Endoscopic therapy combined with photodynamic therapy for intratracheal inflammatory myofibroblastic tumor.

Inflammatory myofibroblastic tumor (IMT) is a rare intermediate tumor that exhibits both benign and malignant behaviors. Whether IMT is an inflammatory or a neoplastic disease remains controversial, and there is currently no standard treatment for this disease. The treatment strategies include surgical tumor removal and drug therapy; however, several patients experience tumor recurrence post-treatment. Herein, we report the endoscopic manifestations and treatment process in a case of IMT. We performed photodynamic therapy (PDT) after endoscopic tumor resection, and no recurrence was found in the patient's re-examination a year later. This indicates that PDT can improve IMT prognosis and reduce its local recurrence, signifying the therapy's potential application value for IMT.

Aloe emodin inhibits telomerase activity in breast cancer cells: transcriptional and enzymological mechanism.

BACKGROUND: Telomerase plays an essential role in cancer cell proliferation. In this study, we investigated inhibition mechanism of aloe emodin (AE) on three different types of breast cancer cell lines, MDA-MB-453, MDA-MB-231 and MCF-7. METHODS: The cells were treated with different concentrations of AE. Relative length of telomere and human telomerase reverse-transcriptase (hTERT) mRNA level was analyzed by quantitative PCR (qPCR). Protein level was assayed by Western blot. Sodium bisulfite methylation sequencing was performed to assess the methylation status of gene promoter. Enzymology kinetics was applied to reveal the interaction between AE and telomerase. Ultraviolet-visible titration and fluorescence resonance energy transfer (FRET) melting experiment were carried out to study the interaction between AE and telomeric DNA. RESULTS: Continuous AE exposure of these cells for 48 h results in shortening of telomeres and inhibition of telomerase. The transcription of hTERT was repressed by activation of E2F1 and inactivation of c-myc proteins. Significant demethylation of CpG islands in hTERT gene promoter was observed in MDA-MB-453 and MCF-7 cells. AE competed with dNTP for occupation of the enzyme active site. AE was a telomeric G-quadruplex structure stabilizer as indicated by titration test and FRET experiments. CONCLUSIONS: AE was a competitive inhibitor of telomerase and a G-quadruplex structure stabilizer. AE decreased the transcription of hTERT gene in the three breast cancer cell lines via up-regulation E2F1 and down-regulation c-myc expressions. The suppressed transcription was also related to the demethylation of the gene promoter.

[Effect of acupuncture on the ultrastructure of neurons and astrocytes in the hippocampal dentate gyrus in rats with Alzheimer's disease induced by Aß1-42].

OBJECTIVE: To observe the effects of acupuncture at "Baihui" (GV 20) and "Shenshu" (BL 23) on the ultrastructure of hippocampal dentate gyrus in rats with Alzheimer's disease. METHODS: Forty SPF Wistar male rats were randomly divided into a normal group, a sham operation group, a model group and an acupuncture group, 10 rats in each one. The rats in the model group and the acupuncture group were treated with injection of 5 µL Aß1-42 at bilateral hippocampus, while the rats in the sham operation group were treated with injection of 5 µL 0.9% NaCl. Three days after modeling, the rats in the acupuncture group were treated with acupuncture at "Baihui" (GV 20) and "Shenshu" (BL 23) for 20 min, once a day, six treatments constituted a course, and totally two courses were given with an interval of 1 day between courses. The rats in the other groups received normal diet and no treatment was given. Before modeling, four days after modeling and after treatment, water maze test was performed to observe the escape latency and the number of crossing platforms. The hippocampal dentate gyrus was collected and transmission electron microscope was applied to observe the ultrastructure changes of neurons and astrocytes. RESULTS: ①Four days after modeling, compared with the normal group and the sham operation group, the escape latency was significantly prolonged and the number of crossing platforms was reduced in the model group (all P<0.01); after treatment, compared with the model group, the escape latency was significantly reduced and the number of crossing platforms was increased in the acupuncture group (both P<0.01). ②In the normal group and the sham operation group, the morphology of neurons and astrocytes was intact, the nuclear and membrane structure were clear, and the morphology of organelles such as mitochondria, endoplasmic reticulum and lysosomes was normal. In the model group, the morphology of neurons was irregular, the nucleus was severely constricted with edema in the cytoplasm, the color of heterochromatin was deepened, the endoplasmic reticulum was expanded, the granulation was removed and the number of mitochondria was decreased, even with malformed-like change in mitochondrial cristae; there was severe edema around astrocytes, few organelles in the cytoplasm, severe swelling of mitochondria and mild expansion of the endoplasmic reticulum. In the acupuncture group, the edema of the neuron and astrocytes was still evident, and the mitochondrial was mildly swollen but relieved compared with that in the model group, and there were no obvious abnormalities in neuronal endoplasmic reticulum and ribosomes. CONCLUSION: Acupuncture could improve the ultrastructure of neurons and astrocytes in the hippocampal dentate gyrus in rats with Alzheimer's disease induced by Aß1-42.

