Antitumor Effects of a New Retinoate of the Fungal Cytotoxin Illudin M in Brain Tumor Models

Abstract

While the fungal metabolite illudin M (1) is indiscriminately cytotoxic in cancer and non-malignant cells, its retinoate 2 showed a greater selectivity for the former, especially in a cerebral context. Illudin M killed malignant glioma cells as well as primary neurons and astrocytes at similarly low concentrations and destroyed their microtubule and glial fibrillary acidic protein (GFAP) networks. In contrast, the ester 2 was distinctly more cytotoxic in highly dedifferentiated U87 glioma cells than in neurons, which were even stimulated to enhanced growth. This was also observed in co-cultures of neurons with U87 cells where conjugate 2 eventually killed them by induction of differentiation based on the activation of nuclear receptors, which bind to retinoid-responsive elements (RARE). Hence, illudin M retinoate 2 appears to be a promising drug candidate.

Keywords: anticancer agents; brain tumors; illudin M; neuronal cells; retinoic acid.

Scheme 1

Synthesis of the retinoate 2 of illudin M (1) and mechanism of action. Reagents and conditions: (a) retinoic acid, C₆H₂Cl₃COCl, DMF, NEt₃, then 1, DMAP, toluene, 16 h, r.t., 41%.

Figure 1

Time-resolved measurement of the absorption at 340 nm (enone group) of 200 µM 1, 2 or solvent (DMSO) mixed with 6.5 mM glutathione in phosphate-buffered saline (PBS). Data are means ± SD of at least four independent measurements every 30 s.

Figure 2

F9 RARE-lacZ reporter cells show concentration-dependent activation upon treatment with 2 for 20 h. Top: bright-field images of cells treated with 2 ((a): 0 µM, (b): 0.3 µM, (c): 0.6 µM, (d): 1.3 µM, (e): 2.5 µM, (f): 5.0 µM), and β-galactosidase activity was visualized by X-Gal staining (scale bar: 100 µm). Bottom: densitometric image analyses (mean ± SD); a greater gray value means stronger RARE binding. The threshold lines at 45 and 175 represent the control and the effect of 10 nM all-trans retinoic acid. * p < 0.001, Student’s t test, groups treated with 2 were compared to control, n = 5.

Figure 3

Upper row: NE-4C neural stem cells differentiate to neurons upon treatment with 10 nM of all-trans retinoic acid (RA). Bottom row: retinoate 2 (10 nM) did not elicit differentiation of NE-4C stem cells. Right column shows immunohistographs after staining for neuron specific ß-tubulin III (green) and for nuclei with DAPI (blue). Scale bar: 50 µm.

Figure 4

Immunostaining of primary neurons derived from E14, 5 forebrains treated with 1 µM of 1, 2 or all-trans retinoic acid (RA) plus 1. Blue: nuclei stained with DAPI; green: tubulin stained with βIII-tubulin specific antibody; red: GFAP stained with α-GFAP antibody. Fluorescence microscope Zeiss Axiovert 200 M, 63× objective, 1024 × 1024 res. Scale bar: 50 µm.

Figure 5

Micrographs of cultures of mouse embryonal neuronal cells untreated (control) or treated with 1 µM of 2; DIV, days in vitro. Scale bar: 100 µm.

Figure 6

U87 glioma cells undergo apoptosis upon 48 h treatment with 10 µM of 2. Cells in the early stages of apoptosis show calcein (green)/Annexin V-Cy3 (red) double labeling. Scale bars: 50 µm; 20 µm for inserts.

Figure 7

(A–D). Annexin V/PI flow cytometry of MZ–54 cells upon treatment with 1 or 2, mean + SEM; Brown–Forsythe ANOVA test with Dunnett’s T3 multiple comparison test (compared with DMSO control); * < 0.05; ** < 0.01; *** < 0.001; **** < 0.0001.

Figure 8

Co-culture of neuronal cells and U87 glioma cells in the absence or presence of 1 µM of 1 or 2. The neuronal aggregates are marked by arrowheads. Scale bar: 100 µm.

Figure 9

Ex vivo tumor growth assay using organotypic brain slice cultures in the absence (DMSO) or presence of 5 µM of 1 or 2. (A) Growth curves of the NCH644^GFP+ tumors over time after treatment with solvent (DMSO, black line) or 5 µM 1 (green line) or 2 (blue line) normalized to the tumor size one day after transplantation (d0) depicted as mean +/− SEM. (B,C) Point plots of the data summarized in (A) after treatment for (B) 5 days or (C) 7 days). Dashed line on y = 1 display the original tumor size. ***: p < 0.001; ****: p < 0.0001; two-Way ANOVA with Tukey’s multiple comparisons test (GraphPad Prism 7).

Figure 10

Ex vivo tumor growth assay using organotypic brain slice cultures in the absence (DMSO) or presence of 5 µM of 1 or 0.1 µM or 0.5 µM 2. (A) Growth curves of the NCH644^GFP+ tumors over time after treatment with solvent (DMSO, black line) or 5 µM 1 (green line), 0.1 µM (light blue line) or 0.5 µM 2 (blue line) normalized to the tumor size one day after transplantation (d0) depicted as mean +/− SEM. (B,C) Point plots of the data summarized in (A) after treatment for (B) 5 days or (C) 7 days). Dashed line on y = 1 display the original tumor size. **: p < 0.001; ***: p < 0.0001; two-way ANOVA with Tukey’s multiple comparisons test (GraphPad Prism 7).