Fig. 6
Inhibition of TG2- TOPOIIα interaction with Glucosamine treatment improved the efficacy of radiotherapy in lung cancer. A: Immunoblots of anti-TG2 IPs. Whole cell lysates were prepared from the irradiated A549 cells (8 Gy) with/without glucosamine treatment, which were subjected to IP with anti-TG2 antibody and immunoblotted with TOPOIIα antibody. B: Representative images of comet assay of 8 Gy irradiated A549 cells in glucosamine (5mM) or DMSO treated groups. Scale bar, 50 μm. Results were further and quantitatively analyzed. C, D: Representative images for immunofluorescence staining of γ-H2AX in DMSO or glucosamine (5mM) treated A549 cells after different times of irradiation. Scale bar, 20 μm. E-G: Colonogenic assay was used to detect the colony formation of glucosamine or DMSO treated A549 cells, H1299 cells and H460 cells after 0, 2, 4 or 8 Gy irradiation. *P < 0.05. **P < 0.01. H: A flow diagram of combined treatments of both glucosamine and radiotherapy. LLC cells (2 × 105cells) mixed in 25 ul Matrigel were injected directly into the right leaf of lung on the first day (D0). The tumor bearing mice were treated with glucosamine (150 mg/kg/d) from D4 to D7 for 3 days, after which the whole lung areas were irradiated with a single dose of 15 Gy. I: Tumor bearing mice were treated with the combined treatments of whole lung irradiation and glucosamine (n = 10). Survival rate of treated mice was monitored for up to 10 days after radiotherapy. J, K: Representative images of the isolated tumors in different animal groups (n = 8), compared with summarized diameters of these isolated tumors. L: Representative images for HE staining, TUNEL and Ki67 IHC staining of tumors in each group (n = 8). M, N: Quantification for the levels of both TUNEL and Ki67 staining on lung tumors after radiotherapy with/without glucosamine treatments. (n = 8) *P < 0.05 and **P < 0.01 versus single radiation group