MS diagnostic model and rapid distinguishing of bioactive limonoids in fruits of Melia toosendan using solid-phase extraction coupled with LC-MS/MS.

INTRODUCTION: Melia toosendan Sieb. et Zucc. has been used as a Chinese folk medicine for roundworm treatment since ancient times. Many diverse limonoids have been isolated from Meliaceae plants, but it remains difficult to isolate and identify other limonoids because of their small natural concentrations. OBJECTIVE: This study was performed to overcome the difficulties associated with fast and accurate identification of limonoids and establish a reliable and sensitive method for the analysis of minor limonoids in M. toosendan fruits. METHODS: An efficient strategy for enrichment, detection, and identification of minor limonoids from M. toosendan fruits using solid-phase extraction with high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (SPE-HPLC-Q-TOF-MS/MS) was developed herein. RESULTS: Characteristic fragmentations and fragmentation ions containing trichilin-, nimbin-, and vilasinin-class limonoid skeletons were initially studied, and characteristic diagnostic ions involved retro Diels-Alder (RDA) reactions or homolytic cleavages, which were used to identify minor limonoids. In total, 13 limonoids, including four new ones, were identified. CONCLUSION: This is the first report on the analysis of M. toosendan fruits to identify limonoids. This novel analysis method may stimulate further research regarding the identification of limonoids in other plant species.

LIN28B/IRS1 axis is targeted by miR-30a-5p and promotes tumor growth in colorectal cancer.

Insulin receptor substrate 1 (IRS1) is a potential oncogene that has been implicated in several malignant tumors. However, the regulatory mechanism of IRS1 remains to be investigated. The aim of our current study is to unveil the mechanism by which IRS1 exerts functions in tumorigenesis of colorectal cancer (CRC). The expression level of IRS1 was found to be higher in CRC cells in comparison with the normal cell. To determine the role of IRS1 in regulating CRC cellular processes, loss-of-function assays were designed and carried out in two CRC cell lines. Both in vitro and in vivo functional assays indicated that silencing of IRS1 suppressed CRC cell survival. Based on bioinformatics prediction and mechanism experiments, IRS1 was identified as a downstream target of miR-30a-5p. Furthermore, RNA-binding protein lin-28 homolog B (LIN28B) was determined to be a stabilizer of IRS1 messenger RNA. More importantly, LIN28B also acted as a target of miR-30a-5p.Through rescue assays, we proved that LIN28B-stablized IRS1 mediated miR-30a-5p-mediated CRC cell growth. In conclusion, this study revealed that LIN28B and LIN28B-stablized IRS1 promoted CRC cell growth by cooperating with miR-30a-5p.

Ciliatasecones A-C, three rearranged limonoids from Toona ciliata var. yunnanensis.

Ciliatasecones A-C (1-3), three rearranged limonoids with a novel ring-seco model and an unprecedented cycle system, were isolated from the root bark of Toona ciliata var. yunnanensis. Ciliatasecones A-B (1-2) share a novel cyclopenta[b]furan ring C/D system through C-9/11-seco and C-11/14 ether linkage. Ciliatasecone C (3) was found to possess a rare rearranged six-membered lactone ring B between C-7 and C-9. Plausible biogenetic pathway speculation indicated that C-9/11 cleavage and oxygen bridge formation played the key roles in the framework rearrangement of 1-3.

The total biosynthesis route of rosmarinic acid in Sarcandra glabra based on transcriptome sequencing.

Sarcandra glabra is a widely distributed and valuable plant in food and daily chemical industries, and is also a common-used medicinal plant for treating inflammatory diseases and tumors. Rosmarinic acid (RA) with significant pharmacological activity is an abundant and important constituent in S. glabra, however, little information about key enzymes involving the biosynthesis of RA in S. glabra is available and the underlying biosynthesis mechanisms of RA in S. glabra remain undeciphered. Therefore, in this study, by full-length transcriptome sequencing analyses of S. glabra, we screened the RA biosynthesis candidate genes based on sequence similarity and conducted enzymatic function characterization in vitro and in vivo. As a result, a complete set of 7 kinds of enzymes (SgPALs, SgC4H, Sg4CL, SgTATs, SgHPPRs, SgRAS and SgC3H) involving the biosynthesis route of RA from phenylalanine and tyrosine, were identified and fully characterized. This research systematically revealed the complete biosynthesis route of RA in S. glabra, which helps us better understand the process of RA synthesis and accumulation, especially the substrate promiscuities of SgRAS and SgC3H provide the molecular biological basis for the efficient biosynthesis of specific and abundant RA in S. glabra. The 7 kinds of key enzymes revealed in this study can be utilized as tool enzymes for production of RA by synthetic biology methods.

