1α,25(OH)2D3 Radiosensitizes Cancer Cells by Activating the NADPH/ROS Pathway

Abstract

The radioresistance of tumors affect the outcome of radiotherapy. Accumulating data suggest that 1α,25(OH)₂D₃ is a potential anti-oncogenic molecule in various cancers. In the present study, we investigated the radiosensitive effects and underlying mechanisms of 1α,25(OH)₂D₃ in vitro and in vivo. We found that 1α,25(OH)₂D₃ enhanced the radiosensitivity of lung cancer and ovarian cancer cells by promoting the NADPH oxidase-ROS-apoptosis axis. Compared to the group that only received radiation, the survival fraction and self-renewal capacity of cancer cells treated with a combination of 1α,25(OH)₂D₃ and radiation were decreased. Both apoptosis and ROS were significantly increased in the combination group compared with the radiation only group. Moreover, N-acetyl-L-cysteine, a scavenger of intracellular ROS, reversed the apoptosis and ROS induced by 1α,25(OH)₂D₃, indicating that 1α,25(OH)₂D₃ enhanced the radiosensitivity of cancer cells in vitro by promoting ROS-induced apoptosis. Moreover, our results demonstrated that 1α,25(OH)₂D₃ promoted the ROS level via activating NADPH oxidase complexes, NOX4, p22^phox, and p47^phox. In addition, knockdown of the vitamin D receptor (VDR) abolished the radiosensitization of 1α,25(OH)₂D₃, which confirmed that 1α,25(OH)₂D₃ radiosensitized tumor cells that depend on VDR. Similarly, our study also evidenced that vitamin D₃ enhanced the radiosensitivity of cancer cells in vivo and extended the overall survival of mice with tumors. In summary, these results demonstrate that 1α,25(OH)₂D₃ enhances the radiosensitivity depending on VDR and activates the NADPH oxidase-ROS-apoptosis axis. Our findings suggest that 1α,25(OH)₂D₃ in combination with radiation enhances lung and ovarian cell radiosensitivity, potentially providing a novel combination therapeutic strategy.

Keywords: 1α,25(OH)2D3; NADPH oxidase; radiosensitivity; reactive oxygen species; vitamin D receptor.

Figure 1

1α,25(OH)₂D₃ enhanced the radiosensitivity of human ovarian cancer and lung cancer cells by ROS-induced apoptosis. (A, B) Results of colony formation assay and sphere formation assay in A549 cancer cells. (C) Results of colony formation assay in SKOV3 cancer cells. (D) Results of limited dilution assay in A549 cancer cells. (E) Effects of 1α,25(OH)₂D₃ and radiation on ROS of A549 cancer cells, related to . (F) Combined effects of 1α,25(OH)₂D₃, radiation and NAC on apoptosis of A549 cancer cells, related to . Line 1 represents the control group, line 2 represents the 1α,25(OH)₂D₃ alone group, Line 3 represents the 1α,25(OH)₂D₃ and NAC group, Line 4 represents the radiation alone group, Line 5 represents the radiation and NAC group, Line 6 represents the 1α,25(OH)₂D₃ and radiation group, Line 7 represents the 1α,25(OH)₂D₃, radiation, and NAC group. Data represents the Mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, n=3.

Figure 2

The enhancing radiosensitivity of 1α,25(OH)₂D₃ depends on VDR. (A, B) The mRNA and protein levels of VDR were examined when treated with shNC and shVDR in SKOV3 cancer cells. (C–E) Colony formation assay was examined in SKOV3 cancer cells when treated with shNC and shVDR. The data represents the Mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, n = 3.

Figure 3

Effects of 1α,25(OH)₂D₃ on ROS and apoptosis depends on VDR. (A, B) ROS was examined in A549 cancer cells when treated with shNC and shVDR. (C–E) Apoptosis percentages was examined in A549 cancer cells when treated with shNC and shVDR. The data represents the Mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, n = 3.

Figure 4

1α,25(OH)₂D₃ enhanced the radiosensitivity of cancer cells by activating the NADPH oxidase-ROS-apoptosis axis. (A) MMP level was examined by Rho 123 in A549 cancer cells. (B) ROS was examined in A549 cancer cells when treated with DPI and Apocynin. (C) MMP level was examined by Rho 123 in A549 cancer cells treated with DPI and Apocynin. (D) The mRNA levels of NOX4, p22^phox and p47^phox were examined in shNC and shVDR A549 cancer cells treated with 1α,25(OH)₂D₃. (E, F) The protein levels of p22^phox and p47^phox were examined in shNC and shVDR A549 cancer cells treated with 1α,25(OH)₂D₃. The data represents the Mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, n = 3.

Figure 5

Vitamin D₃ enhanced the radiosensitivity of ovarian cancer in vivo. (A) The relative luciferase signals were detected in nude mice of control, vitamin D₃, radiation, and combined groups. (B, C) Image and the volume of tumors. (D) The overall survival of nude mice. (E) Serum 25(OH)D levels of nude mice. (F) Representative images of tumors stained for HE, Ki67 and Tunel. The data represents the Mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, n = 3.