Search

Purpose: We have developed a PEGylated transferrin-conjugated liposomes (PTf-Ls) system for the combined tumor imaging and targeted delivery of the IFN-γ–inducible protein-10 (IP-10) gene in a single macromolecular construct. Here, we characterize and analyze the use of this system in a mouse model of breast cancer.Experimental Design: The biophysical and cell transfection properties of PTf-Ls were determined through a series of in vitro experiments. A nude mouse/breast cancer cell line xenograft model (mouse xenograft model) was used to image the tumor internalization of fluorescently labeled PTf-Ls. The clinical use of the system was tested by treating tumor-bearing mice with PTf-Ls loaded with IP-10 plasmid DNA or fluorescent lipoplexes.Results: The resulting 165-nm liposomes (zeta potential = −10.6 mV) displayed serum resistance, low cytotoxicity (in vivo fluorescence and bioluminescence analyses. Tumor fluorescence increased gradually up to 26 hours, whereas background fluorescence decreased to near-baseline levels. Treatment of mice with PTf-Ls entrapped pcDNA3.1-IP-10 suppressed tumor growth in mice by 79% on day 50 and increased the mean survival time of mice. Fluorescent pcDNA-IP-10–entrapped PTf-Ls showed good properties for simultaneous tumor-targeted imaging and gene-specific delivery in an animal tumor model.Conclusions: Our developed transferrin-conjugated liposome system possesses promising characteristics for tumor-targeting, imaging, and gene therapy applications. Clin Cancer Res; 19(15); 4206–17. ©2013 AACR.

Supplementary Figures - PDF file 1800K, Figure S1: Transmission electron microscopy images of P-Ls (A), P-Ls entrapped; Figure S2: High-resolution X-ray photoelectron spectra on nitrogen of Lipid mixture (A), P-Ls (B), PTf-Ls (C) and sulfur atom of PTf-Ls (D); Figure S3: Confocal fluorescence images showing uptake of rhodamine-labeled PTf-Ls by MCF-7 cells. DAPI/nuclei (blue), rhodamine (red); Figure S4: Fluorescence micrographs and differential interference contrast (DIC) images of HeLa cells incubated with PTf-Ls entrapped pEGFP-N1 for 5 h (A), or pretreated for 1 h with 4 microg (B) or 40 microg (C) of transferrin in 1 mL of medium containing 10 % FBS for competition experiment. Images were taken 48 h after transfection; Figure S5: Fluorescence micrographs of HeLa cells transfected with PTf-Ls entrapped pEGFP-N1 (100�x magnification). (A and B) Images taken 48 h after transfection without (A) or with (B) FBS. (C) EGFP expression 24 h after transfection with PTf-Ls entrapped pEGFP-N1 with FBS. (D) EGFP expression 48 h after transfection with PTf-Ls entrapped pEGFP-N1 with FBS; Figure S6: Fluorescence micrographs of HeLa cells incubated with PTf-Ls entrapped pEGFP-N1 for 5 h (I), 24 h (II) or 48 h (III, the transfection medium was changed once at 24 h) (200�x, magnification); Figure S7: Dynamic light scattering particle size distributions for liposomes prepared on 4 June 2011 P-Ls (1) and PTf-Ls (2) or 11 August 2012 P-Ls (2) & PTf-Ls (4), respectively; Figure S8: Cytotoxicity of P-Ls and PTf-Ls measured at 4 h and 24 h after the addition of P-Ls or PTf-Ls to HeLa or MCF-7 cells; Figure S9: Cell cycle profiles of liposome-transfected HeLa cells. Numbers in figure indicate the percentage of cells in each cell cycle phase. Untreated group (A), HeLa cells transfected with P-Ls (B) or PTf-Ls (C)

Supplementary Tables - PDF file 87K, Table S1. Physical properties of liposomes Top: Concentrations of ethanol and Ca2+ for optimized liposome preparation. Bottom: Diameters and surface potentials of P-Ls entrapped pcDNA3.1-IP-10 and PTf-Ls entrapped pcDNA3.1-IP-10 prepared by Method III; Table S2. Chemical composition of liposome surfaces from XPS analysis; Table S3. Serum stability of PTf-Ls entrapped pcDNA3.1-IP-10

Supplementary Methods - PDF file 121K, Supplementary Methods

GC is a fatal disease with high heterogeneity and invasiveness. Recently, SPP1 has been reported to be involved in the tumor progression of multiple human cancers; however, the role of SPP1 in GC heterogeneity and whether it is associated with the invasiveness and mortality of GC remain unclear. Here, we combined multiple RNA sequencing approaches to evaluate the impact of SPP1 on GC. Through bulk RNA sequencing (bulk RNA-seq) and immunohistochemistry (IHC), we found that SPP1 was highly expressed in GC, and high levels of SPP1 were associated with macrophage infiltration, an advanced tumor stage, and higher mortality for advanced GC patients. Furthermore, through simultaneous single-cell and spatial analysis, we demonstrated that SPP1+ macrophages are tumor-specific macrophages unique to cancer and enriched in the deep layer of GC tissue. Cell—cell communication analysis revealed that SPP1/CD44 interactions between SPP1+ macrophages and their localized tumor epithelial cells could activate downstream target genes in epithelial cells to promote dynamic changes in intratumor heterogeneity. Moreover, these activated genes were found to be closely associated with poor clinical GC outcomes and with cancer-related pathways that promote GC progression, as shown by survival analysis and enrichment analysis, respectively. Collectively, our study reveals that tumor-specific SPP1+ macrophages drive the architecture of intratumor heterogeneity to evolve with tumor progression and that SPP1 may serve as a prognostic marker for advanced GC patients, as well as a potential therapeutic target for GC.

