Emerging Role of Decoy Receptor-2 as a Cancer Risk Predictor in Oral Potentially Malignant Disorders.

The aim of this study was to evaluate the expression of the senescence markers, Decoy Receptor 2 (DcR2) and Differentiated Embryo-Chondrocyte expressed gen 1 (DEC1), in oral potentially malignant disorders (OPMDs) to ascertain their possible association with oral cancer risk. The immunohistochemical analysis of DcR2 and DEC1 expression (along with p16 and Ki67 expression) was carried out in 60 patients with clinically diagnosed oral leukoplakia. Fifteen cases (25%) subsequently developed an invasive carcinoma. Correlations between protein marker expression, histological grade and oral cancer risk were assessed. DcR2, DEC1 and Ki67 protein expressions were found to correlate significantly with increased oral cancer risk, and also with an increased grade of dysplasia. Multivariate analysis demonstrated that DcR2 and Ki67 expression are independent predictors of oral cancer development. Our results evidence for the first time the potential of DcR2 as an early biomarker to assess oral cancer risk in patients with oral leukoplakia (HR = 59.7, p = 0.015), showing a superior predictive value to histology (HR = 4.225, p = 0.08). These findings reveal that the increased expression of DcR2 and DEC1 occurred frequently in OPMDs. In addition, DcR2 expression emerges as a powerful biomarker for oral cancer risk assessment in patients with oral leukoplakia.

DCR2, a Cellular Senescent Molecule, Is a Novel Marker for Assessing Tubulointerstitial Fibrosis in Patients with Immunoglobulin A Nephropathy.

BACKGROUND/AIMS: Stress-induced cell senescence, which contributes to cell cycle arrest and is independent of age, plays an important role in chronic kidney disease (CKD) progression. DcR2, as a senescent marker, exclusively expressed in senescent tubular epithelia. The objective of this study was to examine whether urinary DcR2 (uDcR2) could be a potential biomarker for tubulointerstitial fibrosis (TIF) in patients with immunoglobulin A nephropathy (IgAN). METHODS: This study included 210 IgAN patients and 80 healthy volunteers, with uDcR2 levels measured using enzyme-linked immunosorbent assay. We examined the relationship among uDcR2/Cr levels, renal function, and pathological parameters, using regression analysis to identify risk factors for TIF and the area under the curve (AUC) approach to predict TIF. Renal DcR2 expression was quantified by immunohistochemistry. Co-expression of DcR2 with fibrotic markers (α-smooth muscle actin [α-SMA], collagen III) was analyzed by confocal microscopy. RESULTS: Levels of uDcR2/Cr were significantly higher in IgAN patients and in those with more severe TIF, compared with healthy controls. Serum DcR2 levels were similar across groups. The proportion of IgAN patients with stages 1-2 CKD and T0 was highest among those with uDcR2/Cr <130 ng/g. In contrast, the majority of those with uDcR2/Cr >201 ng/g had stages 4-5 CKD and T2. Levels of uDcR2/Cr were positively associated with urinary albumin to creatinine ratio (ACR), urinary N-acetyl-ß-D-glucosaminidase (uNAG)/Cr, and TIF scores and negatively associated with estimated glomerular filtration rate (eGFR). uDcR2/Cr, uNAG, ACR, and eGFR were independent predictors for TIF, with AUC of 0.907 for uDcR2/Cr. This AUC value was higher than that observed for eGFR, uNAG/Cr, or ACR. The sensitivity and specificity of uDcR2/Cr in predicting TIF were 87.0 and 80.5%, respectively. Moreover, uDcR2/Cr levels were positively associated with the percentage of renal DcR2 expression. Renal DcR2 co-localized with α-SMA and collagen III in the kidneys of IgAN patients. CONCLUSIONS: Levels of uDcR2/Cr were closely associated with the severity of TIF and renal function parameters. uDcR2/Cr represents a potential biomarker for predicting TIF in IgAN patients.

Regulation of TNF-Related Apoptosis-Inducing Ligand Signaling by Glycosylation.

