The COX-2/PGE2 Response Pathway Upregulates Radioresistance in A549 Human Lung Cancer Cells through Radiation-Induced Bystander Signaling

Abstract

This study aimed to determine the mechanism underlying the modulation of radiosensitivity in cancer cells by the radiation-induced bystander effect (RIBE). We hypothesized that the RIBE mediates cyclooxygenase-2 (COX-2) and its metabolite prostaglandin E2 (PGE2) in elevating radioresistance in unirradiated cells. In this study, we used the SPICE-QST microbeam irradiation system to target 0.07-0.7% cells by 3.4-MeV proton microbeam in the cell culture sample, such that most cells in the dish became bystander cells. Twenty-four hours after irradiation, we observed COX-2 protein upregulation in microbeam-irradiated cells compared to that of controls. Additionally, 0.29% of the microbeam-irradiated cells exhibited increased cell survival and a reduced micronucleus rate against X-ray irradiation compared to that of non-microbeam irradiated cells. The radioresistance response was diminished in both cell groups with the hemichannel inhibitor and in COX-2-knockout cells under cell-to-cell contact and sparsely distributed conditions. The results indicate that the RIBE upregulates the cell radioresistance through COX-2/PGE2 intercellular responses, thereby contributing to issues, such as the risk of cancer recurrence.

Keywords: COX-2; PGE2; radiation-induced bystander effect; radioresistance.

Figure 1

Timeline of the performed experiment on SPICE-QST proton microbeam irradiation followed by the X-ray irradiation.

Figure 2

A549 cells (HDC) were treated with 10, 50, 100, and 1000 pg/mL of PGE2 for 24 h. Then, COX-2 protein expression was identified using Western blotting (A). A549 cells were treated with 10, 50, 100, and 1000 pg/mL of PGE2 or the vehicle for 24 h. Then, the cells were X-ray irradiated, and cell survival was determined using a colony formation assay. The survival fraction ratio represents the resistance rate against the without PGE2 sample (B). * p-value < 0.05. ** p-value < 0.01.

Figure 3

A COX-2 protein expression, survival fraction, and micronuclei induction rate of A549-GFP HDC samples after with or without microbeam irradiation. A total of 500 protons were irradiated per position, and the number of positions corresponds to the percentage of irradiated cells. Twenty-four hours later, Western blotting revealed an increase in COX-2 protein expression in the bystander cells (A). Regarding cell survival and micronuclei rate, cells were exposed to 4.4 Gy X-rays. Then, the 0.07% and 0.29% IR samples were compared against the without microbeam irradiated sample (0% IR) (B,C). * p-value < 0.05.

Figure 4

A COX-2 protein expression, survival fraction, and micronuclei induction rate of A549-GFP LDC samples after with or without microbeam irradiation. A total of 500 protons were irradiated per position, and the number of positions corresponds to the percentage of irradiated cells. Twenty-four hours later, Western blotting revealed the COX-2 protein expression in the bystander cells (A). Regarding cell survival and micronuclei rate, cells were exposed to 4.4 Gy X-rays. Then, the 0.07% and 0.7% IR samples were compared against the non-microbeam-irradiated sample (0% IR) (B,C).

Figure 5

A survival fraction of A549-GFP HDC samples after 4.4 Gy X-ray exposure. A total of 500 protons were irradiated per position, and the number of positions corresponded to the percentage of irradiated cells. A549-GFP cells in HDC were incubated with a hemichannel inhibitor, 50 µM lanthanum chloride (solid bar), or sham (slant stripe bar). Twenty-four hours later, with or without microbeam-irradiated cells were X-ray irradiated, and the cell survival was detected using colony formation assay. * p-value < 0.05.

Figure 6

A survival fraction and micronuclei induction rate of A549-COX-2-KO HDC samples after 4.4 Gy X-ray exposure. A total of 500 protons were irradiated per position, and the number of positions corresponds to the percentage of irradiated cells. Twenty-four hours later, microbeam-irradiated and non-microbeam-irradiated cells were X-ray irradiated, and the cell survival and micronucleus rates were detected using colony formation and micronucleus formation assays (A,B).

Figure 7

Schematic of the suggested model based on the main outcomes of this study. Responses occur in numerical order from 1 to 5. In microbeam-irradiated A549 cells (1), the expression of COX-2 protein (2) was induced and its metabolite PGE2 (3) was released into the extracellular environment. In HDC, bystander A549 cells were in close contact with each other, allowing PGE2 to act on neighboring bystander A549 cells and stimulate COX-2 expression (4). These COX-2-expressing cells continue to produce PGE2 (5), resulting in a feedback loop and amplification of the COX-2/PGE2 response within the cell population. Consequently, bystander A549 cells exhibit radioresistance. Hemichannels are involved as one of the propagation routes of PGE2, and inhibiting hemichannels diminished radioresistances. PGE2 may also be propagated through GJIC, which is composed of hemichannels. In LDC, in microbeam-irradiated A549 cells (1), COX-2 protein expression (2) was induced. However, the released PGE2 (3) cannot reach the surrounding bystander A549 cells and loses its activity. Thus, no propagation and amplification of the COX-2/PGE2 response were observed, and radioresistance was not induced in bystander A549 cells.