Triterpenoid saponins from Bupleurum marginatum and their anti-liver fibrotic activities.

A new triterpenoid saponin (1), along with five known compounds (2-6), was isolated from Bupleurum marginatum Wall. ex DC, of which compounds 2-4 were obtained for the first time from this plant. The structures were confirmed by the analysis of 1D, 2D NMR, and HR-ESIMS data, and comparison with previous spectral data. Anti-liver fibrotic activities of the isolates were determined as proliferation inhibition of LPS-induced activation of HSC-T6 in vitro.

Upregulation of nemo-like kinase is an independent prognostic factor in colorectal cancer.

AIM: To investigate the expression and oncogenic role of nemo-like kinase (NLK) in colorectal cancer. METHODS: Expression of NLK protein was assessed by immunohistochemistry in tissue specimens from 56 cases of normal colorectal mucosa, 51 cases of colorectal adenoma, and 712 cases of colorectal cancer. In addition, NLK expression was knocked down using a lentivirus carrying NLK small hairpin RNA in colorectal cancer cells. Cell viability methylthiazoletetrazolium assays, colony formation assays, flow cytometry cell cycle assays, Transwell migration assays, and gene expression assays were performed to explore its role on proliferation and migration of colorectal cancer. RESULTS: Expression of NLK protein progressively increased in tissues from the normal mucosa through adenoma to various stages of colorectal cancer. Overexpression of NLK protein was associated with advanced tumor-lymph node-metastasis stages, poor differentiation, lymph node and distant metastases, and a higher recurrence rate of colorectal cancer (P < 0.05). Multivariate analyses showed that NLK expression was an independent prognostic factor to predict overall survival (hazard ratio 2.57, 95% confidence interval: 1.66-3.98; P < 0.001) and disease-free survival (hazard ratio 1.96, 95% confidence interval: 1.40-2.74: P < 0.001) of colorectal cancer patients. Furthermore, knockdown of NLK expression in colorectal cancer cell lines reduced cell viability, colony formation, and migration, and arrested tumor cells at the G0/G1 phase of the cell cycle. At the gene level, knockdown of NLK expression inhibited matrix metalloproteinase-2 expression in colorectal cancer cells. CONCLUSION: NLK overexpression is an independent prognostic factor in colorectal cancer and knockdown of NLK expression inhibits colorectal cancer progression and metastasis.

Isolation and molecular characterization of 1-aminocyclopropane-1-carboxylic acid synthase genes in Hevea brasiliensis.

Ethylene is an important factor that stimulates Hevea brasiliensis to produce natural rubber. 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a rate-limiting enzyme in ethylene biosynthesis. However, knowledge of the ACS gene family of H. brasiliensis is limited. In this study, nine ACS-like genes were identified in H. brasiliensis. Sequence and phylogenetic analysis results confirmed that seven isozymes (HbACS1-7) of these nine ACS-like genes were similar to ACS isozymes with ACS activity in other plants. Expression analysis results showed that seven ACS genes were differentially expressed in roots, barks, flowers, and leaves of H. brasiliensis. However, no or low ACS gene expression was detected in the latex of H. brasiliensis. Moreover, seven genes were differentially up-regulated by ethylene treatment. These results provided relevant information to help determine the functions of the ACS gene in H. brasiliensis, particularly the functions in regulating ethylene stimulation of latex production.

Expression of ITGB1 predicts prognosis in colorectal cancer: a large prospective study based on tissue microarray.

BACKGROUND: ITGB1 is a heterodimeric cell-surface receptor involved in cell functions such as proliferation, migration, invasion and survival. The aim of this study was to assess ITGB1 expression in colorectal cancer and correlate it with clinicopathological features, as well as to evaluate its potential prognostic significance. MATERIALS AND METHODS: In this study, we examined the expression of ITGB1 using tissue microarrays containing analyzed specimens by immunohistochemistry. ITGB1 expression was further correlated with clinicopathological and prognostic data. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. A multivariate study with the Cox's proportional hazard model was used to evaluate the prognostic aspects. RESULTS: ITGB1 expression was present in 88.5% of the analyzed specimens. Significant differences in ITGB1 expression were found between normal mucosa and carcinomas (P<0.001). High ITGB1 expression was associated with poor prognosis, and it independently correlated with shortened overall survival and disease-free survival in colorectal cancer patients (P<0.001). More so, ITGB1 expression, bowel wall invasion, lymph node metastasis and distant metastasis were independent prognostic factors for overall survival. Additionally, significant differences in ITGB1 expression were observed in adenomas and tumors from patients with familial adenomatous polyposis compared to normal colon mucosa (P<0.05) CONCLUSION: The results of this study indicate that ITGB1 overexpression in colorectal tumors is associated with poor prognosis, as well as aggressive clinicopathological features. Therefore, ITGB1 expression could be used as potential prognostic predictor in colorectal cancer patients.

