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Jonathan N. Sachs - One of the best experts on this subject based on the ideXlab platform.
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Noncompetitive Allosteric Antagonism of Death Receptor 5 by a Synthetic Affibody Ligand.
Biochemistry, 2020Co-Authors: Nagamani Vunnam, Sophia Szymonski, Petra Hirsova, Gregory J. Gores, Jonathan N. Sachs, Benjamin J. HackelAbstract:Fatty acid-induced upregulation of Death Receptor 5 (DR5) and its cognate ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key mechanism in the progression of fatty liver disease. Accordingly, inhibition of DR5 signaling represents an attractive strategy for treating fatty liver disease. Ligand competition strategies are prevalent in tumor necrosis factor Receptor antagonism, but recent studies have suggested that noncompetitive inhibition through perturbation of the Receptor conformation may be a compelling alternative. To this end, we used yeast display and a designed combinatorial library to identify a synthetic 58-amino acid affibody ligand that specifically binds DR5. Biophysical and biochemical studies show that the affibody neither blocks TRAIL binding nor prevents the Receptor-Receptor interaction. Live-cell fluorescence lifetime measurements indicate that the affibody induces a conformational change in transmembrane dimers of DR5 and favors an inactive state of the Receptor. The affibody inhibits apoptosis in TRAIL-treated Huh-7 cells, an in vitro model of fatty liver disease. Thus, this lead affibody serves as a potential drug candidate, with a unique mechanism of action, for fatty liver disease.
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Noncompetitive Allosteric Antagonism of Death Receptor 5 by a Synthetic Affibody Ligand.
Biochemistry, 2020Co-Authors: Nagamani Vunnam, Sophia Szymonski, Petra Hirsova, Gregory J. Gores, Jonathan N. Sachs, Benjamin J. HackelAbstract:Fatty acid-induced upregulation of Death Receptor 5 (DR5) and its cognate ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key ...
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soluble extracellular domain of Death Receptor 5 inhibits trail induced apoptosis by disrupting Receptor Receptor interactions
Journal of Molecular Biology, 2017Co-Authors: Nagamani Vunnam, David D. Thomas, Benjamin D. Grant, Jonathan N. SachsAbstract:Abstract Dysregulation of tumor necrosis factor (TNF) Receptor signaling is a key feature of various inflammatory disorders. Current treatments for TNF-related diseases function either by sequestering ligand or blocking ligand–Receptor interactions, which can cause dangerous side effects by inhibiting the Receptors that are not involved in the disease condition. Thus, alternate strategies that target Receptor–Receptor interactions are needed. We hypothesized that the soluble extracellular domain (ECD) of long isoform of Death Receptor 5 (DR5) could block endogenous Receptor assembly, mimicking the biological effect of decoy Receptors that lack the Death domain to trigger apoptosis. Using live-cell fluorescence resonance energy transfer studies, we demonstrated that soluble ECD disrupts endogenous DR5–DR5 interactions. Cell viability assays were used to demonstrate the complete inhibition of TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by the ECD, although TRAIL is still able to bind to the Receptor. Importantly, we used mutagenesis to prove that the inhibition of TRAIL-induced apoptosis by the ECD predominantly comes from the disruption of DR5 oligomerization and not ligand sequestration. Inhibition of Death Receptor activation should have important therapeutic applications in diseases such as nonalcoholic fatty liver disease. More generally, this approach should be generalized to enable the inhibition of other TNF Receptor signaling mechanisms that are associated in a wide range of clinical conditions.
