Endocrinol Metab.  2018 Mar;33(1):121-132. 10.3803/EnM.2018.33.1.121.

Combined Effects of Baicalein and Docetaxel on Apoptosis in 8505c Anaplastic Thyroid Cancer Cells via Downregulation of the ERK and Akt/mTOR Pathways

• Park CH
• Han SE
• Nam-Goong
• Kim YI
• Kim ES

• KMID: 2407129
• DOI: http://doi.org/10.3803/EnM.2018.33.1.121

Abstract

BACKGROUND
Anaplastic thyroid cancer (ATC) is one of the most lethal human malignancies. Docetaxel, a microtubule stabilizer, is a common chemotherapeutic agent used to treat various metastatic cancers. However, prolonged use results in various side effects and drug resistance. Flavonoids, such as baicalein, are accepted chemotherapeutic and dietary chemopreventive agents with many advantages, such as greater accessibility, affordability, and lower toxicity, compared with traditional chemotherapy agents. In this study, we evaluated whether baicalein enhances the effects of docetaxel on apoptosis and metastasis in 8505c ATC cells.
METHODS
The 8505c cells were treated with baicalein or docetaxel individually and in combination. Cell viability was measured by MTT (thiazolyl blue tetrazolium bromide) assay, and apoptosis was detected by fluorescence microscopy of Hoechst-stained cells. The expression of apoptotic (Bax and caspase-3), anti-apoptotic (Bcl-2), angiogenic (vascular endothelial growth factor [VEGF], transforming growth factor β [TGF-β], E-cadherin, and N-cadherin), and signaling (extracellular signal-regulated kinase [ERK] mitogen activated protein kinase [MAPK], Akt, and mammalian target of rapamycin [mTOR]) proteins was determined by Western blot analysis.
RESULTS
The combination of baicalein (50 or 100 µM) and docetaxel (10 nM) significantly inhibited proliferation and induced apoptosis compared with monotherapies. The combination treatment significantly inhibited the expression of Bax, caspase-3, VEGF, TGF-β1, E-cadherin, N-cadherin, and mTOR, but decreased the expression of Bcl-2 and significantly decreased the phosphorylation of ERK and Akt.
CONCLUSION
The combination of baicalein and docetaxel effectively induced apoptosis and inhibited metastasis in 8505c cells through downregulation of apoptotic and angiogenic protein expression and blocking of the ERK and Akt/mTOR pathways in 8505c cells. These results suggest that baicalein enhances the anticancer effects of docetaxel in ATC.

Keyword

Baicalein; Thyroid carcinoma, anaplastic; Angiogenesis; Apoptosis; Combination therapy; Docetaxel; 8505c cells

MeSH Terms

Apoptosis*
Blotting, Western
Cadherins
Caspase 3
Cell Survival
Down-Regulation*
Drug Resistance
Drug Therapy
Endothelial Growth Factors
Flavonoids
Humans
Microscopy, Fluorescence
Microtubules
Neoplasm Metastasis
Phosphorylation
Phosphotransferases
Protein Kinases
Sirolimus
Thyroid Carcinoma, Anaplastic*
Transforming Growth Factors
Vascular Endothelial Growth Factor A
Cadherins
Caspase 3
Endothelial Growth Factors
Flavonoids
Phosphotransferases
Protein Kinases
Sirolimus
Transforming Growth Factors
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 Effects of the combined treatment with baicalein and docetaxel on the viability in 8505c cells. The cells were treated with baicalein (A, C) and docetaxel (B, D) individually or in combination (E, F) for 24 hours (A, B, E) and 48 hours (C, D, F). The cell viability was determined by MTT (thiazolyl blue tetrazolium bromide) assay. The data are presented as the mean±SEM of three independent experiments. aP<0.05; bP<0.1 vs. normal cells.

• 

  Fig. 2 Effects of the combined treatment with baicalein and docetaxel on apoptosis in 8505c cells. The cells were treated with baicalein and docetaxel individually or in combination for 24 hours. (A) Nuclear fragmentation was determined by the observation of Hoechst staining using a fluorescence microscope (×400). The arrows indicate DNA fragmentation in apoptotic cells. Non-treated cells (a); baicalein-treated cells at 20 µM (b), 50 µM (c), and 100 µM (d); docetaxel-treated cells at 10 nM (e); cells treated the combination of docetaxel at 10 nM and baicalein at 20 µM (f), 50 µM (g), and 100 µM (h). The expression of Bax, caspase-3, cleaved caspase-3 (B), and Bcl-2 (C) proteins was measured by Western blotting analysis. The histogram (D, E, F, G) is presented as the mean±SEM of the band density for three independent experiments compared with β-actin expression. aP<0.05; bP<0.01 vs. baicalein alone and docetaxel combined treatment.

• 

  Fig. 3 Effects of the combined treatment with baicalein and docetaxel on metastasis in 8505c cells. The cells were treated with baicalein and docetaxel individually or in combination for 24 hours. The expression of vascular endothelial growth factor (VEGF), transforming growth factor β1 (TGF-β1) (A), E-cadherin, and N-cadherin (D) proteins were measured by Western blotting analysis. The histogram (B, C, E, F) was presented as the mean±SEM of the band density for three independent experiments compared with β-actin expression. aP<0.01; bP<0.001 vs. baicalein alone and docetaxel combined treatment.

• 

  Fig. 4 Effects of the combined treatment with baicalein and docetaxel on the signaling pathway in 8505c cells. The cells were treated with baicalein and docetaxel individually or in combination for 24 hours. The expression of mammalian target of rapamycin (mTOR) (A) and the phosphorylation of extracellular signal-regulated kinase (ERK) and Akt (B) were determined by Western blotting analysis. (C, D, E) Each histogram was presented as the mean±SEM of the band density for three independent experiments compared with β-actin expression (for mTOR) or the total expression (for ERK or Akt). aP<0.05; bP<0.01 vs. baicalein alone and docetaxel combined treatment.

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