Abstract
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to be selective in the induction of apoptosis in cancer cells with minimal toxicity to normal tissues. However, not all cancers are sensitive to TRAIL-mediated apoptosis. Thus, TRAIL-resistant cancer cells must be sensitized first to become responsive to TRAIL. In this study, we observed that pretreatment by acetylsalicylic acid (ASA) augmented TRAIL-induced apoptotic death in human prostate adenocarcinoma LNCaP and human colorectal carcinoma CX-1 cells. Western blot analysis showed that pretreatment of ASA followed by TRAIL treatment activated caspases (8, 9, and 3) and cleaved poly(ADP-ribose) polymerase, the hallmark feature of apoptosis. Most interestingly, at least 12 h of pretreatment with ASA was prerequisite for promoting TRAIL-induced apoptosis and was related to down-regulation of BCL-2. Biochemical analysis revealed that ASA inhibited NF-kappaB activity, which is known to regulate BCL-2 gene expression, by dephosphorylating IkappaB-alpha and inhibiting IKKbeta activity but not by affecting the HER-2/neu phosphatidylinositol 3-kinase-Akt signal pathway. Overexpression of BCL-2 suppressed the promotive effect of ASA on TRAIL-induced apoptosis and changes in mitochondrial membrane potential. Taken together, our studies suggested that ASA-promoted TRAIL cytotoxicity is mediated through down-regulating BCL-2 and by decreasing mitochondrial membrane potential.
Highlights
Recent studies have revealed that Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),2 which is constitutively expressed on murine natural killer cells in the liver, plays an important role in surveillance of tumor metastasis [3]
Our studies demonstrate that acetylsalicylic acid (ASA) augments TRAIL-induced apoptosis by down-regulating BCL-2 gene expression and by decreasing mitochondrial membrane potential, which subsequently leads to an increase in caspase activation
Data from TUNEL assays show that apoptotic cell death occurred when LNCaP cells were pretreated with ASA followed by TRAIL
Summary
Recent studies have revealed that TRAIL,2 which is constitutively expressed on murine natural killer cells in the liver, plays an important role in surveillance of tumor metastasis [3]. Our studies demonstrate that ASA augments TRAIL-induced apoptosis by down-regulating BCL-2 gene expression and by decreasing mitochondrial membrane potential, which subsequently leads to an increase in caspase activation. Following treatment with ASA for 20 h, LNCaP cells were washed three times with PBS and incubated on ice for 15 min in hypotonic buffer A (10 mmol/liter HEPES, pH 7.9, 10 mmol/liter KCl, 0.1 mmol/liter EDTA, 0.1 mmol/ liter EGTA, 1 mmol/liter DTT, 0.5 mmol/liter phenylmethylsulfonyl fluoride, and 0.6% Nonidet P-40).
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