The Protective Role of Troxerutin (Trox) in Counteracting Anaplastic Thyroid Carcinoma (ATC) Progression

Abstract

Anaplastic thyroid carcinoma (ATC) is a rare thyroid neoplasm characterized by aggressiveness and a high mortality rate. Troxerutin (Trox) is a bioflavonoid widely found in various fruits and vegetables with numerous protective effects, including anticancer activities. To evaluate the anti-oxidant and anti-inflammatory effect of Trox, in vitro and in vivo studies were conducted in a model of ATC. Human ATC 8305C cell lines were treated with increasing concentrations of Trox (10 μg/mL, 30 μg/mL, 100 μg/mL, 300 μg/mL), and our results revealed that Trox treatment was able to reduce the viability of ATC cells and migratory capacity, reducing the expression of anti-apoptotic factors, such as B-cell lymphoma (bcl-2), and increasing the expression of pro-apoptotic factors, such as Caspase-3 and BID, activating oxidative stress mediators, such as manganese superoxide dismutase (MnSOD), heme oxygenase-1 (HO-1), glutathione (GSH) and reactive oxygen species modulator 1 (ROMO-1). Furthermore, Trox modulates NF-κB pathway markers, such as NIK and TRAF-6. Further confirmation was obtained through in vivo studies, in which Trox treatment, at doses of 12.5, 25 and 50 mg/kg, reduced morphological alteration, decreasing mast cell accumulation. Therefore, the use of Trox could be considered a promising strategy to counteract the progression of ATC.

Keywords: anaplastic thyroid carcinoma (ATC); anti-oxidant activity; antitumoral activity; chemotherapy; troxerutin.

Figure 1

Trox treatment reduced the viability of FTC-133, 8305C and K1 cell lines. MTT assay revealed that the treatment with Trox at the concentration of 10, 30, 100 and 300 μg/mL significantly reduced viability in the FTC-133, 8305C and K1 cell lines in a concentration-dependent manner at 24 h (A–C) compared to the control group. Data are representative of at least three independent experiments.

Figure 2

Trox treatment reduced the migration capability of 8305C cells. 8305C cells were examined for cell invasion in 60 mm plates by carrying out the scratch test (A–C). The wound was photographed 24 h after the Trox treatments to measure the migratory capacity of the cells. The 100 and 300 μg/mL concentrations of Trox significantly affected the cell migration rate, reducing the invasiveness of 8305C cells compared to untreated cells (B,C).

Figure 3

Proliferation properties were reduced following Trox treatment. Proliferation of 8305C cells was examined by a colony formation assay in six-well plates (A–C). After 10 days, the plate was photographed, and the colonies were counted. Trox at concentrations of 100 and 300 μg/mL reduced the ability of cells to proliferate (B,C), reducing the number of colony formations compared to the control group (A).

Figure 4

Trox treatment reduced the inflammatory response modulating the NF-κB pathway in 8305C cell lysates. The treatment with Trox at the concentrations of 100 and 300 μg/mL significantly reduced NF-κB/IκB-α pathway (Figure 4). IκB-α expression was increased ((B), densitometric analysis (B1)), while NF-κB, NIK and TRAF6 expression was reduced ((A,C,D), densitometric analysis (A1,C1,D1)). In the same way, IL12p70 and IL-17 concentration was evaluated by the ELISA kit assay. Our data demonstrated that Trox, at the concentrations of 100 and 300 μg/mL, reduced the concentration of these pro-inflammatory cytokines (E,F). * p < 0.05 vs. 8305C.

Figure 5

Trox-induced apoptosis processes increased 8305C mortality. Western blot analysis revealed that Trox, at the concentrations of 100 and 300 μg/mL, induced the expression of pro-apoptotic proteins Caspase3 and BID ((A,B), densitometric analysis (A1,B1)) compared with the control group. Bcl-2 expression was reduced after 24 h of treatment with Trox ((C), densitometric analysis (C1)) compared with the control group. * p < 0.05 vs. 8305C; ** p < 0.01 vs. 8305C.

