Role of p53 mutation in the effect of boron neutron capture therapy on oral squamous cell carcinoma

Abstract

Background: Boron neutron capture therapy (BNCT) is a selective radiotherapy, being effective for the treatment of even advanced malignancies in head and neck regions as well as brain tumors and skin melanomas. To clarify the role of p53 gene, the effect of BNCT on oral squamous cell carcinoma (SCC) cells showing either wild- (SAS/neo) or mutant-type (SAS/mp53) p53 was examined.

Methods: Cells were exposed to neutron beams in the presence of boronophenylalanine (BPA) at Kyoto University Research Reactor. Treated cells were monitored for modulations in colony formation, proliferation, cell cycle, and expression of cell cycle-associated proteins.

Results: When SAS/neo and SAS/mp53 cells were subjected to BNCT, more suppressive effects on colony formation and cell viability were observed in SAS/neo compared with SAS/mp53 cells. Cell cycle arrest at the G1 checkpoint was observed in SAS/neo, but not in SAS/mp53. Apoptotic cells increased from 6 h after BNCT in SAS/neo and 48 h in SAS/mp53 cells. The expression of p21 was induced in SAS/neo only, but G2 arrest-associated proteins including Wee1, cdc2, and cyclin B1 were altered in both cell lines.

Conclusion: These results indicate that oral SCC cells with mutant-type are more resistant to BNCT than those with wild-type p53, and that the lack of G1 arrest and related apoptosis may contribute to the resistance. At a physical dose affecting the cell cycle, BNCT inhibits oral SCC cells in p53-dependent and -independent manners.

Figure 1

Suppression of the colony formation of oral SCC cells by BNCT. SAS/neo and SAS/mp53 cells were treated with BNCT, and survival fractions were assessed based on colony formation.

Figure 2

Suppression of the proliferation of oral SCC cells by BNCT. SAS/neo and SAS/mp53 cells were treated with BNCT, and cell viability was measured by the MTT assay. The cell viability of untreated cells was also measured and used as a control. *p < 0.01, SAS/neo vs. SAS/mp53.

Figure 3

Induction of cell cycle arrest by BNCT. A. SAS/neo and SAS/mp53 cells were treated with BNCT and then subjected to flow cytometric analysis. B. Based on an analysis of DNA histograms, the percentages of cells in sub-G₁, G₀/G₁, S, and G₂/M phases were evaluated.

Figure 4

Induction of apoptotic cells with the fragmentation of nuclear DNA by BNCT. SAS/neo and SAS/mp53 cells were treated with BNCT, incubated for 48 h at 37°C, and stained by Hoechst 33342. The proportion of apoptotic cells was determined at various time points. *p < 0.01, SAS/neo vs. BNCT-treated SAS/neo; SAS/mp53 vs. BNCT-treated SAS/mp53.

Figure 5

Altered expression and/or phosphorylation of G1 checkpoint-related proteins by BNCT. SAS/neo and SAS/mp53 cells were treated with BNCT, and the expression of p53 and p21 and phosphorylation of p53 were examined by immunoblot analysis.

Figure 6

Altered expression and/or phosphorylation of G2 checkpoint-related proteins by BNCT. SAS/neo and SAS/mp53 cells were treated with BNCT, and the expression of Wee1, cdc2, and cyclin B1 and phosphorylation of cdc2 were examined by immunoblot analysis.