Canopy Homolog 2 Expression Predicts Poor Prognosis in Hepatocellular Carcinoma with Tumor Hemorrhage.

BACKGROUND/AIMS: Canopy homolog 2 (CNPY2) is a signature gene highly associated with tumor progression, including hepatocellular carcinoma (HCC). The presence of tumor hemorrhage (TH) implies a fast-growing and worse tumor microenvironment. We examined a possible association between CNPY2 levels and TH and evaluated their prognostic values in patients with HCC. METHODS: CNPY2 mRNA and protein levels were respectively determined in two independent cohorts of HCC specimens using quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry of tissue microarrays. Kaplan-Meier survival and Cox regression analyses were executed to evaluate the prognosis of HCC. CNPY2 knockout HCC cell lines were established by the CRISPR/Cas9 gene editing system, and the functional role of CNPY2 in HCC cell proliferation and growth was examined in vitro and in vivo. RESULTS: qRT-PCR showed that CNPY2 expression was significantly higher in HCC tumor tissue than in adjacent non-tumor tissue. Immunohistochemistry of HCC tissue microarrays demonstrated that CNPY2 expression was significantly correlated with TH and clinicopathological features indicating worse HCC progression. The prognostic value of CNPY2 expression and TH was validated by Cox proportional hazards analyses. Furthermore, CNPY2 knockout resulted in the significant suppression of MHCC97H cell proliferation, tumor growth, and hemorrhage. Bioinformatics analysis revealed that CNPY2 was closely associated with the expression levels of 6 positive impact genes in HCC, namely, ROMO1, BOLA2, HSF1, ATG4B, ATF4, and DENR, which are implicated in the regulation of the tumor microenvironment. CONCLUSION: CNPY2 is an oncogene that plays a critical role in the progression of HCC with TH. CNPY2 could be exploited as a novel prognostic marker and potential target for therapeutic intervention in HCC.

Peroxidase from jackfruit: Purification, characterization and thermal inactivation.

Peroxidase (POD) from jackfruit bulb was purified using ammonium sulfate precipitation, hydrophobic interaction and gel filtration columns. The POD was a dimer with a molecular weight of 104kDa. The Km and Vmax values for guaiacol, gallic acid and o­phenylenediamine (OPD) were estimated. OPD was the most suitable substrate. The enzyme showed its maximum activity at pH5.5 and 55-60°C. The activation energy (Ea) of heat inactivation was estimated to be 206.40kJ/mol. The enthalpy, free energy and entropy values for the thermal inactivation were also determined. The POD activity was enhanced by K+, Zn2+, Ba2+, citric acid, malic acid, benzoic acid and EDTA·Na2, but inhibited by Cu2+, Ca2+, glutathione, cysteine and ascorbic acid. Chemical modification indicated a histidine residue was located in the enzyme active site. The POD activity in fruit extracts significantly decreased when heated at 80°C and 90°C. The ferric-reducing antioxidant power, ABTS radical scavenging activity and total phenolics decreased with increasing heating temperature and time.

CLEC1B Expression and PD-L1 Expression Predict Clinical Outcome in Hepatocellular Carcinoma with Tumor Hemorrhage.

