Ouabain Effects on Human Anaplastic Thyroid Carcinoma 8505C Cells

Abstract

Anaplastic thyroid carcinoma (ATC) is a rare, but aggressive, carcinoma derived from follicular cells. While conventional treatments may improve patients' survival, the lethality remains high. Therefore, there is an urgent need for more effective ATC treatments. Cardiotonic steroids, such as ouabain, have been shown to have therapeutic potential in cancer treatment. Thus, we aimed to evaluate ouabain's effects in human anaplastic thyroid cells. For this, 8505C cells were cultured in the presence or absence of ouabain. Viability, cell death, cell cycle, colony formation and migratory ability were evaluated in ouabain-treated and control 8505C cells. The expression of differentiation and epithelial-to-mesenchymal transition (EMT) markers, as well as IL-6, TGFb1 and their respective receptors were also quantified in these same cells. Our results showed that ouabain in vitro decreased the number of viable 8505C cells, possibly due to an inhibition of proliferation. A reduction in migration was also observed in ouabain-treated 8505C cells. In contrast, decreased mRNA levels of PAX8 and TTF1 differentiation markers and increased levels of the N-cadherin EMT marker, as well as IL-6 and TGFb1, were found in ouabain-treated 8505C cells. In short, ouabain may have anti-proliferative and anti-migratory effect on 8505C cells, but maintains an aggressive and undifferentiated profile.

Keywords: anaplastic thyroid cancer; cardiac glycosides; cardiotonic steroids; cytokines; ouabain.

Figure 1

Ouabain decreased 8505C viability in vitro. 8505C cells were cultured in the absence or presence of ouabain at concentrations that ranged from 10⁻⁵–10⁻¹¹ M for 24 h. Next, cell viability was evaluated with MTT assay or using cell counting in a Neubauer chamber using trypan blue. (a) MTT absorbance values of ouabain-treated cultures were normalized to their respective control and data are presented as the mean relative percentage of viable 8505C cells ± SEM after 24 h of culture. (b) Scatter plots represent the absolute number of viable 8505C cells with reference lines showing the medians. At least three independent experiments were performed for each condition. * p < 0.05 using repeated measures ANOVA with Dunnett´s post-test and paired t-test, respectively.

Figure 2

Ouabain did not increase 8505 death or ROS levels. 8505C and NTHY-ori cells were cultured for 24 h with 10⁻⁷ M of ouabain or left untreated. (a) 8505C death was analyzed using Muse Annexin V & Dead Cell Kit (n = 3). Data are expressed as the mean percentage of live, early apoptotic, late apoptotic/dead and total apoptotic cells ± SEM in ouabain-treated and control cultures. (b,c) 8505C and NTHY-ori intracellular ROS levels (CM-H2DCFDA fluorescence) were evaluated with flow cytometry (n = 6). Data are expressed as the mean fluorescence intensity of DCF in 8505C (b) and NTHY-ori (c) cells. * p < 0.05 using repeated measures ANOVA with Dunnett´s post-test.

Figure 3

Effect of ouabain on 8505C cell cycle distribution and 8505C and NTHY-ori CFU formation. 8505C and NTHY-ori cells were cultured in the absence or presence of ouabain 10⁻⁷ M. (a) Cell cycle distribution was analyzed using Muse Cell Cycle Kit. Data are expressed as the mean percentage of 8505C cells ± SEM in G0/G1, S and G2/M phases after 24 h of culture (n = 4). (b–e) Tumor cell colony-forming units were also analyzed after 7 days of culture. Graphs are presented as the number of CFUs and the mean number of cells per colony in 8505C ((b,c), respectively) and NTHY-ori ((d,e), respectively) ouabain-treated and control cultures (n ≥ 4). * p < 0.05 using paired t-test.

Figure 4

Ouabain decreased 8505C migration ability. 8505C cells were cultured in the absence or presence of 10⁻⁷ M of ouabain for 24 h and then allowed to migrate through transwell inserts for 24 h, as described in Materials and Methods section. Bar graph displays transwell migration measurement as mean OD ± SEM of four independent experiments performed in duplicate. A representative image of transwell inserts with 8505C control and ouabain-treated migrating cells stained with crystal violet is also shown. * p < 0.05 using paired t-test.

Figure 5

Effect of ouabain on mRNA expression of PAX8 and TTF1 in 8505C cells. Following 24 h of 8505C culture in the absence or presence of ouabain 10⁻⁷ M, PAX8 and TTF1 mRNA levels were evaluated by RT-PCR. (a,b) Data are presented as the mean ± SEM of mRNA levels of PAX8 (a) and TTF1 (b) of at least three independent experiments, each performed in triplicate. * p < 0.05 using unpaired t-test.

Figure 6

Effect of ouabain on aggressiveness markers expression in 8505C cells. Following 24 h of 8505C culture in the absence or presence of ouabain 10⁻⁷ M, aggressiveness markers expression was evaluated by RT-PCR. (a–d) Data are presented as the mean ± SEM of mRNA levels of Vimentin (a), N-cadherin (b), TWIST (c) and MMP9 (d) of at least three independent experiments, each performed in triplicate. * p < 0.05 using unpaired t-test.

Figure 7

Effect of ouabain on mRNA expression of IL-6, IL-6R, TGFβ1 and TGFRs in 8505C and NTHY-ori cells. Following 24 h of 8505C culture in the absence or presence of ouabain 10⁻⁷ M, IL-6, TGFβ1 and their respective receptors expression were evaluated using RT-PCR. (a–e) Data are presented as the mean ± SEM of mRNA levels of IL-6 (a), IL-6R (b), TGFβ1 (c), TGFRI (d) and TGFRII (e) in control and ouabain-treated 8505C cells. (f) Data are presented as the mean ± SEM of mRNA levels of TGFβ1 in control and ouabain-treated NTHY-ori cells. For this, at least four independent experiments were performed in triplicate. * p < 0.05 using unpaired t-test.

Figure 8

Impact of ouabain on mTOR and AMPK expression in 8505C cells. 8505C cells were cultured in the absence or presence of 10⁻⁷ M of ouabain and, after 24 h, protein levels of mTOR, p-mTOR, AMPKα1, p-AMPKα, AMPKβ and p-AMPKβ were evaluated. (a–c) Graphs show relative p-mTOR/mTOR (a), p-AMPKα/AMPKα1 (b) and p-AMPKβ/ AMPKβ (c) protein levels in control and ouabain-treated 8505C cells. (d–f) Representative blots of at least three independent experiments.

Figure 9

In vivo effect of ouabain. Balb/c nude mice were sub-cutaneously injected with ≈ 107 8505C cells. From the 21st day post-injection, mice were inoculated intra-peritoneally daily with vehicle or ouabain (10⁻⁷ M) for 15 days. (a,b) Graphs show mean weights (a) and tumor volumes (b) ± SEM measured once every three days. (c) Scatter plot represent the tumor weights after 15 days of treatment with reference lines showing the medians.