Fig 1.
Lapatinib works in conjunction with Th1 cytokines to suppress metabolic activity of breast cancer cell lines.
Cultured SK-BR-3, BT-474 and MDA-MB-468 cells were treated either with Th1 cytokines (TNF-α plus IFN-γ; 20 and 12.5ng/ml respectively), Lapatinib (200nM), Lapatinib plus Th1 cytokines or left untreated (control). After 72 hours incubation, resazurin sodium salt was added to each well and further incubated until color change was noted, and optical densities of supernatants read spectrophotometrically at 630nM. Shown are composite data from at least three trials per cell line expressed as mean optical density +/- SEM. Statistical significance was determined by one-way ANOVA followed by the Holm-Sidak multiple comparison test.
Fig 2.
Lapatinib works in conjunction with Th1 cytokines to increase percentage of non-viable cells.
Cultured SK-BR-3, BT-474 and MDA-MB-468 cells were treated either with Th1 cytokines (TNF-α plus IFN-γ; 20 and12.5ng/ml respectively), Lapatinib (200nM), Lapatinib plus Th1 cytokines or left untreated (control). After 72 hours, cells were harvested, stained with Trypan Blue dye and percentage of viable and non-viable cells enumerated microscopically with the aid of a hemocytometer. Shown are composite data from three independent experiments with each cell line expressed as the percentage stained (i.e. non-viable) cells of total cells counted. Statistical significance was determined by one-way ANOVA on percent dead followed by the Holm-Sidak multiple comparison test.
Fig 3.
Lapatinib works in conjunction with Th1 cytokines to maximize indicators of apoptotic cell death.
A) Cultured SK-BR-3, BT-474 and MDA-MB-468 cells were treated either with Th1 cytokines (TNF-α plus IFN-γ; 20 and 12.5ng/ml respectively), Lapatinib (200nM), Lapatinib plus Th1 cytokines or left untreated (control). After 72 hours incubation, cells were treated with 100nMtetramethylrhodamine ethyl ester (TMRE) dye to estimate mitochondrial ΔѰ, as an indicator of apoptotic cell death, as assessed by flow cytometry (lower fluorescent intensity indicates disruption of mitochondrial ΔѰ as a consequence of apoptosis). B) Identically cultured and treated cells were harvested and stained with FITC-Annexin V and propidium iodide (PI) and subjected to FACS analysis. Two-color dot-plot analysis displays percentage of gated cells in each quadrant (double-staining cells represent late apoptotic events).
Fig 4.
Lapatinib and Th1 cytokines suppress HER-family RTK expression and phosphorylation status.
SK-BR-3 and MDA-MB-468 cells were treated with either Th1 cytokines (TNF-α plus IFN-γ; 20 and 12.5ng/ml respectively), 200nM Lapatinib, Th1 cytokines plus Lapatinib, or left untreated (control). A) After 72 hours incubation the cells were harvested, extracted in presence protease and phosphatase inhibitors, and 30μg/well total proteins separated on a 4–15% gradient SDS-PAGE gel prior to electrotransfer onto nitrocellulose membranes. Blots were then probed with anti-HER2, anti-phospho-HER2, anti-HER3 and anti-phospho-HER3 antibodies, and bands detected using chemiluminescence. B) After 24 hours, MDA-MB-468 cells treated as in (A) were supplied with caspase antagonist Z-DEVD-F. After 72 hours total culture cells were harvested and stained with EGFR antibody conjugated to PE/Dazzel 594 and analyzed for EGFR expression via flow cytometry.
Fig 5.
JIMT-1 and MDA-MB-453 cell lines display relative resistance to lapatinib.
Cultured SK-BR-3, JIMT-1, HCC1419, and MDA-MB-453 cells were treated with increasing concentrations of lapatinib (0–4μM). After 72 hours incubation, resazurin sodium salt (Alamar Blue dye) was added to each well and further incubated until color change was noted, and optical densities of supernatants read spectrophotometrically at 630nM. Shown are composite data from at least three trials per cell line expressed as mean optical density +/- SEM.
Fig 6.
Lapatinib resistant lines are resensitized to drug in the presence of Th1 cytokines.
A) Cultured SK-BR-3, HCC1419, JIMT-1, and MDA-MB-453 cells were treated either with Th1 cytokines (TNF-α plus IFN-γ; 20 and 12.5ng/ml respectively), Lapatinib (0–4μM), or Lapatinib plus Th1 cytokines. After 72 hours incubation, resazurin sodium salt was added to each well and further incubated until color change was noted, and optical densities of supernatants read spectrophotometrically at 630nM. B) Statistical analysis at 2μm concentration of lapatinib. Shown are composite data from at least three trials per cell line expressed as mean optical density +/- SEM. Statistical significance was determined by one-way ANOVA followed by the Holm-Sidak multiple comparison test.
Fig 7.
Lapatinib works in conjunction with Th1 cytokines to maximize indicators of apoptotic cell death in drug-resistant cell lines.
Cultured HCC1419, JIMT-1 and MDA-MB-453 cells were treated either with Th1 cytokines (TNF-α plus IFN-γ; 20 and 12.5ng/ml respectively), Lapatinib (2μM), Lapatinib plus Th1 cytokines or left untreated (control). After 72 hours incubationcells were harvested and stained with FITC-Annexin V and propidium iodide (PI) and subjected to FACS analysis. Two-color dot-plot analysis displays percentage of gated cells in each quadrant (double-staining cells represent late apoptotic events).
Fig 8.
Lapatinib does not suppress T lymphocyte activation or function.
A) IFN-γ ELISPOT assayof unfractionated human PBMCs from four unique donors stimulated with MHC class I recall peptides or tetanus toxoid protein in the presence of 2μM lapatinib. B) Composite data of the number of IFN-ɣ spot forming cells(normalized to control)from the same four donors. C) Flow cytometry analysis of allogenic DC:T cell co-cultures from three unique allogeneic stimulator:responder pairs with staining for CD4 (x axes) and CD69 (y axes). Percentage values indicate proportion of double-positive cells. D)ELISA analysis of IFN-ɣ production from four unique allogeneic stimulator:responder pairs in the presence or absence of lapatinib.