Spontaneous tumor hemorrhage (TH) is frequently observed in solid tumors including human hepatocellular carcinoma (HCC). TH implies fast-growing and worse tumor immunological microenvironment; however, the underlying mechanism remains largely unknown. CLEC1B is a signature gene highly associated with tumor progression. PD-L1 expression is a key biomarker predictive of immune checkpoint therapies, which showed astonishing effect on various types of tumor. We assume that, in HCC, TH may closely associate with the expression of these two molecules. In this study, 136 patients with HCC were enrolled. qRT-PCR showed that CLEC1B expression is significantly lower in HCC tumor tissue. Immunohistochemistry of HCC tissue microarrays demonstrated that PD-L1high and CLEC1Blow expressions were significantly correlated with TH and clinicopathological features indicating worse HCC progression. According to univariate/multivariate analysis, a combination of PD-L1high and CLEC1Blow expression was an independent prognostic factor indicating the poor outcome. The prognostic value of PD-L1high and CLEC1Blow was validated by Cox proportional-hazard analyses. Collectively, tumor with TH is closely associated with CLEC1Blow & PD-L1high expression, which may imply high response of PD-L1/PD-1 immune checkpoint therapies. CLEC1B may be a potential therapeutic target for PD-L1/PD-1 immunotherapy. PD-L1high and CLEC1Blow can be a valuable prognosis factor implying worse clinical outcomes.

Purification and characterization of polyphenol oxidase from jackfruit ( Artocarpus heterophyllus ) bulbs.

Polyphenol oxidase (PPO) from jackfruit bulb was purified through acetone precipitation, ion-exchange column, and gel filtration column. PPO was a dimer with the molecular weight of 130 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration. The Km was 8.3 and 18.2 mM using catechol and 4-methylcatechol as substrates, respectively. The optimum pH was 7.0 (catechol as the substrate) or 6.5 (4-methylcatechol as the substrate). The optimum temperature was 8 °C. The enzyme was stable below 40 °C. The activation energy (Ea) of heat inactivation was estimated to be 103.30 kJ/mol. The PPO activity was activated by Mn(2+), SDS, Tween-20, Triton X-100, citric acid, and malic acid but inhibited by K(+), Zn(2+), Mg(2+), Ca(2+), Ba(2+), cetyl trimethyl ammonium bromide (CTAB), kojic acid, tropolone, glutathione (GSH), cysteine (Cys), and ascorbic acid (AA). Cys and AA were effective to reduce browning of jackfruit bulbs during the storage at 8 °C for 15 days.

Overexpression of WDR62 is associated with centrosome amplification in human ovarian cancer.

PURPOSE: To assess the clinical significance of WD40 repeat containing 62 (WDR62), a novel centrosome abnormalities-associated gene, in ovarian cancer. MATERIALS AND METHODS: In this study, WDR62 expression was assessed by western blot (6 ovarian cancer cell lines) and immunohistochemistry (primary epithelial ovarian cancer clinical specimens), and clinical variables were collected by retrospective chart review. Centrosome amplification was assessed by immunofluorescence staining in ovarian cancer cell lines, and by immunohistochemistry staining in ovarian cancer samples. RESULTS: Six ovarian cancer cell lines exhibited significant WDR62 protein overexpression, and amplification of centrosome. High-grade ovarian cancer specimens exhibited significantly stronger nuclear staining of WDR62 than low-grade ovarian carcinoma specimens (80.4% vs 41.3%; P<0.012). High WDR62 expression was strongly associated with supernumerary centrosome count in tumor cells (P < 0.001). CONCLUSION: Our findings suggest that WDR62 overexpression is related to centrosome amplification in ovarian cancer. It may be a novel useful differentiation biomarker and a potential therapy target for OC. Further assessment of WDR62 expression is highly warranted in large, prospective studies.

The expression of Egfl7 in human normal tissues and epithelial tumors.

