Anti-hepatocellular carcinoma activity of Sorbaria sorbifolia by regulating VEGFR and c-Met/apoptotic pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Sorbaria sorbifolia (SS) is a traditional Chinese medicine (TCM) that has been employed anti-hepatocellular carcinoma (HCC) for over 2000 years; yet, its underlying mechanism is still not fully understood. AIM OF THE STUDY: In this study, we evaluated the anti-HCC effect on the freeze-dried powder of the water extract of SS (FDSS) by inhibiting tumor-induced neovascularization, and promoting apoptosis, and elucidated the underlying mechanisms. MATERIALS AND METHODS: HCC cell lines (HepG2 and Huh7 cells) and HepG2 xenograft tumors in zebrafish were employed as in vivo and in vitro models, respectively, to evaluate the anti- HCC-indued neovascularization and apoptosis. In HCC cell lines, CCK-8 assay, wound-healing assay, transwell assay, cell circle assay, apoptosis assay, transmission electron microscopy, and co-culture assay were performed in vitro; in HepG2 xenograft tumor-zebrafish, tumor growth inhibition assay, hematoxylin and eosin (HE) staining, xenograft tumor-zebrafish apoptosis assay, and HCC-indued neovascularization assay were performed to evaluate the effect of FDSS on biological behavior of tumor, HCC-indued neovascularization, and apoptosis. The expression of VEGFR and c-Met/apoptotic pathway-related proteins was detected by western blotting analysis. Assays for c-Met and VEGFR activation were conducted to assess the impact of FDSS in either agonistic or inhibitory roles on these receptor proteins. RESULTS: The findings from our study revealed that FDSS effectively suppresses the proliferation, migration, and invasion of HepG2 and Huh7 cells, as well as inhibiting tumor growth in the HepG2 xenograft zebrafish model by downregulating the expression of p-Met and p-AKT proteins. FDSS decreased the tumor growth associated with promoting apoptosis, including arresting HepG2 and Huh7 cells cycle at G0/G1phase, increasing apoptotic cell numbers and apoptotic bodies in cancer cells, and increasing the apoptotic fluorescence of xenograft tumor zebrafish by downregulating Bcl-2 proteins and upregulating Bax, caspase-9, and caspase-3 levels. We also found that FDSS can inhibit HCC-induced neovascularization and regulate VEGFR. Using an agonist or inhibitor of c-Met and VEGFR in HepG2 cells, we discovered that FDSS can downregulate c-Met and VEGFR protein expression. CONCLUSION: FDSS exerts an anti-HCC effect by inhibiting HCC-indued neovascularization and pro-apoptosis through the inhibition of the action of VEGFR and c-Met/apoptotic pathway.

Associations of urinary and blood cadmium concentrations with all-cause mortality in US adults with chronic kidney disease: a prospective cohort study.

Epidemiological evidence for the relationship between cadmium exposure and mortality in specific chronic kidney disease (CKD) populations remains scarce. We aimed to explore the relationships between cadmium concentrations in urine and blood and all-cause mortality among CKD patients in the USA. This cohort study was composed of 1825 CKD participants from the National Health and Nutrition Examination Survey (NHANES) (1999-2014) who were followed up to December 31, 2015. All-cause mortality was ascertained by matching the National Death Index (NDI) records. We estimated hazard ratios (HRs) and 95% confidence intervals (CIs) for all-cause mortality in relation to urinary and blood cadmium concentrations by Cox regression models. During an average follow-up period of 82 months, 576 CKD participants died. Compared with the lowest quartiles, HRs (95% CIs) for all-cause mortality associated with the fourth weighted quartiles of urinary and blood cadmium concentrations were 1.75 (1.28 to 2.39) and 1.59 (1.17 to 2.15), respectively. Furthermore, the HRs (95% CIs) for all-cause mortality per ln-transformed IQR increment in cadmium concentrations in urine (1.15 µg/g UCr) and blood (0.95 µg/L) were 1.40 (1.21 to 1.63) and 1.22 (1.07 to 1.40), respectively. Linear concentration-response relationships between urinary and blood cadmium concentrations and all-cause mortality were also found. Our findings suggested that increased cadmium concentrations in both urine and blood significantly contributed to enhanced mortality risk in CKD patients, thus highlighting that efforts to reduce cadmium exposure may reduce mortality risk in high-risk populations with CKD.

