LY294002 potentiates the anti-cancer effect of oxaliplatin for gastric cancer via death receptor pathway

AIM:To examine the effects of combined treatment of oxaliplatin and phosphatidylinositol 3’-kinase inhibitor,2-(4-morpholinyl) -8-phenyl-4H-1-benzopyran-4-one(LY294002) for gastric cancer. METHODS:Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide assay.Apoptotic cells were detected by flow cytometric analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay.Western blotting and immuno-precipitation were used to examine protein expres...     

Overcoming cancer cell resistance to Smac mimetic induced apoptosis by modulating cIAP-2 expression

Smac mimetics target cancer cells in a TNFα-dependent manner, partly via proteasome degradation of cellular inhibitor of apoptosis 1 (cIAP1) and cIAP2. Degradation of cIAPs triggers the release of receptor interacting protein kinase (RIPK1) from TNF receptor I (TNFR1) to form a caspase-8 activating complex together with the adaptor protein Fas-associated death domain (FADD). We report here a means through which cancer cells mediate resistance to Smac mimetic/TNFα-induced apoptosis and corresponding strategies to overcome such resistance. These human cancer cell lines evades Smac mimetic-induced apoptosis by up-regulation of cIAP2, which although initially degraded, rebounds and is refractory to subsequent degradation. cIAP2 is induced by TNFα via NF-κB and modulation of the NF-κB signal renders otherwise resistant cells sensitive to Smac mimetics. In addition, other signaling pathways, including phosphatidyl inositol-3 kinase (PI3K), have the potential to concurrently regulate cIAP2. Using the PI3K inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, cIAP2 up-regulation was suppressed and resistance to Smac mimetics-induced apoptosis was also overcome.     

Protection of ghrelin postconditioning on hypoxia/reoxygenation in gastric epithelial cells

AIM: To investigate the protective effect and mechanisms of ghrelin postconditioning against hypoxia/reoxygenation (H/R)-induced injury in human gastric epithelial cells. METHODS: The model of H/R injury was established in gastric epithelial cell line (GES-1) human gastric epithelial cells. Cells were divided into seven groups: normal control group (N); H/R postconditioning group; DMSO postconditioning group (DM); ghrelin postconditioning group (GH); D-Lys3-GHRP-6 + ghrelin postconditioning group (D + GH); capsazepine + ghrelin postconditioning group (C + GH); and LY294002 + ghrelin postconditioning group (L + GH). 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to detect GES-1 cell viability. Hoechst 33258 fluorochrome staining and flow cytometry were conducted to determine apoptosis of GES-1cells. Spectrophotometry was performed to determine release of lactate dehydrogenate (LDH). Protein expression of Bcl-2, Bax, Akt, and glycogen synthase kinase (GSK)-3β was determined by western blotting. Expression of vanilloid receptor subtype 1 (VR1), Akt and GSK-3β was observed by immunocytochemistry. RESULTS: Compared with the H/R group, cell viability of the GH group was significantly increased in a dosedependent manner (55.9% ± 10.0% vs 69.6% ± 9.6%, 71.9% ± 17.4%, and 76.3% ± 13.3%). Compared with the H/R group, the percentage of apoptotic cells in the GH group significantly decreased (12.38% ± 1.51% vs 6.88% ± 0.87%). Compared with the GH group, the percentage of apoptotic cells in the D + GH group, C + GH group and L + GH groups significantly increased (11.70% ± 0.88%, 11.93% ± 0.96%, 10.20% ± 1.05% vs 6.88% ± 0.87%). There were no significant differences in the percentage of apoptotic cells between the H/R and DM groups (12.38% ± 1.51% vs 13.00% ± 1.13%). There was a significant decrease in LDH release following ghrelin postconditioning compared with the H/R group (561.58 ± 64.01 U/L vs 1062.45 ± 105.29 U/L). There was a significant increase in LDH release in the D + GH, C + GH and L + GH groups compared with the GH group (816.89 ± 94.87 U/L, 870.95 ± 64.06 U/L, 838.62 ± 118.45 U/L vs 561.58 ± 64.01 U/L). There were no significant differences in LDH release between the H/R and DM groups (1062.45 ± 105.29 U/L vs 1017.65 ± 68.90 U/L). Compared with the H/R group, expression of Bcl-2 and Akt increased in the GH group, whereas expression of Bax and GSK3β decreased. Compared with the GH group, expression of Bcl-2 decreased and Bax increased in the D + GH, C + GH and L + GH groups, and Akt decreased and GSK-3β increased in the L + GH group. The H/R group also upregulated expression of VR1 and GSK-3β and downregulated Akt. The number of VR1-positive and Akt-positive cells in the GH group significantly increased, whereas the number of GSK-3β-positive cells significantly decreased. These effects of ghrelin were reversed by capsazepine and LY294002.CONCLUSION: Ghrelin postconditioning protected against H/R-induced injury in human gastric epithelial cells, which indicated that this protection might be associated with GHS-R, VR1 and the PI3K/Akt signaling pathway.     

