OBJECTIVETo study the effects of anti-oncogene WWOX on cell growth of cholangiocarcinoma.

METHODSThe expression of WWOX protein was detected with immunohistochemical method-SP in 54 patients with cholangiocarcinoma from July 2005 to May 2010 and 12 samples of normal bile duct tissues. The recombinant WWOX eukaryotic expression plasmid was introduced into RBE cells by liposome-mediated transfection and positive cell clones were selected and amplified. The mRNA and protein expressions in RBE cells stably transfected with WWOX were investigated by quantitative RT-PCR and Western Blot before and after transfection. Cell proliferation was tested by MTT, cell apoptosis was assessed by FCM, the alteration of mitochondria membrane potential (ΔΨm) was detected by JC-1 staining method, cell invasion was determined by Transwell chamber assay. The expression change of bcl-2, bax, FasL, caspase-3 mRNA and protein was detected by quantitative RT-PCR and Western Blot.

RESULTSThe expression of WWOX protein was significantly lower in cholangiocarcinoma than that in normal bile duct tissues and loss of WWOX protein expression was found in 40.7% of cholangiocarcinoma specimens (P < 0.05). RBE cells with stable transfection of WWOX were established. Quantitative RT-PCR showed that the expression of WWOX mRNA was significantly enhanced and Western Blot demonstrated that WWOX protein expression was markedly increased. MTT showed that WWOX gene transfection significantly decreased the proliferation of RBE cells (P < 0.05). FCM analysis showed that the apoptosis rate after transfection was significantly promoted [(1.1 ± 0.6)% vs. (1.7 ± 0.5)% vs. (35.2 ± 4.4)%, P < 0.01], JC-1 staining method indicated that the experimental group was loss of ΔΨm [(12.6 ± 1.9)% vs. (13.6 ± 1.8)% vs. (48.7 ± 2.9)%, P < 0.01], transwell chamber assay showed that the number of transfected cells that passed the transwell membrane was significantly less than those of control groups (77 ± 6 vs. 72 ± 8 vs. 48 ± 6, P < 0.01). Quantitative RT-PCR and Western blotting showed that the expression of bcl-2 mRNA and protein was markedly decreased and the expression of bax, caspase-3 were significantly increased. There was no significant change in the expression of FasL.

CONCLUSIONWWOX exerts its antitumor effect against proliferation through inducing cell apoptosis in cholangiocarcinoma.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Cholangiocarcinoma ; genetics ; metabolism ; pathology ; Genetic Vectors ; Humans ; Oxidoreductases ; genetics ; metabolism ; Plasmids ; genetics ; Transfection ; Tumor Suppressor Proteins ; genetics ; metabolism ; WW Domain-Containing Oxidoreductase

OBJECTIVETo study the relationship between FHIT and WWOX expression and clinicopathological features in hepatocellular carcinoma (HCC).

METHODThe expression of FHIT and WWOX were determined by immunohistochemistry in 142 patients with HCC.

RESULTSAbsent or reduced FHIT and WWOX expression was observed in 68.3% and 77.5% of HCCs, respectively. The expression of FHIT was significantly correlated with that of WWOX (P < 0.01), and progressive loss of FHIT and WWOX expression were observed as tumor differentiation decreased and tumor grade increased (P < 0.05). Absent/reduced FHIT and WWOX expression was associated with tumor invasion and metastasis (P < 0.05). In addition, the expression of FHIT and WWOX in HCC with recrudesce were lower than that in those without recrudesce (P < 0.05).

CONCLUSIONSAbsent/reduced FHIT and WWOX expression is associated with poor prognosis.

Acid Anhydride Hydrolases ; genetics ; Adolescent ; Adult ; Aged ; Carcinoma, Hepatocellular ; genetics ; pathology ; Female ; Gene Expression Profiling ; Humans ; Liver Neoplasms ; genetics ; pathology ; Male ; Middle Aged ; Neoplasm Proteins ; genetics ; Oxidoreductases ; genetics ; Prognosis ; Tumor Suppressor Proteins ; genetics ; WW Domain-Containing Oxidoreductase ; Young Adult

BACKGROUNDWWOX and FHIT are two candidate tumor suppressor genes located in active fragile sites, the damage of which has been associated with the development of breast cancer. The association of the expression of these genes and the development of breast cancer has not been fully explored. We evaluated mRNA and protein expression of WWOX and FHIT in breast tissue with normal histological appearances, atypical ductal hyperplasia, ductal carcinoma in situ, and invasive cancer to see if a progressive decline in expression was present.

METHODSReverse transcription-polymerase chain reaction and Western blotting were used to evaluate the specimens for mRNA and protein expression, including 28 specimens with normal tissue, 28 specimens with atypical ductal hyperplasia, 33 specimens with ductal carcinoma in situ, and 51 specimens with invasive ductal carcinoma.

