Down-regulation of eIF5A-2 prevents epithelial-mesenchymal transition in non-small-cell lung cancer cells

Background

Epithelial-mesenchymal transition (EMT) is believed to be the critical process in malignant tumor invasion and metastases, and has a great influence on improving the survival rate in non-small-cell lung cancer (NSCLC) patients. Recent studies suggested that eukaryotic initiation factor 5A-2 (eIF5A-2) might serve as an adverse prognostic marker of survival. We detected eIF5A-2 in NSCLC A549 cells, and found that the invasive capability correlates with the eIF5A-2 expression.

Methods

Transforming growth factor (TGF)-β1 was used to induce EMT in A549 cells. Western blotting, immunofluorescence, wound healing assay, and transwell-matrigel invasion chambers were used to identify phenotype changes. Western blotting was also used to observe changes of the expression of eIF5A-2. We down-regulated the eIF5A-2 expression using an eIF5A-2 siRNA and identified the phenotype changes by western blotting and immunofluorescence. We tested the change of migration and invasion capabilities of A549 cells by the wound healing assay and transwell-matrigel invasion chambers.

Results

After stimulating with TGF-β1, almost all A549 cells changed to the mesenchymal phenotype and acquired more migration and invasion capabilities. These cells also had higher eIF5A-2 protein expression. Down-regulation of eIF5A-2 expression with eIF5A-2 siRNA transfection could change the cells from mesenchymal to epithelial phenotype and decrease tumor cell migration and invasive capabilities significantly.

Conclusions

The expression of eIF5A-2 was up-regulated following EMT phenotype changes in A549 cells, which correlated with enhanced tumor invasion and metastatic capabilities. Furthermore, in the A549 cell line, the process of EMT phenotype change could be reversed by eIF5A-2 siRNA, with a consequent weakening of both invasive and metastatic capabilities.     

Hydroxychavicol,a polyphenol from Piper betle leaf extract,induces cell cycle arrest and apoptosis in TP53-resistant HT-29 colon cancer cells

This study aims to elucidate the antiproliferative mechanism of hydroxychavicol(HC).Its effects on cell cycle,apoptosis,and the expression of c-Jun N-terminal kinase(JNK)and P38 mitogen-activated protein kinase(MAPK)in HT-29 colon cancer cells were investigated.HC was isolated from Piper betle leaf(PBL)and verified by high-performance liquid chromatography(HPLC),nuclear magnetic resonance(NMR),and gas chromatography-mass spectrometry(GC-MS).The cytotoxic effects of the standard drug 5-fluorouracil(5-FU),PBL water extract,and HC on HT-29 cells were measured after 24,48,and 72 h of treatment.Cell cycle and apoptosis modulation by 5-FU and HC treatments were investigated up to 30 h.Changes in phosphorylated JNK(pJNK)and P38(pP38)MAPK expression were observed up to 18 h.The half maximal inhibitory concentration(IC₅₀)values of HC(30μg/mL)and PBL water extract(380μg/mL)were achieved at 24 h,whereas the IC₅₀of 5-FU(50μmol/L)was obtained at 72 h.Cell cycle arrest at the G0/G1 phase in HC-treated cells was observed from12 h onwards.Higher apoptotic cell death in HC-treated cells compared to 5-FU-treated cells(P<0.05)was observed.High expression of pJNK and pP38 MAPK was observed at 12 h in HC-treated cells,but not in 5-FU-treated HT-29 cells(P<0.05).It is concluded that HC induces cell cycle arrest and apoptosis of HT-29 cells,with these actions possibly mediated by JNK and P38 MAPK.     