BACKGROUND AND AIMS: To investigate the expression of Egfl7 in normal adult human tissues and human epithelial tumors.
 METHODS: RT-PCR and Western blot were employed to detect Egfl7 expression in normal adult human tissues and 10 human epithelial tumors including hepatocellular carcinoma (HCC), lung cancer, breast cancer, prostate cancer, colorectal cancer, gastric cancer, esophageal cancer, malignant glioma, ovarian cancer and renal cancer. Immunohistochemistry and cytoimmunofluorescence were subsequently used to determine the localization of Egfl7 in human epithelial tumor tissues and cell lines. ELISA was also carried out to examine the serum Egfl7 levels in cancer patients. In addition, correlations between Egfl7 expression and clinicopathological features as well as prognosis of HCC and breast cancer were also analyzed on the basis of immunohistochemistry results.
 RESULTS: Egfl7 was differentially expressed in 19 adult human normal tissues and was overexpressed in all 10 human epithelial tumor tissues. The serum Egfl7 level was also significantly elevated in cancer patients. The increased Egfl7 expression in HCC correlated with vein invasion, absence of capsule formation, multiple tumor nodes and poor prognosis. Similarly, upregulation of Egfl7 in breast cancer correlated strongly with TNM stage, lymphatic metastasis, estrogen receptor positivity, Her2 positivity and poor prognosis. 
 CONCLUSIONS: Egfl7 is significantly upregulated in human epithelial tumor tissues, suggesting Egfl7 to be a potential biomarker for human epithelial tumors, especially HCC and breast cancer.

BTB/POZ domain-containing protein 7: epithelial-mesenchymal transition promoter and prognostic biomarker of hepatocellular carcinoma.

UNLABELLED: Epithelial-mesenchymal transition (EMT) is a critical step in the metastasis of hepatocellular carcinoma (HCC). BTB/POZ domain-containing protein 7 (BTBD7) regulates EMT-associated proteins implicated in HCC progression. However, the role(s) of BTBD7 in HCC have not been identified. Using highly metastatic HCC HCCLM3 cells, immortalized L02 hepatocytes, metastatic HCC animal models, and three independent cohorts of HCC patient specimens, we aimed to determine the involvement of BTBD7 in HCC metastasis. We show that BTBD7 messenger RNA and protein was highly expressed in HCC cells and tumor tissues, with such expression being associated with: enhanced cell motility, venous invasion, and poor prognosis. BTBD7 promoted HCC angiogenesis and metastasis in vitro and in vivo, but did not influence cell proliferation or colony formation. BTBD7 enhancement of HCC invasion and EMT phenotype occurred through activation of a RhoC-Rock2-FAK-signaling pathway, resulting in matrix metalloproteinase-2/9 production and microvessel formation. Applying a predictive risk score model, Cox regression analysis revealed that high BTBD7 expression integrated with high microvessel density was a powerful independent predictive factor of HCC clinical outcome. CONCLUSION: The present study identifies BTBD7 as a novel candidate prognostic factor and a potential therapeutic target of HCC. (HEPATOLOGY 2013; 57:2326-2337).

Dickkopf-1 (DKK1) promotes invasion and metastasis of hepatocellular carcinoma.

BACKGROUND: Dickkopf-1 (DKK1) recently has been reported to be involved in the progress of hepatocellular carcinoma, but its concrete role is not clear. The objective of this study is to investigate the clinical significance, biological function and molecular mechanism of DKK1 in the invasion and metastasis of hepatocellular carcinoma. METHODS: The mRNA and protein expression levels of DKK1 in hepatocellular carcinoma were detected and its prognostic significance was assessed. The biological function of DKK1 in hepatocellular carcinoma was investigated by using wound healing, transwell invasion assay and hepatocellular carcinoma metastatic mouse model. RESULTS: DKK1 was predominantly elevated in hepatocellular carcinoma tissues, especially in tissue with vascular invasion. The increased DKK1 expression was correlated with multiple tumour nodes, high Edmondson-Steiner grade and vein invasion, as well as poor overall and disease-free survival of hepatocellular carcinoma. DKK1 could promote hepatocellular carcinoma cell invasion and metastasis in vitro and in vivo. Finally, a positive relationship of DKK1 expression with RhoA and JNK levels was found in both hepatocellular carcinoma cell lines and tissues, suggesting that DKK1 promotes invasion and metastasis of hepatocellular carcinoma possibly through the non-canonical Wnt pathway. CONCLUSIONS: Collectively, our findings suggest that DKK1 could serve as a novel prognostic marker and therapeutic target for hepatocellular carcinoma.

Cellulase hydrolysis of rice straw and inactivation of endoglucanase in urea solution.