Tumor necrosis-factor related apoptosis-inducing ligand, also known as TRAIL or APO2L (Apo-2 ligand), is a cytokine of the TNF superfamily acknowledged for its ability to trigger selective apoptosis in tumor cells while being relatively safe towards normal cells. Its binding to its cognate agonist receptors, namely death receptor 4 (DR4) and/or DR5, can induce the formation of a membrane-bound macromolecular complex, coined DISC (death-signaling inducing complex), necessary and sufficient to engage the apoptotic machinery. At the very proximal level, TRAIL DISC formation and activation of apoptosis is regulated both by antagonist receptors and by glycosylation. Remarkably, though, despite the fact that all membrane-bound TRAIL receptors harbor putative glycosylation sites, only pro-apoptotic signaling through DR4 and DR5 has, so far, been found to be regulated by N- and O-glycosylation, respectively. Because putative N-glycosylation sequons and O-glycosylation sites are also found and conserved in all these receptors throughout all animal species (in which these receptors have been identified), glycosylation is likely to play a more prominent role than anticipated in regulating receptor/receptor interactions or trafficking, ultimately defining cell fate through TRAIL stimulation. This review aims to present and discuss these emerging concepts, the comprehension of which is likely to lead to innovative anticancer therapies.

Requirement for CRIF1 in RNA interference and Dicer-2 stability.

RNA interference (RNAi) is a eukaryotic gene-silencing system. Although the biochemistry of RNAi is relatively well defined, how this pathway is regulated remains incompletely understood. To identify genes involved in regulating the RNAi pathway, we screened for genetic mutations in Drosophila that alter the efficiency of RNAi. We identified the Drosophila homolog of the mammalian CR6-interacting factor 1 (CRIF1), also known as growth arrest and DNA-damage-inducible 45-gamma interacting protein (Gadd45GIP1), as a potential new regulator of the RNAi pathway. Loss-of-function mutants of Drosophila CRIF1 (dCRIF) are deficient in RNAi-mediated target gene knock-down, in the biogenesis of small interfering RNA (siRNA) molecules, and in antiviral immunity. Moreover, we show that dCRIF may function by interacting with, and stabilizing, the RNase III enzyme Dicer-2. Our results suggest that dCRIF may play an important role in regulating the RNAi pathway.

Key arginine residues in R2D2 dsRBD1 and dsRBD2 lead the siRNA recognition in Drosophila melanogaster RNAi pathway.

In Drosophila melanogaster, Dcr-2:R2D2 heterodimer binds to the 21 nucleotide siRNA duplex to form the R2D2/Dcr-2 Initiator (RDI) complex, which is critical for the initiation of siRNA-induced silencing complex (RISC) assembly. During RDI complex formation, R2D2, a protein that contains three dsRNA binding domains (dsRBD), senses two aspects of the siRNA: thermodynamically more stable end (asymmetry sensing) and the 5'-phosphate (5'-P) recognition. Despite several detailed studies to date, the molecular determinants arising from R2D2 for performing these two tasks remain elusive. In this study, we have performed structural, biophysical, and biochemical characterization of R2D2 dsRBDs. We found that the solution NMR-derived structure of R2D2 dsRBD1 yielded a canonical α1-ß1-ß2-ß3-α2 fold, wherein two arginine salt bridges provide additional stability to the R2D2 dsRBD1. Furthermore, we show that R2D2 dsRBD1 interacts with thermodynamically asymmetric siRNA duplex independent of its 5'-phosphorylation state, whereas R2D2 dsRBD2 prefers to interact with 5'-P siRNA duplex. The mutation of key arginine residues, R53 and R101, in concatenated dsRBDs of R2D2 results in a significant loss of siRNA duplex recognition. Our study deciphers the active roles of R2D2 dsRBDs by showing that dsRBD1 initiates siRNA recognition, whereas dsRBD2 senses 5'-phosphate as an authentic mark on functional siRNA.

Chemical shift assignments of dsRBD1 and linker region of R2D2, a siRNA binding protein in the Drosophila RNAi pathway.

In the model organism Drosophila melanogaster, one of the Dicer homologs, Dcr-2, initiates the RNA interference pathway by cleaving long double-stranded RNA into small interfering RNA (siRNA). The Dcr-2:R2D2 heterodimer subsequently binds to the 21-nucleotide siRNA to form the R2D2:Dcr-2 Initiator (RDI) complex, which is critical for initiating the assembly of the RNA-induced silencing complex containing guide siRNA strand. During RDI complex formation, R2D2 senses the stability of the 5' end of the siRNA and a 5'-phosphate group, although the underlying mechanism of siRNA asymmetry sensing and 5'-phosphate recognition by R2D2 is elusive. In this study, we present nearly complete chemical shift assignments of the backbone and the side chain of a construct that comprises the N-terminus dsRBD1 and linker of R2D2 (~ 10.3 kDa; henceforth: R2D2D1L). Our study would further aid in the structural and functional characterization of R2D2.

