Radioprotector WR-2721 and mitigating peptidoglycan synergistically promote mouse survival through the amelioration of intestinal and bone marrow damage.

The identification of an agent effective for the treatment of intestinal and bone marrow injury following radiation exposure remains a major issue in radiological medicine. In this study, we evaluated the therapeutic impact of single agent or combination treatments with 2-(3-aminopropylamino) ethylsulphanyl phosphonic acid (WR-2721) and peptidoglycan (PGN, a toll-like receptor 2 (TLR-2) agonist) on radiation-induced injury of the intestine and bone marrow in lethally irradiated male C57BL/6 mice. A dose of 3 mg of WR-2721 per mouse (167 mg/kg, intraperitoneally) was given 30 min before irradiation, and 30 µg of PGN per mouse (1.7 mg/kg) was injected intraperitoneally 24 h after 10 Gy irradiation. Bone marrow cluster of differentiation (CD)45(+) and CD34(+) markers of multiple haematopoietic lineages, number of granulocyte-erythroid-macrophage-megakaryocyte (GEMM) progenitor colonies, bone marrow histopathology, leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) expression in the intestines, xylose absorption and intestinal histopathology were all assessed at various time-points after irradiation. Furthermore, nuclear factor kappa B (NF-κB) p65 protein in the ileum was stained by immunofluorescent labelling. PGN-treated irradiated mice showed an increase in CD45(+)CD34(+) cells compared with untreated mice 1.25 days after 10 Gy ionizing radiation (IR) (P < 0.05). Furthermore, combined PGN and WR-2721 treatment had an obviously synergistic radio-protective effect in nucleated cells in the bone marrow, including GEMM progenitors and CD45(+)CD34(+) cells 4 days after 10 Gy IR. Single agent PGN or WR-2721 treatment after 10 Gy IR clearly increased Lgr5-positive pit cells (P < 0.05) and xylose absorption (P < 0.05). However only PGN and WR-2721 combination treatment markedly increased villus height (P < 0.05), number of crypts (P < 0.05) and whole-body weights after 10 Gy whole-body irradiation (WBI). The NF-κB p65 subunit was translocated to the nucleus, and phosphate-IκBα (Ser32/Ser36) was detected after stimulation with either PGN or WR-2721, which indicates that these two agents act synergistically through the activation of the NF-κB pathway. Administration of PGN in combination with WR-2721 was demonstrated to have a synergistic effect on the increase in haematopoietic cells and intestinal reconstitution, as well as improved survival in lethally irradiated mice, but resulted in some degree of an immune disorder.

High expression of survivin in sacral chordoma.

Chordoma is a rare and invasive malignant tumor which primarily relies on surgical treatments. Anticipation of its recurrence and patient survival longevity has been a critical issue of the treatments. This retrospective study examined the survivin expression of sacral chordoma in 30 patients undergoing surgery in our hospital from January 2000 to July 2010, and compared it with chordoma recurrence. Survivin expression was 70 % positive in 30 patients. The positive expression of survivin with recurrence was significantly higher than that without recurrence (p = 0.017) and was inversely related to the continuous disease-free survival time (p < 0.001). Survivin expression was associated with recurrence. The correlation suggested that the survivin expression could be used as an independent predictor of recurrence and could be a potential bio-target gene of angiogenesis in sacral chordoma.

Expression of vascular endothelial growth factor and matrix metalloproteinase-9 in sacral chordoma.

Sacral chordoma is a vessel-rich and infiltrative tumor, but the fundamental knowledge of its biological behavior remains unknown. This study was designed to investigate the expression levels and contributions of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) in the angiogenesis and recurrence of sacral chordoma and their correlations. An immunohistochemical method was used to investigate the expression of VEGF, MMP-9, and microvascular density (MVD) in 36 patients with sacral chordoma. Their differences in expressions were statistically analyzed and their correlations with angiogenesis and recurrence were evaluated. The mean MVD of sacral chordomas was significantly higher than that of the adjacent normal tissues (P = 0.033). Immunoreactivity for VEGF and MMP-9 was significantly higher in sacral chordoma tissues than in adjacent normal tissues (P = 0.008, P = 0.005). The mean MVD of VEGF and MMP-9 were statistically higher in positive group than in negative group (P = 0.015, P = 0.004), respectively . Moreover, a significant correlation was found between the VEGF and MMP-9 (P = 0.002). The log-rank test revealed that continuous disease-free survival time (CDFS) was significantly shorter in the MMP-9-positive group than in the MMP-9-negative group (P = 0.019), but the difference in the VEGF-positive group and the VEGF-negative group was not statistically significant (P = 0.938). Our data suggest that VEGF and MMP-9 might act with a synergistic effect and can positively regulate the angiogenesis in sacral chordoma. Positive expression of MMP-9 might indicate the local recurrence of sacral chordoma. The result suggests that some specific drugs which inhibit VEGF, MMP-9, or their receptors may have a good therapeutic effect for sacral chordoma.

Selection of optimal sites for TGFB1 gene silencing by chitosan-TPP nanoparticle-mediated delivery of shRNA.

Most human tumors produce high levels of TGF-beta1, whose autocrine and paracrine actions promote tumor cell invasiveness and metastasis. Currently, many experimental therapies that target TGFB1 have utilized antisense DNA or RNA interference (RNAi). Despite the great potential of RNAi, the selection of effective target sites and proper delivery systems for short hairpin RNA (shRNA) remains a significant issue. Here, we used chitosan nanoparticle-mediated delivery of a shRNA-expressing vector to inhibit TGFB1 expression in the human rhabdomyosarcoma cell line RD. Knockdown of TGFB1 by shRNA resulted in a decrease in RD cell growth in vitro and tumorigenicity in nude mice. The efficiency of TGFB1 gene silencing varied with the selection of targeting sites. These data suggest that chitosan nanoparticle-mediated delivery of an shRNA produces efficient TGFB1 knockdown in rhabdomyosarcoma cells and may be a method of choice for shRNA delivery for gene therapy.