GLABROUS INFLORESCENCE STEMS regulates trichome branching by genetically interacting with SIM in Arabidopsis

Arabidopsis trichomes are large branched single cells that protrude from the epidermis. The first morphological indication of trichome development is an increase in nuclear content resulting from an initial cycle of endoreduplication. Our previous study has shown that the C2H2 zinc finger protein GLABROUS INFLORESCENCE STEMS (GIS) is required for trichome initiation in the inflorescence organ and for trichome branching in response to gibberellic acid signaling, although GIS gene does not play a direct role in regulating trichome cell division. Here, we describe a novel role of GIS, controlling trichome cell division indirectly by interacting genetically with a key endoreduplication regulator SIAMESE (SIM). Our molecular and genetic studies have shown that GIS might indireclty control cell division and trichome branching by acting downstream of SIM. A loss of function mutation of SIM signficantly reduced the expression of GIS. Futhermore, the overexpression of GIS rescued the trichome cluster cell phenotypes of sim mutant. The gain or loss of function of GIS had no significant effect on the expression of SIM. These results suggest that GIS may play an indirect role in regulating trichome cell division by genetically interacting with SIM.     

Partial AZFc duplications not deletions are associated with male infertility in the Yi population of Yunnan Province, China

Objective: There are many reports on associations between spermatogenesis and partial azoospermia factor c (AZFc) deletions as well as duplications; however, results are conflicting, possibly due to differences in methodology and ethnic background. The purpose of this study is to investigate the association of AZFc polymorphisms and male infertility in the Yi ethnic population, residents within Yunnan Province, China. Methods: A total of 224 infertile patients and 153 fertile subjects were selected in the Yi ethnic population. The study was performed by sequence-tagged site plus/minus (STS+/−) analysis followed by gene dosage and gene copy definition analysis. Y haplotypes of 215 cases and 115 controls were defined by 12 binary markers using single nucleotide polymorphism on Y chromosome (Y-SNP) multiplex assays based on single base primer extension technology. Results: The distribution of Y haplotypes was not significantly different between the case and control groups. The frequencies of both gr/gr (7.6% vs. 8.5%) and b2/b3 (6.3% vs. 8.5%) deletions do not show significant differences. Similarly, single nucleotide variant (SNV) analysis shows no significant difference of gene copy definition between the cases and controls. However, the frequency of partial duplications in the infertile group (4.0%) is significantly higher than that in the control group (0.7%). Further, we found a case with sY1206 deletion which had two CDY1 copies but removed half of DAZ genes. Conclusions: Our results show that male infertility is associated with partial AZFc duplications, but neither gr/gr nor b2/b3 deletions, suggesting that partial AZFc duplications rather than deletions are risk factors for male infertility in Chinese-Yi population.     

Association of CASP9, CASP10 gene polymorphisms and tea drinking with colorectal cancer risk in the Han Chinese population

The initiators caspase-9(CASP9) and caspase-10(CASP10) are two key controllers of apoptosis and play important roles in carcinogenesis.This study aims to explore the association between CASPs gene polymorphisms and colorectal cancer(CRC) susceptibility in a population-based study.A two-stage designed population-based case-control study was carried out,including a testing set with 300 cases and 296 controls and a validation set with 206 cases and 845 controls.A total of eight tag selected single nucleotide polymorphisms(SNPs) in CASP9 and CASP10 were chosen based on HapMap and the National Center of Biotechnology Information(NCBI) datasets and genotyped by restriction fragment length polymorphism(RFLP) assay.Multivariate logistic regression models were applied to evaluate the association of SNPs with CRC risk.In the first stage,from eight tag SNPs,three polymorphisms rs4646077(odds ratio(OR) AA+AG:0.654,95% confidence interval(CI):0.406-1.055;P=0.082),rs4233532(OR CC:1.667,95% CI:0.967-2.876;OR CT:1.435,95% CI:0.998-2.063;P=0.077),and rs2881930(OR CC:0.263,95% CI:0.095-0.728,P=0.036) showed possible association with CRC risk.However,none of the three SNPs,rs4646077(OR AA+AG:1.233,95% CI:0.903-1.683),rs4233532(OR CC:0.892,95% CI:0.640-1.243;OR CT:1.134,95% CI:0.897-1.433),and rs2881930(OR CC:1.096,95% CI:0.620-1.938;OR CT:1.009,95% CI:0.801-1.271),remained significant with CRC risk in the validation set,even after stratification for different tumor locations(colon or rectum).In addition,never tea drinking was associated with a significantly increased risk of CRC in testing set together with validation set(OR:1.755,95% CI:1.319-2.334).Our results found that polymorphisms of CASP9 and CASP10 genes may not contribute to CRC risk in Chinese population and thereby the large-scale case-control studies might be in consideration.In addition,tea drinking was a protective factor for CRC.     