In order to optimize the cellulase (from Aspergillus glaucus) hydrolysis of pretreated rice straw, the effects of varying enzyme concentration, temperature, and pH were studied. The best experimental conditions found to degrade the pretreated rice straws were 24 h of incubation at 55 °C and pH 5.0, with an enzyme concentration of 48 mg/L. Urea is one of the important nitrogen sources used in fungi culture, but it is also a denaturant. The model of denaturation of endoglucanase (EG) in urea solutions was established. The denaturation was a slow, reversible reaction. Determination of microscopic rate constants showed k(+0) > k'(+0), indicating that EG was protected by the substrate to a certain extent during denaturation. Comparison with the results from fluorescence emission spectroscopy revealed that the inactivation of EG occurred before the marked conformational changes could be detected.

Purification and properties of endoglucanase from a sugar cane bagasse hydrolyzing strain, Aspergillus glaucus XC9.

An endoglucanase (EG) from Aspergillus glaucus XC9 grown on 0.3% sugar cane bagasse as a carbon source was purified from the culture filtrate using ammonium sulfate, an anion exchange DEAE Sepharose fast flow column, and a Sephadex G-100 column, with a purification fold of 21.5 and a recovery of 22.3%. The ideal time for EG production is on the fourth day at 30 degrees C using bagasse as a substrate. Results obtained indicate that the enzyme was a monomer protein, and the molecular weight was determined to be 31 kDa. The optimum pH and temperature of EG for the hydrolysis of carboxymethylcellulose sodium (CMC-Na) were pH 4.0 and 50 degrees C, respectively. EG was stable over the pH range from 3.5 to 7.5 and at temperatures below 55 degrees C. Kinetic behavior of EG in the hydrolysis of CMC-Na followed Michaelis-Menten kinetics with constant K(m) of 5.0 mg/mL at pH 4.0 and 50 degrees C. The enzyme activity was stimulated by Fe(2+) and Mn(2+) but inhibited by Cd(2+), Pb(2+), and Cu(2+). The EDC chemical modification suggested that at least one carboxyl group probably acted as a proton donor in the enzyme active site.

Cloning and tissue expressions of seven chitinase family genes in Litopenaeus vannamei.

GH18 chitinase is a multi-gene family. The family plays important physiological roles in Crustacea, e.g. ecdysis and defense against pathogen. However, data about GH18 family are rather limited in Crustacea. In the study, different cloning strategies were adopted to clone chitinase genes of Litopenaeus vannamei, which is the most widely cultured shrimp. Seven chitinase family members were identified. Analysis of domain architectures showed the repeated CBM18 modules and catalytic domain of enzymatically inactive chitolectin in Crustacea for the first time. Comparing to the three known groups of crustacean chitinase, four of the seven members are located on new evolutionary clades thus enriched the chitinase family of Crustacea. Tissue expression profiles were investigated in eight tissues. Expression of CHT5 and CHID1 were both detected in the hemocyte by which the innate immunity activity was carried out. The domain architectures, evolutionary relationships and tissue expression patterns all provide reasonable explanation for the existence of multiple genes in crustacean chitinase family.

Decreased expression of inhibitor of growth 4 correlated with poor prognosis of hepatocellular carcinoma.

OBJECTIVE: Inhibitor of growth 4 (ING4) is a candidate tumor suppressor that plays an important role in tumor growth and angiogenesis. Here, we examined the expression of ING4 in hepatocellular carcinoma (HCC) tissues and analyzed its correlation with the progression of HCC. METHODS: Specimens from 136 HCC patients were determined immunohistochemically for ING4 expression. The correlation of ING4 levels with clinicopathologic variables, prognosis, and metastatic potential was analyzed. Among the 136 cases, 36 paired HCC and paracarcinomatous liver tissue specimens were analyzed for ING4 expression levels by real-time quantitative reverse transcription-PCR and Western blotting. MVD was determined by CD34 immunostaining to test whether it correlated with ING4 protein expression level. RESULTS: The ING4 mRNA and protein levels were significantly lower in HCC than paracarcinomatous liver tissue from both real-time quantitative reverse transcription-PCR and Western blotting (P = 0.039 and 0.012, respectively). Importantly, the ING4 protein level correlated with the Edmondson-Steiner grade (P = 0.035), vein invasion (P = 0.015), and microvessel density (P = 0.005). Survival and metastasis analysis indicated that HCC patients with lower ING4 expression had poorer overall survival and disease-free survival than those with high expression (P = 0.0001 and 0.0065; respectively). Multivariable Cox regression analysis revealed that the ING4 expression level was an independent factor for prognosis (hazard risk, 9.63; P = 0.001). CONCLUSIONS: ING4 expression is down-regulated in HCC tissues. ING4 expression level correlates with prognosis and metastatic potential, which suggests that ING4 is a candidate prognostic marker of HCC.