Functional characterization of a Flavonol 3-O-rhamnosyltransferase and two UDP-rhamnose synthases from Hypericum monogynum.

Rhamnosyltransferase (RT) and rhamnose synthase (Rhs) are the key enzymes that are responsible for the biosynthesis of rhamnosides and UDP-l-rhamnose (UDP-Rha) in plants, respectively. How to discover such enzymes efficiently for use is still a problem to be solved. Here, we identified HmF3RT, HmRhs1, and HmRhs2 from Hypericum monogynum, which is abundant in flavonol rhamnosides, with the help of a full-length and high throughput transcriptome sequencing platform. HmF3RT could regiospecifically transfer the rhamnose moiety of UDP-Rha onto the 3-OH position of flavonols and has weakly catalytic for UDP-xylose (UDP-Xyl) and UDP-glucose (UDP-Glc). HmF3RT showed well quercetin substrate affinity and high catalytic efficiency with Km of 5.14 µM and kcat/Km of 2.21 × 105 S-1 M-1, respectively. Docking, dynamic simulation, and mutagenesis studies revealed that V129, D372, and N373 are critical residues for the activity and sugar donor recognition of HmF3RT, mutant V129A, and V129T greatly enhance the conversion rate of catalytic flavonol glucosides. HmRhs1 and HmRhs2 convert UDP-Glc to UDP-Rha, which could be further used by HmF3RT. The HmF3RT and HmRhs1 co-expressed strain RTS1 could produce quercetin 3-O-rhamnoside (quercitrin), kaempferol 3-O-rhamnoside (afzelin), and myricetin 3-O-rhamnoside (myricitrin) at yields of 85.1, 110.7, and 77.6 mg L-1, respectively. It would provide a valuable reference for establishing a better and more efficient biocatalyst for preparing bioactive flavonol rhamnosides by identifying HmF3RT and HmRhs.

The parallel biosynthesis routes of hyperoside from naringenin in Hypericum monogynum.

Hyperoside is a bioactive flavonoid galactoside in both medicinal and edible plants. It plays an important physiological role in the growth of flower buds. However, the hyperoside biosynthesis pathway has not been systematically elucidated in plants, including its original source, Hypericaceae. Our group found abundant hyperoside in the flower buds of Hypericum monogynum, and we sequenced its transcriptome to study the biosynthetic mechanism of hyperoside. After gene screening and functional verification, four kinds of key enzymes were identified. Specifically, HmF3Hs (flavanone 3-hydroxylases) and HmFLSs (flavonol synthases) could catalyze flavanones into dihydroflavonols, as well as catalyzing dihydroflavonols into flavonols. HmFLSs could also convert flavanones into flavonols and flavones with varying efficiencies. HmF3'H (flavonoid 3'-hydroxylase) was found to act broadly on 4'-hydroxyl flavonoids to produce 3',4'-diydroxylated flavanones, dihydroflavonols, flavonols, and flavones. HmGAT (flavonoid 3-O-galactosyltransferase) would transform flavonols into the corresponding 3-O-galactosides, including hyperoside. The parallel hyperoside biosynthesis routes were thus depicted, one of which was successfully reconstructed in Escherichia coli BL21(DE3) by feeding naringenin, resulting in a hyperoside yield of 25 mg/l. Overall, this research not only helped us understand the interior catalytic mechanism of hyperoside in H. monogynum concerning flower development and bioactivity, but also provided valuable insights into these enzyme families.

Integrated molecular networking strategy enhance the accuracy and visualization of components identification: A case study of Ginkgo biloba leaf extract.