Menin and GIP are inversely regulated by food intake and diet via PI3/AKT signaling in the proximal duodenum

Background and Aims:

Ingestion of food stimulates the secretion of incretin peptides glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 to ensure the proper absorption and storage of nutrients. Menin is the 67 kDa protein product of the MEN1 gene recently reported to have a role in metabolism. In this study, we will determine the regulation of menin in the proximal duodenum by food intake and diet in correlation with GIP levels in the proximal duodenum of mice after an 18 h fast followed by 4 and 7 h refeeding and 3 months of high-fat diet.

Methods:

A dual luciferase assay was used to determine GIP promoter activity and ELISA was used to measure the levels of GIP after inhibition of menin through small interfering RNA (siRNA) and exposure to MAPK and AKT inhibitors. Colocalization of menin and GIP were determined by immunofluorescence.

Results:

Menin and GIP expression are regulated by fasting, refeeding and diet in the proximal duodenum. Overexpression of menin in STC-1 cells significantly inhibited GIP mRNA and promoter activity, whereas menin siRNA upregulated GIP levels. Inhibition of GIP expression by the PI3/AKT inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, was abrogated in STC-1 cells with reduced menin levels, whereas the MAPK inhibitor, UO126, inhibited the expression of GIP independent of menin. Exposure of STC-1 cells to GIP reduced menin expression in a dose-dependent manner via PI3K-AKT signaling.

Conclusion:

Feeding and diet regulates the expression of menin, which inversely correlates with GIP levels in the proximal duodenum. In vitro assays indicate that menin is a negative regulator of GIP via inhibition of PI3K-AKT signaling. We show menin colocalizing with GIP in K cells of the proximal gut and hypothesize that downregulation of menin may serve as a mechanism by which GIP is regulated in response to food intake and diet.     

Cytoplasmic localization of wild-type survivin is associated with constitutive activation of the PI3K/Akt signaling pathway and represents a favorable prognostic factor in patients with acute myeloid leukemia