Relationship between carotid intraplaque neovascularization and haemoglobin A1c in diabetic patients.

INTRODUCTION: We aimed to evaluate the characteristics of carotid artery plaques and the relationship between intraplaque neovascularization (IPN) and hemoglobin A1c (HbA1c) in patients of <60 years old with diabetes mellitus (DM) by comparison with diabetes ≥60 years of age. METHODS: One-hundred-and-one patients with DM were studied into two groups: those <60 and those ≥60 years of age. All the patients underwent standard carotid ultrasonography and contrast-enhanced ultrasonography, which we used to evaluate IPN. RESULTS: Diabetic complications were present in 41 of 50 patients (82 %) in the <60-year-old group, of whom 17 (34 %) had diabetes-related vascular complications. Of the 47 plaques in the <60-year-old group, six (13 %) had IPN Grade 0, 16 (34 %) had IPN Grade 1, and 25 (53 %) had IPN Grade 2. The AUC and RAUC of the plaque in the <60-year-old group were significantly higher than those of the ≥60-year-old group (P = 0.012 and 0.031, respectively). There were also differences in the AUC, RAUC and semi-quantitative grades between patients with and without diabetic macrovasculopathy and diabetic peripheral artery disease (all P < 0.05). The AUC, RAUC and semi-quantitative grading of IPN positively correlated with blood glucose and HbA1c (P < 0.05). CONCLUSION: IPN is more common in DM patients who are younger, and have higher blood glucose and HbA1c concentrations, and these plaques are more vulnerable.

The impact of urbanization and consumption patterns on China's black carbon emissions based on input-output analysis and structural decomposition analysis.

Urbanization in China has dramatically increased from 39.10 in 2002 to 58.52% in 2017. Studies have discussed the impacts of urbanization and its corresponding changes in consumption patterns on carbon dioxide emissions; however, little is known about their impacts on black carbon (BC). Therefore, we collected data on the BC emissions of various sectors to calculate the consumption-based BC emissions in China, and we used an input-output analysis (IOA) and structural decomposition analysis (SDA) to explore the impacts of urbanization and changes in consumption patterns on BC emissions from 2002 to 2017, focusing on sectoral BC emissions. The total BC emissions of various sectors first increased and then decreased. BC emissions increased from 1083.47 in 2002 to 2550.83 Gg in 2012. They were then reduced to 2478.63 Gg in 2017. Additionally, with the rise in the urbanization rate, household consumption BC emissions increased from 446.18 in 2002 to 1080.12 Gg in 2017. Urban consumption, rural consumption, and BC emission intensity were the three main contributing factors to household consumption BC emission changes. Transport, storage, postal, and telecommunications services (TSP); farming, forestry, animal husbandry, and fishery (FFA); and residential and other industries (RES) contributed the most to the urbanization-related BC emission increase. In particular, the TSP sector contributed the most to the BC emission increase because of the increasing TSP needs related to urbanization. Therefore, it is necessary to formulate mitigation policies for the TSP sector.

Carotid intraplaque neovascularization predicts atherosclerotic renal artery stenosis in patients with carotid artery stenosis.