Figure 6

Trox reduced the production of free radicals and favored the expression of anti-oxidative proteins. Our data demonstrated via Western blot analysis of the 8305C cell lysates that Trox at the concentrations of 100 and 300 μg/mL favored the expression of MnSOD ((A), densitometric analysis (A1)). The ELISA kit assay showed that Trox increased the concentration of anti-oxidant protein GSH (B) and reduced the concentration of pro-oxidative protein ROMO1 (C). * p < 0.05 vs. 8305C; ** p < 0.01 vs. 8305C.

Figure 7

Trox treatment reduced tumoral progression in thyroid tissue. In the orthotopic model, H/E showed that the ATC group presents morphological alteration than the healthy control group (sham) (A–B1). Trox at doses of 12.5, 25 and 50 mg/kg reduced the characteristics of lymphocytic thyroiditis (C–E1) compared with the ATC group (B,B1) in a dose-dependent manner. Moreover, oral treatment with Trox restored the morphological transition in mice after treatment. The results of histological evaluations are displayed at 2× and 20× magnifications.

Figure 8

Reduction in the number of mast cells following Trox treatment. Trox treatment at doses of 12.5, 25 and 50 mg/kg reduced the accumulation of mast cells ((C–E1), score F) compared to the ATC group that showed a high number of mast cells ((B,B1), score F). The sham group did not present inflamed tissue and was therefore devoid of mast cells ((A,A1), score F). The results of histological evaluations are displayed at 10× and 40× magnifications. *** p < 0.001 vs. sham; ### p < 0.001 vs. ATC. Red circle indicated the mast cells localization (B1–E1).

Figure 9

Trox treatment reduced the inflammatory state of the thyroid tissue. To confirm the in vitro studies, Western blot analysis of the thyroid tissue demonstrated that Trox, especially at the doses of 25 and 50 mg/kg, was able to increase the IκB-α levels ((A), densitometric analysis (A1)) and reduced the expression of NF-κB ((B), densitometric analysis (B1)) compared with the ATC group ((A,B), densitometric analysis (A1,B1)). ## p < 0.01 vs. ATC; ### p < 0.001 vs. ATC.

Figure 10

Treatment with Trox reduced the cascade of pro-inflammatory cytokines release. Western blot analysis demonstrated that Trox at doses of 12.5, 25 and 50 mg/kg increased the levels of IL-12Ap35 ((A), densitometric analysis (A1)) and reduced the expression of IL6 and IL-17A ((B,C), densitometric analysis (B1,C1)) compared with the ATC group ((A–C), densitometric analysis (A1–C1)). ## p < 0.01 vs. ATC; ### p < 0.001 vs. ATC.

Figure 11

Trox-induced apoptotic process. Our data showed that Trox treatment, especially at high doses of 25 and 50 mg/kg, induced the apoptotic process increasing the expression of Caspase3, p53 and BID ((A–C), densitometric analysis (A1–C1)) compared with the ATC group. Moreover, bcl-2 expression was reduced after treatment with Trox ((D), densitometric analysis (D1)) compared with the ATC group. # p < 0.05 vs. ATC; ## p < 0.01 vs. ATC; ### p < 0.001 vs. ATC.

Figure 12

Troxerutin treatment reduced Ki67-positive cells. Ki67 expression was evaluated via histochemistry staining. The ATC group presented a high number of ki67-positive cells ((B), score F) compared with the control group (A), which did not present immunopositivity for the Ki67 marker. Treatment with Trox at doses of 12.5, 25 and 50 mg/kg reduced the number of Ki67-positive cells ((C–E), score F). The results of histological evaluation are displayed at 40× magnifications. *** p < 0.001 vs. sham; # p < 0.05 vs. ATC; ### p < 0.001 vs. ATC.