Community ecological succession of endophytic fungi associates with medicinal compound accumulation in Sophora alopecuroides.

Endophytic fungi of medicinal plants are symbiotic with the host and play an important role in determining metabolites. To understand the relationship between the accumulation of Sophora alopecuroides' medicinal bioactive compounds and the ecological succession of endophytic fungi, here we collected samples from S. alopecuroides at four developmental stages (adult, flowering, podding, and mature) and different organs (roots, stems, leaves, and seeds) at the mature stage. We then used high-performance liquid chromatography-mass spectrometry and high-throughput sequencing on the internal transcribed spacer region to identify the medicinal compounds and endophytic fungal communities in each sample. The endophytic fungal community characteristics and accumulation of medicinally bioactive compounds of S. alopecuroides varied with the host's developmental stages and organs, with the highest total alkaloids content of 111.9 mg/g at the mature stage. Membership analysis and network connection analysis showed a total of 15 core endophytic fungi in different developmental stages and 16 core endophytic fungi in different organs at the mature stage. The unclassified Ascomycota, Aspergillus, and Alternaria were significantly and positively correlated with the medicinal compounds of S. alopecuroides at the mature stage (r > 0.6 or r < -0.6; P < 0.05). In this study, we identified key endophytic fungal resources that affect the content of medicinally bioactive compounds in S. alopecuroides. This discovery could lay the foundation for enhancing the yield of medicinally bioactive compounds in S. alopecuroides and the development and application of functional endophytic fungi.IMPORTANCESophora alopecuroides is a traditional Chinese herbal medicine. The major medicinal chemicals are considered to be quinolizidine alkaloids. Quinolizidine alkaloids have been widely used for the treatment of tumors, dysentery, and enteritis. Previous studies have found that endophytic fungi in S. alopecuroides can promote the accumulation of host quinolizidine alkaloids. However, the relationship between the accumulation of S. alopecuroides' medicinal bioactive compounds and the ecological succession of endophytic fungi remains unclear. In this study, we screened the key endophytic fungal resources affecting the content of medicinally bioactive compounds and laid the foundation for subsequent research on the mechanism by which endophytic fungi promote the accumulation of medicinally bioactive compounds in S. alopecuroides.

Remodeling and spacing factor 1 overexpression is associated with poor prognosis in renal cell carcinoma.

The present study aimed to assess the expression and prognostic significance of remodeling and spacing factor 1 (RSF1; HBXAP) in renal cell carcinoma (RCC). RSF1 expression was analyzed using immunohistochemistry on tissue samples from a consecutive series of 137 patients with RCC who underwent tumor resection between November 2000 and March 2004. The associations between RSF1 expression, clinicopathological factors and patient survival were investigated. Immunohistochemistry revealed that RSF1 was highly expressed in 43.1% (59/137) of the RCC samples. RSF1 expression levels were associated with the T stage of the Tumor-Node-Metastasis grading system. Kaplan-Meier survival analysis indicated that high RSF1 expression in RCC was significantly associated with a poor prognosis. Multivariate analysis revealed that RSF1 expression is an independent prognostic parameter for the duration of overall survival of patients with RCC. The results demonstrated that a high expression level of RSF1 in RCC is associated with advanced tumor stages and a poor prognosis. To the best of our knowledge, the present study provides novel evidence of the biological significance of RSF1 expression in RCC.

Overexpression of Rsf-1 correlates with poor survival and promotes invasion in non-small cell lung cancer.

