AMRI-59 functions as a radiosensitizer via peroxiredoxin I-targeted ROS accumulation and apoptotic cell death induction

Abstract

Previously, we identified AMRI-59 as a specific pharmaceutical inhibitor of peroxiredoxin (PRX) I enzyme activity. In this study, we examined whether AMRI-59 acts as a radiosensitizer in non-small cell lung cancer cells using clonogenic assays. The intracellular mechanisms underlying the radiosensitization effect of AMRI-59 were determined via immunoblotting in addition to measurement of ROS generation, mitochondrial potential and cell death. AMRI-59 activity in vivo was examined by co-treating nude mice with the compound and γ-ionizing radiation (IR), followed by measurement of tumor volumes and apoptosis. The dose enhancement ratios of 30 μM AMRI-59 in NCI-H460 and NCI-H1299 were 1.51 and 2.12, respectively. Combination of AMRI-59 with IR augmented ROS production and mitochondrial potential disruption via enhancement of PRX I oxidation, leading to increased expression of γH2AX, a DNA damage marker, and suppression of ERK phosphorylation, and finally, activation of caspase-3. Notably, inhibition of ROS production prevented ERK suppression, and blockage of ERK in combination with AMRI-59 and IR led to enhanced caspase-3 activation and apoptosis. In a xenograft assay using NCI-H460 and NCI-H1299, combined treatment with AMRI-59 and IR delayed tumor growth by 26.98 and 14.88 days, compared with controls, yielding enhancement factors of 1.73 and 1.37, respectively. Taken together, the results indicate that AMRI-59 functions as a PRX I-targeted radiosensitizer by inducing apoptosis through activation of the ROS/γH2AX/caspase pathway and suppression of ERK.

Keywords: AMRI-59; ROS; non-small cell lung cancer; peroxiredoxin; radiosensitizer.

Figure 1. AMRI-59 induces retardation of NSCLC cell growth in conjunction with IR

‘IR’, radiation only; ‘IR+10 μM’ and ‘IR+30 μM’, combination of radiation with 10 μM and 30 μM AMRI-59, respectively. (A) Chemical structure of AMRI-59 (http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?sid=93087). (B-E) Clonogenic assay for determining the radiosensitization effect of AMRI-59 against NCI-H460 (B, C) and NCI-H1299 (D, E). Representative data from experiments performed in triplicate are shown in each right panel.

Figure 2. AMRI-59 induces NSCLC cell death and enhances apoptosis in conjunction with IR

‘Control’, mock control; ‘10 μM’ and ‘30 μM’, treatment with 10 and 30 μM AMRI-59 only; ‘3 Gy’ and ‘5 Gy’, treatment with 3 and 5 Gy IR only, respectively; ‘3 Gy/10 μM’, ‘3 Gy/30 μM’ and ‘5 Gy/10 μM’, ‘5 Gy/30 μM’, combination with 3 or 5 Gy IR and 10 or 30 μM AMRI-59, respectively; ‘z-VAD’, 20 μM of z-VAD-fmk pre-treatment. (A) PI uptake assay for detection of apoptosis. NCI-H460 or NCI-H1299 cells were treated with various doses of AMRI-59 and IR. (B) Caspase-3 activity detection with ELISA in NSCLCs treated with various doses of AMRI-59 and IR. (C) Immunoblot assay for detection of caspase-3 activation in cells treated with various doses of AMRI-59 and IR. ‘Pro-Cas3’ indicates pro-caspase-3 and ‘Cleaved Cas3’ is the cleaved form of caspase-3. (D) PI uptake assay and (E) Caspase-3 activity detection with or without pre-treatment of z-VAD-fmk for 1 h combined with AMRI-59 and IR. Representative data from experiments performed in triplicate are shown.

Figure 3. AMRI-59 induces ROS accumulation in conjunction with IR in NSCLC cells

‘Control’, mock control; ‘10 μM’ or ‘30 μM’, treatment with 10 or 30 μM AMRI-59 only; ‘3 Gy’ or ‘5 Gy’, treatment with 3 or 5 Gy IR only, respectively; ‘3 Gy/10 μM’, ‘3 Gy/30 μM’ and ‘5 Gy/10 μM’, ‘5 Gy/30 μM’, combination of 3 or 5 Gy IR and 10 or 30 μM AMRI-59, respectively; ‘NAC’, pre-treatment with 5 mM NAC. (A) Immunoblot analysis of oxidation of PRX I in in NCI-H460 and NCI-H1299 cells treated with a combination of AMRI-59 and IR (B, C) ROS determination. ROS detection with FACSorter in IR and AMRI-59-treated NSCLC cells (B), ROS detection with FACSorter in NAC pre-treated cells for 1 h treated with the IR and AMRI-59 combination (C). (D) Mitochondrial potential detection in NSCLC cells co-treated with IR and AMRI-59. (E) Immunoblot of cytochrome c release in NSCLC cells co-treated with IR and AMRI-59.

Figure 4. ROS production modulates apoptotic cell death in NSCLC cells subjected to combination of AMRI-59 and IR

‘Control’, mock control; ‘NAC’, pre-treatment with 5 mM NAC; ‘3 Gy/30 μM’ or ‘5 Gy/30 μM’ combination of 3 or 5 Gy IR and 30 μM AMRI-59; ‘3 Gy/30 μM/NAC’ or ‘5 Gy/30 μM/NAC’, combination of 3 or 5 Gy IR and 30 μM AMRI-59 with 5 mM NAC pre-treatment. (A) PI uptake assay for apoptotic cell death in the NAC-pre-treatment groups for 1 h subjected to AMRI-59 and IR. (B) Caspase-3 activity detection in cells with or without NAC pre-treatment for 1 h subjected to AMRI-59 and IR. Representative data from experiments performed in triplicate are shown.