BACKGROUND AND AIMS: This study aimed to examine whether intraplaque neovascularization (IPN) of carotid plaques, as characterized by contrast-enhanced ultrasound (CEUS), is associated with atherosclerotic renal artery stenosis (ARAS) in patients with normal kidney function. METHODS AND RESULTS: We investigated carotid IPN using CEUS in 198 consecutive patients with normal kidney function with and without ARAS. IPN was graded on the basis of the presence and location of microbubbles within each plaque (0, no visible microbubbles in the plaque; 1, moderate microbubbles confined to the shoulder and/or adventitial side of the plaque; and 2, extensive microbubbles throughout the plaque). The grades of each plaque were averaged to obtain an overall score per patient. ARAS was determined angiographically. We found that a higher CEUS-assessed carotid IPN score was associated with ARAS (Odd Ratio, OR: 7.281; 95% Confidence Interval, 95% CI: 3.246-16.336; P < 0.001). Furthermore, an IPN score >1.75 predicted severe stenosis with a sensitivity of 81% and specificity of 58%. Compared with using the IPN score alone, the addition of the homocysteine (HCY) cutoff value (>22.5 mmol/L) resulted in a stronger predictive value (Area Under Curve, AUC: 0.893 vs 0.834; P < 0.001) for severe ARAS. CONCLUSION: Carotid plaque neovascularization combined with HCY levels is predictive of severe ARAS in patients with normal kidney function. CEUS-assessed carotid IPN is clinically useful for stratification of ARAS in patients with normal kidney function.

Development and External Validation of a Novel Immune Checkpoint-Related Gene Signature for Prediction of Overall Survival in Hepatocellular Carcinoma.

Objective: The purpose of this study was to develop and validate a novel immune checkpoint-related gene signature for prediction of overall survival (OS) in hepatocellular carcinoma (HCC). Methods: mRNA expression profiles and clinical follow-up information were obtained in the International Cancer Genome Consortium database. An external dataset from The Cancer Genome Atlas (TCGA) Liver Hepatocellular Carcinoma database was used to validate the results. The univariate and multivariate Cox regression analyses were performed based on the differentially expressed genes. We generated a four-mRNA signature to predict patient survival. Furthermore, the reliability and validity were validated in TCGA cohort. An integrated bioinformatics approach was performed to evaluate its diagnostic and prognostic value. Results: A four-gene (epidermal growth factor, mutated in colorectal cancer, mitogen-activated protein kinase kinase 2, and NRAS proto-oncogene, GTPase) signature was built to classify patients into two risk groups using a risk score with different OS in two cohorts (all P < 0.0001). Multivariate regression analysis demonstrated the signature was an independent predictor of HCC. Furthermore, the signature presented an excellent diagnostic power in differentiating HCC and adjacent tissues. Immune cell infiltration analysis revealed that the signature was associated with a number of immune cell subtypes. Conclusion: We identified a four-immune checkpoint-related gene signature as a robust biomarker with great potential for clinical application in risk stratification and OS prediction in HCC patients and could be a potential indicator of immunotherapy in HCC. The diagnostic signature had been validated to accurately distinguish HCC from adjacent tissues.

[Isoliquiritigenin can inhibit migration and invasion of human glioma stem cells by down-regulating matrix metalloproteinases].

OBJECTIVE: To investigate the effects of isoliquiritigenin on the migration and invasion of human glioma stem cells and the underlying mechanism. METHODS: The stem cell markers CD133 and Nestin in SHG44 human glioma stem cells were examined with immunofluorescence microscopy. The migration and invasion ability of glioma stem cells was determined by transwell method. The mRNA and protein expression of matrix metalloproteinase (MMP)-2 and MMP-9 were detected by real-time RT-PCR and Western blot, respectively. RESULTS: CD133 and Nestin were positive in SHG44 cells. The number of migrated cells in SHG44 cells treated with 20 and 80 µmol/L isoliquiritigenin for 48 h were significantly lower than that in control group (76±5 and 42±4 vs. 85±6, all P<0.01), and the number of migrated cells in 80 µmol/L isoliquiritigenin group was lower than that in 20 µmol/L isoliquiritigenin group (P<0.01). The numbers of cells crossing through membrane in 20 and 80 µmol/L isoliquiritigenin groups were 190±13 and 130±9, respectively, which were significantly lower than that in control group (230±14, all P<0.01), and the number of crossed cells in the 80 µmol/L isoliquiritigenin group was lower than that in 20 µmol/L isoliquiritigenin group (P<0.01). The mRNA and protein expression levels of MMP-2 and MMP-9 were decreased compared with control group (P<0.05 or P<0.01), and the expression levels in 80 µmol/L isoliquiritigenin group were lower than those in 20 µmol/L isoliquiritigenin group (P<0.05 or P<0.01). CONCLUSIONS: Isoliquiritigenin exhibits antitumor effects on glioma stem cells by inhibiting cell migration and invasion, which may be related to the down-regulation of MMP-2 and MMP-9.

