The effects and mechanisms of exogenous phosphatase and tensin homolog deleted from chromosome ten (PTEN) gene on phosphatase activity-dependent apoptosis of breast cancer cell line MDA468 were investigated. PTEN gene packaged with lipofectin was transferred into breast cancer cell line MDA468 and parental MDA468 cells served as controls. RT-PCR and Western blot were done to detect the expression of target genes. The expression of phosphospecific protein kinase B (PKB/Akt) and focal adhesion kinase (FAK) protein stimulated by epidermal growth factor (EGF) was also detected. Apoptosis was determined by flow cytometry with a double-staining method using FITC-conjugated annexin V and PI. MDA468 cells transfected with PTEN gene could express PTEN mRNA and protein. PTEN decreased the phosphorylation level of AKT protein and down-regulated FAK protein expression in MDA468 stimulated by EGF. The apoptosis rate was 21.68%. PTEN induced breast cancer apoptosis phosphatase activity-dependently. The mechanism is possibly related with phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB)/AKT signaling pathway. Those results may provide new clues on the gene therapy in breast cancer.

The effects and mechanisms of exogenous phosphatase and tensin homolog deleted from chromosome ten (PTEN) gene on phosphatase activity-dependent apoptosis of breast cancer cell line MDA468 were investigated. PTEN gene packaged with lipofectin was transferred into breast cancer cell line MDA468 and parental MDA468 cells served as controls. RT-PCR and Western blot were done to detect the expression of target genes. The expression of phosphospecific protein kinase B (PKB/Akt) and focal adhesion kinase (FAK) protein stimulated by epidermal growth factor (EGF) was also detected. Apoptosis was determined by flow cytometry with a double-staining method using FITC-conjugated annexin V and PI. MDA468 cells transfected with PTEN gene could express PTEN mRNA and protein. PTEN decreased the phosphorylation level of AKT protein and down-regulated FAK protein expression in MDA468 stimulated by EGF. The apoptosis rate was 21.68%. PTEN induced breast cancer apoptosis phosphatase activity-dependently. The mechanism is possibly relatedwith phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB)/AKT signaling pathway. Those results may provide new clues on the gene therapy in breast cancer.

The recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cDNA was cloned into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-PTEN, then homologeous recombination was carried out in the E. coli BJ5183 by contransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy-1. The newly recombined defective adenovirus vector Ad-PTEN containing green fluorescent protein (GFP) was packaged and propagated in 293 cells. After being purified by cesium chloride gradient centrifugation, the adenovirus was transfected into human breast cancer cell line MDA-MB-468 in vitro. The expression of PTEN mRNA and protein in infected human breast cancer cell line MDA-MB-468 was detected by RT-PCR and Western blot respectively. The recombinant defective adenovirus vector carrying PTEN gene was constructed successfully. The viral titer of purified adenovirus was 2. 5 X 10(10) pfu/mL, and about 70% breast cancer cells were infected with Ad-PTEN when multiplicity of infection (MOI) reached 50. The exogenous PTEN mRNA and protein were expressed in MDA-MB-468 cells infected with Ad-PTEN by RT-PCR and Western blot. The recombinant defective adenovirus vector of PTEN gene was constructed successfully using AdEasy-1 system rapidly, which paved a sound foundation for gene study of breast cancer.

Circulating vascular endothelial growth factor-C (VEGF-C) and vascular endothelial growth factor (VEGF) levels in patients with colorectal carcinoma were determined in order to assess their clinical significance as a diagnostic tool for monitoring lymph node metastasis. In 66 patients with colorectal carcinoma and 30 healthy controls, circulating VEGF-C and VEGF levels were assessed by using enzyme-linked immunosorbent assay (ELISA). Serum VEGF-C and VEGF levels were higher in patients with colorectal carcinoma than in healthy controls. Patients with lymph node metastasis had higher serum VEGF-C and VEGF levels than those without lymph node metastasis. The levels of VEGF-C and VEGF were higher in the invasion group than in the non-invasion group. Serum VEGF-C levels reached a sensitivity of 81% and a specificity of 76% with a cutoff value of 1438.0 pg/mL, whereas VEGF levels reached 72% sensitivity and 74% specificity at 240.2 pg/ mL. If 66 patients were divided into 4 groups according to the combined determination of VEGF-C and VEGF levels, the positive predictive value was 85.3%, the negative predictive value was 94.6%, and accuracy was 93.7%. It was suggested that circulating VEGF-C levels might provide additional information for distinguishing the absence from presence of lymph node metastasis in patients with colorectal carcinoma. The combined determination of VEGF-C and VEGF levels could be used as an important index for preoperatively clinical stage of colorectal carcinoma.

