Single-sEV profiling identifies the TACSTD2 + sEV subpopulation as a factor of tumor susceptibility in the elderly.

BACKGROUND: Aging is a very complex physiological phenomenon, and sEVs are involved in the regulation of this mechanism. Serum samples from healthy individuals under 30 and over 60 years of age were collected to analyze differences in sEVs proteomics. RESULTS: Based on PBA analysis, we found that sEVs from the serum of elderly individuals highly express TACSTD2 and identified a subpopulation marked by TACSTD2. Using ELISA, we verified the upregulation of TACSTD2 in serum from elderly human and aged mouse. In addition, we discovered that TACSTD2 was significantly increased in samples from tumor patients and had better diagnostic value than CEA. Specifically, 9 of the 13 tumor groups exhibited elevated TACSTD2, particularly for cervical cancer, colon cancer, esophageal carcinoma, liver cancer and thyroid carcinoma. Moreover, we found that serum sEVs from the elderly (especially those with high TACSTD2 levels) promoted tumor cell (SW480, HuCCT1 and HeLa) proliferation and migration. CONCLUSION: TACSTD2 was upregulated in the serum of elderly individuals and patients with tumors, and could serve as a dual biomarker for aging and tumors.

Mechanism of metamifop resistance in Digitaria ciliaris var. chrysoblephara from Jiangsu, China.

Digitaria ciliaris var. chrysoblephara is one of the most competitive and problematic grass weeds in China. Metamifop is an aryloxyphenoxypropionate (APP) herbicide that inhibits the activity of acetyl-CoA carboxylase (ACCase) of sensitive weeds. Following the introduction of metamifop to China in 2010, it has been continuously used in rice paddy fields, thereby substantially increasing selective pressure for resistant D. ciliaris var. chrysoblephara variants. Here, populations of D. ciliaris var. chrysoblephara (JYX-8, JTX-98, and JTX-99) were observed to be highly resistant to metamifop, with resistance index (RI) values of 30.64, 14.38, and 23.19, respectively. Comparison of resistant and sensitive population ACCase gene sequences revealed that a single nucleotide substitution from TGG to TGC resulted in an amino acid substitution from tryptophan to cysteine at position 2,027 in the JYX-8 population. No corresponding substitution was observed for JTX-98 and JTX-99 populations. The ACCase cDNA of D. ciliaris var. chrysoblephara was successfully obtained by PCR and RACE methods, representing the first amplification of full length ACCase cDNA from Digitaria spp. Investigation of the relative expressions of ACCase gene revealed the lack of significant differences between sensitive and resistant populations before and after herbicide treatments. ACCase activities in resistant populations were less inhibited than in sensitive populations and recovered to the same or even higher levels compared to untreated plants. Whole-plant bioassays were also conducted to assess resistance to other ACCase inhibitors, acetolactate synthase (ALS) inhibitors, auxin mimic herbicide, and protoporphyrinogen oxidase (PPO) inhibitor. Cross-resistance and some multi-resistance were observed in the metamifop-resistant populations. This study is the first to focus on the herbicide resistance of D. ciliaris var. chrysoblephara. These results provide evidence for a target-site resistance mechanism in metamifop-resistant D. ciliaris var. chrysoblephara, while providing a better understanding of cross- and multi-resistance characteristics of resistant populations that will help in the management of herbicide-resistant D. ciliaris var. chrysoblephara.

Prognostic and predictive value of a lncRNA signature in patients with stage II colon cancer.