Molecular networking (MN) is an efficient tool for natural product research. However, single MN might lead to false annotation due to the limited information, and the importance of combining MN with chromatogram is always ignored. In this study, we proposed a comprehensive MN strategy combining feature-based molecular networking (FBMN) and dual ionization mode MS/MS to improve the annotation accuracy and to achieve structural feature visualization in a chemotaxonomic chromatogram. Three steps were taken: (1) employing FBMN and dual ionization mode MS/MS to distinguish isomers and improve components' identification accuracy. (2) Using a 3-level initiative supported by in-house database to evaluate the annotation confidence. As a result, 95 compounds were successfully identified from Ginkgo biloba leaf extract (GBE) and Ginkgo biloba leaf (GBL), and 70 compounds mainly consisting of flavonoid glycosides, ginkgolides, and lignan glycosides were assigned as high-confidence molecules. (3) Building color-dependent chemotaxonomic chromatograms, to achieve component visualization by connecting FBMN with chromatogram in which the peaks of the same color indicated the compounds with similar structural features. Our research provided a new and efficient strategy for component identification and visualization of herbal medicine.

Natural and Pseudonatural Lindenane Heterodimers from Sarcandra glabra by Molecular Networking.

Sarglaoxolane A (1), the first lindenane-normonoterpene heterodimer fused by tetrahydrofuran, was discovered in Sarcandra glabra guided by the first proposed single-node-based molecular networking approach. Moreover, two pseudonatural derivatives (2 and 3) with an oxa-difuranofurone moiety were transformed from 1 and confirmed by X-ray diffraction, and also proven to exist in the plant extract. A combination of molecular networking and biomimetic transformation can significantly promote the discovery and structural elucidation of novel natural products.

Diverse acyclic diterpene derivatives from Aphanamixis sinensis.

Fifteen diterpene derivatives including seven new ones, sinensisins A-G (1, 2, 4, 7, 10, 14, 15), were obtained from the leaves and twigs of Aphanamixis sinensis. Their structures were elucidated by NMR spectroscopic and ECD data analyses. These diverse carbon skeletons containing meroditerpenoids, acyclic diterpenes, and norditerpenoids biogenetically were derived from chain-like diterpenes. Compounds 3, 5, and 6 showed inhibitory effects of nitric oxide (NO) production in LPS-induced RAW 264.7 cells.

Four New Limonoids from the Barks of Toona ciliata.

Four new limonoids, toonayunnanaes F - I (1 - 4), and six known compounds (5 - 10) were isolated from the barks of Toona ciliata. Their structures were elucidated by thoroughly analyzing of NMR and HRMS data, and single-crystal X-ray diffraction of 1. The oxetane ring moiety in 1 was rare in limonoids and other natural products. Compound 1 showed nitric oxide (NO) inhibitory effect with an IC50 38.45 ± 0.41 µM in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages.

Identification of transgelin-2 as a biomarker of colorectal cancer by laser capture microdissection and quantitative proteome analysis.

To search for potential protein markers of colorectal cancer (CRC), the changes in protein expression levels between microdissected tumor cells and normal mucosa epithelia were analyzed by an acetylation stable isotopic labeling method coupled with linear quadrupole ion trap fourier transform mass spectrometry (LTQ-FTMS). In total, 137 proteins were up-regulated or down-regulated significantly in cancer by at least two-fold. Based on gene ontology analysis, the largest part of differential proteins were unknown for both subcellular localization and biological process. In particular, the significant up-regulation of transgelin-2 (TAGLN2) in CRC was validated by Western blot analysis and further evaluated by immunohistochemistry in paired tumor and normal mucosa samples from 120 consecutive CRC patients, 20 adenomas, and eight synchronous hepatic metastases of CRC. TAGLN2 expression was frequently observed in cancer cells, precancerous lesions, and hepatic metastases, whereas in normal epithelia expression was rarely observed. The overexpression of TAGLN2 was associated with lymph node and distant metastasis, advanced clinical stage (P < 0.001), and shorter overall survival in CRCs. Cox regression analysis indicated that high tumor-TAGLN2 expression represents an independent prognostic factor. Consequently, over-expression of TAGLN2 may serve as a new biomarker for predicting progression and prognosis of CRC.

Homeoprotein Cdx2 and nuclear PTEN expression profiles are related to gastric cancer prognosis.