Endocan expression correlated with poor survival in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death in China. We aimed to first present the expression of endocan in HCC tissue and its correlation with the clinicopathological features and overall survival of patients with HCC after curative hepatectomy. Immunohistochemical detection of endocan, CD34, and vascular endothelial growth factor (VEGF) were performed on samples from 100 patients with HCC. Endocan protein was expressed in endothelium of HCC tissue in all specimens, but was not expressed in endothelium of pericarcinomatous liver tissue and normal liver tissue. Microvessel density (MVD) denoted by endocan (endocan-MVD) in HCC was correlated with microscopic venous invasion and VEGF expression (P < 0.05). Survival analysis showed that overall survival of patients was inversely associated with endocan-MVD (P < 0.01). Multivariate analysis showed that endocan-MVD was an independent prognostic marker for overall survival of HCC (P < 0.01). In conclusion, endocan-MVD was a significant factor to predict the prognosis of HCC patients after curative hepatectomy.

[Down-regulated expression of UNC5b related to hepatocellular carcinoma angiogenesis].

OBJECTIVE: To investigate the relationship between UNC5b gene expression and angiogenesis of hepatocellular carcinoma (HCC). METHODS: In situ hybridization was performed to detect the expression of UNC5b mRNA in HCC samples, paracarcinomatous liver tissues samples and normal liver samples. The relationship between UNC5b mRNA expression and the HCC clinicopathological features were also analyzed. Human umbilical artery endothelial cells were isolated and stimulated with HCC tissues homogenate, vascular endothelial growth factor and basic fibroblast growth factor. Then RT-PCR was employed to detect the expression of UNC5b mRNA in normal HUAEC as well as activated HUAEC. RESULTS: In situ hybridization results showed that UNC5b mRNA expression was detected majorly in endothelial cells of all normal liver tissues, and partial PCLTs but was weak or even undetectable in endothelial cells of the corresponding HCC tissues. The expression levels of UNC5b gene in PCLTs were significantly correlated with capsular formation of HCC. Furthermore, RT-PCR results showed that the expression levels of UNC5b mRNA in activated HUAEC were significantly higher than those in normal HUAEC. CONCLUSIONS: Down-regulation of UNC5b gene expression is related to angiogenesis of HCC, which may be associated with the progression of HCC.

[Increased expression of Abi1 in hepatocellular carcinoma and its correlation with poor prognosis of hepatocellular carcinoma].

OBJECTIVE: To investigate the expression of Abi1 in hepatocellular carcinoma (HCC) and the correlation between its expression level and clinical pathological characteristics as well as prognosis of HCC. METHODS: Abi1 expression was determined at both mRNA and protein levels in 40 HCC tissues and their corresponding para carcinomatous liver tissues by RT-PCR and immunohistochemistry. The correlations between Abi1 expression levels and pathological characteristics as well as prognosis were also analyzed. RESULTS: The expression level of Abi1 mRNA in HCC tissues were significantly higher than those in corresponding para carcinomatous liver tissue (P < 0.05), and the expression level of Abi1 mRNA in nodular HCC tissues were also significantly higher than those in solitary large HCC tissues. Immunohistochemistry results showed that Abi1 protein located in cytoplasm of HCC cells and the expression level of Abi1 protein were significant positive correlated with the number of HCC, capsular formation, venous invasion and Edmondson-Steiner grade (P < 0.05). Combined with follow-up data, the results also showed that HCC patients with high Abi1 protein expression had a higher risk of invasion/metastasis and a shorter survival than those with low Abi1 protein expression (P < 0.05). CONCLUSIONS: Expression level of Abi1 is up-regulated in HCC tissues compared with corresponding para carcinomatous liver tissue and the expression level of Abi1 is significantly correlated with the number of tumor, capsular formation, venous invasion, Edmondson-Steiner grade and prognosis of HCC.