Flavopereirine exerts anti-cancer activities in various human thyroid cancer cells

Abstract

Thyroid cancer (TC) stands out as the most prevalent endocrine malignancy globally, with a steadily increasing incidence. Its clinical manifestations include enlarged thyroid nodules, dysphagia, enophthalmos, and various other symptoms. While standard treatments such as thyroidectomy and radioiodine therapy effectively manage most cases of differentiated thyroid cancers (DTC), some recurrent cases of DTC or those involving poorly differentiated thyroid cancers (PDTC) require specialized interventions. However, existing drugs primarily address symptom management without offering a curative solution. Therefore, the development of a new therapeutic agent for these challenging cases is of utmost importance. Flavopereirine, derived from Geissospermum vellosii, has demonstrated promise as a potential anti-cancer agent across various human cancers. However, its specific anti-cancer effects on human thyroid cancer (TC) have remained unclear. Therefore, this study aims to investigate the anti-cancer activity of flavopereirine in human TC. The research findings revealed that flavopereirine effectively hinders the growth of human TC cells, induces cell cycle arrest, promotes apoptosis, and modulates autophagy. Moreover, the study delved into the underlying mechanisms by which flavopereirine influenced signaling pathways. To validate these anti-cancer effects, an in vivo zebrafish model was utilized, confirming the efficacy of flavopereirine against human TC cells. In summary, this study establishes that flavopereirine exhibits notable anti-human TC activities, positioning it as a promising therapeutic candidate for the treatment of human thyroid cancer.

Keywords: Flavopereirine; anti-cancer activity; apoptosis; autophagy; thyroid cancer.

Figure 1

Flavopereirine inhibits cell proliferation in various human thyroid cancer cells. Human thyroid cancer cells lines, (A) IHH-4, (B) WRO, (C) SW579, (D) 8505c, and (E) KMH-2 cells, were treated with flavopereirine, and the cellular survival was examined with CCK-8 assay. DMSO was used as a negative control. Three independent experiments were conducted.

Figure 2

Flavopereirine suppresses colony formation of human PTC and ATC cells. (A) IHH-4, (B) 8505c, and (C) KMH-2 cells were incubated with flavopereirine for 12 days, and the colony formation was determined after 10% crystal violet staining. Three independent experiments were conducted.

Figure 3

Flavopereirine modulates cell cycle in human PTC and ATC cells. (A) IHH-4, (B) 8505c, and (C) KMH-2 cells were administrated with flavopereirine, and the cell cycle regulation was investigated with flowcytometry analysis.

Figure 4

Flavopereirine induces cellular apoptosis in human PTC and ATC cells. (A) IHH-4, (B) 8505c, and (C) KMH-2 cells were incubated with flavopereirine, and the apoptotic cells were examined with flowcytometry analysis after PI/Annexin-V staining. Representative result of three independent experiments.

Figure 5

Flavopereirine induces intrinsic and extrinsic caspase-dependent apoptosis in human PTC and ATC cells. (A) IHH-4, (B) 8505c, and (C) KMH-2 cells were incubated with flavopereirine (10, 15, and 7.5 μM) for 48 h, and the cellular lysate was collected and the proteins in the protein lysate were determined with Western blotting. DMSO was used as a negative control, and Z-VAD-FMK (20 μM) was preincubated for 2 h before flavopereirine treatment.

Figure 6

Blocking caspases activation in human PTC and ATC cells rescues flavopereirine mediated cellular apoptosis. (A) IHH-4, (B) 8505c, and (C) KMH-2 cells were incubated with flavopereirine along (10, 15, and 7.5 μM) or combination treatment with Z-VAD-FMK (20 μM) for 48 h, and the cellular morphology was determined with microscopy (200×), and the apoptotic cells were examined with flowcytometry assay after PI/Annexin-V staining. DMSO was used as a negative control, and Z-VAD-FMK was preincubation for 2 h before flavopereirine administration. Representative result of three independent experiments.

Figure 7

Flavopereirine induces cellular autophagy in human PTC and ATC cells. (A and B) IHH-4, (C and D) 8505c, and (E and F) KMH-2 cells were treated with flavopereirine (A, C and E) for 24 h or the indicated condition. The total cell lysate was collected and the proteins in the lysate were assessed with Western blotting. DMSO was used as the negative control.

Figure 8

Flavopereirine induces autophagy as well as autophagosome formation in human PTC and ATC cells. IHH-4, 8505c and KMH-2 cells were incubated with flavopereirine (10, 15, and 7.5 μM) for 24 h, and (A) the autophagosomes in these cells were evaluated with immunofluorescence staining, and (B) the protein expressions were examined with Western blotting. DMSO was used as the negative control. 3-MA (5 μM) was the autophagy inhibitor, and the rapamycin (30 μM) was a autophagy inducer. Bafilomycin (50 nM) was used to be an autophagic flux blocker.

Figure 9

Flavopereirine modulating signaling pathways in human PTC and ATC cells. (A) IHH-4, 8505c and KHM-2 cells were administrated with flavopereirine for 48 h, and the total cell lysate was collected and the proteins in the lysate were examined with Western blotting. KMH-2 cells were co-incubated with (B) PD98059 and (C) SB203580 to block ERK and p38 activation, and the expressions and activations of ERK and/or p38, caspase-3, and PARP were investigated by Western blots after incubation with flavopereirine for 48 h. (D) KMH-2 cells were transfected with or without constitutively active AKT construct, and the expression and the activation of AKT, caspase-3, and PARP were investigated by Western blots after incubation with flavopereirine for 48 h. DMSO was used as a negative control. GAPDH was a loading control.

Figure 10

Flavopereirine inhibits tumor growth in a zebrafish xenograft model. (A) The toxicity effect of flavopereirine for zebrafish larvae at the indicated concentrations and time. *P < 0.05 compared with the control. (B) IHH-4 and (C) KMH-2 cells labeled with a red fluorescent dye (CM-DiI) were injected into zebrafish yolk sacs. The intensity of red fluorescence is proportional to the tumor size. Thyroid cancer xenograft zebrafish treated with flavopereirine and observed at 24 and 48 h post-treatment. Quantitative analysis of the tumor cell proliferation with or without flavopereirine treatment. *P < 0.05; **P < 0.01 compared with control cells. Data represent means ± SD.