Survivin is over-expressed in most hematologic malignancies but the prognostic significance of the subcompartmental distribution of wild-type or splicing variants in acute myeloid leukemia has not been addressed yet. Using western blotting, we assessed the expression of wild-type survivin and survivin splice variants 2B and Delta-Ex3 in nuclear and cytoplasmic protein extracts in samples taken from 105 patients at the time of their diagnosis of acute myeloid leukemia. Given that survivin is a downstream effector of the PI3K/Akt signaling pathway, survivin expression was also correlated with pSer473-Akt. Wild-type survivin and the 2B splice variant were positive in 76.3% and 78.0% of samples in the nucleus, cytoplasm or both, whereas the Delta-Ex3 isoform was only positive in the nucleus in 37.7% of samples. Cytoplasmic localization of wild-type survivin was significantly associated with the presence of high levels of pSer473-Akt (P<0.001). Inhibition of the PI3K/Akt pathway with wortmannin and {"type":"entrez-nucleotide","attrs":{"text":"Ly294002","term_id":"1257998346","term_text":"LY294002"}}Ly294002 caused a significant reduction in the expression of cytoplasmic wild-type survivin. The presence of cytoplasmic wild-type survivin and pSer473-Akt was associated with a lower fraction of quiescent leukemia stem cells (P=0.02). The presence of cytoplasmic wild-type survivin and pSer473-Akt were favorable independent prognostic factors. Moreover, the activation of the PI3K/Akt pathway with expression of cytoplasmic wild-type survivin identified a subgroup of acute myeloid leukemia patients with an excellent outcome (overall survival rate of 60.0±21.9% and relapse-free survival of 63.0±13.5%). Our findings suggest that cytoplasmic wild-type survivin is a critical downstream effector of the PI3K/Akt pathway leading to more chemosensitive cells and a more favorable outcome in acute myeloid leukemia.     

Activated protein C ligation of ApoER2 (LRP8) causes Dab1-dependent signaling in U937 cells

Binding of activated protein C (APC) to cells triggers multiple beneficial cytoprotective activities that suppress apoptosis, inflammation, and endothelial barrier breakdown. One paradigm for APC''s signaling emphasizes its binding to endothelial cell protein C receptor (EPCR) and subsequent protease activated receptor (PAR)-1 activation. Here we used human monocytic-like U937 cells to evaluate apolipoprotein E receptor 2 (ApoER2)-dependent signaling by APC and found that APC initiated rapid phosphorylation of Tyr-220 in the adaptor protein disabled-1 (Dab1) and of Ser-473 in Akt. APC also induced phosphorylation of Ser-9 in glycogen synthase kinase 3β (GSK3β), which was blocked by the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. Receptor-associated protein (RAP), a general antagonist for binding of ligands to LDL receptor family members, inhibited APC-induced phosphorylation of Dab1 and GSK3β, whereas anti-EPCR or anti-PAR1 blocking antibodies did not. Knocking down ApoER2 by using siRNA-ablated APC induced Dab1 phosphorylation, suggesting that RAP-sensitive APC-induced signaling requires ApoER2. In surface plasmon resonance equilibrium binding studies, APC bound with high affinity to soluble (s) ApoER2 (apparent K_d, ≈30 nM) but not to soluble very low density lipoprotein receptor. RAP blocked APC binding to sApoER2 but not to sEPCR. RAP blocked binding of U937 cells to immobilized APC. RAP also blocked APC''s ability to inhibit endotoxin-induced tissue factor pro-coagulant activity of U937 cells. Thus, we propose that ligation of ApoER2 by APC signals via Dab1 phosphorylation and subsequent activation of PI3K and Akt and inactivation of GSK3β, thereby contributing to APC''s beneficial effects on cells.     

High-fibre diet and Lactobacillus paracasei B21060 in symptomatic uncomplicated diverticular disease

