DNMT3A Cooperates with YAP/TAZ to Drive Gallbladder Cancer Metastasis.

Gallbladder cancer (GBC) is an extremely lethal malignancy with aggressive behaviors, including liver or distant metastasis; however, the underlying mechanisms driving the metastasis of GBC remain poorly understood. In this study, it is found that DNA methyltransferase DNMT3A is highly expressed in GBC tumor tissues compared to matched adjacent normal tissues. Clinicopathological analysis shows that DNMT3A is positively correlated with liver metastasis and poor overall survival outcomes in patients with GBC. Functional analysis confirms that DNMT3A promotes the metastasis of GBC cells in a manner dependent on its DNA methyltransferase activity. Mechanistically, DNMT3A interacts with and is recruited by YAP/TAZ to recognize and access the CpG island within the CDH1 promoter and generates hypermethylation of the CDH1 promoter, which leads to transcriptional silencing of CDH1 and accelerated epithelial-to-mesenchymal transition. Using tissue microarrays, the association between the expression of DNMT3A, YAP/TAZ, and CDH1 is confirmed, which affects the metastatic ability of GBC. These results reveal a novel mechanism through which DNMT3A recruitment by YAP/TAZ guides DNA methylation to drive GBC metastasis and provide insights into the treatment of GBC metastasis by targeting the functional connection between DNMT3A and YAP/TAZ.

Targeted bile acids metabolomics in cholesterol gallbladder polyps and gallstones: From analytical method development towards application to clinical samples.

Bile acids (BAs) are synthesized by the liver from cholesterol through several complementary pathways and aberrant cholesterol metabolism plays pivotal roles in the pathogeneses of cholesterol gallbladder polyps (CGP) and cholesterol gallstones (CGS). To date, there is neither systematic study on BAs profile of CGP or CGS, nor the relationship between them. To explore the metabolomics profile of plasma BAs in healthy volunteers, CGP and CGS patients, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for simultaneous determination of 42 free and conjugated BAs in human plasma. The developed method was sensitive and reproducible to be applied for the quantification of BAs in the investigation of plasma samples. The results show that, compared to healthy volunteers, CGP and CGS were both characterized by the significant decrease in plasma BAs pool size, furthermore CGP and CGS shared aberrant BAs metabolic characteristics. Chenodeoxycholic acid, glycochenodeoxycholic acid, λ-muricholic acid, deoxycholic acid, and 7-ketolithocholic acid were shared potential markers of these two cholesterol gallbladder diseases. Subsequent analysis showed that clinical characteristics including cysteine, ornithine and body mass index might be closely related to metabolisms of certain BA modules. This work provides metabolomic information for the study of gallbladder diseases and analytical methodologies for clinical target analysis and efficacy evaluation related to BAs in medical institutions.

Combining polyp diameter and polyp-to-bile ratio by dynamic contrast-enhanced CT scanning can improve the diagnostic specificity of gallbladder neoplastic polyps.

OBJECTIVE: This study aimed to distinguish between cholesterol and neoplastic gallbladder polyps using dynamic contrast-enhanced CT. METHODS: The dataset retrospectively comprised 222 cases, including 106 cases of cholesterol polyps and 116 cases of neoplastic polyps (59 adenoma and 57 adenocarcinoma). The perception and Hounsfield units of the polyps and gallbladder bile were assessed by contrast-enhanced CT, and the polyp-to-bile ratio (PBR) was calculated. Receiver operating characteristic (ROC) curves and area under the curve analyses were used to assess the diagnostic value of the diameter and PBR for neoplastic polyps. RESULTS: The diameter of cholesterol polyps was significantly smaller than that of neoplastic polyps. The proportion of perceived cholesterol polyps in the plain and arterial phases of CT were significantly lower than those of neoplastic polyps (p < .001). On the contrary, the CT values of gallbladder bile of cholesterol polyps were always significantly higher than those of neoplastic polyps (p < .001). The median PBR values of cholesterol polyps were significantly lower than those of neoplastic polyps (p ≤ .001). ROC analysis showed that diameter and a plain phase PRB had better diagnostic value for neoplastic polyps. Polyp diameter ≥ 11.95 mm and the plain phase PBR ≥1.48 were the optimal cut-off values for diagnosis of neoplastic polyps. Combining a diameter ≥ 12 mm and a PBR in the plain phase ≥1.48 further improved neoplastic polyp diagnostic specificity and positive likelihood ratio (10.453). CONCLUSIONS: Polyp-to-bile ratio in contrast-enhanced CT scanning is a new and convenient index for identifying cholesterol and neoplastic gallbladder polyps.

