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.

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.

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.

[Mechanism of polycomb Bmi1-targeted therapy for colorectal cancer].

OBJECTIVE: To investigate the Bmi1 protein level in human colorectal cancer specimen and associated clinicopathological parameters, and to determine the influence of Bmi1 on the proliferation and apoptosis of colorectal cancer cells. METHODS: Bmi1 protein level was assessed in 85 patients with colorectal cancer and adjacent normal tissue by immunohistochemistry. SW480 cells were transfected with Bmi siRNA plasmid. MTT assay and flow cytometry were used to measure the proliferation and apoptosis of SW480 cells. The expression of Bmi1 and Bcl-2 were measured by Western blot. RESULTS: The positive rate of Bmi1 expression in colorectal cancer tissues was 56.5%(48/85), significantly higher than that in the adjacent noncancerous tissues[17.6%(15/85), P<0.05)]. It was found that the overexpression of Bmi1 was associated with degree of differentiation, status of lymph nodes metastasis, and TNM staging in colorectal cancer(P<0.05). After transfection of SW480 with Bmi1 siRNA, the cell proliferation was inhibited and the apoptosis was significant. The cell proliferation inhibitory rates were 13.1%, 16.5%, and 18.3% at 24 h, 48 h and 72 h after transfection. The apoptotic rates were 15.7%, 45.6%, 40.2%, respectively. Expression of Bmi1 was downregulated after 48 h, as was that of Bcl-2. CONCLUSIONS: Bmi1 expression is associated with the clinicopathological characteristics of colorectal cancer. Blockade of Bmi1 can inhibit the proliferation and accelerate the apoptosis of colorectal cancer cells.

[Clinical value of screening hereditary nonpolyposis colorectal cancer in China with protocol recommended by NCCN guidelines].

OBJECTIVE: To investigate the effect of the protocol recommended by NCCN-2007 on the diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) in China. METHODS: NCCN protocol consists of identifying HNPCC characteristics according to the revised Bethesda Guidelines,genetic counseling with immunohistochemistry and finally genetic testing. Four hundred and nineteen patients diagnosed as colorectal cancer from January 2002 to February 2006 were selected. The hMLH1 and hMSH2 immunostaining were implemented for 90 patients who fulfilled the revised Bethesda Guidelines, in whom 8 patients fulfilling the Amsterdam II (Criteria were classified as group A and the other 82 patients as group B. The frozen tissues were collected from patients who showed loss of hMLH1 or hMSH2 protein expression, then RNA was extracted, and RT-PCR and cDNA sequencing were adopted to detect the germline mutations of hMLH1 and hMSH2. RESULTS: Tumor tissues from 18 patients showed loss of hMLH1 or hMSH2 protein expression (5 patients in group A and 13 in group B). Finally, 21 patients(8 in group A and 13 in group B showed loss expression of MMR protein) were diagnosed as HNPCC, including 2 cases of hMLH1 and 1 case of hMSH2 mutations. These 3 cases with cDNA mutations did not fulfill the Amsterdam II( Criteria, and were finally diagnosed as HNPCC. CONCLUSION: The protocol recommended by NCCN-2007 offers a useful approach to identify HNPCC patients,and reduces the possibility of missed diagnosis of HNPCC.

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.

[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.

[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.

[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.

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.

[Expression and significance of estrogen receptor isoforms in the development of human breast carcinoma].

OBJECTIVE: To investigate the estrogen receptors (ER)alpha and ERbeta expression and their relationship with clinicopathological parameters in human breast carcinoma. METHODS: Samples were obtained from 30 breast carcinoma, reverse transcriptase polymerase chain reaction was used to measure the expression of ERalpha and ERbeta mRNA. RESULTS: ERalpha mRNA level was up-regulated in breast carcinoma tissue compared with adjacent normal tissue (t = 7.399, P < 0.01) while down-regulated in ERbeta. The relative ratio of ERalpha and ERbeta was decreased in normal tissue vs. carcinoma (t = 6.385, P < 0.01), in patients with lymph node metastasis vs. those without lymph node metastasis (t = 2.602, P < 0.05), in late stage carcinoma vs. early stage (t = 3.754, P < 0.05). CONCLUSION: ERalpha and ERbeta play divergent role in the development of human breast carcinoma.