AIM:To investigate in symptomatic uncomplicated diverticular disease the efficacy of symbiotics associated with a high-fibre diet on abdominal symptoms.METHODS:This study was a multicentre,6-mo randomized,controlled,parallel-group intervention with a preceding 4-wk washout period.Consecutive outpatients with symptomatic uncomplicated diverticular disease,aged 40-80 years,evaluated in 4 Gastroenter-ology Units,were enrolled.Symptomatic uncomplicated diverticular disease patients were randomized to two treatment arms A or B.Treatment A(n = 24 patients) received 1 symbiotic sachet Flortec(Lactobacillus paracasei B21060) once daily plus high-fibre diet for 6 mo.Treatment B(n = 21 patients) received high-fibre diet alone for 6 mo.The primary endpoint was regression of abdominal symptoms and change of symptom severity after 3 and 6 mo of treatment.RESULTS:In group A,the proportion of patients with abdominal pain 24 h decreased from 100% at baseline to 35% and 25% after 3 and 6 mo,respectively(P 0.001).In group B the proportion of patients with this symptom decreased from 90.5% at baseline to 61.9% and 38.1% after 3 and 6 mo,respectively(P = 0.001).Symptom improvement became statistically significant at 3 and 6 mo in group A and B,respectively.The proportion of patients with abdominal pain 24 h decreased from 60% to 20% then 5% after 3 and 6 mo,respectively in group A(P 0.001) and from 33.3% to 9.5% at both 3 and 6 mo in group B(P = 0.03).In group A the proportion of patients with abdominal bloating significantly decreased from 95% to 60% after 3 mo,and remained stable(65%) at 6-mo follow-up(P = 0.005) while in group B,no significant changes in abdominal bloating was observed(P = 0.11).After 6 mo of treatment,the mean visual analogic scale(VAS) values of both short-lasting abdominal pain(VAS,mean ± SD,group A:4.6 ± 2.1 vs 2.2 ± 0.8,P = 0.02;group B:4.6 ± 2.9 vs 2.0 ± 1.9,P = 0.03) and abdominal bloating(VAS,mean ± SD,group A:5.3 ± 2.2 vs 3.0 ± 1.7,P = 0.005;group B:5.3 ± 3.2 vs 2.3 ± 1.9,P = 0.006) decreased in both groups,whilst the VAS values of prolonged abdominal pain decreased in the Flortec group,but remained unchanged in the high-fibre diet group(VAS,mean ± SD,group A:6.5 ± 1.5 vs 4.5 ± 2.1,P = 0.052;group B:4.5 ± 3.8 vs 5.5 ± 3.5).CONCLUSION:A high-fibre diet is effective in relievingabdominal symptoms in symptomatic uncomplicated diverticular disease.This treatment may be implemented by combining the high-fibre diet with Flortec.     

Signature of prognostic epithelial–mesenchymal transition related long noncoding RNAs (ERLs) in hepatocellular carcinoma

Reliable biomarkers are of great significance for the treatment and diagnosis of hepatocellular carcinoma (HCC). This study identified potential prognostic epithelial–mesenchymal transition related lncRNAs (ERLs) by the cancer genome atlas (TCGA) database and bioinformatics.The differential expression of long noncoding RNA (lncRNA) was obtained by analyzing the lncRNA data of 370 HCC samples in TCGA. Then, Pearson correlation analysis was carried out with EMT related genes (ERGs) from molecular signatures database. Combined with the univariate Cox expression analysis of the total survival rate of hepatocellular carcinoma (HCC) patients, the prognostic ERLs were obtained. Then use “step” function to select the optimal combination of constructing multivariate Cox expression model. The expression levels of ERLs in HCC samples were verified by real-time quantitative polymerase chain reaction.Finally, we identified 5 prognostic ERLs ({"type":"entrez-nucleotide","attrs":{"text":"AC023157.3","term_id":"6980118","term_text":"AC023157.3"}}AC023157.3, {"type":"entrez-nucleotide","attrs":{"text":"AC099850.3","term_id":"25046596","term_text":"AC099850.3"}}AC099850.3, AL031985.3, {"type":"entrez-nucleotide","attrs":{"text":"AL365203.2","term_id":"9743577","term_text":"AL365203.2"}}AL365203.2, CYTOR). The model showed that these prognostic markers were reliable independent predictors of risk factors (P value <.0001, hazard ratio [HR] = 2.400, 95% confidence interval [CI] = 1.667–3.454 for OS). In the time-dependent receiver operating characteristic analysis, this prognostic marker is a good predictor of HCC survival (area under the curve of 1 year, 2 years, 3 years, and 5 years are 0.754, 0.720, 0.704, and 0.662 respectively). We analyzed the correlation of clinical characteristics of these prognostic markers, and the results show that this prognostic marker is an independent factor that can predict the prognosis of HCC more accurately. In addition, by matching with the Molecular Signatures Database, we obtained 18 ERLs, and then constructed the HCC prognosis model and clinical feature correlation analysis using 5 prognostic ERLs. The results show that these prognostic markers have reliable independent predictive value. Bioinformatics analysis showed that these prognostic markers were involved in the regulation of EMT and related functions of tumor occurrence and migration.Five prognostic types of ERLs identified in this study can be used as potential biomarkers to predict the prognosis of HCC.     