A Gallbladder Cancer Survival Prediction Model Based on Multimodal Fusion Analysis.

BACKGROUND: Gallbladder cancer is the sixth most common malignant gastrointestinal tumor. Radical surgery is currently the only effective treatment, but patient prognosis is poor, with a 5-year survival rate of only 5-10%. Establishing an effective survival prediction model for gallbladder cancer patients is crucial for disease status assessment, early intervention, and individualized treatment approaches. The existing gallbladder cancer survival prediction model uses clinical data-radiotherapy and chemotherapy, pathology, and surgical scope-but fails to utilize laboratory examination and imaging data, limiting its prediction accuracy and preventing sufficient treatment plan guidance. AIMS: The aim of this work is to propose an accurate survival prediction model, based on the deep learning 3D-DenseNet network, integrated with multimodal medical data (enhanced CT imaging, laboratory test results, and data regarding systemic treatments). METHODS: Data were collected from 195 gallbladder cancer patients at two large tertiary hospitals in Shanghai. The 3D-DenseNet network extracted deep imaging features and constructed prognostic factors, from which a multimodal survival prediction model was established, based on the Cox regression model and incorporating patients' laboratory test and systemic treatment data. RESULTS: The model had a C-index of 0.787 in predicting patients' survival rate. Moreover, the area under the curve (AUC) of predicting patients' 1-, 3-, and 5-year survival rates reached 0.827, 0.865, and 0.926, respectively. CONCLUSIONS: Compared with the monomodal model based on deep imaging features and the tumor-node-metastasis (TNM) staging system-widely used in clinical practice-our model's prediction accuracy was greatly improved, aiding the prognostic assessment of gallbladder cancer patients.

Hippocalcin-Like 1 blunts liver lipid metabolism to suppress tumorigenesis via directly targeting RUVBL1-mTOR signaling.

Rationale: Hepatocellular carcinoma (HCC) is one of the most severe cancers worldwide, with few effective targeted therapies for HCC. Lipid metabolic reprogramming is emerged as a hallmark of cancer metabolism that guides response to antitumoral therapies. Such lipid metabolic alteration in cancers is critically regulated by the mammalian target of rapamycin mTOR, which is considered as a promising therapeutic target. Despite efforts, mTOR inhibitors (mTORi) have produced limited response clinically, partly due to incomplete knowledge of mTORC1 addiction in cancers. Methods: CRISPR-Cas9 system was used to establish Hpcal1 null mice. The liver cancer model in mice was generated using Hpcal1-deficient mice with diethylnitrosamine (DEN) /CCL4 or MYC/Trp53-/- via hydrodynamic tail-vein injection. RNA-sequencing (RNA-seq) was used to identify potential signaling pathways. The expression of HPCAL1 and mTOR signaling were determined using quantitative polymerase chain reaction (qPCR), western blot and immunohistochemistry. The role of Hpcal1 in liver tumorigenesis and its response to mTORi was assessed by CCK-8 measurements, colony formation assay and in mouse model. Results: In this study, we identified hippocalcin-like protein 1 (HPCAL1) as an important negative regulator of de novo lipid biosynthesis and mTOR signaling activation, limiting liver tumorigenesis and establishing a metabolic vulnerability of HCC in mice. Genetic loss of HPCAL1 rendered HCC mTORC1-addicted and sensitive to mTORi AZD-8055 in vitro and in vivo. Importantly, HPCAL1 expression was inversely correlated with the levels of mTOR phosphorylation and several critical lipid biosynthesis enzymes in human specimens. Mechanistically, HPCAL1 directly bound to RuvB Like AAA ATPase 1 (RUVBL1), inhibiting the assembly of TEL2-TTI1-TTI2 (TTT)-RUVBL complex and subsequent leading the mTOR signaling suppression. Conclusion: We uncover a metabolic vulnerability and mTOR addiction in HCC with HPCAL1 loss that provides a selective therapeutic window for HCC with mTORC1 hyperactivation using mTORi.