The current staging method is inadequate to identify high-risk recurrence patients with stage II colon cancer (CC). Using a systematic and comprehensive-biomarker discovery and validation method, we aimed to construct a lncRNA-based signature to improve the prognostic prediction of stage II CC. We identified 1,377 differently expressed lncRNAs by analyzing 16 paired stage II CC tumor tissue and adjacent normal mucosal tissue from the TCGA dataset. Subsequently, using a univariable and step multivariable Cox regression model, we trained an 11-lncRNA signature in the training cohort (n = 141), which could divide patients into high-risk and low-risk groups (AUC at 3 years = 0.801, 95% CI: 0.724-0.877; AUC at 5 years = 0.801, 95% CI: 0.718-0.885). Significantly, patients in the high-risk group had poorer recurrence-free survival (RFS) compared with the low-risk group (log-rank test, P < 0.001 in the training cohort). This lncRNA-based signature was further confirmed in the validation cohort (P < 0.001). Multivariate Cox regression and stratified survival analyses showed that the prognostic value of this signature was independent of other clinicopathological risk factors (CEA, T stage, and chemotherapy). Time-dependent receiver operating characteristic (ROC) analysis demonstrated that this signature had better prognostic ability than any other clinical risk factors or single lncRNAs (all P < 0.05). A nomogram was constructed for clinical use, which integrated both the lncRNA-based signature and clinical risk factors (CEA and T stage) and performed well in the calibration plots. Altogether, our lncRNA-based signature was an independent prognostic factor and possessed a stronger predictive power compared with the currently used clinicopathological risk factors when predicting the recurrence of patients with stage II CC. Collectively, this lncRNA-based signature might facilitate individualized treatment decisions and postoperative counseling, ultimately contributing to improved survival.

Decreased CXCR2 expression on circulating monocytes of colorectal cancer impairs recruitment and induces Re-education of tumor-associated macrophages.

Though circulating monocytes are the main source of tumour-associated macrophages (TAMs), the regulatory mechanisms of their recruitment to tumours and further differentiation remain unclear. In the present study, we observed a significant decrease in CXCR2 expression in classical circulating monocytes of patients with colorectal cancer (CRC), particularly those in the late TNM stage. The percentage of CXCR2+ monocytes was negatively associated with systemic inflammatory markers and positively associated with intratumoural immunocyte infiltration. The pro-inflammatory cytokine IFN-γ, which was overexpressed in patients with CRC, down-regulated CXCR2 expression of monocytes/TAMs by promoting GRK-2 expression. In vitro, inhibition of CXCR2 signalling in monocytes led to impaired chemotaxis to the tumour cell line supernatant and lower responsiveness to lipopolysaccharide (LPS) stimulation. Finally, monocytes from patients with CRC with decreased CXCR2 expression showed distinct phenotypes and functions after differentiating into CRC cell line-educated TAMs, including expression of co-stimulatory factors and secretion profile, than those from healthy controls. GRK-2 inhibitor altered the functional characteristics of TAMs. In summary, our findings suggest that CXCR2 expression on circulating monocytes reflects CRC stages and is an important factor determining TAM composition in the tumour microenvironment.

Serum circulating free DNA of syncytin-1 as a novel molecular marker for early diagnosis of non-small-cell lung cancer.

Background: Liquid biopsy has been receiving attention as an emerging detection technology in the clinical application of non-small-cell lung cancer (NSCLC). Methods: We quantified serum circulating free DNA (cfDNA) of syncytin-1 in 126 patients and 106 controls, analyzed the correlation of level with pathological parameters and explored diagnostic utility. Results: The cfDNA of syncytin-1 levels in NSCLC patients were higher than healthy controls (p < 0.0001). These levels were associated with smoking history (p = 0.0393). The area under the curve of cfDNA of syncytin-1 was 0.802, and combination of cfDNA of syncytin-1/cytokeratin 19 fragment antigen 21-1/carcinoembryonic antigen markers improved diagnostic efficiency. Conclusion: The cfDNA of syncytin-1 was detected in NSCLC patients and can be used as a novel molecular marker for early diagnosis.

NLRP3 inflammasome upregulates PD-L1 expression and contributes to immune suppression in lymphoma.