RESULTSCompared with in situ and invasive cancer specimens, both normal and atypical hyperplasia specimens had greater rates of detectable mRNA (WWOX rate ratio = 2.95, 95% CI 1.24 - 7.08; FHIT rate ratio = 4.58, 95% CI 1.82 - 11.81) and Western blotting detectable protein (WWOX rate ratio = 4.12, 95% CI 1.63 - 10.73; FHIT rate ratio = 3.76, 95% CI 1.44 - 10.06). For both proteins, differences between normal and atypical hyperplasia specimens and between in situ and invasive carcinoma specimens were explainable by chance (P > 0.05 for each analysis). Within each histological category, differences among fractions of specimens showed that FHIT and WWOX mRNA and protein expression were explainable by chance (P > 0.05 for each analysis).

CONCLUSIONExpression of FHIT and WWOX decreases along with breast tissue progress from a normal histological appearance to atypical ductal hyperplasia, in situ cancer, and the final invasive cancer.

Acid Anhydride Hydrolases ; analysis ; genetics ; Breast ; pathology ; Breast Neoplasms ; genetics ; Chromosome Fragile Sites ; Female ; Genes, Tumor Suppressor ; Humans ; Hyperplasia ; Neoplasm Proteins ; analysis ; genetics ; Oxidoreductases ; analysis ; genetics ; Tumor Suppressor Proteins ; analysis ; genetics ; WW Domain-Containing Oxidoreductase

BACKGROUNDWhether WW domain containing oxidoreductase (WWOX) gene is a tumor-suppressor is still controversial. Some researchers found that the transcription of the WWOX gene was lacking not only in tumor tissues but also in non-tumorous tissues and sometimes in normal tissues. Hence it is important to explore the role of the expression of the exogenous WWOX gene in the proliferation and apoptosis of primary cultured lung carcinoma cells.

METHODSLipofection technique was used to determine primary cultured lung carcinoma cells containing the highly expressed exogenous WWOX gene and primary cultured cells with vectors as controls. An animal model of lung cancer was made by subcutaneous implantation of tumor cells into nude mice. RT-PCR, Western blotting, flow cytometry, and TUNEL were used to detect the transcription, expression of the exogenous gene and the effect of the expression of targeted genes on the proliferation and apoptosis of the primary cultured lung carcinoma cells.

RESULTSThe growth, clone formation rate (CFR) ((5.33 +/- 1.53)%) of the primary lung cancer cells transfected with the WWOX gene, tumor size and weight were significantly lower than those of the non-transfected lung cancer cells (CFR: (14.33 +/- 1.53)%) and the primary lung cancer cells transfected with blank plasmids (CFR: (11.00 +/- 1.73)%, P < 0.05). The apoptosis level of primary lung cancer cells transfected with the WWOX gene ((40.72 +/- 5.20)%) was significantly higher than that of the non-transfected lung cancer cells ((2.76 +/- 0.02)%) and the primary lung cancer cells transfected with blank plasmids ((2.72 +/- 0.15)%, P < 0.05).

CONCLUSIONThe expression of the exogenous WWOX gene can significantly inhibit the proliferation of lung cancer cells and induce their apoptosis, suggesting that the WWOX gene possesses tumor-suppressing effect.

Animals ; Apoptosis ; Carcinoma ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Lung Neoplasms ; pathology ; Mice ; Mice, Inbred BALB C ; Oxidoreductases ; genetics ; physiology ; Phenotype ; Tumor Suppressor Proteins ; genetics ; physiology ; WW Domain-Containing Oxidoreductase

OBJECTIVETo investigate the expressions of WWOX and CD133 in colorectal cancer (CRC) and their relationship with the clinicopathologic characteristics of CRC.

METHODSThe expressions of WWOX and CD133 proteins were examined by immunohistochemistry in 174 specimens of CRC tissues and 80 normal colorectal mucosa tissues.

RESULTSThe positivity rates of WWOX and CD133 proteins were 41.4% and 53.4% in CRC tissues, respectively, significantly different from the rates in normal colorectal mucosa tissues (87.5% and 5.0%, respectively; P<0.05). WWOX and CD133 protein expressions were signi- ficantly correlated with the histological grades of the tumors, depth of invasion, lymph node metastasis, and Duke's stages (P<0.05). Spearman analysis showed a negative relationship between the WWOX expression and CD133 expression (P<0.05). Kaplan-Meier survival analysis showed that the overall survival time of CRC patients with a positive expression of WWOX was longer than that of patients with a negative expression of WWOX; the overall survival time of patients with a positive expression of CD133 was shorter than that of the negative patients (P<0.05). COX regression analysis identified positive expressions of WWOX and CD133 protein and Duke's stage as the independent prognostic factors of CRC.

CONCLUSIONAbnormal expressions of WWOX and CD133 might be involved in the initiation, development, invasion, and metastasis of CRC. A combined detection of WWOX and CD133 can help in predicting the progression and prognosis of CRC.

AC133 Antigen ; Antigens, CD ; metabolism ; Colorectal Neoplasms ; metabolism ; Disease Progression ; Glycoproteins ; metabolism ; Humans ; Immunohistochemistry ; Kaplan-Meier Estimate ; Lymphatic Metastasis ; Oxidoreductases ; metabolism ; Peptides ; metabolism ; Prognosis ; Tumor Suppressor Proteins ; metabolism ; WW Domain-Containing Oxidoreductase

OBJECTIVETo observe the effects of WWOX on cell attachment in ovarian cancer, and to explore its mechanisms of action.