The aim of the study was to analyze the expression of Cdx2 and nuclear PTEN in relation to clinicopathological features of gastric cancer tissue biopsies in order to determine the value of a combined analysis of Cdx2 and nuclear PTEN expression in distinguishing histological types and prognosis of gastric cancers. The expression of Cdx2 and nuclear PTEN was studied using immunohistochemistry of paraffin-embedded tumor specimens from 99 patients who underwent radical D2 gastrectomy between 1999 and 2001. Cdx2 and nuclear PTEN expression were detected in 39.6% (36 of 91) and 70.3% (64 of 91) of gastric cancer cases, respectively. There was a negative correlation between Cdx2 expression and Lauren classification (p=0.032), and between nuclear PTEN expression and lymph node metastasis (p=0.049). Patients with Cdx2-positive, or nuclear PTEN-positive expression had higher survival rates than those with Cdx2-negative or nuclear PTEN-negative expression (p<0.001 and p=0.003, respectively). Co-expression of Cdx2 and nuclear PTEN showed significantly lower levels in diffuse- or mixed-type cancers than in intestinal-type cancers (p=0.005). Multivariate analysis revealed that Cdx2 expression was an independent prognostic indicator of gastric cancer (p=0.014). These data suggest that combined analysis of Cdx2 and nuclear PTEN expression can have significant value in distinguishing histological types of gastric cancer and assessing prognosis in patients with gastric cancer.

The role of cyclooxygenase-2/prostanoid pathway in visceral pain induced liver stress response in rats.

BACKGROUND: Cyclooxygenase (COX) is the rate-limiting enzyme in the production of prostanoids from arachidonic acid. COX-2 is the inducible enzyme in the COX family, together with the prostanoids forms the COX-2/prostanoid pathway. Research showed that the COX-2/prostanoid pathway is activated in hepatic diseases and liver stress reaction, such as fibrogenesis, portal hypertension, carcinogenesis, and ischemic/reperfusion injury. But there was no report on visceral pain induced liver stress. This study was to investigate the role of the COX-2/prostanoid pathway in liver stress response in rat acute colitis visceral pain liver stress model. METHODS: Fifty-three male SD rats were randomly divided into Naive, Model, NS398 treatment, and Morphine treatment groups. The rat acute colitis visceral pain liver stress model was established under anesthesia by the colonic administration of 0.5 ml of 6% acetic acid using a urethral catheter. NS398 and morphine were administrated 30 minutes prior to model establishment in NS398 and Morphine treatment groups respectively. Spontaneous activities and pain behavior were counted and the extent of colonic inflammation was assessed histologically. Liver tissue levels of Glutathione-S-Transferase (GST) activity, COX-2 mRNA, prostaglandin E2 (PGE2), thromboxane B2 (TXB2) and 6-Ketone-prostaglandin F1alpha (6-K-PGF1alpha) contents were assessed. RESULTS: Thirty minutes after the colonic administration of acetic acid, a significant decrease in spontaneous activities and an increase in pain behaviors were observed in Model group (P < 0.01 and P < 0.05 respectively), accompanied by colonic inflammation. Liver GST activity levels significantly dropped (P < 0.05). Liver COX-2 mRNA expression significantly increased, accompanied by an increase in liver concentrations of PGE2 and TXB2, but no obvious change in 6-K-PGF1alpha concentrations. NS398 and morphine both ameliorated post-stress liver GST activity (P < 0.05 and P < 0.01 respectively), decreased stress-induced COX-2 expression, decreased PGE2 and TXB2 production, but increased liver 6-K-PGF1alpha levels. Morphine attenuation in colonic tissue inflammation was apparent at 24 hours (P < 0.05). CONCLUSIONS: Acute colitis visceral pain liver stress can induce liver injury. Liver injury might have occurred through the activation of the COX-2/prostanoid pathway and increased production of PGE2 and TXB2. Effective analgesia might offer protective effect during visceral pain stress.

[Effect of tumor burden on differentiation of T lymphocytes in the peripheral blood of patients with colorectal cancer].

