Functional analysis of tanshinone IIA that blocks the redox function of human apurinic/apyrimidinic endonuclease 1/redox factor-1

Figures & data

Figure 1 Effects of T2A on the redox function and expression of APE1.

Notes: In (A), (C), (E), and (G), the nuclear extracts of APE1^wt, APE1^shRNA, and APE1^C65S cells were treated with T2A at indicated dose for 30 minutes, respectively. DNA-binding activity of NF-κB (A), AP-1 (C), HIF-1α (E), and SP-1 (G) were assessed by EMSA. In (B), (D), (F), and (H), the quantification of DNA-binding levels of NF-κB (B), AP-1 (D), HIF-1α (F), and SP-1 (H) was performed by densitometry. Each point represents the mean ± standard deviation of three experiments. *P<0.05 indicates statistically significant difference.
Abbreviations: T2A, tanshinone IIA; AP-1, activator protein-1; APE, apurinic/apyrimidinic endonuclease; APE1^wt, wild-type APE1; APE1^shRNA, short hairpin RNA knockdown APE1; APE1^C65S, redox-deficient mutant APE1; NF-κB, nuclear factor-κB; NS, not significant; HIF-1α, hypoxia-inducible transcription factor 1α; EMSA, electrophoretic mobility-shift assay; SP-1, specificity protein-1.

Figure 2 Effects of T2A on AP site incision and interaction activity of APE1.

Notes: (A) 500 pg purified human APE1 protein was exposed to T2A and CRT0044876 at indicated dose prior to measuring DNA strand cleavage activity via the radiolabeled assay. The upper band (substrate) represents uncleaved AP oligonucleotides; whereas, the lower band (product) is the reacted oligonucleotide. (B) 30 ng purified human APE1 protein was exposed to T2A and CRT0044876 at indicated dose prior to measuring AP site interaction activity via the radiolabeled assay. The upper band indicates APE1-bound AP oligonucleotides while the lower band is the unbound AP oligonucleotide. (C), (D) Quantification of cleaved AP oligonucleotides (C) and APE1-bound AP oligonucleotides (D) was performed by densitometry after normalization to DMSO control. Each point represents the mean ± standard deviation of three experiments. *P<0.05 indicates statistically significant difference.
Abbreviations: NS, not significant; AP, apurinic/apyrimidinic; APE, apurinic/apyrimidinic endonuclease; oligo, oligonucleotide; DMSO, dimethyl sulfoxide; T2A, tanshinone IIA; CRT, CRT0044876.

Figure 3 Binding affinity and docking simulation of T2A–APE1 interaction.

Notes: (A) Real-time DPI measurements of change in mass of immobilized APE1 protein after injection of increased concentrations of T2A. (B) Real-time DPI measurements of thickness, density, and mass during addition of T2A to immobilized APE1. (C) The assessment of disassociation and association kinetic constants and the dissociation constant K_D for the interaction between T2A and APE1. (D), (E) The detailed 3D (D) and 2D (E) docking model showing hydrogen-bonding interaction of T2A with Glu-137 of APE1.
Abbreviations: T2A, tanshinone IIA; APE, apurinic/apyrimidinic endonuclease; Asp, asparagine; DPI, dual polarization interferometry; Gln, glutamine; Leu, leucine; Ser, serine; 3D, three-dimensional; 2D, two-dimensional.

Figure 4 Dose- and time-dependent effects of T2A on the growth of HeLa, HCT116, and HUVEC cells.

Notes: HeLa (A) and HCT116 cells (B) were incubated with T2A at concentration of 0 μM, 2 μM, 4 μM, 8 μM, 16 μM, and 32 μM for 24 hours, 48 hours, and 72 hours, respectively. (C) HeLa, HCT116, and HUVEC cells were incubated with T2A at concentration of 0 μM, 5 μM, 10 μM, 20 μM, and 30 μM for 48 hours, respectively. Proliferation was assessed by MTT assay. Each point represents the mean ± standard deviation of three experiments.
Abbreviations: T2A, tanshinone IIA; HeLa, human cervical cancer cell line; HCT116, human colon cancer cell line; HUVEC, human umbilical veins endothelial cells; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide.

