Fig 1: Novel JAG anticancer agents demonstrate a differential and significant anti-aHGG response. (A) Chemical structure of each JAG agent used in this study. (B) U87MG cells (5×103 per well) were seeded overnight and treated the following day with JAG-6A (black), CC-I (blue), JAG-32 (green), or JAG-79 (purple) at =1000 µM. Ninety-six hours posttreatment, loss of cell viability was assessed by CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). Data presented as average of n = 3 independent studies ± SD. (C-F) JAG agent susceptibility was determined for each novel aHGG model. (G) Average EC50 values (±SD) for TMZ and each JAG agent were determined. Two-tailed ANOVAs was conducted, and P values are shown for each comparison; n = 3. (H) Representative microscopy (×10) images of UP-029 cells. UP-029 aHGG cells were seeded (1×104). The next day, cells were treated with indicated agents (10 mM TMZ or 30 µM per JAG agent) and imaged (up to 24 hours) at 37°C under normoxic conditions. Images were recorded using EVOS FL Auto (Life Technologies). (I) Apoptosis assays were conducted for each indicated cell line 24 hours post 30 µM treatment with each JAG agent. Annexin V/PI staining was conducted and apoptosis status was determined using an NC-3000 counter; n = 3 ± SD.
Fig 2: CH004 arrests the cell cycle at the S phase.a CH004 reduces the cell viability of HEK293T and HepG2 cells. HEK293T or HepG2 cells were treated with indicated concentrations of CH004 for 24 h before the cell number was analyzed using the CellTiter96® Aqueous One Solution Cell Proliferation Assay (Promega, Materials and Methods). The means at each concentration of one representative experiment are shown as percentages of DMSO (control, 100%). Means ± SDs (n = 3). b Overexpression of hCBS or knock-down of hCBS counteracts the growth inhibition exerted by CH004 in HEK293T cells. Indicated HEK293T stable cell lines were treated with the CH004 or DMSO for 24 h. Then, the cell viability was quantified with the CellTiter 96® Aqueous One Solution Cell Proliferation Assay under the standard protocol (Promega). The means at each concentration are shown as percentages of their respective DMSO controls (100%). Means ± SDs (n = 3). The efficacy for CBS overexpression and knock-down in HEK293T cells was determined by Western blotting using an anti-CBS antibody (3E1, 1:2000; Abnova). c CH004 induces cell death in HEK293T and HepG2 cells. HEK293T or HepG2 cells were incubated with the indicated concentrations of CH004 or DMSO for 12 h before analysis using a FITC Annexin V Apoptosis Detection Kit (BD bioscience, 556547) on an LSR Fortessa flow cytometer. The means of percentages of dead cells (DMSO, 100%) from three independent samples are shown in d. Means ± SDs (n = 3). e, f Pharmacological inhibition or genetic knock-down of hCBS in HEK293T cells arrests the cell cycle at S phase. HEK293T cells were incubated with indicated compounds for 12 h, and the DNA content was quantified with propidium iodide to analyze the cell cycle distribution by flow cytometry (e). The cell cycle distributions for HEK293T-shNT and HEK293T-shCBS cells were accordingly analyzed and are shown in f. One representative diagram is shown for each condition. The mean percentages of various cell phases from three independent samples are shown in the respective right panels. Means ± SDs (n = 3). Statistical analyses were performed by two-way ANOVA with Bonferroni post-tests. *p < 0.05; **p < 0.01; ***p < 0.001. All the experiments were independently repeated twice and one representative result is present
Fig 3: JAG agents mediate a potent anti-pHGG response with limited toxicity to non-neoplastic astrocytes. (A) KNS42 and (B) SF188 pHGG cells treated with each JAG agent. After 96 hours, loss of cell viability was determined by CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). Representative microscopy (×10) images of (C) KNS42 and SF188 cells. Cells were treated with indicated agents (30 μM per JAG agent) and imaged (up to 24 hours) at 37°C under normoxic conditions. Images and films were recorded using EVOS FL Auto (Life Technologies). (D) Apoptosis assays were conducted for each indicated cell line 24 hours post 30 μM treatment with each JAG agent. Annexin V/PI staining was conducted, and apoptosis status was determined using an NC-3000 counter; n = 3 ± SD. (E and F) Dose-response curves for non-neoplastic CC2565 cells following exposure to TMZ (≤128 mM), vincristine (≤1 mM), or individual JAG agents ≤1000 μM in complete astrocyte growth medium. Data presented as average of n = 3. Error bars indicate ±SD.
Fig 4: Novel biopsy-derived aHGG cell lines display varying sensitivity to the chemotherapeutics temozolomide, gemcitabine, and vincristine. (A) U87MG, (B) UP-029, (C) SEBTA-025, (D) SEBTA-003, and (E) SEBTA-023 were seeded in 96-well plates overnight. The next day, cells were treated with temozolomide (≤128 mM), gemcitabine (≤330 mM), or vincristine (≤1 mM) in DMEM. Ninety-six hours posttreatment, loss of cell viability was assessed by CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). Data presented as mean average of n = 3 ± SD.
Fig 5: Spectral and radioactive properties of [18/19F]-2. (A) Excitation and emission spectra of 5-6 μM solutions of fluorescein (black) and [18/19F]-2 (green) in 1x PBS, pH 7.4 measured on a Cary Eclipse spectrophotometer, with 5 nm slit widths, and excitation at 490 nm. (B) In vitro cell viability tests performed with compound 2 (red line) and fluorescein (control) 24 hour on U87, Hela, MDA-MB-231, and A549 immortal cell lines using a CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS, G3582, Promega). Toxicity is not observed. (C) UV-Vis (280, 350, and 450 nm) UPLC-MS of 2 on a waters Acuity UPLC and a Phenomenex Luna Kinetex 1.7 µm EVO C18 50 x 2.1 mm column (00B-4726-AN), with a 1.5 min, a10-90% H2O:ACN (0.05% TFA) elution gradient indicating a pure synthesis of 2. (D) Reverse phase HPLC of radiolabeled [18F]-2 on a Varian HPLC, using an Waters SunfireTM C18 3.5 μm 4.6 x 50 mm column (186002551), a10-90% H2O:ACN (0.05% TFA) elution gradient and a flow rate of 2 mL/min. (E) 10 µL 3x dilution series of [18/19F]-2 beginning at 70 µM (1.7 pmols) and 170 µCi of [18/19F]-2 (first spot). A 10 µl volume was plated onto glass-backed TLC plate model. Imaging was performed using (i) bright-field; (ii) a UV-sight hand-held 4.5 volt LED black-light (fluorescence); (iii) a Bruker Xtreme optical imaging device (fluorescence: 1 sec acquisition using excitation and emission filters set at λex= 450 nm and λem= 535 nm); (iv) and a phosphorimager.
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