GSTM3 deficiency impedes DNA mismatch repair to promote gastric tumorigenesis via CAND1/NRF2-KEAP1 signaling.

Gastric cancer (GC) is one of the most severe gastric diseases worldwide. However, the molecular basis that drives tumorigenesis and progression is not completely understood, which hinders the efficacy and development of therapeutic options. Glutathione-S-transferases (GSTs) are a group of phase II detoxification enzymes that maintain redox homeostasis; however, their roles in cancers are not well defined. Here, we revealed that the expression of GST family members is significantly impaired in GC tissues. Glutathione-S-transferase mu 3 (GSTM3), a member of GST family, is dramatically downregulated in cancerous tissues and has been identified as an independent prognostic factor in GC associated with tumor differentiation, inhibiting GC cell proliferation and migration in vitro and in vivo. Mechanistically, GSTM3 is transcriptionally activated by NRF2/KEAP1 signaling. As a feedback loop, GSTM3 binds to Cullin-associated and neddylation-dissociated 1 protein (CAND1), an exchange factor for integrating Kelch-like ECH-associated protein 1 (KEAP1) into Cul3-RING ubiquitin ligases (CRL3), to disrupt nuclear factor-erythroid factor 2-related factor 2 (NRF2)/KEAP1 binding and prevent NRF2 ubiquitination and degradation, leading to its activation. A deficiency in glutathione S-Transferase Mu 3 (GSTM3) reduces DNA mismatch repair (MMR) gene expression and increases mutagenesis via CAND1/NRF2 binding. Importantly, GSTM3/NRF2 and KEAP1 were negatively and positively associated with the genomic signature for microsatellite instability, respectively. Clinically, GSTM3, NRF2, and MutS homolog 6 (MSH6) were positively correlated in the GC specimens. This study uncovered a reciprocal regulation between GSTM3 and NRF2 and established a functional and clinical link between GSTM3-NRF2/KEAP1 and MMR during GC cell proliferation and progression, thus providing potential therapeutic targets for GC.

Is Contrast-Enhanced Ultrasound Superior to Computed Tomography for Differential Diagnosis of Gallbladder Polyps? A Cross-Sectional Study.

OBJECTIVE: To compare the clinical value of contrast-enhanced ultrasonography (CEUS) versus computed tomography (CT) for distinguishing neoplastic and non-neoplastic gallbladder polyps. Given whether laparoscopic cholecystectomy is needed, differential diagnosis of neoplastic and non-neoplastic gallbladder polyps is more important than benign and malignant polyps. METHODS: A total of 89 consecutive patients with polypoid lesions of the gallbladder > 10 mm in size without local invasion or distant metastasis during primary screening were enrolled in this prospective and comparative study. All patients who met the inclusion criteria underwent CEUS and CT examinations prior to surgical resection. The enhancement patterns and microvascular imaging types were analyzed on CEUS. The maximum diameter and CT value of the lesions were also recorded and subjected to a comparative analysis. The clinical value of the two diagnostic methods is compared. RESULTS: Of the 89 patients, there were 58 (65.2%) cases of non-neoplastic polyps and 31 (34.8%) cases of neoplastic polyps. The average diameter of neoplastic polyps was significantly higher than that of non-neoplastic polyps (P<0.001). The detection rate using CEUS was 100%. The proportion of perceived non-neoplastic polyps in the nonenhanced and arterial phases were 48.3% and 77.6%, respectively, which were significantly lower than those of neoplastic polyps (93.5%, P<0.001 and 100.0%, P<0.001, respectively). However, in the venous and delayed phases, all cholesterol polyps and neoplastic polyps were perceived. CT showed that non-neoplastic polyps exhibited delayed enhancement. On CEUS 29.0% neoplastic polyps showed a perfusion defect, whereas 6.9% non-neoplastic polyps showed a perfusion defect (P=0.005). The microvascular architecture of the lesions on CEUS was categorized into 4 types: spotty, linear, branched, and spinous, and there were significant differences between the two groups (P<0.001). The sensitivities and specificities were 87.10% and 68.97% for CEUS and 83.87% and 77.59% for CT, respectively (P=0.406). CONCLUSIONS: CEUS and CT are useful for differential diagnosis of neoplastic and nonneoplastic polypoid lesions of the gallbladder. Diagnostic efficacy was comparable between CEUS and CT. Thus, CEUS is preferred over CT in the differential diagnosis of neoplastic and non-neoplastic gallbladder polyps due to its comparable diagnostic efficacy and lack of radiation dose.

