Imatinib enhances docetaxel-induced apoptosis through inhibition of nuclear factor-κB activation in anaplastic thyroid carcinoma cells

Abstract

Background: We previously reported the partial effectiveness of imatinib (also known as STI571, Glivec, or Gleevec) on anaplastic thyroid cancer (ATC) cells. Imatinib is a selective tyrosine kinase inhibitor that has been used for various types of cancer treatments. Recently, several reports have demonstrated that imatinib enhanced the sensitivity of cancer cells to other anticancer drugs. In this study, therefore, we investigated whether imatinib enhances the antitumor activity of docetaxel in ATC cells.

Methods: Two ATC cell lines, FRO and KTC-2, were treated with imatinib and/or docetaxel. Cell survival assay and flow cytometry for annexin V were used to assess the induction of apoptosis. Changes of pro- and antiapoptotic factors were determined by Western blot. Nuclear factor-κB (NF-κB) activity was measured by DNA-binding assay. Tumor growth was also investigated in vivo.

Results: The combined treatment significantly enhanced apoptosis compared with single treatment. ATC cells themselves expressed high levels of antiapoptotic factors, X-linked inhibitor of apoptosis (XIAP), and survivin. The treatment with docetaxel alone further increased their expressions; however, the combined treatment blocked the inductions. Although imatinib alone had no effect on NF-κB background levels, combined treatment significantly suppressed the docetaxel-induced NF-κB activation. Further, the combined administration of the drugs also showed significantly greater inhibitory effect on tumor growth in mice xenograft model.

Conclusions: Imatinib enhanced antitumor activity of docetaxel in ATC cells. Docetaxel seemed to induce both pro- and antiapoptotic signaling pathways in ATC cells, and imatinib blocked the antiapoptotic signal. Thus, docetaxel combined with imatinib emerges as an attractive strategy for the treatment of ATC.

FIG. 1.

Cytotoxic effect of docetaxel and/or imatinib on anaplastic thyroid cancer (ATC) cells. Growth of FRO and KTC-2 cells treated with 1 nM of docetaxel and/or 7 μM of imatinib for 2 and 4 days was determined by cell count assay. Each point represents mean±standard deviation. *p<0.01 versus any other group. Similar results were obtained in three independent experiments.

FIG. 2.

Apoptotic changes in cells treated with drugs. FRO cells were treated with the drugs (1 nM docetaxel and/or 7 μM imatinib) for 16 hours and then double stained with fluorescein isothiocyanate–conjugated annexin V and propidium iodide (PI), then analyzed on an FACS Vantage SE. Fluorescein isothiocyanate and PI emissions were detected in the FL-1 and FL-3 channels, respectively. The cells in left lower, right lower, and right upper quadrant represent viable cells, early apoptotic cells, and terminal stage of apoptotic or necrotic cells, respectively. Data are representative of two independent experiments.

FIG. 3.

Effects of docetaxel and imatinib on apoptotic factors. Cells were treated with the drugs (4 nM docetaxel and/or 7 μM imatinib) for 16 hours [X-linked inhibitor of apoptosis (XIAP) and survivin] or 24 hours [caspase 3 and poly (ADP-ribose) polymerase (PARP)], and whole-cell lysates were examined by Western blotting. β-Actin was used as a loading control. Data are representative of at least two independent experiments.

FIG. 4.

Docetaxel induces nuclear factor-κB (NF-κB) activation, and imatinib inhibits the effect. (A) Cells were treated with 4 nM docetaxel and/or 7 μM imatinib for 24 (FRO) and 48 hours (KTC-2). Nuclear extracts were prepared and subjected to DNA-binding assays. Bars represent mean±SD of three wells. (B) FRO cells were treated with 4 nM docetaxel and/or 7 μM imatinib for 16 hours, and then nuclear extracts (for nuclear p65) or total cell lysates (for IκBα) were examined by Western blotting. β-Actin was used as a loading control. Similar results were obtained in at least two independent experiments.

FIG. 5.

Effect of docetaxel and imatinib in FRO tumor xenograft model. FRO cells were implanted and imatinib was injected i.p. at a dose of 50 mg/kg/day for 14 days, beginning on day 9 after tumor implantation. Docetaxel was injected i.p. at a dose of 5 mg/kg on days 9 and 16. Combined treatment mice were given both drugs. Control group mice received vehicle injection only. Each point represents mean±SD of 10 tumors (in five mice). *p<0.01 versus any other group. i.p., intraperitoneally.

FIG. 6.

A proposed mechanistic model of enhancement of docetaxel-induced apoptosis by imatinib.