Potentiation of TRAIL-Induced Apoptosis in TRAIL-Resistant Cholangiocarcinoma Cells by Curcumin through the Induction of DR5 Membrane Localization and Disruption of the Anti-Apoptotic Complex DR5/DDX3/GSK3ß.

OBJECTIVE: Cholangiocarcinoma (CCA) is a cancer of the bile duct with a poor prognosis. The present study examined the ability of curcumin to sensitize apoptosis in the TNF-related apoptosis-inducing ligand (TRAIL)-resistant CCA cell lines of HuCCA-1 and KKU-213A. METHODS: Apoptosis was measured using a TUNEL assay. Protein expression was determined by immunoblotting. Membrane death receptor 5 (DR5) was detected by flow cytometry. Protein complex was examined by co-immunoprecipitation. RESULT: Curcumin potentiated TRAIL-induced apoptosis in both cell lines, indicating the sensitization to TRAIL-induced apoptosis by curcumin. Additionally, curcumin increased DR5 expression and membrane localization; however, the curcumin/TRAIL combination did not result in further increases in DR5 expression and membrane localization in either cell line. Moreover, the curcumin/TRAIL combination reduced DR5/decoy receptor 2 (DcR2) complexes in both cell lines, suggesting that curcumin may enhance TRAIL-induced apoptosis by disrupting DR5/DcR2 interaction. In addition, levels of the anti-apoptotic complex DR5/ DDX3/GSK3ß were reduced by the curcumin/TRAIL combination in HuCCA-1 but not in KKU-213A cells. This study also demonstrated that the DR5/DcR2 and DR5/DDX3/GSK3ß complexes could be observed under basal conditions, suggesting that these anti-apoptotic complexes may contribute to TRAIL-resistant phenotypes in both cell lines. Pretreatment with the antioxidant N-acetylcysteine attenuated curcumin-enhanced apoptosis by TRAIL, indicating that curcumin sensitized TRAIL-induced apoptosis through an oxidative stress-dependent mechanism. CONCLUSION: The present study demonstrates the potential of using curcumin in combination with TRAIL to yield better TRAIL therapy outcomes in TRAIL-resistant CCA.

Effluent decoy receptor 2 as a novel biomarker of peritoneal fibrosis in peritoneal dialysis patients.

BACKGROUND: Peritoneal fibrosis (PF) is a common complication of peritoneal dialysis (PD), but a specific and sensitive biomarker for PF is lacking. The present study aimed to determine the use of effluent decoy receptor 2 (eDcR2) as a biomarker for PF in PD patients. METHODS: PD patients (n = 248) were recruited, and peritoneal specimens were collected at PD initiation (n = 30) and cessation (n = 33). Enzyme-linked immunoassay was used to measure eDcR2 and the eDcR2 appearance rate (eDcR2-AR) was calculated. The levels of DcR2 mRNA and protein were determined. The correlation of eDcR2 level with peritoneal function, histological parameters and DcR2 expression were analysed. Receiver operating characteristic (ROC) analysis was used to assess the diagnostic performance of eDcR2 for PF, which was defined as a submesothelial thickness 150 µm or more. Co-localisation of DcR2 with a mesothelial marker, fibroblast markers and fibrotic markers were determined. RESULTS: The eDcR2-AR level correlated with PD duration, D/P Cr values, peritoneal Kt/V and peritoneal injury scores, especially submesothelial thickness (r = 0.638, p < 0.001). DcR2 was primarily expressed in peritoneal fibroblasts, and co-localised with α-SMA, vimentin, collagen I and fibronectin, but not with E-cadherin. Peritoneal DcR2 expression had a positive correlation with eDcR2-AR. ROC analysis indicated eDcR2 had an area under the curve of 0.907 for detection of PF (sensitivity: 78.6%, specificity: 100%) and the best cut-off value was 392.5 pg/min. CONCLUSION: The eDcR2-AR level is a potential biomarker for assessing PF in PD patients. Effluent DcR2 was mainly derived from peritoneal fibroblasts and DcR2-positive cells may accelerate PF, suggesting that it may be a potential therapeutic target.

Tubular decoy receptor 2 as a predictor of prognosis in patients with immunoglobulin A nephropathy.

