Versican G3 Modulates Breast Cancer Cell Apoptosis Figure 6. Versican G3 modulated breast cancer cell apoptosis induced by chemotherapeutic drugs through activation of EGFR related signaling. a) 16104 G3- and vector-transfected MT-1, MDA-MB-468, 66c14, 4T07, and 4T1 cells were inoculated and cultured in 10% FBS/ DMEM medium in 96 well culture dishes for 12 hours. After cell attachment, cells were treated with 2 mM Docetaxel for 24 hours. Cell viability was tested by WST-1 assays. b) All cells were treated with 8 mM Doxorubicin, and subjected to WST-1 assays. c) All cells were treated with 10 mM Epirubicin, and subjected to WST-1 assays. Compared with vector control group, n = 6, * p,0.05, **p,0.01, analyzed with t-test. d) Treated with 40 mM C2-ceramide, 2 mM Docetaxel, 8 mM Doxorubicin, 10 mM Epirubicin, 15 mM Cyclophosphamide, or 30 mM Trastuzumab for 6 hours, G3- expressing and vector- expressing 66c14 cells were processed to lysates and subjected to immunoblotting with antibodies to pSAPK/JNK, SAPK/JNK, ERK2, pERK, and b-actin. e) G3-transfected and vector-transfected 66c14 cells (16104) were inoculated and cultured in 10% FBS/DMEM medium in 96 well culture dishes for 12 hours. After cell attachment, we added Docetaxel (2 mM), and EGF (20 ng/ml), AG 1478 (2.0 mM), PD 98059 (50 mM), or SP 600125 (100 nM) cultured for 24 hours. WST-1 Cell Survival Assays were used to analyze cell viability. f) G3-transfected and vector-transfected 66c14 cells (16104) were also treated with Doxorubicin (8 mM), and EGF (20 ng/ml), AG 1478 (2.0 mM), PD 98059 (50 mM), or SP 600125 (100 nM) cultured for 24 hours. Cell viability was analyzed by WST-1 assays. Compared with vector control group, n = 8, * p,0.05, **p,0.01, analyzed with t-test. doi:10.1371/journal.pone.0026396.g006 mediated signaling pathway [49,52,53]. Evidence that EGFR signaling promotes cell proliferation, cell survival and metastasis supports current efforts to identify approaches that inhibit this pathway [54,55,56]. Anti-EGFR immunotherapeutics in cancer treatment is undergoing intensive study [57]. The efficacy of Erlotinib and Gefitinib in treating breast cancer is currently being tested in various phases of clinical trials either as single agent treatment or in combination with other agents such as Docetaxel, Gemcitabine, Paclitaxel [58,59,60]. The overall efficacy of anti- EGFR treatments to date remains moderate and there is desire to Figure 7. The roles of epidermal growth factor-like motifs of versican G3 domain in modulating breast cancer cell apoptosis induced by chemotherapeutic drugs. a)16104 G3-, G3DEGF- and vector-transfected 4T07 were inoculated and cultured in 10% FBS/DMEM medium in 96 well culture dishes for 12 hours. After cell attachment, cells were treated with 40 mM C2-ceramide, 2 mM Docetaxel, 8 mM Doxorubicin, or 10 mM Epirubicin for 24 hours. Cell viability was tested by WST-1 assays. b) After treated with 2 mM Docetaxel or 8 mM Doxorubicin for 1 hour, samples were subjected to Annexin V assays. c) Treated with 2 mM Docetaxel or 8 mM Doxorubicin all cells were processed to lysates and subjected to immunoblotting with antibodies to pSAPK/JNK, SAPK/JNK, ERK2, pERK, GSK-3b (S9P), and b-actin. doi:10.1371/journal.pone.0026396.g007 PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e26396