Antitumor Effect of By-1 from Spent Broth from Submerged Cultures of Stout Camphor Medicinal Mushroom, Taiwanofungus camphoratus (Higher Basidiomycetes), on A549 Adenocarcinoma Cells.

By-1 was obtained from spent broth from submerged cultures of Taiwanofungus camphoratus. This report evaluates the effects of By-1 on plate clone formation, wound healing, cell cycle, activated caspase-3 expression, and ROS release in A549 lung cancer cells. The result of plate clone formation assay revealed that By-1 could dramatically inhibit the viability of A549 cells in vitro. The inhibitory effect of By-1 on cell migration was tested using a wound healing assay. Proliferation rates of A549 cells were significantly inhibited following exposure to By-1 (12.5, 50, and 80 µg/mL). Flow cytometry revealed that the extracts increased, in a concentration-dependent manner, the number of cells in the G0/G1 phases of the cell cycle. The results of the caspase-3 experiment suggested that By-1 could induce A549 cells apoptosis, and this apoptosis was related to the release of reactive oxygen species by the A549 cells. All these results indicate that By-1 has potential in anti-lung cancer drug development.

Expression and prognostic significance of MAP4K4 in lung adenocarcinoma.

Accumulating evidence indicates that mitogen-activated protein 4 kinase 4 (MAP4K4) is frequently overexpressed in many types of human cancers, and plays important roles in transformation, invasiveness, adhesion, and cell migration. The aim of the present study was to explore the expression and prognostic significance of MAP4K4 in lung adenocarcinoma. The results of real-time quantitative PCR and Western blotting analysis revealed an enhanced expression of MAP4K4 in lung adenocarcinomas relative to adjacent non-tumorous lung tissues at both transcriptional and translational levels. Immunohistochemistry showed that 130 of 309 (42%) lung adenocarcinomas had high expression of MAP4K4. MAP4K4 overexpression was significantly correlated with histological grade (p=0.027), pT status (p=0.048), pN status (p=0.006), and pleural invasion (p=0.024). Patients with high MAP4K4 expression had a shorter overall survival compared with those with low MAP4K4 expression, regardless of histological grade, pT status, pN status, or pleural invasion status. Multivariate analysis identified MAP4K4 as an independent prognostic factor for lung adenocarcinoma. In conclusion, our results demonstrate that elevated MAP4K4 expression is closely associated with lung adenocarcinoma progression and has an independent prognostic value in predicting overall survival for patients with lung adenocarcinoma.

Expression and prognostic significance of centromere protein A in human lung adenocarcinoma.

BACKGROUND: Centromere protein A (CENP-A), one of the fundamental components of the human active kinetochore, is frequently upregulated in many cancers and plays important roles in cell cycle regulation, cell survival, and genetic stability. The aim of the present study was to explore the expression and prognostic significance of CENP-A in lung adenocarcinoma. EXPERIMENTAL DESIGN: The expression of CENP-A was detected in 20 fresh human lung adenocarcinoma specimens and corresponding non-tumorous lung tissues by real-time polymerase chain reaction (RT-PCR) and Western blotting analysis. Using immunohistochemistry, we analyzed CENP-A protein expression in additional 309 lung adenocarcinomas. The clinicopathological and prognostic significance of CENP-A expression was analyzed. RESULTS: RT-PCR and Western blotting analysis revealed an enhanced expression of CENP-A in lung adenocarcinomas relative to adjacent non-tumorous lung tissues at both transcriptional and translational levels. Immunohistochemistry showed that 146 of 309 lung adenocarcinomas (47.3%) had high expression of CENP-A. CENP-A overexpression was significantly correlated with pathological grade (P=0.009), pT status (P=0.017), pN status (P=0.002), pleural invasion (P=0.013), high Ki-67 expression (P=0.003), and P53 positivity (P=0.001). Patients with high CENP-A expression had shorter overall survival time compared with those with low CENP-A expression. Multivariate analysis identified CENP-A as an independent prognostic factor for lung adenocarcinoma. CONCLUSION: Our results demonstrate that elevated CENP-A expression is closely associated with lung adenocarcinoma progression and has an independent prognostic value in predicting overall survival for patients with lung adenocarcinoma.

Clinicopathologic significance and function of S-phase kinase-associated protein 2 overexpression in hepatocellular carcinoma.