Inflammation-related factors predicting prognosis of gastric cancer.

Gastric cancer (GC), which is mainly induced by Helicobacter pylori (H. pylori) infection, is one of the leading causes of cancer-related death in the developing world. Active inflammation initiated by H. pylori infection and maintained by inherent immune disorders promotes carcinogenesis and postoperative recurrence. However, the presence with H. pylori in tumors has been linked to a better prognosis, possibly due to the induction of antitumor immunity. Tumor infiltrations of tumor-associated macrophages, myeloid-derived suppressor cells, neutrophils, Foxp3(+) regulatory T cells are correlated with poor prognosis. Tumor infiltrating CD8(+) cytotoxic T lymphocytes, dendritic cells, and CD45RO T cells are generally associated with good prognosis of GC, although some subsets of these immune cells have inverse prognosis prediction values. High ratios of Foxp3(+)/CD4(+) and Foxp3(+)/CD8(+) in tumors are associated with a poor prognosis; whereas high Th1/Th2 ratio in tumors predicts a good prognosis. High levels of interleukin (IL)-6, IL-10, IL-32, and chemokine C-C motif ligands (CCL)7 and CCL21 in circulation, high expression of CXC chemokine receptor 4, chemokine C-C motif receptor (CCR)3, CCR4, CCR5, CCR7, hypoxia-inducible factor-1α, signal transducer activator of transcription-3, cyclooxygenase-2, and orphan nuclear receptor 4A2 in tumors are associated with an unfavorable prognosis. Increased serum levels of matrix metalloproteinases (MMP)-3, MMP-7, and MMP-11 and increased levels of MMP-9, MMP-12, and MMP-21 in tumors are consistently associated with poor survival of GC. Further emphasis should be put on the integration of these biomarkers and validation in large cohorts for personalized prediction of GC postoperative prognosis.

Factors predicting occurrence and prognosis of hepatitis-B-virus-related hepatocellular carcinoma.

Primary liver cancer is an important cause of cancer death, and hepatocellular carcinoma (HCC) accounts for 70%-85% of total liver cancer worldwide. Chronic hepatitis B virus (HBV) infection contributes to > 75% of HCC cases. High serum viral load is the most reliable indicator of viral replication in predicting development of HCC. HBV genotype C is closely associated with HCC in cirrhotic patients aged > 50 years, whereas genotype B is associated with development of HCC in non-cirrhotic young patients and postoperative relapse of HCC. Different HBV subgenotypes have distinct patterns of mutations, which are clearly associated with increased risk of HCC. Mutations accumulate during chronic HBV infection and predict occurrence of HCC. Chronic inflammation leads to increased frequency of viral mutation via cellular cytidine deaminase induction. Mutations are negatively selected by host immunity, whereas some immuno-escaped HBV mutants are active in hepatocarcinogenesis. Inflammatory pathways contribute to the inflammation-necrosis-regeneration process, ultimately HCC. Their hallmark molecules can predict malignancy in HBV-infected subjects. Continuing inflammation is involved in hepatocarcinogenesis and closely related to recurrence and metastasis. HBV load, genotype C, viral mutations and expression of inflammatory molecules in HBV-related HCC tissues are significantly associated with poor prognosis. Imbalance between intratumoral CD8(+) T cells and regulatory T cells or Th1 and Th2 cytokines in peritumoral tissues can predict prognosis of HBV-related HCC. These factors are important for developing active prevention and surveillance of HBV-infected subjects who are more likely to develop HCC, or for tailoring suitable treatment to improve survival or postpone postoperative recurrence of HCC.

[Gene methylation in stool for the screening of colorectal cancer and pre-malignant lesions].

