Isolation of Human Cancer Cell Growth Inhibitory, Antimicrobial Lateritin from a Mixed Fungal Culture

Inhaltsübersicht

Abstract

The purpose of this study was to attempt the reproducible coculture of more than two fungi for biosynthesis of potential antineoplastic substances. Five different fungi were simultaneously inoculated into broth cultures and grown for two weeks. Cancer cell line bioassay-guided fractionation, NMR, and mass spectroscopy led to the isolation and characterization of lateritin. Lateritin inhibited the growth of a mini-panel of human cancer cell lines, gram-positive bacteria, and Candida albicans. Individually, the five fungi did not synthesize detectable levels of lateritin. This study adds to the small but growing body of evidence that mixed fermentation is a viable avenue for natural product drug discovery. In addition, this is the first report of the reproducible coculture of more than two microbes for natural product biosynthesis, and the first report of the human solid tumor cell line and antimicrobial activities of lateritin.

Key words

coculture - mixed fermentation - lateritin - natural product - anticancer agent

Microbial natural products continue to be an excellent resource for pharmaceutical lead discovery. One approach to more fully access the metabolic potential of cultivatable microbes is mixed fermentation, where the presence of neighboring microbes may induce secondary metabolite synthesis. Mixed fermentation for natural product drug discovery is in its infancy, probably because of early fears of lack of reproducibility. However, implementation of this method has resulted in increased antibiotic activity in crude extracts, increased yields of previously described metabolites, increased yields of previously undetected metabolites, analogues of known metabolites resulting from combined pathways, and induction of previously unexpressed pathways for bioactive constituents (for a review, see [1]).

To date, mixed fermentations resulting in the production of bioactive compounds have included two different fungi, two different bacteria, or one bacterium and one fungus [1]. Filamentous fungi are well recognized as a valuable source of unique natural products. In the present study, five different genera of filamentous fungi, all with inactive extracts (murine P388 lymphocytic leukemia cell line [ED₅₀ > 10 µg/mL]), were combined in order to potentially induce new secondary metabolite synthesis. The five environmental isolates Ovadendron sulphureoochraceum, Ascochyta pisi, Emericellopsis minima, Cylindrocarpon destructans, and Fusarium oxysporum were identified to species level by large-subunit (LSU) rRNA gene sequencing. The initial small-scale fermentation (650 mL) of the five fungi grown in mixed culture yielded a P388 ED₅₀ value of 0.21 µg/mL. Activity was confirmed with increasingly larger volumes (consistently active) prior to the 1140-L scale-up. The human cancer cell line activity of the combined fungi was determined to be optimal when the fungi were cultured in solid media for 21 days prior to inoculation and then in mixed broth culture for 14 days. When broths were subcultured at the end of the fermentation period, there was only one fungus, F. oxysporum.

Bioassay-guided fractionation of the scale-up fermentation yielded the natural product lateritin. The isolated lateritin (Supporting Information, Fig. 1S) had a P388 ED₅₀ value of 1.8 µg/mL, and GI_50s for six human tumor cell lines (pancreas BXPC-3, breast MCF-7, CNS SF268, lung-NSC H460, colon KM20L2, and prostate DU-145) ranged from 1.7 µg/mL to 2.0 µg/mL. The following minimum inhibitory concentrations (MICs) were found: Candida albicans ATCC 90 028, 4–8 µg/mL; Micrococcus luteus Presque Isle 456, 2–4 µg/mL; Staphylococcus aureus ATCC 29 213, 4–8 µg/mL; Enterococcus faecalis ATCC 29 212, 8 µg/mL; and Streptococcus pneumoniae ATCC 6303, 8–16 µg/mL. At 64 µg/mL, lateritin did not inhibit the growth of Cryptococcus neoformans ATCC 90 112, Stenotrophomonas maltophilia ATCC 13 637, Escherichia coli ATCC 25 922, Enterobacter cloacae ATCC 13 047, or Neisseria gonorrhoeae ATCC 49 226. Due to a paucity of lateritin, potential activity against filamentous fungi, including the five cocultured filamentous fungi, was not evaluated. No lateritin peaks were detected by reversed-phase HPLC when the five fungi were cultured individually.

