A soluble truncated cadherin induces breast cancer cell apoptosis and growth inhibition.

PURPOSE: To identify the characteristics and function of the truncated cadherin cDNA which encodes a soluble molecule containing the sequence of VE-cadherin extracellular domain repeats from repeat 1 to 4 (designated as CED1-4) and a secreting signal peptide at N terminal. METHODS: A pMSCV/CED1-4 vector was constructed. Recombinant retrovirus ReCED1-4 and ReEmpty were produced by 293 package cells and transfected into MDA-MB435 human breast cancer cells. The expression of CED1-4 in transfectants and their supernatant was analyzed by RT-PCR and Western blot, respectively. MDA-MB435 cell proliferation assays were performed in vitro and in vivo. CED-14-induced apoptosis was demonstrated using Annexin V binding, TUNEL and caspase 3 assays. The expression of integrin beta1 and c-fos mRNA was detected by RT-PCR. RESULTS: The constructed soluble CED1-4 encoded 484 amino acids and a secreting signal peptide (27 amino acids). CED1-4 was expressed by MDA-MB435/CED1-4 cells, and detected in the supernatant of CED1-4 tranfectants. CED1-4 transfection significantly inhibited the growth of MDA-MB435 cells in vitro and in vivo. About 22-fold increase in the early apoptotic cells in MDA-MB435/CED1-4 cells was observed as compared with MDA-MB435/empty cells. Increased activity of caspase 3 in MDA-MB435/CED1-4 cells was more than two times as compared with that of the control cells. Interestingly, integrin beta1 transcriptional level in MDA-MB435/CED1-4 cells was down-regulated as compared with control cells. The resistance of fibronectin to CED1-4 apoptotic inducibility was confirmed by detection of caspase 3. The blockage of c-fos transcriptional expression was detected in MDA-MB435/CED1-4 cells. CONCLUSIONS: The soluble truncated cadherin may be considered an apoptotic inducer and growth inhibitor in the MDA-MB435 breast carcinoma cell line. Down-regulation of integrin beta1 and blockage of c-fos expression may be related to CED1-4-induced apoptosis and growth inhibition.

Anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection.

BACKGROUND: This study was designed to obtain a recombinant retroviral vector containing the human hepatocellular carcinoma-related gene ANGPTL4 (angiopoietin-like 4) cDNA and to evaluate the anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection. METHODS: ANGPTL4 cDNA was cloned in vitro from normal human liver cells HL-7702 by using RT-PCR, and then subcloned into the plasmid vector pMSCV and sequenced. The retroviral plasmid vectors pMSCV-ANGPTL4, pVSV, and pGAG-POL were co-transfected into the packaging cell line 293 EBNA under mediation of lipofectamine. A high-titer retrovirus was obtained as a result, and HepG2 cells were infected with this retrovirus in vitro. Flow cytometry and fluorescence microscopy were used to detect expression of green fluorescence protein (GFP). The expression of ANGPTL4 mRNA in HepG2-ANGPTL4 cells was investigated using RT-PCR. The formation of tumors in nude mice and MTT assays were used to detect the growth of HepG2-ANGPTL4 cells in vivo and in vitro, respectively. RESULTS: The cDNA sequence of the cloned ANGPTL4 gene was consistent with the recently reported sequence. Thus, the recombinant retroviral vector pMSCV-ANGPTL4 was constructed successfully. The titer of the packaged recombinant retrovirus was 1.4 x 10(6) infective viral grains/ml, and the rate of HepG2-ANGPTL4 cells expressing GFP was 68.45%, with an average intensity of fluorescence 31.67 times greater in HepG2-ANGPTL4 cells than in HepG2 cells. The expression of ANGPTL4 mRNA in HepG2-ANGPTL4 cells was higher than in HepG2-pMSCV cells (154% higher) or HepG2 cells (161% higher). The proliferation rate of HepG2-ANGPTL4 cells in vitro was obviously lower than those of HepG2-pMSCV cells and HepG2 cells (P <0.01). The mean volume and weight of tumors seeded from HepG2-ANGPTL4 cells were obviously lower than the mean volume or weight of tumors seeded from HepG2 cells and HepG2-pMSCV cells (P <0.01). CONCLUSION: A stable ANGPTL4-transfected human liver cancer cell line HepG2-ANGPTL4 has been created. The transfer of the human ANGPTL4 gene mediated by a retroviral vector is a possibly effective approach for liver cancer therapy.

[Tyrosine kinases inhibitor A77 1726 inhibits upregulation of collagen generation in fibroblasts induced by IL-13].