Rsf-1 (HBXAP) was recently reported to play roles in tumorigenesis and tumor progression. There have been many reports referred to Rsf-1 overexpression in various cancers and associated with the malignant behavior of cancer cells. However, the molecular mechanism of Rsf-1 in non-small cell lung cancer aggressiveness remains ambiguous. In the present study, we found that there was a significant association between Rsf-1 overexpression and poor overall survival (p = 0.028) in lung cancer. Furthermore, knockdown of Rsf-1 expression in H1299 and H460 cells with high endogenous Rsf-1 expression inhibited cell migration and invasion and downregulated MMP2 expression and nuclear levels of NF-κB. NF-κB inhibitor could also block the effect of Rsf-1 in regulation of MMP2 expression. Further experiments demonstrated that Rsf-1 depletion restrained NF-κB reporter luciferase activity and downregulated bcl-2 and p-IκB protein level. In conclusion, we demonstrated that Rsf-1 was overexpressed in lung cancer and associated with poor survival. Rsf-1 regulated cell invasion through MMP2 and NF-κB pathway.

Secretomes are a potential source of molecular targets for cancer therapies and indicate that APOE is a candidate biomarker for lung adenocarcinoma metastasis.

Identifying patients at high risk of metastasis is a major challenge in lung adenocarcinoma (ADC) therapy, therefore discovery of noninvasive biomarkers and therapeutic targets is urgent. We found significant differences between the secretomes of differentially expressed proteins in lung ADC cell lines, clinical tissue samples and serum plasma samples with high and low metastatic potential. In particular, Apolipoprotein E (APOE) levels were three-times greater in cells with lymph node metastases (LNM) than those without. Our study indicates that APOE is a potential indicator of metastatic lung ADC and that secretomes may offer a valuable resource for biomarkers of lung ADC with LNM.

C-Myc participates in ß-catenin-mediated drug resistance in A549/DDP lung adenocarcinoma cells.

The aim of this study was to investigate c-Myc and ß-catenin-mediated drug resistance in A549/DDP lung adenocarcinoma cells. Cisplatin sensitivity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) toxicity assay. ß-Catenin and c-Myc protein expression following cisplatin treatment were determined using western blotting and immunofluorescence. Flow cytometry was performed to detect cell cycle and apoptosis in A549, A549/DDP, and c-Myc small interfering RNA (siRNA)-transfected A549/DDP cells before and after treatment with different doses of cisplatin. The median inhibitory concentration (IC50 ) in cisplatin-treated A549 and A549/DDP cells was 5.769 ± 0.24 µmol/L and 28.373 ± 0.96 µmol/L, respectively; the cisplatin resistance of A549 cells was about five times that of A549/DDP cells. Endogenous ß-catenin and c-Myc expression in A549/DDP cells were higher than that in A549 cells, and were upregulated in A549/DDP cells (p < 0.05) and downregulated in A549 cells after 48 h cisplatin treatment (p < 0.05). ß-catenin localization transferred from membrane/cytoplasmic/nuclear to cytoplasmic/nuclear, and c-Myc localization transferred from cytoplasmic/nuclear to nuclear in both cell lines following cisplatin treatment. The rate of apoptosis increased in a dose-dependent manner with cisplatin. After 48-h transfection with c-myc siRNA, A549/DDP cells were blocked in the S phase, and G0/G1-phase cells increased. Simultaneously, the apoptotic rate was increased (p < 0.05) and the IC50 decreased significantly (p < 0.05). C-myc, the downstream target gene of ß-catenin, plays an important role in regulating cisplatin resistance in A549/DDP cells. C-Myc siRNA improved the sensitivity of A549/DDP cells to cisplatin.

Inhibition of cytoplasmic GSK-3ß increases cisplatin resistance through activation of Wnt/ß-catenin signaling in A549/DDP cells.

