Hif-1α Inhibitors Could Successfully Inhibit the Progression of Differentiated Thyroid Cancer in Vitro

Abstract

Hypoxia-inducible factor (HIF)-1α plays an important role in cancer progression. In various cancers, including thyroid cancer, overexpression of HIF-1α is related to poor prognosis or treatment response. However, few studies have investigated the role of HIF-1α inhibition in thyroid cancer progression. We evaluated the utility of the HIF-1α inhibitor IDF-11774 in vitro utilizing two thyroid cancer cell lines, K1 and BCPAP. Both cell lines were tested to elucidate the effects of IDF-11774 on cell proliferation and migration using soft agar and invasion assays. Here, we found that a reduction of HIF-1α expression in BCPAP cells was observed after treatment with IDF-11774 in a dose-dependent manner. Moreover, cell proliferation, migration, and anchorage-independent growth were effectively inhibited by IDF-11774 in BCPAP cells but not in K1 cells. Additionally, invasion of BCPAP but not K1 cells was controlled with IDF-11774 in a dose-dependent manner. Our findings suggest that promoting the degradation of HIF-1α could be a strategy to manage progression and that HIF-1α inhibitors are potent drugs for thyroid cancer treatment.

Keywords: HIF-1α; IDF-11774; thyroid cancer.

Figure 1

Expression of HIF-1α in thyroid cancer lines. (A) HIF-1α expression levels in different thyroid cancer cells under normoxia. Thyroid cancer cells (BCPAP and K1) were seeded and incubated in normoxic conditions. Cell lysates were immunoblotted with anti-HIF1α and anti-TUBULIN. (B) HIF-1α expression levels in different thyroid cancer cells under hypoxia (with DMOG for 4 h). Thyroid cancer cells (BCPAP and K1) were seeded and incubated in hypoxic conditions. Cell lysates were immunoblotted with anti-HIF1α and anti-TUBULIN. (C) Western blotting and immunofluorescence analysis of HIF-1α expression of BCPAP and K1 cells after DMOG treatment. (Left) Western blot analysis of HIF1α protein in hypoxia condition. BCPAP and K1 cells were seeded and treated with DMOG at indicated time point. Cell lysates were immunoblotted with anti-HIF1α and anti-TUBULIN. (Right) Immunofluorescence analysis of HIF1α protein under normoxia or hypoxia. BCPAP and K1 cells were stained with HIF1α antibody. Nuclei were counterstained with DAPI. Scale bar represents 50 μm (100×).

Figure 2

Effect of IDF-11774 on the proliferation of BCPAP and K1 cells. (A) IC₅₀ analysis of IDF-11774I on BAPAP and K1 cells. (B) Western blotting analysis of HIF-1α expression in BCPAP and K1 cells after increasing doses of IDF-11774. After pretreatment with DMOG for 4 h, BCPAP and K1 cells were incubated with IDF-11774 for 24 h. Cell lysates were immunoblotted with anti-HIF1α and anti-ACTIN. (C) IDF-11774 treatment decreased cell viability of BCPAP cells in a dose-dependent manner but did not decrease the viability of K1 cells. Cell viability was measured at the indicated time using a CellTiter-Glo system. Data in the graph are presented as mean ± standard deviation of fold increase normalized by cells at day 0 (n = 5, * p < 0.01). (D) IDF-11774 treatment decreased colony formation of BCPAP cells in a dose-dependent manner but did not decrease colony formation in K1 cells. BCPAP and K1 cells were seeded and incubated with IDF for 1–2 weeks. Cell colonies were visualized by staining using crystal violet. (E) IDF-11774 treatment decreased anchorage-independent growth of BCPAP cells in a dose-dependent manner. BCPAP cells seeded and cultured for 3–4 weeks and cell colonies were visualized by staining with crystal violet. Data in the graph are presented as mean ± standard deviation (n = 3, ** p < 0.001).

Figure 3

Effect of IDF-11774 on the migration and invasion properties of BCPAP and K1. (A) The effect of IDF-11774 on the migration of BCPAP and K1 cells was measured using a wound-healing assay. Data in the graph are presented as mean ± standard deviation of relative wound closure normalized by wound area at day 0 (n = 5, ** p < 0.001). (B) Effect of IDF-11774 on the cell invasion of BCPAP and K1 cells in a transwell invasion assay. Cell invasion was estimated, and data are presented as mean ± standard deviation of the number of invaded cells (n = 5, ** p < 0.001).

Figure 4

Alteration of the drug effect on HIF-1α expression after knockdown of HIF-1α and treatment with cycloheximide and velcade. (A) Analysis of HIF-1α expression after IDF-11774 treatment with cycloheximide and velcade. BCPAP cells were incubated with DMOG for 4 h. Cells were treated for 4 h with IDF-11774 alone or with IDF-11774 and velcade. The half-life of HIF1α was estimated by treating with cycloheximide at the indicated time. (B) Immunofluorescence of HIF-1α expression with IDF-11774 and velcade. BCPAP cells were treated with DMOG for 4 h and incubated for 24 h in the presence or absence of IDF-11774. Cells were stained with HIF1α antibody. Nuclei were counterstained with DAPI. Velcade treatment was administered for 5 h before immunostaining. Scale bar represents 50 μm (100×). (C) Analysis of HIF-1α expression in HIF-1α knockdown BCPAP cells. BCPAP cells stably expressing Control-shRNA or HIF1α-shRNAs were detected by Western blot analysis. (D) HIF-1α knockdown attenuated the effect of cell viability of BCPAP cells treated with IDF-11774. BCPAP Cells stably expressing shRNA were incubated with DMSO or IDF-11774. Cell viability was measured at the indicated time using the CellTiter-Glo system. Data in the graph are presented as mean ± standard deviation of fold increase normalized by cells at day 0 (n = 5, ** p < 0.001).