Abstract Objective analysis of Cyclin D1, STAT3 and its target gene product and Caspase  3 expression in ovarian cancer tissue, explore the STAT3 its target gene products in the pathogenesis of ovarian cancer. Collected after surgical removal of ovarian carcinoma 40 cases, Western blot analysis of 40 cases of ovarian cancer tissue, normal ovarian tissue of STAT3 and its target gene product Cyclin D1 and Caspase  3 expression. P  STAT3, Cyclin D1 and Caspase  3 expression levels in ovarian cancer tissues was significantly higher than that of normal tissue (tumor vs. normal, P = 0.028,0.035, 0.047); of p  STAT3 and Cyclin D1 expression level with FIGO stage (P = 0.026, P = 0.041). the of p  STAT3 and Cyclin D1 expression in ovarian cancer tissues was linearly correlated (r = 0.412, P <0.05). Conclusion STAT3 signal transduction pathway may play an important role in the process of ovarian cancer, the detection of STAT3 and its target gene product expression in ovarian cancer may reflect the malignant potential of the tumor.

Key words ovarian cancer; signal transduction; proliferation; apoptosis

  Constitutive Activation of STAT3 Pathway and Overexpression of Target Gene Products Correlate with Malignant Potential in Human Ovarian Carcinoma

    HOU Tao1, ZHANG Ying1, MA Xiang  Tao2, FU Jing3

    1.Department of Obstetrics and Gynecology, Beijing Haidian Hospital, Beijing 100080, China, 2.Department of Surgery, 3.Department of PathologyAbstract: Objective The purpose of this study was conducted to investigate the expression of Signal transducer and activator of transcription 3 ( STAT3) and target gene products including Cyclin D1 and Caspase  3 in human ovarian carcinoma tissues, and to explore the mechanisms in tumorigenesis of ovarian carcinoma. Methods Primary ovarian cancers and normal ovarian tissue were obtained from 40 patients undergoing ovarian cancer resection. Western blot analysis was used to measure the expression of STAT3, p  STAT3, Cyclin D1, and Caspase  3 in cancerous tissues, normal ovarian tissues. Results p  STAT3, Cyclin D1, and Caspase  3 protein levels were increased in ovarian cancer compared with adjacent normal tissue (P = 0.028,0.035,0.047). Overexpression of p  STAT3 and Cyclin D1 were correlated with FIGO stage in ovarian cancer (P = 0.026,0.041). It was found that Cyclin D1 was in a positive linear correlation fashion with p  STAT3 in tumor (r = 0.412, P <0.05). Conclusion These findings provide evidence that STAT3 signaling pathway may play an important role in the tumorigenesis of ovarian carcinoma, and detecting the expression of STAT3 and its target gene products may predict the malignant potential of ovarian carcinoma.

    Key words: Ovarian neoplasm; Signal transduction; Proliferation; Apoptosis

 The STAT3 transcription signal transducer and activator of family (signal transducers and activators of transcription, STATs), an important member of this pathway accept the stimulation of growth factors, cytokines, extracellular signal the role specific DNA fragments in the nucleus, regulation target gene transcription, affect cell proliferation, differentiation and apoptosis [1]. The STAT3 abnormal activation is closely related with the progression and prognosis of a variety of malignant tumors, but STAT3 signal transduction pathway and its target gene product research is still in its infancy with ovarian cancer [2]. In this study, Western blot analysis of STAT3 (p  STAT3) and its target gene product Cyclin D1 and Caspase 3 expression in ovarian cancer tissue, analysis of STAT3 and its target gene product and the relationship between the malignant potential of ovarian cancer.

    1 Materials and methods

    1.1 Materials

    The PVDF membrane was used in the Western blot were purchased from Millipore Corporation, for developing a film commercially available from Eastman Kodak Company. In this study, all antibodies were purchased from Santa Cruz. Prestained standard molecular weight proteins were purchased from GIBCO / BRL Company. ECL chemiluminescence kit from Amersham. The concentrated protein analysis solution from Bio  Rad. Other reagents were involved in the study of molecular biology purity purchased from Sigma.

