Thianthrenation-Enabled Pyrrolidin-2-yl and Tetrahydrofuran-2-yl Methylation of (Hetero)Arenes.

The mild and efficient palladium-catalyzed pyrrolidin-2-yl and tetrahydrofuran-2-yl methylation of (hetero)arenes has been developed. A wide range of (hetero)arenes underwent the regioselective thianthrenation to generate the arylthianthrenium triflate, and the developed Pd-catalyzed alkene carboamination and carboalkoxylation reactions afforded the corresponding biologically important pyrrolidine and tetrahydrofuran derivatives. Mechanistic studies indicated that this reaction proceeds through a syn-heteropalladation mechanistic pathway. The demonstrated late-stage functionalization and enantioselective reaction will help to promote the potential application of the established method in organic synthesis and related fields.

Prognostic and Therapeutic Significance of Adhesion-regulating Molecule 1 in Estrogen Receptor-positive Breast Cancer.

INTRODUCTION: Adhesion-regulating molecule 1 (ADRM1) is a polyubiquitin receptor on the 26S proteasome. ADRM1 is upregulated in many cancers. In this study, we evaluated the potential prognostic and predictive value of ADRM1 in breast cancer. MATERIALS AND METHODS: Individual and pooled survival analyses were performed on 19 independent breast cancer microarray datasets. Gene signatures enriched by ADRM1 were also analyzed in pooled datasets. RESULTS: Gene set enrichment analysis revealed that high expression of ADRM1 was significantly associated with aggressive breast cancer. Our findings revealed that ADRM1 mRNA levels were significantly associated with estrogen receptor (ER) status, progesterone receptor status, tumor size, lymph node status, histologic grade, and molecular subtypes. We also found that higher mRNA ADRM1 expression was significantly correlated with poor survival in patients with breast cancer. The prognostic power of ADRM1 mRNA was similar to the 70-gene wound response genes and 21 gene recurrence score; it was superior to TNM staging. The prognostic value of ADRM1 was better in ER-positive (ER+) breast cancer cases than in ER-negative breast cancer cases. In cases involving stage II breast cancer, radiotherapy significantly reduced the relative risk of OS in the ADRM1-low subgroup. CONCLUSION: ADRM1 mRNA levels were significantly related to poor outcome in our breast cancer sample population. It could serve as a prognostic biomarker, especially in ER+ breast cancer and Luminal A breast cancer cases, as well as a predictive biomarker for ER+ breast cancer.

Retraction Note: Antisense oligonucleotides targeting midkine induced apoptosis and increased chemosensitivity in hepatocellular carcinoma cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Retraction Note: Antisense oligonucleotides targeting midkine inhibit tumor growth in an in situ human hepatocellular carcinoma model.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Downregulation of PTPRK Promotes Cell Proliferation and Metastasis of NSCLC by Enhancing STAT3 Activation.

OBJECTIVE: The receptor-type tyrosine-protein phosphatase κ (PTPRK) is a candidate tumor suppressor involved in the tumorigenesis of various organs. However, its expression and biological roles in non-small-cell lung cancer (NSCLC) have not yet been investigated. METHODS: PTPRK expression in NSCLC tissues and cell lines was examined using real-time PCR and western blotting. In addition, the effects of PTPRK on cell migration, invasion, and proliferation were evaluated in vitro. Furthermore, we explored whether the downregulation of PTPRK led to STAT3 activation in NSCLC cell lines by western blotting. The expression of phospho-STAT3Tyr705 in primary human NSCLC tissues was evaluated by immunohistochemistry. RESULTS: The results showed that PTPRK expression was frequently reduced in NSCLC tissues with lymph node metastasis and cell lines. The inhibition of PTPRK expression resulted in increased proliferation, invasion, and migration of NSCLC cells in vitro. Additionally, after silencing of PTPRK, phospho-STAT3Tyr705 was significantly increased in NSCLC cells. Moreover, the phospho-STAT3Tyr705 levels of NSCLC tissues were positively correlated with lymph node metastasis and significantly inversely correlated with the expression of PTPRK (p < 0.05). CONCLUSIONS: These results suggested that PTPRK functions as a novel tumor suppressor in NSCLC, and its suppressive ability may be involved in STAT3 activation.

