Cytochrome-c mediated a bystander response dependent on inducible nitric oxide synthase in irradiated hepatoma cells

Abstract

Background: Radiation-induced bystander effect (RIBE) has important implication in tumour radiotherapy, but the bystander signals are still not well known.

Methods: The role of cytochrome-c (cyt-c) and free radicals in RIBE on human hepatoma cells HepG2 was investigated by detecting the formation of bystander micronuclei (MN) and the generation of endogenous cyt-c, inducible nitric oxide (NO) synthase (iNOS), NO, and reactive oxygen species (ROS) molecules.

Results: When HepG2 cells were cocultured with an equal number of irradiated HepG2 cells, the yield of MN in the nonirradiated bystander cells was increased in a manner depended on radiation dose and cell coculture time, but it was diminished when the cells were treated with cyclosporin A (CsA), an inhibitor of cyt-c release. Meanwhile the CsA treatment inhibited radiation-induced NO but not ROS. Both of the depressed bystander effect and NO generation in the CsA-treated cells were reversed when 5 μM cyt-c was added in the cell coculture medium. But these exogenous cyt-c-mediated overproductions of NO and bystander MN were abolished when the cells were pretreated with s-methylisothiourea sulphate, an iNOS inhibitor.

Conclusion: Radiation-induced cyt-c has a profound role in regulating bystander response through an iNOS-triggered NO signal but not ROS in HepG2 cells.

Figure 1

(A) Dose response of the yield of MN in the irradiated HepG2 cells and bystander HepG2 cells that were cocultured with irradiated cells for 12 h. (B) Time response of bystander MN formation in HepG2 cells that were cocultured with 3 Gy γ-irradiated HepG2 cells. ^**P<0.01, ^*** P<0.001 compared with the control without irradiation.

Figure 2

Influence of CsA and exogenous cyt-c on the MN formation of 3 Gy γ-irradiated HepG2 cells and bystander HepG2 cells, which was cocultured with 3 Gy γ-irradiated cells for 12 h. ^**P<0.01, ^***P<0.001 compared with the control without irradiation or to the indicated group with drug treatment.

Figure 3

Typical fluorescence image of the distribution of cyt-c in HepG2 cells (A) and percentage of cyt-c-positive cells in the HepG2 population (B) 12 h after 3 Gy γ irradiation. In some experiments, HepG2 cells were pretreated with 5 μM CsA for 1 h before the irradiation. Green fluorescence indicated cyt-c, blue fluorescence indicated nuclei. Scale bar, 20 μm. ^***P<0.001 compared with the control without irradiation or CsA treatment. The colour reproduction of this figure is available at the British Journal of Cancer online.

Figure 4

Time course of expression of ROS, NO, and iNOS postirradiation. (A) The column plots showed the relative intensity of DCFH and DAF-FM that were calculated as the mean intensity of experimental group cells compared with the mean intensity of control cells. (B) The relative expression level of iNOS examined by western analysis that was normalised to α-tubulin first and then the ratio of each normalised value to the control value was calculated. ^*P<0.05, ^**P<0.01 compared with the control without irradiation.

Figure 5

Influence of exogenous cyt-c on the induction of NO and ROS in HepG2 cells. (A) Relative intensity of NO-induced DAF-FM fluorescence in HepG2 cells 12 h postirradiation. (B) Relative intensity of ROS-induced DCFH fluorescence in HepG2 cells 2 and 12 h postirradiation. In some experiments, the irradiated HepG2 cells were pretreated with 5 μM CsA before irradiation. ^***P<0.001 compared with the control without irradiation and ^**P<0.01, ^***P<0.001 between indicated groups.

Figure 6

Cyt-c-mediated RIBE through NO in an iNOS-dependent pathway. Influence of iNOS inhibitor SMT on radiation-induced bystander MN (A) and NO production (B). In some experiments, the irradiated HepG2 cells were treated with 5 μM CsA for 1 h before irradiation and/or 5 μM cyt-c for 12 h after radiation. ^***P<0.001 compared with the control without irradiation. (C) Effect of CsA and cyt-c on the expression of iNOS induced by radiation.