2010;70:8822C8831. was activated in response to lapatinib treatment to induce IL-6 expression. Furthermore, our data showed that microRNA-7 directly binds and inhibits Raf-1 3UTR activity, and that down-regulation of miR-7 by lapatinib contributes to the activation of Raf-1 signaling pathway and the induction of IL-6 expression. Our results not only revealed IL-6 as a key regulator of lapatinib-induced metastasis, but also explored the requirement of miR7/Raf-1/MAPK/AP-1 axis in lapatinib-induced IL-6 expression. < 0.01; ***: < 0.001; #: < 0.05 as compared with control group. Elevation of IL-6 contributes to the lapatinib-enhanced aggressiveness of TNBC cells Up-regulation of IL-6 has been reported to enhance the migration ability of various tumor cells [21, 26, 32]. In consistence with our previous findings [15], lapatinib-treated clones (231/Lap#6 and 231/Lap#12) showed higher migration ability than their parental cells in transwell migration assays (Physique ?(Figure2A).2A). We next examined whether IL-6 secretion is usually involved in lapatinib-induced migration ability of TNBCs through an autocrine regulation. To this end, 231/Lap#6 cells were incubated with or without specific anti-IL-6 antibody in the transwells. The results showed that their migration ability was dramatically inhibited by anti-IL-6 antibody (Physique ?(Figure2B).2B). To further confirm this obtaining, 231 and 231/Lap#6 cells were cultured for 24 GW9508 hrs, and then the incubated media were collected and defined as conditioned media 231-CM and 231/Lap#6-CM, respectively. As illustrated in Physique ?Determine2C,2C, these conditioned media were added into the lower chamber of transwells, and 231 cells in GW9508 serum-free medium were seeded in the upper chamber for migration assay. Incubation with 231/Lap#6-CM induced higher migration ability of 231 cells than that with 231-CM did (Physique ?(Figure2D)2D) and this effect was attenuated by anti-IL6 antibody (Figure ?(Figure2E).2E). Furthermore, the migration ability of 231 cells was also enhanced when recombinant IL-6 protein was added into 231-CM (Physique ?(Figure2F).2F). These results indicated that lapatinib enhances the migration ability of TNBC cells through IL-6 up-regulation. Open in a separate window Physique 2 IL-6 is usually involved in the lapatinib-enhanced aggressiveness of 231 cellA. In transwell migration assay, the 231 and 231/Lap cells were seeded in upper chamber with serum-free medium. The migrated cells through the membrane were stained with crystal violet and counted. B. The 231/Lap#6 cells were treated with or without anti-IL-6 antibody (30 ng/ml) in the upper chamber for Rabbit polyclonal to AHCYL1 24 hrs, and then migrated cells were counted in transwell migration assay. C. and D. As illustrated in (C), the incubated media from 231 cells and 231/Lap#6 cells were collected and defined as conditioned media 231-CM and 231/Lap#6-CM, respectively. These conditioned media were added into the lower chamber. The 231 cells were seeded in the upper chamber with serum-free medium. The migrated 231 cells was further analyzed and quantified. E. The 231/Lap#6 cells were treated with or without neutralizing IL-6 antibody (30 ng/ml). The lower chamber was filled with 231/Lap#6-CM. The migrated 231/Lap#6 cells were further analyzed and quantified. F. The 231 cells were treated with or without recombinant IL-6 protein (30 ng/ml) in the lower chamber of transwell migration assay. The quantitative results were expressed as mean S.E.M of three independent experiments. *: < 0.05; ***: < 0.001 as compared with control group. MAPK/AP-1 axis mediated lapatinib-induced IL-6 production in GW9508 TNBC cells We next investigated how the IL-6 expression is usually up-regulated GW9508 by lapatinib. Accumulating evidences showed that IL-6 expression is mainly controlled by MAPK pathway [33C35], and sustained activation of MAPK pathways was observed in response to lapatinib resistance in HER2-positive breast cancer cells [36]. We thus examined the involvement of MAPK pathways in the up-regulation of IL-6 expression in 231/Lap cells. As shown in Figure ?Determine3A,3A, activities of ERK1/2, p38, and JNK were higher in 231/Lap cells compare to 231 cells. Pharmacological inhibitors of individual MAPKs (PD98059, AZD6244 and U0126 for ERK1/2; SP600125 for JNK; SB203580 and SB202190 for p38) suppressed the IL-6 expression at both protein (Physique ?(Figure3B)3B) and mRNA levels (Figure ?(Physique3C),3C), suggesting that lapatinib induces IL-6 expression in part through activation of MAPK pathways. Open in a separate window Physique 3 Lapatinib induced.