Five-week-old female BALB/c nude mice were obtained from the Shanghai Experimental Animal Center of Chinese Academy of Sciences (Shanghai, China). trastuzumab, saracatinib == Introduction == Overexpression of human epidermal growth factor receptor-2 (HER2 or ErbB2), a member of the ErbB family of receptor tyrosine kinases (RTKs), is found in many solid tumors.1,2Trastuzumab, a humanized monoclonal antibody (mAb) directed against ErbB2, inhibits ErbB2-mediated signaling, induces Amyloid b-Protein (1-15) antibody-dependent cellular cytotoxicity, and prevents cleavage of the extracellular domain name of ErbB2.3,4Trastuzumab was approved by the US Food and Drug Administration (FDA) for clinical use for patients with ErbB2-overexpressing metastatic breast malignancy in 1998, and for ErbB2-positive metastatic gastric and gastro-esophageal junction cancer in 2010 2010. Despite the effectiveness of trastuzumab, the majority of trastuzumab-responsive patients develop resistance within one year of treatment initiation.5,6Thus, there is an urgent need to overcome trastuzumab resistance. The nonreceptor tyrosine kinase c-SRC (SRC) is usually overexpressed in a wide range of tumors.7The aberrant activation of SRC regulates multiple functions during tumor progression, such as apoptosis, proliferation, cell adhesion, cell migration and invasion, angiogenesis, and metastasis.8SRC interacts with multiple RTKs and facilitates RTK-mediated signaling.9-12The combination of SRC inhibitors and trastuzumab has been demonstrated to be effective in suppressing the in vitro and in vivo growth of trastuzumab-resistant breast cancer cell lines.13In gastric cancer, SRC activity has been found to be increased.14,15A recent study has shown that inhibition of SRC activity suppresses the in vitro and in vivo growth of gastric cancer cell lines.16However, the antitumor activity of trastuzumab plus SRC inhibitor in gastric cancer has not yet been reported. In this study, we investigated the antitumor effect of trastuzumab in combination with the SRC inhibitor saracatinib on both trastuzumab-sensitive and -resistant gastric cancer cell lines, and we examined its mechanism of action. == Results == == Characterization of trastuzumab-resistant NCI-N87 cell line == We treated the NCI-N87 cell line with 10 g/ml of trastuzumab for nine months to obtain trastuzumab-resistant sub-line NCI-N87R. NCI-N87R was significantly more resistant to trastuzumab Rabbit polyclonal to AK2 treatment than the parental cell line Amyloid b-Protein (1-15) both in vitro and in vivo (Fig. 1A and B). We found that the amount of EGFR-ErbB2 and ErbB2-ErbB3 heterodimers was enhanced in the NCI-N87R cell line compared with the parental cell line (Fig. 1C). NCI-N87R cells also showed an increase in EGFR and ErbB3 phosphorylation (Fig. 1D). The phosphorylation of AKT and MAPK was also markedly enhanced in the NCI-N87R cell line (Fig. 1D). Moreover, NCI-N87R cells showed increased phosphorylation of SRC at Tyr416 and of the SRC downstream target paxillin at Tyr118 (Fig. 1D). These results suggest that SRC activation and increased ErbB signaling may be acquired resistance Amyloid b-Protein (1-15) mechanisms. Physique 1.Characterization of trastuzumab-resistant gastric cancer cell line NCI-N87R. (A) MTS assay evaluating cell proliferation of NCI-N87 and NCI-N87R cell lines upon treatment with trastuzumab (10 g/ml). Error bars, SD ***P< 0.0001. (B) Tumor volume of NCI-N87 and NCI-N87R xenografts after treatment with control IgG, trastuzumab, saracatinib, or trastuzumab plus saracatinib. Data are shown as means SEM ***P< 0.0001, Mann-Whitney test. (C) Co-immunoprecipitation assay detecting ErbB2/EGFR and ErbB2/ErbB3 heterodimerization in the NCI-N87 and NCI-N87R cell lines. (D) Immunoblots comparing major cell signaling changes between NCI-N87 and NCI-N87R cell lines. == Trastuzumab and saracatinib synergistically inhibit the growth of both trastuzumab-sensitive and trastuzumab-resistant gastric cancer cell lines == We examined the inhibitory effects of saracatinib on NCI-N87 and NCI-N87R cell lines. The results showed that saracatinib suppressed the in vitro proliferation of these two cell lines in a dose-dependent manner (Fig. 2A). Remarkably, the antiproliferative activity of saracatinib was comparable in trastuzumab-sensitive and trastuzumab-resistant gastric cancer cell lines (Fig. 2A). Next, we evaluated and compared the ability of saracatinib and trastuzumab, either alone or in combination, to inhibit the in vitro growth of NCI-N87 and NCI-N87R cell lines. As shown inFigure 2B, saracatinib plus trastuzumab exhibited a significantly greater antiproliferative activity against NCI-N87 cells than either agent alone. Similar results were obtained with NCI-N87R cells (Fig. 2B). To further investigate whether the combination of saracatinib and trastuzumab is usually synergistic, we treated NCI-N87 and NCI-N87R cell lines with various clinically relevant concentration ranges of saracatinib and trastuzumab. Data were analyzed using the method of Chou and Talalay to establish.