Effect of photodynamic therapy on expression of HRAS, NRAS and caspase 3 genes at mRNA levels, apoptosis of head and neck squamous cell carcinoma cell line

Photodiagnosis Photodyn Ther. 2021 Mar:33:102142. doi: 10.1016/j.pdpdt.2020.102142. Epub 2020 Dec 8.

Abstract

Objectives: This study aimed to assess the effect of photodynamic therapy (PDT) on expression of CASP3, NRAS and HRAS genes at mRNA levels, and apoptosis of head and neck squamous cell carcinoma (HNSCC) cell line.

Materials and methods: In order to complete the present in vitro study, HNSCC cell line (NCBI C196 HN5) purchased from Pasteur Institute. Cells were divided into four groups; Group 1: photodynamic treatment (laser + methylene blue (MB) as photosensitizer), group 2: MB, group 3: laser (with 660 nm wavelength), and group 4: control (without any treatment). To determine the optimal concentration of MB, in a pilot study, toxicity of MB in different concentration was assessed using MTT assay. Cells in group 1, 2 and 3 was treated at optimal concentration of MB (1.6 μg/mL). Gene expression at mRNA levels was assessed after 24 h incubation, using real-time (qRT)-PCR. The expression of BAX and BCL2 genes at the mRNA levels was analyzed to evaluate apoptosis. 2-ΔΔCt values of BCL2, BAX, CASP3, NRAS, and HRAS in groups was analyzed using ANOVA. Tukey's HSD and Games Howell test was used to compare between two groups.

Results: Over-expression of BAX (p < 0.001), CASP3 (p < 0.001) and down-regulation of BCL2 (p = 0.004), HRAS (p = 0.023) and NRAS (p = 0.045) were noted in group 1 (PDT), compared with the control group. Treatment by laser alone induce down-regulation of CASP3 (p < 0.05), BAX (p < 0.05), BCL2 (p > 0.05), HRAS (p > 0.05) and NRAS (p > 0.05).

Conclusion: PDT caused down-regulation of NRAS, HRAS and BCL2 and over-expression of CASP3 and BAX genes at mRNA levels in HNSCC cell line. The present study raises the possibility that the role of MB on BCL2 down-regulation and BAX and CASP3 over-expression was higher than laser alone while it seems that laser alone was more effective than MB in HRAS and NRAS down-regulation.

Keywords: Apoptosis; Head and neck squamous cell carcinoma; Methylene blue; Photodynamic therapy; Proto-oncogene; Tumor suppressor genes.

MeSH terms

  • Apoptosis
  • Caspase 3
  • Cell Line
  • Cell Line, Tumor
  • GTP Phosphohydrolases / genetics
  • GTP Phosphohydrolases / pharmacology
  • Head and Neck Neoplasms* / drug therapy
  • Head and Neck Neoplasms* / genetics
  • Humans
  • Membrane Proteins / genetics
  • Photochemotherapy* / methods
  • Photosensitizing Agents / pharmacology
  • Pilot Projects
  • Proto-Oncogene Mas
  • Proto-Oncogene Proteins p21(ras) / genetics
  • Proto-Oncogene Proteins p21(ras) / pharmacology
  • RNA, Messenger
  • Squamous Cell Carcinoma of Head and Neck / drug therapy
  • Squamous Cell Carcinoma of Head and Neck / genetics

Substances

  • MAS1 protein, human
  • Membrane Proteins
  • Photosensitizing Agents
  • Proto-Oncogene Mas
  • RNA, Messenger
  • Caspase 3
  • GTP Phosphohydrolases
  • NRAS protein, human
  • HRAS protein, human
  • Proto-Oncogene Proteins p21(ras)