Antitumor effects of anlotinib in thyroid cancer

Abstract

There is no effective treatment for patients with poorly differentiated papillary thyroid cancer or anaplastic thyroid cancer (ATC). Anlotinib, a multi-kinase inhibitor, has already shown antitumor effects in various types of carcinoma in a phase I clinical trial. In this study, we aimed to better understand the effect and efficacy of anlotinib against thyroid carcinoma cells in vitro and in vivo. We found that anlotinib inhibits the cell viability of papillary thyroid cancer and ATC cell lines, likely due to abnormal spindle assembly, G2/M arrest, and activation of TP53 upon anlotinib treatment. Moreover, anlotinib suppresses the migration of thyroid cancer cells in vitro and the growth of xenograft thyroid tumors in mice. Our data demonstrate that anlotinib has significant anticancer activity in thyroid cancer, and potentially offers an effective therapeutic strategy for patients of advanced thyroid cancer type.

Keywords: anlotinib; apoptosis; migration; multi-kinase inhibitor; thyroid cancer.

Figures

Figure 1

Anlotinib inhibits thyroid cancer cells proliferation in vitro. (A) Thyroid cancer cells BCPAP and 8505C were treated with anlotinib (0, 1, 5 and 10 µM) for 24 h, and then photographed with inverted contrast microscopy (100× magnification). Scale bars: 50 µm. (B) Representative images of colony formation assays using BCPAP and 8505C cells treated with (0, 1, 5 and 10 µM) for 8 days. (C and D) Quantification of the colony formation assays in BCPAP and 8505C cells. Data are shown as mean ± s.d. (n ≥ 3).

Figure 2

Anlotinib induces G2/M phase arrest in thyroid cancer cells. (A and C) BCPAP cells (A) and 8505C cells (C) were treated with DMSO or 10 µM anlotinib for 24 h and stained with PI. Cell cycle distribution was assessed with flow cytometry and quantified by Modifit software. (B and D) The percentage of cells in sub-G1, G1, S and G2/M phase were calculated and plotted. Data are shown as mean ± s.d. (n ≥ 3).

Figure 3

Anlotinib leads to abnormal spindle formation in thyroid cancer cells. (A and C) BCPAP cells (A) and 8505C cells (C) were treated with anlotinib (0, 1, 5 and 10 µM) for 24 h. The morphological changes of nuclei were examined using Hoechst 33342 staining. Scale bars: 100 µm. The blowup images from the red boxes show nuclear condensation and structural changes after exposure to anlotinib. (B and D) Effect of anlotinib on mitotic spindle formation in thyroid cancer cells. Cells were treated with or without 10 µM anlotinib for 24 h and subjected to immunofluorescence staining for TUBULIN. The right plots show the percentage of cells with abnormal spindle in the metaphase cells treated with 0, 1, 5 and 10 µM anlotinib. Data are shown as mean ± s.d. (n ≥ 3).

Figure 4

Anlotinib induces apoptosis in thyroid cancer cells. (A and D) BCPAP (A) and 8505C (D) cells were treated with DMSO and 10 µM anlotinib for 36 h. Cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. The low right (LR) quadrant of the histograms indicates the early apoptotic cells, and the upper right quadrant indicates the late apoptotic cells. (B and E) Quantification results of experiments described in A and D. Data are shown as mean ± s.d. (n ≥ 3). (C and F) BCPAP (C) and 8505C (F) cells were treated with anlotinib (0, 1, 5, 10 and 15 µM) for 36 h. Cell lysates were prepared and subjected to Western blot to detect the expression of TP53, cleaved caspase 3 (CL-caspase 3) and cleaved PARP (CL-PARP).

Figure 5

Anlotinib induces apoptosis partly through activating the TP53 pathway. (A and B) siRNA target TP53 and control RNA were transfected into thyroid cancer cells. Twenty-four hours after transfection, the cells were treated with or without 10 µM anlotinib for 24 h. Cell lysates were prepared and subjected to Western blot. (C and D) Thyroid cancer cells described in (A and B) were stained with Annexin V-FITC/PI and analyzed with flow cytometry. Data are shown as mean ± s.d. (n ≥ 3).

Figure 6

The effects of anlotinib on the migration of thyroid cancer cells. (A and B) Representative images of wound-healing assays using BCPAP (A) and 8505C (B) cells in the presence of 0, 1, 5 and 10 µM anlotinib. Scale bars: 25 µm. The plots on the right show the quantification of wound healing assays. Data are shown as mean ± s.d. (n ≥ 3). (C and D) The expression and cellular distribution of F-actin in BCPAP (C) and 8505C cells (D). Cells were treated with DMSO or 10 µM anlotinib for 24 h, and then stained with rhodamine-phalloidin (red) for F-actin. Hoechst 33342 was applied to counter-stain the nucleus (blue). Scale bars: 100 µm.

Figure 7

Anlotinib suppresses tumor growth in vivo. (A) Images of dissected tumors from nude mice injected with K1 cells of DMSO treated group (n = 5) and anlotinib treated group (n = 5). (B) Tumor weights of control and anlotinib-treated groups. (C) Tumor volumes with or without anlotinib treatment. (D) HE staining, Ki67, TP53 and CL-caspase 3 IHC staining of the tumors with or without anlotinib treatment. Scale bars: 100 µm.