The F-box protein S-phase kinase-associated protein 2 is frequently overexpressed in human cancers. Herein, we aimed to investigate the expression pattern, clinical significance, and biological function of S-phase kinase-associated protein 2 in hepatocellular carcinoma. Analysis by reverse transcriptase polymerase chain reaction, Western blot, and immunohistochemistry revealed that S-phase kinase-associated protein 2 was aberrantly overexpressed in hepatocellular carcinomas relative to adjacent nontumor liver tissues. This overexpression was significantly associated with advanced tumor stage, increased histologic grade, vascular invasion, and intrahepatic metastasis, as well as worse overall survival and higher early recurrence rate. Knockdown of the endogenous S-phase kinase-associated protein 2 expression in 1 hepatocellular carcinoma cell line, Huh7, by RNA interference reduced cell proliferation, blocked the cell cycle at G1 phase, and increased apoptosis. S-phase kinase-associated protein 2 silencing resulted in a deregulation of multiple cell-cycle regulatory proteins in Huh7 cells, as detected by quantitative real-time polymerase chain reaction arrays. Furthermore, high S-phase kinase-associated protein 2 immunoreactivity was found to be significantly correlated with reduced expression of P27, P21, and cell-cycle checkpoint kinase 2, as well as with increased expression of transcription factors Dp-1, cyclin D2, and cyclin D1 in hepatocellular carcinoma tissues. These data demonstrate that S-phase kinase-associated protein 2 expression is closely linked to tumor progression and represents an independent predictor of poor prognosis in hepatocellular carcinoma. S-phase kinase-associated protein 2 is involved in hepatocellular carcinoma cell proliferation through regulating numerous genes involved in cell-cycle progression, thereby providing a potential therapeutic target for this malignancy.

Expression and clinicopathologic significance of glypican 3 in hepatocellular carcinoma.

Glypican 3 (GPC3) is a glycosylphosphatidylinositol-anchored membrane protein and plays an important role in regulation of cell growth, differentiation, and migration. The aims of this study were to investigate the expression of GPC3 in human liver, biliary tract, and pancreatic tumors and to evaluate its diagnostic role in differentiating hepatocellular carcinoma (HCC) from other hepatic mimickers. Immunohistochemistry was performed on a large collection of surgically resected samples from 941 primary liver tumors, 50 metastatic adenocarcinomas, and 30 normal livers as well as primary adenocarcinomas of the pancreas (n = 17), gallbladder (n = 30), and extrahepatic bile duct (n = 20). The relationship of GPC3 expression and clinicopathologic features in patients with HCC was determined. We found that 516 (52%) of the 991 liver neoplastic tissue samples demonstrated positive staining for GPC3. A high incidence of GPC3 expression (492/757; 65%) was observed in HCC, whereas intrahepatic cholangiocarcinomas, adenocarcinomas, and benign liver lesions displayed rare positive cases. There were significant correlations between GPC3 expression and clinicopathologic characteristics, including histologic grade (P < .001), intrahepatic metastasis (P = .007), and positive serum hepatitis B surface antigen (P = .042), in patients with HCC. In conclusion, our results confirm the high expression of GPC3 in HCC and suggest its potential diagnostic value as a clinical marker for this disease.

ShRNA-targeted MAP4K4 inhibits hepatocellular carcinoma growth.

PURPOSE: Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is overexpressed in many types of cancer. Herein, we aimed to investigate its expression pattern, clinical significance, and biological function in hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: MAP4K4 expression was examined in 20 fresh HCCs and corresponding nontumor liver tissues. Immunohistochemistry for MAP4K4 was performed on additional 400 HCCs, of which 305 (76%) were positive for hepatitis B surface antigens. The clinical significance of MAP4K4 expression was analyzed. MAP4K4 downregulation was performed in HCC cell lines HepG2 and Hep3B with high abundance of MAP4K4, and the effects of MAP4K4 silencing on cell proliferation in vitro and tumor growth in vivo were evaluated. Quantitative real-time PCR arrays were employed to identify the MAP4K4-regulated signaling pathways. RESULTS: MAP4K4 was aberrantly overexpressed in HCCs relative to adjacent nontumor liver tissues. This overexpression was significantly associated with larger tumor size, increased histologic grade, advanced tumor stage, and intrahepatic metastasis, as well as worse overall survival and higher early recurrence rate. Knockdown of the MAP4K4 expression reduced cell proliferation, blocked cell cycle at S phase, and increased apoptosis. The antitumor effects of MAP4K4 silencing were also observed in vivo, manifested as retarded tumor xenograft growth. Furthermore, multiple tumor progression-related signaling pathways including JNK, NFκB, and toll-like receptors were repressed by MAP4K4 downregulation. CONCLUSIONS: MAP4K4 overexpression is an independent predictor of poor prognosis of HCC patients, and inhibition of its expression might be of therapeutic significance.

