Structural scaffold for eIF4E binding selectivity of 4E‐BP isoforms: crystal structure of eIF4E binding region of 4E‐BP2 and its comparison with that of 4E‐BP1

To clarify the higher eukaryotic initiation factor 4E (eIF4E) binding selectivity of 4E‐binding protein 2 (4E‐BP2) than of 4E‐BP1, as determined by Trp fluorescence analysis, the crystal structure of the eIF4E binding region of 4E‐BP2 in complex with m⁷GTP‐bound human eIF4E has been determined by X‐ray diffraction analysis and compared with that of 4E‐BP1. The crystal structure revealed that the Pro47‐Ser65 moiety of 4E‐BP2 adopts a L ‐shaped conformation involving extended and α‐helical structures and extends over the N‐terminal loop and two different helix regions of eIF4E through hydrogen bonds, and electrostatic and hydrophobic interactions; these features were similarly observed for 4E‐BP1. Although the pattern of the overall interaction of 4E‐BP2 with eIF4E was similar to that of 4E‐BP1, a notable difference was observed for the 60–63 sequence in relation to the conformation and binding selectivity of the 4E‐BP isoform, i.e. Met‐Glu‐Cys‐Arg for 4E‐BP1 and Leu‐Asp‐Arg‐Arg for 4E‐BP2. In this paper, we report that the structural scaffold of the eIF4E binding preference for 4E‐BP2 over 4E‐BP1 is based on the stacking of the Arg63 planar side chain on the Trp73 indole ring of eIF4E and the construction of a compact hydrophobic space around the Trp73 indole ring by the Leu59‐Leu60 sequence of 4E‐BP2. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Modulation of pro- and anti-apoptotic factors in human melanoma cells exposed to histone deacetylase inhibitors

Valproic acid (VPA, 2-propylpentanoic acid) is an established drug in the long-term therapy of epilepsy. Recently, VPA was demonstrated to inhibit histone deacetylases (HDACs) class I enzyme at therapeutically relevant concentrations, thereby, mimicking the prototypical histone deacetylase inhibitors, tricostatin A (TSA) or suberoylanilide hydroxamic acid (SAHA). In the present study, we investigated the cellular effects of VPA, TSA and SAHA on four human melanoma cell lines (WM115, WM266, A375, SK-Mel28) with particular reference to the modulation of regulators of apoptosis, including Bcl-2, BclXL, Mcl-1, Apaf-1, BclXs, NOXA, TRAIL-R1, TRAIL-R2, caspase 8, and survivin). Firstly, we found that VPA induced apoptosis in two of the four human melanoma cell lines, while both TSA and SAHA exhibited an antiproliferative and apoptotic effects in all four cell lines, a different expression of Bcl-2 and BclX(L/S) occurred. On the other hand, SAHA and VPA modulated differently pro- and anti-apoptotic factors. In particular, the treatment with VPA enhanced the level of expression of survivin only in VPA-resistant cell lines, whereas down-regulation of survivin was induced by VPA and SAHA in VPA-sensitive cells. In the latter, since activation of caspase 8 was documented, a receptor-mediated apoptosis was suggested. Taken together, our results suggest that HDAC inhibitors may represent a promising therapeutic strategy to treat melanoma.     

Gene expression profile of human lymphoid CEM cells sensitive and resistant to glucocorticoid-evoked apoptosis

Three closely related clones of leukemic lymphoid CEM cells were compared for their gene expression responses to the glucocorticoid dexamethasone (Dex). All three contained receptors for Dex, but only two responded by undergoing apoptosis. After a time of exposure to Dex that ended late in the interval preceding onset of apoptosis, gene microarray analyses were carried out. The results indicate that the expression of a limited, distinctive set of genes was altered in the two apoptosis-prone clones, not in the resistant clone. That clone showed altered expression of different sets of genes, suggesting that a molecular switch converted patterns of gene expression between the two phenotypes: apoptosis-prone and apoptosis-resistant. The results are consistent with the hypothesis that altered expression of a distinctive network of genes after glucocorticoid administration ultimately triggers apoptosis of leukemic lymphoid cells. The altered genes identified provide new foci for study of their role in cell death.     

4E-BP1 degradation and eIF4E truncation occur spatially distinctly in the porcine uterine epithelia and are features of noninvasive implantation in the pig

