Programmed Cell Death 4 Enhances TRAIL Sensitivity In Gastric Cancer Cells And The Mechanisms

Introduction Tumor necrosis factor related apoptosis induced ligand (TRAIL) induces apoptosis inmany types of cancers with limited cytotoxicity on normal cells. Therefore, it has emerged as a promising antineoplastic agent. However, some cancer cells are resistant to TRAIL and the mechanism for this is unknown. Some studies showed that resistance to TRAIL is caused not only by differential expression of TRAIL receptors but by some intracellular inhibitors that act downstream of TRAIL receptors. Furthermore, the resistance of cancer cells to TRAIL can be reversed with chemotherapeutic agents, RNA synthesis inhibitors and protein synthesis inhibitors. To elucidate the intracellular mechanisms that control TRAIL sensitivity may help to develop TRAIL-targeted cancer therapies.In light of the importance of TRAIL to cancer therapy and the challenge from its resistance, we focused on identifying factors that control the sensitivity to TRAIL. Programmed cell death 4 (PDCD4) is a novel tumor suppressor that inhibits tumorigenesis, tumor progression and invasion. The expression of PDCD4 protein and/or mRNA was lost in progressed carcinomas of the lung, breast, colon, prostate, brain, liver compared to PDCD4 expression in adjacent normal tissues. Loss of PDCD4 in progressed tumors correlated with the pathological grading and prognosis of tumors. PDCD4 protein localizes mostly in the nuclei of confluent or quiescent normal fetal fibroblasts. It has also been shown that PDCD4 is predominantly a nuclear protein that can be exported from the nucleus to cytoplasm. PDCD4 has a similar domain to MA3 and inhibits protein translation by binding to helicase and inhibiting the helicase activity of eukaryotic translation initiation factor 4A (eIF4A), a component of translation complex. Furthermore, PDCD4 acts as a target for tumor therapy through inducing apoptosis and inhibiting angiogenesis or enhancing the sensitivity of tumors to antitumor drugs or radiotherapy.Now that PDCD4 is important to tumor progression and therapy, we focused on investigating whether PDCD4 regulates the sensitivity of TRAIL and the mechanism. The PI3K/Akt pathway regulates a number of normal cellular processes, including cell proliferation, survival, and motility through phosphorylation of multiple downstream targets. FLICE-inhibitory protein (FLIP) is one of the critical regulators of apoptosis triggered by TRAIL. High levels of FLIP were found in many types of tumors. FLIP shares a high degree of homology with caspase-8, and it competes with caspase-8 in binding to Fas associated DD-containing protein (FADD), which results in inhibition of apoptosis.Previously, we have explored the therapeutic effects of TRAIL on gastric cancer and hepatomas and identified some effectors that regulate the sensitivity to TRAIL. Here, we showed that TRAIL induced apoptosis in several gastric cancer cell lines. Overexpression of PDCD4 sensitized BGC823 cells to TRAIL. However, treatment of MKN28 cells with PDCD4 shRNA lowered the sensitivity of MKN28 cells to TRAIL. Importantly, PDCD4 regulated endogenous FLIP levels at both the transcriptional and translational levels. Furthermore, the activation of PDCD4 was regulated by the PI3K/Akt pathway, and inhibition of Akt by LY2940042 enhanced PDCD4 activity. Finally, we showed that PDCD4 controlled the sensitivity of gastric cancer to TRAIL by regulating FLIP protein levels. It is suggested that PDCD4 might be an important target for gastric cancer therapy.Materials and methods1. Cell lines and antibodiesHuman gastric carcinoma cell lines MKN28, BGC823, and SGC7901 were maintained in RPMI1640(Gibco,USA) supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere containing 5% CO2. Soluble rhuTRAIL was purchased from ProSpec(Israel). Anti-PDCD4, anti-TRAIL, anti-p-Akt, anti-caspase-8/cleaved-caspase-8, and anti-FLIP antibodies were purchased from SantaCruz Biotechnology Inc. (USA). Anti-β-actin and horseradish peroxidase (HRP)-coupled anti-rabbit antibodies, nitrocellulose membrane, HRP-coupled anti-mouse antibody, and enhanced chemiluminescence (ECL) solutions were purchased from Bioster(China).2. Plasmid construction and transfectionPDCD4 cDNA was cut from pBluescriptR-PDCD4 (Invitrogen, USA)using restriction endonucleases BamH I and EcoR I. PDCD4 gene was subcloned into pIRES2-EGFP plasmid, and then transfected into E. coli DH5α. E. coli DH5αwere maintained in LB plates supplemented with 30μg/ml kanamycin, 20μl x-gal and 2μl IPTG. White clones were selected and maintained in LB liquid medium. The plasmid was extracted, sequenced and then transfected into gastric cancer cells using Liperfactamine 2000(Invitrogen) according to the manufacture's protocol. The cell lines overexpressing PDCD4 protein were screened with G418 (800μg/ml) for three weeks3. Targeted down-regulation of PDCD4 by shRNAShRNA fragment targeting PDCD4 (5'GTGCTTCTGAGTATGTCTA3') was obtained from a database of short hairpin RNA gene-silencing constructs. ShRNA was subcloned into p-RNAT-U6. 1/Neo carrier and transfected into MKN28 cells using Liperfactamine 2000.Meanwhile, non-transfected cells and control shRNA transfected cells were used as negative controls. The cell line exhibiting PDCD4 downregulation was screened with G418 for three weeks.4. Western blot analysisCells were lyzed on ice for 30 min in a lysis buffer [50 mmol/L Tris-HCl, 150mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and protease inhibitors]. 50μg proteins were denatured in 2×loading buffer at 100°C for 5min, separated on SDS-PAGE gel, and transferred onto nitrocellulose membrane. The proteins were then detected using specific antibodies and appropriate secondary antibodies and visualized by radioautography using ECL.5. Reverse transcription-polymerase chain reactionTotal RNA was isolated using Trizol reagent (Invitrogen) according to manufacture's protocol. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using specific primers by the RT-PCR kit (Promega, USA) according to the manufacture's protocol.β-actin was used as loading control. Amplified products were separated on agarose gels.6. Cell viability assay and apoptosis assessmentCell viability was determined by 3- (4, 5-dimethylthiazolyl) -2, 5-diphenyltetrazolium bromide (MTT) assay. Gastric carcinoma cells were plated on 96-well plates (15×103 cells per well). After the indicated treatments, the cells were incubated with 0.5mg/ml MTT reagent for 2-3 h and lyzed with dimethyl sulfoxide (DMSO). Absorbance was measured at 490nm in a microplate reader (Bio-Rad, Richmond, CA, USA). The cell survival rate was calculated by dividing the absorbance of the treated cells by that of the non-treated cells. Apoptosis was detected by DAPI (Beyontime, China) staining according to the manufacture's protocol.7. Statistical analysisData were expressed as mean±SD of three or more independent measurements. Paired data were subjected to two-tailed Students t test. A P value less than 0. 05 was considered statistically significant.Results1. The expression of PDCD4 and FLIP correlates with the sensitivity of gastric cancer cells to TRAILWestern blot analysis showed that the level of PDCD4 was the lowest in BGC823 cells and the highest in MKN28 cells. MTT assay demonstrated that BGC823 cells were resistant to TRAIL in a dose- and time-dependent manner, whereas MKN28 cells were sensitive to TRAIL. Apoptosis analysis showed that MKN28 cells were sensitive to TRAIL-induced apoptosis, while BGC823 cells were resistant to TRAIL-induced apoptosis in a time- and dose-dependent manner.2. Upregulation of PDCD4 expression enhances the sensitivity of BGC823 cells toTRAILBGC823 cells were transfected with pIRES2-PDCD4 plasmid carrying the green fluorescence reporting gene EGFP. Single cell clones with green fluorescence was screened with G418, which overexpressed PDCD4 protein stably. MTT assay indicated that BGC823 cells overexpressing PDCD4 were more sensitive than the non-transfected cells to TRAIL. Moreover, the apoptotic index (AI) was significantly