The NLRP3 inflammasome plays a pro-tumorigenic role in various malignancies. However, its potential role in lymphomagenesis remains unclear. In this study, we identified an immunosuppressive state in patients with diffuse large B cell lymphoma (DLBCL), which was characterized by markedly elevated interleukin (IL)-18 levels in lymphoma tissues and positive correlation with programmed death ligand 1 (PD-L1) expression. Furthermore, NLRP3 inflammasome activation in DLBCL cell lines upregulated PD-L1 and reduced the proportion of cytotoxic T cells. NLRP3 inflammasome blockade in vivo suppressed lymphoma growth and ameliorated anti-tumor immunity by downregulating PD-L1 in the tumor microenvironment and decreasing the proportion of PD-1/TIM-3-expressing T cells, myeloid-derived suppressor cells, tumor-associated macrophages, and regulatory T cells. Further in vivo studies revealed IL-18 as the main effector cytokine involved in the negative regulation of anti-lymphoma immunity. Interestingly, NLRP3 blockers combined with anti-PD-L1 treatment exerted antagonistic effects during lymphoma therapy. Altogether, our findings indicate that NLRP3 inflammasome promotes immunosuppression by modulating PD-L1 and immune cells. Accordingly, this study highlights the prognostic and therapeutic values of the NLRP3 inflammasome in lymphoma.

Genome-wide DNA methylation analysis by MethylRad and the transcriptome profiles reveal the potential cancer-related lncRNAs in colon cancer.

Colon cancer (CC) is characterized by global aberrant DNA methylation that may affect gene expression and genomic stability. A series of studies have demonstrated that DNA methylation could regulate the expressions of not only protein-coding genes but also ncRNAs. However, the regulatory role of lncRNA genes methylaton in CC remains largely unknown. In the present study, we systemically characterize the profile of DNA methylation, especially the aberrant methylation of lncRNAs genes using MethylRAD technology. A total of 132 999 CCGG/8487 CCWGG sites were identified as differentially methylated sites (DMSs), which were mainly located on the introns and intergenic elements. Moreover, 1,359 CCGG/1,052 CCWGG differentially methylated genes (DMGs) were screened. Our results demonstrated that aberrant methylation of lncRNA genes occurred most frequently, accounting for 37.5% and 44.3% in CCGG and CCWGG DMGs respectively. In addition, 963 lncRNA DMGs were co-analyzed with 1328 differentially expressed lncRNAs which were identified from TCGA database. We found that 15 lncRNAs might be CC-related lncRNAs. ZNF667-AS1 and MAFA-AS1 were down-regulated in CC, which might be silenced by hypermethylation. Besides, 13 lncRNAs were hypomethylated and up-regulated in CC. Moreover, our results validated the expression and methylation level of CC-related lncRNAs by RT-qPCR and pyrosequencing assay. In conclusion, we performed a genome-wide DNA methylation analysis by MethylRAD to acquire both CCGG and CCWGG DMSs and DMGs in CC. The results screened lncRNA DMSs as potential biomarkers and identified 15 lncRNAs as CC-related lncRNAs. This study provided novel therapy targets and valuable insights into molecular mechanism in tumorigenesis and development of CC.

The utilization pattern of serum tumor markers in lung cancer patients: A population-based retrospective descriptive study.

