SF1126 induces G1 arrest and inhibits proliferation. Cells were either untreated or treated with 40-μM SF1126 for 48 h, at which point they were fixed and stained with propidium iodide. DNA content was then analyzed by flow cytometry. Representative cell cycle profiles are shown for a SK-parental and SK-HRp2, and b BT-parental and BT-HRp3 cells. The percentages of c SK-parental and SK-HRp2cells, and d BT-parental and BT-HRp3 cells in G0/G1, S, or G2/M are shown. SF1126 induced cell cycle arrest at the G1 phase with reduced S phase (proliferating fraction) in all cells

SF1126 inhibits proliferation of parental and trastuzumab-resistant HER2-over-expressing breast cancer cells. a Parental SKBR3 HER2-overexpressing breast cancer cells (SK-parental), SKBR3-derived trastuzumab-resistant pool 2 (SK-HRp2) and clone 3 (SK-HRc3), and b parental BT474 HER2-overexpressing breast cancer cells (BT-parental), BT474-derived trastuzumab-resistant pool 2 (BT-HRp2) and pool 3 (BT-HRp3) were treated with twofold serial dilutions of SF1126 ranging from 7.5 to 120 μM for 6 days. Cell proliferation was measured using the MTS colorimetric assay, and is expressed as a percentage of untreated cells per line. Error bars represent standard deviation between six replicates. SF1126 inhibited the proliferation of parental and trastuzumab-resistant SKBR3 and BT474 HER2-overexpressing breast cancer cells

SF1126 inhibits PI3K signaling. a,b SK-parental and SK-HRp2, and c, d BT-parental, BT-HRp2 or BT-HRc3 cells were treated with 0, 10, 20, or 40-μM SF1126 for 30 min. Total protein lysates (50 µg) were immunoblotted for a, c phosphorylated serine 473 (p-S473) Akt, total Akt, phosphorylated Thr202/Tyr204 ERK1/2, and total ERK1/2, and for b, d phosphorylated threonine 308 (p-T308) Akt and total Akt. Actin served as a loading control. Parental and resistant SKBR3 cells showed reduced p-S473Akt with 40-μM SF1126 and reduced p-T308Akt with lower doses of 10–20 μM SF1126. BT-parental and resistant cells showed reduced p-S473Akt and p-T308Akt at 10–20 μM SF1126. ERK1/2 phosphorylation was not significantly altered by SF1126

SF1126 induces apoptosis of parental and trastuzumab-resistant cells. a SK-parental, SK-HRp2, BT-parental, and BT-HRp3 cells were either untreated or treated with 40-μM SF1126 for 48 h, at which point they were fixed and stained with propidium iodide. DNA content was analyzed by flow cytometry. The percentages of cells with sub-diploid DNA content are shown, with error bars representing standard deviation between duplicates. SF1126 increased the percentage of cells in sub-G1, consistent with induction of apoptosis. b Cells were untreated or treated with 20-μM SF1126 for 48 h, at which point the drug-containing media were removed and replaced with drug-free media. Cells were maintained for an additional 6 days, then stained with methylene blue and photographed with the Odyssey Imaging System. Experiments were done in duplicate at least two times. Representative cultures are shown for each group. SF1126 inhibited colony survival. c SK-parental and SK-HRp2cells, and d BT-parental and BT-HRp2 cells were treated with 0, 10, 20, or 40-μM SF1126 for 24 h. Total protein lysates (50 µg) were immunoblotted for PARP, cleaved caspase 3, and survivin. Actin served as a loading control. SF1126 induced cleavage of PARP and caspase 3, and caused downregulation of survivin. These results indicate that SF1126 promotes growth arrest and apoptosis of HER2-over-expressing breast cancer cells

Combination SF1126 and trastuzumab synergistically inhibit proliferation of trastuzumab-resistant cells. a SK-HRp2 and b BT-HRp3 cells were treated with 0, 3.25, 7.5, 15, or 30-μM SF1126 (SF); 0, 1, 2, 4, or 8 μg/mL trastuzumab (HCT); or combination SF + HCT. After 6 days, MTS colorimetric assays were performed to measure cell proliferation. Values are expressed as a percentage of untreated cells per line. Error bars represent standard deviation between six replicates. The combination of trastuzumab with SF1126 inhibited proliferation of SK-HRp2 and BT-HRp3 more than SF1126 alone. (Results of statistical analysis are shown in Table 1.) c SK-HRp2, SK-HRc3, BT-HRp2, and BT-HRp3 were plated in soft agar assays and untreated (U) or treated with trastuzumab (H) at 4 μg/mL, SF1126 (S) at 15 μM, or the combination of drugs (H + S) at these doses. After 2 weeks of maintaining cells on drugs, colonies were stained with crystal violet and counted under a microscope in four random non-overlapping fields. The average number of colonies was calculated and is shown as a percentage of the average number of colonies in the untreated group per line. Error bars represent standard deviation of colony numbers between selected fields. SF1126 alone reduced colony growth by 50–90%, and combination with trastuzumab achieved stronger growth inhibition than SF1126 alone