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Death Receptor 5 Activation Is Energetically Coupled to Opening of the Transmembrane Domain Dimer
Biophysical Journal, 2017Co-Authors: Nagamani Vunnam, Cecily K. Campbell-bezat, Andrew K. Lewis, Jonathan N. SachsAbstract:Abstract The precise mechanism by which binding of tumor necrosis factor ligands to the extracellular domain of their corresponding Receptors transmits signals across the plasma membrane has remained elusive. Recent studies have proposed that activation of several tumor necrosis factor Receptors, including Death Receptor 5, involves a scissorlike opening of the disulfide-linked transmembrane (TM) dimer. Using time-resolved fluorescence resonance energy transfer, we provide, to our knowledge, the first direct biophysical evidence that Death Receptor 5 TM-dimers open in response to ligand binding. Then, to probe the importance of the closed-to-open TM domain transition in the overall energetics of Receptor activation, we designed point-mutants (alanine to phenylalanine) in the predicted, tightly packed TM domain dimer interface. We hypothesized that the bulky residues should destabilize the closed conformation and eliminate the ∼3 kcal/mol energy barrier to TM domain opening and the ∼2 kcal/mol energy difference between the closed and open states, thus oversensitizing the Receptor. To test this, we used all-atom molecular dynamics simulations of the isolated TM domain in explicit lipid bilayers coupled to thermodynamic potential of mean force calculations. We showed that single point mutants at the interface altered the energy landscape as predicted, but were not enough to completely eliminate the barrier to opening. However, the computational model did predict that a double mutation at i , i +4 positions at the center of the TM domain dimer eliminates the barrier and stabilizes the open conformation relative to the closed. We tested these mutants in cells with time-resolved fluorescence resonance energy transfer and Death assays, and show remarkable agreement with the calculations. The single mutants had a small effect on TM domain separation and cell Death, whereas the double mutant significantly increased the TM domain separation and more than doubled the sensitivity of cells to ligand stimulation.
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Soluble Extracellular Domain of Death Receptor 5 Inhibits TRAIL-Induced Apoptosis by Disrupting Receptor–Receptor Interactions
Journal of Molecular Biology, 2017Co-Authors: Nagamani Vunnam, David D. Thomas, Benjamin D. Grant, Jonathan N. SachsAbstract:Abstract Dysregulation of tumor necrosis factor (TNF) Receptor signaling is a key feature of various inflammatory disorders. Current treatments for TNF-related diseases function either by sequestering ligand or blocking ligand–Receptor interactions, which can cause dangerous side effects by inhibiting the Receptors that are not involved in the disease condition. Thus, alternate strategies that target Receptor–Receptor interactions are needed. We hypothesized that the soluble extracellular domain (ECD) of long isoform of Death Receptor 5 (DR5) could block endogenous Receptor assembly, mimicking the biological effect of decoy Receptors that lack the Death domain to trigger apoptosis. Using live-cell fluorescence resonance energy transfer studies, we demonstrated that soluble ECD disrupts endogenous DR5–DR5 interactions. Cell viability assays were used to demonstrate the complete inhibition of TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by the ECD, although TRAIL is still able to bind to the Receptor. Importantly, we used mutagenesis to prove that the inhibition of TRAIL-induced apoptosis by the ECD predominantly comes from the disruption of DR5 oligomerization and not ligand sequestration. Inhibition of Death Receptor activation should have important therapeutic applications in diseases such as nonalcoholic fatty liver disease. More generally, this approach should be generalized to enable the inhibition of other TNF Receptor signaling mechanisms that are associated in a wide range of clinical conditions.
Toshiyuki Sakai - One of the best experts on this subject based on the ideXlab platform.
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Myeloid zinc finger 1 mediates sulindac sulfide-induced upregulation of Death Receptor 5 of human colon cancer cells
Scientific Reports, 2014Co-Authors: Mano Horinaka, Tatsushi Yoshida, Mitsuhiro Tomosugi, Shusuke Yasuda, Yoshihiro Sowa, Toshiyuki SakaiAbstract:A combined therapy of sulindac sulfide and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising strategy for the treatment of cancer. Sulindac sulfide had been shown to induce the expression of Death Receptor 5 (DR5), a Receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism underlying the upregulation of DR5 has not yet been elucidated. We demonstrate here that myeloid zinc finger 1 (MZF1) mediates the induction of DR5 by sulindac sulfide. Sulindac sulfide induced the expression of DR5 at the protein and mRNA levels in colon cancer SW480 cells. Furthermore, sulindac sulfide increased DR5 promoter activity. We showed that sulindac sulfide stimulated DR5 promoter activity via the -301 to -253 region. This region contained a putative MZF1-binding site. Site-directed mutations in the site abrogated the enhancement in DR5 promoter activity by sulindac sulfide. MZF1 directly bound to the putative MZF1-binding site of the DR5 promoter and the binding was increased by sulindac sulfide. The expression of MZF1 was also increased by sulindac sulfide, and MZF1 siRNA attenuated the upregulation of DR5 by sulindac sulfide. These results indicate that sulindac sulfide induces the expression of DR5 by up-regulating MZF1.