Toxicologic evaluation of repetitive 4-week intravenous injections of midkine antisense oligonucleotide nanoliposomes in rats.

Midkine antisense oligonucleotide (MK-ASODN) nanoliposomes have previously been shown to have inhibitory activity against hepatocellular carcinoma growth. Herein we report the 4-week sub-chronic toxicity of MK-ASODN nanoliposomes in SD rats. The adverse effects included loss of body weight gain and food consumption, peri-rhinal bleeding, piloerection, peri-anal filth, and kidney, liver, spleen, thymus, lung, and injection site lesions at high doses. Macroscopic changes were observed in the kidneys of the high-dose group, accompanied by a variation in urine protein and white blood cells, blood urea nitrogen, and serum creatinine. The increased spleen and liver coefficient, and the variation in circulating white blood cells, lymphocytes, and eosinophils in the high-dose group demonstrated that inflammation was caused by MK-ASODN nanoliposomes and was consistent with the macroscopic changes in the spleen and liver. The main necropsy findings of the animals that died were macroscopic changes in the lung. No severe toxic effects or mortalities occurred in the low- and medium-dose groups. However, a No Adverse Effect Level (NOAEL) was not identified since there were changes in organs deemed to be adverse at all dose levels. Thus, the maximum tolerated dose of MK-ASODN nanoliposomes for rats was considered to be 6 mg/kg/day.

Serum Lipidomics Profiling to Identify Biomarkers for Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide, which ranks top in both incidence and mortality. To broaden our understanding of the lipid metabolic alterations in NSCLC and to identify potential biomarkers for early diagnosis, we performed nontargeted lipidomics analysis in serum from 66 early-stage NSCLC, 40 lung benign disease patients (LBD), and 40 healthy controls (HC) using Ultrahigh Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (UHPLC-Q-TOF/MS). The identified biomarker candidates of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) were further externally validated in a cohort including 30 early-stage NSCLC, 30 LBD, and 30 HC by a targeted lipidomic analysis. We observed a significantly altered lipid metabolic profile in early-stage NSCLC and identified panels of PCs and PEs to distinguish NSCLC patients and HC. The levels of PCs and PEs were found to be dysregulated in glycerophospholipid metabolism, which was the top altered pathway in early-stage NSCLC. Receiver operating characteristic (ROC) curve analysis revealed that panels of PCs and PEs exhibited good performance in differentiating early-stage NSCLC and HC. The levels of PE(16:0/16:1), PE(16:0/18:3), PE(16:0/18:2), PE(18:0/16:0), PE(17:0/18:2), PE(18:0/17:1), PE(17:0/18:1), PE(20:5/16:0), PE(18:0/18:1), PE(18:1/20:4), PE(18:0/20:3), PC(15:0/18:1), PC(16:1/20:5), and PC(18:0/20:1) in early-stage NSCLC were significantly increased compared with HC (p<0.05). Overall, our study has thus highlighted the power of using comprehensive lipidomic approaches to identify biomarkers and underlying mechanisms in NSCLC.

Simultaneous quantification of serum monounsaturated and polyunsaturated phosphatidylcholines as potential biomarkers for diagnosing non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is one of the most common malignancies worldwide. In this study, we investigated Ultrahigh Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry and Gas Chromatography Time-of-Flight/Mass Spectrometry-based non-targeted metabolomic profiles of serum samples obtained from early-stage NSCLC patients and healthy controls (HC). Metabolic pathways and the biological relevance of potential biomarkers were extensively studied to gain insights into dysregulated metabolism in NSCLC. The identified biomarker candidates were further externally validated via a targeted metabolomics analysis. The global metabolomics profiles could clearly distinguish NSCLC patients from HC. Phosphatidylcholine (PC) levels were found to be dysregulated in glycerophospholipid (GPL) metabolism, which was the top altered pathway in early-stage NSCLC. Compared with those in HC, significant increases in the levels of saturated and monounsaturated PCs such as PC (15:0/18:1), PC (18:0/16:0) and PC (18:0/20:1) were observed in NSCLC. Additionally, relative to those in HC, the levels of 9 polyunsaturated PCs, namely, PC (17:2/2:0), PC (18:4/3:0), and PC (15:0/18:2), and so on were significantly decreased in NSCLC patients. A panel of 12 altered PCs had good diagnostic performance in differentiating early-stage NSCLC patients from HC, and these PCs may thus be used as serum biomarkers for the early diagnosis of NSCLC.