BACKGROUND: Accelerated senescence of renal tubular epithelial cells (RTECs) might contribute to immunoglobulin A nephropathy (IgAN) progression. This study aimed to determine whether the RTEC senescence marker, decoy receptor 2 (DcR2), could predict prognosis in IgAN. METHODS: We included a retrospective cohort of 105 patients with biopsy-proven IgAN. Tubular DcR2 expression was assessed at renal biopsy and the Oxford histological MEST-C score [mesangial hypercellularity (M), endocapillary proliferation (E), segmental sclerosis (S), interstitial fibrosis/tubular atrophy (T) and crescents (C)] defined disease severity. IgAN progression was defined as a composite of end-stage renal disease or a 30% decline in the estimated glomerular filtration rate (eGFR), analyzed using Kaplan-Meier and Cox regression analyses. RESULTS: Tubular DcR2 was overexpressed in IgAN. Numbers of DcR2 and p16 double-positive RTECs increased with increasing severity of tubular atrophy/interstitial fibrosis (T lesion). Patients with ≥25% tubular DcR2 expression experienced worse proteinuria, T lesions and a lower eGFR. Cumulative renal survival was significantly lower in patients with ≥25% DcR2 positivity. Multivariate regression analyses showed that ≥25% tubular DcR2 expression was significantly associated with worse eGFR slopes (the rate of renal function decline; P = 0.003) and the incidence of the composite outcome (P = 0.001) in IgAN. The addition of tubular DcR2 to a model with clinical data at biopsy (mean arterial pressure, proteinuria and eGFR) or MEST-C score significantly improved the 5-year risk prediction of IgAN progression, as confirmed by receiver operating characteristic curve analyses. CONCLUSIONS: Tubular DcR2 expression detected at biopsy was a strong independent predictor for IgAN progression and might have prognostic value in addition to established risk markers.

An Aedes aegypti-Derived Ago2 Knockout Cell Line to Investigate Arbovirus Infections.

Mosquitoes are known as important vectors of many arthropod-borne (arbo)viruses causing disease in humans. These include dengue (DENV) and Zika (ZIKV) viruses. The exogenous small interfering (si)RNA (exo-siRNA) pathway is believed to be the main antiviral defense in arthropods, including mosquitoes. During infection, double-stranded RNAs that form during viral replication and infection are cleaved by the enzyme Dicer 2 (Dcr2) into virus-specific 21 nt vsiRNAs, which are subsequently loaded into Argonaute 2 (Ago2). Ago2 then targets and subsequently cleaves complementary RNA sequences, resulting in degradation of the target viral RNA. Although various studies using silencing approaches have supported the antiviral activity of the exo-siRNA pathway in mosquitoes, and despite strong similarities between the siRNA pathway in the Drosophila melanogaster model and mosquitoes, important questions remain unanswered. The antiviral activity of Ago2 against different arboviruses has been previously demonstrated. However, silencing of Ago2 had no effect on ZIKV replication, whereas Dcr2 knockout enhanced its replication. These findings raise the question as to the role of Ago2 and Dcr2 in the control of arboviruses from different viral families in mosquitoes. Using a newly established Ago2 knockout cell line, alongside the previously reported Dcr2 knockout cell line, we investigated the impact these proteins have on the modulation of different arboviral infections. Infection of Ago2 knockout cell line with alpha- and bunyaviruses resulted in an increase of viral replication, but not in the case of ZIKV. Analysis of small RNA sequencing data in the Ago2 knockout cells revealed a lack of methylated siRNAs from different sources, such as acute and persistently infecting viruses-, TE- and transcriptome-derived RNAs. The results confirmed the importance of the exo-siRNA pathway in the defense against arboviruses, but highlights variability in its response to different viruses and the impact the siRNA pathway proteins have in controlling viral replication. Moreover, this established Ago2 knockout cell line can be used for functional Ago2 studies, as well as research on the interplay between the RNAi pathways.

Chemotherapeutic Agents Sensitize Resistant Cancer Cells to the DR5-Specific Variant DR5-B more Efficiently than to TRAIL by Modulating the Surface Expression of Death and Decoy Receptors.