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Aclarubicin enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis through Death Receptor 5 upregulation.
Cancer Science, 2011Co-Authors: Mano Horinaka, Miki Wakada, Tatsushi Yoshida, Takumi Shiraishi, Susumu Nakata, Mitsuhiro Tomosugi, Sae Yoshikawa, Toshiyuki SakaiAbstract:Anthracycline drugs are potent anti-tumor agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a Death ligand with promising anti-cancer effects. However, some tumor types develop resistance to TRAIL. We examined the effect of aclarubicin (ACR), an anthracycline, in combination with TRAIL. The combination of TRAIL and ACR synergistically induced apoptosis in human acute lymphoblastic leukemia Jurkat cells and human lung cancer A549 cells. In contrast, another anthracycline, doxorubicin (DOX), only slightly sensitized Jurkat cells and A549 cells to TRAIL-induced apoptosis, with weaker enhancement of Death Receptor 5 (DR5) expression than ACR. The RNase protection assay, real time RT-PCR and western blot demonstrated that ACR upregulated the expression of a TRAIL Receptor, DR5. Caspase inhibitors and dominant negative DR5 efficiently reduced the apoptotic response to the treatment with ACR and TRAIL, indicating that the combined effect depends on caspase activities and the interaction between TRAIL and its Receptor. ACR but not DOX increased the activity of the DR5 gene promoter in Jurkat cells carrying a mutation in the p53 gene, suggesting that ACR upregulates DR5 expression through p53-independent transcription. These results suggest the combination of TRAIL and ACR to be a promising treatment for malignant tumors. (Cancer Sci 2012; 103: 282–287)
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Inhibition of tissue transglutaminase sensitizes TRAIL-resistant lung cancer cells through upregulation of Death Receptor 5.
FEBS Letters, 2010Co-Authors: Manuela Frese-schaper, Toshiyuki Sakai, Julian A. Schardt, Giovanni L. Carboni, Ralph A. Schmid, Steffen FreseAbstract:Tissue transglutaminase (TG2) is implicated in cellular processes such as apoptosis and cell migration. Its acyl transferase activity cross-links certain proteins, among them transcription factors were described. We show here that the TG2 inhibitor KCC009 reversed resistance to tumor necrosis factor-related apoptosis-inducing factor (TRAIL) in lung cancer cells. Sensitization required upregulation of Death Receptor 5 (DR5) but not of Death Receptor 4. Upregulation of DR5 involved the first intron of the DR5 gene albeit it was independent from p53 and nuclear factor kappa B. In conclusion, inhibition of tissue transglutaminase provides an interesting strategy for sensitization to TRAIL-induced apoptosis in p53-deficient lung cancer cells.
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Death Receptor 5 promoter-enhancing compounds isolated from Catimbium speciosum and their enhancement effect on TRAIL-induced apoptosis
Bioorganic & Medicinal Chemistry, 2009Co-Authors: Takashi Ohtsuki, Toshiyuki Sakai, Hiroyuki Kikuchi, Takashi Koyano, Thaworn Kowithayakorn, Masami IshibashiAbstract:The TRAIL/Death-Receptor signaling pathway has been considered a promising target for selective cancer therapy, although some malignant tumors exhibit TRAIL resistance. We previously found that isoflavonoid enhanced TRAIL-induced apoptosis in TRAIL-resistant cells, which is achieved through up-regulation of Death Receptor 5 (DR5). In our screening program targeting DR5 promoter enhancement activity, activity-guided fractionations of the extract of Catimbium speciosum led to the isolation of six compounds. Of the isolates, cardamomin (6), the most potent compound, enhanced the expressions of DR5 and DR4 and decreased the Bcl-xL level in TRAIL-resistant DLD1 cells. The combination of 6 and TRAIL synergistically enhanced TRAIL-induced apoptosis against TRAIL-resistant cells upon the activation of caspase-8, 9, and 3. In addition, enhancement of apoptosis by 6 was inhibited by human recombinant DR5/Fc and DR4/Fc chimera proteins, TRAIL-neutralizing fusion proteins, indicating that 6 sensitize TRAIL-resistant cells to TRAIL through the induction of DR5 and DR4. Also, up-regulation of DR5 by 6 paralleled that of CCAAT/enhancer-binding protein-homologous protein (CHOP).
