Journal: Journal of neurosurgery

Article Title: Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell–like spheroids, and tumor xenografts through multiple cell signaling pathways

doi: 10.3171/2016.8.JNS161197

Figure Lengend Snippet: SFN reduces survival in GBM cell lines but does not affect the survival of the normal human brain cells or normal human MSCs. Established U87MG, SF767, U118, U373 cell lines (A), and MHBT32, MHBT161, GBM10, and GBM43 early-passage GBM primary cultures (B) were treated with 5 to 50 μM SFN for 48 hours. Cell survival was determined by the methylene blue cytotoxicity assay (*p < 0.0001; **p < 0.0001; ***p < 0.0001; ****p < 0.0001for SFN vs vehicle [n = 6]). C: Normal brain cells were treated with 1 to 50 μM SFN for 48 hours, and cell survival was measured by the methylene blue cytotoxicity assay (*p < 0.0001 vs vehicle [n = 6]; bars and whiskers indicate median survival ± SD, respectively). D: MSCs were treated with 10 and 30 μM SFN for 24 hours. Cell survival was determined by the methylene blue cytotoxicity assay (p > 0.05 for SFN vs vehicle).

Article Snippet: Authenticated human GBM cell lines U87, U373, U118, and SF767 were obtained from American Type Culture Collection.

Techniques: Cytotoxicity Assay

Journal: Journal of neurosurgery

Article Title: Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell–like spheroids, and tumor xenografts through multiple cell signaling pathways

doi: 10.3171/2016.8.JNS161197

Figure Lengend Snippet: Molecular profile of the GBM cell lines

Article Snippet: Authenticated human GBM cell lines U87, U373, U118, and SF767 were obtained from American Type Culture Collection.

Techniques: Amplification, Expressing, Mutagenesis

Journal: Journal of neurosurgery

Article Title: Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell–like spheroids, and tumor xenografts through multiple cell signaling pathways

doi: 10.3171/2016.8.JNS161197

Figure Lengend Snippet: SFN triggered apoptosis as well as caspase 3, caspase 7, and caspase 9 activation in SFN-treated GBM cells. SFN induced apoptosis in U87MG (A) and M-HBT-32 cells (B) treated with 5 to 30 μM for 24 hours. Apoptotic cells with condensed and fragmented nuclei were identified by DAPI staining. A minimum of 300 cells were counted for each treatment, and the percentage of apoptotic cells was calculated (*p < 0.0001 vs vehicle [n = 6]). C and D: U87MG and MHBT32 cells were treated with SFN for 24 hours and processed for Western blotting using antibodies to caspase 3, caspase 7, and caspase 9 as described in the Methods.

Article Snippet: Authenticated human GBM cell lines U87, U373, U118, and SF767 were obtained from American Type Culture Collection.

Techniques: Activation Assay, Staining, Western Blot

Journal: Journal of neurosurgery

Article Title: Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell–like spheroids, and tumor xenografts through multiple cell signaling pathways

doi: 10.3171/2016.8.JNS161197

Figure Lengend Snippet: SFN mediated ROS formation and the effect of NAC. A: U87-MG cells were plated at a density of 5 × 105 in 60-mm dishes, allowed to attach overnight, and exposed to either 5 to 30 μM SFN for 24 hours or pretreated with 10 mM NAC for 3 hours and 5 to 30 μM SFN for 24 hours, and ROS was determined as described in the Methods. B: U87 cells were treated with 10 mM NAC, and the survival fractions of the NAC-treated cells compared with the untreated control sample were determined as described in the Methods section (*p < 0.001; **p < 0.0001 for SFN + NAC vs SFN [n = 3]). C: U87 cells were pretreated with 1 mM CCCP, rotenone, or CCCP + rotenone, respectively, then treated with 10 μM SFN, and ROS was determined as described in the Methods (*p < 0.01 for SFN + CCCP vs SFN alone; **p < 0.0001 for SFN + rotenone vs SFN alone; **p < 0.0001 for SFN + rotenone + CCCP vs SFN alone [n = 3]). Bars and whiskers indicate median survival ± SD, respectively.

Article Snippet: Authenticated human GBM cell lines U87, U373, U118, and SF767 were obtained from American Type Culture Collection.