Yizhuo Lu,1 Jia Cheng,2 Wangyu Cai,2 Huiqin Zhuo,2 Guoyang Wu,1 Jianchun Cai2 1Department of General Surgery, Zhongshan Hospital Xiamen University, Institute of Gastrointestinal Oncology, School of Medicine, Xiamen University, Xiamen Municipal Key Laboratory of Gastrointestinal Oncology, Xiamen, Fujian, 361004, People’s Republic of China; 2Department of Gastrointestinal Surgery, Zhongshan Hospital Xiamen University, Institute of Gastrointestinal Oncology, School of Medicine, Xiamen University, Xiamen Municipal Key Laboratory of Gastrointestinal Oncology, Xiamen, Fujian, 361004, People’s Republic of ChinaCorrespondence: Jianchun CaiDepartment of Gastrointestinal Surgery, Zhongshan Hospital Xiamen University, Institute of Gastrointestinal Oncology, School of Medicine, Xiamen University, Xiamen Municipal Key Laboratory of Gastrointestinal Oncology, Room 203, No. 146, Hubin South Road, Siming District, Xiamen, Fujian Province, 361004, People’s Republic of ChinaTel +86 592-2993191Email jianchuncai0592@163.comBackground: Circular RNA VPS33B (circVPS33B) has been revealed to be upregulated in gastric cancer (GC) tissues. However, the role of circVPS33B in infiltrative GC is indistinct.Methods: Expression of circVPS33B was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation, migration, and invasion of infiltrative GC cells (XGC-1) were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), plate clone, wound-healing, or transwell assays. Protein levels were detected by Western blotting. Measurements of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were executed using an XF96 extracellular flux analyzer. Glucose uptake and lactate production were analyzed by glycolysis assay. The regulatory mechanism of circVPS33B had been explored by bioinformatics analysis, dual-luciferase reporter assay, and/or RNA pull-down assay. In vivo tumorigenesis assay was executed to verify the oncogenicity of circVPS33B.Results: CircVPS33B was upregulated in infiltrative GC tissues and cells. CircVPS33B silencing decreased tumor growth in vivo and inhibited proliferation, migration, invasion, EMT, and Warburg effect of infiltrative GC cells in vitro. Mechanically, circVPS33B regulated heterogeneous nuclear ribonucleoprotein K (HNRNPK) expression via sponging miR-873-5p. Furthermore, miR-873-5p inhibitor offset circVPS33B knockdown-mediated effects on malignant behaviors and Warburg effect of infiltrative GC cells. HNRNPK overexpression reversed the inhibitory impact of miR-873-5p mimic on malignant behaviors and Warburg effect of infiltrative GC cells.Conclusion: CircVPS33B accelerated Warburg effect and tumor growth through regulating the miR-873-5p/HNRNPK axis in infiltrative GC, manifesting that circVPS33B might be a potential target for infiltrative GC treatment.Keywords: circVPS33B, miR-873-5p, HNRNPK, infiltrative GC

Gastric Cancer (GC) is one of the main causes leading to death. PMP22, as a member of the GAS3 family of tetraspan proteins, it is associated with a variety of neurological diseases. Recently, more and more studies have shown that PMP22 play a great role in the physiological processes such as cells adhesion, migration, proliferation and tumorigenesis, but the involvement and functional mechanisms of PMP22 in Gastric carcinoma are not investigated clearly. In this study, we found that the PMP22 was overexpressed in the GC cells and tissue. Knockdown of PMP22 inhibits cell growth. Over-expressed PMP22 inhibits the etoposide-induced apoptosis, meanwhile knockdown of PMP22 promotes the etoposide-induced proliferation suppression, and increases cell apoptosis in GC cells. Furthermore, PMP22 enhanced the inhibition of the p53 transcriptional activities and down-regulated the p53 targeting genes, including p21, BAX and PUMA with or without treatment of etoposide. Finally, our results showed that PMP22 reduced the etoposide-induced tumor growth suppression in nude mice. Taken together, our research provided an anti-apoptotic properties alternative mechanism for PMP22 in gastric carcinoma and suggested PMP22 can be a potential target for the treatment of gastric cancer.

