Boric Acid Affects the Expression of DNA Double-Strand Break Repair Factors in A549 Cells and A549 Cancer Stem Cells: An In Vitro Study

Abstract

DNA double-strand break (DSB) repair genes interact with tumor stemness- and resistance-associated processes in cancer stem cells (CSCs). Therefore, targeting DNA DSB genes in cancer treatment is important for the CSC phenotype. Although the anti-cancer effect of boric acid (BA) has been studied, its effect on DNA DSB is unclear. Moreover, no studies investigate BA's effects on DNA DSB of lung cancer stem cells (LC-SCs). To fill the gap, we aimed to assess the effects of BA on A549 cancer stem cells. CSCs were isolated from human non-small cell lung cancer cells (A549) and characterized by flow cytometry. Different concentrations of BA (at doses ranging from 1 to 100 mM) were applied to cancer stem cells. Cytotoxic activities were determined using the cell viability assay (MTT assay) at 24 and 48 h. Expression levels of DNA DSB genes that BRCA1, BRCA2, RAD51, KU70/80, ATM, and XRCC4 were evaluated by RT-qPCR. Additionally, immunofluorescence staining analysis was exploited for caspase-3 and E-cadherin. ATM expression increased significantly (p < 0.001). No significant change was observed in the expression of other genes. Moreover, BA up-regulated caspase-3 and E-cadherin expression. Consequently, we can say that BA affects DNA DSB and the apoptotic abilities of LC-SCs.

Keywords: Boric acid; Cytotoxicity; DNA DSB; Lung cancer stem cell.

Fig. 1

The morphology of A549 (human non-small lung cancer cells) and A549 derived cancer stem cells (LC-SCs). A: A549 cells (scale bar = 200 µm); B–C: LC-SCs (scale bar = 200 µm and scale bar = 100 µm, respectively); D: Tumorspheres formation of LC-SCs (scale bar = 100 µm)

Fig. 2

Identification of LC-SCs by flow cytometry. Cell surface expression of CD44, CD133, CD117, and CD338 in the A549 cell line. Flow cytometry analysis detected CD44, CD133, CD117, and CD338 cell populations

Fig. 3

Cell viability of LC-SCs. Cell viability was measured with the MTT assay. LC-SCs were exposed to different concentrations of BA (1, 10, 12.5, 25, 50, 75, and 100 mM) for 24 and 48 h. (BA, boric acid). (mean ± SD; n = 3; *P < 0.05, **P < 0.01, and ***P < 0.001; ns means not significant)

Fig. 4

mRNA expressions of DNA DSB repair genes. RT-qPCR was performed as described in the previous study [22]. GAPDH was used as a control. ATM expression increased significantly (p < 0.001). (mean ± SD; n = 3; *P < 0.05, **P < 0.01, and ***P < 0.001; ns means not significant)

Fig. 5

Immunocytochemistry analysis of caspase-3 and E-cadherin expression. BA increased the expression of caspase-3 and E-cadherin. After treatment with BA for 24 and 48 h, apoptosis in A549 cells and LC-SCs was assessed immunocytochemically. A–F: caspase-3 expression in green; G–L: E-cadherin expression in red. Nuclei were labeled with DAPI (blue). (Scale bar = 100 µm)