Clin Exp Otorhinolaryngol.  2012 Jun;5(2):86-93. 10.3342/ceo.2012.5.2.86.

Growth Inhibitory Effect of Palatine Tonsil-derived Mesenchymal Stem Cells on Head and Neck Squamous Cell Carcinoma Cells

• Lim YS
• Lee JC
• Lee YS
• Lee BJ
• Wang SG

• KMID: 2117502
• DOI: http://doi.org/10.3342/ceo.2012.5.2.86

Abstract

OBJECTIVES
Mesenchymal stem cells (MSCs) play an important role in the development and growth of tumor cells. However, the effect of human MSCs on the growth of human tumors is not well understood. The purpose of this study is to confirm the growth effect of palatine tonsil-derived MSCs (TD-MSCs) on head and neck squamous cell carcinoma (HNSCC) cell lines and to elucidate the mechanism of their action.
METHODS
TD-MSCs were isolated from patient with chronic tonsillitis and tonsillar hypertrophy. Two human HNSCC cell lines (PNUH-12 and SNU-899) were studied and cocultured with isolated palatine tonsil-derived MSC. The growth inhibitory effect of MSCs on HNSCC cell lines was tested through methylthiazolyldiphenyl-tetrazolium (MTT) assay. The apoptosis induction effect of MSCs on cell lines was assessed with flow cytometry and reverse transcriptase (RT)-PCR.
RESULTS
Palatine tonsil-derived MSCs exhibited a growth inhibitory effect on both cell lines. Cell cycle analysis showed an accumulation of tumor cells predominantly in G0/G1 phase with an increase in concentration of TD-MSCs, which was confirmed by increased mRNA expression of cell cycle negative regulator p21. Apoptosis of tumor cells increased significantly as concentration of cocultured TD-MSCs increased. Additionally, mRNA expression of caspase 3 was upregulated with increased concentration of TD-MSCs.
CONCLUSION
TD-MSCs have a potential growth inhibitory effect on HNSCC cell lines in vitro by inducing apoptotic cell death and G1 phase arrest of cell lines.

Keyword

Apoptosis; Mesenchymal stem cells; Palatine tonsil; Squamous cell carcinoma

MeSH Terms

Apoptosis
Carcinoma, Squamous Cell
Caspase 3
Cell Cycle
Cell Death
Cell Line
Flow Cytometry
G1 Phase
Growth and Development
Head
Humans
Hypertrophy
Mesenchymal Stromal Cells
Neck
Palatine Tonsil
RNA, Messenger
RNA-Directed DNA Polymerase
Tonsillitis
Caspase 3
RNA, Messenger
RNA-Directed DNA Polymerase

Figure

• 

  Fig. 1 Characterization and differentiation of tonsil derived-mesenchymal stem cells (TD-MSCs) analyzed by flow cytometry and microscopy. (A) The mesenchymal stem cell (MSC)-specific markers CD73, CD90, and CD105 were expressed in TD-MSCs, whereas the hematopoietic stem-cell markers CD45, CD31, and CD117 were not. (B) TD-MSCs showed a fibroblast-like morphology and had the ability for adipogenic, osteogenic, and chondrogenic differentiation in differentiation induction medium. Differentiated cells were stained with oil red O, alizarin red S, and alcian blue.

• 

  Fig. 2 Tonsil derived-mesenchymal stem cells (TD-MSCs) shows methylthiazolyldiphenyl-tetrazolium (MTT) metabolism inhibitory effect on head and neck squamous cell carcinoma (HNSCC) tumor cells by number-dependent manner. 5×104 cells of each head and neck cancer cell line (PNUH-12 and SNU 899) were incubated with mitomycin C (MMC)-treated TD-MSC or fibroblast at different cell ratio (1:20, 1:10, 1:5, 1:2), and cell viability was evaluated by the MTT assay (A) and (B). Results are mean inhibitory rate±SD of three independent experiments. *P<0.05 (compared with fibroblast treated control).

• 

  Fig. 3 Tonsil derived-mesenchymal stem cells (TD-MSCs) induced cell cycle arrest of head and neck squamous cell carcinoma (HNSCC) tumor cells. 5×104 tumor cells were seeded onto 6-well plate and incubated for 18 hours. TD-MSCs and fibroblasts were cocultured for 48 hours at the cell ratio of 1:20, 1:10, 1:5, 1:2. Cells were harvested, washed twice with phosphate-buffered saline and measured by flow cytometry analysis. An accumulation of tumor cells increased in G0/G1 phase with an increase in concentration of TD-MSCs, which was statistically significant (PNUH-12, 33.1% vs. 50.6%; SNUH-899, 36.6% vs. 58.5%; P<0.05) on each tumor cell line (A) and (B). The increase of G0/G1 phase in TD-MSC treated SNU-899 cell line was more than in fibroblast treated control at the cell ratio of 1:5 and 1:2 (B). *P<0.05 (compared with fibroblast treated control).

• 

  Fig. 4 Cell cycle analysis shows apoptosis of tumor cells increased significantly as the cell ratio of cocultured tonsil derived-mesenchymal stem cells (TD-MSCs) increased on each tumor cell line (A) and (B). TD-MSCs exhibited significantly increased apoptosis of tumor cells more than fibroblasts at the cell ratio of 1:10, 1:5, and 1:2. *P<0.05, **P<0.001 (compared with fibroblast treated control).

• 

  Fig. 5 Upregulated mRNA expression of caspase 3 is demonstrated with increased concentration of tonsil derived-mesenchymal stem cells (TD-MSCs) at each tumor cell line (A) and (B), which could support the apoptosis of tumor cells. *P<0.05 (compared with fibroblast treated control).

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