Apigenin inhibits hepatoma cell growth through alteration of gene expression patterns.

Apigenin, a common plant flavonoid, has been shown to possess anti-tumor properties; however, the underlying molecular mechanisms are still not completely understood. In the present study, we investigated the effects of apigenin on human hepatoma Huh7 cell proliferation, cell cycle distribution, apoptosis, and colony formation in vitro, as well as on the tumorigenicity of Huh7 cells in vivo. To get more insight into the mechanism of apigenin action, we performed genome-wide expression profiling of apigenin-treated Huh7 cells using cDNA microarrays (Agilent Whole Human Genome Oligo Microarray) that contain 41,000 genes. Ten of the most differentially expressed genes (≧5-fold changes) were selected for further evaluation by quantitative RT-PCR (qPCR) and Western blot analyses. Notably, apigenin (5-20 µg/ml) remarkably inhibited Huh7 cell proliferation and colony formation as compared to the vehicle control, which was in a dose-dependent manner. Accompanying with the decreased growth, apigenin-treated cells showed a cell cycle arrest at G2/M phase and an increased rate of apoptosis. Moreover, the xenografts derived from Huh7 cells were significantly (p<0.05) retarded by the delivery of apigenin (50 µg/mouse/day) relative to the control counterparts. Gene expression profile analysis revealed that 1336 genes were up-regulated and 428 genes were down-regulated by apigenin. The down-regulation of interleukin-4 receptor and ubiquitin specific protease 18 and the up-regulation of SLC27A3 and chemokine (C-C motif) receptor 2 were further confirmed by the qPCR and Western blot results. In conclusion, apigenin exhibits inhibitory effects on hepatoma cell growth, which is likely mediated through alteration of gene expression profiles.

Analysis of Keshan disease investigation result in Yunnan province in 2008 / 中国地方病学杂志

Objective In order to master the current situation of Keshan disease in Yunnan province and to provide scientific basis for Keshan disease control and prevention. Methods Eighteen villages were selected as the investigation sites in 6 counties across all the Keshan disease wards in Yunnan province,where the residents were investigated. Then,the villages census data was collected,clinical examination aiming mainly on cardiovascular system was carried out,including electrocardiography and X-ray to the suspected patients. Correct diagnose of Keshan disease was made by the Diagnostic Standard of Keshan Disease(GB 17021-1997). At the same time,10 food samples and 10 hair samples for detecting selenium content in every investigation site. Results There were 9818 residents investigated in the 18 investigation sites in 6 counties,and 34 eases of Keshan disease were found,the total incidence rate was 0.35%(34/9818). Among the 34 Keshan disease eases,32 cases were latent Keshan disease,the incidence rate was 0.33%(32/9818); 2 cases were chronic Keshan disease,the incidence rate was 0.02%(2/9818). There was no any acute and sub acute cases be found. Most Keshan disease cases aged from 5 to 14,67.65% (23/34). Abnormal ECG rate was 6.90% (677/9818). Among 56 X-ray films,47 cases had a cardiothoracic ratio less than or equal to 0.50,83.93%(47/56),5 cases from 0.51 to 0.55,8.93%(5/56),4 cases from 0.56 to 0.60,7.14%(4/56). Selenium content was detected in 180 food samples and 180 hair samples. The average food selenium content (mg/kg) was 0.013±0.010,the lowest content in Yongsheng county (0.006± 0.001),the highest content in Tonghai county(0.027±0.009). The average hair selenium eontentwas(0.252± 0.078)mg/kg,with the lowest(0.145±0.043)mg/kg in Yoagsbeng county,the highest (0.297±0.062)mg/kg in Tonghai county. Conclusions The detected ratio of Keshan disease is low in Yunnan province. Most of Keshan disease patients age from 5 to 14. It was presented that the Keshan disease infectious agents were still strong and active. The foodstuffs and hair Selenium content is low in food and hair sample,and varies in different investigation site. It is necessary to supply selenium for prevent Keshan disease in the severe areas.