Hepatocellular carcinoma (HCC) accounts for ~85% of primary liver cancer cases and is a leading cause of mortality worldwide. Effective early diagnosis is difficult for HCC; however, effective biomarkers may be beneficial for diagnosis. In the current study, serum samples, and HCC and adjacent tissue samples were obtained from patients with HCC for the detection of biomarkers using 2‑D gel electrophoresis (2‑DE) and matrix‑assisted laser desorption/ionization‑time of flight (TOF)/TOF mass spectrometry. The crude serum samples did not need to be prepared for removal of high abundance proteins. The mRNA expression levels of HCC‑associated proteins were detected in tissues using reverse transcription‑quantitative PCR. Statistical analysis and database matching were used to identify the differentially expressed proteins detected in the serum and tissue groups. Immunohistochemistry (IHC) was performed to detect the expression of significant proteins in HCC and adjacent tissues. The results revealed ~800 protein spots on a 2‑DE gel that were detected in serum samples, and 1,200 spots were identified in the tissue samples. The protein and mRNA expression levels of oxysterol binding protein‑like 11 (OSBPL11) in HCC serum and tissue samples were consistent. Pathway analysis demonstrated that members of the apolipoprotein family, particularly apolipoprotein E (APOE), and RAS family members were closely associated in HCC, either directly or via ferratin heavy polypeptide 1. IHC results demonstrated that the APOE protein serves an important role in liver cancer development. The lysis buffer used in the current study was effective for serum protein separation in 2‑DE sample preparation. In addition, the present study revealed that downregulated OSBPL11 may be a potential indicator for HCC, and the apolipoprotein family, particularly APOE, and the RAS family may cooperatively serve an important role.

In lung cancer, antiangiogenic strategies targeting tumor-derived endothelial cells (TECs) afford a survival advantage, but the characteristics of TECs have not been comprehensively elucidated. Herein, high-purity (> 98%) TECs were obtained, and these cells retained expression of EC markers and exhibited high viability. ITRAQ-2DLC-MS/MS was performed to profile the proteome and the heterogeneity of ECs. Only 31 of 1820 identified proteins were differentially expressed between adenocarcinoma (ADC)- and squamous cell carcinoma (SCC)-derived TECs (TEC-A and TEC-S, respectively), and cadherin-2 (CDH2) was the most significantly upregulated protein in TEC-A samples. Positive immunostaining for CDH2 (score > 3) was significantly more frequent in the endothelium of ADC tissues than in that of SCC tissues. Loss- or gain-of-function analysis showed that CDH2 significantly promoted in vitro and in vivo angiogenesis and sensitivity to the antagonist exherin. The MAPK/ERK and MAPK/JNK signaling pathways may play crucial roles in CDH2-induced HIF-1α/VEGF-mediated angiogenesis. Moreover, high CDH2 expression in TECs was significantly associated with tumor stage, visceral pleural metastasis, and decreased overall survival in patients with ADC but not SCC. Together, these data indicate the importance of CDH2 in angiogenesis and highlight its potential both for antiangiogenic therapy and as a candidate prognostic marker for ADC. Electronic supplementary material The online version of this article (10.1186/s12943-019-0987-1) contains supplementary material, which is available to authorized users.

Background: We aimed to investigate the effect of PI3K/Akt signaling pathway on PRAS40Thr246 phosphorylation in gastric cancer cells. Methods: The study was conducted from April 2017 to January 2018 in Zhongshan Hospital, Xiamen University, Xiamen, China. Gastric cancer cells were divided into three groups: gastric cancer cell group, LY294002 group and MK-2206 group. Specific tests were conducted accordingly. Results: Inhibition of PI3K/Akt signaling pathway activation and PRAS40Thr246 phosphorylation could inhibit proliferation and invasion and promote apoptosis of gastric cancer cells, and PRAS40Thr246 phosphorylation could activate PI3K/Akt signaling pathway. Conclusion: The levels of PI3K/Akt signaling pathway related proteins and p-PRAS40Thr246 were significantly increased in gastric cancer cells. p-PRAS40-Thr246 was able to reflect the activation of the PI3K/Akt signaling pathway, reflecting the sensitivity of the PI3K/AKT signaling pathway to inhibitors.

Background: Circular RNA VPS33B (circVPS33B) is upregulated in gastric cancer (GC) tissues. However, the role of circVPS33B in infiltrative GC is indistinct. Methods: Expression of circVPS33B, miR-873-5p, and heterogeneous nuclear ribonucleoprotein K (HNRNPK) mRNA was detected using quantitative real-time polymerase chain reaction (qRT-PCR). The proliferation, colony formation, migration, and invasion of infiltrative GC cells (XGC-1) were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), plate clone, wound healing, or transwell assays. Several protein levels were examined by western blotting. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of XGC-1 cells were evaluated by XF96 extracellular flux analyzer. Glucose uptake and lactate production were analyzed by glycolysis assay. The relationship between circVPS33B or HNRNPK and miR-873-5p was verified by dual-luciferase reporter and/or RNA pull-down assays. In vivo tumorigenesis assay was executed for verifying the in vitro results.Results: CircVPS33B and HNRNPK were upregulated while miR-873-5p was downregulated in infiltrative GC tissues and XGC-1 cells. CircVPS33B silencing decreased tumor growth in vivo and inhibited proliferation, colony formation, migration, invasion, and Warburg effect of XGC-1 cells in vitro. CircVPS33B regulated HNRNPK expression via sponging miR-873-5p. The inhibitory influence of circVPS33B knockdown on the malignancy and Warburg effect of XGC-1 cells was overturned by miR-873-5p inhibitor. HNRNPK overexpression reversed the repression of the malignancy and Warburg effect of XGC-1 cells caused by miR-873-5p mimic.Conclusions: CircVPS33B accelerated infiltrative GC progression through regulating the miR-873-5p/HNRNPK axis, manifesting that circVPS33B might be a promising target for infiltrative GC treatment.