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Proteasome inhibitor MG132 induces Death Receptor 5 through CHOP in prostate cancer cell
Cancer Research, 2006Co-Authors: Tatsushi Yoshida, Miki Wakada, Takumi Shiraishi, Susumu Nakata, Yoichi Mizutani, Tsuneharu Miki, Toshiyuki SakaiAbstract:2385 TNF-related apoptosis-inducing ligand (TRAIL) is one of promising candidates for cancer therapeutics due to its ability to induce apoptosis in cancer cells with little or no toxicity in normal cells. However, some tumor types exhibit resistance to TRAIL. Thus it is important to overcome this resistance. Combined treatment with a proteasome inhibitor and TRAIL is a promising strategy to overcome TRAIL resistance in cancer cells. Proteasome inhibitors induce the expression of Death Receptor 5 (DR5), a Receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism of DR5 up-regulation has not been elucidated. In this study, we report that CHOP is a regulator of DR5 induction by proteasome inhibitor MG132. First, we show that the combination of MG132 and TRAIL effectively induced apoptosis even in hormone refractory prostate cancer DU145 cells. DU145 cells have a mutation of tumor suppressor p53 gene. Therefore our result suggests that the combination of MG132 and TRAIL is efficacious in malignant tumors carrying p53 mutation. MG132 induced DR5 expression at a protein and mRNA level in DU145 cells, indicating that the induction was also independent of p53. Furthermore, MG132 increased DR5 promoter activity. Using a series of deletion-mutant plasmids containing DR5 promoters of various sizes, we found that MG132 stimulated the promoter activity through the region of - 289 to - 253. This region contained a CHOP-binding site. Site-directed mutation of the site abrogated the promoter activity enhanced by MG132. An electrophoretic mobility shift assay demonstrated that CHOP directly bound to the MG132-responsive site on the DR5 promoter. Expression of the CHOP protein was increased with MG132 along with DR5 up-regulation. In addition, MG132 also increased CHOP mRNA and promoter activity. Furthermore, CHOP siRNA attenuated the DR5 up-regulation due to MG132. These results indicate that the proteasome inhibitor MG132 induces DR5 expression through CHOP up-regulation.
Nagamani Vunnam - One of the best experts on this subject based on the ideXlab platform.
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Noncompetitive Allosteric Antagonism of Death Receptor 5 by a Synthetic Affibody Ligand.
Biochemistry, 2020Co-Authors: Nagamani Vunnam, Sophia Szymonski, Petra Hirsova, Gregory J. Gores, Jonathan N. Sachs, Benjamin J. HackelAbstract:Fatty acid-induced upregulation of Death Receptor 5 (DR5) and its cognate ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key mechanism in the progression of fatty liver disease. Accordingly, inhibition of DR5 signaling represents an attractive strategy for treating fatty liver disease. Ligand competition strategies are prevalent in tumor necrosis factor Receptor antagonism, but recent studies have suggested that noncompetitive inhibition through perturbation of the Receptor conformation may be a compelling alternative. To this end, we used yeast display and a designed combinatorial library to identify a synthetic 58-amino acid affibody ligand that specifically binds DR5. Biophysical and biochemical studies show that the affibody neither blocks TRAIL binding nor prevents the Receptor-Receptor interaction. Live-cell fluorescence lifetime measurements indicate that the affibody induces a conformational change in transmembrane dimers of DR5 and favors an inactive state of the Receptor. The affibody inhibits apoptosis in TRAIL-treated Huh-7 cells, an in vitro model of fatty liver disease. Thus, this lead affibody serves as a potential drug candidate, with a unique mechanism of action, for fatty liver disease.
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Noncompetitive Allosteric Antagonism of Death Receptor 5 by a Synthetic Affibody Ligand.
Biochemistry, 2020Co-Authors: Nagamani Vunnam, Sophia Szymonski, Petra Hirsova, Gregory J. Gores, Jonathan N. Sachs, Benjamin J. HackelAbstract:Fatty acid-induced upregulation of Death Receptor 5 (DR5) and its cognate ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key ...
