J Korean Assoc Oral Maxillofac Surg.  2016 Dec;42(6):337-344. 10.5125/jkaoms.2016.42.6.337.

The influence of p53 mutation status on the anti-cancer effect of cisplatin in oral squamous cell carcinoma cell lines

• Jo DW
• Kim YK
• Yun PY

• KMID: 2364000
• DOI: http://doi.org/10.5125/jkaoms.2016.42.6.337

Abstract

OBJECTIVES
The purpose of this study was to evaluate the anti-cancer activity of cisplatin by studying its effects on cell viability and identifying the mechanisms underlying the induction of cell cycle arrest and apoptosis on oral squamous cell carcinoma (OSCC) cell lines with varying p53 mutation status.
MATERIALS AND METHODS
Three OSCC cell lines, YD-8 (p53 point mutation), YD-9 (p53 wild type), and YD-38 (p53 deletion) were used. To determine the cytotoxic effect of cisplatin, MTS assay was performed. The cell cycle alteration and apoptosis were analyzed using flow cytometry. Western blot analysis was used to detect the expression of cell cycle alteration- or apoptosis-related proteins as well as p53.
RESULTS
Cisplatin showed a time- and dose-dependent anti-proliferative effect in all cell lines. Cisplatin induced G2/M cell accumulation in the three cell lines after treatment with 0.5 and 1.0 µg/mL of cisplatin for 48 hours. The proportion of annexin V-FITC-stained cells increased following treatment with cisplatin. The apoptotic proportion was lower in the YD-38 cell line than in the YD-9 or YD-8 cell lines. Also, immunoblotting analysis indicated that p53 and p21 were detected only in YD-8 and YD-9 cell lines after cisplatin treatment.
CONCLUSION
In this study, cisplatin showed anti-cancer effects via G2/M phase arrest and apoptosis, with some difference among OSCC cell lines. The mutation status of p53 might have influenced the difference observed among cell lines. Further studies on p53 mutation status are needed to understand the biological behavior and characteristics of OSCCs and to establish appropriate treatment.

Keyword

Cisplatin; p53; Oral squamous cell carcinoma; Apoptosis

MeSH Terms

Apoptosis
Blotting, Western
Carcinoma, Squamous Cell*
Cell Cycle
Cell Cycle Checkpoints
Cell Line
Cell Survival
Cisplatin*
Epithelial Cells*
Flow Cytometry
Immunoblotting
Cisplatin

Figure

• 

  Fig. 1 Cell viability after cisplatin treatment. All cell lines showed a significant reduction in viability with time and dose. YD-8, YD-9, and YD-38 cell lines were incubated with various concentrations of cisplatin (0.5, 1.0, 2.0, 3.0, 5.0, and 10.0 µg/mL) for 24 hours (A), 48 hours (B), and 72 hours (C). Cell viability was determined via MTS assay. The data are expressed as the mean percentage of viable cells±standard error (n=3).

• 

  Fig. 2 Cisplatin induces G2/M arrest in the YD-8, YD-9, and YD-38 cell lines. The cells were incubated with 0.5 and 1.0 µg/mL cisplatin (cis) for 48 hours, fixed and stained with propidium iodide, and analyzed for DNA content.

• 

  Fig. 3 Induction of apoptosis in cisplatin-treated YD-8, YD-9, and YD-38 cell lines. The cells were harvested after 48 hours of incubation with 1.0 and 5.0 µg/mL of cisplatin. Apoptosis was determined by staining the cells with annexin V-FITC and propidium iodide (PI) labeling.

• 

  Fig. 4 Effects of cisplatin on cell cycleregulating molecules in the YD-8, YD-9, and YD-38 cell lines. The cells were treated with cisplatin at 0.5, 1.0, and 2.0 µg/mL and the expression levels of proteins were examined by Western blotting 48 hours after treatment.

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