Angiogenesis and vascular maturation play important roles in tumorigenesis and tumor development. The expression of neuropilin 1 (NRP1) is closely associated with angiogenesis in tumors; however, the molecular mechanisms of action in angiogenesis and tumor maturation, as well as the potential clinical value of NRP1 remain unclear. The importance of NRP1 expression in tumor progression was determined using The Cancer Genome Atlas (TCGA) database analysis. Gain- and loss-of-function experiments of NRP1 were performed in vascular endothelial cells (ECs) to investigate the functions in angiogenesis. CCK-8, flow cytometry, Transwell experiments and a series of in vitro experiments were used to detect cell functions. A combination of angiogenesis antibody arrays and RNA-Seq analyses were performed to reveal the proangiogenic mechanisms of action. The function of semaphorin 4D (SEMA4D) was also investigated separately. NRP1 mRNA levels were significantly increased in primary tumors compared with normal tissues based on TCGA data (P

According to the growth pattern, gastric cancer (GC) could be classified into expanding-type GC and infiltrative-type GC (Ming’s classification). The growth pattern of GC is often related to the malignant degree, invasion, metastasis, and other pathological characteristics of tumors. MicroRNAs (miRNAs) play important roles in modulating gene expression during the GC development. In this study, miR-29s were significantly correlated with the gastric carcinogenesis and Ming’s classification. Biological function of miR-29s is most closely related to the pathway of extracellular matrix (ECM)-receptor interaction. ECM structural assembly, cell movement, and cell adhesion are the main functional categories of target genes in this pathway. Among these targets, the COL4A1 gene ranked at the top in the association analysis of combined miR-29s biological function and GC subtype, and miR-29s inhibited its translation by binding to the 3′ UTR region. Infiltrative-type GC cells secrete a higher level of COL4A1 protein than do expanding-type GC cells. The expression of COL4A1 in GC is correlated with clinicopathological features. Downregulation of COL4A1 expression significantly inhibited the migration and invasion of GC cells. High COL4A1 expression was correlated with poor prognosis in survival analysis. The miR-29s regulatory network may affect the development of growth patterns and pathological progress of GC by regulating the function of COL4A1., Graphical Abstract, According to the growth pattern, gastric cancer (GC) could be classified into expanding-type GC and infiltrative-type GC. Cai and colleagues show that miR-29s were differentially expressed in these two types of GC and mainly regulated the ECM-receptor interaction pathway by targeting COL4A1 to inhibit the migration and invasion of GC cells.

// Huifang Peng 1, * , Hongwei Jin 2, * , Huiqin Zhuo 3, 4 and Heqing Huang 1, 5, 6 1 State Key Laboratory of Stress Cell Biology, School of Life Science, Xiamen University, Xiamen, Fujian 361004, China 2 Xiamen Center of Clinical Laboratory, Zhongshan Hospital, Xiamen University, Xiamen, Fujian 361004, China 3 Department of Gastrointestinal Surgery, Zhongshan Hospital, Xiamen University, Xiamen, Fujian 361004, China 4 Institute of Gastrointestinal Oncology, Medical College of Xiamen University, Xiamen University, Xiamen, Fujian 361004, China 5 State Key Laboratory of Marine Environmental Science, College of Oceanography and Environmental Science, Xiamen University, Xiamen, Fujian 361004, China 6 The Key Laboratory for Chemical Biology of Fujian Province, College of Chemistry & Chemical Engineering, Xiamen University, Xiamen, Fujian 361004, China * These authors have contributed equally to this work Correspondence to: Heqing Huang, email: hqhuang@xmu.edu.cn Huiqin Zhuo, email: zhuohuiqin@xmu.edu.cn Keywords: cisplatin, transferrin, targeted drug delivery, ovarian cancer, antitumor treatment Received: May 12, 2016 Accepted: April 02, 2017 Published: April 21, 2017 ABSTRACT Cisplatin is a widely used anticancer drug, while non-targeted delivery, development of drug resistance, and serious side effects significantly limit its clinical use. In order to improve the tumor-targeting properties of cisplatin, transferrin (Tf) was employed as a carrier to transfer cisplatin into cancer cells via transferrin receptor 1 (TfR1) mediated endocytosis. The binding ability of cisplatin and Tf could be improved by pretreating Tf with 10% ethanol, and the binding number of cisplatin for each Tf molecule could reach to 40 without structural or functional impairment of Tf. The Tf-cisplatin complex could be delivered into human ovarian carcinoma cells high efficiently. In tumor-bearing nude-mice model, the Tf-cisplatin complex inhibited tumor growth in vivo more effectively than free cisplatin, with less toxicity in other tissues. Tumor targeting efficiency of the Tf-cisplatin complex was supported by in vivo and ex vivo imaging and platinum residues detected in each ex vivo organ. These data suggested that Tf-cisplatin was more effective and less drug-resistance than cisplatin, with targeting to tumor cells. Therefore, Tf-mediated delivery of cisplatin is a potential strategy for targeted delivery into tumor cells.