increased in the transfected BGC823 cells. These data suggest that overexpression of PDCD4 enhances the sensitivity of BGC823 cells to TRAIL.3. Inhibition of PDCD4 expression by shRNA reduces the sensitivity of MKN28 cells to TRAILPDCD4 shRNA was delivered to MKN28 cells that were sensitive to TRAIL and had increased levels of PDCD4 protein to obtain a cell line with stable inhibition of PDCD4 expression. The resultant cells were treated with TRAIL.MTT assay and apoptosis analysis demonstrated a decreased sensitivity of the transfected MKN28 cells to TRAIL and a decreased apoptotic index of these cells. The findings demonstrate that inhibition of PDCD4 expression lowers the sensitivity of MKN28 cells to TRAIL.4. PDCD4 regulates FLIP protein level in gastric cancer cellsIn order to investigate whether PDCD4 regulates FLIP expression, FLIP and caspase-8 expression under different conditions were analyzed by Western blotting. It was found that FLIP protein expression was downregulated, while caspase-8 expression was upregulated in BGC823 cells overexpressing PDCD4, compared to normal BGC823 cells. The FLIP level was further decreased in BGC823 cells as early as 4h after treatment with TRAIL. In contrast, the FLIP level maintained at a high level in the non-transfected BGC823 cells even after TRAIL treatment. Meanwhile, caspase-8 expression was upregulated in the transfected BGC823 cells, but not in the non-transfected BGC823 cells after TRAIL treatment. It was noteworthy that FLIP levels decreased in MKN28 cells after treatment with TRAIL, but not in MKN28 cells transfected with PDCD4 shRNA. All these data support the hypothesis that PDCD4 controls the sensitivity of gastric cancer cells to TRAIL by regulating FLIP protein level.In order to investigate whether FLIP levels are transcriptionally regulated, RT-PCR was performed to determine the level of FLIP mRNA. Upregulation of PDCD4 expression decreased the FLIP mRNA level in BGC823 cells significantly, and inhibition of PDCD4 expression by shRNA raised the FLIP mRNA level. In order to determine whether proteasomal degradation is also involved in the regulation of FLIP protein expression, BGC823 and MKN28 cells were treated with the proteasome inhibitor MG132. MG132 treatment completely abolished the changes in FLIP protein expression caused by PDCD4. These results suggest that PDCD4 triggers FLIP degradation through the proteasome.5. PDCD4 is regulated by the PI3K/Akt pathway in gastric cancer cellsIn order to identify the regulators upstream of PDCD4, Akt activity was examined by Western blotting in BGC823, SGC7901, and MKN28 cells. Of the three cell lines, BGC823 cells were found to have the highest level of active Akt, and MKN 28 cells to have the lowest level. The results suggested that the activity of Akt tended to be inversely proportional to the expression of PDCD4 in gastric cancer cells. In order to examine whether inhibition of Akt activity enhances PDCD4 and makes BGC823 cells sensitive to TRAIL, PDCD4 activity was examined following inhibition of Akt activity by the PI3K inhibitor LY294002. The results demonstrated that the activity of PDCD4 was enhanced and BGC823 cells became sensitive to TRAIL after inhibition of Akt. These data suggest that PDCD4 is regulated by the PI3K/Akt pathway.Conclusion1. In gastric cancer cells, the expression of PDCD4 was related to the sensitivity of TRAIL.The more expression of PDCD4, the more sensitive to TRAIL.2. PDCD4 gene transfection sensitized TRAIL-resistant BGC823 cells to TRAIL induced apoptosis in both time and dose dependent manner.3. PDCD4 shRNA desensitized TRAIL-sensitive MKN28 cells to TRAIL in both time and dose dependent manner.4. PDCD4 enhanced TRAIL induced apoptosis through regulating FLIP levels and caspase-8 activity in gastric cancer cells. This regulation was realized by both transcriptional and post-transcriptional mechanisms.5. PDCD4 expression was involved in PI3K/Akt pathway. Inhibition of Akt by LY294002 enhanced PDCD4 expresssion and sensitized gastric cancer cells to TRAIL.