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Volume was determined by the following equation, where L is length and W is width of the tumor: V was calculated as follows. Antitumor efficacy was determined by the percentage tumor growth inhibition.Five minutes later, all animals were euthanized by cervical dislocation under isoflurane anesthesia, and lung tissues were collected immediately and then subjected to imaging for another min.Fishers exact test was used to determine whether there was any difference in the incidence of lung metastasis between control and erlotinibTasosartan treated groups.A, SUM cells were transfected with empty vector or CAMEK for h.Western blot analysis was done to determine the level of activated ERK after CAMEK transfection.B, beginning hafter CAMEK transfection, SUM cells were treated with the indicated concentrations of erlotinib for another h, and then the proliferation inhibitory effect of erlotinib was quantified by WST assay.C, SUM cells were treated with control siRNA or ERK siRNA for h.Western blot analysis was done to determine the expression level of ERK after siRNA knockdown.The intensity ratio of ERK protein to its internal control band was relative to the ratio from the negative control siRNA, which was normalized as. D, beginning hafter siRNA knockdown, SUM cells were treated with the indicated concentrations of erlotinib for another h.Proliferation inhibitory effects of erlotinib were quantified by WST assay.We first tested the expression levels of EGFR and HER in two IBC cell lines, SUM and KPL.Western blot analysis showed that SUM cells have high expression of EGFR and low expression of HER and that KPL cells have high expression of both EGFR and HER. We then tested whether the EGFR pathway is intact in these two IBC cell lines by treating cells with EGF stimulation.Phosphorylation of EGFR was upregulated by EGF stimulation in both cell lines. Erlotinib inhibits proliferation and anchorageindependent growth of IBC cells, and this inhibitory activity of erlotinib is ERK dependent.Because EGFR siRNA knockdown inhibited IBC cell proliferation, we further studied the biological effect of EGFR TKI erlotinib on IBC cells.To study the effect of erlotinib on anchorageindependent growth of IBC, SUM and KPL cells were plated in soft agar and examined for differences in colony formation.We found that erlotinibtreated cells developed much fewer colonies in soft agar than did untreated cells. Because SUM cells have active EGFR Tasosartan pathways, we studied the role of ERK in SUM.We induced ERK activation by transiently transfecting CAMEK and then treated them with erlotinib.We found that ERK siRNA knockdown cells were more sensitive to erlotinib than control siRNA knockdown cells. Furthermore, inhibition of ERK activity by MEK inhibitors, PD and U, also sensitized SUM cells to erlotinib. The role of ERK in another IBC cell line, KPL, was also studied.We performed ERK siRNA knockdown in KPL cells and then treated them with erlotinib. We found that ERK siRNA knockdown cells were more sensitive to erlotinib than were controlsiRNA knockdown cells. Erlotinib inhibits the motility and invasiveness of SUM cells and reverses the mesenchymal phenotype of IBC cells in threedimensional culture.Because patients with IBC are at high risk for recurrence in the form of metastatic disease, we first examined the motility of SUM cells by transwell migration assay after erlotinib treatment.

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