METHODSAttachment assay was used to assess the adhesion of wwox-transfected PEO1 cells and vector-transfected PEO1 cells that were constructed, as well as PEO1 parent cells. Alpha/beta integrin-mediated cell adhesion assays were designed to identify cells surface integrins in PEO1 clone cells. Integrin function blocking experiments were designed to further determine integrins in PEO1 clone cells according to the integrin that was selected in integrin expression profiling. FACS analysis was used to further detect the level of integrin alpha3 on the cell membrane.

RESULTSAttachment assay showed that adhesion of WWOX-transfected PEO1 cells to fibronectin was significantly slower than that in vector-transfected controls or PEO1 parent cells, cultured on the pre-coated fibronectin for 2 hours (P<0.01). The level of membranous integrins alpha2 and alpha3 in the WWOX-transfected PEO1 cells was significantly decreased, as compared with that in vector-transfected controls (P<0.05), but there was no association with the level of functioning integrins betal or beta2 in clone cells (P>0.05). The attachment assays were repeated after pre-incubating the cells with integrin alpha2 or alpha3 function-blocking antibodies. These results showed that blocking integrin alpha3 significantly reduced the binding to fibronectin of all the PEO1 clonal lines, as compared with cells pre-incubated with a non-specific IgG antibody (P<0.05). In contrast, preincubation with alpha2 blocking antibody had very little effect on fibronectin binding in these cells (P>0.05). FACS analysis showed that membranous integrin alpha3 expression revealed a marked reduction in WWOX-transfected cells than that in vector-transfected cells.

CONCLUSIONWWOX acts as an ovarian tumor suppressor by modulating the interaction between tumor cells and the extracellular matrix, decreasing integrin activity and adhesion of tumor cells to fibronectin. This suggests an important role for loss of WWOX tumor suppressor in promoting attachment and adhesion of ovarian cancer cells on locoregional peritoneum, and further resulting in enhancing locoregional peritoneal tumor spread.

CD18 Antigens ; metabolism ; Cell Adhesion ; Female ; Fibronectins ; metabolism ; Humans ; Integrin alpha2 ; metabolism ; Integrin alpha3 ; metabolism ; Integrin beta1 ; metabolism ; Ovarian Neoplasms ; metabolism ; pathology ; Oxidoreductases ; genetics ; metabolism ; Protein Binding ; Transfection ; Tumor Suppressor Proteins ; genetics ; metabolism ; WW Domain-Containing Oxidoreductase

OBJECTIVETo explore the clinicopathologic characteristics and biological markers of breast carcinomas in young women.

METHODSImmunohistochemical SP method was used to study breast cancer susceptibility gene (BRCA1) and WWOX in breast carcinomas of patient ≤ 35 years of age (107 cases) and ≥ 60 years of age (112 cases). The findings were correlated with clinicopathological features. In addition, PCR amplification and direct sequencing were performed to detect the BRCA1 gene mutation of exons 2 and 20 using fresh frozen tissue samples in other 10 patients who were ≤ 35 years of age.

RESULTSThe positive rate of BRCA1 protein expression was higher in the young age group [65.4% (70/107)] than that of the old age group [35.7% (40/112)]. ER, PR, HER2, and WWOX protein expression and proliferation marker Ki-67 were no statistically different in the two groups (all P > 0.05). BRCA1 expression was significantly correlated with pTNM and axillary lymph node metastasis (both P < 0.05), but not with ER, PR, HER2 and WWOX protein expression (all P > 0.05). Ki-67 and histological grading showed no statistical correlation (P > 0.05). WWOX protein expression showed no correlation with clinicopathologic characteristics (all P > 0.05). Mutation of exons 2 and 20 of the BRCA1 gene was not detected in any of 10 cases studied.

CONCLUSIONBRCA1 cytoplasmic expression statistically correlates with the development and prognosis of breast cancer of young patients.

Adult ; Age Factors ; Aged ; Aged, 80 and over ; BRCA1 Protein ; genetics ; metabolism ; Breast Neoplasms ; metabolism ; pathology ; surgery ; Carcinoma, Ductal, Breast ; metabolism ; pathology ; surgery ; Exons ; Female ; Genes, BRCA1 ; Humans ; Ki-67 Antigen ; metabolism ; Lymphatic Metastasis ; Middle Aged ; Mutation ; Neoplasm Staging ; Oxidoreductases ; metabolism ; Receptor, ErbB-2 ; metabolism ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism ; Tumor Suppressor Proteins ; metabolism ; WW Domain-Containing Oxidoreductase ; Young Adult

Adenocarcinoma ; metabolism ; pathology ; Adult ; Aged ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Female ; Humans ; Immunohistochemistry ; Lung ; chemistry ; pathology ; Lung Neoplasms ; metabolism ; pathology ; Male ; Middle Aged ; Oxidoreductases ; metabolism ; Sex Factors ; Smoking ; Tumor Suppressor Proteins ; metabolism ; WW Domain-Containing Oxidoreductase