TRAIL is considered a promising antitumor agent because it causes apoptosis of transformed cells without affecting normal cells. However, many types of tumors are cytokine resistant, and combination therapy with various chemotherapeutic drugs is being developed to overcome the resistance. We have demonstrated that the combination of TRAIL with doxorubicin, bortezomib, and panobinostat dramatically reduced the viability of TRAIL-resistant A549 and HT-29 cells. Chemotherapy even more efficiently sensitized cells to the DR5-specific mutant variant of TRAIL DR5-B, which does not have an affinity for decoy receptors. Bortezomib and doxorubicin greatly enhanced the surface expression of the death receptors DR5 and DR4, while panobinostat increased expression of DR5 and suppressed expression of DR4 in both cell lines. All drugs increased surface expression of the decoy receptors DcR1 and DcR2. Unlike the combined treatment, if the cells were pretreated with chemotherapy for 24 h, the cytotoxic activity of TRAIL was less pronounced, while sequential treatment of cells enhanced the effectiveness of DR5-B. The same results were obtained with agonistic anti-DR5 antibodies. Thus, the effectiveness of TRAIL was rather limited due to changes in the ratio of death and decoy receptors and DR5-specific agonists may be preferred in combination antitumor therapy regimens.

Interaction between N-cadherin and decoy receptor-2 regulates apoptosis in head and neck cancer.

N-cadherin is a neural cell adhesion molecule that aberrantly occurs in head and neck cancers to promote cancer cell growth. However, the underlying mechanisms remain unclear. Here we report that N-cadherin increases cancer cell growth by inhibiting apoptosis. Apoptosis eliminates old, unnecessary, and unhealthy cells. However, tumor cells have the ability of avoiding apoptosis that increases cancer cell growth. Recent studies have found that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in tumor cells by reacting with four distinct cell surface receptors: TRAIL-R1 (DR-4), TRAIL-R2 (DR-5), TRAIL-R3 (DcR-1), and TRAIL-R4 (DcR-2). Among these TRAIL receptors, the death receptors DR-4 and DR-5 transmit apoptotic signals owing to the death domain in the intracellular portion. Conversely, the decoy receptors DcR-1 and DcR-2 lack a complete intracellular portion, so neither can transmit apoptotic signals. DcR-1 or DcR-2 overexpression suppresses TRAIL-induced apoptosis. In this study, N-cadherin overexpression increased DcR-2 expression and decreased DR-5 expression. In contrast, knockdown of N-cadherin expression upregulated DR-5 expression and downregulated DcR-2 expression. A significantly positive relationship between N-cadherin and DcR-2 expression was also found in HNSCC specimens. Those specimens with a lower apoptotic index showed a higher expression of N-cadherin and/or DcR-2. In addition, we demonstrated that N-cadherin interacts directly with DcR-2. Notably, DcR-2 induces cancer cell survival through the cleavage of caspases and PARP by activating MAPK/ERK pathway and suppressing NF-kB/ p65 phosphorylation, which has a very important role in resistance to chemotherapy.

High-dose HOOK effect in urinary DcR2 assay in patients with chronic kidney disease.

BACKGROUND: Urinary DcR2 (uDcR2) is a biomarker for the early detection the tubulointerstitial injury (TII) in patients with chronic kidney disease (CKD), but the high-dose hook effect may lead to falsely low or even negative results when using an enzyme-linked immunosorbent assay (ELISA). This study aimed to investigate if the high-dose hook effect exists with ELISA testing, and to uncover a potential approach for reducing this effect. METHODS: 72 CKD patients were recruited and categorized into four groups based on TII scores. uDcR2 was measured in undiluted and serially diluted (two-, four-, eight- and 16-fold dilutions) urine using an ELISA kit. The results from the assay were normalized to urinary creatinine. We evaluated the correlation between uDcR2/cre levels at different dilutions and renal histological parameters. Receiver operating characteristic (ROC) curves were generated to examine the value of uDcR2/cre for predicting TII. RESULTS: uDcR2/cre levels in the undiluted urine were significantly higher in patients with CKD than those in the control. However, higher TII scores did not yield higher levels of uDcR2/cre in the undiluted urine. After serial dilution, uDcR2/cre levels were highest with the four-fold dilution. A positive correlation was found between uDcR2/cre levels at different dilutions and TII scores, with the highest correlation coefficient and the largest AUC being observed at the four-fold dilution. CONCLUSIONS: The high-dose hook effect was apparent during ELISA testing of uDcR2 in CKD patients, yet dilution of the urine samples neutralized this effect. However, the use of a four-fold dilution of urine for uDcR2/cre testing may eliminate the high-dose hook effect and make it possible to effectively monitor the severity of TII in CKD patients.

The potential evasion of immune surveillance in mucosa associated lymphoid tissue lymphoma by DcR2-mediated up-regulation of nuclear factor-κB.