OBJECTIVE: To evaluate association between DNA methylation of MAL, CDKN2A, and MGMT in stool and development of colorectal cancer, and to evaluate the screening value of these biomarkers in colorectal cancer and pre-malignant lesions. METHODS: Morning stool specimens were collected from 69 patients with colorectal cancer, 24 with colon adenoma, 19 with hyperplastic polyps, and 26 healthy controls. DNA was extracted and treated with bisulfite. Methylation-specific PCR(MSP) was performed for methylation analysis of MAL, CDKN2A and MGMT in DNA samples. Associations between clinicopathological features and gene methylation were analyzed. The sensitivity of diagnosis by combining three methylation markers was compared with fecal occult blood test(FOBT). RESULTS: The methylation frequencies of MAL, CDKN2A and MGMT were 78.3%, 52.5% and 55.1% in colorectal cancer, 58.3%, 41.7% and 37.5% in colon adenomas, 26.3%, 15.8% and 10.5% in hyperplastic polyps, and 3.8%, 0 and 3.8% in healthy controls, respectively. Significant differences in three genes were found between colorectal cancer and hyperplastic polyp, colorectal cancer and healthy control, colon adenoma and hyperplastic polyp, colon adenoma and healthy control(all P<0.05). The diagnostic sensitivity by combining three methylation markers was 92.8% in colorectal cancer, 70.8% in colon adenomas, significantly higher than FOBT examination (29.0% in colorectal cancer and 25.0% in colon adenomas, all P<0.05). No significant associations existed between three genes methylation of the three genes and clinical characteristic including sex, age, tumor location, lymph node metastases and TNM stage (all P>0.05). CONCLUSION: DNA methylations levels of MAL, CDKN2A, and MGMT in stools are significantly higher in colorectal cancer and colon adenoma, which may serve as an noninvasive approach for the screening of colorectal cancer and pre-malignant lesions.

Molecular characterization of HbCDPK1, an ethephon-induced calcium-dependent protein kinase gene of Hevea brasiliensis.

Calcium-dependent protein kinases (CDPKs), as major primary Ca(2+) sensors, have been implicated in the regulation of stress and developmental signals in plants. In this study, a novel CDPK gene, designated HbCDPK1, was isolated from Hevea brasiliensis. The HbCDPK1 cDNA had 2,400 bp with an open reading frame of 1,671 bp encoding 556 amino acids, and the deduced HbCDPK1 protein contained four characteristic domains identified in CDPKs, showing a high level of sequence similarity to CDPKs from other plants. Expression analysis revealed more significant accumulation of the transcripts of HbCDPK1 in latex than in the leaves, bark, and roots in H. brasiliensis. In addition, transcription of HbCDPK1 was strongly induced by mechanical wounding, jasmonic acid (JA), and ethephon. Recombinant HbCDPK1 was expressed in E. coli, and its activity was assayed. The assay indicated that HbCDPK1 had the kinase and Ca(2+)-binding activity in vitro as a calcium-dependent protein. The potential roles of the HbCDPK1 are discussed as to latex production and rubber biosynthesis.

Expression profiling of a novel calcium-dependent protein kinase gene, LeCPK2, from tomato (Solanum lycopersicum) under heat and pathogen-related hormones.

A full-length cDNA LeCPK2 (GenBank GQ205414) from tomato (Solanum lycopersicum) encoding a calcium-dependent protein kinase (CDPK) was cloned by in silico cloning using NtCPK5 (AY971376) as a virtual probe. The deduced amino acid sequence of LeCPK2 contained the kinase, autoinhibitory, and calmodulin-like domains typical of CDPKs. Expression profiling indicated that LeCPK2 expressed predominantly in flowers and responded divergently to heat and cold stress, in which obvious mRNA accumulation was detected at 4 h under 42 degrees C stress, but no change in LeCPK2 mRNA levels was observed in 6 h at 4 degrees C. Mechanical wounding and phytohormones including ethylene, methyl jasmonate, and salicylic acid were also observed to arouse the expression of LeCPK2 in a similar pattern. mRNA accumulation was enhanced at 30 min and reached a maximum at 3 h, followed by a decrease to the normal level. All the results suggest that LeCPK2 is a novel versatile isoform of tomato CDPKs.