BACKGROUND: The trends in usage of tumor markers, including CEA, SCC, NSE, Cyfra21-1, and ProGRP, in Chinese lung cancer patients in the real-world setting are not fully investigated. METHODS: A retrospective descriptive study was conducted using the database of Qilu Hospital of Shandong University, China between January 2013 and December 2017, involving patients primarily diagnosed with NSCLC or SCLC. Utilization trends by first discharge year, utilization rates within different durations before and after first discharge date, and combined utilization patterns of multiple tumor markers were analyzed. RESULTS: The utilization of all these tumor markers showed increased from 2013 to 2017. CEA, Cyfra21-1, and NSE were the most frequently detected, which increased slightly from around 50% in 2013 to around 78% in 2017 in NSCLC and from around 70% in 2013 to around 92% in 2017 in SCLC. CEA, Cyfra21-1, and NSE were the most commonly measured within 3 months before first diagnosis with approximately 65% in NSCLC and 80% in SCLC, and ProGRP had the lowest utilization (around 30%). CEA, NSE, and Cyfra21-1 had the highest utilization rates after first diagnosis with both around 80% in NSCLC or SCLC. Combined usage of five tumor markers was ranked the first pattern in combined utilization. CONCLUSIONS: This study suggests CEA, Cyfra21-1, and NSE are the most frequently detected before or after first diagnosis of NSCLC or SCLC. However, SCC and ProGRP tests appeared to have relatively low usages. The utilization pattern was consistent with recommendations of guideline, but underutilization still existed.

Novel long non-coding RNA LINC02323 promotes epithelial-mesenchymal transition and metastasis via sponging miR-1343-3p in lung adenocarcinoma.

BACKGROUND: We have previously developed a unique metastasis-associated signature consisting of six long non-coding RNAs (lncRNAs), including a novel lncRNA, namely LINC02323. In the present study, we aimed to investigate the underlying roles of LINC02323 in the migration, invasion and TGF-ß-induced epithelial-mesenchymal transition (EMT) of lung adenocarcinoma (LUAD) cells. METHODS: The distribution of LINC02323 was detected by the nuclear-plasma separation experiment. Cell proliferation was assessd by MTT assay, and cell migration and invation were detected by transwell assays. EMT was detected by RT-qPCR and western blotting. Interaction between miRNA and LINC02323 was predicted by starBase v2.0 and confirmed by the double luciferase reporting system. RESULTS: LINC02323 was distributed in the cytoplasm and nucleus. The overexpression or deletion of LINC02323 did not affect the proliferation of LUAD cells, while significantly affected the migration and invasion of LUAD cells. TGF-ß-induced EMT process was significantly affected by both RNA interference (RNAi) and overexpression of LINC02323. The predicted results showed that there were binding sites between LINC02323 and miR-1343-3p. The expression of LINC02323 was found to be negatively correlated with miR-1343-3p in LUAD by analyzing The Cancer Genome Atlas (TCGA) database. The double luciferase reporting system, RT-qPCR and western blotting experiments confirmed that LINC02323 could bind to miR-1343-3p, which bound to TGF-ß receptor 1 (TGFBR1). Inhibition of miR-1343-3p reversed LINC02323 silencing-mediated suppression of migration, invasion and EMT. CONCLUSIONS: LINC02323 acts as a competing endogenous RNA (ceRNA), which sponged miR-1343-3p to upregulate the TGFBR1 expression and promote the EMT and metastasis in LUAD. KEY POINTS: SIGNIFICANT FINDINGS OF THE STUDY: LINC02323 promotes epithelial-mesenchymal transition and metastasis via sponging miR-1343-3p in lung adenocarcinoma. WHAT THIS STUDY ADDS: LINC02323 is a key molecule in the process of invasion and metastasis of LUAD and might be used as a potential target in metastatic cancer.

Identification of an exosomal long non-coding RNAs panel for predicting recurrence risk in patients with colorectal cancer.

Recurrence is a major cause of cancer-related deaths in colorectal cancer (CRC) patients, but the current strategies are limited to predict this clinical behavior. Our aim is to develop a recurrence prediction model based on long non-coding RNAs (lncRNAs) in exosomes of serum to improve the prediction accuracy. In discovery phase, 11 lncRNAs were found to be associated with CRC recurrence in tissues using high-throughput lncRNAs microarray and reverse transcription quantitative real-time PCR. And, 9 of them were correlated with their expression levels of serum exosomes. In training phase, a model based on 5-exosomal lncRNAs (exolncRNAs) panel was constructed, and showed high distinguish capability for recurrent CRC patients. ROC showed the panel was superior to serum CEA and CA19-9 in prediction of CRC recurrence. In both training and test sets, high-risk patients defined by the 5-exolncRNAs panel had poor recurrence free and overall survival. And, COX model showed it was an independent factor for CRC prognosis. Moreover, there was a significant relationship in detection of 5-exolncRNAs between plasma samples and paired serum samples. In summary, the 5-exolncRNAs panel robustly stratifies CRC patients' risk of recurrence, enabling more accurate prediction of prognosis.

