Low extracellular pH augments TRAIL-induced apoptotic death through the mitochondria-mediated caspase signal transduction pathway

Abstract

Tumor necrosis factor-related apoptosis inducing ligand (TRAIL/APO-2L), a member of the tumor necrosis factor (TNF) gene family, is considered as one of the most promising cancer therapeutic agents due to its ability to selectively kill tumor cells. Although microenvironments of solid tumors (hypoxia, nutrient deprivation, and low pH) often affect the effectiveness of chemotherapy, few studies have been reported on the relationship between tumor microenvironments and TRAIL. In this study, we investigated whether low extracellular pH affects TRAIL-induced apoptotic death. When human prostate carcinoma DU145 cells were treated with 200 ng/ml His-tagged TRAIL for 4 h, the survival was approximately 10% at pH 6.3–6.6 and 61.3% at pH 7.4. Similar results were observed in human colorectal carcinoma CX-1 cell line. The TRAIL-mediated activation of caspase, cytochrome c release, and poly (ADP-ribose) polymerase (PARP) cleavage was promoted at low extracellular pH. Immunoprecipitation followed by western blot analysis shows that low extracellular pH enhances the association of truncated Bid with Bax during treatment with TRAIL. Western blot analysis also shows that the low extracellular pH-enhanced TRAIL cytotoxicity does not involve modulation of the levels of TRAIL receptors (DR4, DR5, and DcR2), FLIP, inhibitor of apoptosis (IAP), and Bcl-2. Overexpression of Bcl-2 effectively prevented low extracellular pH-augmented TRAIL cytotoxicity. Taken together, we propose that TRAIL-mediated cytotoxicity is greatly enhanced in low pH environments by promoting caspase activation.

Introduction

It is well known that severe architectural and functional abnormalities are commonly observed in the capillary network that develops during tumor growth [1]. These abnormalities cause insufficient blood supply and development of a pathophysiological tumor microenvironment. Previous studies with the micropore chamber sampling procedure [2] and tumor-isolated preparations [3] reveal differences in the constituents of serum (vascular compartment) compared to interstitial fluid (interstitial compartment). Vascular and interstitial compartments are two major compartments of the extracellular space of solid tumors. The tumor interstitial compartment is characterized by low oxygen tensions (hypoxia) [4], low glucose concentrations [5], high lactate concentrations [6], [7], and low extracellular pH [8]. These characteristic features, which occur transiently or chronically, can markedly affect the therapeutic response [9], [10]. Recently, we demonstrated that low glucose augments the effect of tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a potent anticancer agent, which induces apoptosis [11]. This low glucose-induced augmentation is mediated through the ceramide–Akt–FLIP pathway [11]. In this study, we investigated whether low extracellular pH alters TRAIL-induced cytotoxicity. We believe that understanding the role of low extracellular pH in TRAIL-mediated cytotoxicity will be important for predicting the effectiveness of this anticancer agent and developing new drug targets.

TRAIL/APO-2L is a type II integral membrane protein belonging to the tumor necrosis factor (TNF) family. TRAIL is a 281-amino acid protein, related most closely to Fas–APO-1 ligand. Like Fas ligand (FasL) and TNF, the C-terminal extracellular region of TRAIL (amino acids 114–281) exhibits a homotrimeric subunit structure [12]. However, unlike FasL and TNF, it induces apoptosis in a variety of tumor cell lines more efficiently than normal cells [13]. This differential cytotoxicity suggests that TRAIL is a good candidate for use as an effective anticancer agent. However, recent studies reveal that a polyhistidine-tagged TRAIL induces apoptosis in normal human hepatocytes in culture [14]. This is probably due to an aberrant conformation and subunit structure of TRAIL in the presence of low zinc concentrations [15]. In contrast, native-sequence, non-tagged recombinant TRAIL, when produced under optimized zinc concentrations, is markedly more active against tumor cells than the polyhistidine-tagged ligand, but has minimal toxicity toward human hepatocytes in vitro [15]. Moreover, preclinical studies in mice and primates have shown that administration of TRAIL can induce apoptosis in human tumors, but no cytotoxicity to normal organs or tissue [16]. In addition, unlike TNF and FasL, TRAIL mRNA is expressed constitutively in many tissues [12], [17]. Recent studies also reveal that TRAIL, which is constitutively expressed on murine natural killer cells in the liver, plays an important role in surveillance of tumor metastasis [18]. Although TRAIL is an apoptosis-inducing member of the TNF gene family on the basis of amino acid homology to the TNF and FasL [12], [17], unlike TNF and FasL, TRAIL infusion does not cause a lethal inflammatory response.

The apoptotic signal induced by TRAIL is transduced by its binding to the death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5), which are members of the TNF receptor superfamily. Both DR4 and DR5 contain a cytoplasmic death domain that is required for TRAIL receptor-induced apoptosis. TRAIL also binds to TRAIL-R3 (DcR1) and TRAIL-R4 (DcR2), which act as decoy receptors by inhibiting TRAIL signaling [19], [20], [21], [22], [23], [24]. Unlike DR4 and DR5, DcR1 does not have a cytoplasmic domain and DcR2 retains a cytoplasmic fragment containing a truncated form of the consensus death domain motif [21]. The relative resistance of normal cells to TRAIL has been explained by the presence of many the decoy receptors on normal cells [25], [26]. Recently, this hypothesis has been challenged based on results showing poor correlations between DR4, DR5, and DcR1 expression and sensitivity to TRAIL-induced apoptosis in normal and cancerous breast cell lines [27] and melanoma cell lines [28]. This discrepancy indicates that other factors such as death inhibitors including FLICE-inhibitory protein (FLIP) [28], Fas-associated protein (FAP-1) [29], Bcl-2 [30], Bcl-X_L[30], Bruton's tyrosine kinase (BTK) [31], silencer of death domain (SODD) [32], toso [33], inhibitor of apoptosis (IAP) [34], X-linked inhibitor of apoptosis (XIAP) [35], and survivin [36] are also involved in differential sensitivity to TRAIL. Previous studies show that chemotherapeutic agents [27], [28], [37] and ionizing radiation [38] can sensitize TRAIL-induced cytotoxicity by decreasing intracellular levels of FLIP [28] or increasing DR5 gene expression in response to genotoxic stress [37], [38], [39]. In this study, we observed that low extracellular pH augments TRAIL-induced apoptotic death by promoting the mitochondria-mediated caspase signal transduction pathway via facilitating the interaction between truncated Bid and Bax rather than changes in the levels of TRAIL receptors, FLIP, IAP, and Bcl-2.