Inhibition of Phosphatidylinositol 3′-Kinase/AKT Signaling Promotes Apoptosis of Primary Effusion Lymphoma Cells

Abstract

Purpose: Phosphatidylinositol 3V -kinase (PI3V -kinase) can be activated by the K1 protein of Kaposi sarcoma^associated herpes virus (KSHV). However, the role of PI3V -kinase in KSHVassociated primary effusion lymphoma (PEL) is not known. To assess this, we studied survival and apoptosis in PEL cell lines following inhibition of PI3V -kinase. Experimental Design: Constitutive activation of several targets of PI3-kinase and apoptotic proteins were determined by Western blot analysis using specific antibodies.We used LY294002 to block PI3V -kinase/AKTactivation and assess apoptosis by flow cytometric analysis. Results: Blocking PI3V -kinase induced apoptosis in PEL cells, including BC1, BC3, BCBL1, and HBL6, whereas BCP1was refractory to LY294002-induced apoptosis. LY294002-induced apoptosis did not seem to involve Fas/Fas-L but had an additive effect to CH11-mediated apoptosis. We also show that AKT/PKB is constitutively activated in all PELs and treatment with LY294002 causes complete dephosphorylation in all cell lines except BCP1where a residual AKT phosphorylation remained after 24 hours of treatment. FKHR and GSK3 were also constitutively phosphorylated in PELs and treatment with LY294002 caused their dephosphorylation. Although inhibition of PI3V -kinase induced cleavage of BID in all cell lines, cytochrome c was released from the mitochondria and caspase-9 and caspase-3 were activated in LY294002-induced apoptotic BC1but not in resistant BCP1. Similarly, XIAP, a target of AKT, was down-regulated after LY294002 treatment only in sensitive PEL cells. Conclusions: Our data show that the PI3V -kinase pathway plays a major role in survival of PEL cells and suggest that this cascade may be a promising target for therapeutic intervention in primary effusion lymphomas.

HGF/c-Met pathway has a prominent role in mediating antiapoptotic signals through AKT in epithelial ovarian carcinoma

“…Cells were then washed twice with PBS and resuspended in 500 ml of mitochondrial incubation buffer, and mitochondrial membrane potential (% of green and red aggregates) was determined by flow cytometry as described previously. 18,33 Assays for Cytochrome c Release The release of cytochrome c from the mitochondria was assayed as described earlier. 18,33 Briefly, cells were treated with and without PHA665752 as described in figure legends, harvested, and resuspended in hypotonic buffer.…”

“…18,33 Assays for Cytochrome c Release The release of cytochrome c from the mitochondria was assayed as described earlier. 18,33 Briefly, cells were treated with and without PHA665752 as described in figure legends, harvested, and resuspended in hypotonic buffer. Cells were homogenized and cytosolic fraction was isolated by differential centrifugation and 20 mg of protein from cytosolic fraction of each sample was analyzed by immunoblotting using an anti-cytochrome c antibody.…”

“…16 There is strong evidence for a functional relationship between c-Met and XIAP through AKT in cancer cells: XIAP expression is found to be regulated by HGF/c-Met through AKT activity in several cancers. [17][18][19] In this study, we assessed the prevalence of c-Met protein expression, its relation to activated AKT, and its downstream antiapoptotic targets such as XIAP and Bcl-XL in a large cohort of Saudi epithelial ovarian carcinomas (EOCs) using tissue microarray (TMA) technology. We next examined the effect of c-Met inhibition on the EOC cell growth both in vitro and in vivo.…”

Curcumin suppresses constitutive activation of nuclear factor-κB and requires functional Bax to induce apoptosis in Burkitt's lymphoma cell lines

“…Cells were harvested and percentage apoptosis was measured by flow cytometry after staining with fluorescein-conjugated Annexin V and propidium iodide (PI; Molecular Probes) as described previously (25). For cell cycle analysis, cells were washed once with PBS and resuspended in 500 AL hypotonic staining buffer and analyzed by flow cytometry as described previously (26,27).…”

Primary Effusion Lymphoma Diagnosed by Pericardiocentesis

“…Two potential experimental therapies are bortezomib, a proteasome inhibitor, and drugs such as LY294002, which inhibit the phosphatidylinositol 3′-kinase/AKT pathway. Both therapies have been shown to induce apoptosis in PEL cell lines (25,26). Matta demonstrated that the activity of bortezomib was associated with inhibition of nuclear factor kappa-B pathways, upregulation of p53, p21, and p27, and activation of the caspase cascade (27).…”