Isoliquiritigenin inhibits the proliferation and induces the differentiation of human glioma stem cells.

Glioma stem cells (GSCs) have been proven to be resistant to various therapeutic strategies, such as temozolomide chemotherapy and radiotherapy, leading to glioma recurrence. Isoliquiritigenin (ISL), a menber of the flavonoids isolated from liquorice has been found to be a potent stimulator of cell differentiation and has potential application for treating various types of cancer including human brain glioma. However, the antitumor activity of ISL on GSCs and the signaling pathway underlying its therapeutic effects are poorly understood. In the present study, GSCs were isolated from SHG44 human glioma cells by serum-free culture and treated with ISL or DAPT (a Notch/γ-secretase inhibitor). It was found that ISL dose-dependently inhibited GSC growth after 72 h of treatment and decreased the formation of tumor spheres. Meanwhile, GSCs differentiated into astrocytes and neurons. Furthermore, these therapeutic effects were accompanied by downregulation of Notch1 and Hes1 at the protein and mRNA levels. Taken together, our results demonstrated that ISL exhibits antitumor effects on GSCs by inhibiting proliferation and inducing differentiation. The therapeutic effect may be related to downregulation of the Notch1 signaling pathway. Application of ISL presents potential benefits for the treatment of human brain glioma.

An inflammatory myofibroblastic tumor in the transplanted liver displaying quick wash-in and wash-out on contrast-enhanced ultrasound: A case report.

RATIONALE: Inflammatory myofibroblastic tumor (IMT) is an uncommon mesenchymal neoplasm, and its presence in a grafted liver is exceedingly rare. PATIENT CONCERNS: A 54-year-old woman was admitted to our hospital with a half-month history of intermittent melena. She had undergone deceased-donor liver transplantation (LT) for hepatitis B virus related liver cirrhosis without hepatocellular carcinoma 5 months previously. DIAGNOSIS: Laboratory examination showed impaired liver and renal functions and Epstein-Barr virus (EBV) infection, but tumor markers within normal ranges. Gastroscopy showed esophageal varices. Ultrasound and computed tomography angiography revealed an ill-defined and irregular solitary lesion in the porta hepatis, encasing both the portal vein and the hepatic artery. The lesion was characterized by arterial hyper-enhancement and hypo-enhancement in the remaining phases with contrast-enhanced ultrasound (CEUS). The lesion was finally confirmed as an IMT by ultrasound-guided biopsy. INTERVENTION: The patient received conservative treatment, including immunosuppression, endoscopic variceal ligation, antibiotics, steroids, and antiviral agents. OUTCOME: The patient's gastrointestinal bleeding was controlled, but the symptoms associated with portal hypertension worsened. Attempts to perform a transjugular intrahepatic portosystemic shunt were unsuccessful, and she unfortunately died soon after. LESSONS: A differential diagnosis of IMT should be considered in LT recipients presenting with EBV infection, normal tumor markers, and a de novo hepatic lesion with quick wash-in and wash-out on CEUS. Ultrasound is associated with the advantages of convenience and nonionizing radiation, and should thus be the priority approach for monitoring transplanted liver.

Contrast-enhanced ultrasound improves accurate identification of appendiceal mucinous adenocarcinoma in an old patient: A case report.

