Zhong X et al. (2018), Eukaryotic Translation Initiation Factor 4A Dow...

ECB-ART-46347

Cancers (Basel) 2018 May 22;105:. doi: 10.3390/cancers10050153.

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Eukaryotic Translation Initiation Factor 4A Down-Regulation Mediates Interleukin-24-Induced Apoptosis through Inhibition of Translation.

Zhong X , Persaud L , Muharam H , Francis A , Das D , Aktas BH , Sauane M .

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Dysregulated activity of helicase eIF4A drives transformation to and maintenance of cancer cell phenotype by reprogramming cellular translation. Interleukin 24 (IL-24) is a tumor-suppressing protein, which has the ability to inhibit angiogenesis, sensitize cancer cells to chemotherapy, and induce cancer cell-specific apoptosis. In this study, we found that eIF4A is inhibited by IL-24. Consequently, selective reduction of translation was observed for mRNAs harboring strong secondary structures in their 5''-untranslated regions (5''UTRs). These mRNAs encode proteins, which function in cell survival and proliferation. Consistently, overexpression of eIF4A conferred cancer cells with resistance to IL-24-induced cell death. It has been established that inhibition of eIF4A triggers mitochondrial-mediated apoptosis. We showed that IL-24 induces eIF4A-dependent mitochondrial depolarization. We also showed that IL-24 induces Sigma 1 Receptor-dependent eIF4A down-regulation and mitochondrial depolarization. Thus, the progress of apoptosis triggered by IL-24 is characterized by a complex program of changes in regulation of several initiation factors, including the eIF4A.

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Species referenced: Echinodermata
Genes referenced: eif4a LOC115919910 LOC373385 LOC574811 LOC575742 LOC576121 LOC582436 LOC587430 LOC587482 LOC590297 mmp7 ndufs6 tubgcp2