A redox probe screens MTHFD1 as a determinant of gemcitabine chemoresistance in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a type of solid tumor derived from the bile duct epithelium that features universal gemcitabine resistance. Here, we utilized a gene-encoded ROS biosensor probe (HyPer3 probe) to sort subpopulations with different redox statuses from CCA cells. The isolated HyPer-low subpopulation CCA cells, which exhibited relatively lower cellular ROS levels, exhibited higher chemoresistance to gemcitabine than HyPer-high subpopulation CCA cells in vitro and in vivo. Mechanistically, increased expression of MTHFD1 was found in HyPer-low cells. Knocking down MTHFD1 in HyPer-low cells enhanced cellular ROS and restored sensitivity to gemcitabine. Furthermore, the MTHFD1 inhibitor antifolate compound methotrexate (MTX) increased cellular ROS, and combining gemcitabine with MTX effectively suppressed cholangiocarcinoma cell growth. In summary, the MTHFD1 level mediated the heterogeneous cellular redox status in CCA, which resulted in chemoresistance to gemcitabine. Our data suggest a novel strategy for CCA chemotherapy.

Effect of metabolic syndrome components on the risk of malignancy in patients with gallbladder lesions.

Background: Gallbladder lesions have become more common nowadays. But there is limited evidence-based guidance on surveillance of these patients for malignancy. Predicting malignancy could help clinicians better manage this condition and improve the prognosis. We evaluated the independent and joint effects of metabolic syndrome components on the risk of malignancy among patients with gallbladder lesions. Methods: Using a multicenter database, consecutive patients with pathologically confirmed gallbladder lesions between 2012 and 2019 were identified. Univariate and multivariate logistic regression analyses were used to evaluate the effects of metabolic syndrome components (diabetes, hypertension, dyslipidemia and obesity) as additive or combined indicators for the risk of malignancy. Unadjusted and adjusted odds ratios were calculated. Results: Of the 625 patients, 567 patients were identified with benign gallbladder lesions and 58 patients with gallbladder cancer (GBC). GBC group had less obesity but more dyslipidemia. Among all metabolic syndrome components, only dyslipidemia was significantly associated with GBC (odds ratio 2.674, 95% confidence interval 1.173-6.094). Dyslipidemia was an independent risk factor for malignancy (adjusted odds ratio 2.164, 95% confidence interval 1.165-4.021), regardless of whether the other risk factors and metabolic syndrome components were combined. Patients with decreased high-density lipoprotein had 3.035-fold higher risk of malignancy (adjusted odds ratio 3.035, 95% confidence interval 1.645-5.600). Conclusions: Dyslipidemia is associated with a 2.674-fold increase in the risk of malignancy in patients with gallbladder lesions. Dyslipidemia is an independent risk factor for malignancy, regardless of the presence of the other risk factors and metabolic syndrome components.

The longitudinal change of circulating tumor cell during chemotherapy and its correlation with disease features, treatment response and survival profile of advanced gallbladder carcinoma.