Gastric cancer (GC) is one of the most common malignant tumors worldwide. Peripheral myelin protein 22 (PMP22) is a 22-kDa tetraspan glycoprotein that is predominantly expressed by myelinating Schwann cells. However, recent studies have shown that PMP22 is closely related to cell proliferation and tumorigenesis in different cancers. In this study, we discovered a new miRNA that regulates PMP22 and gastric cancer cell prolifration. Our bioinformatics analysis suggested that there is a conserved miRNA recognition site for miR-139-5p on the 3' UTR of PMP22. Interestingly, our results showed overexpression of miR-139-5p significantly suppressed growth and prolifration in GC cells and inhibited tumor growth in nude mice xenografted with GC cells. MiR-139-5p suppressed the activity of a luciferase reporter containing the PMP22-3' UTR, and the ectopic expression of PMP22 rescued the miR-139-5p-mediated inhibition of cell proliferation in GC cells. Mechanistically, miR-139-5p may negatively regulate PMP22 to repress cell proliferation by targeting the NF-κB signaling pathway in gastric cancer. Finally, overexpression of miR-139-5p significantly inhibited tumor growth in nude mice xenografted with GC cells.and the miR-139-5p levels were inversely correlated with PMP22 expression in nude mice tumor. Taken together, our data suggest an important regulatory role of miR-139-5p in gastric cancer, suggesting that miR-139-5p and PMP22 might be important diagnostic or therapeutic targets for gastric cancer and other human diseases.

Cachexia is a complex metabolic derangement syndrome that affects approximately 50-80% of cancer patients. So far, few works have been reported to provide a global overview of gastric cancer cachexia (GCC)-related metabolic changes. We established a GCC murine model by orthotopicly implanting BGC823 cell line and conducted NMR-based metabolomic analysis of gastric tissues, sera, and gastrocnemius. The model with typical cachexia symptoms, confirmed by significant weight loss and muscle atrophy, showed distinctly distinguished metabolic profiles of tumors, sera, and gastrocnemius from sham mice. We identified 20 differential metabolites in tumors, 13 in sera, and 14 in gastrocnemius. Tumor extracts displayed increased pyruvate and lactate, and decreased hypoxanthine, inosine, and inosinate, indicating significantly altered glucose and nucleic acid metabolisms. Cachectic mice exhibited up-regulated serum lactate and glycerol, and down-regulated glucose, which were closely related to hyperlipidemia and hypoglycemia. Furthermore, gastrocnemius transcriptomic and metabolomic data revealed that GCC induced perturbed pathways mainly concentrated on carbohydrate and amino acid metabolism. Specifically, cachectic gastrocnemius exhibited increased α-ketoglutarate and decreased glucose. In vitro study indicated that α-ketoglutarate could prompt myoblasts proliferation and reduce glucose deficiency-induced myotubes atrophy. Overall, this work provides a global metabolic overview to understand the metabolic alterations associated with GCC-induced muscle atrophy.

Table S2. Clinical data for 218 lung carcinoma specimens examined in this study. (DOC 274 kb)

Materials/Methods. (DOC 46 kb)

Table S1 Proteins differentially expressed in lung adenocarcinoma-derived endothelial cells (TEC-A) in comparison to squamous cell carcinoma-derived endothelial cells (TEC-S). (DOC 68 kb)

Table S3. Expression of CDH2 in tumor-derived endothelial cell and its correlation with clinicopathological features of patients with NSCLC. (DOC 82 kb)

The current study aimed to develop a rapid, robust and adequately sensitive method for simultaneous determination of the concentration of tramadol and its active metabolites in zebrafish. The pharmacokinetic and elimination pattern of tramadol and its major phase I metabolites following oral or intramuscular administration in zebrafish tissues was achieved using electrospray ionization‑quadrupole‑time of flight/mass spectrometry (ESI‑Q‑TOF/MS) and gas chromatography/mass spectrometry (GC‑MS). Following administration, the metabolisms were detected in the brain, eyes, muscle and gill tissues within 1 h. Two tramadol metabolites, O‑ and N‑desmethyltramadol, were detected in brain tissue, with N‑desmethyltramadol detected at a higher level. Following GC‑MS detection the curve indicated an initial rapid phase, corresponding to the detection of the tramadol within 1 min, and reached peak value in the brain at 5 min. Faster drug clearance was detected in low‑dose groups, and concentration had dropped around the to initial level (1.11 µg) at 20 min, but was detectable for up to 3 h. However, it took 80 min to fall back to the initial value (1.73 µg) in the high‑dose groups, and tramadol was detectable for up to 4 h. This study developed and validated a simple and high throughput analytical procedure to determine the distribution and pharmacokinetic profiles of tramadol, and its primary metabolites in tissues of zebrafish.