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	Figure 1. IL-24-mediatd down-regulation of eIF4A is necessary to mediate apoptosis. (A) Melanoma (HO-1), breast (MCF-7), prostate (DU-145) and cervical cancer cells (HeLa) were treated for 24 h with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell). Cell extracts were subjected to Western blot analysis to detect eIF4A, eIF4G, eIF4E and β-actin protein. (B) Cells were treated as described in (A), and cell viability was determined by trypan blue exclusion assay 4 days after treatment (left panel). Numbers represent the ratio of specific treatments to values in control cells (Ad.vector). Results of cell counting by trypan blue exclusion assay are plotted as mean ± SD of three independent experiments. , p < 0.001 comparted to Ad.vector. Cells were treated as described before, and then assayed for cell death using Annexin V staining a measure of apoptosis, and it was determined 48 h later by FACS analysis using the CellQuest software (Becton Dickinson). An average of three independent experiments is shown ± SD (right panel) , p < 0.001 comparted to Ad.vector. (C) HeLa cells overexpressing eIF4A or control cells were treated with Ad.IL-24 (100 pfu per cell), and cell viability was determined by trypan blue exclusion assay. Numbers represent the ratio of specific treatments to values in control cells (Ad.vector). An average of three independent experiments is shown ± SD (left panel). , p < 0.05 comparted to Ad.vector. Cells were treated as described in upper panel, and then assayed for cell death using Annexin V staining, and a measure of apoptosis was determined by FACS (right panel) , p < 0.05 comparted to Ad.vector. Cells were treated as described in upper panel, cell extracts were subjected to Western blot analysis to detect cleaved caspase-3 and β-actin protein. (D) Melanoma (HO-1), breast (MCF-7), prostate (DU-145) and cervical cancer cells (HeLa) were treated for 24 h with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell). Cell extracts were subjected to Western blot analysis to detect cleaved caspase-3 and β-actin protein.
	Figure 2. IL-24 appears to reduces translation of mRNAs harboring structured 5′UTRs. HeLa cells were transfected with firefly (FF) reporters harboring: (A) ATP5O 5′UTR with a proximal portion of TISU element before the initiation codon (ATP5O [5′UTR]-FF); (B) ATP5O 5′UTR followed by a stem-loop structure (ATP5O (5′UTR)-SL-FF); (C) IRF7 5′UTR (IRF7 (5;UTR)-FF); (D) ATP5O 5′UTR with a full TISU element (ATP5O (TISU)-FF); (E) NDUFS6 5′UTR (NDUFS6 (5′UTR)-FF); or (F) UQCC2 5′UTR (UQCC2 (5′UTR)-FF). As a control, cells were cotransfected with a Renilla reporter. Cells were treated for 24 h with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell). Each experiment was performed in independent triplicates, each consisting of three replicates, and data are shown as mean ± SD. p < 0.001 comparted to Ad.vector. RLU, relative light units.
	Figure 3. IL-24 treatment leads to reduction of mRNAs harboring structured 5′UTRs. (A) HeLa cells were treated with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell) for 72 h, and lysates were prepared and probed with antibodies specific to cdc25, c-myc, ODC, XIAP, GAPDH, α-tubulin, and β-actin. (B) HeLa cells were incubated with Ad.IL24 (100 pfu per cell) or Ad.vector (100 pfu per cell), and expression of cdc25, c-myc, ODC, XIAP, GAPDH, α-tubulin, and β-actin mRNA was determined by real-time PCR.
	Figure 4. IL-24 treatment leads to mitochondrial dysfunction. (A) HeLa cells were treated with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell) for 72 h. Mitochondrial membrane potential (MMP) was analyzed by monitoring TMRE fluorescence intensity. Results are presented as mean ± SD (n = 3) , p < 0.05 comparted to Ad.vector. (B) HeLa cells were treated with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell) for 72 h, and lysates were prepared and probed with antibodies specific to Bcl2, Mcl1, BIRC5, and β-actin (Left panel). HeLa cells were incubated with Ad.IL24 (100 pfu per cell) or Ad.vector (100 pfu per cell), and expression of Bcl2, Mcl1, BIRC5, and β-actin mRNA was determined by real-time PCR (right panel). (C) HeLa cells overexpressed eIF4A or control cells were treated with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell) for 72 h. Mitochondrial membrane potential (MMP) was analyzed by monitoring TMRE fluorescence intensity. Results are presented as mean ± SD (n = 3) , p < 0.05 compared to Ad.vector. (D) HeLa cells overexpressing eIF4A cells were transfected with firefly (FF) reporters harboring ATP5O 5′UTR followed by a stem-loop structure (ATP5O (5′UTR)-SL-FF). As a control, cells were cotransfected with a Renilla reporter. Cells were treated for 24 h with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell). Luminescence was monitored 48 h post-transfection. Data for Firefly luminescence normalized to Renilla luminescence. Each experiment was performed in independent triplicates, each consisting of three replicates, and data are shown as mean ± SD. *, p < 0.005 comparted to Ad.vector. RLU, relative light units.
	Figure 5. IL-24-dependent Sigma 1 Receptor (Sig1R) mediate eIF4A down-regulation, translation of mRNAs harboring structured 5′UTRs and mitochondrial dysfunction. (A) HeLa cells were transfected with firefly (FF) reporter harboring ATP5O 5′UTR followed by a stem-loop structure (ATP5O [5′UTR]-SL-FF). Cells were cotransfected with a Renilla reporter. Cells were incubated with the Sig1R agonist (SKF10047) or overexpression of Sig1R with or without Ad.IL-24 (100 pfu per cell). Data for firefly luminescence normalized to Renilla luminescence. Each experiment was performed in independent triplicates, each consisting of three replicates, and data are shown as mean ± SD. , p < 0.05. RLU, relative light units. (B) HeLa cells were treated as described in (A). Mitochondrial membrane potential (MMP) was analyzed by monitoring TMRE fluorescence intensity. Results are presented as mean ± SD (n = 3). , p < 0.05. (C) HeLa cells were incubated with the indicated conditions. Cells were collected, protein purified, and subjected to western blot analysis to detect eIF4A, eIF4G and eIF4E protein. (D) HeLa cells were treated as described in (A), and lysates were prepared and probed with antibodies specific to cdc25, c-myc, ODC, XIAP, Bcl2, Mcl1, BIRC5, and β-actin. (E) HeLa cells were treated as described in A, and lysates were prepared and probed with antibodies specific to cleaved caspase-3 and β-actin.

References [+] :

