The effect of silencing the Tip60 gene on the response to radiotherapy in breast cancer cells

Abstract

Since patients with triple-negative breast cancer do not respond to hormone therapy, the main treatment method is the combination of chemotherapy and radiotherapy. Because the DNA of the tumor cell is the target in both some chemotherapeutics and radiotherapy, problems may occur in individuals with a high DNA repair pathway. It is suggested that high expression of the Tip60 gene, which has an important role in repairing DNA damage, will increase the repair of DNA double-strand breaks in tumor cells, especially during radiotherapy treatment, thus reducing the response to treatment and adversely affecting treatment. In this study, for the first time, the role of the silenced and active Tip60 gene in response to radiotherapy in MDA-MB-231 and MCF-7 cells was investigated. For this purpose, the Tip60 gene was silenced by applying siRNA to the cell lines and UV was applied. In the study, cytotoxicity and DNA breaks were measured by MTT and COMET methods, and mRNA and protein expression values were measured by PCR and Raman spectrophotometer in silenced, unsilenced, UV-treated, and non-UV-treated cell lines. According to the results of the study, increased DNA damage was observed in MCF-7 cell lines in which the Tip60 gene was silenced, and radiotherapy was applied, compared to the cell lines with the Tip60 gene active. It was observed that DNA damage in MDA-MB-231 cell lines was less than in cell lines with the active Tip60 gene.

Keywords: Comet; MCF-7; MDA-MB-231; MTT; PCR; Protein expression; Tip60; siRNA.

Fig. 1

PCR amplification plots of β-ACTB andTip60. A and B. β-ACTB, C. unsilenced Tip60, D. silenced Tip60 gene.

Fig. 2

Relative evaluation of the cells with the 2^−ΔΔCt method by normalizing the expression levels of Tip60 in cell lines with and without the tip60 gene silenced.

Fig. 3

Protein analysis results by Surface Enriched Raman Spectroscopy in MCF-7 and MDA-MB-231 cell lines.

Fig. 4

Percentage of viability in MCF 7 and MDA-MB-231 cell lines after silencing of the Tip60 gene and before and after UV treatment.

Fig. 5

DNA damage in MCF-7 and MDA-MB 231 cells was examined by comet assays. The results show DNA damage in cells in terms of tail density.

Fig. 6

Comet images of UV-treated MCF-7 cells with the Tip60 gene activated.

Fig. 7

A. Comet images of MDA-MB- 231 cell with activated Tip60 gene and UV-treated, B. Comet images of Tip60 gene silenced and UV-treated MDA-MB- 231 cell.