[Inverse correlation between Snail and E-cadherin expression in carcinoma cell lines and invasive ability in vitro].

OBJECTIVE: Malignant transformation of epithelial cell frequently coincides with loss of E-cadherin. Here we study the expression of Snail and E-cadherin and correlate their expression with cell differentiation and in vitro invasion. METHODS: The expression and localization of Snail and E-cadherin were studied by Northern blot and laser confocal microscopy in two normal cell lines (MDCK, NIH 3T3) and six carcinoma cell lines (A431, MCF-7, MDA-MB-453, HepG2, MDA-MB-435s, MDA-MB-231). Boyden chamber assay was done to detect the invasive ability of cells in vitro. RESULTS: Snail mRNA and protein were detected in fibroblasts NIH 3T3 and poorly differentiated carcinoma cell lines HepG2, MDA-MB-435s and MDA-MB-231. On the contrary, E-cadherin mRNA and protein were detected in normal epithelial cell line MDCK and well differentiated carcinoma cell lines A431 and MDA-MB-453. In MCF-7 cells, Snail and E-cadherin expressions were revealed both at mRNA and protein levels. The cells with higher expression of Snail had stronger ability of invasion than those with lower expression of Snail. CONCLUSION: There is an inverse correlation between Snail and E-cadherin expressions and their expressions are correlated with cell differentiation and tumor invasiveness.

[High glucose stimulates synthesis of fibronectin in human mesangial cells via serum and glucocorticoid induced protein kinase-1 signaling pathway].

OBJECTIVE: To investigate the role of serum and glucocorticoid induced kinase-1 (SGK(1)) pathways in fibronectin (FN) synthesis in human mesangial cell (HMC) under high glucose condition and the mechanism by which SGK(1) contributes to glomerulosclerosis in diabetic nephropathy (DN). METHODS: HMCs were cultured and transfected with (P)IRES2-EGFP-(S422D) SGK(1) mutant (SD), plasmid containing SGK(1) dominant activation mutant, or blank plasmid. Non-transfected HMCs were used as control group. Then the HMCs were divided into 6 groups: transfected with SD + high glucose (SD-HG, 25 mmol/L D-glucose), transfected with FP + high glues (FP + HG), non-transfected + high glucose (NT-HG), transfeted with SD + normal glucose (SD-NG, 5.5 mmol/L D-glucose), transfected with FP + normal glues (FP + NG), and non-transfected + normal glucose (NT-NG). Eight hours after the glucose stimulation, RT-PCR was used to examine the SGK(1) mRNA expression and fibronectin (FN). Western blotting was used to detect the fibronectin (FN) protein expression. RESULTS: The SGK(1) mRNA expression of the SD + HG group was 0.709, significantly higher than those of the FP + HG and NT + HP groups (0.497 and 0.491, both P < 0.01). The SGK(1) protein expression of the SD + HG group was 1,178,497, significantly higher than those of the FP + HG and NT + HP groups (193,875 and 195,597 respectively, both P < 0.01). The FN mRNA expression of the SD + HG group was 0.749, significantly higher than those of the FP + HG and NT + HP groups (0.463 and 0.475 respectively, both P < 0.01). The FN protein expression of the SD + HG group was 659,550, significantly higher than those of the FP + HG and NT + HG groups (342,354 and 340,428 respectively, both P < 0.01). There were not significant differences in the expressions of FN mRNA and protein among different NG groups. CONCLUSION: SGK(1) may be involved in the signal transduction leading to the increase of fibronectin production in DN and therefore may play an active part in glomerulosclerosis in DN.

[Effects of chemokine receptor and its ligand on migration of ovarian cancer cells].