The current study aimed to investigate the relation of circulating tumor cell (CTC) with clinicopathological features. In addition, its longitudinal change during chemotherapy and its correlation with prognosis in advanced gallbladder carcinoma (GBC) patients were explored. Totally 45 unresectable, locally advanced or metastatic GBC patients who underwent chemotherapy were enrolled in this prospective study. The CTC in 7.5 ml blood was detected at pre-treatment and 3 months post-treatment. CTC was almost detectable in all advanced GBC patients before treatment, whose count was positively correlated with metastatic disease (vs. local advanced disease) (P=0.002), number of organs with metastases (P=0.006), and CA199 level (P=0.002). After treatment, CTC count declined from 4.0 (range: 0.0-83.0) at pre-treatment to 2.0 (range: 0.0-36.0) at post-treatment (P=0.003). Interestingly, pre-treatment CTC count (P=0.270) was of no difference, while post-treatment CTC count was lower (P=0.038) in objective-response patients compared to that in non-objective-response patients; meanwhile, both pre-treatment CTC count (P=0.017) and post-treatment CTC count (P<0.001) were lower in disease-control patients compared with those in non-disease-control patients. Importantly, pre-treatment CTC count ≥2 (versus <2) was only correlated with worse progression-free survival (PFS) (P=0.014) but not overall survival (OS) (P=0.057); while pre-treatment CTC count ≥5 (versus <5), post-treatment CTC count ≥2 (versus <2), post-treatment CTC count ≥5 (versus <5), CTC count up (versus equal/down) were all correlated with poor PFS and OS (all P<0.050). In conclusion, higher CTC count during chemotherapy correlates with worse treatment response, PFS and OS in advanced GBC patients, which implies that CTC measurement may optimize the prognostication and individualized treatment in these patients.

Risk factors and socio-economic burden in pancreatic ductal adenocarcinoma operation: a machine learning based analysis.

BACKGROUND: Surgical resection is the major way to cure pancreatic ductal adenocarcinoma (PDAC). However, this operation is complex, and the peri-operative risk is high, making patients more likely to be admitted to the intensive care unit (ICU). Therefore, establishing a risk model that predicts admission to ICU is meaningful in preventing patients from post-operation deterioration and potentially reducing socio-economic burden. METHODS: We retrospectively collected 120 clinical features from 1242 PDAC patients, including demographic data, pre-operative and intra-operative blood tests, in-hospital duration, and ICU status. Machine learning pipelines, including Supporting Vector Machine (SVM), Logistic Regression, and Lasso Regression, were employed to choose an optimal model in predicting ICU admission. Ordinary least-squares regression (OLS) and Lasso Regression were adopted in the correlation analysis of post-operative bleeding, total in-hospital duration, and discharge costs. RESULTS: SVM model achieved higher performance than the other two models, resulted in an AU-ROC of 0.80. The features, such as age, duration of operation, monocyte count, and intra-operative partial arterial pressure of oxygen (PaO2), are risk factors in the ICU admission. The protective factors include RBC count, analgesic pump dexmedetomidine (DEX), and intra-operative maintenance of DEX. Basophil percentage, duration of the operation, and total infusion volume were risk variables for staying in ICU. The bilirubin, CA125, and pre-operative albumin were associated with the post-operative bleeding volume. The operation duration was the most important factor for discharge costs, while pre-lymphocyte percentage and the absolute count are responsible for less cost. CONCLUSIONS: We observed that several new indicators such as DEX, monocyte count, basophil percentage, and intra-operative PaO2 showed a good predictive effect on the possibility of admission to ICU and duration of stay in ICU. This work provided an essential reference for indication in advance to PDAC operation.

Changes in plasma bile acids are associated with gallbladder stones and polyps.

BACKGROUND: The development of gallbladder disease (GBD) is related to bile acid (BA) metabolism, and the rate of BA circulation increases the risk of biliary cancer. However, it is unclear whether patterns of circulating bile acids (BAs) change in patients with benign GBDs such as gallbladder stones and polyps. Herein, we compared and characterised plasma BA profiles in patients with cholecystolithiasis and non-neoplastic polyps with healthy controls, and explored relationships between plasma BA profiles, demographics, and laboratory test indices. METHODS: A total of 330 subjects (13 healthy controls, 292 cholecystolithiasis and 25 non-neoplastic polyps) were recruited and plasma BA profiles including 14 metabolites from patients with pathologically confirmed cholecystolithiasis and non-neoplastic polyps were compared with controls. BAs were quantitated by liquid chromatography and mass spectrometry, and statistical and regression analyses of demographics and laboratory test indices were performed. RESULTS: Females displayed a higher burden of GBD than males (63.36% cholecystolithiasis, 60% non-neoplastic polyps). Cholecystolithiasis and non-neoplastic polyps were associated with increased plasma total secondary BAs, while levels of primary BAs were lower than in healthy controls. Plasma ursodeoxycholic acid (UDCA), tauroursodeoxycholic acid (TUDCA), glycyurdeoxycholic acid (GUDCA), taurochenodeoxycholic acid (TCDCA) and glycochenodeoxycholic acid (GCDCA) were decreased significantly in GBDs, and ursodeoxycholic acid (UDCA) was negatively correlated with white blood cell count and neutrophil percentage. CONCLUSIONS: Secondary BA levels were higher in patients with cholecystolithiasis and non-neoplastic polyps. White blood cell count and percentage of neutrophil in peripheral blood were negatively correlated with UDCA, indicating an anti-inflammation effect of UDCA.