Identification of key gene modules and hub genes of human mantle cell lymphoma by coexpression network analysis.

PURPOSE: Mantle cell lymphoma (MCL) is a rare and aggressive subtype of non-Hodgkin lymphoma that is incurable with standard therapies. The use of gene expression analysis has been of interest, recently, to detect biomarkers for cancer. There is a great need for systemic coexpression network analysis of MCL and this study aims to establish a gene coexpression network to forecast key genes related to the pathogenesis and prognosis of MCL. METHODS: The microarray dataset GSE93291 was downloaded from the Gene Expression Omnibus database. We systematically identified coexpression modules using the weighted gene coexpression network analysis method (WGCNA). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis were performed on the modules deemed important. The protein-protein interaction networks were constructed and visualized using Cytoscape software on the basis of the STRING website; the hub genes in the top weighted network were identified. Survival data were analyzed using the Kaplan-Meier method and were compared using the log-rank test. RESULTS: Seven coexpression modules consisting of different genes were applied to 5,000 genes in the 121 human MCL samples using WGCNA software. GO and KEGG enrichment analysis identified the blue module as one of the most important modules; the most critical pathways identified were the ribosome, oxidative phosphorylation and proteasome pathways. The hub genes in the top weighted network were regarded as real hub genes (IL2RB, CD3D, RPL26L1, POLR2K, KIF11, CDC20, CCNB1, CCNA2, PUF60, SNRNP70, AKT1 and PRPF40A). Survival analysis revealed that seven genes (KIF11, CDC20, CCNB1, CCNA2, PRPF40A, CD3D and PUF60) were associated with overall survival time (p < 0.05). CONCLUSIONS: The blue module may play a vital role in the pathogenesis of MCL. Five real hub genes (KIF11, CDC20, CCNB1, CCNA2 and PUF60) were identified as potential prognostic biomarkers as well as therapeutic targets with clinical utility for MCL.

A novel cysteine-sparing G73A mutation of NOTCH3 in a Chinese CADASIL family.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic disease leading to stroke and vascular dementia. CADASIL is an inherited small blood vessel disease caused by mutations in the gene encoding the neurogenic locus notch homolog protein 3 (NOTCH3). NOTCH3 is large type I membrane receptor mainly expressed in vascular smooth muscle cells and pericytes. Most identified mutations result in insert or deletion of a cysteine residue within the EGF-like repeats. To date, some cases with a cysteine-sparing mutant have been described. Genetic analysis revealed a novel mutation in NOTCH3 in a CADASIL family. Molecular analysis revealed its potential pathogenic mechanism in causing CADASIL. In this paper, we present a Chinese family with a novel cysteine-sparing mutation in exon 3 (c.218G>C, p.G73A) of the NOTCH3 gene. Family carriers of the same mutation presented with symptoms and imaging abnormalities characteristic of CADASIL. The location of glycine 73 in between C5-C6 disulfide bond of EGF-like domain 1 shows high conservation from humans to zebra fish. It has previously been suggested that the aggregate-prone property of mutant NOTCH3 contributes to a cytotoxic effect in the pathogenic mechanism underlying CADASIL. Here, we investigated the pathogenic mechanism of the new mutation in vitro using HEK293 cells transfected with either a wild-type (WT) or c.218G>C (p.G73A) NOTCH3ECD plasmids, and we found p.G73A NOTCH3ECD was more prone to form aggregation and resistant to degradation. Moreover, the p.G73A NOTCH3ECD compromised cell viability by promoting apoptosis. Two known CADASIL mutants R133C and R75P showed similar results with G73A mutants. Our study here identified G73A as a new mutation in NOTCH3 to cause CADASIL and revealed that the G73A mutation and two known mutants R75P and R133C decreased NOTCH3 protein turnover and induced cell death.

