Figure 1.
Inhibition of leukemia cell proliferation by lapatinib.
K562 CML cells were left untreated or were treated with 0.1% dimethyl sulfoxide (DMSO, vehicle), DMSO with different doses of lapatinib (2.5, 5, or 10 µM), or 12-O-Tetradecanoylphorbol 13-acetate (TPA) 10 µM for 1 to 3 days as indicated. (A) Cell numbers for each treatment were counted using trypan-blue dye exclusion assay. (B) Relative numbers of viable cells were detected using MTT assay. (C–F) Inhibition of cell proliferation by lapatinib in leukemia cell lines, but not in primary CD14+ mononuclear or bone marrow cells. 1×105/ml CML MEG-01 (C), AML NB4 (D), HL-60 cells (E), K562, or 5×105/ml human CD14+ mononuclear and mouse bone marrow cells (F) were untreated (unTx), treated with DMSO vehicle (0), or DMSO with different doses of lapatinib (µM) for 2 (C), 3 (D–E), or 1–3 days (F). The raw data (B and F) or relative percentage of growth inhibition (C–E) were assessed and calculated using both trypan-blue dye exclusion and MTS assays (C–E) or MTT assays (F) as indicated in each figure as described in (A–B). Optical density (OD) values from DMSO-treated control cells were used as a standard (0% cell death), and the relative percentage of growth inhibition was calculated using the following method: [(mean OD values from DMSO-treated cells – mean OD values from drug-treated cells)/mean OD values from DMSO-treated cells]×100. The data are expressed as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (t-test) between lapatinib-treated and DMSO control cells.
Figure 2.
Induction of apoptosis by lapatinib in K562 and HL-60 cells.
(A) K562 or HL-60 cells were left untreated or treated with various concentrations of lapatinib or TPA as indicated for 1–3 days. Cells were collected and resuspended in propidium iodide (PI)-containing hypotonic buffer, and then the percentage of apoptotic cells with DNA ladders (hypodiploid cells) was analyzed by flow cytometry. The data are expressed as means ± SEMs. (B) K562 cells were left untreated or treated with lapatinib for 3 days; cells were then collected, resuspended in both AnnexinV and PI containing buffer, and analyzed by flow cytometry. The percentages of PI(+)/annexin(+) or PI(−)/annexin(+) cells are indicated in each figure. (C) Induction of both apoptotic and non-apoptotic cell death by lapatinib in K562 cells. After DMSO or 10 µM lapatinib treatment for 1–3 days for K562, or 3 days for HL-60, cells were split into two tubes and resuspended in PI-containing phosphate buffered saline (upper panel in left figure, total dead cells) or PI-containing hypotonic buffer (lower panel in left figure, apoptotic cells), respectively, for simultaneously detecting total dead cells without intact plasma membranes or apoptotic cells as described in (A). The graph in the right panel represents the data as: the percentage of total dead cells (empty bars) and the percentages of apoptotic cells (solid bars). After drug treatment for 3 days, HL-60 cells were attached on slides using cytospin apparatus and observed after staining with Liu's stain (right panel of 2C). (D) After DMSO, 2.5, 5 or 10 µM lapatinib treatment for 8 or 16 h, K562 cells were stained with both PI and DiOC6(3). The mitochondrial transmembrane potential of the cells was analyzed by flow cytometry. *P<0.05, **P<0.01, ***P<0.001 (t-test) between treated and DMSO control cells.
Figure 3.
Induction of autophagy in lapatinib-treated K562 cells.
(A) Induction of morphological changes by lapatinib in K562 cells. After drug treatment for 3 days, cells were attached on slides using cytospin apparatus and observed microscopically after staining with Liu's stain. Arrows indicate cells with giant contours, characteristic of megakaryocytic differentiation. (B) Expression profiles of autophagic factors in lapatinib-treated K562 cells. K562 cell lysates were prepared 3 days (for LC3) or as indicated (for Beclin-1) after DMSO, 5, or 10 µM lapatinib exposure, then, the amounts of LC3, Beclin-1, and actin were examined by immunoblotting using antibodies against the respective proteins. LC3-II is an autophagic marker. (C–E) Abrogation of lapatinib-induced sub G1, but not growth inhibition, by pancaspase inhibitor z-VAD-fmk. After K562 cells were treated with lapatinib alone or lapatinib plus 20 µM z-VAD-fmk for 48 h, growth inhibition (C), percentage of sub G1 cells (D), or LC3I to LC3II conversion (LC3I/II) (E) was assessed by MTS assay, flow cytometry, or immunoblotting as described in Fig. 2 or Fig. 3B. Increased LC3II formation is indicated by arrows. *P<0.05, ***P<0.001 (t-test).
Figure 4.
Rescue of K562 cells from lapatinib-induced cytotoxicity by the autophagy inhibitor 3-methyladenine (3-MA).
K562 cells were left untreated or were treated with various concentrations of lapatinib in the presence or absence of 1.25-mM or 2.5-mM 3-MA for 24 (A) or 48 h (B) as indicated in each figure. Relative amounts of viable cells were detected using the MTS assay, and the relative percentage of growth inhibition was calculated as described in Fig. 1. *P<0.05, **P<0.01 (t-test).
Figure 5.
Protection of K562 cells from lapatinib-induced cytotoxicity by knockdown of autophagy-related proteins.
After transduction with shRNA expression lentivirus as indicated in each figure, K562 cells were selected and kept in puromycin-containing medium. Cells were treated with DMSO or lapatinib for 72 (A) or 48 h (B), and then the relative percentages of growth inhibition were detected using the MTS assay (A, left panel and B, upper panel) or trypan blue exclusion assay (A, right panel and B, lower panel) and calculated as described in Fig. 1. Knockdown efficiency of ATG7, beclin-1, and ATG5-12 conjugates or the loading control, actin, were examined by immunoblotting using antibody against the respective proteins as described in Fig. 3. *P<0.05, **P<0.01, ***P<0.001 (t-test).
Figure 6.
Induction of megakaryocytic differentiation by lapatinib in K562 cells.
(A) After 10-µM lapatinib or 1-µM TPA treatment, K562 cells were collected, stained with anti-CD61-FITC, and the fluorescent intensity of FITC in the live cells was analyzed. The percentage of CD61 positive cells for each figure is indicated. (B) Data from separate experiments of drug exposure for 1–3 days in (A) are expressed as mean induction folds of CD61 positive cells as follows: percentage of CD61 positive cells (lapatinib or TPA)/percentage of CD61 positive cells (DMSO). (C) Data from separate experiments of lapatinib exposure for 1–3 days in (A) are expressed as percentage induction of mean fluorescence as follows: [mean fluorescence (lapatinib or TPA)/mean fluorescence (DMSO)]×100. *P<0.05 (t-test).