MiR-1260b promotes the migration and invasion in non-small cell lung cancer via targeting PTPRK.

OBJECTIVE: Non-small cell lung cancer (NSCLC) accounts for 80-85% of lung cancer cases which cause most of cancer-related deaths globally. As our previous study discovered miR-1260b can be regarded as a specific signature for metastasis in NSCLC patients. However, the molecular mechanisms of miR-1260b underlying NSCLC progression and metastasis remain dismal. METHODS: The expression of miR-1260b in NSCLC tissues and cell lines were examined by real-time PCR, the effects of miR-1260b on cell migration, invasion and proliferation were evaluated in vitro. Furthermore, luciferase reporter assay was performed to identify the targets of miR-1260b, and the association between miR-1260b and its target gene was determined by real-time PCR and western blot assay. RESULTS: The results showed that miR-1260b was significantly upregulated in NSCLC cell lines. The inhibition of miR-1260b expression decreased the migratory and invasive rates in A549 cells while miR-1260b overexpression had the opposite effect. Furthermore, PTPRK was identified as a direct target of miR-1260b, and PTPRK expression was inversely correlated with miR-1260b in NSCLC cell lines and clinical tissues. CONCLUSIONS: These results suggested that miR-1260b may play an important role in NSCLC metastasis progression and could serve as a putative target for diagnosis and treatment of NSCLC.

Simultaneous detection of human CYP2C19 polymorphisms and antibiotic resistance of Helicobacter pylori using a personalised diagnosis kit.

OBJECTIVES: A personalised diagnosis kit for Helicobacter pylori that employs visual gene chip technology for the simultaneous detection of CYP2C19 polymorphisms and clarithromycin/levofloxacin antibiotic resistance was evaluated. METHODS: Gastric antrum mucosa biopsy specimens of 394 patients were tested using the kit. DNA sequencing and antibiotic susceptibility testing of the H. pylori were also performed. RESULTS: In total, 267 (67.8%) of the 394 specimens were positive for H. pylori using the kit and DNA sequencing, and 136 (34.5%) were positive by culturing. For human CYP2C19 and the bacterial 23S rRNA and gyrA genes, the concordance rates were 92.4% (364/394), 96.6% (258/267) and 97.0% (259/267) between the kit and DNA sequencing results, respectively. For clarithromycin and levofloxacin resistance, the concordance rates were 90.4% (123/136) and 81.6% (111/136) between the kit and antibiotic susceptibility testing results. CONCLUSIONS: The personalised diagnosis kit for H. pylori provides useful information for the choice of proton pump inhibitor and antibiotic in combination therapy.

AEG-1 mRNA expression in non-small cell lung cancer is associated with increased tumor angiogenesis.

Astrocyte-elevated gene-1 (AEG-1) is implicated in the oncogenesis and angiogenesis of various types of human malignant disease. However, the angiogenesis roles of AEG-1 in non-small cell lung cancer (NSCLC) remain to be further elucidated. In the present study, the expression level of AEG-1 mRNA in seven human lung cell lines and 89 paired tissue samples (tumor tissues (TTs) and pair-matched normal adjacent tissues (PMNATs)) from NSCLC patients was detected by real-time PCR. Staining of vascular endothelial growth factor (VEGF) and intratumoral microvessel density (iMVD, labeled by CD105) were assessed by immunohistochemistry. Furthermore, cell migration and invasion were evaluated by wound healing assay and transwell assays. AEG-1 mRNA level was significantly higher in human lung cancer cells and TTs than that in human normal bronchial epithelial cell line 16HBE and PMNATs, respectively (P<0.001). Higher AEG-1 mRNA level in patients with NSCLC was correlated with clinical stages (P=0.028), differentiation (P=0.042), and lymph node metastasis (P=0.004). Moreover, Upregulated AEG-1 mRNA expression level was associated with higher tumor angiogenesis, reflected by the increase of VEGF expression and iMVD counting (P=0.021, P<0.001). However, 95D cell line transfected with AEG-1 siRNA oligos (siAEG-1) exhibited no significant decrease of cell invasion or migration capacities when compared with the control cells (P>0.05).These results suggested that AEG-1 may play important roles at the transcription level in malignant transformation and tumor angiogenesis in NSCLC, and anti-AEG-1 mRNA expression may be a novel potential strategy for anti-angiogenic therapy of NSCLC.

