The absorbance of the plates was measured on a microplates reader at a wavelength of 570 nm
The absorbance of the plates was measured on a microplates reader at a wavelength of 570 nm. TMZ-resistant characteristic. Open in a separate window Number?1. Gliobalastoma PQM130 cells with high manifestation of ObR represent TMZ-resistant characteristic. (A) Circulation cytometry to type ObR+ cells in U87 gliobalastoma cells. ObR+ and ObR? cells were cultured for 72 h in the presence of indicated concentration of TMZ. MTT assay was performed to assess the anti-proliferative effects of TMZ (B), Annexin V and PI staining FACS was used to assess apoptosis of cells (C). (D) U87 cells with a low dose of TMZ in tradition press for 4 wk to establish TMZ-resistant cells, and the image showed that nude mice bearing U87 and U87R cells xenografts were treated with 100 M TMZ; 4 wk later on, the xenografts were weighted. (E) The mRNA and (F) protein expression levels of ObR were elevated in U87R cells. ObR+ gliobalastoma cells possess stem/progenitor cell properties Preclinical studies suggested that glioblastoma stem/progenitor cells were highly resistant to standard chemotherapeutic PQM130 medicines, including TMZ.15,16 We therefore hypothesized that ObR+ cells might show intrinsic properties of stem/progenitor cells. For this purpose, we 1st evaluated the manifestation of CD133, Nestin, and GFAP in ObR+ and U87R cells by using immunofluorescence. Interestingly, both ObR+ cells and U87R cells coexpressed ObR, CD133, and Nestin, whereas bad for GFAP, a biomaker for mature astrocyte. Consistent with the immunofluorescence data, western blot analysis showed that CD133 and Nestin were high manifestation significantly in ObR+ cells and U87R cells, but GFAP displayed almost no manifestation in both cells (Fig.?2A and B). The isolated ObR+ U87 cells formed clones efficiently, whereas ObR? cells failed to do this (Fig.?2C). In addition, ObR+ cells were much more invasive than ObR? cells (< 0.05, Fig.?2D). We next inoculated nude mice subcutaneously with 104 ObR+ cells and ObR? cells to investigate their tumorigenicity in vivo. A significant difference in tumor incidence was observed between the mice inoculated with the 2 2 cells. The ObR+ cells produced tumors in 100% mice, whereas ObR? cells produced tumors in only 25% mice 5 wk after injection (Fig.?2E). Collectively, these results suggested that ObR+ cells displayed gliobalastoma stem/progenitor cells properties. Open in a separate window Number?2. Stem/progenitor cell properties of ObR+ gliobalastoma cells. (A) Immunofluorescence analysis of ObR+ and U87R cells stained with ObR, CD133, Nestin, and GFAP. (B) Western blot analysis of CD133, Nestin, and GFAP in ObR+ and U87R cells. (C) Representative image of the plates comprising colonies derived from ETS2 1000 ObR? and ObR+ cells. (D) Cells invasiveness of ObR+ and ObR? cells using the Matrigel invasion assay. (E) Representative PQM130 mice with subcutaneous tumors and tumorigenicity of 104 sorted cells derived from ObR+ and ObR? cells. It has been demonstrated previously that gliobalastoma stem/progenitor cells have self-renewal properties. When the ObR+ cells and ObR? cells were cultured in DMEM supplemented with 10% FBS, 7 d later on, the percentage of ObR+ cells remained more than 80%, and after 2 wk, ObR+ portion remained almost 60% by FACS analysis. In contrast, ObR? cells managed their ObR status (Fig.?3A). To evaluate the effect of leptin on U87R cells oncogenesis, nude mice implanted with U87R cells (4 104) were randomly assigned and received i.p. injections of either PBS or leptin (2 g/g body weight) every other day time for a total 5 times. Two weeks later on, the tumor volume of leptin-treated mice grew more rapidly compared with the PBS group (Fig.?3B), suggesting that leptin could aggravate the cells with high manifestation of ObR-mediated malignancy. Open in a separate window Number?3. The maintenance of self-renewal in ObR+ cells. (A) Percentage of sorted ObR+ and ObR? cells after culturing for numerous instances as analyzed by circulation cytometry. (B) Leptin stimulated tumor development by implanted U87R cells. nude mice implanted with U87R (4 104) cells were randomly assigned and received i.p. injections of either PBS or leptin (2 g/g body weight) every other day time for 2 wk. Correlation of ObR and CD133 manifestation in glioma cells To investigate the medical significance, immunohistochemical analysis was conducted by using 15 units of malignant glioma as main and recurrence tumors. These main tumors were surgically resected, TMZ treated, and then the recurrent tumors were surgically resected again. The scores of ObR were evaluated as 0 to 3 and increased significantly in the recurrence tumors compared with the primary.