Involvement of phosphatidate phosphatase in the biosynthesis of triacylglycerols in Chlamydomonas reinhardtii

Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerols and inorganic orthophosphates. This reaction is integral in the synthesis of triacylglycerols. In this study, the mRNA level of the PAP isoform CrPAP2 in a species of Chlamydomonas was found to increase in nitrogen-free conditions. Silencing of the CrPAP2 gene using RNA interference resulted in the decline of lipid content by 2.4%–17.4%. By contrast, over-expression of the CrPAP2 gene resulted in an increase in lipid content by 7.5%–21.8%. These observations indicate that regulation of the CrPAP2 gene can control the lipid content of the algal cells. In vitro CrPAP2 enzyme activity assay indicated that the cloned CrPAP2 gene exhibited biological activities.     

萎叶提取物增强5-氟尿嘧啶对结肠癌细胞HT29和HCT116的生长抑制作用

研究目的：探讨萎叶（PB）提取物对5-氟尿嘧啶（5-FU）抑制结肠癌细胞HT29和HCT116生长的影响。研究方法：HT29和HCT116细胞分别给予PB、5-FU以及两种药物联合治疗24小时,应用等效线图法分析PB和5-FU的药效学相互作用,AnnexinV／PI染色法检测HT29和HCT116细胞的凋丁L=情况,高效液相色谱法排除PB和5-FU间任何可能的相互化学作用。重要结论：联合PB,低剂量5-FU可以在短时间内起到细胞毒作用,而单独应用PB或5-FU治疗较联合治疗可以诱导更多细胞发生凋亡。进一步采用等效线图法分析显示PB和5-Fu的联合作用在抑制结肠癌细胞HT29和HCT116的生长中分别体现出协同和拮抗作用。因此可以认为在HT29细胞中,PB使得较低剂量5-FU发挥最大抑制结肠癌细胞生长效果,然而在HCT116细胞中,PB没有显著降低5-FU的药物浓度,说明PB和5-FU的相互作用不仅仅体现在诱导细胞凋亡方面。     

舒尼替尼对结肠成纤维细胞体内外抑制机制研究(英文)

研究目的:探索小分子酪氨酸激酶抑制剂舒尼替尼(sunitinib)对结肠肿瘤微环境中的肿瘤相关成纤维细胞的作用及其机制。创新要点:舒尼替尼通过抑制肿瘤间质成纤维细胞的生长,间接发挥抗肿瘤效应,为结肠癌综合治疗的提供新途径。研究方法:通过细胞周期分析和细胞增殖测定进行舒尼替尼体外抑制肿瘤细胞的研究。采用Western-blot检测磷酸化血小板衍生生长因子β受体(PDGFR-β)、蛋白激酶B(Akt)及细胞外信号调节蛋白激酶(ERK)的蛋白水平。通过注射肠腺癌细胞株SW620和结肠成纤维细胞构建的裸鼠移植瘤模型来研究舒尼替尼的体内抑瘤效果。重要结论:舒尼替尼可有效抑制结肠癌来源的原代结肠成纤维细胞生长,该抑制作用主要通过抑制血小板衍生生长因子(PDGF)信号通路得以实现。     

FLT3 and NPM1 mutations in Chinese patients with acute myeloid leukemia and normal cytogenetics