Figure 5. The combination of AMRI-59 and IR induces enhanced DNA damage

‘Control’, mock control; ‘30 μM’, 30 μM AMRI-59 only; ‘3 Gy’, or ‘5 Gy’, 3 or 5 Gy IR only, respectively; ‘3 Gy/30 μM’ or, ‘5 Gy/30 μM’ combination of 3 or 5 Gy IR and 30 μM AMRI-59. (A) Immunohistochemical assay showing formation of γH2AX foci (arrow) in IR only, AMRI-59 only and combined AMRI-59 and IR groups. White bars in each image indicate 20 μM. (B) Immunoblot assay for detection of elevated γH2AX in IR only, AMRI-59 only and combined AMRI-59 and IR groups. Representative data from experiments performed in triplicate are shown.

Figure 6. Combination of AMRI-59 and IR promotes apoptotic cell death via elimination of ERK activity

‘Control’, mock control; ‘3 Gy’ or ‘5 Gy’, 3 or 5 Gy IR only, respectively; ‘10 μM’ or ‘30 μM’, treatment with 10 or 30 μM AMRI-59 only; ‘3 Gy/30 μM’ or ‘5 Gy/30 μM’ combination of 3 or 5 Gy IR and 30 μM AMRI-59; ‘3 Gy/30 μM/PD’ or ‘5 Gy/30 μM/PD’, combination of 3 or 5 Gy IR and 30 μM AMRI-59 with 10 μM PD98059 pre-treatment. ‘PD’, 10 μM PD98059 pre-treatment only. (A) Immunoblot assay for detection of phosphorylated and basal ERK. (B) PI uptake assay for detection of apoptotic death in cells with or without PD98059 pre-treatment for 1 h. (C) Caspase-3 activity detection in cells subjected to AMRI-59 and IR with or without PD pre-treatment for 1 h. (D) Immunoblot assay for detection of phosphorylated and basal ERK with NAC pre-treatment for 1 h. Representative data from experiments performed in triplicate are shown.

Figure 7. Attenuation of CREB-1 activity is a step in the apoptotic cell death pathway triggered by the combination of AMRI-59 and IR

‘Control’, mock control; ‘CREB inh’, pre-treatment with 10 μM CREB-1 inhibitor only; ‘NAC’, pre-treatment with 5 mM NAC; ‘3 Gy/30 μM’ or ‘5 Gy/30 μM’, combination of 3 or 5 Gy IR and 30 μM AMRI-59; ‘3 Gy/30 μM/NAC’ or ‘5 Gy/30 μM/NAC’, combination of 3 or 5 Gy IR and 30 μM AMRI-59 with 5 mM NAC pre-treatment; ‘3 Gy/30 μM/CREB inh’ or ‘5 Gy/30 μM/CREB inh’, combination of 3 or 5 Gy IR and 30 μM AMRI-59 with 10 μM CREB inhibitor pre-treatment. (A) EMSA assay for detection of activated CREB-1 in cells subjected to AMRI-59 and IR with or without CREB inhibitor pre-treatment for 1 h. (B) PI uptake assay for detection of apoptotic death in cells treated with a combination of AMRI-59 and IR with or without CREB-1 inhibitor pre-treatment. (C) EMSA assay for detection of activated CREB-1 in cells treated with a combination of AMRI-59 and IR treatment with or without NAC pre-treatment for 1 h. Representative data from experiments performed in triplicate are shown.

Figure 8. Combination of AMRI-59 and IR enhances apoptotic cell death in vivo

‘Control’, mock control; ‘Drug only’, 100 mg/kg AMRI-59 only; ‘Radiation only’, Treatment of NCI-H460 or NCI-H1299 with 3 or 5 Gy IR only; ‘Drug/Radiation’, combination of 3 or 5 Gy IR and 100 mg/kg AMRI-59 for treatment of NCI-H460 or NCI-H1299, respectively. (A) Images indicate schedules for in vivo experiments. Mice were injected with NCI-H460 (2 x 10⁶) or NCI-H1299 (1 x 10⁷) cells and divided into four treatment groups (5 mice/group). (B) Calculation of xenograft sizes. (C) TUNEL assay with dark brown dots considered TUNEL-positive cells. (D) Quantitative analysis of TUNEL-positive cells. Representative data from experiments performed in triplicate are shown.

Figure 9. Scheme of the intracellular mechanism of the radiosensitization effect of AMRI-59

The N-terminal conserved cysteine (Cys⁵²-SH) of PRX I was selectively oxidized by H₂O₂ to cysteine sulfenic acid (Cys⁵²-SOH), which then reacted with Cys¹⁷³-SH of the other subunit to form an intermolecular disulfide that was subsequently reduced by an appropriate electron donor. AMRI-59 disrupts the oxidation stage of catalytic site Cys-SH by H₂O_2, and result to accumulation of intracellular ROS. In this study, IR treatment also promotes ROS production, and then induces synergic ROS increase. The increased ROS promotes increase of γ-H2AX level and decrease of ERK and CREB-1 activation, which are ones of main components of cell survival signaling. Suppression of cell survival signaling promotes apoptotic cell death resulted from caspase-3 activation.