Necrosis Factor-related Apoptosis-inducing Ligand Resistance Research Articles

Rocaglamide overcomes tumor necrosis factor-related apoptosis-inducing ligand resistance in hepatocellular carcinoma cells by attenuating the inhibition of caspase-8 through cellular FLICE-like-inhibitory protein downregulation.

The enhancement of apoptosis is a therapeutic strategy used in the treatment of cancer. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent. However, hepatocellular carcinoma (HCC) cells exhibit marked resistance to the induction of cell death by TRAIL. The present study investigated whether rocaglamide, a naturally occurring product isolated from the genus Aglaia, is able to sensitize resistant HCC cells to TRAIL-mediated apoptosis. Two HCC cell lines, HepG2 and Huh-7, were treated with rocaglamide and/or TRAIL and the induction of apoptosis and effects on the TRAIL signaling pathway were investigated. The in vivo efficacy of rocaglamide was determined in TRAIL-resistant Huh-7-derived tumor xenografts. Rocaglamide significantly sensitized the TRAIL-resistant HCC cells to apoptosis by TRAIL, which resulted from the rocaglamide-mediated downregulation of cellular FLICE-like inhibitory protein and subsequent caspase-8 activation. Furthermore, rocaglamide markedly inhibited tumor growth from Huh-7 cells propagated in severe combined immunodeficient mice, suggesting that chemosentization also occurred in vivo. These data suggest that rocaglamide acted synergistically with TRAIL against the TRAIL-resistant HCC cells. Thus, it is concluded that rocaglamide as an adjuvant to TRAIL-based therapy may present a promising therapeutic approach for the treatment of HCC.

New pyranonaphthoquinones and a phenazine alkaloid isolated from Streptomyces sp. IFM 11307 with TRAIL resistance-overcoming activity

Four new pyranonaphthoquinones (1-4) were isolated from the liquid culture of Streptomyces sp. IFM 11307. Additionally, one new phenazine derivative (5), along with the known phenazine-1,6-dicarboxylic acid (6) were identified. The chemical structure of compounds 1-6 was elucidated by 1D and 2D NMR spectroscopy together with CD spectral analysis. Compounds 1-4 significantly overcame tumor necrosis factor-related apoptosis-inducing ligand resistance in human gastric adenocarcinoma cell lines.

Epidermal Growth Factor Receptor-mediated Tissue Transglutaminase Overexpression Couples Acquired Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance and Migration through c-FLIP and MMP-9 Proteins in Lung Cancer Cells

Acquired chemoresistance not only blunts anticancer therapy but may also promote cancer cell migration and metastasis. Our previous studies have revealed that acquired tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance in lung cancer cells is associated with Akt-mediated stabilization of cellular caspase 8 and Fas-associated death domain (FADD)-like apoptosis regulator-like inhibitory protein (c-FLIP) and myeloid cell leukemia 1 (Mcl-1). In this report, we show that cells with acquired TRAIL resistance have significantly increased capacities in migration and invasion. By gene expression screening, tissue transglutaminase (TGM2) was identified as one of the genes with the highest expression increase in TRAIL-resistant cells. Suppressing TGM2 dramatically alleviated TRAIL resistance and cell migration, suggesting that TGM2 contributes to these two phenotypes in TRAIL-resistant cells. TGM2-mediated TRAIL resistance is likely through c-FLIP because TGM2 suppression significantly reduced c-FLIP but not Mcl-1 expression. The expression of matrix metalloproteinase 9 (MMP-9) was suppressed when TGM2 was inhibited, suggesting that TGM2 potentiates cell migration through up-regulating MMP-9 expression. We found that EGF receptor (EGFR) was highly active in the TRAIL-resistant cells, and suppression of EGFR dramatically reduced TGM2 expression. We further determined JNK and ERK, but not Akt and NF-κB, are responsible for EGFR-mediated TGM2 expression. These results identify a novel pathway that involves EGFR, MAPK (JNK and ERK), and TGM2 for acquired TRAIL resistance and cell migration in lung cancer cells. Because TGM2 couples TRAIL resistance and cell migration, it could be a molecular target for circumventing acquired chemoresistance and metastasis in lung cancer.