This study investigated expression profiles of tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) pathway components and mechanisms underlying TRAIL-induced apoptosis in mucosa associated lymphoid tissue (MALT) lymphoma. Genetic aberrations including translocations and trisomies were assessed by reverse transcription polymerase chain reaction and fluorescence in situ hybridization. Expression of TRAIL, death receptors 4 and 5, decoy receptors 1 and 2, and FADD-like interleukin-1ß-converting enzyme (FLICE) inhibitory protein was analyzed by immunohistochemistry. All 32 patients under study showed some alterations in TRAIL pathway mainly involving loss of death receptors (37.5%), gain of decoy receptors (3.1%) or both (59.4%). Decoy receptor 2 (DcR2) was highly expressed in patients with normal cytogenetic status as compared to those with cytogenetic aberrations (p = 0.005). Moreover, DcR2 expression correlated significantly with nuclear factor-κB (NF-κB) expression (R = 0.372, p = 0.047). High expression of DcR2 in patients with normal cytogenetic status and its significant correlation with NF-κB expression provides a potential clue to evasion of immune surveillance in cytogenetically normal MALT lymphomas.

DR4 specific TRAIL variants are more efficacious than wild-type TRAIL in pancreatic cancer.

Current treatment modalities for pancreatic carcinoma afford only modest survival benefits. TRAIL, as a potent and specific inducer of apoptosis in cancer cells, would be a promising new treatment option. However, since not all pancreatic cancer cells respond to TRAIL, further improvements and optimizations are still needed. One strategy to improve the effectiveness of TRAIL-based therapies is to specifically target one of the 2 cell death inducing TRAIL-receptors, TRAIL-R1 or TRAIL-R2 to overcome resistance. To this end, we designed constructs expressing soluble TRAIL (sTRAIL) variants that were rendered specific for either TRAIL-R1 or TRAIL-R2 by amino acid changes in the TRAIL ectodomain. When we expressed these constructs, including wild-type sTRAIL (sTRAIL(wt)), TRAIL-R1 (sTRAIL(DR4)) and TRAIL-R2 (sTRAIL(DR5)) specific variants, in 293 producer cells we found all to be readily expressed and secreted into the supernatant. These supernatants were subsequently transferred onto target cancer cells and apoptosis measured. We found that the TRAIL-R1 specific variant had higher apoptosis-inducing activity in human pancreatic carcinoma Colo357 cells as well as PancTu1 cells that were additionally sensitized by targeting of XIAP. Finally, we tested TRAIL-R1 specific recombinant TRAIL protein (rTRAIL(DR4)) on Colo357 xenografts in nude mice and found them to be more efficacious than rTRAIL(wt). Our results demonstrate the benefits of synthetic biological approaches and show that TRAIL-R1 specific variants can potentially enhance the therapeutic efficacy of TRAIL-based therapies in pancreatic cancer, suggesting that they can possibly become part of individualized and tumor specific combination treatments in the future.

Synergistic Effect of Subtoxic-dose Cisplatin and TRAIL to Mediate Apoptosis by Down-regulating Decoy Receptor 2 and Up-regulating Caspase-8, Caspase-9 and Bax Expression on NCI-H460 and A549 Cells.

UNLABELLED: Objective(s) : Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in tumor cells, more than half of tumors including non-small cell lung cancer (NSCLC) exhibit TRAIL-resistance. The purpose of this study was to determine whether subtoxic-dose cisplatin and TRAIL could synergistically enhance apoptosis on NSCLC cells and investigate its underlying mechanisms. MATERIALS AND METHODS: NCI-H460 and A549 cells were treated with TRAIL alone, cisplatin alone or combination treatment in this study. The cytotoxicity was evaluated according to Sulforhodamine B assay, and apoptosis was examined using Hoechst 33342 staining and flow cytometry. The mRNA and protein levels of TRAIL receptors and apoptotic proteins including caspase-8, caspase-9, Bcl-2 and Bax were determined by RT-PCR and Western blotting, respectively. RESULTS: Our results showed that NCI-H460 cells were sensitive to TRAIL, whereas A549 cells were resistant. However, subtoxic-dose cisplatin could enhance the both cells to TRAIL-mediated cell proliferation inhibition and apoptosis. The underlying mechanisms might be associated with the down-regulation of DcR2 and up-regulation of Caspase-8, Caspase-9 and Bax. CONCLUSION: Subtoxic-dose cisplatin could enhance both TRAIL- sensitive and TRAIL- resistant NSCLC cells to TRAIL-mediated apoptosis. These findings motivated further studies to evaluate such a combinatory therapeutic strategy against NSCLC in the animal models.