Systematic Identification and Assessment of Therapeutic Targets for Breast Cancer Based on Genome-Wide RNA Interference Transcriptomes.

With accumulating public omics data, great efforts have been made to characterize the genetic heterogeneity of breast cancer. However, identifying novel targets and selecting the best from the sizeable lists of candidate targets is still a key challenge for targeted therapy, largely owing to the lack of economical, efficient and systematic discovery and assessment to prioritize potential therapeutic targets. Here, we describe an approach that combines the computational evaluation and objective, multifaceted assessment to systematically identify and prioritize targets for biological validation and therapeutic exploration. We first establish the reference gene expression profiles from breast cancer cell line MCF7 upon genome-wide RNA interference (RNAi) of a total of 3689 genes, and the breast cancer query signatures using RNA-seq data generated from tissue samples of clinical breast cancer patients in the Cancer Genome Atlas (TCGA). Based on gene set enrichment analysis, we identified a set of 510 genes that when knocked down could significantly reverse the transcriptome of breast cancer state. We then perform multifaceted assessment to analyze the gene set to prioritize potential targets for gene therapy. We also propose drug repurposing opportunities and identify potentially druggable proteins that have been poorly explored with regard to the discovery of small-molecule modulators. Finally, we obtained a small list of candidate therapeutic targets for four major breast cancer subtypes, i.e., luminal A, luminal B, HER2+ and triple negative breast cancer. This RNAi transcriptome-based approach can be a helpful paradigm for relevant researches to identify and prioritize candidate targets for experimental validation.

Phospholipid Scramblase 1 Interacts with Midkine and Regulates Hepatic Cancer Cell Proliferation and Migration.

BACKGROUND: Mounting evidence indicates that nuclear targeting by growth factors plays an indispensable role on their biological activities. Midkine (MK) is a multifunctional growth factor and has been discovered to play important roles in carcinogenesis. MK has been reported to localize to the nucleus and nucleolus of HepG2 cells and is involved in cell proliferation and apoptosis. METHODS: The interaction was reconfirmed by in vitro pull down and in vivo coimmunoprecipitation (Co-IP), also by the colocalization in the HepG2 cells. The proliferation and migration was determined by MTT and trans-well assay. RESULTS: PLSCR1 was identified as a novel MK-interacting protein. Notably, PLSCR1 interacted with MK in the cell nucleus and regulated hepatic carcinoma cell proliferation and migration. CONCLUSIONS: This study suggests that PLSCR1 positively regulates hepatic carcinoma cell proliferation and migration through interacting with MK, thus deepening our understanding on the regulation of midkine during hepatic carcinoma growth and metastasis.

Overexpression of miR-1260b in Non-small Cell Lung Cancer is Associated with Lymph Node Metastasis.

Lymph node (LN) metastasis is often an early event in the progression of malignant tumors and it contributes to the majority of cancer mortalities. MiRNAs play key roles in tumor metastasis. This study aimed to investigate the specific miRNAs as putative indicators of metastasis early diagnosis for non-small cell lung cancer (NSCLC). In this study, five NSCLC cases with LN metastasis and four cases without metastasis (NLN) were enrolled for Agilent Human miRNA array. The interested differentially expressed miRNA was validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in the LN metastasis (n = 46) and NLN (n = 39) groups. The microarray results revealed that three miRNAs (miR-1260b, miR-423-3p, miR-23a-5p) were differentially expressed in LN metastasis group compared with NLN group. The expression of miR-1260b was tested by qRT-PCR and the mean relative expression fold change (2(-ΔΔCt)) in LN metastasis was significantly higher than that in the NLN group (3.942, 1.743 respectively, P = 1.179E-04). The patients with tumor-node-metastasis (TNM) stage III were identified more frequently in LN metastasis group (P = 1.772E-11) and with a higher expression level of miR-1260b (5.126, P = 1.147E-06). In addition, the LN metastasis cases were associated with a poorly differentiated degree (P = 0.007). The overexpression of miR-1260b in NSCLC with LN metastasis can be regarded as a specific signature for early progression and prognosis of NSCLC.