Phosphoinositide 3-kinase/Akt and nuclear factor κB pathways are involved in tumor necrosis factor-related apoptosis-inducing ligand resistance in human gastric cancer cells

Human gastric cancer cells are generally believed to be less sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, but the events responsible for this resistance are as yet unclear. In this study, we investigated the role of the phosphoinositide 3-kinase (PI3K)/Akt, nuclear factor κB (NF-κB) and extracellular signal-regulated kinase (ERK) signaling pathways in the TRAIL resistance of gastric cancer cells. TRAIL failed to induce observable apoptosis in the three cell lines. Further investigation revealed that TRAIL engagement led to the activation of PI3K/Akt as well as of NF-κB. The inhibition of PI3K/Akt by a specific inhibitor facilitated TRAIL-induced apoptosis. Blockage of TRAIL-induced NF-κB activation by transient transfection with a phosphorylation-defective mutant IκB also enhanced the sensitivity of cells towards TRAIL. Meanwhile, ERKs were highly activated in the resting cells and were not further activated by TRAIL treatment. However, the inhibition of ERK activity by PD98059 also enhanced the apoptosis-inducing ability of TRAIL. Our data demonstrated that the activation of PI3K/Akt and NF-κB by TRAIL is responsible for resistance to TRAIL in human gastric cancer cells. Blockage of survival signals significantly enhances the apoptosis induced by TRAIL.

Constituents of Pongamia pinnata Isolated in a Screening for Activity to Overcome Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Resistance

In a search for natural products with activity to overcome tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-resistance, we performed the bioassay-guided fractionation of a semi mangrove, Pongamia pinnata, collected from Bangladesh, and isolated a new compound, (2S)-(2″,3″:7,8)-furanoflavanone (1), along with six known flavonoids (2-7). Two of the compounds significantly overcame TRAIL-resistance in human gastric adenocarcinoma (AGS) cell lines.

Acquired Activation of the Akt/Cyclooxygenase-2/Mcl-1 Pathway Renders Lung Cancer Cells Resistant to Apoptosis

Acquired apoptosis resistance plays an important role in acquired chemoresistance in cancer cells during chemotherapy. Our previous observations demonstrated that acquired tumor necrosis factor-related apoptosis-inducing ligand resistance in lung cancer cells was associated with Akt-mediated stabilization of cellular FLICE-like inhibitory protein (c-FLIP) and Mcl-1. In this report, we determined that these cells also have acquired resistance to apoptosis induced by chemotherapeutics such as cisplatin and doxorubicin (Adriamycin), which was detected in vitro in cell cultures and in vivo in xenografted tumors. We further found that cyclooxygenase-2 (COX-2) is dramatically overexpressed in cells with acquired apoptosis resistance. COX-2 seems to be a crucial mediator in acquired apoptosis resistance because suppressing COX-2 activity with a chemical inhibitor or reducing COX-2 protein expression level with COX-2 small interfering RNA dramatically alleviated resistance to therapeutic-induced apoptosis. Inhibiting Akt markedly suppressed COX-2 expression, suggesting COX-2 is a downstream effector of this cell survival kinase-mediated apoptosis resistance. Furthermore, the expression of Mcl-1 but not c-FLIP was significantly reduced when COX-2 was suppressed, and knockdown of Mcl-1 substantially sensitized the cells to apoptosis. Our results establish a novel pathway that consists of Akt, COX-2, and Mcl-1 for acquired apoptosis resistance, which could be a molecular target for circumventing acquired chemoresistance in lung cancer.

Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway.

Evidence for Two Modes of Development of Acquired Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance: INVOLVEMENT OF Bcl-xL

Previous studies have shown that repeated application of TRAIL induces acquired resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Using human prostate adenocarcinoma DU-145 and human pancreatic carcinoma MiaPaCa-2 cells as a model, we now demonstrate for the first time that two states of acquired TRAIL resistance can be developed after TRAIL treatment. Data from survival assay and Western blot analysis show that acquired TRAIL resistance was developed within 1 day and gradually decayed within 6 days after TRAIL treatment in both cell lines. After TRAIL treatment, the level of Bcl-xL increased and reached a maximum within 2 days and gradually decreased in both cell lines. Bcl-xL-mediated development of acquired TRAIL resistance was suppressed by knockdown of Bcl-xL expression. Protein interaction assay revealed that during the development of TRAIL resistance, Bcl-xL dissociated from Bad and then associated with Bax. Overexpression of mutant-type Bad (S136A), which prevents this dissociation, partially suppressed the development of acquired TRAIL resistance. Thus, our results suggest that (a) dissociation of Bad from Bcl-xL and (b) an increase in the intracellular level of Bcl-xL are responsible for development of acquired TRAIL resistance.