BACKGROUND: Adenocarcinoma of appendiceal origin is far rarer than other colorectal carcinomas and its preoperative diagnosis is challenging. To our knowledge, utility of contrast-enhanced ultrasound (CEUS) to diagnose it is much less. METHOD: A 61-year-old man presented with abdominal pain in the right lower quadrant for 20 days. In order to fulfill an accurately preoperative diagnosis, he received laboratory and imaging tests such as carcinoembryonic antigen (CEA), computer tomography (CT), CEUS and endoscope. DIAGNOSIS AND INTERVENTION: He was initially suspected of suffering appendicitis, while his white blood cell count was normal and carcinoembryonic antigen (CEA) in serum was remarkably increased. Both routine ultrasound and computer tomography (CT) examinations supported suppurative appendicitis. The overall data, however, failed to excluded neoplastic pathology thoroughly. Therefore, CEUS was carried out and showed an inhomogeneous enhancement intra the lesion located in the body of the appendix, which made our consideration of neoplasm. The result of the follow-up biopsy guided by endoscope was consistent with appendiceal tumor. The patient received laparoscopic right hemicolectomy. Histopathology confirmed as well differentiated mucinous adenocarcinoma of appendix origin. His postoperative course was uneventful, and he had a regular diet again without any complaint. RESULT: Serum CEA was remarkably increased (12.00 ng/mL). Both routine ultrasound and CT examinations supported suppurative appendicitis. However, CEUS examination showed an inhomogeneous enhancement intra the lesion located in the body of the appendix, which made our consideration of neoplasm. The follow-up biopsy guided by endoscope and surgical specimens confirmed as well differentiated mucinous adenocarcinoma of appendix origin. CONCLUSION: Most mucinous adenocarcinoma mimicking appendicitis results in difficult diagnosis preoperatively. Clinician and radiologist should be aware of it when old patient presented with appendicitis especially along with high level of CEA.

Positive correlation between neovascularization degree of carotid atherosclerosis determined by contrast-enhanced ultrasound and level of serum C-reactive protein.

BACKGROUND: Neovascularization is one of the most important risk factors for unstable plaque. This study was designed to correlate plaque thickness, artery stenosis and levels of serum C-reactive protein with the degree of intraplaque enhancement determined by contrast-enhanced ultrasound. PATIENTS AND METHODS: Contrast-enhanced ultrasound was performed on 72 carotid atherosclerotic plaques in 48 patients. Contrast enhancement within the plaque was categorized as grade 1, 2 or 3. Maximum plaque thickness was measured in short-axis view. Carotid artery stenosis was categorized as mild, moderate or severe. RESULTS: Plaque contrast enhancement was not associated with the degree of artery stenosis or with plaque thickness. Serum C-reactive protein levels were positively correlated with the number of new vessels in the plaque. C-reactive protein levels increased in the three groups(Grade 1: 3.72±1.79mg/L; Grade 2: 7.88±4.24 mg/L; Grade 3: 11.02±3.52 mg/L), with significant differences among them (F=10.14, P<0.01), and significant differences between each two groups (P<0.05). Spearman’s rank correlation analysis showed that serum C-reactive protein levels were positively correlated with the degree of carotid plaque enhancement (Rs =0.69, P<0.01). CONCLUSIONS: The combination of C-reactive protein levels and intraplaque neovascularization detected by contrast-enhanced ultrasound may allow more accurate evaluation of plaque stability.

Identification of molecular determinants from Moloney leukemia virus 10 homolog (MOV10) protein for virion packaging and anti-HIV-1 activity.

Discovery of novel antiretroviral mechanism is essential for the design of innovative antiretroviral therapy. Recently, we and others reported that ectopic expression of Moloney leukemia virus 10 (MOV10) protein strongly inhibits retrovirus replication. MOV10, a putative RNA helicase, can be packaged into HIV-1 virions by binding to the nucleocapsid (NC) region of Gag and inhibit viral replication at a postentry step. Here, we report critical determinants for MOV10 virion packaging and antiviral activity. MOV10 has 1,003 amino acids and seven helicase motifs. We found that MOV10 packaging requires the NC basic linker, and Gag binds to the N-terminal amino acids 261-305 region of MOV10. Our predicted MOV10 three-dimensional structure model indicates that the Gag binding region is located in a structurally exposed domain, which spans amino acids 93-305 and is Cys-His-rich. Simultaneous mutation of residues Cys-188, Cys-195, His-199, His-201, and His-202 in this domain significantly compromised MOV10 anti-HIV-1 activity. Notably, although MOV10-Gag interaction is required, it is not sufficient for MOV10 packaging, which also requires its C-terminal all but one of seven helicase motifs. Moreover, we have mapped the minimal MOV10 antiviral region to amino acids 99-949, indicating that nearly all MOV10 residues are required for its antiviral activity. Mutations of residues Cys-947, Pro-948, and Phe-949 at the C terminus of this region completely disrupted MOV10 anti-HIV-1 activity. Taken together, we have identified two critical MOV10 packaging determinants and eight other critical residues for anti-HIV-1 activity. These results provide a molecular basis for further understanding the MOV10 antiretroviral mechanism.

