C1 treatment accumulates GSCs in G2/M and triggers subsequent mitotic catastrophe.

<p>A, Proliferation assays on two glioblastoma cell lines (U87 and U251). U87 and U251 cells were treated with 5.7 μM C1 or DMSO. Cells were trypsinized and estimated by counting, in duplicate, after 72 h of treatment. Two different experiments were conducted with similar results. B, Time-Lapse on mitotic U251 cells stably expressing GFP was performed in the absence (DMSO) or in the presence of C1 (at 1 μM). The compound was added to the cell culture just before imaging and then cells were continuously imaged. Three independent experiments were conducted and 10 to 15 fields were followed in each. None of the followed mitotic cells divided in two daughter cells. Representative field is imaged, DNA is in blue and the merge shows GFP and DNA. Several polyploid cells were present in the image. Arrows and arrowheads in upper panels of DMSO and C1- treated cell indicate the same cells through time-lapse. The elapse times are indicated on each photo, in some assays, a zoom of one cell is shown (the red bar represents 5 μm) this cell is present on the former field and labelled with an arrow). Images in bottom panels show DNA only (left) and DNA overlap with GFP (right) after 72-hour treatment with DMSO or C1 (the red bars on each panel represent 20 μm). Arrows in the bottom panel of C1-treated cells indicate mitotic catastrophe by C1 treatment. C, Pictures demonstrate pre-mitotic phase (left panel), mitotic (mid panel) process by full karyokinesis and cytokinesis and after cell division (right panel). MELK expression was determined with immunocytochemistry of GBM1600 cells with anti-MELK antibody (red), chromatin staining with Hoechst stain (blue). Picture of pre-mitosis shows GBM1600 cells highly expressed MELK at pre-mitosis phase (400× magnification). Then GBM1600 cells were treated with 5 μM C1 or control and were subjected to immunocytochemistry 3 days later with anti-MELK and chromatin staining (640× magnification). Data were confirmed by three independent experiments. C1 treated cells are micronucleated at metaphase and followed multinuclear chromatin condensation (mid panel). Right panel show multinuclear asymmetric divided chromatin of C1 treated cell compared with DMSO treated cell. D, Flow cytometric analysis of C1- and DMSO-treated GBM1600 cells with Propidium Iodide at 3 days after treatment shows 62.7% of C1-treated cells resulted in the G2/M arrest, whereas the control cells have 19.3% of the G2/M arrested cells.E, Graph indicating the proportions of live, early apoptotic, and late apoptotic U251 cells with varying doses of C1 or DMSO.</p

Genes in the DNA damage-induced response pathway are downregulated in Siomycin A-treated GSCs.

<p>cDNA microarray of GBM146, GBM157, and GBM206 samples treated with 1 μM Siomycin A or control (DMSO) were subjected to cluster (A) and canonical pathway analyses (D) using Ingenuity software. Log (<i>p</i>Value) of most significantly downregulated pathways are shown (<i>p</i><0.05). The most downregulated and upregulated genes in Siomycin A-treated GSCs are shown in (B) and (C), respectively. Expression of FOXM1, MELK, Aurora A/B, and Survivin were significantly decreased by Siomycin A treatment compared with DMSO treatment.</p

C1 treatment inhibits GSC proliferation <i>in vitro</i> and <i>in vivo</i>.

<p>A, Graph of neurosphere forming assay indicating the relative neurosphere numbers of C1-treated patient-derived GBM samples (GBM146, GBM157, and GBM206) and normal neural progenitors (16wf). B, CD133(+) and (−) cells, separated from GBM157-derived sphere cultures, were treated with 1 μM C1 or DMSO (control) under the identical serum-free conditions for 48 hours. The effect on CD133(+) cells was assessed by the neurosphere number per well, and the effect on CD133(−) cells was assessed by the % change of the total cell number in comparison to the control sample. C, Schematic showing organotypic slice cultures explanted GBM tissues and treated with C1 or DMSO (control) for 16 hours and evaluated with H&E, Ki67, and Nestin staining. D, Immunohistochemistry of C1- or DMSO-treated GBM slice cultures with anti-Ki-67 monoclonal antibody (Original magnification, ×200). E, Graph indicating the numbers of neurospheres (left) or total cells (right) in serum-free medium derived from C1- or DMSO-treated slice cultures for 16 hours. F, Schematic drawing of the effect of C1 treatment for the mouse intracranial GBM models derived from GSCs. Cells from GBM157 spheres were injected intracranially into immunocompromised mice (C1 mice: n = 4, control mice: n = 12). At day 7 post transplantation, C1 was injected intratumorally at quantities of 2.5 pmol (n = 3), 25 pmol (n = 4), or 250 pmol (n = 5). G, Representative images for immunohistochemistry with Ki-67 staining of GBM slice cultures treated with 25 pmol C1 or DMSO at day 10 of treatment. Ki-67 positive cells in each group were analyzed automated digital image analysis (Original magnification, ×200). H, Representative images for immunohistochemistry with human-specific Nestin antibody using GBM157-derived mouse intracranial tumors treated with varying doses of C1 or DMSO intratumoral injection (bar: 1 mm). I, Graph indicates tumor sizes in each group as determined by Nestin staining intensities analyzed using automated digital image analysis.</p