Lapatinib, a Dual Inhibitor of Epidermal Growth Factor Receptor (EGFR) and HER-2, Enhances Radiosensitivity in Mouse Bladder Tumor Line-2 (MBT-2) Cells In Vitro and In Vivo

Abstract

BACKGROUND The aim of this study was to evaluate the effect of lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR) and HER-2, on the radiosensitivity of murine bladder tumor line-2 (MBT-2) cells in vitro and in vivo. MATERIAL AND METHODS MBT-2 cells were pretreated with lapatinib at doses ranging from 200-1,000 nM for 30 min followed by radiation at doses ranging from 2.5-10 Gy for 30 min. A clonogenic assay (colony formation assay) assessed cell survival. Western blot measured phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated HER-2 (p-HER2) and the apoptosis marker, PARP. The C3H/HeN mouse tumor xenograft model underwent subcutaneous injection of MBT-2 cells; mice were divided into four groups, treated with lapatinib (200 mg/kg), radiation (15 Gy), a combination of both, and with vehicle (control). RESULTS Lapatinib pretreatment, combined with radiation, decreased MBT-2 cell survival, and suppressed radiation-activated levels of p-EGFR and p-HER-2. MBT-2 cells treated with a 10 Gy dose of radiation and 1000 nM of lapatinib showed combination index (CI) values of <1 indicating synergy. Increased expression of γ-H2AX, indicated increased apoptosis. In mice with tumor xenografts, a daily dose of lapatinib (200 mg/kg/day) for seven days combined with radiation on the fourth day suppressed tumor growth to a greater degree than radiation alone. CONCLUSIONS Lapatinib treatment enhanced the radiation sensitivity in an in vitro and in vivo murine bladder cancer model by decreasing radiation-mediated EGFR and HER-2 activation, and by causing DNA damage leading to cell apoptosis.

Figure 1

Results of the clonogenic assay (colony formation assay) following radiosensitization of the mouse bladder tumor line-2 (MBT-2) cells and treatment with lapatinib. (A) Quantitative results of the clonogenic assay (colony formation assay) assay for mouse bladder tumor line-2 (MBT-2) cells treated with lapatinib in combination with radiation. Lapatinib treatment doses ranged from 200–1,000 nM for 30 min followed by radiation doses ranging from 2.5–10 Gy. The cells were fixed after 7 days, and cell colonies (defined as >50 cells) were counted per well. The cell colonies for each dose were presented as a percentage of the control group. Results are the mean (n=3), ± standard deviation (SD). (B) The combination index (CI) for selected doses of lapatinib with radiation are calculated and plotted as a function of the MBT-2 cell fraction affected (Fa). A value of CI<1 indicates synergism.

Figure 2

Radiation treatment increased the levels of phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated HER-2 (p-HER2) at low and high doses in mouse bladder tumor line-2 (MBT-2) cells in a time-dependent manner. MBT-2 cells were exposed to radiation doses ranging from 2.5–10 Gy, followed by preparation of cell lysates. Western blot analysis shows a time-dependent increase in phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated HER-2 (p-HER2).

Figure 3

Lapatinib treatment down-regulated the radiation-mediated levels of phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated HER-2 (p-HER2) in mouse bladder tumor line-2 (MBT-2) cells in a dose-dependent manner. Pretreatment of mouse bladder tumor line-2 (MBT-2) cells with lapatinib (100 nM and 200 nM) for 24 h followed by radiation of varying doses range from 2.5–10 Gy. The cell lysates were obtained after 2 h. Western blots were performed for phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated HER-2 (p-HER2). β-actin was used as loading control. Lapatinib treatment down-regulated the radiation-mediated levels of p-EGFR, p-AKT, p-HER2 in MBT-2 cells in a dose-dependent manner.

Figure 4

Lapatinib treatment increased radiation-mediated apoptosis in mouse bladder tumor line-2 (MBT-2) cells. (A) MBT-2 cells pre-treated with lapatinib (200 nM) for 30 min followed by radiation at a dose of 10 Gy. The cell cycle distribution studies were done 6 h after cell treatment with lapatinib, and radiation in combination with lapatinib. Data presented is the mean (n=3) ±SD. * P<0.05. (B) MBT-2 cells pre-treated with lapatinib (50 nM and 100 nM) for 30 min followed by radiation at doses between 2.5–10 Gy. After 6 h, cell lysates were prepared. Western blot analysis was performed to detect the presence of the apoptotic marker, poly (ADP-ribose) polymerase (PARP).

Figure 5

Lapatinib increased radiation-mediated DNA damage in radiation-exposed mouse bladder tumor line-2 (MBT-2) cells. (A) The γ-H2AX count, representing radiation-induced DNA damage, was performed for 150 cells/group. The results are represented as the mean number of foci or cells per group with lapatinib (50 nM and 100 nM) for 30 min followed by radiation at doses between 2.5–10 Gy. (B) The MBT-2 cells received pretreatment with lapatinib (50 nM and 100 nM) for 30 min followed by radiation at doses between 2.5–10 Gy. The cell lysates underwent Western blot to detect phosphorylated γ-H2AX (p- γ-H2AX) and H2AX.

Figure 6

Lapatinib treatment combined with radiation resulted in an increased tumor suppressive effect in the mouse bladder tumor line-2 (MBT-2) xenografts. (A) The mouse tumor xenograft model was created by injecting MBT-2 cells subcutaneously in C3H/HeN mice and were divided into four groups, treated with lapatinib (200 mg/kg), radiation (15 Gy), and a combination of both, and with vehicle (control). Values are shown as mean tumor volumes per group. (B) The mice with tumors were sacrificed on the 8^th day. Photomicrographs of sections of the tumor tissues, treated with radiation, lapatinib, and combined radiation and lapatinib were recorded ×200 magnification, following immunohistochemical staining with primary antibodies for HER-2 and EGFR (A1–H1).