Identification of a single amino acid required for APOBEC3 antiretroviral cytidine deaminase activity.

During studies of APOBEC3 (A3) anti-human immunodeficiency virus type 1 (anti-HIV-1) mechanisms, we identified a single cysteine at position 320 (C320) that disrupts A3DE activity. This residue is located in the recently identified DNA binding domain in A3G. Replacing C320 with a corresponding tyrosine from A3F (Y307) increased A3DE antiviral activity more than 20-fold. Conversely, replacing A3F Y307 with a cysteine or inserting a similar cysteine into A3B or A3G disrupted the anti-HIV activity of A3. Further investigation uncovered that C320 significantly reduces A3DE catalytic activity.

Moloney leukemia virus 10 (MOV10) protein inhibits retrovirus replication.

Moloney leukemia virus 10 (MOV10) protein is a superfamily-1 RNA helicase, and it is also a component of the RNA-induced silencing complex. Recent studies have shown that MOV10 plays an active role in the RNA interference pathway. Here, we report that MOV10 inhibits retrovirus replication. When it was overexpressed in viral producer cells, MOV10 was able to reduce the infectivity of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus, and murine leukemia virus. Conversely, when MOV10 expression was reduced by small interfering RNAs, HIV-1 infectivity was increased. Consistently, silencing of MOV10 expression in a human T cell line enhanced HIV-1 replication. Furthermore, we found that MOV10 interacts with HIV-1 nucleocapsid protein in an RNA-dependent manner and is packaged into virions. It blocks HIV-1 replication at a postentry step. In addition, we also found that HIV-1 could suppress MOV10 protein expression to counteract this cellular resistance. All of these results indicate that MOV10 has a broad antiretroviral activity that can target a wide range of retroviruses, and it could be actively involved in host defense against retroviral infection.

Identification of APOBEC3DE as another antiretroviral factor from the human APOBEC family.

A tandem arrayed gene cluster encoding seven cytidine deaminase genes is present on human chromosome 22. These are APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3DE, APOBEC3F, APOBEC3G, and APOBEC3H. Three of them, APOBEC3G, APOBEC3F, and APOBEC3B, block replication of human immunodeficiency virus type 1 (HIV-1) and many other retroviruses. In addition, APOBEC3A and APOBEC3C block intracellular retrotransposons and simian immunodeficiency virus (SIV), respectively. In opposition to APOBEC genes, HIV-1 and SIV contain a virion infectivity factor (Vif) that targets APOBEC3F and APOBEC3G for polyubiquitylation and proteasomal degradation. Herein, we studied the antiretroviral activities of the human APOBEC3DE and APOBEC3H. We found that only APOBEC3DE had antiretroviral activity for HIV-1 or SIV and that Vif suppressed this antiviral activity. APOBEC3DE was encapsidated and capable of deaminating cytosines to uracils on viral minus-strand DNA, resulting in disruption of the viral life cycle. Other than GG-to-AG and AG-to-AA mutations, it had a novel target site specificity, resulting in introduction of GC-to-AC mutations on viral plus-strand DNA. Such mutations have been detected previously in HIV-1 clinical isolates. In addition, APOBEC3DE was expressed much more extensively than APOBEC3F in various human tissues and it formed heteromultimers with APOBEC3F or APOBEC3G in the cell. From these studies, we concluded that APOBEC3DE is a new contributor to the intracellular defense network, resulting in suppression of retroviral invasion.