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.

[Effects of selective cyclooxygenase-2 inhibitor NS-398 on 5-fluorouracil chemotherapy and progression of colon cells: an experimental study].

OBJECTIVE: To investigate the effects of selective cyclooxygenase-2 (COX-2) inhibitor NS-398 on 5-fluorouracil (5-Fu) chemotherapy and on the progression of colon cells. METHODS: Colon cancer cells of HT-29 and SW480 lines were cultured. Selective COX-2 inhibitor NS-398, 5-Fu, or NS-398 combining with 5-Fu were added into the cultures to be co-cultured for 24, 48, and 72 hours respectively. RT-PCR and ELISA analysis were performed to detect the level of COX-2 mRNA expression and prostaglandin 2 (PGE2) concentration in the cells of both HT-29 and SW480 lines. The proliferation and apoptosis of the two cell lines were observed with MTT assay and flow cytometry. RESULTS: Expression of COX-2 mRNA were negative in SW480 line and positive in HT-29 line. Compared with SW480 line, the HT-29 line showed an obvious decline of PGE2 concentration following NS-398 treatment. Both NS-398 and 5-Fu inhibited the cells' proliferation and induced apoptosis in a dose-dependent manner, and a more significant inhibition was found when the cells were co-treated with NS-398 and 5-Fu. Although, there was no significant difference between these in inducing apoptosis. CONCLUSION: Selective COX-2 inhibitor NS-398 can inhibit the proliferation of colon cancer cells and induce apoptosis thereof. The mechanism of NS-398 against colon cancer may be independent upon the expression levels of COX-2 mRNA and PGE2 of colon cancer. NS-398 may be a subsidiary drug in 5-Fu chemotherapy in treating colon cancer.

[Relationship of Stat3 and its target gene products with malignancy in human colorectal carcinoma].

BACKGROUND & OBJECTIVE: Signal transducers and activators of transcription 3 (Stat3) pathway can be activated by cytokines and growth factors, and activation of Stat3 is involved in modulating cell proliferation, differentiation, and apoptosis. Stat3 has been classified as an oncogene because Stat3 can mediate malignant transformation of cultured cells. This study was conducted to investigate the expression of Stat3 and its target gene products including Cyclin D1 and Bcl-x(L) in human colorectal carcinoma (CRC) tissues and cells, and to explore the mechanisms in tumorigenesis of CRC. METHODS: The expression of Stat3, p-Stat3, Cyclin D1, and Bcl-x(L) in 45 cases of cancerous tissues, adjacent normal tissues, and two colon cancer cell lines including SW480 and HCT116 was measured by Western blot analysis. The expression pattern of Stat3 and its activated form p-Stat3 was determined by immunohistochemical staining. The relationship of the expression of p-Stat3, Cyclin D1, and Bcl-x(L) in CRC with various clinicopathological characteristics was analyzed statistically. RESULTS: The protein expression rates of p-Stat3, Cyclin D1, and Bcl-x(L) in colorectal cancer and adjacent normal mucosa were 57.8%, 64.4%, 68.9%, and 42.2%, 35.6%, 31.1%,respectively; and their protein levels (A value) were 114263+/-53598, 58321+/-24872, 71032+/-43425 in colorectal cancer and 55971+/-28762, 22563+/-11160, 37281+/-14622 in adjacent normal mucosa (P< 0.05). Overexpression of p-Stat3 was correlated with clinical stage and nodal metastasis in colorectal cancer (P = 0.026 and P= 0.018, respectively). Elevated levels of Cyclin D1 were associated with nodal metastasis (P= 0.041). It was found that Cyclin D1 was in a positive linear correlation fashion with p-Stat3 in tumor (r = 0.382, P< 0.05). Activated Stat3 was also detected in both colon cancer cell lines SW480 and HCT116. CONCLUSION: Stat3 signaling pathway may play an important role in the tumorigenesis of colorectal carcinoma. Determination of Stat3 and its target gene products can be used to indicate the malignancy degree of colorectal carcinoma.