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soluble extracellular domain of Death Receptor 5 inhibits trail induced apoptosis by disrupting Receptor Receptor interactions
Journal of Molecular Biology, 2017Co-Authors: Nagamani Vunnam, David D. Thomas, Benjamin D. Grant, Jonathan N. SachsAbstract:Abstract Dysregulation of tumor necrosis factor (TNF) Receptor signaling is a key feature of various inflammatory disorders. Current treatments for TNF-related diseases function either by sequestering ligand or blocking ligand–Receptor interactions, which can cause dangerous side effects by inhibiting the Receptors that are not involved in the disease condition. Thus, alternate strategies that target Receptor–Receptor interactions are needed. We hypothesized that the soluble extracellular domain (ECD) of long isoform of Death Receptor 5 (DR5) could block endogenous Receptor assembly, mimicking the biological effect of decoy Receptors that lack the Death domain to trigger apoptosis. Using live-cell fluorescence resonance energy transfer studies, we demonstrated that soluble ECD disrupts endogenous DR5–DR5 interactions. Cell viability assays were used to demonstrate the complete inhibition of TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by the ECD, although TRAIL is still able to bind to the Receptor. Importantly, we used mutagenesis to prove that the inhibition of TRAIL-induced apoptosis by the ECD predominantly comes from the disruption of DR5 oligomerization and not ligand sequestration. Inhibition of Death Receptor activation should have important therapeutic applications in diseases such as nonalcoholic fatty liver disease. More generally, this approach should be generalized to enable the inhibition of other TNF Receptor signaling mechanisms that are associated in a wide range of clinical conditions.
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Death Receptor 5 Activation Is Energetically Coupled to Opening of the Transmembrane Domain Dimer
Biophysical Journal, 2017Co-Authors: Nagamani Vunnam, Cecily K. Campbell-bezat, Andrew K. Lewis, Jonathan N. SachsAbstract:Abstract The precise mechanism by which binding of tumor necrosis factor ligands to the extracellular domain of their corresponding Receptors transmits signals across the plasma membrane has remained elusive. Recent studies have proposed that activation of several tumor necrosis factor Receptors, including Death Receptor 5, involves a scissorlike opening of the disulfide-linked transmembrane (TM) dimer. Using time-resolved fluorescence resonance energy transfer, we provide, to our knowledge, the first direct biophysical evidence that Death Receptor 5 TM-dimers open in response to ligand binding. Then, to probe the importance of the closed-to-open TM domain transition in the overall energetics of Receptor activation, we designed point-mutants (alanine to phenylalanine) in the predicted, tightly packed TM domain dimer interface. We hypothesized that the bulky residues should destabilize the closed conformation and eliminate the ∼3 kcal/mol energy barrier to TM domain opening and the ∼2 kcal/mol energy difference between the closed and open states, thus oversensitizing the Receptor. To test this, we used all-atom molecular dynamics simulations of the isolated TM domain in explicit lipid bilayers coupled to thermodynamic potential of mean force calculations. We showed that single point mutants at the interface altered the energy landscape as predicted, but were not enough to completely eliminate the barrier to opening. However, the computational model did predict that a double mutation at i , i +4 positions at the center of the TM domain dimer eliminates the barrier and stabilizes the open conformation relative to the closed. We tested these mutants in cells with time-resolved fluorescence resonance energy transfer and Death assays, and show remarkable agreement with the calculations. The single mutants had a small effect on TM domain separation and cell Death, whereas the double mutant significantly increased the TM domain separation and more than doubled the sensitivity of cells to ligand stimulation.