OBJECTIVE: To analyze the effect of tumor burden on the differentiation of T1 and T2 cells and its implication in patients with colorectal cancer. METHODS: Peripheral venous blood samples were obtained from 20 patients with primary colorectal cancer before and 7 days after the operation, radical operation in 17 patients and palliative resection in 3 patients. Twenty sex and age-matched patients with benign diseases treated in the same period were used as controls. T1 and T2 cells in the peripheral blood were evaluated by detecting the intracellular interferon-gamma and interleukin-4 production with 4-color flow cytometry. Lymphocyte subsets in the peripheral blood were also measured by flow cytometry. RESULTS: At the time of diagnosis, the percentage of T1 and T2 cells in the peripheral lymphocytes of the case group was lower significantly than that of the control group (P = 0.006, and P = 0.017). There was no significant difference in the T, CD4(+) T, CD8(+) T, B, and NK cells between the two groups. After getting rid of the tumor burden, the percentage of T1 cells increased, however, not significantly (P > 0.05). And the percentage of T2 cells increased significantly (P = 0.020). The percentages of T1 cell of the patients with the tumor > or = 5 cm and of the patients with poorly differentiated tumors were significantly lower than those of the patients with the tumor < 5 cm and of the patients with well or moderately differentiated tumors (P = 0.064, and P = 0.072). The percentage of T1 cells in the patients with lymph node metastasis and stage III approximately IV tumor were lower significantly than those of the patients without lymph node metastasis and those with stage I approximately II tumor (P = 0.033 and P = 0.033). No significant differences were found between the population of T1 cells and such factors as tumor size, serosa invasion, and distant metastasis. CONCLUSION: Tumor load suppresses the differentiation of T1 and T2 cells in the patients with colorectal cancer significantly, and may be an important factor in the development of immunosuppression. After getting rid of the tumor burden, the percentages of T1 and T2 increase in a short time, and the immunologic function is improved. Determination of T1 may be helpful to indicate the prognosis of colorectal cancer.

Increased expression of mitogen-activated protein kinase and its upstream regulating signal in human gastric cancer.

AIM: To investigate the expression of mitogen-activated protein kinases (MAPKs) and its upstream protein kinase in human gastric cancer and to evaluate the relationship between protein levels and clinicopathological parameters. METHODS: Western blot was used to measure the expression of extracellular signal-regulated kinase (ERK)-1, ERK-2, ERK-3, p38 and mitogen or ERK activated protein kinaseMEK-1 proteins in surgically resected gastric carcinoma, adjacent normal mucosa and metastatic lymph nodes from 42 patients. Immunohistochemistry was employed for their localization. RESULTS: Compared with normal tissues, the protein levels of ERK-1 (integral optical density value 159 526+/-65 760 vs 122 807+/-65 515, P = 0.001), ERK-2 (168 471+/-95 051 vs 120 469+/-72 874, P<0.001), ERK-3 (118 651+/-71 513 vs 70 934+/-68 058, P<0.001), P38 (104 776+/-51 650 vs 82 930+/-40 392, P = 0.048) and MEK-1 (116 486+/-45 725 vs 101 434+/-49 387, P = 0.027) were increased in gastric cancer tissues. Overexpression of ERK-3 was correlated to TNM staging (average ratio of integral optic density (IOD)(tumor): IOD(normal) in TNM I, II, III, IV tumors was 1.43+/-0.34, 5.08+/-3.74, 4.99+/-1.08, 1.44+/-1.02, n = 42, P = 0.023) and serosa invasion (4.31+/-4.34 vs 2.00+/-2.03, P = 0.037). In poorly differentiated cancers (n = 33), the protein levels of ERK-1 and ERK-2 in stage III and IV tumors were higher than those in stage I and II tumors (2.64+/-3.01 vs 1.01+/-0.33, P = 0.022; 2.05+/-1.54 vs 1.24+/-0.40, P = 0.030). Gastric cancer tissues with either lymph node involvement (2.49+/-2.91 vs 1.03+/-0.36, P = 0.023; 1.98+/-1.49 vs 1.24+/-0.44, P = 0.036) or serosa invasion (2.39+/-2.82 vs 1.01+/-0.35, P = 0.022; 1.95+/-1.44 vs 1.14+/-0.36, P = 0.015) expressed higher protein levels of ERK-1 and ERK-2. In Borrmann II tumors, expression of ERK-2 and ERK-3 was increased compared with Borrmann III tumors (2.57+/-1.86 vs 1.23+/-0.60, P = 0.022; 5.50+/-5.05 vs 1.83+/-1.21, P = 0.014). Borrmann IV tumors expressed higher p38 protein levels. No statistically significant difference in expression of MAPKs was found when stratified to tumor size or histological grade (P>0.05). Protein levels of ERK-2, ERK-3 and MEK-1 in metastatic lymph nodes were 2-7 folds higher than those in adjacent normal mucosa. The immunohistochemistry demonstrated that ERK-1, ERK-2, ERK-3, p38 and MEK-1 proteins were mainly localized in cytoplasm. The expression of MEK-1 in gastric cancer cells metastasized to lymph nodes was higher than that of the primary site. CONCLUSION: MAPKs, particularly ERK subclass are overexpressed in the majority of gastric cancers. Overexpression of ERKs is correlated to TNM staging, serosa invasion, and lymph node involvement. The overexpression of p38 most likely plays a prominent role in certain morphological subtypes of gastric cancers. MEK-1 is also overexpressed in gastric cancer, particularly in metastatic lymph nodes. Upregulation of MAPK signal transduction pathways may play an important role in tumorigenesis and metastatic potential of gastric cancer.