[Variability of Reverse Transcriptase Gene and S Gene in Lamivudine-treated Chronic Hepatitis B Patients].

We wished to undertake molecular characterization of the reverse transcriptase (RT) gene and overlapping surface (S) gene in lamivudine-treated patients with chronic infection with the hepatitis B virus (HBV). Sequencing analyses of the HBV RT/S gene of isolates from 25 chronic hepatitis B (CHB) patients with the YMDD mutation and 30 treatment-naïve CHB patients were undertaken. In patients with the YMDD mutation, rtM2041 was the major type of mutation (20/25, 80%). rtL80I was present in most of the patients with rtM204I (14/20, 70%). rtL180M coexisted with rtM204V (5/5, 100%). Patients with the YMDD mutation had a significantly higher prevalence of mutation of the RT gene than treatment-naïve CHB patients (P < 0.05). Classical primary resistance and secondary/compensatory mutations were detected at only five sites (rtL80, rtV173, rtL180, rtM204, rtM250) in CHB patients with the YMDD mutation. The frequency of nucleos(t)ide analog resistance (NAr) mutation within the RT gene in patients with the YMDD mutation was significantly higher than that in treatment-naïve patients (P < 0.05). Amino-acid mutations within the RT gene were also associated with other types of NAr in patients with the YMDD mutation. The rate of amino-acid variants within the S gene region was significantly higher in patients with the YMDD mutation than that in treatment-naïve patients (P < 0.05). sM133L and sG145R variants were also present in patients with the YMDD mutation. These observations suggest that CHB patients with the YMDD mutation also have NAr mutations related to other NA drugs, which might lead to cross-resistance in CHB patients. Variants present in the S gene region could cause changes in the antigenicity of HBsAg, which could result in a false-negative diagnosis of HBsAg and immune in escape of the HBV.

Role of midkine-progranulin interaction during angiogenesis of hepatocellular carcinoma.

Midkine (MK) is a heparin-binding growth factor involved in growth, survival, migration, and differentiation of various target cells and dysregulation of MK signaling is implicated in a variety of inflammatory diseases and cancers. Although MK has been reported to act on endothelial cells and to have proangiogenic effects, the exact role of MK in angiogenesis is poorly defined. Progranulin (PGRN) is a secreted glycoprotein that functions as an important regulator of development, cell cycle progression, cell motility, tumorigenesis, angiogenesis. We screened the PGRN from the hepatic cancer cell cDNA library which was interacted with MK, and confirmed the binding by co-immunoprecipitation and co-localization. During our study, the interaction between MK and PGRN had the important role on the HUVECs proliferation, migration, and tubulogenesis, which indicated the interaction may regulate the angiogenesis, also the in vivo angiogenesis model CAM showed the promotion effect stimulated by MK and PGRN. These findings provide the first evidence linking the association of MK and PGRN and may identify the mechanism of MK during the hepatocellular carcinoma angiogenesis.

Inhibitory effect of midkine-binding peptide on tumor proliferation and migration.