The recombinant defective adenovirus vector carrying human PTEN tumor suppres sor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cDNA was cloned into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-PTEN, then homologeous recombination was carried out in the E. coli BJ5183 by contransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy-1. The newly recombined defective adenovirus vector AdPTEN containing green fluorescent protein (GFP) was packaged and propagated in 293 cells. After being purified by cesium chloride gradient centrifugation, the adenovirus was transfected into human breast cancer cell line MDA-MB-468 in vitro. The expression of PTEN mRNA and protein in infected human breast cancer cell line MDA-MB-468 was detected by RT-PCR and Western blot respectively. The recombinant defective adenovirus vector carrying PTEN gene was constructed successfully. The viral titer of purified adenovirus was 2.5×1010 pfu/mL, and about 70 % breast cancer cells were infected with Ad PTEN when multiplicity of infection (MOI) reached 50. The exogenous PTEN mRNA and protein were expressed in MDA-MB-468 cells infected with Ad-PTEN by RT-PCR and Western blot. The recombinant defective adenovirus vector of PTEN gene was constructed successfully using AdEasy-1 system rapidly, which paved a sound foundation for gene study of breast cancer.

Circulating vascular endothelial growth factor-C (VEGF-C) and vascular endothelial growth factor (VEGF) levels in patients with colorectal carcinoma were determined in order to assess their clinical significance as a diagnostic tool for monitoring lymph node metastasis. In 66 patients with colorectal carcinoma and 30 healthy controls, circulating VEGF-C and VEGF levels were assessed by using enzyme-linked immunosorbent assay (ELISA). Serum VEGF-C and VEGF levels were higher in patients with colorectal carcinoma than in healthy controls. Patients with lymph node metastasis had higher serum VEGF-C and VEGF levels than those without lymph node metastasis.The levels of VEGF-C and VEGF were higher in the invasion group than in the non-invasion group.Serum VEGF-C levels reached a sensitivity of 81% and a specificity of 76% with a cutoff value of 1438.0 pg/mL, whereas VEGF levels reached 72% sensitivity and 74% specificity at 240.2 pg/ mL. If 66 patients were divided into 4 groups according to the combined determination of VEGF-C and VEGF levels, the positive predictive value was 85.3%, the negative predictive value was 94.6%, and accuracy was 93.7%. It was suggested that circulating VEGF-C levels might provide additional information for distinguishing the absence from presence of lymph node metastasis in patients with colorectal carcinoma. The combined determination of VEGF-C and VEGF levels could be used as an important index for preoperatively clinical stage of colorectal carcinoma.

Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-kappaB and the expression of chemokine MOB-1, MCP-lgenes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTC in vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-kappaB was monitored by using electrophoretic mobility shift assay. The results showed that as compared with control group, M3 cholinergic receptor agonist (10(-3) mol/L, 10(-4) mol/L carbachol) could induce a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10(-3) mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-kappaB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10(-3) mol/L carbachol) in vitro for 30 min. Either M3 cholinergic receptor antagonist (10(-5) mol/L atropine) or NF-kappaB inhibitor (10(-2) mol/L PDTC) could obviously inhibit the activation of NF-kappaB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P<0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-lgenes in pancreatic acinar cells in vitro through the activation of NF-kappaB.

Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-kappaB and the expression of chemokine MOB-1, MCP-lgenes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTC in vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-kappaB was monitored by using electrophoretic mobility shift assay. The results showed that as compared with control group, M3 cholinergic receptor agonist (10(-3) mol/L, 10(-4) mol/L carbachol) could induce a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10(-3) mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-kappaB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10(-3) mol/L carbachol) in vitro for 30 min. Either M3 cholinergic receptor antagonist (10(-5) mol/L atropine) or NF-kappaB inhibitor (10(-2) mol/L PDTC) could obviously inhibit the activation of NF-kappaB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P<0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-lgenes in pancreatic acinar cells in vitro through the activation of NF-kappaB.
Adaptor Proteins, Signal Transducing ; Carbachol ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; Chemokine CCL2 ; biosynthesis ; genetics ; Chemokines ; biosynthesis ; genetics ; Humans ; NF-kappa B ; biosynthesis ; genetics ; Pancreas, Exocrine ; metabolism ; Pancreatitis ; etiology ; RNA, Messenger ; biosynthesis ; genetics ; Receptor, Muscarinic M3 ; agonists ; physiology ; Signal Transduction

To study the expression of vascular endothelial growth factor C (VEGF-C) in colorectal carcinoma and its relationship with lymph node metastasis, the expression of VEGF-C protein in colorectal carcinoma tissues obtained from 94 patients who underwent radical resection was immunohistochemically detected. Meanwhile, the expression of VEGF-C mRNA in 4 colorectal carcinoma cell lines was examined by reverse transcription polymerase chain reaction (RT-PCR). VEGF-C protein was found to be expressed in 53.2% of patients. The expression was more frequently detected in tumors with lymph node metastasis than in those without metastasis (P<0.01), and there was significant correlation between its expression and lymphatic invasion, TNM stage (P<0.01). However, no significant correlation was found between its expression and the age, gender, tumor location, depth of invasion and vascular invasion. 2 of the 4 colorectal carcinoma cell lines, including LoVo and LoVo-5FU, expressed VEGF-C mRNA. The expression of VEGF-C is closely related to lymph node metastasis, and it might take part in the tumor lymphangiogenesis.
Adenocarcinoma ; metabolism ; secondary ; Aged ; Carcinoma, Papillary ; metabolism ; secondary ; Colorectal Neoplasms ; metabolism ; pathology ; Female ; Humans ; Lymph Nodes ; pathology ; Lymphangiogenesis ; Lymphatic Metastasis ; Male ; Middle Aged ; RNA, Messenger ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor C ; biosynthesis ; genetics

The effects of estradiol and tamoxifen on the proliferation of estrogen receptor positive cells and the relationship between the tamoxifen tolerance and cell origin were investigated. The tissues of human endometrium and breast cancer were randomly selected following dissection for primary cell culture. After the breast cancer cells and endometrial cells were treated with 1 x 10(-8) mol/L estradiol and/or 1 x 10(-6) tamoxifen, 3H-labelled thymine nucleotide was used to trace the kinetics of cell proliferation. There was no significant difference in the inhibition on the human endometrial cells between tamoxifen-treated group (6.3%) and control group (6.4%), but tamoxifen could significantly inhibit the proliferation of the human breast cancer cells (45.84%) as compared with control group (52.72%). Moreover, tamoxifen could significantly stimulate the proliferation of tamoxifen resistant breast cancer cells (9.64%) as compared with control group (6.32%). Estradiol could significantly stimulate the proliferation of all the three kinds of cells as compare with control group. The combined use of estradiol and tamoxifen could inhibit the proliferation of the endometrial cells and breast cancer cells as compared with estradiol used alone, but on the tamoxifen resistant breast cancer cells, they could more significantly stimulate the proliferation than E2. It was concluded that E2 could stimulate the proliferation of these three kinds of cells. However, the inhibitive effects of tamoxifen on the proliferation of these cells were dependent on the estradiol.
Antineoplastic Agents, Hormonal/*pharmacology ; Breast Neoplasms/*pathology ; Cell Division/drug effects ; Cells, Cultured ; Drug Interactions ; Endometriosis/pathology ; Endometrium/*pathology ; Estradiol/*pharmacology ; Tamoxifen/*pharmacology ; Tumor Cells, Cultured