Article Figures & Data
Figures
- Figure 1.
Heat stress potentiates apoptosis due to TRAIL, but not to chemotherapeutic agents, in malignant mesothelioma cells. A, heat stress (43°C for 60 min, recovery 60 min) enhances apoptosis due to TRAIL (1 or 2.5 ng/mL for 20 h) in M28 mesothelioma cells. Heat stress had no effect on apoptosis due to the ribotoxic agent anisomycin or to the DNA damaging agents etoposide, gemcitabine, or cisplatin. B, after activation of SPR by heat, the potentiation of apoptosis was maximal when TRAIL (1 ng/mL for 8 h) was added after a 1-h recovery and was still present after a 12-h recovery. C, in primary nonmalignant human mesothelial cells, heat stress (HS) had no effect on subsequent apoptosis due to TRAIL (1 ng/mL for 8 h). Columns, mean of at least three experiments done in triplicate; bars, SE. *, P < 0.05 versus TRAIL alone.
- Figure 2.
Role of PI3K/Akt in the potentiation of TRAIL-induced apoptosis by heat stress. A, the high baseline expression of p-Akt in M28 mesothelioma cells [phospho-Akt (Ser473)] is decreased at the end of heat stress. One representative experiment. Columns, mean of three experiments; bars, SE. *, P < 0.05 versus control. B, activation of SPR by heat also significantly inhibits the phosphorylation of p70S6K, a kinase downstream of Akt, measured at the end of heat stress. One representative experiment. Columns, mean of three experiments; bars, SE. *, P < 0.05 versus control. C, LY294002 (50 μmol/L for 1 h) significantly inhibits the high constitutive Akt phosphorylation in M28 mesothelioma cells. One representative experiment. Columns, mean of three experiments; bars, SE. *, P < 0.05 versus control. D, TRAIL alone (1 ng/mL for 8 h) induces minimal apoptosis in M28 mesothelioma cells. However, pretreatment with LY294002 (50 μmol/L for 1 h) or heat stress enhances the effect of TRAIL (1 ng/mL for 8 h). Columns, mean of three experiments done in triplicate; bars, SE. *, P < 0.05 versus TRAIL alone.
- Figure 3.
Heat stress and 17-AAG dissociate Hsp90 from PDK-1. A, M28 mesothelioma cells exposed to heat stress (43°C for 60 min, recovery 60 min) were subjected to lysis and immunoprecipitation (IP) with an antibody specific for PDK-1. The resultant immunoprecipitates were then examined via immunoblotting (IB) for Hsp90 and PDK-1. At baseline, PDK-1 associated with Hsp90. At 1 h after heat stress, the PDK-1 has mostly dissociated from Hsp90. In the absence of antibody, no nonspecific association is seen. One representative experiment of three. Columns, mean of at least three experiments; bars, SE. *, P < 0.05 versus no heat stress control. B, 17-AAG leads to a dissociation of Hsp90 from the PDK-1. M28 cells were exposed to 17-AAG (1 μg/mL for 1 h) and subjected to lysis and immunoprecipitation with an antibody specific for PDK-1. The resultant immunoprecipitates then were examined via immunoblotting for Hsp90 and PDK-1. One representative experiment. Columns, mean of three experiments; bars, SE. *, P < 0.05 versus controls. C, pretreatment with 17-AAG (2 μg/mL for 1 h) significantly decreases p-Akt. One representative experiment. Columns, mean of three experiments; bars, SE. *, P < 0.05 versus controls. D, TRAIL alone (1 ng/mL for 8 h) induces minimal apoptosis in M28 mesothelioma cells. However, 17-AAG (1 μg/mL) enhances the apoptosis due to TRAIL (1 ng/mL for 8 h). Columns, mean of three experiments done in triplicate; bars, SE. *, P < 0.05 versus TRAIL alone.
- Figure 4.
A constitutively active PDK-1 (CA-PDK-1) maintains Akt activity, thereby blocking the ability of heat stress or 17-AAG to potentiate TRAIL-induced apoptosis. A, M28 mesothelioma cells were subjected to mock transfection or transfection with a constitutively active PDK-1 plasmid. Forty-eight hours later, the cells were exposed to 17-AAG (1 μg/mL for 1 h). The active PDK-1 was able to prevent the decrease in p-Akt seen with 17-AAG. B, transfection of M28 cells with constitutively active PDK-1 prevented the ability of heat stress or 17-AAG (1 μg/mL for 8 h) to potentiate TRAIL-induced apoptosis. Columns, mean of three experiments done in triplicate; bars, SE. *, P < 0.05 versus TRAIL alone; **, P < 0.05 versus controls without constitutively active PDK-1.
- Figure 5.
Heat stress increases TRAIL-induced caspase 8 and Bid cleavage and decreases the expression of c-FLIP. A and B, M28 mesothelioma cells were exposed to heat stress (43°C for 60 min, recovery 60 min) followed by no treatment for 7 h or TRAIL (1 ng/mL) for 4 or 7 h. Heat stress led to an increase in TRAIL-induced caspase 8 cleavage as judged by an increased detection of the large 43-kDa fragment of caspase 8. In addition, heat stress induced an increase of the TRAIL-induced cleavage of Bid, as judged by a decrease in whole Bid. Actin was used as a standard to confirm equivalent protein loading. One representative experiment. Columns, mean of at least three experiments; bars, SE. *, P < 0.05 versus TRAIL alone. C, heat stress (43°C for 60 min, recovery 60 min) decreases expression of c-FLIP. One representative experiment. Columns, mean of at least three experiments; bars, SE. *, P < 0.05 versus controls.
- Figure 6.
Heat stress requires Bid for full potentiation of TRAIL-induced apoptosis. A, M28 mesothelioma cells were transfected with either random or Bid-specific small interfering RNAs, showing almost complete inhibition of Bid protein expression by Bid-specific small interfering RNA at 48 h after transfection. B, M28 cells transfected 48 h previously with Bid-specific or random small interfering RNA were exposed to heat stress and TRAIL. The absence of Bid inhibited the effect of heat stress on subsequent TRAIL-induced apoptosis. Columns, mean of at least three experiments done in triplicate; bars, SE. *, P < 0.05 versus cells transfected with the random small interfering RNA.
Volume 67, Issue 6