A Suggested New Bacteriophage Genus, “Kp34likevirus”, within the Autographivirinae Subfamily of Podoviridae

Klebsiella pneumoniae phages vB_KpnP_SU503 (SU503) and vB_KpnP_SU552A (SU552A) are virulent viruses belonging to the Autographivirinae subfamily of Podoviridae that infect and kill multi-resistant K. pneumoniae isolates. Phages SU503 and SU552A show high pairwise nucleotide identity to Klebsiella phages KP34 ({"type":"entrez-nucleotide","attrs":{"text":"NC_013649","term_id":"294661411","term_text":"NC_013649"}}NC_013649), F19 ({"type":"entrez-nucleotide","attrs":{"text":"NC_023567","term_id":"612508640","term_text":"NC_023567"}}NC_023567) and NTUH-K2044-K1-1 ({"type":"entrez-nucleotide","attrs":{"text":"NC_025418","term_id":"725949105","term_text":"NC_025418"}}NC_025418). Bioinformatic analysis of these phage genomes show high conservation of gene arrangement and gene content, conserved catalytically active residues of their RNA polymerase, a common and specific lysis cassette, and form a joint cluster in phylogenetic analysis of their conserved genes. Also, we have performed biological characterization of the burst size, latent period, host specificity (together with KP34 and NTUH-K2044-K1-1), morphology, and structural genes as well as sensitivity testing to various conditions. Based on the analyses of these phages, the creation of a new phage genus is suggested within the Autographivirinae, called “Kp34likevirus” after their type phage, KP34. This genus should encompass the recently genome sequenced Klebsiella phages KP34, SU503, SU552A, F19 and NTUH-K2044-K1-1.     

PI3K expression and PIK3CA mutations are related to colorectal cancer metastases

AIM: To assess the significance of phosphatidylinositol 3-kinase (PI3K) in colorectal cancer (CRC) and toxicity of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 in CRC cells with different metastatic abilities.METHODS: Sixty formalin-fixed and paraffin-embedded CRC tumor specimens were investigated. Adjacent normal colonic mucosa specimens from 10 of these cases were selected as controls. PI3K protein was detected by immunohistochemistry and PIK3CA mutations were investigated by gene sequencing analysis. A flow-cytometry-based apoptosis detection kit was used to determine PI3K inhibitor-induced apoptosis in CRC cell lines SW480 and SW620. Expression of phosphorylated protein kinase B in CRC cell lines was detected by Western blotting.RESULTS: There was a significant difference in the proportion of primary lesions (30%, 18/60) vs metastatic lesions (46.7%, 28/60) that were positive for PI3K (P < 0.05). Mutations were detected in exon 9 (13.3%) and exon 20 (8.3%). Out of 60 cases, seven mutations were identified: two hotspot mutations, C.1633G>A resulting in E545A, and C.3140A>G resulting in H1047R; two novel missense mutations C.1624G>A and C.3079G>A; and three synonymous mutations (C.1641G>A, C.1581C>T and C.3027T>A). Exposure of SW480 cells to PI3K inhibitor for 48 h resulted in a significant increase of apoptotic cells in a dose-dependent manner [3.2% apoptotic cells in 0 μmol/L, 4.3% in 5 μmol/L, 6.3% in 10 μmol/L (P < 0.05), and 6.7% in 20 μmol/L (P < 0.05)]. Moreover, PI3K inhibitor induced a similar significant increase of apoptotic cells in the SW620 cell line for 48 h [3.3% apoptotic cells in 0 μmol/L, 13.3% in 5 μmol/L (P < 0.01), 19.2% in 10 μmol/L (P < 0.01), and 21.3% in 20 μmol/L (P < 0.01)].CONCLUSION: High PI3K expression is associated with CRC metastasis. PI3K inhibitor induced apoptosis in CRC cells and displayed strong cytotoxicity for highly metastatic cells. PI3K inhibition may be an effective treatment for CRC.     

