The Potential Therapeutic Value of Aspirin in Anaplastic Thyroid Cancer

Abstract

Background: several experimental findings and epidemiological observations indicated that aspirin/acetylsalicylic acid (ASA) may be endowed with anticancer effects against a variety of human malignancies, including thyroid carcinomas. Among these, undifferentiated/anaplastic thyroid carcinoma (ATC) is one of the most aggressive and lethal human cancers, refractory to all currently available therapies. Methods: we here evaluated in a preclinical setting the effects of ASA on a panel of three ATC-derived cell lines: the CAL-62, the 8305C, and the 8505C. Results: the data obtained demonstrated the ability of ASA to inhibit, in a dose- and time-dependent manner, the proliferation of all ATC cell lines investigated, with IC₅₀ values comprised between 2.0 and 4.3 mM. Cell growth was restrained with the same efficacy when the ASA treatment was applied to three-dimensional soft-agar cultures. In addition, ASA significantly reduced migration and invasion in two of the three ATC cell lines. We finally investigated the effects of ASA on the MAPK and PI3K/Akt signaling pathways, which are often altered in ATC. The results showed that the phosphorylation status of the Akt1/2/3 kinases was significantly reduced following ASA treatment, while ERK1/2 phosphorylation was either unaffected or slightly upregulated. Conclusions: our findings support epidemiological evidence on the anticancer potential of ASA. On this basis, further investigations should be carried out to assess the usefulness of ASA as adjuvant therapy in patients affected by ATC.

Keywords: anaplastic thyroid cancer; aspirin; therapy.

Figure 1

Dose-dependent inhibition of anaplastic thyroid cancer (ATC)-derived cell lines proliferation by ASA. Cells were treated with increasing doses of acetylsalicylic acid (ASA) (from 0.05 to 10 mM) for 72 h. Data are reported as the mean ± standard deviation (SD).

Figure 2

Time-dependent inhibition of ATC-derived cell lines proliferation by ASA. Cells were seeded in 96-well plates, treated with ASA (10 mM for CAL-62, 5 mM for 8305C and 8505C), and measured at 24-h time intervals. Data are reported as the mean ± SD.

Figure 3

Apoptotic effects of ASA on ATC-derived cell lines. Cells were seeded in 96-well plates and treated with ASA for 48 h. At the end of the incubation time, apoptosis was assessed using the Cell Death Detection ElisaPLUS kit to determine cytoplasmic histone-associated DNA fragments. The enrichment was calculated as the absorbance ratio between treated and non-treated cells. Bars represent the mean ± standard error (SE) of three independent experiments. * p < 0.05.

Figure 4

Effects of ASA on the anchorage-independent growth of ATC cells. Cells were grown in a soft agar gel mixed with cell culture medium ± ASA (10 mM for CAL-62, 5 mM for 8305C and 8505C) for two weeks. Photos were finally acquired, and colonies having diameter ≥50 μm were counted. Bars represent the mean ± SE of three independent experiments. N.D., not detectable. ** p < 0.01; *** p < 0.001.

Figure 5

Effects of ASA on ATC cell migration in adherent cultures. Scratch areas were measured with the ImageJ software at different time intervals, and the speed of cell migration was calculated. Bars represent the mean ± SE of three independent experiments. **, p < 0.01.

Figure 6

Effects of ASA on ATC cell invasion. Cells were seeded onto PET membranes precoated with ECM in a serum-free medium and incubated for 12 h ± ASA at IC₅₀ concentrations. The complete medium was used as a chemoattractant. After removal of non-migrated cells, invading cells were fixed and stained with Crystal Violet. Bars represent the mean ± SE of three independent experiments. *, p < 0.05.

Figure 7

Phosphorylation status of Akt and MAPK in ATC-derived cell lines treated with ASA. Cells were incubated for 24 h ± ASA (10 mM for CAL-62, 5 mM for 8305C and 8505C), then protein extracts were prepared and analyzed by Western blot. Panel (A) Images from western blot. Panel (B) densitometric analyses. Bars represent the mean ± SE of three independent experiments. *, p < 0.05; ***, p < 0.001. Original western blots are presented in File S1.