Feasibility and safety of single-incision laparoscopic cholecystectomy versus conventional laparoscopic cholecystectomy in an ambulatory setting.

BACKGROUND: Single-incision laparoscopic surgery has emerged as an alternative to conventional laparoscopic cholecystectomy (LC) in the clinical setting. Limited information is available on the possibility of performing single-incision laparoscopic surgery as an ambulatory procedure. This study aimed to determine the feasibility and safety of single-incision laparoscopic cholecystectomy (SILC) versus conventional LC in an ambulatory setting. METHODS: Ninety-one patients were randomized to SILC (n = 49) or LC (n = 42). The success rate, operative duration, blood loss, hospital stay, gallbladder perforation, drainage, delayed discharge, readmission, total cost, complications, pain score, vomiting, and cosmetic satisfaction of the two groups were then compared. RESULTS: There were significant differences in the operative time (46.89 ±â€¯10.03 min in SILC vs. 37.24 ±â€¯10.23 min in LC; P < 0.001). As compared with LC, SILC was associated with lower total costs (8012.28 ±â€¯752.67 RMB vs. 10258.91 ± 1087.63 RMB; P < 0.001) and better cosmetic satisfaction (4.94 ± 0.24 vs. 4.74 ± 0.54; P = 0.031). There were no significant differences between-group in terms of general data, success rate, blood loss, hospital stay, gallbladder perforation, drainage, delayed discharge, readmission, complications, pain score, and vomiting (P > 0.05). CONCLUSIONS: Ambulatory SILC is safe and feasible for selected patients. The advantages of SILC as compared with LC are improved cosmetic satisfaction and lower total costs.

DSCAM-AS1 promotes tumor growth of breast cancer by reducing miR-204-5p and up-regulating RRM2.

Breast cancer (BC) is a common malignancy worldwide. More than 3 700 000 women die of BC every year. DSCAM-AS1 was overexpressed several kinds of cancer and miR-204-5p was lowly expressed, which indicated that miR-204-5p had anti-tumor activity and DSCAM-AS1 had pro-tumor activity. We intended to analyze DSCAM-AS1, miR-204-5p, and ribonucleotide reductase M2 (RRM2). Microarray analysis and quantitative Real Time fluorescence Polymerase Chain Reaction (qRT-PCR) were employed to determine DSCAM-AS1 and miR-204-5p expression. Luciferase reporter assay was applied to examine the target relationship between DSCAM-AS1, miR-204-5p, and RRM2. Cell Counting Kit-8 (CCK-8 assay), transwell assay, and flow cytometry were used to detect cell proliferation, invasion, and apoptosis. The expression of DSCAM-AS1, miR-204-5p, and RRM2 were confirmed by Western blot. We also conducted in vivo assay to verify the effect of DSCAM-AS1. DSCAM-AS1 was up-regulated, while miR-204-5p was down-regulated in BC tissues and cells. DSCAM-AS1 directly targeted miR-204-5p. DSCAM-AS1 promoted the proliferation and invasion of BC cells by reducing miR-204-5p and inhibiting miR-204-5p expression. DSCAM-AS1 expression was related to the expression of RRM2, and miR-204-5p could reverse the function of DSCAM-AS1. RRM2 was up-regulated in BC cells, and miR-204-5p inhibited RRM2 expression by targeting RRM2. Overexpression of RRM2 stimulated proliferation and cell invasion and impeded apoptosis. In vivo experiments showed that knockdown of DSCAM-AS1 decreased the tumorigenesis of BC cells, increased the expression of miR-204-5p. DSCAM-AS1 promoted proliferation and impaired apoptosis of BC cells by reducing miR-204-5p and enhancing RRM2 expression. DSCAM-AS1/miR-204-5p/RRM2 may serve as novel therapeutic targets for BC.