Figure 5 Effect of APE1 knockdown or its redox mutation on the T2A activities in cell growth.

Notes: (A) APE1^wt, APE1^shRNA, and APE1^C65S cells were treated with T2A at indicated dose for 48 hours, respectively. Proliferation was assessed by MTT assay. (B) APE1^wt, APE1^shRNA, and APE1^C65S cells were treated with T2A at indicated dose for 24 hours, respectively. Protein level of cleaved PARP (c-PARP) was assessed by Western blot assay. (C) Quantification of c-PARP protein level by densitometry after normalization to β-actin. Each point represents the mean ± standard deviation of three experiments. *P<0.05 and **P<0.01 denote statistically significant difference.
Abbreviations: APE, apurinic/apyrimidinic endonuclease; T2A, tanshinone IIA; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; PARP, poly adenosine diphosphate ribose polymerase; NS, not significant; APE1^wt, wild-type APE1; APE1^shRNA, short hairpin RNA knockdown APE1; APE1^C65S, redox-deficient mutant APE1.

Figure 6 Effects of T2A on genotoxic potential of chemical agents and ionizing radiation in HeLa and HCT116 cells.

Notes: HeLa (A) and HCT116 (C) cells were pretreated with T2A at 2 μM and 4 μM and then incubated with either of agents at indicated dose for an additional 48 hours. Proliferation was assessed by MTT assay. Each point represents the mean ± standard deviation of three experiments. *P<0.05 and **P<0.01 denote statistically significant difference. (B), (D) The products of SR (T) × SR (E), SR (T) × SR (C), SR (T) × SR (S), SR (T) × SR (H), SR (T) × SR (P), and SR (T) × SR (I) represent the survival rate of HeLa (B) and HCT116 (D) cells treated with T2A (SR [T]) at 2 μM and 4 μM multiplied by the survival rate of Eto (SR [E]), MMC (SR [C]), MMC (SR [S]), HU (SR [H]), Cpt (SR [P]), and ionizing radiation (SR [I]) treatment at indicated dose, respectively. These products are > the survival rate of HeLa (B) and HCT116 (D) cells treated with combined T2A and Eto (SR [T + E]), T2A and MMC (SR [T + C]), T2A and MMS (SR [T + S]), T2A and Cpt (SR [T + P]), T2A and ionizing radiation (SR [T + I]) at each concentration of T2A tested, respectively, except for the survival rate of cells treated with combined T2A and HU (SR [T + H]).
Abbreviations: T2A, tanshinone IIA; NS, not significant; SR, survival rate; Eto, etoposide; MMC, mitomycin C; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; MMS, methyl methanesulfonate; Cpt, camptothecin; HU, hydroxyurea; IR, ionizing radiation; HeLa, human cervical cancer cell line; HCT116, human colon cancer cell line.

Figure S1 Representative Western blot images showing APE1 endogenous and exogenous protein levels in control (HeLa cells), APE1^shRNA, APE1^wt, and APE1^C65S cells.

Abbreviations: APE, apurinic/apyrimidinic endonuclease; HeLa, human cervical cancer cell line; APE1^wt, wild-type APE1; APE1^shRNA, short hairpin RNA knockdown APE1; APE1^C65S, redox-deficient mutant APE1.

Figure S2 Increasing amounts of E3330 or T2A were incubated for 30 minutes with the nuclear extracts of HeLa cells.

Note: DNA-binding activity of NF-κB was assessed by EMSA.
Abbreviations: T2A, tanshinone IIA; NF-κB, nuclear factor-κB; EMSA, electrophoretic mobility-shift assay; HeLa, human cervical cancer cell line.

Figure S3 View of T2A docked into the X-ray crystallographic structure of APE1.

Abbreviations: T2A, tanshinone IIA; APE, apurinic/apyrimidinic endonuclease.

Figure S4 HeLa cells were treated with T2A at indicated dose for 24 hours.

Notes: Whole-cell extracts were employed for Western blot analysis of APE1 protein levels. Detection of β-actin protein levels served as the loading control.
Abbreviations: T2A, tanshinone IIA; APE, apurinic/apyrimidinic endonuclease; HeLa, human cervical cancer cell line.