Poly(ethylene oxide)-Li10SnP2S12 Composite Polymer Electrolyte Enables High-Performance All-Solid-State Lithium Sulfur Battery.

Composite polymer electrolyte membranes are fabricated by the incorporation of Li10SnP2S12 into the poly(ethylene oxide) (PEO) matrix using a solution-casting method. The incorporation of Li10SnP2S12 plays a positive role on Li-ionic conductivity, mechanical property, and interfacial stability of the composite electrolyte and thus significantly enhances the electrochemical performance of the solid-state Li-S battery. The optimal PEO-1%Li10SnP2S12 electrolyte presents a maximum ionic conductivity of 1.69 × 10-4 S cm-1 at 50 °C and the highest mechanical strength. The possible mechanism for the enhanced electrochemical performance and mechanical property is analyzed. The uniform distribution of Li10SnP2S12 in the PEO matrix inhibits crystallization and weakens the interactions among the PEO chains. The PEO-1%Li10SnP2S12 electrolyte exhibits lower interfacial resistance and higher interfacial stability with the lithium anode than the pure PEO/LiTFSI electrolyte. The Li-S cell comprising the PEO-1%Li10SnP2S12 electrolyte exhibits outstanding electrochemical performance with a high discharge capacity (ca. 1000 mA h g-1), high Coulombic efficiency, and good cycling stability at 60 °C. Most importantly, the PEO-1%Li10SnP2S12-based cell possesses attractive performance with a high specific capacity (ca. 800 mA h g-1) and good cycling stability even at 50 °C, whereas the PEO/LiTFSI-based cell cannot be successfully discharged because of the low ionic conductivity and high interfacial resistance of the PEO/LiTFSI electrolyte.

Increasing tolerance to bispyribac-sodium is able to allow glutathione homeostasis to recover in indica rice compared with japonica rice.

The high activity and broad weed spectrum of BS has made it widely used in China. However, accidental crop injuries, particularly occurring in Jiangsu, Hunan, Hubei and Heilongjiang provinces in recent years, have resulted in limiting the application of BS in China. In this study, glutathione homeostasis was measured in the contrasting sensitivity of indica and japonica rice cultivar after bispyribac-sodium (BS) treatment. The results showed that japonica rice cultivar Nanjing 9108 was more sensitive to BS than indica rice Nanjing 11 and indica-hybrid cultivar Guangliangyou 6326. In response to the exposure of BS in all rice cultivars, especially Nanjing 9108, the perturbation of glutathione homeostasis occurred, including the decreased reduced glutathione (GSH) and increased oxidized glutathione (GSSG). These results were supported by increased activities of glutathione S-transferases (GSTs) in Nanjing 11 and Guangliangyou 6326. Further tests revealed that when Nanjing 11 was pretreated with the glutathione-depleting agents L-buthionine-sulfoximine (BSO) or diethylmaleate (DEM), the GSH levels, the activity of GSTs, and the gene expression levels of GR and GSTs decreased, finally increasing the phytotoxicity of BS. The aforementioned DEM inhibitory responses were further rescued by exogenously applied GSH. In contrast, the pretreatment of glutathione or N-acetyl-L-cysteine (NAC) not only increased the contents of GSH, the activities of GSTs, and the expression level of GR and GSTs gene, but also alleviated BS phytotoxicity in Nanjing 9108. In both cultivars, DEM increased phytotoxicity and GSH partially reversed this. This study suggests that increasing tolerance to BS was able to allow glutathione homeostasis to recover in indica rice cultivar compared with japonica rice cultivar.