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Soluble Extracellular Domain of Death Receptor 5 Inhibits TRAIL-Induced Apoptosis by Disrupting Receptor–Receptor Interactions
Journal of Molecular Biology, 2017Co-Authors: Nagamani Vunnam, David D. Thomas, Benjamin D. Grant, Jonathan N. SachsAbstract:Abstract Dysregulation of tumor necrosis factor (TNF) Receptor signaling is a key feature of various inflammatory disorders. Current treatments for TNF-related diseases function either by sequestering ligand or blocking ligand–Receptor interactions, which can cause dangerous side effects by inhibiting the Receptors that are not involved in the disease condition. Thus, alternate strategies that target Receptor–Receptor interactions are needed. We hypothesized that the soluble extracellular domain (ECD) of long isoform of Death Receptor 5 (DR5) could block endogenous Receptor assembly, mimicking the biological effect of decoy Receptors that lack the Death domain to trigger apoptosis. Using live-cell fluorescence resonance energy transfer studies, we demonstrated that soluble ECD disrupts endogenous DR5–DR5 interactions. Cell viability assays were used to demonstrate the complete inhibition of TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis by the ECD, although TRAIL is still able to bind to the Receptor. Importantly, we used mutagenesis to prove that the inhibition of TRAIL-induced apoptosis by the ECD predominantly comes from the disruption of DR5 oligomerization and not ligand sequestration. Inhibition of Death Receptor activation should have important therapeutic applications in diseases such as nonalcoholic fatty liver disease. More generally, this approach should be generalized to enable the inhibition of other TNF Receptor signaling mechanisms that are associated in a wide range of clinical conditions.
Tatsushi Yoshida - One of the best experts on this subject based on the ideXlab platform.
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Myeloid zinc finger 1 mediates sulindac sulfide-induced upregulation of Death Receptor 5 of human colon cancer cells
Scientific Reports, 2014Co-Authors: Mano Horinaka, Tatsushi Yoshida, Mitsuhiro Tomosugi, Shusuke Yasuda, Yoshihiro Sowa, Toshiyuki SakaiAbstract:A combined therapy of sulindac sulfide and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising strategy for the treatment of cancer. Sulindac sulfide had been shown to induce the expression of Death Receptor 5 (DR5), a Receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism underlying the upregulation of DR5 has not yet been elucidated. We demonstrate here that myeloid zinc finger 1 (MZF1) mediates the induction of DR5 by sulindac sulfide. Sulindac sulfide induced the expression of DR5 at the protein and mRNA levels in colon cancer SW480 cells. Furthermore, sulindac sulfide increased DR5 promoter activity. We showed that sulindac sulfide stimulated DR5 promoter activity via the -301 to -253 region. This region contained a putative MZF1-binding site. Site-directed mutations in the site abrogated the enhancement in DR5 promoter activity by sulindac sulfide. MZF1 directly bound to the putative MZF1-binding site of the DR5 promoter and the binding was increased by sulindac sulfide. The expression of MZF1 was also increased by sulindac sulfide, and MZF1 siRNA attenuated the upregulation of DR5 by sulindac sulfide. These results indicate that sulindac sulfide induces the expression of DR5 by up-regulating MZF1.
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Aclarubicin enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis through Death Receptor 5 upregulation.
Cancer Science, 2011Co-Authors: Mano Horinaka, Miki Wakada, Tatsushi Yoshida, Takumi Shiraishi, Susumu Nakata, Mitsuhiro Tomosugi, Sae Yoshikawa, Toshiyuki SakaiAbstract:Anthracycline drugs are potent anti-tumor agents. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a Death ligand with promising anti-cancer effects. However, some tumor types develop resistance to TRAIL. We examined the effect of aclarubicin (ACR), an anthracycline, in combination with TRAIL. The combination of TRAIL and ACR synergistically induced apoptosis in human acute lymphoblastic leukemia Jurkat cells and human lung cancer A549 cells. In contrast, another anthracycline, doxorubicin (DOX), only slightly sensitized Jurkat cells and A549 cells to TRAIL-induced apoptosis, with weaker enhancement of Death Receptor 5 (DR5) expression than ACR. The RNase protection assay, real time RT-PCR and western blot demonstrated that ACR upregulated the expression of a TRAIL Receptor, DR5. Caspase inhibitors and dominant negative DR5 efficiently reduced the apoptotic response to the treatment with ACR and TRAIL, indicating that the combined effect depends on caspase activities and the interaction between TRAIL and its Receptor. ACR but not DOX increased the activity of the DR5 gene promoter in Jurkat cells carrying a mutation in the p53 gene, suggesting that ACR upregulates DR5 expression through p53-independent transcription. These results suggest the combination of TRAIL and ACR to be a promising treatment for malignant tumors. (Cancer Sci 2012; 103: 282–287)