Analysis of Keshan Disease surveillance data in Yunnan Province in 2007 / 中国地方病学杂志

Objective To study the current incidence of Keshan disease in Yunnan Province,and provide scientific basis for Keshan disease(KD) prevention and control. Methods Based on the Scheme of KD Surveillance, 16 villages in 11 counties were chosen as surveillance sites by the historical data. An survey was made to the residents in the 16 surveillance sites by filling in the questionnaire, inquiry medical history, clinical examination, electrocardiogram and 2 meters post-anterior chest X-ray for suspected cases. KD cases were diagnosed according to the Diagnostic Criteria for Keshan Disease(GB 17021-1997). The prevalence data of KD in the whole province were collected from the KD case report in 2007 and the trace surveys. Results There were 6877 residents in 16 surveillance sites of 11 surveillance counties and totally 39 KD cases were diagnosed with a detection ratio of 0.57% (39/6877). The detection ratio of latent and chronic KD were 0.41%(28/6877) and 0.16%(11/6877), respectively and no acute or subacute cases were found. The cases aged 5 to 14 years old accounting for 66.67% (26/39). Electrocardiogram examination of 6877 residents were made and 5.25% (361/6877) abnormal electrocardiograms were detected in the 16 surveillance sites. Fifty-five people were checked by chest X-ray and there were 31 cases with heart-chest ratio ≤0.50, 16 cases with heart-chest ratio from 0.51 to 0.55 and 8 cases with heart-chest ratio from 0.56 to 0.60. The prevalence rate and incidence rate of chronic KD were 4.24 per 100 000 and 0.50 per 100 000 in Yunnan. No acute or subacute cases were found and the latent cases were listed. The prevalence rate and incidence rate were 7.76 per 100 000 and 1.18 per 100 000 in the 16 surveillance sites. Conclusions The incidence of KD is low incidence in Yunnan Province. Higher ineidence of chronic KD was detected in the some areas and the corresponding control measures need to be adopted.

Clinical features of unexpected sudden death clustered in 7 families in Yunnan Province / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the clinical features of unexpected sudden death (SUD) clustered in families in Yunnan province.</p><p><b>METHODS</b>This retrospective study analyzed the clinical features of SUD occurred between July to September 2005 in 7 families in Yunnan province.</p><p><b>RESULTS</b>All 16 SUD patients shared common clinical features such as fatigue and repeated syncope and one group of SUD patients (n = 8 from 4 families) presented with the gastric intestinal tract manifestations including nausea, vomiting, abdominal pain and diarrhea with suspected dietary history and abnormal laboratory enzyme findings (GOT/GPT, CK/CKMB, LDH/LDH1 etc.). In SUD patients without gastric intestinal tract manifestations (n = 8 from 3 families), there were no clear symptoms before death and repeated ventricular tachycardia and ventricular fibrillation were recorded in one survivor. There was no clear evidence for the involvements of hereditary and infectious factors for observed SUD.</p><p><b>CONCLUSION</b>The reason for the unexpected sudden death clustered in 7 families in Yunnan remains unclear. Repeated syncope and fatigue served as the common clinical features in the presence or absence of gastric intestinal tract manifestations in all SUD cases. Further studies are needed to clarify the pathology and detailed clinical manifestations of SUD occurred in this area.</p>

Investigation and analysis on the incidence of "Yunnan unknown-cause sudden cardiac death" during / 中华流行病学杂志

<p><b>OBJECTIVE</b>To understand the epidemiologic and clinical characteristics of the "Yunnan unknown-cause sudden cardiac death (YUSCD)".</p><p><b>METHODS</b>Cases of YUSCD occurred in 2002-2004 were investigated with cross-sectional study. Clinical manifestation was observed and analyzed.</p><p><b>RESULTS</b>The YUSCD areas were mountainous and most of the YUSCD cases appeared between June and August. Most of the YUSCD cases were young farmers and showed family/village clustering nature. Most of the patients died quickly with only few recovered. The overall incidence of YUSCD was 1.83% with mortality as 1.51%. The fatality rate of YUSCD appeared to be 78.1%.</p><p><b>CONCLUSION</b>The YUSCD cases had a clustering feature along with time and area. The clinical manifestation of YUSCD could not be specifically identified, making the clinical diagnosis and treatment difficult when practicing in the fields that called for further studies on epidemiology, clinical work and etiology.</p>