Cisplatin-based chemotherapy is recommended as the first-line therapy for advanced non-small cell lung cancer (NSCLC). However, acquired cisplatin resistance is ubiquitous in patients with NSCLC, but the molecular mechanism of such resistance remains ambiguous. The present study sought to examine the role of the Wnt/ß-catenin signaling pathway in cisplatin resistance by assessing the phosphorylation and subcellular distribution of GSK-3ß in a human lung adenocarcinoma cell line, A549, and its cisplatin-resistant subline, A549/DDP. Total GSK-3ß, phosphorylated GSK-3ß(ser9) and phosphorylated GSK-3ß(tyr216) in cytoplasmic and nuclear fractions of A549/DDP and A549 cells were examined by western blot analysis. The regulation of cisplatin resistance, apoptosis, ß-catenin and survivin protein expression by inhibition of cytoplasmic GSK-3ß were determined by MTT assay, flow cytometry analysis, immunofluorescence technique and western blot analysis. In the present study, cytoplasmic levels of p-GSK-3ß(ser9) were significantly increased in A549/DDP cells as compared with A549 cells (P<0.01), and these levels were further increased by cisplatin treatment in A549/DDP cells (P<0.01). In contrast, cytoplasmic levels of p-GSK-3ß(ser9) were reduced in A549 cells after treatment with cisplatin (P<0.01). However, cytoplasmic levels of p-GSK-3ß(tyr216) were significantly decreased in A549/DDP cells as compared with A549 cells (P<0.01), and these levels were further decreased by cisplatin treatment in A549/DDP cells (P<0.01). Conversely, cytoplasmic levels of p-GSK-3ß(tyr216) were raised in A549 cells after treatment with cisplatin (P<0.01). Analysis of downstream effectors of the Wnt/ß-catenin signaling pathway revealed upregulation of ß-catenin and survivin expression in A549/DDP cells treated with cisplatin as compared to untreated cells. In A549 cells, cisplatin treatment decreased the expression of ß-catenin and survivin. Furthermore, phosphorylation of GSK-3ß at serine 9 by LiCl and transient interference of GSK-3ß by siRNA increased ß-catenin and survivin protein expression in A549/DDP cells. Low exogenous and endogenous cytoplasmic GSK-3ß expression enhanced the IC50 and inhibited apoptosis. In conclusion, activation of the Wnt/ß-catenin signaling pathway and upregulated survivin expression due to cytoplasmic GSK-3ß inhibition might lead to cisplatin resistance in NSCLC.

Expression of ezrin correlates with malignant phenotype of lung cancer, and in vitro knockdown of ezrin reverses the aggressive biological behavior of lung cancer cells.

Ezrin, one of the ezrin-radixin-moesin proteins, is involved in the formation of cell membrane processes such as lamellipodia and filopodia and acts as a membrane-cytoskeleton linker. Its aberrant expression correlates with development and progression of several human cancers. However, the expression of ezrin and its role in lung cancer are currently unknown. In this study, we performed ezrin small interfering RNA transfection in two lung cancer cell lines and examined the effects on malignant phenotypes in cancer cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, wound healing, and chamber transwell assays. Ezrin knockdown significantly reduced the proliferation, migration, and invasion of lung cancer cells in vitro. To address the possible mechanisms, we evaluated the expression of adhesion molecules E-cadherin and ß-catenin by Western blot and reverse transcriptase-polymerase chain reaction analyses. The results demonstrated that downregulation of ezrin reduced ß-catenin and increased E-cadherin at the protein level but had no effects on their mRNA levels, suggesting posttranscriptional regulation of these two adhesion molecules. Immunofluorescence assays revealed that ezrin knockdown restored membranous expression of E-cadherin and decreased cytoplasmic ß-catenin in lung cancer cells. In addition, ezrin expression was immunohistochemically evaluated on 135 normal and 183 lung cancer tissues. The expression of ezrin was significantly higher in cancer samples than paired autologous normal lung tissues. In normal bronchial epithelium, ezrin was mainly localized on the apical membrane, while in lung cancers and metastatic foci, ezrin was primarily distributed in cytoplasm. Among lung cancer tissues, expression of ezrin was higher in the invasive front of primary lesions and the highest in lymphatic metastasis. Statistical analysis demonstrated that ezrin expression correlated significantly with lymphatic metastasis and advanced TNM stage. Our data suggest that ezrin may play a crucial role in governing the biological behavior of lung cancer.

[Cloning of the promoter region of Leuciscus Merzbacheri beta-actin gene and detection of its function].

