1'-O-methyl-averantin isolated from the endolichenic fungus Jackrogersella sp. EL001672 suppresses colorectal cancer stemness via sonic Hedgehog and Notch signaling

Abstract

Endolichenic fungi are host organisms that live on lichens and produce a wide variety of secondary metabolites. Colorectal cancer stem cells are capable of self-renewal and differentiation into cancer cells, which makes cancers difficult to eradicate. New alternative therapeutics are needed to inhibit the growth of tumor stem cells. This study examined the ability of an extract of Jackrogersella sp. EL001672 (derived from the lichen Cetraria sp.) and the isolated compound 1'-O-methyl-averantin to inhibit development of cancer stemness. The endolichenic fungus Jackrogersella sp. EL001672 (KACC 83021BP), derived from Cetraria sp., was grown in culture medium. The culture broth was extracted with acetone to obtain a crude extract. Column chromatography and reverse-phase HPLC were used to isolate an active compound. The anticancer activity of the extract and the isolated compound was evaluated by qRT-PCR and western blotting, and in cell viability, spheroid formation, and reporter assays. The acetone extract of EL001672 did not affect cell viability. However, 1'-O-methyl-averantin showed cytotoxic effects against cancer cell lines at 50 μg/mL and 25 μg/mL. Both the crude extract and 1'-O-methyl-averantin suppressed spheroid formation in CRC cell lines, and downregulated expression of stemness markers ALDH1, CD44, CD133, Lgr-5, Msi-1, and EphB1. To further characterize the mechanism underlying anti-stemness activity, we examined sonic Hedgehog and Notch signaling. The results showed that the crude extract and the 1'-O-methyl-averantin inhibited Gli1, Gli2, SMO, Bmi-1, Notch-1, Hes-1, and the CSL complex. Consequently, an acetone extract and 1'-O-methyl-averantin isolated from EL001672 suppresses colorectal cancer stemness by regulating the sonic Hedgehog and Notch signaling pathways.

Figure 1

1′-O-methyl-averantin, isolated from a crude extract of EL001672, suppresses cancer stemness. (A) Images of the endolichenic fungus EL001672 isolated from Cetraria sp., and a flow chart illustrating isolation of the pure compound 1′-O-methyl-averantin. (B) Chemical structure of 1′-O-methyl-averantin. (C) IC₅₀ values of 1′-O-methyl-averantin against the CSC221, CaCo2, DLD1, and HCT116 cell lines. (D) Viability of cancer cell lines treated with EL001672 crude extract and 1′-O-methyl-averantin. DMSO used as a control. 50, 25, 12.5 and 6.125 µg/ml 1′-O-methyl averantin represent a concentration of 129.49, 64.74, 32.37 and 16.19 µM, respectively. Data represent the mean ± standard error of the mean, *p < 0.05; **p < 0.01; ***p < 0.001 (compared with DMSO-treated cells).

Figure 2

EL001672 extract and 1′-O-methyl-averantin inhibit colorectal cancer cell stemness. (A–C) Representative images of spheroid formation by CSC221, DLD1, and HCT116 cells treated with EL001672 extract or 1′-O-methyl-averantin for 14 days, and quantitative analysis of the number of spheroids formed following each treatment. 10, 5 and 2.5 µg/ml 1′-O-methyl averantin represent a concentration of 25.90, 12.95 and 6.47 µM, respectively. Data represent the mean ± standard error of the mean, *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

A crude extract of EL001672 and 1′-O-methyl-averantin suppress expression of cancer stemness markers. CSC221 cells were treated for 48 h with a crude extract of EL001672 or 1′-O-methyl-averantin for mRNA and protein expressions. (A,B) Quantitative analysis of mRNA encoding cancer stem markers aldehyde dehydrigebase-1 (ALDH1), cluster of differentiation 133 (CD133), CD44, Lgr5, Musashi-1, and EphB1. CSC221 cells were treated for 48 h with a crude extract of EL001672 or 1′-O-methyl-averantin. (C,E) Immunoblots are shown. Relative expression of each target protein after cells were exposed to a crude extract of EL001672 (D) or 1′-O-methyl-averantin (F). 10, 5 and 2.5 µg/ml 1′-O-methyl averantin represent a concentration of 25.90, 12.95 and 6.47 µM, respectively. Data represent the mean ± SEM. *p < 0.05; **p < 0.01 (compared with DMSO-treated CSC221 cells).