BACKGROUND & OBJECTIVE: Chemokine receptors express on many tumor cells, and closely correlate with migration and metastasis of tumor cells. This study was to investigate expressions of chemokine(C-X-C) receptor 4 (CXCR4) and chemokine (C-X-C motif) ligand 12 (CXCL12) in human ovarian epithelial tumor cells, and their effects on migration of tumor cells. METHODS: Expression of CXCR4 mRNA and protein in 15 specimens of epithelial ovarian cancer tissue, ovarian cancer cell line CAOV3, endothelial cell line HUVEC, and 10 specimens of normal ovary tissue were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot. Expression of CXCL12 mRNA in retroperitoneal lymph nodes, and smooth muscle of fallopian tube from the same 15 epithelial ovarian cancer patients was tested by RT-PCR, quantity of CXCL12 in ascites of 15 patients was assayed using ELISA. Boyden Transwells was used to analyze effects of CXCL12, and cancerous ascites on chemotaxis of CAOV3, and HUVEC cells. RESULTS: (1) Expression levels of CXCR4 mRNA in ovary cancer tissues, CAOV3 cells, and HUVEC cells were 2.30+/-1.12, 1.89+/-1.20, and 1.68+/-1.11, respectively; those of CXCR4 protein were 1.35+/-0.14, 1.86+/-0.34, and 1.96+/-0.23, respectively; CXCR4 mRNA and protein can't be detected in normal ovarian tissues. (2) In 15 ovarian cancer patients, concentrations of CXCL12 in ascites were 632-9 326 pg/ml, and CXCL12 mRNA level in retroperitoneal lymph nodes was 1.14+/-0.87, CXCL12 mRNA can't be detected in smooth muscle of fallopian tube. (3) Recombinant human CXCL12 induced migration of CAOV3, and HUVEC cells, the chemotactic indices (CI) were 3.9+/-1.2, and 4.1+/-1.6, significantly higher than those of control (1.0+/-0.4, and 1.1+/-0.7) (P<0.05)u cancerous ascites induced migration of CAOV3 cells with CI of 1.9+/-0.8, significantly higher than that of control (P<0.05). CONCLUSION: CXCR4 and CXCL12 may play roles in metastasis of epithelial ovarian cancer by promoting migration of tumor cells and endothelial cells.

[Effect of transcriptional factor snail on epithelial-mesenchymal transition and tumor metastasis].

BACKGROUND & OBJECTIVE: Transcription factor Snail mediates epithelial-mesenchymal transition (EMT), and is associated with tumor metastasis. This study was designed to observe the enhancive effect of Snail and the reverse effect of antisense-Snail on EMT of tumor cells, and explore the role of Snail in tumor metastasis. METHODS: Snail cDNA was transfected into canine renal epithelial cell line MDCK; antisense-Snail was transfected into human breast cancer cell line MDA-MB231. The expression of epithelial markers E-cadherin, beta-catenin and Cytokeratin 18, mesenchymal marker Fibronectin, metastasis-related marker matrix metalloproteinase-2 (MMP-2), and RhoA were detected by Western blot. The metastatic potential of tumor cells was examined by in vitro cell wound model and Boyden chamber invasion assay. RESULTS: The invasion potential of MDCK cells was enhanced after transfection of Snail. The expression of E-cadherin, beta-catenin, and Cytokeratin 18 was significantly lower in Snail-transfected MDCK cells than in control cells (P < 0.05); the expression of Fibronectin, MMP-2, and RhoA was significantly higher in Snail-transfected cells than in control cells (P < 0.05). Inhibiting the expression of Snail with antisense-Snail in MDA-MB231 cells led to opposite results. CONCLUSION: Snail promotes EMT in normal epithelial cells, and inhibiting the expression of Snail may reverse EMT and suppress tumor metastasis.

[Correlation of expression of RhoA (RhoC and their effector ROCK-1 with malignant phenotype of ovarian cancer cells in vitro].

OBJECTIVE: To investigate the expression of RhoA, RhoC and their effector ROCK-1 in four ovarian cancer cell lines in vitro and their correlation with invasiveness. METHODS: Expression of RhoA, RhoC and ROCK-1 mRNA and protein in four ovarian cancer cell lines SW626, Skov-3, A2780 and Caov-3 was detected by RT-PCR and Western blot assay. Invasion assay was done in Boyden chamber. RESULTS: The expression levels of RhoA, RhoC and ROCK-1 mRNA and protein varied in the four different cell lines examined. The expression level of RhoC, but not RhoA and ROCK-1, was significantly correlated with the invasive capability of these cells in vitro (r = 0.95, P < 0.01). Expression of RhoA at the level of transcription was not correlated with that at the translation level. The expression of RhoA and RhoC did not correlate with that of ROCK-1. CONCLUSION: Expression level of RhoC may serve as an independent parameter in evaluating metastasis and become a new target in inhibiting ovarian cancer metastasis.

Association between Nm23-H1 gene expression and metastasis of ovarian carcinoma.