To utilize the gene resources of Leuciscus merzbacheri, a 2,398 bp (SZ21) DNA fragment including the 5'-flanking region and partial open reading frame of the beta-actin gene was obtained through PCR amplification. SZ21 includes a regulatory sequence which contains the 5'-proximal promoter, the first, the second and the third exons and the partial fourth exon sequence. The 5'-proximal promoter region is critical for transcription activity including the CAAT box, TATA box and CArG box. The analysis of putative transcription binding sites of the promoter by on-line software revealed the presence of the critical transcription binding sites (such as E-box, RU49, ZBPF, CEBP and CREB). CMV promoter for eukaryote vector pEGFP-N1-AFP III was destroyed by Aat II digestion. SZ21 regulatory sequence was inserted into the vector pEGFP-N1-AFP III (with destroyed CMV) that can express green fluorescence protein, and beta2 pEGFP-N1-AFP III recombination vector was constructed. Linearized beta2 pEGFP-N1-AFP III was transfected into BHK-21 cell through lipofectin. EGFP expression of the transgenic cell was observed by micro fluoroscope. The results indicated that the cloned Leuciscus merzbacheri beta-actin gene promoter SZ21 has the activity to switch on the EGFP expression in mammal cell, and has a con-tinued starting expression activity passing on from generation to generation in green fluorescence cell. In addition, the SZ21 target fragment was detected in the BHK-21 green fluorescence cell genomic DNA by PCR. This suggested that the SZ21 promoter of beta-actin gene has effective transcription activity and can promote the expression of foreign gene.

[Effects of comprehensive interventions in community on smoking, chronic bronchitis, and asthma in rural areas of Beijing].

OBJECTIVE: To study the effects of comprehensive interventions in community on smoking, chronic bronchitis, and asthma in rural areas of Beijing. METHODS: Twenty-three villages in rural areas of Beijing were randomly divided into interventional (13 villages) and control villages (10 villages) in 1992. Comprehensive interventions including education of former-smokers and improvement of living environment were carried out in the interventional villages, and none was done in the control villages. In April 2000, surveys on smoking, chronic bronchitis, and asthma were carried out among 34,436 participants aged 15 or more in the interventional and control villages. During the same period, knowledge on prevention from chronic obstructive pulmonary diseases (COPD), living environments, and smoking were assessed among 1658 high-risk individuals of COPD at baseline and following-up period. RESULTS: The scores of knowledge and improvement on living environments in the interventional villages were significantly higher than those in control villages (P < 0.001). The decrease rate of smoking and current smoking rate in the interventional villages were significantly higher than in the control villages (0.4% vs -0.8%, P < 0.001; 2.4% vs 1.3%, P < 0.001) in men, while not different in women (P > 0.05). Among never smokers at baseline, the accumulated incidence of smoking among people aged 15 to 24 from 1993 to 2000 was significantly lower in the interventional villages than in the control villages in men (18.9% vs 23.7%, P = 0.005) and in women (0% vs 0.7%, P = 0.005). Daily cigarettes smoked by smokers in the interventional villages were less than in control villages in both men (14.8 +/- 7.0 vs 17.2 +/- 8.2 cigs daily, P < 0.001) and women (12.8 +/- 6.9 vs 13.4 +/- 7.2 cigs daily, P = 0.088). The increase of prevalence of chronic bronchitis in the interventional villages was less than in the control villages (men: 0.9% vs 1.3%, P = 0.012; women: 0.1% vs 0.3%, P = 0.003). After the age factor is adjusted, odds ratio (OR) for accumulated incidence of chronic bronchitis from 1993 to 2000 in the interventional villages were 0.80 (95%CI: 0.60-1.07) for men, 0.76 (95%CI: 0.45-1.28) in women. The OR for asthma was not significant in both men and women. CONCLUSIONS: Comprehensive interventions in community may improve knowledge of COPD prevention and living environments, decrease the smoking rate, cigarettes smoked per day, and incidence of chronic bronchitis, but have no significant effects on asthma.