Dissecting the roles of DR4, DR5 and c-FLIP in the regulation of geranylgeranyltransferase I inhibition-mediated augmentation of TRAIL-induced apoptosis

Abstract

Background: Geranylgeranyltransferase I (GGTase I) has emerged as a cancer therapeutic target. Accordingly, small molecules that inhibit GGTase I have been developed and exhibit encouraging anticancer activity in preclinical studies. However, their underlying anticancer mechanisms remain unclear. Here we have demonstrated a novel mechanism by which GGTase I inhibition modulates apoptosis.

Results: The GGTase I inhibitor GGTI-298 induced apoptosis and augmented tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human lung cancer cells. GGTI-298 induced DR4 and DR5 expression and reduced c-FLIP levels. Enforced c-FLIP expression or DR5 knockdown attenuated apoptosis induced by GGTI-298 and TRAIL combination. Surprisingly, DR4 knockdown sensitized cancer cells to GGTI298/TRAIL-induced apoptosis. The combination of GGTI-298 and TRAIL was more effective than each single agent in decreasing the levels of IkappaBalpha and p-Akt, implying that GGTI298/TRAIL activates NF-kappaB and inhibits Akt. Interestingly, knockdown of DR5, but not DR4, prevented GGTI298/TRAIL-induced IkappaBalpha and p-Akt reduction, suggesting that DR5 mediates reduction of IkappaBalpha and p-Akt induced by GGTI298/TRAIL. In contrast, DR4 knockdown further facilitated GGTI298/TRAIL-induced p-Akt reduction.

Conclusions: Both DR5 induction and c-FLIP downregulation contribute to GGTI-298-mediated augmentation of TRAIL-induced apoptosis. Moreover, DR4 appears to play an opposite role to DR5 in regulation of GGTI/TRAIL-induced apoptotic signaling.

Figure 1

GGTI-298 as a single agent (A and B) or combined with TRAIL (C and D) inhibits the growth (A and C) and induces apoptosis (B and D) of human NSCLC cells. A and C, The indicted cell lines were seeded in 96-well cell culture plates and treated the next day with the given concentrations of GGTI-298 (A) or with the given concentrations of GGTI-298 alone, TRAIL alone, and their combinations as indicated (C). After 3 days (A) or 24 h (C), cell number was estimated using the SRB assay. Data are the means of four replicate determinations; Bars, ± SDs. B and D, The indicated cell lines were treated with 15 μM GGTI-298 for 48 h (B) or with the indicated doses of TRAIL alone, 15 μM GGTI-298 alone, and their respective combinations for 24 h (D). Apoptosis were then determined by Annexin V staining.

Figure 2

GGTI-298 modulates the levels of DR4, DR5, c-FLIP, p-Akt and survivin in human NSCLC cells. A, The given cell lines were treated with 15 μM GGTI-298 for 16 h. B, A549 cells were treated with the indicated concentrations of GTI-298 for 16 h. C, A549 cells were treated with 15 μM GGTI-298 for the given times. After the aforementioned treatments, the cells were subjected to preparation of whole-cell protein lysates and subsequent Western blotting. LE, longer exposure; UP, unprenylated.

Figure 3

RhoB is upregulationed by GGTI-298 (A) and in part mediates DR5 upregulation by GGTI-298 (B). A, A549 cells were treated with 15 μM GGTI-298 for the given times and then subjected to preparation of whole-cell protein lysates and subsequent Western blotting. B, A549 cells were cultured in a 6-well plate and the next day transfected with the indicated siRNAs. After 36 h, the cells were treated with 15 μM GGTI-298 for 12 h and then subjected to preparation of whole-cell protein lysates for Western blotting. Ctrl, control; LE, longer exposure.

Figure 4

Comparing the effects of GGTI-DU40 and SN-DU40 on modulation of DR5, DR4 and c-FLIP (A), on decreasing cell survival (B), and on TRAIL-induced apoptosis (C and D) in human NSCLC cells. A and D, The indicated cell lines were treated with the given concentrations of GGTI-DU40 or SN-DU40 for 8 h (A) or with DMSO, 10 μM GGTI-DU40 or SN-DU40 alone, 20 ng/ml TRAIL alone of the combination of TRAIL with GGTI-DU40 or SN-DU40 for 6 h (D). The cells were then subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis for the proteins as indicated. CF, cleaved form. B and C, The indicated cell lines were seeded in 96-well plates and treated with the given concentrations of GGTI-DU40 or SN-DU40 (B) or with 20 ng/ml TRAIL alone, the given concentrations of GGTI-DU40 or SN-DU40 alone, or the combination of GGTI-DU40 or SN-DU40 with TRAIL (C). After 3 days (A) or 24 h (C), the cells were subjected to the SRB assay for measurement of cell number. Data are means of four replicate determinations. Bars, ± SDs.