BACKGROUND & OBJECTIVE: Metastasis is the leading cause of treatment failure and death of ovarian cancer. However, The molecular mechanisms associated with acquisition of metastatic ability in ovarian cancer are poorly understood. This study aimed at selecting the ovarian carcinoma cell lines with high frequency metastasis and studing the association between nm23-H1 gene expression in the model of ovarian carcinoma cells so as to provide the evidence for systematical experimental studying and clinical practice. METHODS: Each ovarian cancer cell line was transplanted subcutaneously into the flank of nude mice, and the metastatic behavior was evaluated by counting the number of lung tumor foci at different time. The metastatic tumors were cultured in vitro, then established substrain and transplanted subcutaneously three times. The mRNA and protein level of nm23 in 8 human ovarian cancer cell lines were examined. RESULTS: Four cell lines have high frequency metastatic potentiality. The subpopulation of cells with high frequency metastasis could be screened by injection several times. The expression of nm23 mRNA and protein in human ovarian cancer cells is inversely related to metastatic behavior in experimental animals (r=0.96, P=0.0001). CONCLUSION: The difference of metastatic potential, which was determined by genetic and molecular levels, was significant among different type of cell lines and subtypes. The expression of nm23 mRNA and protein in human ovarian carcinomas were correlated closely with the reduced metastatic behavior in experimental animals and may serve as a sensitive prognostic indicator of ovarian cancer.

[Decrease of chronic graft-versus-host disease by adding anti-human thymocyte globulin to the conditioning regimen].

OBJECTIVE: To observe the influence of adding anti-human thymocyte globulin (ATG) into conditioning regimen on graft-versus-host disease (GVHD) and life quality of the patients of allo-peripheral blood stem cell transplantation (PBSCT). METHODS: Patients were distributed into study (19 cases) and control (24 cases) groups at random. Median dose of rabbit ATG was added to the conditioning regimen based on the fludara, busufan and cyclophosphamide (FBC) in study group, and no ATG in the control group. Acute and chronic GVHD disease and Karnofsky scores were compared between two groups after allo-PBSCT. RESULTS: The patients in the study group received a mean of 6.0 (3 - 9) x 10(8)/kg mononucleated cells and 5.5 (4.5 - 7.5) x 10(6)/kg in the control group. The mean CD(34)(+) cells number was 5.5 (3.0 - 6.5) x 10(5)/kg in the study and 5.0 (3.0 - 7.0) x 10(6)/kg in the control group respectively. Eighteen patients in the study group and in the control group were successfully engrafted. The mean time of absolute neutrophil count recovered more than 500/ micro l was 13 days and 12 days respectively. Acute GVHD occurred in 6 patients of the study group, and 15 of the control group. Seven patients suffered from chronic GVHD and 14 got 90 Kanrofsky scores in a mean of 250 days follow-up in the study group, and 19 patients GVHD and 4 patients respectively in a mean of 440 days follow-up in the control group. There was a significant difference for acute and chronic GVHD and life quality between the two groups. CONCLUSIONS: Addition of anti-thymocyte globulin to the FBC conditioning regimen had no effect on stem cells engraftment but could decrease acute and chronic GVHD and improve patients life quality.

[Reversion of ovarian carcinoma metastasis by adeno-associated virus-mediated gene transfer of nm23H1 in orthotopic implantation model].

