Development of trastuzumab-resistant human gastric carcinoma cell lines and mechanisms of drug resistance

Trastuzumab has been successfully employed for the treatment of Her-2-positive gastric cancer. However, there are problems with both primary and secondary resistance to trastuzumab. In this study, we employed the human gastric carcinoma cell line NCI-N87 with high Her-2 expression to create trastuzumab-resistant NCI-N87/TR cells by stepwise exposure to increasing doses of trastuzumab. Western blotting and Real-time PCR were conducted to detect protein and gene levels. Compared with NCI-N87 cells, the expression of P-IGF-1R and P-AKT proteins was significantly increased in NCI-N87/TR cells (both P = 0.000), while PTEN gene and protein expression showed a significant decrease (both P = 0.000). In addition, mutations of the PTEN gene were detected at exons 5, 7, and 8. The sensitivity of NCI-N87/TR cells to trastuzumab was increased by transfection with the PTEN gene, or by incubation with a PI3K inhibitor (LY294002) or an IGF-IR inhibitor (AG1024), as well as siRNA targeting PI3K p110 or IGF-1R. Taken together, our findings showed that activation of the PI3K-AKT signaling pathway was one of the major mechanisms leading to resistance of NCI-N87/TR gastric cancer cells to trastuzumab, which was probably associated with PTEN gene down-regulation and mutation, as well as with over-activity of the IGF-1R signaling pathway.