Genome-wide identification of a novel miRNA-based signature to predict recurrence in patients with gastric cancer.

The current tumor node metastasis (TNM) staging system is inadequate for identifying high-risk gastric cancer (GC) patients. Using a systematic and comprehensive-biomarker discovery and validation approach, we attempted to build a microRNA (miRNA)-recurrence classifier (MRC) to improve the prognostic prediction of GC. We identified 312 differentially expressed miRNAs in 446 GC tissues compared to 45 normal controls by analyzing high-throughput data from The Cancer Genome Atlas (TCGA). Using a Cox regression model, we developed an 11-miRNA signature that could successfully discriminate high-risk patients in the training set (n = 372; P < 0.0001). Quantitative real-time polymerase chain reaction-based validation in an independent clinical cohort (n = 88) of formalin-fixed paraffin-embedded clinical GC samples showed that MRC-derived high-risk patients succumb to significantly poor recurrence-free survival in GC patients (P < 0.0001). Cox and stratification analysis indicated that the prognostic value of this signature was independent of clinicopathological risk factors. Time-dependent receiver operating characteristic (ROC) analysis revealed that the area under the curve of this signature was significantly larger than that of TNM stage in the TCGA (0.733 vs. 0.589 at 3 years, P = 0.004; 0.802 vs. 0.635 at 5 years, P = 0.005) and validation cohort (0.835 vs. 0.689 at 3 years, P = 0.003). A nomogram was constructed for clinical use, which integrated both MRC and clinical-related variables (depth of invasion, lymph node status and distance metastasis) and did well in the calibration plots. In conclusion, this novel miRNA-based signature is superior to currently used clinicopathological features for identifying high-risk GC patients. It can be readily translated into clinical practice with formalin-fixed paraffin-embedded specimens for specific decision-making applications.

Cell Cycle Arrest and Apoptosis Induction Activity of Nitidine Chloride on Acute Myeloid Leukemia Cells.

BACKGROUND: Acute myeloid leukemia (AML) is the most common hematological malignancy in adults, characterized by distorted proliferation and development of myeloid cells and their precursors in the bone marrow. Nitidine chloride, a naturally occurring alkaloid, has been identified to possess antitumor activity. However, the effects of nitidine chloride on acute myeloid leukemia cells and its underlying mechanisms have not been elucidated. Here we investigated the cellular and molecular mechanism of the anti-leukemic effects of nitidine chloride. METHODS AND RESULTS: Nitidine chloride treatment for 48 consecutive hours exhibited a timedependent and dose-dependent growth inhibition activity against AML cells by inducing cell cycle arrest and apoptosis. Moreover, nitidine chloride downregulated Cyclin B1, CDK1 and Bcl-2, upregulated p27 and Bax, inactivated PARP, activated Caspase-3 in AML cells. We further demonstrated that growth inhibition activity of nitidine chloride in AML cells is partially via inhibiting the phosphorylation of AKT and ERK. CONCLUSION: In conclusion, our data suggest that nitidine chloride could be an effective therapeutic agent against AML via cell cycle arrest and apoptosis.

MiR-424 and miR-27a increase TRAIL sensitivity of acute myeloid leukemia by targeting PLAG1.

Although microRNAs have been elaborated to participate in various physiological and pathological processes, their functions in TRAIL resistance of acute myeloid leukemia (AML) remain obscure. In this study, we detected relatively lower expression levels of miR-424&27a in TRAIL-resistant and semi-resistant AML cell lines as well as newly diagnosed patient samples. Overexpression of miR-424&27a, by targeting the 3'UTR of PLAG1, enhanced TRAIL sensitivity in AML cells. Correspondingly, knockdown of PLAG1 sensitized AML cells to TRAIL-induced apoptosis and proliferation inhibition. We further found that PLAG1 as a transcription factor could reinforce Bcl2 promoter activity, causing its upregulation at the mRNA level. Both downregulated PLAG1 and elevated expression of miR-424&27a led to Bcl2 downregulation and augmented cleavage of Caspase8, Caspase3 and PARP in the presence of TRAIL. Restoration of Bcl2 could eliminate their effects on AML TRAIL sensitization. Overall, we propose that miR-424&27a and/or PLAG1 might serve as novel therapeutic targets in AML TRAIL therapy.