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Proteasome inhibitor MG132 induces Death Receptor 5 through CHOP in prostate cancer cell
Cancer Research, 2006Co-Authors: Tatsushi Yoshida, Miki Wakada, Takumi Shiraishi, Susumu Nakata, Yoichi Mizutani, Tsuneharu Miki, Toshiyuki SakaiAbstract:2385 TNF-related apoptosis-inducing ligand (TRAIL) is one of promising candidates for cancer therapeutics due to its ability to induce apoptosis in cancer cells with little or no toxicity in normal cells. However, some tumor types exhibit resistance to TRAIL. Thus it is important to overcome this resistance. Combined treatment with a proteasome inhibitor and TRAIL is a promising strategy to overcome TRAIL resistance in cancer cells. Proteasome inhibitors induce the expression of Death Receptor 5 (DR5), a Receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism of DR5 up-regulation has not been elucidated. In this study, we report that CHOP is a regulator of DR5 induction by proteasome inhibitor MG132. First, we show that the combination of MG132 and TRAIL effectively induced apoptosis even in hormone refractory prostate cancer DU145 cells. DU145 cells have a mutation of tumor suppressor p53 gene. Therefore our result suggests that the combination of MG132 and TRAIL is efficacious in malignant tumors carrying p53 mutation. MG132 induced DR5 expression at a protein and mRNA level in DU145 cells, indicating that the induction was also independent of p53. Furthermore, MG132 increased DR5 promoter activity. Using a series of deletion-mutant plasmids containing DR5 promoters of various sizes, we found that MG132 stimulated the promoter activity through the region of - 289 to - 253. This region contained a CHOP-binding site. Site-directed mutation of the site abrogated the promoter activity enhanced by MG132. An electrophoretic mobility shift assay demonstrated that CHOP directly bound to the MG132-responsive site on the DR5 promoter. Expression of the CHOP protein was increased with MG132 along with DR5 up-regulation. In addition, MG132 also increased CHOP mRNA and promoter activity. Furthermore, CHOP siRNA attenuated the DR5 up-regulation due to MG132. These results indicate that the proteasome inhibitor MG132 induces DR5 expression through CHOP up-regulation.
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luteolin induces apoptosis via Death Receptor 5 upregulation in human malignant tumor cells
Oncogene, 2005Co-Authors: Mano Horinaka, Miki Wakada, Ryoko Nakanishi, Hoyoku Nishino, Hiroshi Matsui, Tatsushi Yoshida, Takumi Shiraishi, Susumu Nakata, Toshiyuki SakaiAbstract:Luteolin, a naturally occurring flavonoid, induces apoptosis in various cancer cells. Little is known however concerning the underlying molecular mechanisms responsible for this activity. In this report, we reveal a novel mechanism by which luteolin-induced apoptosis occurs, and show for the first time that the apoptosis by luteolin is mediated through Death Receptor 5 (DR5) upregulation. Luteolin markedly induced the expression of DR5, along with Bcl-2-interacting domain cleavage and the activation of caspase-8, -10, -9 and -3. In addition, suppression of DR5 expression with siRNA efficiently reduced luteolin-induced caspase activation and apoptosis. Human recombinant DR5/Fc also inhibited luteolin-induced apoptosis. On the other hand, luteolin induced neither DR5 protein expression nor apoptosis in normal human peripheral blood mononuclear cells. These results suggest that DR5 induced by luteolin plays a role in luteolin-induced apoptosis, and raises the possibility that treatment with luteolin might be promising as a new therapy against cancer.
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Proteasome inhibitor MG132 induces Death Receptor 5 through CCAAT/enhancer-binding protein homologous protein.
