The c-MYC-ABCB5 axis plays a pivotal role in 5-fluorouracil resistance in human colon cancer cells

c-MYC overexpression is frequently observed in various cancers including colon cancer and regulates many biological activities such as aberrant cell proliferation, apoptosis, genomic instability, immortalization and drug resistance. However, the mechanism by which c-MYC confers drug resistance remains to be fully elucidated. In this study, we found that the c-MYC expression level in primary colorectal cancer tissues correlated with the recurrence rate following 5-fluorouracil (5-FU)-based adjuvant chemotherapy. Supporting this finding, overexpression of exogenous c-MYC increased the survival rate following 5-FU treatment in human colon cancer cells, and knockdown of endogenous c-MYC decreased it. Furthermore, c-MYC knockdown decreased the expression level of ABCB5, which is involved in 5-FU resistance. Using a chromatin immunoprecipitation assay, we found that c-MYC bound to the ABCB5 promoter region. c-MYC inhibitor (10058-F4) treatment inhibited c-MYC binding to the ABCB5 promoter, leading to a decrease in ABCB5 expression level. ABCB5 knockdown decreased the survival rate following 5-FU treatment as expected, and the ABCB5 expression level was increased in 5-FU-resistant human colon cancer cells. Finally, using a human colon cancer xenograft murine model, we found that the combined 5-FU and 10058-F4 treatment significantly decreased tumorigenicity in nude mice compared with 5-FU or 10058-F4 treatment alone. 10058-F4 treatment decreased the ABCB5 expression level in the presence or absence of 5-FU. In contrast, 5-FU treatment alone increased the ABCB5 expression level. Taken together, these results suggest that c-MYC confers resistance to 5-FU through regulating ABCB5 expression in human colon cancer cells

Original Article Hypoxically preconditioned human peripheral blood mononuclear cells improve blood flow in hindlimb ischemia xenograft model

Abstract: Transplantation of peripheral blood mononuclear cells (PBMNCs) is a promising therapeutic approach for the treatment of hindlimb ischemia. However, insufficient angiogenesis in ischemic hindlimb after cell transplantation reduces the importance and practicality of this approach. Previously, we demonstrated using mouse models that hypoxic preconditioning augmented the cellular functions of rodent PBMNCs, such as increased cell adhesion capacity and accelerated neovascularization in ischemic hindlimb. To test the clinical application of this therapeutic strategy in this study, we investigated whether the protocol of hypoxic preconditioning, which was established in a condition of 2% O 2 for 24 h, can be made available for human PBMNCs (hPBMNCs). In addition, we grafted preconditioned hPBMNCs in a hindlimb ischemia mouse model. Hypoxic preconditioning enhanced cell adhesion capacity and oxidative stress resistance in hPBMNCs. We also observed an up-regulation of platelet endothelial cell adhesion molecule-1 (PECAM-1) in hPBMNCs by hypoxic preconditioning. Furthermore, preconditioned hPBMNCs significantly recovered limb blood flow in ischemic mice after transplantation. These results indicate that our established preconditioning protocol is available for hPBMNCs to effectively reinforce multiple cellular functions. Taken together with our series of study, we believe that this simple but powerful therapeutic strategy will be helpful in curing patients with severe hindlimb ischemia

Treatment of Cutaneous Ulcers with Multilayered Mixed Sheets of Autologous Fibroblasts and Peripheral Blood Mononuclear Cells

Background/Aims: We have developed a mixed-cell sheet consisting of autologous fibroblasts and peripheral blood mononuclear cells with a high potency for angiogenesis and wound healing against refractory cutaneous ulcers in mouse and rabbit models. To increase the effectiveness of the mixed sheet, we developed a multilayered mixed sheet. Methods: We assessed the therapeutic effects of multilayered sheets on cutaneous ulcers in mice. Growth factors and chemokines were assessed by enzyme-linked immunosorbent assay. Angiogenesis and fibroblast migration were measured by using tube formation and migration assays. Wound healing rate of cutaneous ulcers was evaluated in mice with diabetes mellitus. Results: The concentration of secreted vascular endothelial growth factor, hepatocyte growth factor, transforming growth factor, C-X-C motif chemokine ligand (CXCL)-1, and CXCL-2 in multilayered sheets was much higher than that in single-layered mixed-cell sheets (single-layered sheets) and multilayered sheets of fibroblasts alone (fibroblast sheets). The supernatant in multilayered sheets enhanced angiogenic potency and fibroblast migration compared with single-layered and fibroblast sheets in an in vitro experiment. The wound healing rate in the multilayered sheet-treated group was higher compared with the no-treatment group (control) at the early stage of healing. Moreover, both vessel lumen area and microvessel density in tissues treated with multilayered sheets were significantly increased compared with tissues in the control group. Conclusion: Multilayered sheets promoted wound healing and microvascular angiogenesis in the skin by supplying growth factors and cytokines. Accordingly, our data suggest that multilayered sheets may be a promising therapeutic material for refractory cutaneous ulcers