Effect of X-ray irradiation on hepatocarcinoma cells and erythrocytes in salvaged blood

Abstract

The broad clinical acceptance of intraoperative blood salvage and its applications in cancer surgery remain controversial. Until now, a method that can safely eliminate cancer cells while preserving erythrocytes does not exist. Here, we investigated whether X-ray generated from linear accelerator irradiation at a certain dose can kill hepatocarcinoma cells while preserving erythrocytes. HepG2, SK-Hep1 or Huh7 cells were mixed into the aliquots of erythrocytes obtained from healthy volunteers. After the mixed cells were exposed to 30 Gy and 50 Gy X-rays irradiation, the viability, clonogenicity, DNA synthesis and tumorigenicity of the tumor cells were determined by the MTT assay, plate colony formation, 5-ethynyl-2'-deoxyuridine incorporation, and subcutaneous xenograft implantation into immunocompromised mice. The ATP, 2,3-DPG, free Hb, osmotic fragility, blood gas variables in erythrocytes and morphology of erythrocytes at 0 h, 12 h, 24 h, 48 h, 72 h after irradiation were analyzed. X-ray irradiation at 30 Gy effectively inhibited the viability, proliferation, and tumorigenicity of HepG2, SK-Hep1 and Huh7 cells without noticeably damaging the ability of oxygen-carrying, membrane integrity and morphology of erythrocytes. Theses results suggest that X-ray at 30 Gy irradiation might be safe to eliminate hepatocarcinoma cells while preserving erythrocytes in salvaged blood.

Figure 1

X-ray irradiation inhibited the viability of tumor cell lines in vitro. After X-ray irradiation, cell viability was determined using MTT assay in separated HepG2 (A), Huh7 (B), and SK-Hep1 (C) cells after culturing for 24 h, 48 h and 72 h. The cell viability in these three cell lines exposed to 30 Gy and 50 Gy X-rays irradiation after culturing for 24 h is shown in panel D. Date are means ± SEM; n = 6. Con: the dose of irradiation was 0 Gy. **p < 0.01 vs. the control group at the same culture time; ^# p < 0.05, ^## p < 0.01 vs. the same treated group cultured for 24 h.

Figure 2

X-ray irradiation inhibited DNA synthesis in tumor cell lines in vitro. DNA synthesis (scale bar = 200 μm) was detected by 5-ethynyl-2′-deoxyuridine incorporation (EdU) incorporation assay in X-ray irradiated HepG2 cells after culturing for 24 h (A). DNA synthesis was detected by EdU incorporation assay in X-ray irradiated HepG2 (B), Huh7 (C), and SK-Hep1 (D) cells after culturing for 24 h and 72 h. DNA synthesis in these three cell lines exposed to 30 Gy and 50 Gy X-ray irradiation after culturing for 24 h is shown in panel E. Date are means ± SEM; n = 6. Con: the dose of irradiation was 0 Gy. **p < 0.01 vs. the control group at the same culture time; ^# p < 0.05, ^## p < 0.01 vs. the same treated group cultured for 24 h.

Figure 3

X-ray irradiation induced death in HepG2 cells in vitro. After 0 Gy (A), 30 Gy (B) and 50 Gy (C) X-rays irradiation, HepG2 cells were cultured for 7 d. HepG2 cells were cultured for 14 d after exposure to 30 Gy X-ray irradiation (D). Scale bar = 50 μm.

Figure 4

X-ray irradiation inhibited the growth of xenograft tumors in immunocompromised mice. The subcutaneous xenograft tumors developed by non-irradiated HepG2, Huh7 and SK-Hep1 cells in immunocompromised mice (A). The body weights of immunocompromised mice subcutaneously xenotransplanted with HepG2 (B), Huh7 (C) and SK-Hep1 (D) cells. The volume of xenograft tumors in immunocompromised mice subcutaneously xenotransplanted with non-irradiated tumor cells (E). Date are means ± SEM; n = 8 mice in each group.

Figure 5

Effects of X-ray irradiation on erythrocytes in vitro. ATP (A), 2,3-DPG (B), and free Hb (C) were detected in X-ray irradiated erythrocytes after culturing for 0 h, 12 h, 24 h, 48 h and 72 h. Osmotic fragility (D) was assayed in X-ray irradiated erythrocytes after culturing for 0 h. Data are means ± SEM; n = 14. ^** p < 0.01 vs. Con (0 Gy at 0 h after irradiation).