Glucosamine Enhances TRAIL-Induced Apoptosis in the Prostate Cancer Cell Line DU145

Abstract

Background: Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively kills tumor cells in cancer patients. However, patients often develop TRAIL resistance; thus, agents that can sensitize cells to TRAIL therapy would be beneficial clinically. Methods: Immunoblotting, flow cytometry, confocal microscopy, qPCR and caspase 8 activity assays were used to investigate whether glucosamine (GlcN) can sensitize cancer cells to TRAIL thereby enhancing apoptosis and potentially improving clinical response. Results: GlcN sensitized DU145 cells to TRAIL-induced apoptosis but did not increase death receptor 5 (DR5) cell surface expression. Once treated, these cells responded to TRAIL-induced apoptosis through both extrinsic and intrinsic apoptotic pathways as evidenced by the cleavage of both caspases 8 and 9. The combination of GlcN and TRAIL suppressed the expression of key anti-apoptotic factors cFLIP, BCL-X_L, MCL-1 and XIAP and translocated BAK to the mitochondrial outer membrane thereby facilitating cytochrome C and SMAC release. In addition to the activation of apoptotic pathways, TRAIL-mediated inflammatory responses were attenuated by GlcN pretreatment reducing nuclear NF-kB levels and the expression of downstream target genes IL-6 and IL-8. Conclusions: GlcN/TRAIL combination could be a promising strategy for treating cancers by overcoming TRAIL resistance and abrogating TRAIL-induced inflammation.

Keywords: DR5; DU145; ER stress; TRAIL; apoptosis; cancer; glucosamine.

Figure 1

Glucosamine (GlcN) increases expression of multiple apoptosis effectors and synergizes tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. (A) Sensitivity of different cell lines to GlcN-induced deglycosylation as measured by EGFR. C4-2B, DU145, HeLa and PC3 cells were cultured in the presence of 2 mM GlcN for 24 h. Whole-cell lysates were subjected to immunoblotting using an EGFR antibody. The open arrow indicates the N-glycosylated EGFR receptor and the filled arrow indicates the deglycosylated receptor. (B) DU145 cells were grown in 2 mM GlcN for the indicated time and whole-cell extracts were prepared and analyzed by immunoblotting using antibodies against DR4 and DR5 and multiple unfolded protein response (UPR) indicators (Bip, XBP1s, ATF6, p-EIF2a, ATF4, CHOP) and pan-actin. (C) Untreated (G–/T–) cells or cells treated with 2 mg/mL GlcN for 24 h (G+T–), 50 ng/mL TRAIL for 8 h (G–T+) or a combination of both GlcN for 24 h then TRAIL for 8 h (G+T+) were stained with annexin V/PI and then analyzed by flow cytometry analysis. Bars represent the mean value of three independent assays, **p < 0.01. (D) Whole-cell extracts were analyzed by immunoblotting with cleaved caspases 8, 9 and 3, PARP and GAPDH after treatment as in 1C. (E) Flow cytometry analysis of cell surface DR4/DR5 treated with 2 mM GlcN.

Figure 2

GlcN/TRAIL treatment in DU145 cells modulates both extrinsic and intrinsic apoptotic regulators. (A) Cells were treated as in Figure 1C and whole-cell extracts were prepared and analyzed by immunoblotting with antibodies against c-FLIP, BID, BCL-XL, MCL-1, XIAP, cIAP-1 and GAPDH. (B) Confocal imaging of DU145 cells treated the same as in A and stained with BAK (green), TOMM20 (red) and DAPI (blue), magnification 63×. (C) Cells were treated as described in A and cytosolic extracts were probed with antibodies against SMAC, cytochrome C and GAPDH.

Figure 3

GlcN/TRAIL suppresses TRAIL-activated NF-kB signaling via inhibiting RIP1 and phosphorylation of NF-kB. (A) Cells were treated with TRAIL (50 ng/mL) for the designated time and nuclear extracts were prepared and analyzed by immunoblotting using antibodies against phospho-NF-kB and TOPO-1. (B) Cells were treated as in A, and total RNA was prepared for qRT-PCR analysis of IL-6 and IL-8 expression. Values represent the mean of two independent assays. (C) Cells were treated as in Figure 1C, nuclear extracts were prepared and analyzed by immunoblotting with antibodies against phospho-NF-kB, and TOPO-1. The bottom panel represents densitometric scanning of the results normalized by TOPO-1 levels. Bars represent the mean value of three independent assays, * p < 0.05. (D) Cells were treated as in Figure 1C, whole-cell extracts were prepared and analyzed by immunoblotting by using antibodies against RIP1 and GAPDH. The bottom panel represents densitometric scanning of the results normalized by GAPDH levels. Bars represent the mean value of three independent assays. * p < 0.05; ** p < 0.01.

Figure 4

Suppression of caspase 8 activity leads to the rescue of GlcN/TRAIL-induced apoptotic cell death. DU145 cells were treated as in Figure 1C in the absence (-) or presence (+) of the caspase 8 inhibitor Z-IETD-FMK. (A) After treatment, cells were stained with annexin V/PI and then analyzed by flow cytometry analysis. Bars represent the mean value of three independent assays, *** p < 0.001. (B) Whole-cell extracts were prepared and analyzed by immunoblotting with antibodies against cleaved caspases 3 and 8, and phospho-NF-kB. (C) Caspase 8 activity was measured. Bars represent the mean value of three independent assays, * p < 0.05.

Figure 5

GlcN increases the flux toward apoptosis versus inflammation (anti-apoptotic) in combination treatment with TRAIL. The addition of GlcN/TRAIL increases overall caspase levels and activates molecules associated with the intrinsic apoptotic pathway (i.e., SMAC and cytochrome C). GlcN/TRAIL also reduces inflammatory pathways by reducing NF-kB levels which are typically stimulated by TRAIL treatment alone.