[Molecular mechanism of cyclooxygenase-2 inhibitor in inhibition of proliferation of colon cancer cells by modulating Stat5 signal transduction pathway].

OBJECTIVE: To investigate the role of NS398, a selective cyclooxygenase (COX)-2 inhibitor, in proliferation and apoptosis of colorectal cancer, and to reveal the mechanism of inhibiting colon cancer by NS-398 Independent of COX-2. METHODS: Human colon cancer cells of the line SW480 were cultured and then divided into 2 groups: experimental group and control group. NS398 of the concentrations of 12.5, 25, 50, 75, 100, and 125 micromol/L was added into the culture fluid of the experimental group. MTT assay was used to observe the proliferation of the cells, flow cytometry was used to test the cell cycle, RT-PCR analysis was performed to examine COX-2 mRNA expression, and Western blotting analysis was performed to detect the expression of Stat5, peroxisome proliferators-activated receptors (PPARs), cyclin D1 and Bcl-x(L). RESULTS: Expression of COX-2 mRNA was not detected in the SW480 colon cancer cells. 72 hours after the addition of NS398 75 micromol/L the proliferative level of the SW480 cells was decreased; the rate of the cells at the G(1) stage increased from 31.2% to 40.6%, and the rate of cells at the S stage decreased from 52.8% to 41.2%. The expression of Stat5, PPARdelta, cyclin D1 and Bcl-x(L) decreased along with the elongation of time of NS398 action. CONCLUSION: COX-2 inhibitor, such as NS-398 inhibits the colon cancer cell proliferation and induces apoptosis of colon cancer cells with the possible mechanism of inhibiting the proliferation and inducing the apoptosis of colon cancer cells through a pathway independent of COX-2.

[Correlation between Stat3 signal transduction pathway and expression of cyclooxygenase-2 in colorectal cancer cells].

OBJECTIVE: To explore the expression of Stat3 signal transduction pathway and expression of cyclooxygenase-2 (COX-2) in colorectal cancer cells and their correlation with the clinicopathological parameters of colorectal cancer, and to reveal the mechanism of Stat3 signal transduction pathway regulating the expression of cCOX-2 in colorectal cancer cells. METHODS: RT-PCR was used to detect the mRNA expression of Stat3 and COX-2 and immunohistochemistry was used to detect the protein expression of Stat3 and COX-2 in 50 specimens excised during operation from 50 patients with colorectal cancer aged 58 (37-75). Human colorectal cancer cells of the HT-29 strain were cultured. MTT method was used to examine the growth of the cells. AG490, an inhibitor of the upstream kinase JAK2 of Stat3 was added in to the culture fluid. 24, 48, and 72 hours later flow cytometry was used to examine the apoptosis of the cells. RESULTS: The mRNA expression levels of Stat3 and COX-2 in the colorectal cancer tissues were 1.97 and 1.88 times those of the adjacent normal tissues (both P < 0.05). The mRNA expression levels of Stat3 and COX-2 in the colorectal cancer tissues of the patients with lymph node metastasis were both significantly higher than those of the patients without lymph node metastasis (both P < 0.05). The mRNA expression levels of Stat3 and COX-2 in the colorectal cancer tissues of the patients of cancer with low differentiation were both significantly higher than those of the patients with high differentiation (both P < 0.05). Pearson correlation analysis showed that Stat3 mRNA expression was linearly correlated with COX-2 mRNA expression (r = 0.749, P < 0.01). The protein expression levels of Stat3 and COX-2 in the colorectal caner tissues were significantly higher than those in the adjacent normal tissues (both P < 0.01). AG490 time and dose-dependently inhibited the growth of the HKC cells, induced apoptosis of the HKC cells, and down-regulated the protein expression of Stas3 and COX-2. CONCLUSION: Overexpression of Stat3 and COX-2 may play a critical role in the development of colorectal cancer. Stat3 pathway influences the proliferation and apoptosis of colorectal cells and COX-2 gene expression. Blockade of the Stat3 signal pathway inhibits the proliferation and promotes the apoptosis of colorectal cancer cells.

[The effect of transfecting Stat3beta cDNA on human breast cancer].