Lateritin had previously been isolated from Gibberella lateritium and Isaria japonica [2], [3], but not from any of the five fungi used here. Such bioactive N-methylated peptides are of interest, as N-methylation increases membrane permeability [4], [5] and proteolytic resistance [5], [6], [7], which may increase therapeutic usefulness [8]. There are two reports of biological activity for lateritin: inhibition of rat liver acyl-coA : cholesterol acyltransferase [2] and induction of apoptotic death in human leukemia (HL-60) cells [3]. A stereoisomer of lateritin, bassiatin, was isolated from the fungus Beauveria bassiana [9]. In the B. bassiana study, lateritin was synthesized from N-carbobenzoxy-N-methylphenylalanine and (±)-2-hydroxy-3-methyl-butyric acid benzyl ester. Bassiatin, but not lateritin, inhibited ADP-induced aggregation of rabbit platelets [9]. Synthesis of lateritin and bassiatin using Mitsunobu cyclization of hydroxyacid acyclic precursors was reported in 2005 [8]. Both synthetic methods suffered from low yields at key steps [8].

The present report may provide the first description of a biologically active natural product produced in a mixed fermentation with more than two microbes. To our knowledge, this is also the first demonstration of the solid tumor cell line cytotoxicity of lateritin and its anti-gram-positive and anti-Candida activities. Our research priorities now include determining whether F. oxysporum is responsible for lateritin synthesis, and discovering the minimum number of fungi and the specific external signals that are required to induce lateritin synthesis in this mixed culture. Scale-up biosynthesis of lateritin will help elucidate whether the antifungal property of lateritin contributes to the elimination of four of the five fungal species during mixed fermentation.

Materials and Methods

The five filamentous fungi were isolated from soil samples collected (G. R. P.) in 1999 and 2000 along the banks of rivers or lakes in Canada, Alaska, and Montana. Isolated colonies were subcultured and fermented, and the extracts were screened against the murine P388 lymphocytic leukemia cell line. Fungi were identified by large-subunit (LSU) rRNA gene sequencing (Accugenix). Specific sequences were identified with PE Applied Biosystems MicroSeq analysis software and database. Voucher specimens (MIC 5759, MIC 5620, MIC 5835, MIC 5638, MIC 5789) are held at Arizona State University (R. K. P.).

Activity peak experiments and large-scale fermentations were performed in one-half-strength malt extract broth (Difco) at room temperature with shaking (125 rpm). Each 6-L flask for large-scale fermentation (1140 L) contained 4 L of media. Flasks were inoculated with 308 µL of an OD_600nm = 0.5 of each fungus and incubated for two weeks.

General chemical procedures can be found in the Supporting Information. The cancer cell line-inhibitory CH₂Cl₂ extract (45 g) of the scale-up fermentation broth (1140 L) was passed in CH₃OH through a Sephadex LH-20 column (5 × 90 cm). One bioactive (human cancer cell line bioassay) fraction (1.05 g, GI₅₀ = 0.1 µg/mL) was chromatographed on a Sephadex LH-20 column (3 × 50 cm) using n-hexane-DCM‐CH₃OH (5 : 1 : 1, 1000 mL) as eluent, which led to an active fraction (67.3 mg). Every 50-mL elute was collected as a fraction after the first colored line eluted out, which led to an active fraction (fraction 3–6, 67.3 mg). Further separation of the active fraction was performed using reversed-phase HPLC. Initially, analytical HPLC of the fraction was conducted on an HP 1100 series instrument with both ELSD and UV detectors to locate the target peaks. The fraction was then separated on a C8 column (4.6 × 250 mm) and eluted with CH₃OH‐H₂O (50 : 50 to 98 : 2 in 60 min) at a flow rate of 1 mL/min, affording a 5.3-mg specimen. The structure was determined to be that of lateritin based on NMR and mass spectral analyses (see Supporting Information).

Inhibition of mouse leukemia P388 cells was assayed according to established protocols [10]. Inhibition of human cancer cell line growth was evaluated with the National Cancer Institute's sulforhodamine B assay [11]. Antimicrobial activity of lateritin was assessed using the Clinical Laboratory Standards Institute broth microdilution assays [12], [13]. Assays were run at least in duplicate, using recommended controls.

Supporting information

Detailed extraction procedures and the structure of lateritin are available as Supporting Information.

Acknowledgements

Financial support (acknowledged with appreciation) was provided by grants R01 CA90441-02–05, 2R56 CA090441-06A1, and 5R01 CA090441-07 from the Division of Cancer Treatment, Diagnosis and Centers, National Cancer Institute, DHHS; by the Arizona Biomedical Research Commission; by the Fannie E. Rippel Foundation; by Dr. Alec D. Keith; and by the Robert B. Dalton Endowment Fund. For other assistance, we thank Drs. Jean-Charles Chapuis, Dennis L. Doubek, Fiona Hogan, and John C. Knight, as well as Mr. Lee Williams.

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