// Yihua Pei 1, * , Qin Yao 1, * , Sibo Yuan 2 , Bozhen Xie 3 , Yan Liu 4 , Chunsheng Ye 5 , Huiqin Zhuo 2 1 Central Laboratory, The Affiliated Zhongshan Hospital, Xiamen University, Xiamen, Fujian 361004, China 2 Department of Gastrointestinal Surgery, The Affiliated Zhongshan Hospital, Xiamen University, Xiamen, Fujian 361004, China 3 Department of Spine Surgery, The Affiliated Zhongshan Hospital, Xiamen University, Xiamen, Fujian 361004, China 4 Department of Pathology, The Affiliated Zhongshan Hospital, Xiamen University, Xiamen, Fujian 361004, China 5 Department Otolaryngology, The Affiliated Zhongshan Hospital, Xiamen University, Xiamen, Fujian 361004, China * These authors have contributed equally to this work Correspondence to: Yihua Pei, email: peiyihua8@sina.com Huiqin Zhuo, email: zhuohuiqin@xmu.edu.cn Keywords: hepatoblastoma, GATA4, miR125b, DKK3 Received: January 19, 2016 Accepted: October 14, 2016 Published: October 24, 2016 ABSTRACT GATA4 is a zinc finger DNA-binding protein that plays an important role in mammalian liver development. However, the effects of GATA4 in hepatoblastoma (HB), a common liver cancer in pediatric patients, remain largely unknown. In this study, we demonstrate that GATA4 promotes growth and survival in the Huh6 human hepatoblastoma cell line. GATA4 expression was high in Huh6 cells, and its knockdown decreased expression of Dickkopf-related protein 3 (DKK3), a gene that may contribute to premature or undifferentiated phenotypes in HB. GATA4 also directly bound to the promoter regions of the miRNA miR125b and inhibited its expression in Huh6 cells. DKK3 was a direct target of miR125b in Huh6 cells. Inhibition of miR125b or overexpression of DKK3 promoted proliferation, survival, migration, and invasion in Huh6 cells. This is the first report to demonstrate that GATA4 promotes oncogenesis by inhibiting miR125b-dependent suppression of DKK3 expression. This GATA4/miR125b/DKK3 axis may be a major regulator of growth, migration, invasion, and survival in hepatoma cells, and is therefore a potential therapeutic target or biomarker for progression in HB patients.

Gastric cancer (GC) is one of the most malignant tumors with a poor prognosis. Alterations in metabolic pathways are inextricably linked to GC progression. However, the underlying molecular mechanisms remain elusive. We performed NMR-based metabolomic analysis of sera derived from a rat model of gastric carcinogenesis, revealed significantly altered metabolic pathways correlated with the progression of gastric carcinogenesis. Rats were histologically classified into four pathological groups (gastritis, GS; low-grade gastric dysplasia, LGD; high-grade gastric dysplasia, HGD; GC) and the normal control group (CON). The metabolic profiles of the five groups were clearly distinguished from each other. Furthermore, significant inter-metabolite correlations were extracted and used to reconstruct perturbed metabolic networks associated with the four pathological stages compared with the normal stage. Then, significantly altered metabolic pathways were identified by pathway analysis. Our results showed that oxidative stress-related metabolic pathways, choline phosphorylation and fatty acid degradation were continually disturbed during gastric carcinogenesis. Moreover, amino acid metabolism was perturbed dramatically in gastric dysplasia and GC. The GC stage showed more changed metabolite levels and more altered metabolic pathways. Two activated pathways (glycolysis; glycine, serine and threonine metabolism) substantially contributed to the metabolic alterations in GC. These results lay the basis for addressing the molecular mechanisms underlying gastric carcinogenesis and extend our understanding of GC progression.

Increasing evidence indicates that tumor-derived endothelial cells (TECs) are more relevant for the study of tumor angiogenesis and for screening antiangiogenic drugs than normal ECs (NECs). In this data article, high-purity (>98%) primary CD105+ NECs and TECs purified from a mouse Lewis lung carcinoma model bearing 0.5 cm tumors were identified using 2D-PAGE and Matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS). All the identified proteins were categorized functionally by Gene Ontology (GO) analysis, and gene-pathway annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG). Finally, protein–protein interaction networks were also built. The proteomics and bioinformatics data presented here provide novel insights into the molecular characteristics and the early modulation of the TEC proteome in the tumor microenvironment. Keywords: Tumor-derived endothelial cells, Lung cancer, 2D-PAGE, MALDI-TOF, MS/MS

Cancer cells metabolize different energy sources to generate biomass rapidly. The purine biosynthetic pathway was recently identified as an important source of metabolic intermediates for these processes. However, very little was known about the regulatory mechanisms of purine metabolism in hepatocellular carcinoma (HCC). We explored the role of dual-specificity tyrosine (Y) phosphorylation-regulated kinase 3 (Dyrk3) in HCC metabolism. Dyrk3 was significantly down-regulated in HCC compared with normal controls. Its introduction in HCC cells markedly suppressed tumor growth and metastasis in xenograft tumor models. Mass spectrometric analysis of metabolites suggests that the effect of Dyrk3 on HCC occurred at least partially through down-regulating purine metabolism, as evidenced by the fact that inhibiting purine synthesis reverted the HCC progression mediated by the loss of Dyrk3. We further provide evidence that this action of Dyrk3 knockdown requires nuclear receptor coactivator 3 (NCOA3), which has been shown to be a coactivator of activating transcription factor 4 (ATF4) to target purine pathway genes for transcriptional activation. Mechanistically, Dyrk3 directly phosphorylated NCOA3 at Ser-1330, disrupting its binding to ATF4 and thereby causing the inhibition of ATF4 transcriptional activity. However, the phosphorylation-resistant NCOA3-S1330A mutant has the opposite effect. Interestingly, the promoter activity of Dyrk3 was negatively regulated by ATF4, indicating a double-negative feedback loop. Importantly, levels of Dyrk3 and phospho-NCOA3-S1330 inversely correlate with the expression of ATF4 in human HCC specimens. Conclusion: Our findings not only illustrate a function of Dyrk3 in reprograming HCC metabolism by negatively regulating NCOA3/ATF4 transcription factor complex but also identify NCOA3 as a phosphorylation substrate of Dyrk3, suggesting the Dyrk3/NCOA3/ATF4 axis as a potential candidate for HCC therapy.

