Article Figures & Data
Figures
- Fig. 1.
A, growth-inhibitory effects of lapatinib in gastric and esophageal cancer cell lines. Cells were treated with lapatinib at ranges from 0.001 to 10 μmol/L. N87 and OE19 (two HER2-amplified cell lines) were the most sensitive cell lines to lapatinib. This data is the result of experiments repeated in triplicate. B, HER2 gene copy number was analyzed using FISH. Samples were counterstained with DAPI and scored as positive for amplification if they had either (A) greater than four copies of HER2/neu per chromosome 17 centromere in at least 20 cells, or had (B) uncountable clusters of signals suggestive of an amplicon in 20 or more cells. HER2/Neu amplification was seen in two cell lines: OE19 and N87. SNU1 is representative of nonamplification.
- Fig. 2.
Cell signaling after treatment with lapatinib and erlotinib in gastric cancer cell lines: all cell lines were treated with 1 μmol/L of lapatinib. A, no appreciable differences in EGFR expression were seen after treatment with lapatinib but pEGFR was decreased in the two most sensitive cell lines. No differences in total HER2 expression were seen after treatment with lapatinib but pHER2 was also decreased in the two most sensitive cell lines. B, downregulation of pAKT was seen in the most lapatinib-sensitive cell lines. Similarly, downregulation of pERK was seen in the most lapatinib-sensitive cell lines.
- Fig. 3.
Lapatinib induces G0-G1 cell cycle arrest and apoptosis in HER2-amplified gastric cancer. A, N87 and OE19 cells were treated with 1 μmol/L of lapatinib and cells were analyzed for DNA content by flow cytometry. The proportion of cells that undergo apoptosis is increased and is maintained both at 48 h and 5 d after treatment. B, N87 and OE19 both show an increase of the G0-G1 fraction after lapatinib treatment. No appreciable differences in the cell cycle are seen in SNU1 and SNU5 after treatment with lapatinib.
- Fig. 4.
The effects of lapatinib are distinct from the effects of anti-EGFR blockade by erlotinib or anti-HER2 blockade by trastuzumab. Cell signaling after treatment with erlotinib in gastric cancer cell lines: all cell lines were treated with 1 μmol/L of erlotinib. Some downregulation of pEGFR was seen in the most erlotinib-sensitive cell lines and no effect was seen on total EGFR expression. As in lapatinib-sensitive cells, downregulation of pERK was seen in the most erlotinib-sensitive cell lines. No effects in pHER2 or total HER2 expression were seen after treatment with erlotinib.
- Fig. 5.
The combination of lapatinib and trastuzumab is synergistic in HER2-amplified gastric cancer. A, in this experiment, the lapatinib concentrations ranged from 3.125 to 100 nmol/L for OE19 and 0.625 to 20 nmol/L for N87. For trastuzumab, doses ranged from 0.3125 to 10 μg/mL for both cell lines. Growth inhibition was calculated as a percentage of the untreated controls. Experiments were done in duplicate for each cell line. B, mean CI values for lapatinib-trastuzumab combinations in two HER2-amplified gastric cancer cell lines. Error bars indicate the 95% CI of the mean value. CI values were derived from variables of the median effects plots, and statistical tests were used to determine significance. In this analysis, synergy is defined as CI values significantly lower than 1.0, antagonism as CI values significantly higher than 1.0, and additivity as CI values equal to 1.0. Synergistic interactions were observed in both cell lines ranging from 0.228 (P < 0.0001) in OE19 to 0.226 (P < 0.0005) in N87. C, cells were treated with lapatinib at 100 nmol/L (OE19) or 20 nm (N87), trastuzumab (10 μg/mL) or the combination. Cells were analyzed for DNA content by flow cytometry. Both N87 and OE19 show a statistically significant increase in apoptosis with combination therapy than with either lapatinib or trastuzumab alone. There is also an increase in G0-G1 cell cycle arrest in the combination than with either lapatinib or trastuzumab alone. D, comparisons of signaling on downstream markers with lapatinib alone, trastuzumab alone or the combination. In this experiment, lapatinib treatment was at 100 nmol/L (OE19) or 20 nm (N87) combined with trastuzumab at 10 μg/mL. Minimal downregulation of pAKT, pERK are seen with lapatinib alone in both cell lines. Trastuzumab alone also has no effect on any of these markers. The combination of the two drugs causes a significant downregulation of pAKT and pERK in both HER2-amplified cell lines but is most pronounced in N87, the cell line with the highest level of amplification. No significant changes were seen with the combination in either pEGFR or pHER2 downregulation.
- Fig. 6.
Antitumor activity of lapatinib and trastuzumab on N87-bearing xenografts. N87 cells (5 × 106 cells with 50% Matrigel) were injected s.c. into nude mice and mice were randomized into one of four groups (n = 11/group). Treatment started at day 4 after injection with either lapatinib (100 mg/kg daily for 3 wk), trastuzumab (10 mg/kg i.p. twice weekly) or sterile PBS (i.p. twice weekly) or the combination of lapatinib and trastuzumab at the doses above. Tumors were measured biweekly. Student's t test was used to compare tumor sizes between the groups and results are presented as mean. Error bars represent SD of the mean. Student's t test was done at the end of the experiment. All comparisons were statistically significant between the following groups: combo versus L or T alone, P < 0.05; combo versus control, P < 0.001; L or T versus control, P < 0.001.
Tables
- Table 1.
Lapatinib and erlotinib concentrations that achieve IC50 and the corresponding k-Ras, and HER2 molecular status in gastric and esophageal cancer cells
Cell line Lapatinib IC50 (mean ± SE, μmol/L) Erlotinib IC50 growth (mean ± SE, μmol/L) Trastuzumab, growth inhibition (%) HER2 amplification status K-Ras mutation N87 0.01 ± 0.04 3.32 ± 0.37 15.50 ± 6.08 Amplified WT OE19 0.09 ± 0.02 2.31 ± 0.50 34.53 ± 5.20 Amplified WT NUGC4 0.35 ± 0.03 0.24 ± 0.04 0 Not amplified WT NUGC3 2.24 ± 0.55 0.70 ± 0.01 0 Not amplified WT FU97 4.86 ± 0.34 4.76 ± 0.96 0 Not amplified WT SNU16 8.58 ± 0.69 6.50 ± 1.31 0 Not amplified WT IM95 >10 >10 2.80 Not amplified WT IM95m >10 >10 7.85 Not amplified WT MKN74 >10 >10 10.60 Not amplified WT MKN1 >10 0.96 ± 048 5.83 Not amplified WT KATOIII >10 5.98 ± 0.98 4.18 Not amplified WT AGS >10 >10 5.01 Not amplified G12D (exon 1) SNU1 >10 >10 0 Not amplified G12D (exon 1) SNU5 >10 >10 0 Not amplified WT NOTE: Fourteen gastric and esophageal cancer cell lines were treated with lapatinib, erlotinib, and trastuzumab as described. Lapatinib and erlotinib reported as concentrations that achieve IC50 whereas the effects of trastuzumab are reported as a percentage of growth inhibited. HER2 amplification status and k-Ras mutation status of the cell lines as measured by FISH and PCR, respectively.
Volume 16, Issue 5
