RT - PCR 26106 cells were harvested, and total RNA was extracted with the Qiagen RNeasy mini kit. Two micrograms of total RNA were used to synthesize cDNA, a portion of which (equal to 0.2 mg RNA) was used in a PCR with two appropriate primers. PCR products were analyzed on agarose gel and detected using ethidium bromide staining as previously described [38,39]. Results Versican G3 domain enhanced tumor cell survival in serum free medium by up-regulating pERK and GSK-3b (S9P) A greater viability in low serum and serum-free conditions in the presence of versican G3 was observed in human breast cancer cells [19]. To investigate the expression of versican G3 domain on breast cancer cell survival, G3-transfected or vector-transfected 66c14 cells were cultured in serum free DMEM medium. G3 transfected cells grew faster than vector cells in the initial 4 days. After 4 days, a great number of vector cells floated in the medium, while the G3 transfected cells appeared well attached (Fig. 1a). Annexin V assays confirmed that cell death occurred through apoptosis (Fig. 1b). G3-transfected 66c14 cells showed a greater viability during 14 days of culture in serum free medium (Fig. 1c). Versican G3 domain enhanced mouse breast cancer cell line 66c14, 4T07 and human breast cancer cell line MT1 and MDA- MB-468 survival in serum free medium (Fig. 1d). However expression of G3 in 4T1 cell line, which is demonstrated to have high levels of endogeneous versican [20], didn’t change the cell proliferation significantly. Flow cytometer confirmed that the percentage of cells in S, G2 and M stages were much higher in G3- transfected cells than in vector cells (Fig. 1e). Immunoblotting indicated that versican G3 enhanced cell survival in serum free medium by increasing expression of pERK, GSK-3b (S9P) and CDK2 (Fig. 2a). Versican G3 enhanced cell survival could be prevented by selective EGFR inhibitor AG 1478 and selective MEK (ERK kinase) inhibitor PD 98059 (Fig. 2b). Immunoblotting showed that both AG 1478 and PD 98059 enhanced expression of pSAPK/JNK in G3 expressing cells, and partly prevented G3 enhanced expression of pERK. Whereas only PD 98059 blocked G3 enhanced expression of GSK-3b (S9P) (Fig. 2c). Selective JNK inhibitor SP 600125 enhanced expression of GSK-3b (S9P). Versican G3 enhanced breast cancer cell apoptosis induced by C2-ceramide through expression of pSAPK/ JNK and caspase-3 66c14 cells expressing versican G3 demonstrated lower cell viability compared with vector control groups when cultured in C2-ceramide (Fig. 3a, 3c, 3d). Annexin V assays confirmed that cell death occurred through apoptosis (Fig. 3b). C2-ceramide is a synthetic lipid, a potent apoptosis inducing substance that has been described as a second messenger of TNF and other stimuli. Immunoblotting showed that the G3 construct enhanced tumor cell apoptosis induced by C2-ceramide through expressing high levels of pSAPK/JNK and caspase-3 (Fig. 3e). During this procedure, G3-transfected cells expressed high level of pERK (Fig. 3e). Lower cell viability was also recorded in G3-expressing MT-1, MDA-MB-468, 4T07, and 4T1 cells after treatment with C2-ceramide (no significance in 4T1 cell line, Fig. 4a). To investigate whether versican G3 promotes cell apoptosis through the EGFR/JNK pathway, we cultured the G3- and vector- transfected 66c14 cells with C2-ceramide, EGF, AG 1478, PD 98059, or SP 600125. We found that versican G3 enhanced cell Versican G3 Modulates Breast Cancer Cell Apoptosis apoptosis induced by C2-ceramide, an observation inhibited by EGFR inhibitor AG 1478 and SAPK/JNK inhibitor SP 600125 (Fig. 4b). During treatment with C2-ceramide, G3-transfected cells expressed increased pSAPK/JNK and caspase-3, which were also induced by EGF, findings blocked by AG 1478 and SP 600125 but not by PD 98059 (Fig. 4c). SP 600125 also enhanced G3 transfected cells expression of GSK-3b (S9P) when treated with C2-ceramide (Fig. 4c). Versican G3 modulated effects on breast cancer cell apoptosis induced by chemotherapeutic agents through the activation of EGFR related signaling In order to investigate the effects of versican G3 domain on breast cancer cell apoptosis induced by chemotherapeutic drugs, we chose 5 frequently used compounds. Docetaxel is a clinically well established anti-mitotic chemotherapy medication used mainly for the treatment of breast, ovarian, and non-small cell lung cancer [40,41]. Doxorubicin and Epirubicin are anthracy- cline antibiotics and work through intercalating DNA strands that result in complex formation that inhibits DNA and RNA synthesis. They also trigger DNA cleavage by topoisomerase II, resulting in mechanisms that lead to cell death. Both agents are commonly used in the treatment of a wide range of cancers [42]. Cyclophosphamide, a nitrogen mustard alkylating agent, from the oxazophorines group [43] was also evaluated. Finally, Trastuzumab is a humanized monoclonal antibody that acts on the HER2/neu receptor and is used principally as an anti-cancer therapy in breast cancer patients whose tumors overexpress this receptor [44]. Analysis by light microscopy revealed that G3-transfected 4T07 cells showed increased cell apoptosis induced by Docetaxel, however, there was a reduction in cell apoptosis when treated with Doxorubicin, or Epirubicin. There was no appreciable difference between G3-transfected cells and the vector- cells after they were treated with Cyclophosphamide or Trastuzumab (Fig. 5a). Annexin V apoptosis assays confirmed that apoptosis was enhanced in G3 expressing cells when treated with Docetaxel, while apoptosis decreased when cultured with Doxorubicin and Epirubicin. WST-1 assays showed that versican G3- transfected MT-1, MDA-MB-468, 66c14, 4T07 cells expressed lower viability when treated with Docetaxel while higher viability was observed when cells were cultured in Doxorubicin and Epirubicin (Fig. 6a, 6b, 6c). However there is no significance for 4T1 cells when treated with Docetaxel, and also no significance for MDA-MB-468 when treated with Doxorubicin. The expression of endogenous versican probably makes the effect of function of exogenously expression of versican G3 not so obviously. Higher expression of versican in 4T1 cell line than other three mouse breast cancer cell lines supports above explanation [20]. MDA-MB-468, a human breast cancer cell line with a very high number of EGF receptors [45], shows less EGFR enhanced when trasfected with versican G3 domain. This may be the main reason why the G3 expressing MDA-MB-468 shows less chemical sensitivity to chemicals. Immunoblotting showed that G3-expressing cells increased p- ERK expression in the chemically treated and non-treated samples. When treated with C2-ceramide or Docetaxel, G3- expressing cells expressed a dramatically high level of pSAPK/ JNK, while Doxorubicin and Epirubicin did not significantly impact expression of pSAPK/JNK in G3-expressing cells (Fig. 6d). WST-1 Cell Survival Assays showed that versican G3 enhanced cell apoptosis induced by Docetaxel, an observation blocked by AG 1478 and SP 6000125 (Fig. 6e); it was also observed that cell apoptosis decreased in the presence of Doxorubicin, a finding blocked by AG 1478 and PD 98059 (Fig. 6f). PLoS ONE | www.plosone.org 3 November 2011 | Volume 6 | Issue 11 | e26396