Figure 4

The crude extract of EL001672 and 1′-O-methyl-averantin block the sonic Hedgehog (SHH) signaling pathway. (A,D) Expression of mRNA encoding Gli1, Gli2, SMO, and Bmi-1 by CSC221 cells after treatment with the indicated concentrations of crude extract or 1′-O-methyl-averantin and incubated 48 h. (B–E) Western blot analysis of Gli1, Gli2, SMO, and Bmi-1 protein levels in CSC221 cells treated with indicated concentrations of crude extract and 1′-O-methyl-averantin and incubated 48 h. (C–F) Quantitative analysis of protein expression. 10, 5 and 2.5 µg/ml 1′-O-methyl averantin represent a concentration of 25.90, 12.95 and 6.47 µM, respectively. Data represent the mean ± standard error of the mean, *p < 0.05; **p < 0.01; ***p < 0.001; difference compared with DMSO-treated.

Figure 5

Inhibitory effect of 1′-O-methyl-averantin on Gli-overexpression models. (A) Gli-luc reporter assays: NIH 3T3 cells stably incorporating Gli-dependent firefly luciferase and constitutive Renilla luciferase reporters were treated with 2.5, 5 or 10 μg/mL crude extract, 1′-O-methyl-averantin, 5 μg/mL GANT61 (a Gli inhibitor), and Vismodegib-GDC-0449 (a SMO inhibitor) and incubated 48 h. (B) Gli-luc reporter assays in HEK293T. HEK293T cells transfected with si-negative control and/or si-SMO for 12 h, and then cells were exposed to Gli-luc plasmid and Gli1 or Gli2 plasmid transfection for 12 h. After that cell treated with DMSO or 1′-O-methyl-averantin for 48 h. (C,D) Quantitative analysis of mRNA expression levels of Gli1 and SMO on HEK293T cells. HEK293T cells transfected with si-negative control and/or si-SMO for 12 h, and then cells were exposed to Gli1 plasmid and/or mock transfection for 12 h. After that cell treated with DMSO or 1′-O-methyl-averantin for 48 h. 10, 5 and 2.5 µg/ml 1′-O-methyl averantin represent a concentration of 25.90, 12.95 and 6.47 µM, respectively. Data represent the mean ± standard error of the mean, *p < 0.05; **p < 0.01; ***p < 0.001; difference compared with DMSO-treated.

Figure 6

Effect of a crude extract of EL001672 and 1′-O-methyl-averantin on the NOTCH signaling pathway. (A,B) Quantitative analysis of Notch-1 and Hes-1 (hairy and enhancer of split-1) mRNA expression by CSC221 cells treated with the indicated concentrations of crude extract or 1′-O-methyl-averantin and incubated 48 h. (C,D) CSL (CBF1/Su(H)/Lag-1)-luc and Hes-1 reporter assays in HEK293T cells treated with 2.5, 5, or 10 μg/mL crude extract, 1′-O-methyl-averantin, 5 μg/mL GANT61 (a Gli inhibitor), and Vismodegib-GDC-0449 (a SMO inhibitor) and incubated 48 h. 10, 5 and 2.5 µg/ml 1′-O-methyl averantin represent a concentration of 25.90, 12.95 and 6.47 µM, respectively. Data represent the mean ± standard error of the mean. *p < 0.05; **p < 0.01; ***p < 0.001; difference compared with each groups.