Morphologic features of sudden cardiac death in Yunnan province, with emphasis on myocarditis / 中华病理学杂志

<p><b>OBJECTIVES</b>To study the pathologic feature of sudden cardiac death in Yunnan province and to investigate the role of myocarditis.</p><p><b>METHODS</b>During the period from 1991 to 2006, there were 29 cases of sudden cardiac death with autopsy performed. Fourteen of these cases were diagnosed to have myocarditis based on Dallas criteria and World Heart Federation's consensus. The clinical and pathologic findings were reviewed. The cardiac conduction system was examined in details by serial sectioning in 3 cases.</p><p><b>RESULTS</b>Fourteen cases suffered with myocarditis, which accounted for 48% of all cases of sudden cardiac death studied. The age of the deceased ranged from 8 to 68 years (mean = 30 years), with male-to-female ratio equaled to 9:5. Lymphocytic myocarditis and neutrophil myocarditis were the two major types, affecting 11 and 3 cases, respectively. The inflammatory infiltrates were often patchy rather than diffuse. The inflammatory foci were detected only in 8% to 42% (average = 20%) of the paraffin sections of the heart tissue. These lesions were usually located in the lateral wall of left ventricle and occasionally in interventricular septum and right ventricular wall. Myocardial injury was mild in most cases while patchy myocytolysis or coagulation necrosis was observed only in a few cases. Most of the lesions were relatively new and histologic evidence of myocardial repairing sometimes coexisted. Pericarditis and subacute endocarditis were also identified in 4 and 1 cases, respectively. Atrioventricular node was involved by myocarditis in 1 of the 3 cases examined for cardiac conduction system. Two cases showed gross evidence of cardiac dilatation (either left ventricle or biventricular). Respiratory tract and pulmonary infection was present in 5 cases.</p><p><b>CONCLUSIONS</b>Myocarditis represents one of the major pathologic changes of sudden cardiac death occurring in Yunnan province. The inflammation is usually focal. Further studies are required for delineation of possible etiologies which may include virus, bacteria or exogenous toxin.</p>

Retrospective study on 116 unexpected sudden cardiac deaths in Yunnan, China / 中华流行病学杂志

<p><b>OBJECTIVE</b>To identify the epidemiological and clinical features of unexpected sudden cardiac deaths (SUD) in Yunnan.</p><p><b>METHODS</b>Choosing the old SUD cases from Xiangyun, Heqing, Nanjian and Dayao counties and using the standardized verbal autopsy Form, we interviewed the family members of the cases, witnesses and doctors as well as reviewing their medical files to get relative information.</p><p><b>RESULTS</b>We identified 116 SUDs in 21 villages from 1984 to 2004. The village-specific annually standardized incidence rates were ranged from 0.2/1000 to 8.9/1000 (median = 0.8/1000). 66% and 29% of the SUDs occurred in July and August respectively. The incidence rates of SUD were higher (1.6/1000, chi(2) = 16, P < 0.01) in 10 - 39 year-olds, and higher in females than in males (RR = 1.6, 95% CI: 1.1 - 2.3). Seventy percent of SUD occurred in families having clustering nature and 60% of the additional cases in the family were occurred within 24 hours (median = 20 hours) after the first SUD identified in the family. SUD occurred in 23 families followed the first affected family in a village during the same season. In these 23 families, 61% of the first SUD occurred within 8 days after the first SUD in the first affected family. 68% and 66% of the SUDs did not have any complaints or signs during the last 3 weeks or from 3 weeks to 2 days prior to the onset of the disease. 63% of the SUDs had cardiac symptoms within the last 2 days prior to the onset with major symptoms as dizziness, nausea, faintness, unconsciousness, weakness and palpitation. The median duration from acute onset to death was 2 hours.</p><p><b>CONCLUSIONS</b>The extreme time-space clustering of SUD in families and in villages suggested that the risk factors occurred in specific time and location. Familial clustered SUD cases had common exposure pattern. Sudden onset of acute cardiac symptoms often followed by sudden death. Epidemiological study on new cases was necessary to identify risk factors and to develop hypothesis for causation. In July 2005, we instituted a special SUD surveillance system for all the affected counties together with 10 counties which had no reported cases.</p>