Endocrinol Metab.  2021 Oct;36(5):1095-1110. 10.3803/EnM.2021.1155.

Lobeglitazone, A Peroxisome Proliferator-Activated Receptor-Gamma Agonist, Inhibits Papillary Thyroid Cancer Cell Migration and Invasion by Suppressing p38 MAPK Signaling Pathway

• Jin JQ
• Han JS
• Ha J
• Baek HS
• Lim DJ

• KMID: 2521957
• DOI: http://doi.org/10.3803/EnM.2021.1155

Abstract

Background
Peroxisome proliferator-activated receptor-gamma (PPAR-γ) ligands have been widely shown to correlate with epithelial-mesenchymal transition (EMT) and cancer progression. Lobeglitazone (LGZ) is a novel ligand of PPAR-γ; and its role in EMT and metastasis in papillary thyroid carcinoma (PTC) is poorly understood. We aimed to investigate the role of LGZ in metastatic behavior of PTC cells.
Methods
Half maximal inhibitory concentration (IC₅₀) values of LGZ in BRAF-mutated PTC cell lines (BCPAP and K1) were determined using MTT assay. Rosiglitazone (RGZ), the PPAR-γ ligand was used as a positive control. The protein expression of PPAR-γ, cell-surface proteins (E-cadherin, N-cadherin), cytoskeletal protein (Vimentin), transcription factor (Snail), p38 mitogenactivated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2 pathway, and matrix metalloproteinase (MMP)-2 expression were measured using Western blotting. Changes in E-cadherin expression were also determined using immunocytochemistry. Cell migration and invasion were analyzed using wound healing and Matrigel invasion assays.
Results
Treatment with LGZ or RGZ significantly inhibited transforming growth factor-beta1 (TGF-β1)-induced EMT-associated processes such as fibroblast-like morphological changes, EMT-related protein expression, and increased cell migration and invasion in BCPAP and K1 cells. LGZ restored TGF-β1-induced loss of E-cadherin, as observed using immunocytochemistry. Furthermore, LGZ and RGZ suppressed TGF-β1-induced MMP-2 expression and phosphorylation of p38 MAPK, but not ERK1/2. Although there was no change in PPAR-γ expression after treatment with LGZ or RGZ, the effect of downstream processes mediated by LGZ was hampered by GW9662, a PPAR-γ antagonist.
Conclusion
LGZ inhibits TGF-β1-induced EMT, migration, and invasion through the p38 MAPK signaling pathway in a PPAR-γ-dependent manner in PTC cells.

Keyword

Thyroid cancer; papillary; PPAR gamma; Neoplasm metastasis

Figure

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  Fig. 1 Identification of lobeglitazone (LGZ) as a selective peroxisome proliferator-activated receptor (PPAR) agonist. (A) The half maximal inhibitory concentration (IC50) values of LGZ in papillary thyroid carcinoma (PTC) cells at 24 and 48 hours were measured using MTT assay. Data error bars correspond to the standard error of the mean (n=6). (B) Western blot assay shows the low protein level of PPAR-γ and no significant change in response to rosiglitazone (RGZ) or LGZ treatment, compared with control. BCPAP and K1 cells are treated with RGZ 10 μM or LGZ 10 μM for 24 hours. 3T3-L1 cell line is used as a positive control. (C) Western blot shows the protein levels of CCAAT/enhancer binding protein alpha (C/EBPα). Stimulation with transforming growth factor-beta1 (TGF-β1; 20 ng/mL) in the presence or absence of RGZ (10 μM) or LGZ (10 μM) at 24 hours in BCPAP and K1 cells.

• 

  Fig. 2 Transforming growth factor-beta1 (TGF-β1) induces epithelial-mesenchymal transition (EMT) in papillary thyroid carcinoma (PTC) cells. (A) BCPAP cells are treated with different concentrations of TGF-β1 for 48 hours and (B) K1 cells for 24 hours. Western blot analysis of E-cadherin, N-cadherin, Vimentin, and Snail protein expression in PTC cells. β-Actin is used as a control. Statistical analysis is performed using the Student’s t test from three independent experiments repeated in triplicate, and error bars correspond to the standard error of the mean and P values are indicated in each panel. aP<0.05; bP<0.01 vs. control.

• 

  Fig. 3 Lobeglitazone (LGZ) and rosiglitazone (RGZ) inhibit transforming growth factor-beta1 (TGF-β1)-induced epithelial-mesenchymal transition in papillary thyroid carcinoma (PTC) cells. (A) Western blot analysis of E-cadherin, N-cadherin, Vimentin, Snail protein expression in PTC cells stimulated with TGF-β1 (20 ng/mL) in the presence or absence of peroxisome proliferator-activated receptor gamma ligands RGZ or LGZ at different concentrations for 24 and 48 hours. β-Actin is used as loading control. (B) The morphological changes in PTC cells following treatments at 24- and 48-hour time point. The images are acquired at 100× magnification. (C) Confocal microscopy images of E-cadherin (Alexa Fluor® 488, Green Fluorescent) expression under different stimuli in PTC cells. Human embryonic kidney 293 cells are used as positive control. Cell’s nuclei are stained with 4′,6-diamidino-2-phenylindole (DAPI). Data are expressed as mean±standard error of the mean for three experiments.