Cancer Research, 2005Co-Authors: Tatsushi Yoshida, Mano Horinaka, Miki Wakada, Takumi Shiraishi, Susumu Nakata, Yoichi Mizutani, Tsuneharu Miki, Toshiyuki SakaiAbstract:Combined treatment with a proteasome inhibitor and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is a promising strategy for cancer therapy. Proteasome inhibitors induce the expression of Death Receptor 5 (DR5), a Receptor for TRAIL, and sensitize cancer cells to TRAIL-induced apoptosis; however, the molecular mechanism of DR5 up-regulation has not been elucidated. In this study, we report that CCAAT/enhancer-binding protein homologous protein (CHOP) is a regulator of DR5 induction by proteasome inhibitor MG132. MG132 induced DR5 expression at a protein and mRNA level in prostate cancer DU145 cells. Furthermore, MG132 increased DR5 promoter activity. Using a series of deletion mutant plasmids containing DR5 promoters of various sizes, we found that MG132 stimulated the promoter activity via the region of −289 to −253. This region contained a CHOP-binding site. Site-directed mutation of the site abrogated the promoter activity enhanced by MG132. An electrophoretic mobility shift assay showed that CHOP directly bound to the MG132-responsive site on the DR5 promoter. Expression of the CHOP protein was increased with MG132 along with DR5 up-regulation. Furthermore, CHOP small interfering RNA attenuated the DR5 up-regulation due to MG132. These results indicate that the proteasome inhibitor MG132 induces DR5 expression through CHOP up-regulation.
Gregory J. Gores - One of the best experts on this subject based on the ideXlab platform.
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Noncompetitive Allosteric Antagonism of Death Receptor 5 by a Synthetic Affibody Ligand.
Biochemistry, 2020Co-Authors: Nagamani Vunnam, Sophia Szymonski, Petra Hirsova, Gregory J. Gores, Jonathan N. Sachs, Benjamin J. HackelAbstract:Fatty acid-induced upregulation of Death Receptor 5 (DR5) and its cognate ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key mechanism in the progression of fatty liver disease. Accordingly, inhibition of DR5 signaling represents an attractive strategy for treating fatty liver disease. Ligand competition strategies are prevalent in tumor necrosis factor Receptor antagonism, but recent studies have suggested that noncompetitive inhibition through perturbation of the Receptor conformation may be a compelling alternative. To this end, we used yeast display and a designed combinatorial library to identify a synthetic 58-amino acid affibody ligand that specifically binds DR5. Biophysical and biochemical studies show that the affibody neither blocks TRAIL binding nor prevents the Receptor-Receptor interaction. Live-cell fluorescence lifetime measurements indicate that the affibody induces a conformational change in transmembrane dimers of DR5 and favors an inactive state of the Receptor. The affibody inhibits apoptosis in TRAIL-treated Huh-7 cells, an in vitro model of fatty liver disease. Thus, this lead affibody serves as a potential drug candidate, with a unique mechanism of action, for fatty liver disease.
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Noncompetitive Allosteric Antagonism of Death Receptor 5 by a Synthetic Affibody Ligand.
Biochemistry, 2020Co-Authors: Nagamani Vunnam, Sophia Szymonski, Petra Hirsova, Gregory J. Gores, Jonathan N. Sachs, Benjamin J. HackelAbstract:Fatty acid-induced upregulation of Death Receptor 5 (DR5) and its cognate ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key ...
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bile acids up regulate Death Receptor 5 trail Receptor 2 expression via a c jun n terminal kinase dependent pathway involving sp1
Journal of Biological Chemistry, 2004Co-Authors: Hajime Higuchi, Steven F Bronk, Annette Grambihler, Ali Canbay, Gregory J. GoresAbstract:Abstract Bile acids up-regulate Death Receptor 5 (DR5)/TRAIL-Receptor 2 (TRAIL-R2) expression thereby sensitizing hepatocytes to TRAIL-mediated apoptosis. However, the precise mechanism by which bile acids enhance DR5/TRAIL-R2 expression is unknown. Although several bile acids enhanced DR5/TRAIL-R2 expression, deoxycholic acid (DCA) was the most potent. DCA stimulated JNK activation and the JNK inhibitor SP600125 blocked DCA-induced DR5/TRAIL-R2 mRNA and protein expression. Reporter gene analysis identified a 5′-flanking region containing two Sp1 binding sites within the DR5/TRAIL-R2 promoter as bile acid responsive. Sp1 binding to one of the two sites was enhanced by DCA treatment as evaluated by electrophoretic mobility shift assays and chromatin immunoprecipitation studies. JNK inhibition with SP600125 also blocked binding of Sp1 to the DR5/TRAIL-R2 promoter. Finally, point mutations of the Sp1 binding site attenuated promoter activity. In conclusion, Sp1 is a bile acid-responsive transcription factor that mediates DR5/TRAIL-R2 gene expression downstream of JNK.