Techniques: Control

Journal: Journal of neurosurgery

Article Title: Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell–like spheroids, and tumor xenografts through multiple cell signaling pathways

doi: 10.3171/2016.8.JNS161197

Figure Lengend Snippet: SFN induced DNA SSBs and triggered the induction of γ-H2AX and DSBs in U87 glioblastoma cells. A: Control vehicle-treated U87-MG cells (a) and cells treated with 10 and 30 μM SFN, respectively (b and c), were processed for the comet assay under alkaline conditions as described in the Methods. B: The comet assay was performed under neutral conditions. Control U87 cells (a) and cells treated with 10 and 30 μM SFN, respectively (b and c), were processed for the comet assay under neutral conditions. C: Under neutral conditions, SFN treatment increased the DNA tail length. At least 200 comet cells were counted and compared with vehicle-treated cells (*p < 0.001 for 10, 30, and 50 μM SFN vs vehicle alone [n = 3]). D: U87 cells (3 × 105 cells) were treated with the vehicle (0.05% DMSO) (Lane 1) or 10 μM SFN (Lane 2) for 24 hours. Cell lysates (50 μg protein) were analyzed via Western blot using the antiphospho-H2AX antibody (Ser139) and anti–β-actin.

Article Snippet: Authenticated human GBM cell lines U87, U373, U118, and SF767 were obtained from American Type Culture Collection.

Techniques: Control, Single Cell Gel Electrophoresis, Western Blot

Journal: Journal of neurosurgery

Article Title: Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell–like spheroids, and tumor xenografts through multiple cell signaling pathways

doi: 10.3171/2016.8.JNS161197

Figure Lengend Snippet: SFN triggers apoptosis in CD133-positive GSCs. A: The frequency of CD133-positive cells in unsorted GBM cell populations. The GBM cells were stained with the CD133 antibody conjugated with phycoerythrin (anti–CD133-PE) and analyzed by flow cytometry. B: The CD133-positive cells were isolated from U87 cells by FACS and cultured in sphere-forming neural growth media, as described in the Methods (a). The spheroids formed after 7 days and were processed for fluorescence microscopy for CD133 detection (b) and CD133 and DAPI staining (c). C: SFN induced apoptosis in GBM CD133-positive cells. The CD133-positive stem cells were treated with 30 and 70 μM SFN for 16 hours; after harvesting the cells, apoptosis was determined by DAPI staining. At least 3 microscopic fields of 300 cells were scored per condition per experiment by counting the total number of CD133-positive cells, and the percentage of those that showed a fragmented or dense DAPI-stained nucleolus representing the apoptotic cells was calculated (*p < 0.0001 vs vehicle [n = 3]).

Article Snippet: Authenticated human GBM cell lines U87, U373, U118, and SF767 were obtained from American Type Culture Collection.

Techniques: Staining, Flow Cytometry, Isolation, Cell Culture, Fluorescence, Microscopy

Journal: Journal of neurosurgery

Article Title: Sulforaphane suppresses the growth of glioblastoma cells, glioblastoma stem cell–like spheroids, and tumor xenografts through multiple cell signaling pathways

doi: 10.3171/2016.8.JNS161197

Figure Lengend Snippet: Confocal microscopy of U87 spheroids and the effect of SFN or SFN + NAC on the survival of the spheroids. A: The spheroids were stained with the CD133 or SOX2 antibody, and the confocal images were acquired with a confocal/2-photon Olympus FLOVIEW FV1000 MPE system, as described in the Methods (a, DAPI staining; b, CD133 expression; c, SOX2 expression; d, overlapping CD133 and SOX2 expression; e, isotype control antibody [magnification ×20]; scale bar = 100 μm). B: SFN inhibits the survival of U87 spheroids. The U87 primary spheroids were isolated as described in the Methods and dispersed to single cells by treating them with Accutase for 15 to 20 minutes. The cells were spun down by centrifugation at 1000g for 4 minutes and seeded in fresh sphere-forming media in 96-well plates at a range of 20 to 50 cells per well. After 2 to 3 days, the spheroids containing 6 to 8 cells were formed, which were not treated (a, control), treated with 5 to 30 μM SFN (b–d,) pretreated with 1 mM NAC for 3 hours (e), or pretreated with 1 mM NAC and then treated 5 to 30 μM SFN (f–h) for 8 to 10 days. C: SFN inhibits growth and reduces the survival of the spheroids isolated from U87 cells, and NAC prevents SFN’s effect (*p < 0.001 SFN vs vehicle alone; **p < 0.001 SFN + NAC [n = 3]). Tumor primary neurospheres from M-HBT-161 (D) and GBM43 (E) early-passage primary cultures isolated and treated with SFN as described above. The untreated spheroids (a) or spheroids treated with 10, 30, and 50 μM SFN (b, c, and d, respectively) inhibits growth and reduces the survival of spheroids isolated from early-passage GBM primary cultures.

Article Snippet: Authenticated human GBM cell lines U87, U373, U118, and SF767 were obtained from American Type Culture Collection.

Techniques: Confocal Microscopy, Staining, Expressing, Control, Isolation, Centrifugation