Fig 1: In vivo analysis of the inhibitory effect of ATF-Fc, trastuzumab and ATF-Fc plus trastuzumab on SK-BR-3 cell proliferation. A SK-BR-3 human breast cancer xenograft BALB/C (neu) nude mouse model was established. (A) The curve represents the change in tumor volume following treatment with ATF-Fc, trastuzumab or ATF-Fc plus trastuzumab. The ATF-Fc (10 mg/kg) in combination with trastuzumab (6 mg/kg) significantly inhibited SK-BR-3 cell growth. After 30 days, the tumor volume in the group treated with ATF-Fc plus trastuzumab was significantly decreased, as compared with in the control group and in the groups treated with ATF-Fc or trastuzumab alone (*P<0.05 and **P<0.01) (B) Change in tumor weight following treatment of the nude mice with ATF-Fc, trastuzumab or ATF-Fc plus trastuzumab. Treatment with ATF-Fc plus trastuzumab significantly reduced the SK-BR-3 tumor volume compared with ATF-Fc alone, trastuzumab alone and the control. ATF-Fc, amino-terminal fragment of uPA -Fc fragment of human immunoglobulin G1.
Fig 2: Effect of ATF-Fc and trastuzumab on apoptosis of SK-BR-3 cells. Apoptosis was analyzed using Annexin-V in the SK-BR-3 cells following treatment for 72 h. Flow cytometry was used to determine the percentage of cells positive for annexin-V and/or 7-AAD. (A) Representative dot plots of three independent analyses. Early apoptotic cells were identified as Annexin-V positive and 7-AAD negative. Late apoptotic cells were identified as positive for Annexin V and 7-AAD staining. (B) The apoptotic rate of tumor cells was significantly increased in the ATF-Fc (50 µg/ml) plus trastuzumab (10 mg/ml) treatment group, as compared with in the ATF-Fc (50 µg/ml) or trastuzumab (10 mg/ml) alone groups (**P<0.01). 7-AAD, 7-amino-actinomycin D; ATF-Fc, amino-terminal fragment of uPA-Fc fragment of human immunoglobulin G1; FITC, fluorescein isothiocyanate.
Fig 3: Expression of uPAR and HER-2 in various cell lines. (A) The uPAR expression levels in EC-109, MCF-7 and SK-BR-3 cells were determined by flow cytometry. The uPAR expression rate was 6.5, 56.32 and 69.87%. (B) The SK-BR-3 cells exhibited significantly increased levels of expression compared with EC-109 cells (**P<0.01). (C) The HER-2 protein expression was determined via immunohistochemistry in EC-109, MCF-7 and SK-BR-3 cells. HER-2 was not present in EC-109 and MCF-7 cells. By contrast, the SK-BR-3 cells exhibited brown staining, indicating positive expression. (D) The HER-2 gene expression was detected by fluorescence in situ hybridization. The SK-BR-3 cells exhibited positive amplification with a HER-2/CEP-17 ratio of 4.38, whereas the EC-109 and MCF-7 cells were negative for amplification and had HER-2/CEP-17 ratios of 1.41 and 1.26, respectively. FITC, fluorescein isothiocyanate; HER-2, human epidermal growth factor receptor; uPAR, urokinase plasminogen activator receptor; uPA, urokinase-type plasminogen activator.
Fig 4: Extracellular level of uPA in EC-109, MCF-7 and SK-BR-3 cells. The extracellular level of uPA in EC-109, MCF-7 and SK-BR-3 cells was determined via ELISA. Each experiment was repeated three times. The data are presented as the mean ± standard deviation. The uPA expression levels in the three types of cells were 12.32±1.18, 63.77±9.26 and 78.62±6.09 pg/ml, respectively. The SK-BR-3 and MCF-7 cells exhibited significantly increased levels of uPA expression compared with EC-109 cells (**P<0.01). uPA, urokinase-type plasminogen activator.
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