Bhat, Targeting the translation machinery in cancer. 2015, Pubmed

	Figure 1. IL-24-mediatd down-regulation of eIF4A is necessary to mediate apoptosis. (A) Melanoma (HO-1), breast (MCF-7), prostate (DU-145) and cervical cancer cells (HeLa) were treated for 24 h with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell). Cell extracts were subjected to Western blot analysis to detect eIF4A, eIF4G, eIF4E and β-actin protein. (B) Cells were treated as described in (A), and cell viability was determined by trypan blue exclusion assay 4 days after treatment (left panel). Numbers represent the ratio of specific treatments to values in control cells (Ad.vector). Results of cell counting by trypan blue exclusion assay are plotted as mean ± SD of three independent experiments. , p < 0.001 comparted to Ad.vector. Cells were treated as described before, and then assayed for cell death using Annexin V staining a measure of apoptosis, and it was determined 48 h later by FACS analysis using the CellQuest software (Becton Dickinson). An average of three independent experiments is shown ± SD (right panel) , p < 0.001 comparted to Ad.vector. (C) HeLa cells overexpressing eIF4A or control cells were treated with Ad.IL-24 (100 pfu per cell), and cell viability was determined by trypan blue exclusion assay. Numbers represent the ratio of specific treatments to values in control cells (Ad.vector). An average of three independent experiments is shown ± SD (left panel). , p < 0.05 comparted to Ad.vector. Cells were treated as described in upper panel, and then assayed for cell death using Annexin V staining, and a measure of apoptosis was determined by FACS (right panel) , p < 0.05 comparted to Ad.vector. Cells were treated as described in upper panel, cell extracts were subjected to Western blot analysis to detect cleaved caspase-3 and β-actin protein. (D) Melanoma (HO-1), breast (MCF-7), prostate (DU-145) and cervical cancer cells (HeLa) were treated for 24 h with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell). Cell extracts were subjected to Western blot analysis to detect cleaved caspase-3 and β-actin protein.
	Figure 2. IL-24 appears to reduces translation of mRNAs harboring structured 5′UTRs. HeLa cells were transfected with firefly (FF) reporters harboring: (A) ATP5O 5′UTR with a proximal portion of TISU element before the initiation codon (ATP5O [5′UTR]-FF); (B) ATP5O 5′UTR followed by a stem-loop structure (ATP5O (5′UTR)-SL-FF); (C) IRF7 5′UTR (IRF7 (5;UTR)-FF); (D) ATP5O 5′UTR with a full TISU element (ATP5O (TISU)-FF); (E) NDUFS6 5′UTR (NDUFS6 (5′UTR)-FF); or (F) UQCC2 5′UTR (UQCC2 (5′UTR)-FF). As a control, cells were cotransfected with a Renilla reporter. Cells were treated for 24 h with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell). Each experiment was performed in independent triplicates, each consisting of three replicates, and data are shown as mean ± SD. p < 0.001 comparted to Ad.vector. RLU, relative light units.
	Figure 3. IL-24 treatment leads to reduction of mRNAs harboring structured 5′UTRs. (A) HeLa cells were treated with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell) for 72 h, and lysates were prepared and probed with antibodies specific to cdc25, c-myc, ODC, XIAP, GAPDH, α-tubulin, and β-actin. (B) HeLa cells were incubated with Ad.IL24 (100 pfu per cell) or Ad.vector (100 pfu per cell), and expression of cdc25, c-myc, ODC, XIAP, GAPDH, α-tubulin, and β-actin mRNA was determined by real-time PCR.
	Figure 4. IL-24 treatment leads to mitochondrial dysfunction. (A) HeLa cells were treated with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell) for 72 h. Mitochondrial membrane potential (MMP) was analyzed by monitoring TMRE fluorescence intensity. Results are presented as mean ± SD (n = 3) , p < 0.05 comparted to Ad.vector. (B) HeLa cells were treated with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell) for 72 h, and lysates were prepared and probed with antibodies specific to Bcl2, Mcl1, BIRC5, and β-actin (Left panel). HeLa cells were incubated with Ad.IL24 (100 pfu per cell) or Ad.vector (100 pfu per cell), and expression of Bcl2, Mcl1, BIRC5, and β-actin mRNA was determined by real-time PCR (right panel). (C) HeLa cells overexpressed eIF4A or control cells were treated with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell) for 72 h. Mitochondrial membrane potential (MMP) was analyzed by monitoring TMRE fluorescence intensity. Results are presented as mean ± SD (n = 3) , p < 0.05 compared to Ad.vector. (D) HeLa cells overexpressing eIF4A cells were transfected with firefly (FF) reporters harboring ATP5O 5′UTR followed by a stem-loop structure (ATP5O (5′UTR)-SL-FF). As a control, cells were cotransfected with a Renilla reporter. Cells were treated for 24 h with Ad.IL-24 (100 pfu per cell) or Ad.vector (100 pfu per cell). Luminescence was monitored 48 h post-transfection. Data for Firefly luminescence normalized to Renilla luminescence. Each experiment was performed in independent triplicates, each consisting of three replicates, and data are shown as mean ± SD. *, p < 0.005 comparted to Ad.vector. RLU, relative light units.
	Figure 5. IL-24-dependent Sigma 1 Receptor (Sig1R) mediate eIF4A down-regulation, translation of mRNAs harboring structured 5′UTRs and mitochondrial dysfunction. (A) HeLa cells were transfected with firefly (FF) reporter harboring ATP5O 5′UTR followed by a stem-loop structure (ATP5O [5′UTR]-SL-FF). Cells were cotransfected with a Renilla reporter. Cells were incubated with the Sig1R agonist (SKF10047) or overexpression of Sig1R with or without Ad.IL-24 (100 pfu per cell). Data for firefly luminescence normalized to Renilla luminescence. Each experiment was performed in independent triplicates, each consisting of three replicates, and data are shown as mean ± SD. , p < 0.05. RLU, relative light units. (B) HeLa cells were treated as described in (A). Mitochondrial membrane potential (MMP) was analyzed by monitoring TMRE fluorescence intensity. Results are presented as mean ± SD (n = 3). , p < 0.05. (C) HeLa cells were incubated with the indicated conditions. Cells were collected, protein purified, and subjected to western blot analysis to detect eIF4A, eIF4G and eIF4E protein. (D) HeLa cells were treated as described in (A), and lysates were prepared and probed with antibodies specific to cdc25, c-myc, ODC, XIAP, Bcl2, Mcl1, BIRC5, and β-actin. (E) HeLa cells were treated as described in A, and lysates were prepared and probed with antibodies specific to cleaved caspase-3 and β-actin.