    1.2 patient information and tissue specimens

    Collecting Beijing Haidian Hospital, Obstetrics and Gynecology, December 2000 to 2006 12 surgically resected ovarian cancer specimens 40 cases, aged 33 to 69 years, with a median age of 48 years old; postoperative pathologic grade: G1 14 cases, G2 11 cases, G3 15 cases; tumor stage according to the International Federation of Gynecology and Obstetrics (FIGO) stage, divided into Ⅰ 8 cases, Ⅱ seven cases, Ⅲ 21 cases, Ⅳ four cases. All patients underwent cytoreductive surgery, 15 ~ 20min after the surgical removal of the tumor acquisition ovarian cancer tissue, normal ovarian tissue as a control (from the same period of pathologically confirmed benign ovarian lesions) and immediately placed in liquid nitrogen and stored. Before surgery in patients who did not receive chemotherapy or radiotherapy and signed informed consent.

    1.3 Research Methods

    1.3.1 tissue protein extract

    Tissue lysis lysis buffer (150mmol / L NaCl; 1% peroxygen sodium cholate; by 1% Triniton X-100; 0.1% sodium dodecyl sulfonate; 10 mM Tris, pH 7.2; 1 mmol / L, n sodium orthovanadate; 1 mmol / L phenylmethanesulfonyl fluoride; 1 mmol / L NaF; 0.1mmol / L aprotinin, 1mmol / L leupeptin). Lysate 13 000r/min centrifuged at 4 ℃ 30min [3].

    1.3.2 Method for determination of the protein concentration (Bradford method)

    With bovine serum albumin (BSA) as a standard, according to a protein quantification kit (Bio-Rad, USA) protein quantification standard curves, and in spectrophotometer 595nm metering density value calculated extract protein concentration.

    1.3.3 Western blot

    Conducting before the Western blot, the protein extract with 2 x sodium dodecyl sulfonate (SDS) sample buffer 1:1 mixture (125 mmol / L Tris-HCl, pH 6.8; 4% sodium dodecyl sulfo sodium; 20% glycerol; 10% 2  mercapto-ethanol), after the heated water bath of 100 ° C for 5min. To take the total protein 50μg after the separation of the 10% polyacrylamide gel electrophoresis and transferred to PVDF membranes. Electrophoresis prestained standard molecular weight proteins in the polyacrylamide gel was added as an indicator. Transferred to a membrane with TBST buffer (10mmol / L Tris. HCl, pH 7.5,150 mmol / L NaCl, by 0.5% Tween-20) with 5% bovine serum albumin closed 30min. Closed after an anti of STAT3, p  STAT3, Cyclin D1, Caspase-3 working concentration of 1:1 000 GAPDH as an internal reference and incubated overnight at 4 ° C under conditions with TBST (each 5min) washing the membrane, with horseradish peroxidase-conjugated secondary antibody incubation 30min, the working concentration of 1:1000. Then hybridization signals were detected using the ECL chemiluminescence kit [4].

    1.3.4 absorbance measurements

    Phospho Imager image analyzer (Molecular Dynamics, USA) strip was measured the absorbance (A value), the value of A represents the relative expression level of the protein.

    1.3.5 immunohistochemical method

    The tissue specimens are fixed in 4% formalin, embedded in paraffin. Ovarian carcinoma and normal ovarian tissue was cut into 5 μm thick, adhered on the slide, dried in air, and then in an oven heated to 60 ℃ for 2 h. Slice dewaxing in xylene by gradient ethanol hydration, inhibition of endogenous peroxidase with 0.3% hydrogen peroxide  methanol solution. Antigen Retrieval is carried out below: the slices in 10 mM citrate buffer (pH 6.0) for 10min after washing with PBS (pH7.4) buffer and the slices cooled, and then sliced ​​with 5% serum in the moist chamber at room temperature incubated 30 min to block non-specific binding sites. Sections at 4 ℃ overnight, diluted 1:100 were incubated with an anti-STAT3 and p  STAT3. DAB color hematoxylin, negative control with TBS to replace the primary antibody.