The implantation of the blastocyst into the endometrium is an indispensable premise for successful embryonic development. This process is regulated by maternal and embryonic signals that influence gene expression at the translational level, among other processes. Recently, we have shown that proteolytical cleavage of the prototypical 25‐kDa, mRNA cap‐binding protein eIF4E produces a stable variant with a molecular mass of approximately 23 kDa exclusively in the porcine endometrium during implantation. This is accompanied by dephosphorylation and reduction of the abundant repressor 4E‐BP1. Here, we investigate the distribution of the truncated eIF4E and of 4E‐BP1 in the porcine uterine tissue, their binding in native samples, and we analyzed eIF4E‐, eIF4G‐, and 4E‐BP1‐specific proteolytic activities. Our results show that in pigs, the truncated eIF4E is located in the endometrial luminal epithelium during implantation. Neither glandulary tissue nor stroma expressed any truncated eIF4E. The reduced abundance of 4E‐BP1 during implantation is mainly the result of decay in the glandular epithelia. Moreover, steroid replacements, in vitro protease assays, and cell lysate fractionation showed that eIF4E cleavage and 4E‐BP1 decay both depended on the ovarian steroid hormones estradiol and progestrone, but these effects are the result of different proteolytic activities. Although eIF4G cleavage also depends on calcium, stimulation by these steroids could not be established. We propose that the translation initiation process in the endometrium is differently regulated by the truncated eIF4E, utilizing different abundances of 4E‐BP1 and binding dynamic of eIF4E/4E‐BP1 in distinct forms of implantation. Mol. Reprod. Dev. 78:895–905, 2011. © 2011 Wiley Periodicals, Inc.     

HPV E6/E7 promotes aerobic glycolysis in cervical cancer by regulating IGF2BP2 to stabilize m6A-MYC expression

Enhanced aerobic glycolysis constitutes an additional source of energy for tumor proliferation and metastasis. Human papillomavirus (HPV) infection is the main cause of cervical cancer (CC); however, the associated molecular mechanisms remain poorly defined, as does the relationship between CC and aerobic glycolysis. To investigate whether HPV 16/18 E6/E7 can enhance aerobic glycolysis in CC, E6/E7 expression was knocked down in SiHa and HeLa cells using small interfering RNA (siRNA). Then, glucose uptake, lactate production, ATP levels, reactive oxygen species (ROS) content, extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were evaluated. RNA-seq was used to probe the molecular mechanism involved in E6/E7-driven aerobic glycolysis, and identified IGF2BP2 as a target of E6/E7. The regulatory effect of IGF2BP2 was confirmed by qRT-PCR, western blot, and RIP assay. The biological roles and mechanisms underlying how HPV E6/E7 and IGF2BP2 promote CC progression were confirmed in vitro and in vivo. Human CC tissue microarrays were used to analyze IGF2BP2 expression in CC. The knockdown of E6/E7 and IGF2BP2 attenuated the aerobic glycolytic capacity and growth of CC cells, while IGF2BP2 overexpression rescued this effect in vitro and in vivo. IGF2BP2 expression was higher in CC tissues than in adjacent tissues and was positively correlated with tumor stage. Mechanistically, E6/E7 proteins promoted aerobic glycolysis, proliferation, and metastasis in CC cells by regulating MYC mRNA m⁶A modifications through IGF2BP2. We found that E6/E7 promote CC by regulating MYC methylation sites via activating IGF2BP2 and established a link between E6/E7 and the promotion of aerobic glycolysis and CC progression. Blocking the HPV E6/E7-related metabolic pathway represents a potential strategy for the treatment of CC.     

D-Pinitol treatment induced the apoptosis in human leukemia MOLT-4 cells by improved apoptotic signaling pathway

Cancer is still remain as a global burden with the 18.1 million and 9.6 million new cases and mortlities, respectively estimated globally. Leukemia may arise at all ages varied from the infants to elders. In this exploration, we planned to evaluate the antiproliferative effect of D-pinitol on human leukemia MOLT-4 cells. Anticancer potential of D-pinitol was examined using MTT assay. Reactive oxygen species (ROS) generation was studied by fluorescence microscopic method using DCFH-DA staining. Apoptotic morphological alterations were determined by dual staining (acridine orange and ethidium bromide). Western blot and ELISA methods were employed to study apoptotic protein expression. D-pinitol treatment significantly induced cytotoxicity in human leukemia MOLT-4 cells. We observed that D-pinitol induces the generation of ROS in MOLT-4 cells. Further, we noticed that D-pinitol significantly induced apoptosis in a dosage dependent manner. Moreover, western blot and ELISA based analysis revealed that D-pinitol elevated the Bax, Caspase-3, Caspase-9 and attenuated the Bcl-2 expression in leukemic cancer cell. Our findings suggest that D-pinitol treatment induces the apoptosis in human leukemic cells by generating intracellular ROS and modulating apoptotic protein expression.     

In vitro and in vivo activity of 4-thio-uridylate against JY cells, a model for human acute lymphoid leukemia

We have previously reported the in vitro anti-proliferative effect of 4-thio-uridylate (s⁴UMP) on OCM-1 uveal melanoma cells. Here, we assessed the efficacy of s⁴UMP on JY cells. Treatment of JY cells with s⁴UMP suppressed their colony forming activity and induced apoptosis; healthy human bone marrow granulocyte–macrophage progenitor cells were 14-fold less sensitive to the nucleotide. In vivo effectiveness of s⁴UMP was determined using xenograft SCID mouse model. s⁴UMP decreased the cell number and colony forming activity of the total cell content of the femur of SCID mice transplanted with JY cells without affecting the bone marrow of healthy mice. These results suggest that s⁴UMP alone or in combination with other clinically approved anti-leukemic remedies should be further explored as a potential novel therapeutic agent.