p53 upregulates PLCε-IP3-Ca2+ pathway and inhibits autophagy through its target gene Rap2B

The tumor suppressor p53 plays a pivotal role in numerous cellular responses as it regulates cell proliferation, metabolism, cellular growth, and autophagy. In order to identify novel p53 target genes, we utilized an unbiased microarray approach and identified Rap2B as a robust candidate, which belongs to the Ras-related GTP-binding protein superfamily and exhibits increased expression in various human cancers. We demonstrated that p53 increases the intracellular IP3 and Ca2+ levels and decreases the LC3 protein levels through its target gene Rap2B, suggesting that p53 can inhibit the autophagic response triggered by starvation via upregulation of the Rap2B-PLCε-IP3-Ca2+ pathway. As a confirmed target gene of p53, we believe that further investigating potential functions of Rap2B in autophagy and tumorigenesis will provide a novel strategy for cancer therapy

Rap2B promotes proliferation, migration and invasion of human breast cancer through calcium-related ERK1/2 signaling pathway

Rap2B, a member of GTP-binding proteins, is widely upregulated in many types of tumors and promotes migration and invasion of human suprarenal epithelioma. However, the function of Rap2B in breast cancer is unknown. Expression of Rap2B was examined in breast cancer cell lines and human normal breast cell line using Western blot analysis. Using the CCK-8 cell proliferation assay, cell cycle analysis, and transwell migration assay, we also elucidated the role of Rap2B in breast cancer cell proliferation, migration, and invasion. Results showed that the expression of Rap2B is higher in tumor cells than in normal cells. Flow cytometry and Western blot analysis revealed that Rap2B elevates the intracellular calcium level and further promotes extracellular signal-related kinase (ERK) 1/2 phosphorylation. By contrast, calcium chelator BAPTM/AM and MEK inhibitor (U0126) can reverse Rap2B-induced ERK1/2 phosphorylation. Furthermore, Rap2B knockdown inhibits cell proliferation, migration, and invasion abilities via calcium related-ERK1/2 signaling. In addition, overexpression of Rap2B promotes cell proliferation, migration and invasion abilities, which could be neutralized by BAPTM/AM and U0126. Taken together, these findings shed light on Rap2B as a therapeutic target for breast cancer

Integration of Consonant and Pitch Processing as Revealed by the Absence of Additivity in Mismatch Negativity

Consonants, unlike vowels, are thought to be speech specific and therefore no interactions would be expected between consonants and pitch, a basic element for musical tones. The present study used an electrophysiological approach to investigate whether, contrary to this view, there is integrative processing of consonants and pitch by measuring additivity of changes in the mismatch negativity (MMN) of evoked potentials. The MMN is elicited by discriminable variations occurring in a sequence of repetitive, homogeneous sounds. In the experiment, event-related potentials (ERPs) were recorded while participants heard frequently sung consonant-vowel syllables and rare stimuli deviating in either consonant identity only, pitch only, or in both dimensions. Every type of deviation elicited a reliable MMN. As expected, the two single-deviant MMNs had similar amplitudes, but that of the double-deviant MMN was also not significantly different from them. This absence of additivity in the double-deviant MMN suggests that consonant and pitch variations are processed, at least at a pre-attentive level, in an integrated rather than independent way. Domain-specificity of consonants may depend on higher-level processes in the hierarchy of speech perception

CPEB2 inhibit cell proliferation through upregulating p21 mRNA stability in glioma

Abstract Glioma is the most common primary malignant brain tumor in adults and remains an incurable disease at present. Thus, there is an urgent need for progress in finding novel molecular mechanisms that control the progression of glioma which could be used as therapeutic targets for glioma patients. The RNA binding protein cytoplasmic polyadenylate element-binding protein 2 (CPEB2) is involved in the pathogenesis of several tumors. However, the role of CPEB2 in glioma progression is unknown. In this study, the functional characterization of the role and molecular mechanism of CPEB2 in glioma were examined using a series of biological and cellular approaches in vitro and in vivo. Our work shows CPEB2 is significantly downregulated in various glioma patient cohorts. Functional characterization of CPEB2 by overexpression and knockdown revealed that it inhibits glioma cell proliferation and promotes apoptosis. CPEB2 exerts an anti-tumor effect by increasing p21 mRNA stability and inducing G1 cell cycle arrest in glioma. Overall, this work stands as the first report of CPEB2 downregulation and involvement in glioma pathogenesis, and identifies CPEB2 as an important tumor suppressor gene through targeting p21 in glioma, which revealed that CPEB2 may become a promising predictive biomarker for prognosis in glioma patients

MDA-7/IL-24 induces Bcl-2 denitrosylation and ubiquitin-degradation involved in cancer cell apoptosis.

