Chronic exposure to the gibberellin derivative GA-13315 sensitizes breast cancer MCF-7 cells but not colon cancer HCT116 cells to irinotecan

Abstract

13-Chlorine-3,15-dioxy-gibberellic acid methyl ester (GA-13315) is a gibberellin derivative that exhibits selective cytotoxicity to multidrug resistant MCF-7/ADR cells and reverses drug resistance when administered at subtoxic doses in combination with chemotherapy drugs. The present study aimed to investigate the impact of chronic GA-13315 exposure on the chemosensitivity of MCF-7 and HCT116 cell lines. Cells were administered a subtoxic dose of 1 µM GA-13315 for 12 weeks and the sensitivity of the cells to GA-13315, irinotecan and cisplatin, was assessed. The Cell Counting Kit-8 assay results demonstrated that the chronic exposure did not induce resistance to GA-13315, in either MCF-7 or HCT116 cells. Notably, MCF-7 cells were sensitized to irinotecan following exposure to GA-13315; however, HCT116 cells were not. The sensitizing effect of GA-13315 was associated with the alterations of topoisomerase 1 (Top1) protein expression, tyrosyl DNA phosphodiesterase 1 and checkpoint kinase 1. Further analysis indicated that GA-13315 caused DNA fragmentation; however, DNA damage was not mediated by a Top1-dependent molecular mechanism, as GA-13315 was revealed not to be a Top1 poison, despite inhibiting the catalytic activity of Top1. Taken together, the results of the present study indicated that GA-13315 may be used for sensitizing MCF-7 cells to irinotecan, as the chronic exposure of GA-13315 to MCF-7 cells still showed sensitizing effects to irinotecan.

Keywords: chemosensitivity; chronic exposure; multidrug resistance; topoisomerase I.

Figure 1.

Chemical structure of 13-chlorine-3,15-dioxy-gibberellic acid methyl ester.

Figure 2.

Effect of 13-chlorine-3,15-dioxy-gibberellic acid methyl ester on the expression of proteins involved in the chemosensitivity of tumor cell lines. (A) Protein expression levels were normalized to β-actin and quantified using ImageJ v1.49 software, and (B) data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. 0 weeks (control); ^#P<0.05 vs. 4 weeks; ^φP<0.05 vs. 8 weeks. Top1, topoisomerase I; Tdp1, tyrosyl DNA phosphodiesterase 1; Chk1, checkpoint kinase 1; Bcl-2, B-cell lymphoma-2.

Figure 3.

Effect of 13-chlorine-3,15-dioxy-gibberellic acid methyl ester on the mRNA expression levels of genes involved in the chemosensitivity of tumor cell lines. Reverse transcription-quantitative PCR analysis was performed to determine the mRNA expression levels of (A) Top1, (B) Chk1, (C) Bax and (D) Bcl-2 in MCF-7 and HCT116 cells. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. 0 week (control); ^#P<0.05 vs. 4 weeks; ^φP<0.05 vs. 8 weeks. Top1, topoisomerase I; Chk1, checkpoint kinase 1; Bcl-2, B-cell lymphoma-2.

Figure 4.

Effect of GA-13315 on DNA fragmentation. Cells were treated with irinotecan, cisplatin and GA-13315 for 48 h at the IC₅₀ concentration, underwent chronic GA-13315 exposure and were subjected to single-cell electrophoresis. DNA fragmentation was indicated by the comet tail visualized by DAPI staining (magnification, ×40). GA-13315, 13-chlorine-3,15-dioxy-gibberellic acid methyl ester.

Figure 5.

Effect of GA-13315 on the catalytic activity of (A) Top1 and (B) Top1-mediated DNA cleavage. GA-13315 inhibited the catalytic activity of Top1 in a dose-dependent manner, which was demonstrated by the increased amount of supercoiled DNA as the concentration of GA-13315 increased. GA-13315 did not affect Top1-mediated DNA cleavage, evidenced by the lack of nicked DNA in the presence of GA-13315. CPT-11 (50 µM) was used as the reference drug. The control test was performed using 0.1% DMSO instead of GA-13315. GA-13315, 13-chlorine-3,15-dioxy-gibberellic acid methyl ester; M, marker; Ctrl, control.