9-cis-retinoic acid elevates MRP3 expression by inhibiting sumoylation of RXRα to alleviate cholestatic liver injury.

AIMS: Vitamin A and its metabolites has been found to be protective against cholestatic liver injury, but the exact underlying mechanisms involved in cholestatic liver injury remain unclear. The objective of this study was to determine the function and mechanisms of 9-cis-retinoic acid, the metabolite of vitamin A, in cholestatic liver injury. METHODS: The bile duct ligated (BDL) mice were treated with 9-cis-retinoic acid by intravenous injection through the tail for 10 days. The liver function and histology were assessed in the matched group and experimental group. The expression of MRP3 in liver tissue was tested by qRT-PCR, Western blotting, and IHC. Effect of RXRα sumoylation on MRP3 expression was investigated at a cellular level. Influence of 9-cis-retinoic acid on RXRα sumoylation was also tested in cells. RESULTS: Our findings showed that 9-cis-retinoic acid significantly decreases the serum ALT and AST level, alleviates hepatic necrosis of the BDL-mice. We also identified MRP3, an important protective hepatobiliary transporter in cholestasis, was elevated by 9-cis-retinoic acid in vivo and in vitro. 9-cis-retinoic acid weakened the sumoylation of RXRα, which promotes the cytoplasmic location of RXRα and lightens the interaction of RXRα and RARα. Inhibition of RXRα and RARα interaction increased MRP3 expression. CONCLUSIONS: 9-cis-retinoic acid alleviates cholestatic liver injury by elevating MRP3 expression through its mechanism of inhibiting sumoylation of RXRα.

The Effects of Angelica Sinensis Polysaccharide on Tumor Growth and Iron Metabolism by Regulating Hepcidin in Tumor-Bearing Mice.

BACKGROUND/AIMS: Iron plays a fundamental role in cell biology and its concentration must be precisely regulated. It is well documented that excess iron burden contributes to the occurrence and progression of cancer. Hepcidin secreted by liver plays an essential role in orchestrating iron metabolism. In the present study, we aimed to investigate the ability of angelica sinensis polysaccharide (ASP) to decrease iron burden in tumor-bearing mice and the mechanism of ASP regulation hepcidin expression. METHODS: Western blot, RT-PCR, immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) were used to detect the regulation of hepcidin and related cytokines by ASP. The role of ASP in tumor proliferation was investigated using in vivo assays. Iron depositions and iron concentrations in organs were determined by hematoxylin-eosin (H&E) staining and atomic absorption spectrophotometer. RESULTS: We found that ASP could inhibit tumor growth in mice xenografted with 4T1 and H22 cancer cells. In vivo experiments also showed that ASP could potently regulate hepcidin expression in liver and serum and decrease iron burden in liver, spleen and grafted tumors in mouse model. Treatment with ASP in hepatic cell lines reproduced comparable results in decreasing hepcidin as in mouse liver. Furthermore, we found that ASP markedly suppressed the expression of interleukin-6 (IL-6), JAK2, p-STAT3, and p-SMAD1/5/8 in liver, suggesting that JAK/STAT and BMP-SMAD pathways were involved in the regulation of hepcidin expression by ASP. We also found down-regulation of iron-related cytokines in ASP treated mice. CONCLUSION: The present study provides new evidence that ASP decreases hepcidin expression, which can reduce iron burden and inhibit tumor proliferation. These findings might aid ASP developed as a potential candidate for cancer treatment in patients with iron overload.

Umbilical Cord Mesenchymal Stem Cells Conditioned Medium Promotes Aß25-35 phagocytosis by Modulating Autophagy and Aß-Degrading Enzymes in BV2 Cells.

