Tumor cell metabolism is a promising target for various cancer treatments. Apart from aerobic glycolysis, cancer cell growth is dependent on glutamine (Gin) supply, leading to their survival and differentiation. Therefore, we examined whether treatment with TNF-related apoptosis-inducing ligand (TRAIL) sensitizes MDA-MB-231 cells to apoptosis under GIn deprivation condition (TRAIL/GIn deprivation). GIn deprivation decreased cell proliferation as expected, but did not induce remarkable cell death. TRAIL/GIn deprivation, however, significantly increased growth inhibition and morphological shrinkage of MDA-MB-231 cells compared to those induced by treatment with either Gin deprivation or TRAIL alone. Moreover, TRAIL/GIn deprivation upregulated the apoptotic sub-G(1) phase accompanied with a remarkable decrease of pro-caspase-3, pro-caspase-9, and anti-apoptotic xIAP, and Bcl-2. Increased cleavage of PARP and pro-apoptotic Bid protein expression suggests that TRAIL/GIn deprivation triggers mitochondrion-mediated apoptosis in MDA-MB-231 cells. Additionally, TRAIL/GIn deprivation upregulated the expression of endoplasmic reticulum (ER) stress markers such as ATF4 and phosphorylated eIF2 alpha, thereby enhancing the C/EBP homologous protein (CHOP) protein level. Transient knockdown of CHOP partically reversed TRAIL/GIn deprivation-mediated apoptosis. Accordingly, TRAIL/GIn deprivation enhanced the expression of death receptor 5 (DR5) and transient knockdown of DR5 completely restored TRAIL/GIn deprivation-mediated apoptosis. Taken together, our results suggest that GIn deprivation conditions can be used for the development of new therapies for TRAIL-resistant cancers. (C) 2017 Elsevier Inc. All rights reserved.