CP100356 Hydrochloride,a P-Glycoprotein Inhibitor,Inhibits Lassa Virus Entry: Implication of a Candidate Pan-Mammarenavirus Entry Inhibitor

Lassa virus (LASV)—a member of the family Arenaviridae—causes Lassa fever in humans and is endemic in West Africa. Currently, no approved drugs are available. We screened 2480 small compounds for their potential antiviral activity using pseudotyped vesicular stomatitis virus harboring the LASV glycoprotein (VSV-LASVGP) and a related prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV). Follow-up studies confirmed that {"type":"entrez-nucleotide","attrs":{"text":"CP100356","term_id":"2269319269"}}CP100356 hydrochloride ({"type":"entrez-nucleotide","attrs":{"text":"CP100356","term_id":"2269319269"}}CP100356), a specific P-glycoprotein (P-gp) inhibitor, suppressed VSV-LASVGP, LCMV, and LASV infection with half maximal inhibitory concentrations of 0.52, 0.54, and 0.062 μM, respectively, without significant cytotoxicity. Although {"type":"entrez-nucleotide","attrs":{"text":"CP100356","term_id":"2269319269"}}CP100356 did not block receptor binding at the cell surface, it inhibited low-pH-dependent membrane fusion mediated by arenavirus glycoproteins. P-gp downregulation did not cause a significant reduction in either VSV-LASVGP or LCMV infection, suggesting that P-gp itself is unlikely to be involved in arenavirus entry. Finally, our data also indicate that {"type":"entrez-nucleotide","attrs":{"text":"CP100356","term_id":"2269319269"}}CP100356 inhibits the infection by other mammarenaviruses. Thus, our findings suggest that {"type":"entrez-nucleotide","attrs":{"text":"CP100356","term_id":"2269319269"}}CP100356 can be considered as an effective virus entry inhibitor for LASV and other highly pathogenic mammarenaviruses.     

LY294002对人胃腺癌细胞株BGC-823糖酵解水平的影响及其机制探讨

背景:有氧糖酵解是肿瘤细胞的特征性表型之一,靶向糖酵解有望成为一种有效的肿瘤治疗策略。M2型丙酮酸激酶(PKM2)在肿瘤组织中呈高表达,参与肿瘤细胞的有氧糖酵解。缺氧诱导因子-1α(HIF-1α)是调控肿瘤细胞糖酵解相关基因表达的重要转录因子,而PI3K信号在肿瘤细胞中可上调HIF-1α表达。目的:探讨PI3K特异性抑制剂LY294002对人胃腺癌细胞增殖和糖酵解水平的影响及其可能机制。方法:以不同浓度LY294002(10～100μmol/L)在不同条件下(常氧或缺氧)处理人胃腺癌细胞株BGC-823,CCK-8实验和流式细胞分析检测细胞增殖和细胞凋亡,蛋白质印迹法检测p-Akt、p-mTOR、HIF-1α、PKM2表达,比色法检测反映糖酵解水平的胞内乳酸脱氢酶活性和胞外乳酸浓度。结果:LY294002可呈浓度和时间依赖性地抑制BGC-823细胞增殖,在较高浓度时可诱导细胞早期凋亡。LY294002可呈浓度依赖性地抑制p-Akt、p-mTOR、HIF-1α表达,在较高浓度时可抑制PKM2表达,同时降低糖酵解水平。缺氧诱导可消除LY294002对HIF-1α、PKM2表达和糖酵解水平的抑制作用。结论:LY294002可通过阻断PI3K/Akt/mTOR信号通路抑制人胃腺癌细胞增殖及其糖酵解水平,而HIF-1α介导了糖酵解的抑制过程。