c-Jun NH2-terminal kinase-dependent upregulation of DR5 mediates cooperative induction of apoptosis by perifosine and TRAIL

Abstract

Background: Perifosine, an alkylphospholipid tested in phase II clinical trials, modulates the extrinsic apoptotic pathway and cooperates with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to augment apoptosis. The current study focuses on revealing the mechanisms by which perifosine enhances TRAIL-induced apoptosis.

Results: The combination of perifosine and TRAIL was more active than each single agent alone in inducing apoptosis of head and neck squamous cell carcinoma cells and inhibiting the growth of xenografts. Interestingly, perifosine primarily increased cell surface levels of DR5 although it elevated the expression of both DR4 and DR5. Blockade of DR5, but not DR4 upregulation, via small interfering RNA (siRNA) inhibited perifosine/TRAIL-induced apoptosis. Perifosine increased phosphorylated c-Jun NH2-terminal kinase (JNK) and c-Jun levels, which were paralleled with DR4 and DR5 induction. However, only DR5 upregulaiton induced by perifosine could be abrogated by both the JNK inhibitor SP600125 and JNK siRNA. The antioxidants, N-acetylcysteine and glutathione, but not vitamin C or tiron, inhibited perifosine-induced elevation of p-c-Jun, DR4 and DR5. Moreover, no increased production of reactive oxygen species was detected in perifosine-treated cells although reduced levels of intracellular GSH were measured.

Conclusions: DR5 induction plays a critical role in mediating perifosine/TRAIL-induced apoptosis. Perifosine induces DR5 expression through a JNK-dependent mechanism independent of reactive oxygen species.

Figure 1

The combination of perifosine and TRAIL exhibits enhanced effects on decreasing cell survival (A), inducing apoptosis (B) and inhibiting colony formation (C) in HNSCC cell lines and augments xenograft growth inhibition (D). A, The indicated cell lines were seeded in 96-well plates and the next day treated with the indicated concentrations of TRAIL alone, perifosine (PRFS) alone, or their respective combinations (P + T). After 24 h, the cells were subjected to estimation of cell number using the SRB assay. The data are means ± SD of four replicate determinations. B, M4e cells were treated with PBS control, 5 μM perifosine, 10 ng/ml TRAIL and the combination of perifosine and TRAIL. After 24 h, apoptotic (sub-G1) cells were measured by flow cytometry. C, M4e cells plated in 12-well cell culture plates were treated with 1 μM perifosine, 10 ng/ml TRAIL, or their combination. The same treatments were repeated every 3 days. After 12 days, the plates were stained for cell colonies with crystal violet dye, and photographs of colonies taken using a digital camera. D, Four groups of mice were treated with PBS (n = 11), perifosine (n = 11), TRAIL (n = 13) and perifosine combined with TRAIL (n = 13), respectively, as described in "Materials and Methods". After about 3 weeks, the tumors were removed from the sacrificed mice and weighed. The data are means ± SD.

Figure 2

Perifosine induces DR4 and DR5 expression (A and B) at the transcriptional level (C and D). A and B, The indicated cell lines were treated with the given concentrations of perifosine (PRFS) for 16 h. C, M4e cells were pre-treated with the given concentrations of Act D for 30 min and then co-treated with 10 μM perifosine for 8 h. After the aforementioned treatments, the cells were subjected to preparation of whole-cell protein lysates and subsequent detection of the indicated proteins using Western blot analysis. D, M4e cells were treated with indicated concentrations of perifosine for 7 h and then subjected to preparation of cellular total RNA and subsequent RT-PCR for detection of DR4, DR5, and GAPDH (internal loading control).

Figure 3

siRNA-mediated inhibition of perifosine-induced upregulation of DR5 (A), but not DR4 (B), attenuates augmented induction of apoptosis by the combination of perifosine and TRAIL. M4e cells were transfected with control (Ctrl), DR5 (A) or DR4 (B) siRNA. After 48 h, the cells were treated without and with the combination of 5 μM perifosine and 12.5 ng/ml TRAIL (PRFS/TRAIL). After 24 h, the cells were harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis to detect DR5, DR4 and caspase cleavage (A and C) and for detection of apoptosis with annexin V-flow cytometry (B and D). Columns in B and C are means ± SD of duplicated determinations.

Figure 4

Perifosine increases cell surface levels of DR5 (A), but not DR4 (B), in M4e cells. M4e cells were treated with PBS or 10 μM perifosine for 12 h and then harvested for analysis of cell surface DR5 and DR4 by immunofluorescent staining and subsequent flow cytometry.

Figure 5

Perifosine activates JNK signaling (A), which mediates DR5 induction by perifosine (B and C). A, M4e cells were treated with 10 μM perifosine for the times indicated. B, M4e cells were pre-treated with the given concentrations of SP600125 for 30 min and then co-treated with 10 μM perifosine for 12 h. C, M4e cells were transfected with control (Ctrl) or JNK siRNA for 48 h and then treated with PBS or perifosine for additional 12 h. After the aforementioned treatments, the cells were subjected to preparation of whole-cell protein lysates and subsequent detection of the indicated proteins using Western blot analysis.

Figure 6

Effects of NAC and other antioxidants on perifosine-induced DR4 and DR5 expression (A-C) and on enhancement of TRAIL-induced apoptosis by perifosine (D). A, M4e cells were pre-treated with the given concentrations of NAC for 30 min and then co-treated with 10 μM perifosine for 12 h; B, M4e cells were pre-treated with 0.5 mM Vitamin C (Vit C), or 0.5 mM tiron for 30 min and then co-treated with 10 μM perifosine for 10 h; C, M4e cells were pre-treated with the given concentrations of GSH for 30 min and then co-treated with 10 μM perifosine for 10 h. D, M4e cells were pre-treated with 5 mM NAC for 30 min and co-treated with 5 μM perifosine, 12.5 ng/ml and the combination of perifosine and TRAIL for an additional 12 h. After the aforementioned treatments, the cells were subjected to preparation of whole-cell protein lysates and subsequent detection of the indicated proteins using Western blot analysis.

Figure 7

Effects of perifosine on ROS generation (A) and GSH levels (B and C) and effects of DEM on DR4 and DR5 expression in the absence and presence of NAC (D). A, M4e cells were loaded with DCF-DA and then exposed to the indicated concentrations of perifosine (PRFS) or H₂O₂ (positive control). At the indicated times post addition of the agents, ROS generation was monitored as described in "Materials and Methods". B and C, M4e cells were treated with the indicated concentrations of perifosine or DEM (B) for 9 h or treated with 10 uM for the given times or 1 mM DEM for 9 h (C). The cells were then subjected to GSH assay as described in "Materials and Methods". D, M4e cells were treated with 1 mM DEM in the absence and presence of 5 mM NAC for the indicated times (NAC was added to the cells 30 min before addition of perifosine). The cells were then lysed for preparation of whole-cell protein lysates and subsequent detection of the indicated proteins using Western blot analysis.