Sulfasalazine, an inhibitor of the cystine-glutamate antiporter, reduces DNA damage repair and enhances radiosensitivity in murine B16F10 melanoma

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Abstract

The sodium-independent cystine-glutamate antiporter plays an important role in extracellular cystine uptake. It comprises the transmembrane protein, xCT and its chaperone, CD98. Because glutathione is only weakly cell membrane permeable, cellular uptake of its precursor, cystine, is known to be a key step in glutathione synthesis. Moreover, it has been reported that xCT expression affects the progression of tumors and their resistance to therapy. Sulfasalazine is an inhibitor of xCT that is known to increase cellular oxidative stress, giving it anti-tumor potential. Here, we describe a radio-sensitizing effect of sulfasalazine using a B16F10 melanoma model. Sulfasalazine decreased glutathione concentrations and resistance to H2O2 in B16F10 melanoma cells, but not in mouse embryonic fibroblasts. It synergistically enhanced the cyto-killing effect of X-irradiation in B16F10 cells. It inhibited cellular DNA damage repair and prolonged cell cycle arrest after X-irradiation. Furthermore, in an in vivo transplanted melanoma model, sulfasalazine decreased intratumoral glutathione content, leading to enhanced susceptibility to radiation therapy. These results suggest the possibility of using SAS to augment the treatment of radio-resistant cancers.

Figures

  • Fig 1. Murine B16F10 melanoma cells highly express xCT. B16F10 cells and MEF were subjected to RT-qPCR and western blot analyses of xCT expression. (A) RT-qPCR analysis for xCT mRNA expression in both cell types. (B) Image of a representative western blot for xCT in both cell types. (C) Western blot images were analyzed and the relative xCT expression was calculated for both cell types. Error bars = SD, p<0.05.
  • Fig 2. SAS decreases cellular resistance to H2O2. The cellular viability, GSH levels, ROS levels, and sensitivity to H2O2 were evaluated in B16F10 cells and MEF after SAS treatment. (A) Cellular viability after treatment with SAS (24 h). (B) Cellular ROS measurement after SAS treatment (24 h). ROS levels were analyzed by DCFDA staining. (C) Quantitative analysis of intracellular GSH concentration after SAS treatment (200 μM). (D) The effect of SAS on H2O2 cytotoxicity. Cells were treated with SAS (200 μM, 24 h) followed by treatment with H2O2 (24 h). Error bars = SD, p<0.05.
  • Fig 3. Knockdown of xCT decreases cellular resistance to H2O2. The cellular viability, GSH levels, ROS levels, and sensitivity to H2O2 were evaluated in B16F10 cells after siRNA treatment. (A) xCT expression after siSLC7A11 treatment. (B) Cellular ROS measurement after siRNA (72 h). ROS levels were analyzed by DCFDA staining. (C) Quantitative analysis of intracellular GSH concentration after siRNA. (D) The effect of SAS on H2O2 cytotoxicity. Cells were treated with siRNA (72 h) followed by treatment with H2O2 (24 h). Error bars = SD, p<0.05.
  • Fig 4. SAS sensitizes B16F10 cells to X-irradiation. A colony formation assay was used to determine the cytotoxicity and the radio-sensitizing effect of SAS treatment (24 h). (A) Colony formation assay for treatment with SAS alone. B16F10 cells were seeded and treated with SAS at the concentrations shown. (B) Colony formation assay for treatment with SAS plus X-irradiation. After pretreatment with SAS (200 μM, 24 h), B16F10 cells were subjected to X-irradiation at the doses shown. (C) Evaluation of MC fraction after X-irradiation. B16F10 cells were stained for γ-tubulin after 8 Gy of irradiation with or without SAS treatment. IR, X-irradiation. (D) Quantification of MC fractions after manual counting. IR, irradiation, error bars = SD, p<0.05.
  • Fig 5. SAS inhibits cellular DNA damage repair. The effect of SAS on DNA damage repair capacity was evaluated in B16F10 cells after X-irradiation at a dose of 1 Gy (IR). At the time points shown in figure, cells were immunostained for the DSB marker, γH2AX. Representative images of γH2AX foci after irradiation are shown, following pretreatment with (A) DMSO or (B) SAS. Scale bar = 10 μm. (C) Quantification of the numbers of γH2AX foci. (D) Relative tail moment was analyzed by comet assay at 60 min after 1 Gy of X-irradiation. Error bars = SD, p<0.05.
  • Fig 6. SAS prolongs cell cycle arrest. The effect of SAS on cell cycle perturbation after X-irradiation (IR) was determined. Populations of (A) G0/G1phase, (B) S-phase, and (C) G2/M-phase were analyzed by propidium iodide staining and flow cytometry. Error bars = SD, p<0.05.
  • Fig 7. SAS sensitizes transplanted B16F10 tumors to irradiation. The radio-sensitizing effect of SAS was evaluated on tumors formed by B16F10 cells inoculated into the hind legs of C57BL/6N mice. (A) Images of xCT immunostaining in a representative tumor. The transplanted B16F10 tumor was excised and immunostained for xCT. White T, tumor region; scale bar = 200 μm. (B) Intratumoral GSH concentration with and without SAS treatment. After 12 days, transplanted B16F10 tumors were treated with SAS (24 h, 250 mg/kg intraperitoneally). Error bars = SD, p<0.05. (C) Representative images of γH2AX staining at 3 h after 1 Gy X-irradiation with or without pretreatment with SAS. IR, irradiation, scale bar = 200 μm. (D) Experimental radiotherapy to validate the effect of SAS. B16F10 tumor-bearing mice were treated with SAS for 3 days at 24 h intervals, and then X-irradiated at a dose of 4 Gy (n = 4). Tumor volumes were measured as described in the Materials and Methods section. IR, irradiation, error bars = SD, p<0.05.

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Nagane, M., Kanai, E., Shibata, Y., Shimizu, T., Yoshioka, C., Maruo, T., & Yamashita, T. (2018). Sulfasalazine, an inhibitor of the cystine-glutamate antiporter, reduces DNA damage repair and enhances radiosensitivity in murine B16F10 melanoma. PLoS ONE, 13(4). https://doi.org/10.1371/journal.pone.0195151

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