ZD6474 induces growth arrest and apoptosis of GIST-T1 cells, which is enhanced by concomitant use of sunitinib

Abstract

ZD6474 (Zactima, AstraZeneca, Macclesfield, UK) is an orally available, small-molecule inhibitor of vascular endothelial growth factor receptor-2 and epidermal growth factor receptor tyrosine kinases, with additional activity versus rearranged during transfection (RET). This study explored the effect of ZD6474 in gastrointestinal stromal tumor-T1 (GIST-T1) cells that possess a gain of function mutation in exon 11 of the c-KIT gene. ZD6474 induced growth arrest and apoptosis of GIST-T1 cells in association with blockade of c-Kit and its downstream effectors, including Akt and extracellular signal-regulated kinase (ERK). ZD6474 treatment also blocked the mammalian target of rapamycin (mTOR), which lies downstream of Akt and ERK. Interestingly, when ZD6474 was combined with sunitinib (SU11248; Sutent, Pfizer, Kalamazoo, MI, USA), a class III and V receptor tyrosine kinase inhibitor, the ZD6474-mediated growth inhibition was potentiated in association with further down-regulation of the mTOR targets p-p70S6K and p-4E-BP-1. The combination of ZD6474 and sunitinib should be investigated further.

Figure 1

ZD6474 induces growth arrest and apoptosis of GIST‐T1 cells. (A) MTT assay. GIST‐T1 cells were plated in 96‐well plates (2 × 10⁵ cells/mL) and cultured with various concentrations of ZD6474 (0.1–10 µM). After 3 days, cells were treated with MTT for 4 h, and absorbance was measured. Results represent the mean ± SD of three experiments performed in triplicate. (B) Colony forming assay. GIST‐T1 cells were cultured in 24‐well plates (500 cells per plate) with various concentrations of ZD6474 (0.1–5000 nM). Colonies (> 40 cells) were counted after 14 days of incubation. Results are expressed as a mean percentage relative to control plates containing 0.001% DMSO (control diluent). Each point represents a mean ± SD of three independent experiments, each performed in triplicate. (C) TUNEL assay. GIST‐T1 cells were plated in 24‐well plates and cultured either with or without ZD6474 (2 µM and 5 µM); 48 h later, apoptosis was measured by TUNEL assay. Results represent the mean ± SD of two experiments, each performed in triplicate. (D) Western blot analyses. GIST‐T1 cells were cultured in the presence of ZD6474 (0.1 or 2 µM). After 48 h, cells were harvested and proteins were extracted and subjected to Western blot analyses. The polyvinylidene fluoride membrane was probed sequentially with anti‐PARP, ‐Bcl‐2, ‐Bcl‐xL, ‐p53 and ‐β‐actin antibodies. Band intensities were measured using densitometry. Two repeated experiments yielded similar results.

Figure 2

RT‐PCR analyses of VEGF and VEGFRs in GIST‐T1 cells. Expression of c‐KIT, VEGF and three major receptors for VEGF family ligands, Flt‐1 (VEGFR‐1), KDR (VEGFR‐2) and Flt‐4 (VEGFR‐3) in GIST‐T1 cells was analyzed by RT‐PCR. HUVEC; human umbilical vein endothelial cells.

Figure 3

ZD6474 (ZD) inhibits auto‐phosphorylation of c‐KIT and its downstream effectors in GIST‐T1 cells. (A) c‐KIT. GIST‐T1 cells were cultured in the presence of various concentrations of ZD6474 (0.1–5 µM). After 48 h, cells were harvested and proteins were extracted. The c‐KIT protein was immunoprecipitated and subjected to Western blot analyses. The membrane was probed sequentially with the antiphosphotyrosine antibody (top) and the antic‐KIT antibody (bottom). (B) c‐KIT effectors. GIST‐T1 cells were cultured in the presence of ZD6474 (0.1–2 µM). After 48 h, cells were harvested and proteins were extracted and subjected to Western blot analyses. The membrane was probed sequentially with anti‐p‐STAT3, ‐p‐STAT5, ‐p‐Akt, ‐p‐ERK, ‐STAT3, ‐STAT5, ‐Akt, and ‐ERK antibodies. Band intensities were measured using densitometry. Two repeated experiments yielded similar results.

Figure 4

ZD6474 (ZD) blocked EGF‐stimulated tyrosine phosphorylation of EGFR and ERK in GIST‐T1 cells. GIST‐T1 cells were serum starved for 24 h and exposed to EGF (50 ng/mL) or ZD6474 (2 µM) individually or in combination. After 30 min, cells were harvested and proteins were extracted. (A) EGFR. EGFR protein was immunoprecipitated and subjected to Western blot analyses. The membrane was probed sequentially with the antiphosphotyrosine antibody (top) and the anti‐EGFR antibody (bottom). (B) ERK. Extracted proteins were subjected to Western blot analyses. The membrane was probed sequentially with anti‐p‐ERK (Tyr²⁰²/Tyr²⁰⁴) and ‐ERK antibodies. Band intensities were measured using densitometry.

Figure 5

ZD6474 (ZD) potentiates the effect of sunitinib (SU) in GIST‐T1 cells. (A) Combination index. GIST‐T1 cells were plated in 96‐well plates (2 × 10⁵/mL) and cultured with ZD6474 (0.1–5 µM) or sunitinib (0.1–100 nM) either alone or in combination. After 3 days, cell viability was measured by MTT assay. The combination index (CI) of ZD6474 and sunitinib was calculated using the median effect method. CI values less than 1 indicate synergy, a CI = 1 indicates an additive effect, and a CI greater than 1 indicates antagonism between the 2 agents. (B) Western blot analyses. GIST‐T1 cells were exposed to ZD6474 (0.1 or 2 µM). After 60 mins cells were harvested and cell lysates were prepared and subjected to Western blot analyses. The membrane was probed sequentially with anti‐p‐p70S6K (Thr³⁸⁹), anti‐p‐4E‐BP‐1 (Thr⁷⁰), anti‐p70S6K and anti‐4E‐BP‐1 antibodies. Two identical experiments yielded similar results. (C) Western blot analyses. GIST‐T1 cells were exposed to ZD6474 (1 µM) or sunitinib (10 nM) either alone or in combination. After 60 min, cells were harvested and cell lysates were prepared and subjected to Western blot analyses. The membrane was probed sequentially with anti‐p‐p70S6K (Thr³⁸⁹), anti‐p‐4E‐BP‐1 (Thr⁷⁰), anti‐p70S6K and anti‐4E‐BP‐1 antibodies. Two identical experiments yielded similar results.