Mesenchymal stem cell (MSC) therapy is a promising prospect for the treatment of Alzheimer's disease (AD); however, the underlying mechanisms by which MSCs mediate positive effects are still unclear. We speculated that MSCs mediate microglial autophagy and enhance the clearance of Aß. To test this hypothesis, we cultured BV2 microglial cells with umbilical cord mesenchymal stem cells conditioned medium (ucMSCs-CM) in the presence or absence of Aß25-35 oligomers. We investigated BV2 cell proliferation, cell death, and Aß25-35 phagocytosis as well as protein expression levels of LC3, Beclin-1, p62, insulin-degrading enzyme (IDE), and neprilysin (Nep) with western blotting. The results showed that ucMSCs-CM inhibited the proliferation and decreased cell death of BV2 cells induced by Aß25-35. ucMSCs-CM also promoted the phagocytosis of Aß25-35 by BV2 cells and changed the expression of autophagy-related proteins LC3, Beclin-1, and p62. Treatment also upregulated the expression of Aß-degrading enzymes IDE and Nep. Furthermore, the culture medium in BV2 cells with Aß25-35 and ucMSCs-CM prevented neuronal cell SH-SY5Y from cell death compared to control medium without ucMSCs-CM. Altogether, these data suggested that ucMSCs-CM protect microglial and neuronal cells from Aß25-35-induced cell death and promote Aß phagocytosis by modulating autophagy and enhancing the expression of Aß-degrading enzymes in microglia.

Hem-o-lok Clips Migration: An Easily Neglected Complication after Laparoscopic Biliary Surgery.

Clip migration into the common bile duct (CBD) is a rare but well-established phenomenon of laparoscopic biliary surgery. The mechanism and exact incidence of clip migration are both poorly understood. Clip migration into the common bile duct can cause recurrent cholangitis and serve as a nidus for stone formation. We present a case, a 54-year-old woman, of clip-induced cholangitis resulting from surgical clip migration 12 months after laparoscopic cholecystectomy and laparoscopic common bile duct exploration (LC+LCBDE) with primary closure.

A coumarin-based fluorescence resonance energy transfer probe targeting matrix metalloproteinase-2 for the detection of cervical cancer.

Cervical cancer is one of the most common gynecological malignancies worldwide. At present, the methods used for cervical cancer screening have many disadvantages. Matrix metalloproteinase-2 (MMP-2) is low or absent in normal cervical epithelial cells while overexpressed in cervical intraepithelial neoplasia and cervical cancer, which provides the possibility for the detection of cervical cancer based on MMP-2. The development of a strategy with high sensitivity and specificity for cervical cancer detection would be expected to improve the cure rate. Connecting a fluorophore [7-(diethylamino)-2-oxo-2H-chromene-3-succinimidyl ester] with a quencher {4-[4-(dimethylamino)phenylazo]benzoic acid N-succinimidyl ester} via an MMP-2 substrate peptide GPLGVRGKGG, a new coumarin-based fluorescence resonance energy transfer probe targeting MMP-2 was prepared to examine cervical cancer by cell imaging. The cervical cancer cell lines showed green fluorescence with intensities in the order of CaSki > SiHa > C33A > HeLa, which indicated that the cervical cancer cell lines possessed significantly differential expression levels of MMP-2. Quantitative (real­time) PCR, RT-PCR, and western blot experiments were consistent with such results. The fluorescence detection limit of the probe for MMP-2 was estimated to be 0.05 ng/ml. Therefore, this MMP-2 probe potentially provides a sensitive and specific visual method for cervical cancer screening, diagnosis and prognostic judgment.

Umbilical Cord Mesenchymal Stem Cells Combined With a Collagenfibrin Double-layered Membrane Accelerates Wound Healing.

The aim of this study was to examine the effects of human umbilical cord mesenchymal stem cells (hUCMSCs) in combination with a collagen-fibrin double-layered membrane on wound healing in mice. A collagen-fibrin double-layered membrane was prepared, and the surface properties of the support material were investigated using a scanning electron microscope. Twenty-four mice were prepared for use as full-thickness skin wound models and randomly divided into 3 groups: group A, a control group in which the wounds were bound using a conventional method; group B, a group treated with hUCMSCs combined with a collagen membrane; and group C, a group treated with hUCMSCs combined with a collagen-fibrin double-layered membrane. The postoperative concrescence of the wounds was observed daily to evaluate the effects of the different treatments. Scanning electron microscope observation showed the collagen-fibrin scaffolds exhibited a highly porous and interconnected structure, and wound healing in the double-layered membrane group was better than in groups A or B. Treatment with hUCMSCs combined with a collagen-fibrin double-layered membrane accelerated wound healing.