Mutations of fms-like tyrosine kinase 3 (FLT3) and nucleophosmin (NPM1) exon 12 genes are the most common abnormalities in adult acute myeloid leukemia (AML) with normal cytogenetics. To assess the prognostic impact of the two gene mutations in Chinese AML patients, we used multiplex polymerase chain reaction (PCR) and capillary electrophoresis to screen 76 AML patients with normal cytogenetics for mutations in FLT3 internal tandem duplication (FLT3/ITD) and exon 12 of the NPM1 gene. FLT3/ITD mutation was detected in 15 (19.7%) of 76 subjects, and NPM1 mutation in 20 (26.3%) subjects. Seven (9.2%) cases were positive for both FLT3/ITD and NPM1 mutations. Significantly more FLT3/ITD aberration was detected in subjects with French-American-British (FAB) M1 (42.8%). NPM1 mutation was frequently detected in subjects with M5 (47.1%) and infrequently in subjects with M2 (11.1%). FLT3 and NPM1 mutations were significantly associated with a higher white blood cell count in peripheral blood and a lower CD34 antigen expression, but not age, sex, or platelet count. Statistical analysis revealed that the FLT3/ITDpositive group had a lower complete remission (CR) rate (53.3% vs. 83.6%). Survival analysis showed that the FLT3/ITD-positive/NPM1 mutation-negative group had worse overall survival (OS) and relapse-free survival (RFS). The FLT3/ITD-positive/NPM1 mutation-positive group showed a trend towards favorable survival compared with the FLT3/ITD-positive/NPM1 mutation-negative group (P=0.069). Our results indicate that the FLT3/ITD mutation might be a prognostic factor for an unfavorable outcome in Chinese AML subjects with normal cytogenetics, while NPM1 mutation may be a favorable prognostic factor for OS and RFS in the presence of FLT3/ITD.     

Carfilzomib通过上调Gadd45a表达抑制肺腺癌细胞生长（英文）

目的:最近有研究表明卡非佐米(Carfilzominb,CFZ)能有效抑制肺腺癌细胞生长,但是其中的内在机制仍然需要进一步研究。本文针对CFZ抑制肺腺癌生长机制进行了系统研究。创新点:揭示了蛋白酶体抑制剂抗实体肿瘤的新机制,为这类药物用于实体肿瘤治疗提供了有利依据。同时Gadd45a可做为候选指标用于蛋白酶体抑制剂抗肿瘤疗效的预测。方法:应用流式细胞术检测CFZ对肺腺癌细胞周期和凋亡的影响;通过MTS比色法及平板克隆形成实验分析CFZ对肺腺癌细胞生长的抑制作用;使用蛋白质印迹法(western blot)和定量聚合酶链反应(qPCR)检测相关基因表达水平的改变。结论:CFZ通过AKT/FOXO3a通路上调Gadd45a基因的表达,诱导肺腺癌细胞周期阻滞和凋亡,从而发挥抗肿瘤效应。     

Effect of lipopolysaccharide on the hemocyte apoptosis of Eriocheir sinensis

In the present study, we investigated the possible toxicity mechanism of lipopolysaccharide (LPS) extracted from Gram-negative bacteria in Eriocheir sinensis hemocytes. Apoptotic hemocytes and reactive oxygen species (ROS) production induced by the LPS were monitored by the combination of flow cytometry and microscope observation. It was shown that LPS induced serious damage on the DNA and morphological changes in hemocytes, including cell shrinkage, fracture of nucleus membrane, margination, condensation and fragmentation of chromatin, and formation of apoptotic bodies indicating obvious hemocyte apoptosis. As compared with the control group, the apoptotic cell ratio increased to 30.61% and 39.01% after 1-h exposure and 57.72% and 75.01% after 2-h exposure to 1 and 10 μg/ml LPS, respectively (P<0.05). Significant outburst of ROS production was observed in LPS-treated hemocytes with approximately 176.6% of relative dichlorofluorescein mean fluorescence at 1-h exposure, followed by a drastic decline (P<0.05). These results indicated that LPS would induce oxidative stress on hemocytes from E. sinensis and cause ROS burst, DNA damage, and subsequently apoptosis. The process of ROS-mediated apoptosis might be one of the potential toxicity mechanisms of LPS on crustacean hemocytes.