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  Fig. 4 Lobeglitazone (LGZ) and rosiglitazone (RGZ) inhibit transforming growth factor-beta1 (TGF-β1)-induced phosphorylation of p38 but not extracellular signal-regulated kinase (ERK) 1/2. (A) TGF-β1 induces activation of p38 and ERK1/2 in BCPAP and K1 cells in a time-dependent manner. (B) LGZ and RGZ inhibit TGF-β1-induced phosphorylation of p38. Cell are pretreated with RGZ or LGZ for 30 minutes and stimulated with or without TGF-β1 (20 ng/mL) for 15 minutes. Lower panel: relative protein expression rate was normalized using the value of p-P38/T-P38. (C) LGZ and RGZ have no suppressive effects on the TGF-β1-induced phosphorylation of ERK1/2. Cell are pretreated with RGZ or LGZ for 30 minutes and stimulated with or without TGF-β1 (20 ng/mL) for 15 minutes. Lower panel: relative protein expression rate was normalized using the value of p-ERK1/2/T-ERK. Statistical analysis is performed using the Student’s t test from three independent experiments repeated in triplicate; Error bars correspond to the standard error of the mean, P values are indicated in each panel. aP<0.05 vs. control; bP<0.05 vs. TGF-β1-treated.

• 

  Fig. 5 Lobeglitazone (LGZ) and rosiglitazone (RGZ) inhibit cell migration, invasion, and matrix metalloproteinase 2 (MMP-2) protein expression induced by transforming growth factor-beta1 (TGF-β1) in papillary thyroid carcinoma (PTC) cells. (A) Wound healing cell migration assay shows that LGZ and RGZ inhibit PTC cell migration induced by TGF-β1. Left: representative images of scratched and recovering wounded areas at 0 and 24 hours. Right: quantitative analysis of the rate of wound closure. Cells treated with LGZ and RGZ show significantly lower migration ability than those treated with TGF-β1 (20 ng/mL). (B) Cells are trypsinized and plated in the Matrigel-coated transwell chambers to evaluate invasion. Left: representative images of PTC cells following different treatments at 24 hours. Right: quantitative analysis of the invasive cell number. Cells treated with peroxisome proliferator-activated receptor gamma (PPAR-γ) ligands show significantly lower invasion ability than those treated with TGF-β1. (C) Protein expression level of MMP-2 in PTC cells treated with TGF-β1 in the presence or absence of PPAR-γ ligands. Relative protein expression rate isnormalized using β-actin as a loading control. Data are expressed as mean±standard error of the mean for three experiments. Statistical analysis is performed using the Student’s t test, P values are indicated in each panel. aP<0.05 vs. control; bP<0.05 vs. TGF-β1-treated.

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  Fig. 6 Effects of lobeglitazone (LGZ) inhibition of phosphorylation of p38 mitogen-activated protein kinase (MAPK) and matrix metalloproteinase 2 (MMP-2) expression induced by transforming growth factor-beta1 (TGF-β1) are peroxisome proliferator-activated receptor gamma (PPAR-γ) dependent. (A) Western blot showing the effect of GW9662 on LGZ-mediated inhibition of the TGF-β1 activation of p38 MAPK and MMP-2 expression in papillary thyroid carcinoma (PTC) cells. PTC cells are pre-treated with GW9662 for 2 hours and cells are treated with presence or absence of LGZ 5 μM for 30 minutes, then stimulated with TGF-β1 (20 ng/mL) for 15 minutes. (B) The effect of GW9662 on LGZ-mediated inhibition of TGF-β1-induced MMP-2 protein expression in PTC cells by Western blot assay. PTC cells are pre-treated with GW9662 for 2 hours and cells are treated with presence or absence of LGZ 5 μM for 30 minutes, then stimulated with TGF-β1 at the indicated concentrations for 24 and 48 hours. Relative protein expression rate is normalized using β-actin as a loading control. Statistical analysis was performed using the Student’s t test from three independent experiments repeated in triplicates; Error bars correspond to the standard error of the mean; P values are indicated in each panel. aP<0.05 vs. control; bP<0.05 vs. TGF-β1-treated; cP<0.05 vs. TGF-β1 with LGZ-treated.

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  Fig. 7 Schematic representation of transforming growth factor-beta1 (TGF-β1)-induced epithelial-mesenchymal transition (EMT) and role of lobeglitazone (LGZ), a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist in BRAFV600E-mutated papillary thyroid carcinoma cells. LGZ suppresses the p38 mitogen-activated protein kinase (MAPK) signaling pathway and finally reduces migration and invasion of papillary thyroid carcinoma (PTC) cells induced by TGF-β1. “→” indicates promotion; “⊥” indicates inhibition. ERK, extracellular signal-regulated kinase.

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