OBJECTIVE: To explore the feasibility of reverse effect of recombinant nm23H(1) adeno-associated virus (rAAV-nm23H(1)) in ovarian carcinoma metastatic orthotopic implantation nude model. METHODS: Using DNA recombination technique to construct AAV main plasmid pUF(1)-nm23H(1). rAAV-nm23H(1) and rAAV-Laz were produced by co-transfection of rAAV system in package cell 293 by phosphate-calcium deposit method, and then the transfection efficiency was measured. The titer was measured by dot hybridization. Ovarian carcinoma cell line SW626 was used in the establishment of the ovarian carcinoma orthotopic implantation nude model. The biologic feature of the model was observed and the expression of nm23H(1) in the tumor was measured by Western blot. Three groups of ovarian carcinoma orthotopic implantation nude model were applied by PBS (as control) (12 mice), rAAV-Laz (13 mice), rAAV-nm23H(1) (13 mice) intraperitoneally on the day 10 after transplantation. Then the effect of rAAV-nm23H(1) on survival and liver metastatic incident rates in models were observed. RESULTS: rAAV-nm23H(1) and rAAV-Laz were constructed and identified successfully. The titers were both 1 x 10(10) virus particles/ml. The transfection efficiency was 70%. The observation of biological feature showed the orthotopic implantation model was metastatic. The expression of nm23H(1) in AAV-nm23H(1) group was higher significantly than control and rAAV-Laz group (P < 0.05). Survival time of rAAV-nm23H(1) group was 136.67 days, compared with blank control group 106.67 days and rAAV-Laz group 107.06 days. Kaplan-Meier analysis showed rAAV-nm23H(1) group survived longer than control and rAAV-Laz group (P = 0.0051, P = 0.018 P < 0.05). The control and rAAV-Laz groups showed no statistically difference in survival time (P = 0.059 P > 0.05). The metastatic incident rate of control, rAAV-Laz and rAAV-nm23H(1) group was 75%, 61.5%, and 30.8% respectively, the rate of rAAV-nm23H(1) group was lower significantly than that of control and rAAV-Laz group (P = 0.03, P = 0.01). There was no significant difference between control and rAAV-Laz group in liver metastatic rate (P > 0.05). CONCLUSION: SW626 line which expressed lower level of nm23H(1) showed high-frequency metastasis properties, rAAV-nm23H(1) could reverse its metastasis in ovarian carcinoma orthotopic transplantation model.

[Correlation of expression of vascular endothelial growth factor and matrix matalloproteinase-2 to invasion of ovarian tumor cells in vitro].

BACKGROUND & OBJECTIVE: Vascular endothelial growth factor(VEGF) and matrix metalloproteinase-2 (MMP-2) are the important factors in tumor metastasis. To the author's knowledge, their relationship has not been addressed to date. This study was designed to investigate the in vitro invasion and the expression level of VEGF and MMP-2 in ovarian tumor cells, and to evaluate the correlation of expression of VEGF and MMP-2 in the ovarian tumor metastasis. METHOD: Boyden chamber in vitro invasion assay was used to detect the invasive capacity in vitro in two ovarian tumor cell lines CaOV-3 and COC1. The expression levels of VEGF and MMP-2 in CaOV-3 and COC1 were evaluated by semiquantitative RT-PCR and Western blot. The activity of MMP-2 in two cell lines was detected by substrate zymography. RESULTS: Boyden chamber in vitro invasion assay indicated that the mean invasion percentage of CaOV-3 cells (21.9 +/- 1.5) was significantly higher than that of COC1 cells (8.8 +/- 0.9) (P < 0.05). Contrast to COC1 cells, CaOV-3 cells expressed significantly higher levels of mRNA of VEGF and MMP-2 as well as protein by RT-PCR and Western blot. Substrate zymography showed that the MMP-2 activity of CaOV3 cells was two times than that of COC1 cells. CONCLUSION: The in vitro invasive ability of ovarian tumor cells appeared to be positive correlated to high expression of VEGF and MMP-2. There may be relationship between VEGF and MMP-2 in the process of metastasis of ovarian carcinoma.

[Monitoring impact of measurement of urinary beta-human chorionic gonadotropin on orthotopic implantation model of human ovarian carcinoma in athymic nude mice].

BACKGROUND & OBJECTIVES: Tumor orthotopic transplantation model has become the main carrier for tumor animal experiment. However, there was no convenient and reliable method to monitor tumor growth in animal body. This study was designed to evaluate the monitoring impact of urinary beta-human chorionic gonadotropin (beta-HCG) on tumor growth in orthotopic implantation model. METHODS: Pclone-beta-HCG stably transfected human ovarian carcinoma cell line A2780-CG was made as the orthotopic implantation model. The urinary beta-HCG/creatine ratio was continuously determined and plotted into curve, so as to indirectly know the rule of tumor growth in nude mice and the monitoring value of the ratio for intraperitoneal chemotherapy by cisplatin. RESULTS: The tumorigenesis time in beta-HCG system transfected tumor cell A2780-CG was similar to that in the original cell line. The content of urinary beta-HCG/creatine had a positive correlation to tumor weight in animal body (r = 0.98); After chemotherapy with cisplatin, the beta-HCG/creatine ratio showed progressively decreased. CONCLUSION: beta-HCG system could be used to sensitively and non-invasively monitor the tumor growth in orthotopic implantation model of human ovarian carcinoma in nude mice. Meanwhile, it provides a new convenient monitoring method for tumor treatment in the model.