MDA-7/IL-24 was involved in the specific cancer apoptosis through suppression of Bcl-2 expression, which is a key apoptosis regulatory protein of the mitochondrial death pathway. However, the underlying mechanisms of this regulation are unclear. We report here that tumor-selective replicating adenovirus ZD55-IL-24 leads to Bcl-2 S-denitrosylation and concomitant ubiquitination, which take part in the 26S proteasome degradation. IL-24-siRNA completely blocks Bcl-2 ubiquitination via reversion of Bcl-2 S-denitrosylation and protects it from proteasomal degradation which confirmed the significant role of MDA-7/IL-24 in regulating posttranslational modification of Bcl-2 in cancer cells. Nitric oxide (NO) is a key regulator of protein S-nitrosylation and denitrosylation. The NO donor, sodium nitroprusside (SNP), down-regulates Bcl-2 S-denitrosylation, attenuates Bcl-2 ubiquitination and subsequently counteracts MDA-7/IL-24 induced cancer cell apoptosis, whereas NO inhibitor 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (PTIO) shows the opposite effect. At the same time, these NO modulators fail to affect Bcl-2 phosphorylation, suggesting that NO regulates Bcl-2 stability in a phosphorylation-independent manner. In addition, Bcl-2 S-nitrosylation reduction induced by ZD55-IL-24 was attributed to both iNOS decrease and TrxR1 increase. iNOS-siRNA facilitates Bcl-2 S-denitrosylation and ubiquitin-degradation, whereas the TrxR1 inhibitor auranofin prevents Bcl-2 from denitrosylation and ubiquitination, thus restrains the caspase signal pathway activation and subsequent cancer cell apoptosis. Taken together, our studies reveal that MDA-7/IL-24 induces Bcl-2 S-denitrosylation via regulation of iNOS and TrxR1. Moreover, denitrosylation of Bcl-2 results in its ubiquitination and subsequent caspase protease family activation, as a consequence, apoptosis susceptibility. These findings provide a novel insight into MDA-7/IL-24 induced growth inhibition and carcinoma apoptosis

Effect of ZD55-IL-24 on Bcl-2 S-nitrosylation and ubiquitination.

<p>(<b>A</b>) Hela, A375 and 7860 cells in response to ZD55-IL-24 (20 MOI) were prepared for immunoprecipitation using anti-Bcl-2 antibody. The resulting immune complexes were analyzed for anti-S-nitrosocysteine by Western blotting and stood for Bcl-2 S-nitrosylation level at the different time points 12 h, 24 h, 36 h and 48 h, respectively. (<b>B</b>) Time course of Bcl-2 ubiquitination in Hela cells was detected by immunoprecipitation using anti-Bcl-2 antibody and then followed by immunoblotting with anti-ubiquitin antibody. (<b>C</b>) Effect of IL-24-siRNA (100 nM) upon IL-24, Bcl-2 expression, Bcl-2 S-nitrosylation and ubiquitination in Hela cells was detected by Western blotting and co-immunoprecipitation. β-actin was used as a loading control. The corresponding bands were scanned and the optical density (O.D.) was determined as the fold change versus control group. Data are means±standard deviation (S.D.) from three independent experiments (n = 3); *<i>p</i><0.05 versus control group; ^#<i>p</i><0.05 versus scrambled siRNA group.</p

Effect of NO modulators on caspase signal pathway and Hela cell viability.

<p>(A) Hela cells were pretreated with ZD55-IL24 (20 MOI) for 24 h and then added with NO donor SNP(2 mM), NO inhibitor PTIO(300 µM), SNP co-administration of reducing agent DTT(10 mM) and proteasomal inhibitor MG132(10 µM) for 6 h respectively. Procaspase-9, cleaved caspase-9, procaspase-3, cleaved caspase-3 were detected by Western blotting. (B) Effect of NO modulators SNP, PTIO, SNP+DTT and proteasome inhibitor MG132 on early apoptosis and late apoptosis in Hela cells treated with ZD55-IL-24 were detected by staining with Annexin V-FITC (green color) and Propidium Iodide (red color) respectively. (C) Hela cells were stained with Annexin V-FITC and Propidium Iodide and immediately analyzed by flow cytometry. (D) Effects of NO modulators and MG132 on Hela cell viability were determined by MTT assay. The corresponding bands were scanned and the optical density (O.D.) was determined as the fold change versus control group. Data are means±standard deviation (S.D.) from three independent experiments (n = 3). ^#<i>p</i><0.05 versus ZD55-EGFP group; *<i>p</i><0.05 versus ZD55-IL-24 group; ^@<i>p</i><0.05 versus DMSO group.</p

Schematic model of IL-24 induced cancer cell apoptosis.