AIM: To observe the inhibitory effects of tyrosine kinases inhibitor A77 1726 on collagen generation induced by IL-13 in fibroblasts. METHODS: The inhibition rate of fibroblast proliferation with different concentration of A77 1726 was observed by MTT method. The fibroblasts were divided into the experimental group (A77 1726 50 micromol/L and IL-13 100 microg/L) and the control group (IL-13 100 microg/L). After 24, 48 and 72 hours, the inhibitory effects of A77 1726 on collagen secretion of fibroblasts investigated by hydroxyproline release assay. The inhibitory effects of A77 1726 on collagen type I alpha1 gene mRNA expression in fibroblasts were examinated by RT-PCR. The influence of A77 1726 on collagen type I synthesis in fibroblasts was analyzed by Western blot. RESULTS: The proliferation of fibroblasts was inhibited by A77 1726. Total collagen generation was down-regulated after 48 h and 72 h stimulation of A77 1726 (P<0.05). The expression level of collagen type I alpha1 gene mRNA was obviously lower in the experimental group than in the control group after 48 h and 72 h stimulation of A77 1726 (P<0.05). Collagen type I production of fibroblasts treated with A77 1726 for 48 and 72 hours was decreased obviously in the experimental group than in the control group (P<0.05). CONCLUSION: Tyrosine kinases inhibitor A77 1726 has an inhibitory effect on collagen protein synthesis in fibroblasts induced by IL-13.

[Effects of interleukin 13 on the differentiation and expression of transcription factor c-fos of HEL cells].

OBJECTIVE: To study the effects of IL-13 on the differentiation and expression of transcription factor c-fos of human erythroleukemia cell line (HEL) cells. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was used to observe the mRNA expression of IL-13 receptor a 1, GP i b, vWF and c-fos, and Western blot and cytometry were used to analyse their protein expression. RESULTS: IL-13 receptor a 1 was expressed on HEL cells. IL-13 (100 ng/ml ) up-regulated the mRNA expression of GP II b and vWF. The ratio of luminous absorption (LA) of GP I b to p-actin bands ( AB) was 1. 303 in control group, whereas was 2. 912 in experiment group; being 2. 23-fold higher than that in control group (P < 0. 05). The ratio of LA to AB for vWF was 0.217 in control group, and 0. 506 in experiment group; indicating a 2. 33-fold increase in experiment group (P <0. 05). The protein expression of GP I b and vWF was significantly increased in experiment group, compared with that in control group. IL-13 inducing the increased expression of c-fos mRNA and protein of HEL cells peaked at 30 min and 60 min, respectively. The ratio of LA to AB for c-fos was also increased at 30 min and 60 min (P <0. 05). CONCLUSION: IL-13 prompts the differentiation of HEL cells and up-regulates the expression of c-fos.

[Effects of MAPK antagonist on TPO stimulated UT2 cells proliferation and differentiation].

OBJECTIVE: To explore the effects of MAPK antagonist on TPO stimulated UT7 cell proliferation and differentiation, and to elucidate the mechanism of TPO functioning on UT7 cells. METHODS: EGFP pMSCV and MEK 1 pMSCV MEK 1 plasmids were transferred into UT7 cells. Phosphorylated MEK1 of UT7 cells was examined by Western blot. The proliferation and CD41 expression of UT7 cells transfected with mutant (ser222A) MEK1 or exposed to PD98059 were examined. RESULTS: (1) 60.73% EGFP positive cells were obtained in retroviral vector MEK1 pMSCV transfected UT7cells. (2) In different time of TPO stimulating UT7 cells, the level of phosphorylated MEK1 was lower in experiment group than in control group. In experiment group, the level of phosphorylated MEK1 was decreased after stimulated by TPO for 1 hour, and almost disappeared after stimulated for 3 hours. (3) The effect of TPO on UT7 cell proliferation was inhibited by PD98059 and the transfected mutation MEK1 gene. The proliferation rate was 98.58% in DMSO control group, 39.00% in PD98059 group (P < 0.05), 102.13% in EGFP pMSCV group, and 48.94% in MEK1pMSCV (P < 0.05). (4) The CD41 expression on UT7 was inhibited by PD98059 and the transfected mutation MEK1 gene. CONCLUSION: Phosphorylation of MEK1 in UT7 cells can be induced by TPO. There was a relationship between the TPO stimulating time and phosphorylation of MEK1. The effects of TPO on UT7 cell proliferation and CD41 expression is mediated by MAPK signal transduction pathway.