    1.3.6 Statistical Methods

    Application SPSS 12.0 software to complete p  SATA3, Cyclin D1, of Caspase  3 with various clinical and pathological features with t-test analysis using Pearson correlation analysis, P value of less than 0.05 was considered statistically significance.

    2 Results

    P  STAT3 in ovarian cancer tissue, Cyclin D1 and Caspase  3 protein expression level was significantly higher than that of normal ovarian tissue (tumor vs normal, P = 0.028,0.035,0.047), as shown in Figure 1. Ovarian cancer tissue of p  STAT3 and Cyclin D1 expression levels with FIGO stage (P <0.05). Pearson correlation analysis showed that of p  STAT3 and Cyclin D1 expression in ovarian cancer tissue was a linear correlation (r = 0.412, P <0.05), and p  STAT3 Caspase  3 expression in ovarian cancer tissue no correlation 0.05)。">(r = 0.127, P> 0.05).

    T: Ovarian Cancer Organization, N: normal ovarian tissue

    Figure 1 STAT3 pathway members expressed in ovarian cancer and normal ovarian tissue

    3 Discussion

    JAKs / STATs signal transduction pathway and cell proliferation, differentiation and apoptosis are closely related, the pathway abnormal activation can lead to abnormal cell proliferation and malignant transformation. Mammals STATs family consists of seven members: STAT1 ~ STAT4, STAT5a, STAT5b and STAT6. STAT3 as an important member of the STATs pathway, the expression and activation of myeloid leukemia, prostate cancer, head and neck squamous cell carcinoma and gastrointestinal cancer is closely related to [5, 6].

    STAT3 tyrosine kinase as upstream through the regulation of target genes to induce the expression of some of the key product to affect the occurrence of tumors, an important target gene products including Cyclin D1 and Caspase family members. Cyclin D1 (Cyclin D1) play an important role in the regulation of the cell cycle, usually combined with the cyclin-dependent kinase CDK4 or CDK6, control the process from G1 to S phase. The STATs can start with Cyclin D1 specific binding sites on the sub-combination of Cyclin D1 transcription regulation of the cell cycle [7]. Duan et al [8] found that the high p  STAT3 increased expression of Cyclin D1 expression in ovarian cancer cell lines, p  STAT3 cell lines with low expression of Cyclin D1 expression decreased. Caspase (Caspase) aspartic acid residues the Kitt sex cysteine ​​protease, plays a key role in the process of apoptosis. Has an important role in the enzymatic modification of Bcl  2 protein family, more than ten species have been found to belong to this protease family members, including Caspase 1 ~ 10, etc., while Caspase3 is an important member of this family [9 ]. The study found that p  STAT3, Cyclin D1 and FIGO staging Late related (P <0.05), Pearson correlation analysis showed that of p  STAT3 and Cyclin D1 expression in ovarian cancer tissues showed a linear correlation (r = 0.482, P < 0.05),其可能机制是失活状态的STAT3不能与Cyclin D1启动子结合,从而抑制">0.05), while of p  STAT3 Caspase 3 expression in ovarian cancer tissue was no correlation (r = 0.127, P> 0.05), the possible mechanisms inactivation of STAT3 can not with Cyclin D1 promoter binding, thus inhibiting Cyclin D1 expression, inhibition of cell proliferation.

    Short of STAT3 signaling pathway in ovarian cancer transcriptional regulation mechanism is unclear, STAT3 abnormal activation and apoptosis of ovarian cancer relationship needs to be further clear. Found in experimental animal models and clinical observation of tumor cell tolerance the chemotherapy with abnormally high levels of STAT3 and Caspase members, blocking STAT3 signaling pathway drug resistant tumor cells can induce apoptosis [8]. In-depth study of STAT3 signal transduction pathway mechanism may be theoretical and experimental foundation for the treatment of ovarian cancer [9, 10].