Figure 4

G28UCM shows cytotoxic activity in developed HER2 + and FASN + trastuzumab and lapatinib-resistant cells. A. Development of trastuzumab- and lapatinib-resistant AU565-derived cells. AU565 parental cells (with ○) and AU565 cells cultured for six months in 2 μM of trastuzumab (AU565TR, with ●) were both treated with 2 μM of trastuzumab for 2, 3, 6, 8 and 10 days. Cells were trypsinized and counted by trypan blue exclusion. Results are shown as the percentages of viable cells compared with untreated control cultures for each cell line and period-time. All experiments were repeated at least two times. AU565 parental cells (with ○) and AU565 cells cultured for one month in 3.5 μM and the next five months in 7 μM of lapatinib (AU565LR, with ●) were both treated in with different concentrations of lapatinib (1 to 10 μM) for 48 h. Circles represent the percentage of surviving cells after 48 h in lapatinib treatment, which was determined using an MTT assay. Results are expressed as percentage of surviving cells from three independent experiments performed in triplicate (mean ± SD). B. FASN and HER2 expression levels in parental and resistant cells. AU565 parental and resistant cells (AU565TR and AU565LR) were lysed for protein and immunoblotted for FASN, T-HER2, p-HER2. Blots were reprobed for β-actin as loading control. Gels shown are representative of those obtained from three independent experiments. C. Cytotoxicity in AU565 parental and resistant cells (AU565TR and AU565LR) following G28UCM treatment. AU565 parental and resistant cells were treated along with different concentrations of G28UCM (1 to 40 μM), trastuzumab (1 to 50 μM) or lapatinib (1 to 50 μM). Results represent IC30 values of G28UCM, trastuzumab and lapatinib in AU565 parental and resistant cells (AU565TR and AU565LR), which was determined using an MTT assay. Results are expressed as mean IC30 values ± SD from three independent experiments performed in triplicate.