Wnt signaling is involved in 6-benzylthioinosine-induced AML cell differentiation.

BACKGROUND: We previously demonstrated that 6-benzylthioinosine (6-BT) could induce the differentiation of a subset of acute myeloid leukemia (AML) cell lines and primary AML cells regardless of their cytogenetics. In this study we investigated whether Wnt signaling pathways played roles in 6-BT-induced differentiation of AML cells. METHODS: We induced differentiation of HL-60 leukemic cells and primary AML cells in vitro using 6-BT. Real-time PCR (qPCR), western blot, and luciferase assays were used to examine the molecules' expression and biological activity in canonical and noncanonical Wnt signaling pathways. AML cell differentiation was measured by the Nitroblue tetrozolium (NBT) reduction assay. RESULTS: 6-BT regulated the expression of both canonical and non-canonical Wnt signaling molecules in HL-60 cells. Both 6-BT and all-trans-retinoic-acid (ATRA) reduced canonical Wnt signaling and activated noncanonical Wnt/Ca2+ signaling in HL-60 cells. Pre-treatment of HL-60 cells with an inhibitor of glycogen synthase kinase-3ß (GSK-3ß), which activated canonical Wnt signaling, partly abolished the differentiation of HL-60 cells induced by 6-BT. Pre-treatment of HL-60 cells with an inhibitor of protein kinase C (PKC), resulting in inactivation of non-canonical Wnt/Ca2+ signaling, abolished 6-BT-induced differentiation of HL-60 cells. Several molecules in the non-canonical Wnt/Ca2+ pathway were detected in bone marrow samples from AML patients, and the expression of FZD4, FZD5, Wnt5a and RHOU were significantly reduced in newly diagnosed AML samples compared with normal controls. CONCLUSIONS: Both canonical and non-canonical Wnt signaling were involved in 6-BT-induced differentiation of HL-60 cells, and played opposite roles in this process. Wnt signaling could be involved in the pathogenesis of AML not only by regulating self-renewal of hematopoietic stem cells, but also by playing a role in the differentiation of AML cells.

The expression of VEGF and Dll4/Notch pathway molecules in ovarian cancer.

BACKGROUND: VEGF and Dll4/Notch pathways play important roles in tumor angiogenesis. The purpose of this study is to investigate the expression of these two pathway molecules in ovarian cancer and their possible relationships in carcinogenesis. METHODS: Twenty-eight specimens of human ovarian carcinoma, 18 of benign ovarian and 20 of healthy ovarian tissues were subjected to immunohistochemical analysis for VEGF, VEGFR1, and VEGFR2, Dll4, Notch1, and Notch3 expression. Microvessel density (MVD) was evaluated by counting the number of CD34-stained microvessels in each pathologic specimen. RESULTS: The expression of VEGF, VEGFR1, Dll4, Notch1, or Notch3 in ovarian tumor tissues was higher than that in normal ovary tissues as well as that in benign ovarian tumor tissues (P<0.05). In the tumor tissues, Dll4 was positively correlated with VEGFR1 expression and Notch1 was positively associated with VEGFR2 and MVD. Moreover, VEGFR2 expression was positively associated with ascites and distant metastasis (R=0.401, P=0.034). CONCLUSIONS: Dll4 represents a potential biomarker and therapeutic target for ovarian angiogenesis. VEGFR2 is significantly related to ovarian metastasis and invasion. Therefore testing the key molecules of these two pathways expression may have some diagnostic and prognostic value for ovarian cancer.