Figure 5

Enforced expression of ectopic FLIP_S attenuates the effects of the GGTI-298 and TRAIL combination on decreasing cell survival (A) and inducing apoptosis (B). A, The indicated transfectants were seeded in 96-well plates and treated with the indicated concentrations of GGTI-298 plus 20 ng/ml TRAIL. After 24 h, the cells were subjected to the SRB assay for measurement of cell number. Data are means of four replicate determinations. Bars, ± SDs. B, The indicated transfectants were treated with DMSO, 15 μM GGTI-298, 20 ng/ml TRAIL, and the combination of GGTI-298 and TRAIL for 24 h and then subjected to detection of apoptosis by Annexin V staining. GGTI, GGTI-298.

Figure 6

Modulation of c-FLIP levels by enforcing expression of ectopic c-FLIP (A and B) or siRNA-mediated downregulation of c-FLIP (C and D) regulates cell sensitivity to GGTI-298-induced apoptosis. A and B, The indicated transfectants were treated with the given concentrations of GGTI-298 (A) or 15 μM GGTI-298 (B) for 48 h and then subjected to estimation of cell number using the SRB assay (A) or detection of apoptosis by Annexin V staining (B). C and D, H226 cells seeded in a 6-well plate (C) or a 96-well (D) plate were transfected with control (Ctrl) or c-FLIP siRNA. Twenty-four hours later, the cells were exposed to 30 μM (C) or the indicated concentrations (D) of GGTI-298. After 24 h, the cells were subjected to preparation of whole-cell protein lysates and subsequent detection of the indicated proteins using Western blotting (C) or to estimation of cell number by the SRB assay (D). Data in A and D are means of four replicate determinations. Bars, ± SDs. CF, cleaved form.

Figure 7

Blockage of DR5 induction abrogates augmented induction of apoptosis by GGTI-298 and TRAIL combination (A), whereas inhibition of DR4 induction sensitizes cells to GGTI-298 plus TRAIL-induced apoptosis (A -C). A and B, A549 cells were cultured in a 6-well plate and the next day transfected with the indicated siRNAs twice in a 24 h interval. Twenty four hours after the second transfection, cells were treated with 15 μM GGTI-298 plus 20 ng/ml TRAIL (A and B, right panel) or 15 μM GGTI-298 only (B, left panel) for 8 h and then subjected to preparation of whole-cell protein lysates for Western blotting (A and B, left panels) or detection of apoptosis by Annexin V staining (A and B, right panels). C, H157 cells were cultured in a 6-well plate and the next day transfected with the indicated siRNAs twice in a 24 h interval. Twenty four hours after transfection, the cells were treated with 15 μM GGTI-298, 20 ng/ml TRAIL or GGTI-298 plus TRAIL. After 8 h (left panel) and 24 h (right panel), the cells were harvested for preparation of whole-cell protein lysates for Western blotting (left panel) or for detection of apoptosis by the Annexin V staining (right panel), respectively. Data are means of duplicate experiments; Bars, ± SEs. Ctrl, control; GGTI, GGTI-298; G + T, GGTI-298 plus TRAIL; CF, cleaved from.

Figure 8

The combination of GGTI-298 and TRAIL augments Akt inhibition, IκBα degradation (A) and NF-κB activation (B), which are differentially modulated by DR4 and DR5 (C). A, A549 cells were treated with the combination of 15 μM GGTI-298 and 20 ng/ml TRAIL for the given times (upper panel) or with DMSO (D), 15 μM GGTI-298 (G) alone, 20 ng/ml TRAIL (T) alone or GGTI-298 and TRAIL combination (G/T) for 2 h (lower panel). The cells were then subjected to preparation of whole-cell protein lysates and subsequent detection of the indicated proteins using Western blotting. B, A549 cells stably transfected with NF-κB-luc reporter gene were treated with DMSO, 15 μM GGTI-298 alone, 20 ng/ml TRAIL alone or GGTI-298 and TRAIL combination for 3 h. The cells were then lysed for NF-κB luciferase assay, which was finally normalized by protein content. The columns are means of triplicate determinations; Bars, ± SDs. C, A549 cells were cultured in 6-well plates and the next day transfected with the indicated siRNAs twice in a 48 h interval. Twenty-four hours after the second transfection, the cells were treated with 15 μM GGTI-298 plus 20 ng/ml TRAIL (G/T) for the indicated time and then subjected to preparation of whole-cell protein lysates and subsequent detection of the given proteins using Western blotting.