Decoy Receptor-2 Small Interfering RNA (siRNA) Strategy Employing Three Different siRNA Constructs in Combination Defeats Adenovirus-Transferred Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Resistance in Lung Cancer Cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells. However, studies have indicated that more than half of human tumors exhibit TRAIL resistance. Although the mechanism of TRAIL resistance is not understood, it represents a barrier to any TRAIL-mediated gene therapy approach. In addition, no correlation between TRAIL receptor (TRAIL-R) expression profile and TRAIL resistance has been demonstrated in cancer cells. In this study, three different lung cancer cell lines and three different primary cell cultures established from patients with lung cancer (two patients with squamous cell lung carcinoma and one with adenocarcinoma) were screened for sensitivity to adenoviral delivery of TRAIL. Whereas TRAIL-resistant primary lung cell cultures and the A549 lung cancer cell line exhibited high levels of surface decoy receptor-2 (DcR2/TRAIL-R4) expression, TRAIL-sensitive lung cancer cell lines (HBE and H411) failed to express it. A DcR2 short interfering RNA (siRNA) approach involving three different siRNA constructs in combination downregulated DcR2/TRAIL-R4 expression and sensitized lung cancer cells to TRAIL-induced apoptosis. Immunohistochemical staining of samples from 10 patients with lung carcinoma suggested that high-level DcR2/TRAIL-R4 expression is a common phenotype observed in patients with non-small cell lung carcinoma.

Decoy Receptor-2 Small Interfering RNA (siRNA) Strategy Employing Three Different siRNA Constructs in Combination Defeats Adenovirus-Transferred Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Resistance in Lung Cancer Cells

Decoy Receptor-2 Small Interfering RNA (siRNA) Strategy Employing Three Different siRNA Constructs in Combination Defeats Adenovirus-Transferred Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Resistance in Lung Cancer Cells

Mcl-1 mediates tumor necrosis factor-related apoptosis-inducing ligand resistance in human cholangiocarcinoma cells.

Cholangiocarcinomas are usually fatal neoplasms originating from bile duct epithelia. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising agent for cancer therapy, including cholangiocarcinoma. However, many cholangiocarcinoma cells are resistant to TRAIL-mediated apoptosis. Thus, our aim was to examine the intracellular mechanisms responsible for TRAIL resistance in human cholangiocarcinoma cell lines. Three TRAIL-resistant human cholangiocarcinoma cell lines were identified. All of the cell lines expressed TRAIL receptor 1/death receptor 4 (TRAIL-R1/DR4) and TRAIL-R2/DR5. Expression of TRAIL decoy receptors and the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) was inconsistent across the cell lines. Of the antiapoptotic Bcl-2 family of proteins profiled (Bcl-2, Bcl-x(L), and Mcl-1), Mcl-1 was uniquely overexpressed by the cell lines. When small-interfering-RNA (siRNA) technology was used to knock down expression of Bcl-2, Bcl-x(L), and Mcl-1, only the Mcl-1-siRNA sensitized the cells to TRAIL-mediated apoptosis. In a cell line stably transfected with Mcl-1-small-hairpin-RNA (Mcl-1-shRNA), Mcl-1 depletion sensitized cells to TRAIL-mediated apoptosis despite Bcl-2 expression. TRAIL-mediated apoptosis in the stably transfected cells was associated with mitochondrial depolarization, Bax activation, cytochrome c release from mitochondria, and caspase activation. Finally, flavopiridol, an anticancer drug that rapidly down-regulates Mcl-1, also sensitized cells to TRAIL cytotoxicity. In conclusion, these studies not only demonstrate that Mcl-1 mediates TRAIL resistance in cholangiocarcinoma cells by blocking the mitochondrial pathway of cell death but also identify two strategies for circumventing this resistance.