BACKGROUND: To investigate the inhibitory effect of midkine-binding peptides on human umbilical vein endothelial cells (HUVECs) proliferation and angiogenesis of xenograft tumor. METHODS: The midkine-binding peptides were panned by Ph.D.-7(™) Phage Display Peptide Library Kit, and the specific binding activities of positive clones to target protein were examined by phage ELISA. The effect of midkine-binding peptides on proliferation of HUVECs was confirmed by MTT test. The xenograft tumor model was formed in BALB/c mice with the murine hepatocarcinoma cells H22 (H22). Microvessel density (MVD) was analyzed by immunohistochemistry of factor VIII staining. RESULTS: Midkine-binding peptides have the inhibitory effects on tumor angiogenesis, a proliferation assay using human umbilical vein endothelial cells (HUVECs) indicated that particular midkine-binding peptides significantly inhibited the proliferation of the HUVECs. Midkine-binding peptides were also observed to efficiently suppress angiogenesis induced by murine hepatocarcinoma H22 cells in BALB/c nude mice. CONCLUSION: The midkine-binding peptides can inhibit solid tumor growth by retarding the formation of new blood vessels. The results indicate that midkine-binding peptides may represent potent anti-angiogenesis agents in vivo.

Biomarker identification and pathway analysis by serum metabolomics of lung cancer.

Lung cancer is one of the most common causes of cancer death, for which no validated tumor biomarker is sufficiently accurate to be useful for diagnosis. Additionally, the metabolic alterations associated with the disease are unclear. In this study, we investigated the construction, interaction, and pathways of potential lung cancer biomarkers using metabolomics pathway analysis based on the Kyoto Encyclopedia of Genes and Genomes database and the Human Metabolome Database to identify the top altered pathways for analysis and visualization. We constructed a diagnostic model using potential serum biomarkers from patients with lung cancer. We assessed their specificity and sensitivity according to the area under the curve of the receiver operator characteristic (ROC) curves, which could be used to distinguish patients with lung cancer from normal subjects. The pathway analysis indicated that sphingolipid metabolism was the top altered pathway in lung cancer. ROC curve analysis indicated that glycerophospho-N-arachidonoyl ethanolamine (GpAEA) and sphingosine were potential sensitive and specific biomarkers for lung cancer diagnosis and prognosis. Compared with the traditional lung cancer diagnostic biomarkers carcinoembryonic antigen and cytokeratin 19 fragment, GpAEA and sphingosine were as good or more appropriate for detecting lung cancer. We report our identification of potential metabolic diagnostic and prognostic biomarkers of lung cancer and clarify the metabolic alterations in lung cancer.

Development of hybrid-type modified chitosan derivative nanoparticles for the intracellular delivery of midkine-siRNA in hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most of the patients with HCC lose the surgical opportunity at the time of diagnosis. Some novel therapeutic modalities, like gene therapy, are promising for the treatment of HCC. However, the success of gene therapy depends on two aspects: efficient gene materials and gene delivery vectors. The present study was to develop new chitosan-based nanoparticles for a midkine-siRNA (anti-HCC gene drug) delivery. METHODS: The novel gene delivery vector (MixNCH) was synthesized by hybrid-type modification of chitosan with 2-chloroethylamine hydrochloride and N, N-dimethyl-2-chloroethylamine hydrochloride. The chemical structure of MixNCH was characterized by FT-IR and 1HNMR. The cytotoxicity of MixNCH was determined by MTS assay. The gene condensation ability and size, zeta potential and morphology of MixNCH/MK-siRNA nanoparticles were measured. The in vitro transfection and gene knockdown efficiency of midkine by MixNCH/MK-siRNA nanoparticles was detected by qRT-PCR and Western blotting. Gene knockdown effect at the molecule level on the proliferation of HepG2 in vitro was determined by MTS assay. RESULTS: MixNCH was successfully acquired by aminoalkylation modification of chitosan. The MixNCH could condense MK-siRNA well above the weight ratio of 3. The average size of MixNCH/MK-siRNA nanoparticles was 100-200 nm, and the surface charge was about +5 mV. Morphologically, MixNCH/MK-siRNA nanoparticles were in regular spherical shape with no aggregation. Regarding to the in vitro transfection of nanoparticles, the MixNCH/MK-siRNA nanoparticles reduced MK mRNA level to 14.03%+/-4.03%, which were comparable to Biotrans (8.94%+/-3.77%). MixNCH/MK-siRNA effectively inhibited the proliferation of HepG2 in vitro. CONCLUSION: MixNCH/MK-siRNA nanoparticles could be effective for the treatment of hepatocellular carcinoma.