DAP5 ameliorates cisplatin-induced apoptosis of renal tubular cells.

BACKGROUND: Nephrotoxicity of cisplatin limits its clinical application. Cisplatin-induced acute renal tubular epithelial cell apoptosis is one of the major mechanisms of cisplatin nephrotoxicity. Here, the role and regulation of death-associated protein 5 (DAP5) in cisplatin-induced tubular cell apoptosis were investigated. METHODS: After upregulation of DAP5 expression by plasmid transfection and downregulation of DAP5 expression by small interfering RNA in human kidney tubular epithelial cell line (HKC) cells, the degree of cell apoptosis was assessed by flow cytometric analysis. The expression of Bax and Bcl-2 proteins was detected by Western blot analysis. The relationship between the PI3K/Akt/mTOR signaling pathway and DAP5 was also evaluated. RESULTS: During cisplatin-induced apoptosis in HKC cells, DAP5 underwent proteolytic fragmentation, yielding an 86-kDa species, DAP5/p86. Overexpression of DAP5/p97 and DAP5/p86 increased the translation of Bcl-2 and reduced the extent of cisplatin-induced apoptosis. Knockdown of DAP5 expression using small interfering RNA decreased the translation of Bcl-2 and increased the degree of apoptosis. Neither manipulation affected the expression of Bax. DAP5 expression was positively regulated by the PI3K/Akt/mTOR signaling pathway. CONCLUSION: Collectively, the results from the present study revealed a new role for DAP5 in cisplatin-induced apoptosis: DAP5/p97 and DAP5/p86 enhanced the translation of the anti-apoptotic protein Bcl-2 and inhibited cisplatin-induced apoptosis. The PI3K/Akt/mTOR signaling pathway may positively regulate the expression of DAP5.

Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G.

Assembly of the eIF4E/eIF4G complex has a central role in the regulation of gene expression at the level of translation initiation. This complex is regulated by the 4E-BPs, which compete with eIF4G for binding to eIF4E and which have tumor-suppressor activity. To pharmacologically mimic 4E-BP function we developed a high-throughput screening assay for identifying small-molecule inhibitors of the eIF4E/eIF4G interaction. The most potent compound identified, 4EGI-1, binds eIF4E, disrupts eIF4E/eIF4G association, and inhibits cap-dependent translation but not initiation factor-independent translation. While 4EGI-1 displaces eIF4G from eIF4E, it effectively enhances 4E-BP1 association both in vitro and in cells. 4EGI-1 inhibits cellular expression of oncogenic proteins encoded by weak mRNAs, exhibits activity against multiple cancer cell lines, and appears to have a preferential effect on transformed versus nontransformed cells. The identification of this compound provides a new tool for studying translational control and establishes a possible new strategy for cancer therapy.

RNA-mediated sequestration of the RNA helicase eIF4A by Pateamine A inhibits translation initiation.

Eukaryotic initiation factor 4A (eIF4A) is a member of the DEAD-box family of putative RNA helicases whose members are involved in many aspects of RNA metabolism. eIF4A is thought to facilitate binding of 43S preinitiation complexes to mRNAs by unwinding secondary structures present in the 5' untranslated region. Pateamine A, a small-molecule inhibitor of translation initiation, acts in an unusual manner by stimulating eIF4A activity. Herein, we report the elucidation of pateamine's mode of action. We demonstrate that Pateamine A is a chemical inducer of dimerization that forces an engagement between eIF4A and RNA and prevents eIF4A from participating in the ribosome-recruitment step of translation initiation.

N-acetyl-cysteine abolishes hydrogen peroxide-induced modification of eukaryotic initiation factor 4F activity via distinct signalling pathways.

During the oxidative stress generated by hydrogen peroxide (H2O2) in nerve growth factor (NGF)-differentiated PC12 cells, eIF4E binding protein (4E-BP1) and initiation factor 4E (eIF4E) phosphorylated levels decrease significantly, and an enhancement of the association of 4E-BP1 to eIF4E, which in turn decreases eIF4F formation is observed. The treatment with N-acetyl-cysteine (NAC) completely abolishes the H2O2-induced decrease in eIF4E phosphorylated levels, whereas the decrease in 4E-BP1 phosphorylated levels and eIF4F activity inhibition are significantly but not fully reversed. Rapamycin, the mammalian target of rapamycin (FRAP/mTOR) inhibitor, prevents the effect of NAC on H2O2-induced eIF4F complex formation inhibition. Besides the inhibitor induces a similar decrease in 4E-BP1 phosphorylated levels to that promote by H2O2. However, rapamycin has no effect on the NAC-induced recovery in phosphorylated eIF4E levels. Neither the MAP kinase inhibitors, PD98056 and SB203580, or the protein phosphatase 2A inhibitor, okadaic acid, mimic NAC effect on the H2O2-induced eIF4E dephosphorylation. Altogether our findings suggest that the effects caused by oxidative stress on eIF4s factors depends on two MAP kinase-independent signal transduction pathways, being at least one of them rapamycin-dependent.

Inhibition of eukaryotic translation initiation by the marine natural product pateamine A.

Translation initiation in eukaryotes is accomplished through the coordinated and orderly action of a large number of proteins, including the eIF4 initiation factors. Herein, we report that pateamine A (PatA), a potent antiproliferative and proapoptotic marine natural product, inhibits cap-dependent eukaryotic translation initiation. PatA bound to and enhanced the intrinsic enzymatic activities of eIF4A, yet it inhibited eIF4A-eIF4G association and promoted the formation of a stable ternary complex between eIF4A and eIF4B. These changes in eIF4A affinity for its partner proteins upon binding to PatA caused the stalling of initiation complexes on mRNA in vitro and induced stress granule formation in vivo. These results suggest that PatA will be a valuable molecular probe for future studies of eukaryotic translation initiation and may serve as a lead compound for the development of anticancer agents.

[Study on the expression of eIF families after elemene treatment].

OBJECTIVE: To investigate the activity of Caspase-3 and the expression of eukaryotic initiation factor families, bFGF and VEGF after elemene on laryngeal carcinoma HEp-2 cells. METHOD: The HEp-2 cells after elemene treatment were analyzed utilizing Westernblot and reverse transcriptase polymerase chain reaction (RT-PCR). The activity of Caspase-3 was assessed by colorimetric assay. RESULT: The activity of Caspase-3 was enhanced after elemene treatment. The protein expression of eIF4E, eIF4G, bFGF and VEGF were significantly inhibited by elemene; and the mRNA expression of bFGF and VEGF were inhibited either. CONCLUSION: Elemene can effectively inhibit the growth of HEp-2 cells and result in the alteration of activity of Caspase-3. There were significant correlations between the decreased expression of protein eIF4E, eIF4G, bFGF and VEGF. The mechanism of eIF4E and eIF4G decrease the expression of bFGF and VEGF is post-transcriptional.