An Endolichenic Fungi-Derived Fatty Acid, cis-10-Nonadecenoic acid, Suppresses Colorectal Cancer Stemness

Abstract

Endolichenic fungi (ELF), symbionts of lichens, have been reported to produce diverse bioactive secondary metabolites with promising pharmaceutical potential. In this study, we isolated and identified an ELF, EL001668 (KACC 83020BP), from Cetraria laevigata Rass., and assessed its crude extract and bioactive compounds against colorectal cancer (CRC) stem cell activity. cis-10-nonadecenoic acid (c-NDA), isolated through bioactivity-guided fractionation exerted substantial inhibitory effects on CRC stemness, such as the suppression of spheroid formation and the downregulation of the key stem cell markers ALDH1, CD44, and CD133. Comparative analysis with the omega-3 fatty acids EPA and DHA, with well-established properties, showed that c- NDA exerted comparable or superior inhibitory effects against the markers and phenotypic traits of stemness. Besides, the crude extract of EL001668 exhibited greater suppression of certain markers in comparison to the individual compounds. These findings suggest that c-NDA, in conjunction with ELF-derived compounds, holds potential as a novel therapeutic candidate targeting CRC stem cells. Taken together, the current study demonstrated that c-NDA, similar to EPA and DHA, may possess adjunct or complementary effects in cancer treatment and other diseases.

Keywords: Bioactivity-guided fractionation; Colorectal cancer stem cells; Endolichenic fungi; Fatty acids; Natural products; cis-10-nonadecenoic acid.

Fig. 1

Isolation of a bioactive compound from EL001668 crude extract. (A) Flowchart illustrating the separation of the purified compound c-NDA isolated from Cetraria laevigata Rass. (B) HPLC chromatograms (detected at 210 nm) of (a) blank; (b) the isolated bioactive compound c-NDA.

Fig. 2

The EL001668 bioactive component, c-NDA, suppresses spheroid formation. (A) CSC221 cells treated with EL001668 crude extracts and their fractions. (B) Treatments of cells with bioactive components of EL001668, c-NDA, Fraction 5A (Fr5-A), and the crude extract for further validation. After 10-14 days of incubation, the number of spheroid formations after each treatment was analyzed quantitatively. *p<0.05; **p<0.01; ***p<0.001; n=3.

Fig. 3

Comparing the effects of EPA and DHA with c-NDA on spheroid formation. (A) Chemical structures of EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid). (B) Cell viability of CSC221 treated with the EL001668 crude extract, c-NDA, EPA, and DHA at 1.56-100 µg/mL concentrations. DMSO was used as a control. (C) CSC221 cells were treated with the crude extract of EL001668, c-NDA, EPA, and DHA. After 10-14 days of incubation, the number of spheroid formations after each procedure was analyzed quantitatively. *p<0.05; **p<0.01; ***p<0.001; n=3.

Fig. 4

Effects of the fatty acid constituents of EL001668 crude extract and of purified fatty acids on cancer stemness markers. (A) CSC221 cells were treated for 48 h with the crude extract of EL001668, c-NDA, DHA, or EPA at 5 µg/mL for mRNA expression analysis. Quantitative assessment of mRNA levels for cancer stem cell markers, such as ALDH1, BMI-1, CD133, CD44, and Lgr5. *p<0.05; **p<0.01; ***p<0.001; NS, no significant difference between groups; n=3. (B) Western blot analysis demonstrating the protein expression levels of ALDH1, CD44, CD133, Lgr-5, Musashi-1, BMI-1. Quantitative analysis of Western blot data. The bar graph illustrates the relative expression levels of the indicated proteins, normalized to the loading control. *p<0.05; **p<0.01; ***p<0.001, n=2.

Fig. 5

A schematic illustration demonstrating that c-NDA treatment impacts colorectal cancer stem-like properties by downregulating key markers associated with tumor progression, post-transcriptional regulation, epigenetic modification, self-renewal, and proliferation. Schematic representation created with BioRender.com (https://biorender.com).