<p>(<b>I</b>) IL-24 inhibits iNOS expression leading to reduction of NO turnover and attenuation of Bcl-2 S-nitrosylation. (<b>II</b>) Trx denitrosylates S-nitrosylated Bcl-2 through its dithiol moiety, thereby forming a reduced Bcl-2 and oxidized Trx; oxidized Trx is reduced (and therefore reactivated) by the seleno-flavoprotein Trx reductase(TrxR) and NADPH, suggesting that TrxR through reducing oxidized Trx may facilitate Bcl-2 denitrosylation. (<b>III</b>) Under basal condition, Bcl-2 S-nitrosylation stabilizes protein structure and resists to the ubiquitin-proteasome degradation. Formation of heterodimers with proapoptotic protein such as Bax, inhibition of cytochrome c release and caspase protease family activation, and regulation of mitochondrial transmembrane potential are some of mechanisms by which Bcl-2 exerts its anti-apoptotic effect. (<b>IV</b>) In response to IL-24, Bcl-2 S-denitrosylation via both iNOS decrease (<b>a</b>) and TrxR1 increase (<b>b</b>) facilitates Bcl-2 ubiquitination, which finally is degraded by the 26S proteasome. Bax triggers release of cytochrome c and activation of caspase protease family, which mediated the intracellular proteolysis that is characteristic of cell apoptosis.</p

Effect of iNOS on Bcl-2 S-nitrosylaiton, ubiquitination and protein expression and caspase signal pathway.

<p>(A) Time course analysis of iNOS expression in Hela, A375 and 7860 cells treated with ZD55-IL-24 (20 MOI). (B) Hela, A375 and 7860 cells were transfected with iNOS-siRNA (100 nM) for 12 h, then treated with ZD55-IL-24 for 12 h. Effect of iNOS-siRNA on iNOS expression was analyzed by Western blotting. (C) Effect of iNOS-siRNA on Bcl-2 protein expression was analyzed by Western blotting. (D) Effect of iNOS-siRNA on Bcl-2 S-nitrosylation were detected by immunoprecipitation using anti-Bcl-2 antibody and then followed by immunoblotted with anti-S-nitrosocysteine antibody. (E) Effect of iNOS-siRNA on ubiquitin-Bcl-2 was detected by immunoprecipitation using anti-Bcl-2 antibody and then followed by immunoblotting with anti-ubiquitin antibody. (F) Effect of iNOS-siRNA on caspase-9, caspase-3 and PARP in Hela cells was detected by Western blotting. The corresponding bands were scanned and the optical density (O.D.) was determined as the fold change versus control group. Data are means±standard deviation (S.D.) from three independent experiments (n = 3). *<i>p</i><0.05 versus control group; ^#<i>p</i><0.05 versus scrambled siRNA group.</p

Effect of TrxR1 on Bcl-2 S-nitrosylaiton, ubiquitination and protein expression and caspase signal pathway.

<p>(<b>A</b>) Time course analysis of TrxR1 expression in Hela, A375 and 7860 cells treated with ZD55-IL-24 (20 MOI). (<b>B</b>) Hela, A375 and 7860 cells were treated with ZD55-IL-24 for 24 h and then added with TrxR1 inhibitor auranofin (5 µM) for 6 h. Effect of TrxR1 inhibitor auranofin on TrxR1 protein expression was detected by Western blotting. (C) Effect of TrxR1 inhibitor auranofin on Bcl-2 protein expression in Hela, A375 and 7860 cells was detected by the western blot assay. (D) Effect of TrxR1 inhibitor auranofin on Bcl-2 S-nitrosylation was detected by immunoprecipitation using anti-Bcl-2 antibody and then followed by immunoblotting with anti-S-nitrosocysteine antibody. (E) Effect of TrxR1 inhibitor auranofin on Bcl-2 ubiquitination in Hela cells was detected by immunoprecipitation using anti-Bcl-2 antibody and then followed by immunoblotting with anti-ubiquitin antibody. (F) Effect of TrxR1 inhibitor auranofin on caspase-9, 3 and PARP was detected by Western blotting. β-actin was used as a loading control. The corresponding bands were scanned and the optical density (O.D.) was determined as the fold change versus control group. Data are means±standard deviation (S.D.) from three independent experiments (n = 3). *<i>p</i><0.05 versus ZD55-EGFP; ^#<i>p</i><0.05 versus DMSO group.</p