Programmed cell death 4 (PDCD4) mediates the sensitivity of gastric cancer cells to TRAIL-induced apoptosis by down-regulation of FLIP expression

Abstract

Tumor necrosis factor-related apoptosis induced ligand (TRAIL) is an important apoptosis inducer in a variety of tumor cells. In the present study, we determined the underlying molecular mechanisms by which certain gastric cancer cells are resistant to TRAIL. We first detected expression of programmed cell death 4 (PDCD4) in three gastric cancer cell lines and identified its association with the sensitivity of gastric cancer cells to TRAIL. We then stably transfected PDCD4 cDNA or shRNA into these gastric cell lines. Our data showed that restoration of PDCD4 expression induced TRAIL sensitivity, whereas knockdown of PDCD4 expression reduced the sensitivity of these tumor cells to TRAIL treatment. PDCD4 was able to suppress expression of FLICE-inhibiting protein (FLIP), a negative regulator of apoptosis. Knockdown of FLIP expression using FLIP shRNA had similar effects as those of restored PDCD4 expression. Furthermore, the proteasome inhibitor MG132 was able to inhibit expression of FLIP mRNA and protein and upregulate the sensitivity of these cells to TRAIL treatment. Taken together, the results from the current study demonstrated that PDCD4 plays an important role in mediating the sensitivity of gastric cancer cells to TRAIL-induced apoptosis through FLIP suppression. Therefore, the proteasome inhibitor MG132 should be further evaluated for combination therapy with TRAIL.

Introduction

Tumor necrosis factor-related apoptosis induced ligands (TRAIL) are considered promising anticancer agents as they can induce different cancer cells to undergo apoptosis and have very few side effects in non-malignant cells [1], [2]. Mechanistically, TRAIL-induced apoptosis occurs through binding to their cognate receptors, TRAIL-R1 (DR4/Apo-2A) and TRAIL-R2. However, certain tumor cells have been found to be only partially sensitive or completely resistant to TRAIL despite functional TRAIL receptors being expressed on the tumor cells. To better understand the underlying molecular mechanisms responsible for these phenomena, previous studies have demonstrated that the resistance to TRAIL-induced apoptosis could be due to impairment of DR4 and DR5 being transported to the cell surface [3] or to certain intracellular inhibitors that suppress the downstream molecular pathways of TRAIL receptors [4]. Thus, the resistance of various cancer cells to TRAIL can be reversed through a combination of chemotherapeutic agents [1] or inhibitors of RNA and protein synthesis [4]. Such combination treatment truly enhanced sensitivity of different cancers to TRAIL, lending credence to the notion of effective TRAIL-based antitumor therapy.

Programmed cell death 4 (PDCD4) is a novel tumor suppressor gene whose protein product plays a role in suppression of tumorigenesis and tumor progression and invasion [5], [6], [7]. Previous studies showed that overexpression of PDCD4 inhibited tumor promoter TPA-induced neoplastic transformation in JB6 cells and P-positive cells [8], [9], [10]. Lost expression of PDCD4 mRNA and protein has been identified in a number of different human cancers (such as lung, breast, colon, prostate [11], brain [12] and liver [13]) and was associated with higher grades of pathology and worse prognosis [11], [14]. PDCD4 protein is predominantly localized in the nuclei of confluent or quiescent normal fetal fibroblasts [15], but it can be exported from the nuclei into the cytoplasm in vitro by a leptomycin B-sensitive mechanism upon serum withdrawal [16]. PDCD4 protein functions as an inhibitor that suppresses protein translation through its binding to and inhibiting of the helicase activity of eukaryotic translation initiation factor 4A (eIF4A), and the latter is a component of the protein translation complex [5], [17], [18], [19], [20], [21]. It was reported that PDCD4 promoted tumor cell apoptosis after TGF-β treatment in human hepatocellular carcinoma Huh7 cells [13]. PDCD4 can also inhibit tumor cell growth by suppression of carbonic athydrase type II expression [11], [22]. Based on its function in tumor cells, including induction of tumor cell apoptosis, inhibition of tumor angiogenesis, and increase in sensitivity of tumor cells to antitumor drugs or radiotherapy, PDCD4 protein has become an attractive candidate for antitumor therapy [23], [24].

FLICE-inhibitory protein (FLIP) is an important apoptosis regulator that prevents apoptosis induced by death receptors and TRAIL [25]. FLIP shares a high degree of homology with caspase-8 and competes with caspase-8 to displace its binding to Fas-associated DD-containing protein (FADD), resulting in inhibition of apoptosis [26]. In this study, we hypothesized that PDCD4 may play a role in suppression of FLIP for PDCD4-induced apoptosis in TRAIL-resistant gastric cancer cells.