OBJECTIVE: To study the effect of transfecting Stat3beta cDNA on human breast cancer. METHODS: Human breast cancer cells of the line SK-BR-3 were cultured and divided into 3 groups: Stat3beta transfection group (to be transfected with plasmid pIRES-Stat3beta containing Stat3beta by transient transfection technique), lipofectin reagent transfection group pIRES-EGFP transfection group, and control group. The positively transfected cells were isolated by fluorescence-activated cell sorter. Flow cytometry was used to analyze the cell cycles and cell apotosis. Western blotting was used to detect the expression of STAT3 protein. MTT method was used to examine the proliferation of the cells. RESULTS: Forty-eight hours after exposure to the plasmid pIRES-Stat3beta the transfection rate of the SK-BR-3 cells was 13.79%. SK-BR-3 cells expressed STA3 protein during proliferation. In comparison with the SK-BR-3 cells of other 3 group, the proliferation of the cells transfected with pIRES-Stat3beta was significantly decreased. Forty-eight hours after transfection, 81.09% of the cells transfected with the plasmid pIRES-Stat3beta accumulated at the G(0)/G(1) stage, a rate significantly higher than those of the other groups, and displayed a significantly higher rate of apoptosis. CONCLUSION: Transfection of plasmid pIRES-Stat3beta containing Stat3beta blocks the Stat3 pathway, thus inhibiting the proliferation and augment the apoptosis of human breast cancer cells and providing a novel gene therapy target.

[Change in apoptosis and its mechanism in intestinal epithelial cells under oxidative stress].

OBJECTIVE: To investigate change in apoptosis level and its mechanism of intestinal epithelial cells under oxidative stress. METHODS: HT-29 cells were cultured in vitro, which were treated with hydrogen peroxide (H2O2), to simulate the intestinal epithelial cells injured by reactive oxidative species. The cells viability was observed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, cell apoptosis and apoptosis associated proteins were evaluated by flow cytometry and Western blot. RESULTS: Cells viability of HT-29 was decreased by H2O2 which showed dose-dependent and time-dependent patterns (all P<0.05). The cell apoptotic ratios were increased with the concentration of H2O2 increased and the time of the stimulation prolonged compared with the controls (all P<0.05). Although the expression of the Bax was increased when HT-29 cells were stimulated with different concentrations of H2O2 for 24 hours, the expression of Bcl-2 was decreased. While HT-29 cells were stimulated with 500 micromol/L H2O2, the expression of the Bax was increased and that of Bcl-2 was decreased overtime. CONCLUSION: These data suggest that oxidative stress appears to be related to the apoptosis in intestinal epithelial cells under stress. The imbalance of Bcl-2/Bax expression might result in intestinal epithelial cell apoptosis in oxidative stress.

Constitutive activation of Stat3 signaling pathway in human colorectal carcinoma.

AIM: Signal transducers and activators of transcription (STATs) are a family of transcription factors activated in response to cytokines and growth factors. Constitutive activation of Stat3 has been observed in a growing number of tumor-derived cell lines, as well as tumor specimens from human cancers. The purpose of this study was to investigate the expression of p-Stat3, activated form of Stat3, and its downstream mediators including cyclin D1 and Bcl-x(L) in colorectal carcinoma (CRC), and to explore the possible mechanism of Stat3 signaling pathway in the tumorigenesis of colorectal carcinoma. METHODS: Tissue samples from 45 patients of primary colorectal carcinoma were selected for studying Stat3 signaling pathway protein expression. Western blot analysis was used to measure the expression of p-Stat3, cyclin D1, and Bcl-x(L) proteins in colorectal carcinomas. Furthermore, the expression patterns of these proteins were analyzed for their distribution at the cellular level by immunohistochemical staining of the tissues. RESULTS: Protein levels of p-Stat3, cyclin D1, and Bcl-x(L) were increased in colorectal carcinomas compared with adjacent normal mucosae (P<0.05). Elevated levels of p-Stat3 were correlated with the nodal metastasis and the stage (P<0.05). Overexpression of cyclin D1 was associated with the nodal metastasis (P<0.05). There was also a significant correlation between the expressions of p-Stat3 and cyclin D1 (r=0.382, P<0.05). CONCLUSION: Constitutive activation of Stat3 may play an important role in the tumorigenesis of colorectal carcinoma, and the detailed mechanism of Stat3 signaling pathway in CRC deserves further investigation.