Additional file 4: Table S2. Detail information of enriched clusters.

RIOK1 has recently been shown to play important roles in cancers, but its posttranslational regulation is largely unknown. Here we report that RIOK1 is methylated at K411 by SETD7 methyltransferase and that lysine-specific demethylase 1 (LSD1) reverses its methylation. The mutated RIOK1 (K411R) that cannot be methylated exhibits a longer half-life than does the methylated RIOK1. FBXO6 specifically interacts with K411-methylated RIOK1 through its FBA domain to induce RIOK1 ubiquitination. Casein kinase 2 (CK2) phosphorylates RIOK1 at T410, which stabilizes RIOK1 by antagonizing K411 methylation and impeding the recruitment of FBXO6 to RIOK1. Functional experiments demonstrate the RIOK1 methylation reduces the tumor growth and metastasis in mice model. Importantly, the protein levels of CK2 and LSD1 show an inverse correlation with FBXO6 and SETD7 expression in human colorectal cancer tissues. Together, this study highlights the importance of a RIOK1 methylation-phosphorylation switch in determining colorectal and gastric cancer development.

RioK1 has recently been shown to play important roles in cancers, but its posttranslational regulation is largely unknown. Here we report that RioK1 is methylated at K411 by SETD7 methyltransferase, and that lysine-specific demethylase 1 (LSD1) reverses its methylation. The mutated RioK1 (K411R) that cannot be methylated exhibits a longer half-life than does the methylated RioK1. FBXO6 specifically interacts with K411-methylated RioK1 through its FBA domain to induce RioK1 ubiquitination. Casein kinase 2 (CK2) phosphorylates RioK1 at T410, which stabilizes RioK1 by antagonizing K411 methylation and impeding the recruitment of FBXO6 to RioK1. Functional experiments demonstrate the RioK1 methylation reduces the tumor growth and metastasis in CRC and GC. Importantly, the protein levels of CK2 and LSD1 show an inverse correlation with FBXO6 and SETD7 expression in human CRC tissues. Therefore, this study highlights the importance of a RioK1 methylation-phosphorylation switch in determining CRC and GC development.

Burkitt lymphoma (BL) has been reported to be strongly associated with Epstein-Barr virus (EBV) infection. The fact that EBV is generally present in cancer cells but rarely found in healthy cells represents an opportunity for targeted cancer therapy. One approach is to activate the lytic replication cycle of the latent EBV. Nuclear factor (NF)-κB is thought to play an essential role in EBV lytic infection. Elevated NF-κB levels inhibit EBV lytic replication. Parthenolide (PN) is a sesquiterpene lactone found in medicinal plants, particularly in feverfew (Tanacetum parthenium). The aim of the present study was to analyze the effect of PN on the survival of Raji EBV-positive lymphoma cells. Raji cells were treated with 0, 4 or 6 µmol/l PN for 48 h. MTT assay and western blot analysis were performed to evaluate the findings. Results showd that PN suppressed the growth of the EBV-positive BL cell line, Raji, and activated the transcription of BZLF1 and BRLF1 by inhibiting NF-κB activity. Most notably, when PN was used in combination with ganciclovir (GCV), the cytotoxic effect of PN was amplified. These data suggest that the induction of lytic EBV infection with PN in combination with GCV may be a viral‑targeted therapy for EBV-associated BL.

To investigate heterogeneity of endothelial cells (ECs) in the tumor microenvironment and biomarkers for antitumor angiogenesis therapy, high-purity (> 98%) normal (NECs) and tumor-derived CD105 + ECs (TECs) were purified from a mouse Lewis lung carcinoma model bearing 0.5 cm tumors by immunomagnetic separation. Proteomics analysis revealed that 48 proteins (28 upregulated and 20 downregulated) were differentially regulated by at least 1.5-fold in TECs, and that these proteins were involved in metabolism, energy pathways, protein folding, cell growth and/or functioned as structural constituents of the cytoskeleton. Upregulation of heat shock protein 60 (Hspd1) and transgelin-2 (Tagln2) was revealed in TECs, and by immunohistochemistry (IHC) in paired tissues from 30 consecutive lung cancer (LC) patients. Higher expression levels of Hspd1, Tagln2 were detected in microvascular ECs of paratumor and tumor tissues than in paired normal counterparts. Stronger Tagln2 staining was associated with clinical stage, tumor size, and histological neural invasion. Higher Hspd1 (area under the curve [AUC], 0.82) and lower Tagln2 (AUC, 0.90) levels were detected in LC patient sera. Pearson correlation analysis revealed a positive correlation between serum Hspd1 and Tagln2 levels. In conclusion, higher Tagln2 levels were associated with tumor development, lymph node metastasis, and neural invasion in LC and may thus serve as a potential biomarker of tumor angiogenesis. Significance High-purity endothelial cells (normal and tumor derived) were prepared to characterize ECs heterogeneity in the tumor microenvironment and to explore biomarkers of early stages of tumor development by proteomics. Candidate proteins Hspd1 and Tagln2, were further verification in the sera and tumor tissues of lung cancer patients. Moreover, higher Tagln2 was significantly associated with clinical tumor development, metastasis, and neural invasion. All these results indicated a crucial role for Tagln2 in TECs for tumor development and metastasis.

