane potential and release of cyto-chrome c from mitochondria into the cytosol. In addition, combined TRAIL and wortmannin treatment resulted in cleavage of several proteins: PARP, Akt, p21/WAF1, and MDM2 as well as dephosphorylation of Akt. The proteolysis of p21/WAFI and Akt, which are known survival factors, presumably amplify the apoptotic cascade in LNCaP. Transfection of FADD-DN in LNCaP resulted in inhibition of caspase activation as well as in resistance to combined treatment with TRAIL and wortmannin. CONCLUSIONS: These results suggest that caspase-8 activation is necessary but not sufficient for TRAIL-mediated apoptosis and is presumably blocked downstream of caspase-8 by the PI3K/Akt pathway. Copyright 2002 Wiley-Liss, Inc.
BACKGROUND: The differential sensitivity of tumor cells to TRAIL-induced apoptosis may be mediated by different intracellular inhibitors of apoptosis, and only a few reports have described the pathway(s) that are activated in response to TRAIL in prostate cells. METHODS: LNCaP was transfected with a dominant-negative form of FADD (FADD-DN) and cells were selected in the presence of hygromycin. Cell viability was estimated by calcein assay. Apoptosis was estimated by caspase activation using both fluorogenic substrates and Western blot analysis of activated caspases. To detect cytochrome c release, mitochondria-free cytosol was prepared and Western blot analysis was performed. RESULTS: LNCaP is resistant to TRAIL but TRAIL transiently induces DEVDase activity and activation of caspase-8; caspase-2, -3, -7, and -9 were not activated. Wortmannin, an inhibitor of the PI3K/Akt pathway, converted the phenotype of LNCaP from TRAIL-resistant to -sensitive. In the presence of wortmanninTRAIL induced activation of caspase-2, -3, -7, -8, and -9, as well as dissipation of mitochondrial transmembrane potential and release of cyto-chrome c from mitochondria into the cytosol. In addition, combined TRAIL and wortmannin treatment resulted in cleavage of several proteins: PARP, Akt, p21/WAF1, and MDM2 as well as dephosphorylation of Akt. The proteolysis of p21/WAFI and Akt, which are known survival factors, presumably amplify the apoptotic cascade in LNCaP. Transfection of FADD-DN in LNCaP resulted in inhibition of caspase activation as well as in resistance to combined treatment with TRAIL and wortmannin. CONCLUSIONS: These results suggest that caspase-8 activation is necessary but not sufficient for TRAIL-mediated apoptosis and is presumably blocked downstream of caspase-8 by the PI3K/Akt pathway. Copyright 2002 Wiley-Liss, Inc.
Authors: Xinwen Yang; Jianhua Wang; Cunren Liu; William E Grizzle; Shaohua Yu; Shuangqin Zhang; Stephen Barnes; William J Koopman; John D Mountz; Robert P Kimberly; Huang-Ge Zhang Journal: Am J Pathol Date: 2005-09 Impact factor: 4.307
Authors: Stanley F Barnett; Deborah Defeo-Jones; Sheng Fu; Paula J Hancock; Kathleen M Haskell; Raymond E Jones; Jason A Kahana; Astrid M Kral; Karen Leander; Ling L Lee; John Malinowski; Elizabeth M McAvoy; Debbie D Nahas; Ronald G Robinson; Hans E Huber Journal: Biochem J Date: 2005-01-15 Impact factor: 3.857
Authors: Joseph E Aslan; Huihong You; Danielle M Williamson; Jessica Endig; Robert T Youker; Laurel Thomas; Hongjun Shu; Yuhong Du; Robert L Milewski; Matthew H Brush; Anthony Possemato; Kam Sprott; Haian Fu; Kenneth D Greis; Douglas N Runckel; Arndt Vogel; Gary Thomas Journal: Mol Cell Date: 2009-05-14 Impact factor: 17.970