In this study, we investigated the part of Akt in intrinsic resistance to TRAIL, which is common in EOC cells. that TRAIL-induced caspase-8 activation was observed in both sensitive and resistant cell lines, Bid cleavage occurred only in sensitive cells or in SKOV3ip1 cells treated with LY294002. Bid manifestation was low in resistant cells and Akt activation downregulated its manifestation. Depletion of Bid by siRNA in OVCAR3 cells was associated with a decrease in TRAIL-mediated apoptosis. Overexpression of Bid only in SKOV3ip1 cells enhanced TRAIL-induced apoptosis. Simultaneous blockade of Akt pathway further improved TRAIL-induced apoptosis. Thus, Akt functions upstream of mitochondria and inhibits TRAIL-induced apoptosis by reducing Bid protein levels and possibly inhibiting its cleavage. (launch in OVCAR3 and SKOV3ip1 cells. Mitochondrial outer membrane permeabilization was assessed from the uptake of a lipophilic cationic dye where reddish fluorescence represents intact mitochondria membrane and green fluorescence represents apoptotic mitochondria. Treatment of OVCAR3 Ac2-26 cells with TRAIL, increased the number of green-labeled mitochondria (Number 5a) and consequently the percentage of apoptotic mitochondria as compared with SKOV3ip1 cells (Number 5b). Heavy membrane, enriched in mitochondria and cytosolic fractions, were isolated from OVCAR3 and SKOV3ip1 cells, after treatment with TRAIL. Cytochrome was recognized in the cytosol of OVCAR3 cells as early as 2?h after TRAIL treatment whereas cytochrome was not detected in SKOV3ip1 even after 8?h of TRAIL treatment (Number 5c). These results suggest the mitochondrial cell death pathway is definitely inhibited in resistant cells. Open in a separate window Number 5 Lack of mitochondrial activation in TRAIL-resistant cells. (a) OVCAR3 and SKOV3ip1 cells were cultured for 24?h without TRAIL and the mitochondrial membrane integrity was assessed using MitoLight apoptotic detection kit staining. In treated cells, new culture medium comprising TRAIL (100?ng/ml) was added for 5?h before subjected to MitoLight apoptotic detection kit staining. Only TRAIL-treated cells are demonstrated. The reddish fluorescence (remaining panels) represents dimeric dye that has accumulated in the intact mitochondria membrane representing non-apoptotic cells. The green fluorescence (middle panels) represents cytoplasmic swimming pools of monomeric-lipophilic-cationic dye indicating the the lack of ability of mitochondria to concentrate the dye and consequently shows apoptotic cells. Right panels represent overlays of remaining and middle panels. (b) Percentage of apoptotic mitochondria in OVCAR3 and SKOV3ip1 Ac2-26 cells during TRAIL treatment. (c) OVCAR3 and SKOV3ip1 cells were treated with TRAIL for different times and levels of cytochrome in cytosolic (C) and membrane (M) fractions were determined by western blot. COX IV was used like a mitochondrial marker and loading control. tBid is not recognized in resistant cells TRAIL-induced Bid cleavage generates a truncated form of Bid (tBid) that promotes the insertion of Bax into the mitochondrial outer membrane. As demonstrated in Number 6a, TRAIL (100?ng/ml) treatment resulted in a reduction in full-length Bid and the appearance of tBid overtime in sensitive cells but not in resistant cells suggesting that Akt interfere with caspase-8-mediated Bid cleavage. To further support this observation, SKOV3ip1 and COV2 cells were pre-incubated with “type”:”entrez-nucleotide”,”attrs”:”text”:”LY204002″,”term_id”:”1257488338″,”term_text”:”LY204002″LY204002 (5?) in the presence or absence of TRAIL. When TRAIL was combined with LY294002, there was a reduction of full-length Bid, but we did not detect tBid presumably, Rabbit Polyclonal to CRABP2 because the levels of tBid are too low to be recognized by immunoblot (Number 6b). Overexpression of Akt1 in CaOV3 cells prevented TRAIL-induced Bid cleavage (Number 6c). These results suggest that Akt inhibits TRAIL-induced activation of the mitochondrial cell death pathway by preventing the build up of tBid at levels adequate to induce apoptosis. Open in a separate window Number 6 Effect of Akt on Bid cleavage. (a) Immunoblot analysis for the assessment of Bid cleavage. Sensitive and resistant cells were treated with TRAIL (100?ng/ml) for various instances and Bid cleavage was determined by the decrease of full-length Bid protein and the appearance of tBid about european blot using anti-Bid antibodies. (b) TRAIL-resistant SKOV3ip1 and COV2 cells incubated with LY294002 (5?) or remaining untreated for 1?h before adding TRAIL for 8?h. Cell lysates were analyzed by western blotting using the indicated antibodies. (c) CaOV3 cells expressing the bare vector (CaOV3-EV) or Akt1 (CaOV3-Akt1) were treated with TRAIL for 8?h. Cell lysates were analyzed as explained above. Tubulin was Ac2-26 used to ensure equivalent loading. Akt activation decreases Bid protein levels We compared levels of Bid protein in our TRAIL-sensitive and -resistant cell lines..