Immunotherapy is one of the most promising new therapies for hepatocellular carcinoma (HCC) in recent years. In this study, folate-conjugated chitosan nanoparticles (FA-CS-NPs) were loaded with mouse interferon-γ-inducible protein-10 (IP-10) plasmid, which were used for immunotherapy in HCC. H22 tumor-bearing mice were treated with FA-CS-NPs entrapped IP-10 plasmid and targeting efficiency was observed by optical imaging in vivo. Flow cytometry was used to measure the number of myeloid-derived suppressor cells (MDSCs) in the tumor and CD4+CD25+FoxP3+ T-regulatory cells (CD4+CD25+FoxP3+ Tregs) in the spleen. The enzyme-linked immunospot (ELISPOT) assay was used to quantify the number of interferon-γ (IFN-γ)-positive cells. IP-10 expression, tumor vessel density, cell proliferation and apoptosis were evaluated by immunohistochemistry. It was shown that FA-CS-NPs entrapped IP-10 plasmid displayed anti-tumor activity with inhibition of tumor growth and prolonging the survival time in H22 tumor-bearing mice. Treatment of H22 tumor-bearing mice with FA-CS-NPs entrapped IP-10 plasmid inhibited angiogenesis and promoted IP-10 expression and induced apoptosis in the tumor. FA-CS-NPs entrapped IP-10 plasmid-treated mice also had a lower proportion of Tregs in the spleen, a higher proportion of MDSCs in the tumor and higher number of IFN-γ-positive cells in the spleen compared with the mice from the other experimental groups. These data suggested that the gene delivery system of folate-conjugated chitosan nanoparticle loaded with IP-10 plasmid may be a promising strategy for immunotherapy of HCC.

In lung cancer, antiangiogenic treatment targeting tumor endothelial cells (ECs) provides a survival advantage. To fully elucidate the behavior of ECs in a tumor microenvironment, high-purity (98%) normal, paratumor-, and tumor-derived CD105(+) ECs were purified from lung squamous cell carcinoma by incubating cells with anti-CD105 antibody-coated magnetic beads. These cells exhibited typical EC characteristics. Totally, 1765 proteins were identified with high confidence by isobaric stable isotope tags and two-dimensional LC/MS/MS (iTRAQ-2DLC/MS/MS). In particular, 178 and 162 proteins were differentially expressed in paratumor- and tumor-derived ECs, respectively, compared to normal ECs. The up- and down-regulation trends showed good interassay correlation. Using gene ontology, they were classified into genes involved in major reprogramming of cellular metabolic processes, oxidative stress response, redox homeostasis, apoptosis, and platelet degranulation/activation. Moreover, tumor angiogenesis-initiating ECs appeared to acquire distinct properties. For example, cell migration and regulation of smooth muscle cell migration of paratumor-derived ECs were significantly faster than that of normal and tumor-derived ECs. Among them, two migration-associated proteins, neuropilin 1 and platelet-derived growth factor receptor β predominantly expressed in ECs of paratumor from 16 patients with lung squamous cell carcinoma, were identified as potential biomarkers for antiangiogenic therapy.

Purpose: We have developed a PEGylated transferrin-conjugated liposomes (PTf-Ls) system for the combined tumor imaging and targeted delivery of the IFN-γ–inducible protein-10 (IP-10) gene in a single macromolecular construct. Here, we characterize and analyze the use of this system in a mouse model of breast cancer. Experimental Design: The biophysical and cell transfection properties of PTf-Ls were determined through a series of in vitro experiments. A nude mouse/breast cancer cell line xenograft model (mouse xenograft model) was used to image the tumor internalization of fluorescently labeled PTf-Ls. The clinical use of the system was tested by treating tumor-bearing mice with PTf-Ls loaded with IP-10 plasmid DNA or fluorescent lipoplexes. Results: The resulting 165-nm liposomes (zeta potential = −10.6 mV) displayed serum resistance, low cytotoxicity ( Conclusions: Our developed transferrin-conjugated liposome system possesses promising characteristics for tumor-targeting, imaging, and gene therapy applications. Clin Cancer Res; 19(15); 4206–17. ©2013 AACR.

e19069Background: Although the cytotoxic effects of novel CRM1 inhibitor, small molecule selective inhibitors of nuclear export (SINE) on Mantle Cell Lymphoma (MCL) cells have now been established,...