Background
Internal tissues of plants are habitats of a class of beneficial endosymbiotic microorganisms (predominantly bacteria and fungi) called endophytes that have been observed in all plants investigated to date [
1]. In this plant-endophyte relationship, plants are hosts which generally offer nourishment and protection while endophytes improve plant defense, health and stress tolerance by solubilizing phosphates, fixing nitrogen, secreting siderophores, hydrolytic enzymes, antimicrobials or by producing plant hormones such as indole-3-acetic acid [
2,
3].
In comparison to free living fungi, crude extracts of fungal endophytes are an underexplored but rich source of bioactive and chemically diverse secondary metabolites which include terpenoids, alkaloids, phenols, furandiones, dimeric anthrones and benzopyroanones [
4,
5]. This is evidenced by a detailed review of 46 genera and 111 species of fungal endophytes producing cytotoxic secondary metabolites by Chen et al. [
6]. In order to increase the likelihood of isolating fungal endophytes that produce medicinally important secondary metabolites, documented medicinal plants that are used in traditional medicine are targeted [
5].
Datura stramonium is a medicinal plant that is known for producing over 64 tropane alkaloids of which atropine, scopolamine and hyoscyamine are predominantly found in relatively high concentrations [
7,
8]. While ethnomedical uses of
D. stramonium include inhalation of smoke from burnt leaves to relieve symptoms of asthma, bronchitis, sedation, epilepsy and psychosis to name just a few [
8], exploration into the use of tropane alkaloids as potentially anticancer lead compounds has been ongoing since the early 2000s [
9]. Bacterial and fungal endophytes have been previously isolated from
D. stramonium in studies focusing on the potential use of endophytic extracts as biocontrol agents for controlling plant and human pathogens [
10‐
13], in vitro α-glucosidase inhibitors and antioxidant agents [
14]. To the best of our knowledge, this is the first study that reports the cytotoxic activity of crude extracts endophytic fungi from
D. stramonium on human A549 lung carcinoma and UMG87 glioblastoma cell lines. The results of the bioactive crude extract observed in this study may form a foundation for developing a fungal-derived drug for glioblastoma multiforme treatment.
Discussion
Medicinal plants with known ethnopharmacological properties are proven sources for isolation of endophytes that produce secondary metabolites with novel and medically significant bioactivities [
21,
37]. The surface sterilization method of isolating endophytes is highly effective to reduce contamination of epiphytes when sodium hypochlorite is employed [
38]. In this study, efficacy of surface sterilization was validated by plating on PDA the last rinse water used in the surface sterilization process as a control. No microbial growth was observed on these plates.
The Shannon-Wiener diversity index (
H´) for the isolated endophytes was calculated and found to be 3.44, indicating a high species diversity among the fungal endophyte community in
D. stramonium. Greatest diversity was observed in the leaves where the highest number of isolates were recovered with the
Alternaria genus being the most prevalent. This genus has been previously reported as an endophyte in
D. stramonium [
39], while also being a pathogen in other plants of a different species which include cereals, strawberries and tomatoes [
40]. Interestingly, pathotypes of the
Alternaria genus mostly occur as foliar pathogens which produce host-selective toxins (HSTs) to target the above-mentioned susceptible plants [
41]. Both the endophytes and pathotypes of this genus are rarely isolated from the seeds and roots, and less frequently from the stems [
40‐
43].
The three endophytes from the
Alternaria genus (
A. tenuissima KTDL2,
A. alternata KTDL3 and
Alternaria sp. KTDL7) produced varying shades of dark brown pigmented hyphae due to melanin production, a pigment known to improve stress tolerance of plant hosts by trapping and eliminating oxygen radicals generated during abiotic stress [
44]. The endophyte
Bipolaris sp. KTDS5 appeared to have a mixture of black pigmented and nonpigmented white colonies, where the black pigment was also evidence of fungal melanin production [
45]. Endophytes that produce melanized septate hyphae and microsclerotia-like structures are commonly known as “Dark Septate Endophytes” (DSE), they are collectively thought to improve nutrient acquisition and stress tolerance in plants [
46].
R. mucilaginosa KTDS2 had pink colonies and
G. castaneus KTDL1,
Colletotrichum sp. KTDL4,
Talaromyces sp. KTDL6,
S. schenckii KTDL8,
Trichoderma sp. KTDL11 and
P. crystallinus KTDS1 all had a cream-white appearance. Pigments in fungi are chiefly produced in the mevalonate pathway and include carotenoids such as lycopene, γ-carotene, β-carotene, cantaxanthin, astaxanthin, neurosporaxanthin and torulene [
47]. Besides contributing to the metabolism of the host plant, natural pigments produced by (endophytic) fungi have great potential in the food and beverage industry where synthetic pigments are often toxic and carcinogenic [
48]. The specific individual roles played by each fungal isolate in the plant-endophyte relationship with
D. stramonium are still yet to be better understood.
Among the isolated fungal endophytes in this study, significant and selective cytotoxic activity was observed from the crude extract of
Alternaria sp. KTDL7 on UMG87 glioblastoma cells in the MTS assay. The highest cytotoxic activity of this crude extract was observed at 50 and 100 μg/mL, indicating a dose-response dependent activity. Still on the same fungal extract and cell line, an interesting observation was noted whereby the actual cell viability of the 50 μg/mL treatment (2.68%) was found to be 1.61% lower than that of double the concentration, the 100 μg/mL (4.29%) treatment (Fig.
1). Upon testing the two means with multivariate analysis of variance, no significant statistical difference in their activity was found as the
p-value
P > 0.05. Mechanisms underlying the selective cytotoxicity observed from the crude extract of
Alternaria sp. KTDL7 were not investigated as this was beyond the scope of this study.
In the xCELLigence assay, the cytotoxic activity of the crude extract of
Alternaria sp. KTDL7 at 100 μg/mL on UMG87 glioblastoma cells was observed to be much higher and not comparable to that of the same extract at 50 μg/mL (Fig.
3). The resulting differences in the behavior of this extract when assayed in the xCELLigence and MTS assay can be explained by the fact that both assays target different markers. The xCELLigence assay determines cell viability indirectly by measuring impedance in 96 well plates, thus cells adhered to the bottom of the wells with micro-electrodes will increase electrical resistance which is recorded as a high cell index. Detachment of cells from the bottom of the plate will result lower electrical resistance, hence lower cell index values. The MTS assay targets the activity of mitochondrial activity of living cells.
Auranofin was used in this study as a positive control in both the MTS and xCELLigence assays. Originally, this drug was approved for the treatment of rheumatoid arthritis. Continued studies however have shown that auranofin (in its individual and combination treatments with other agents) exhibits anticancer activity by inhibiting thioredoxin reductase [
49], and thus inducing apoptosis, among other anticancer mechanisms. A number of cancer cell lines that have shown susceptibility to auranofin include MCF-7 human breast cancer cells [
50,
51], Hep3B human hepatocellular carcinoma cells [
52], LNcap and 22RV1 human prostate cancer cells [
53], SKOV3 ovarian cancer cells [
54], HCT116 and HT-29 colorectal cancer cells [
55], human glioblastoma multiforme cells [
56], and 10 non-small lung cancer cell lines [
57]. The cytotoxic mechanism of action of auranofin on UMG87 glioblastoma cells is still yet to be fully explained, however its xCELLigence profile in this study lead to the assumption that it has an intracellular target, most likely a gene involved in metabolism since low doses of the drug induced resistance and hypermetabolism (Fig.
3).
Considering the gap in knowledge about bioactive extracts from endophytic fungi, it became necessary to perform secondary metabolite profiling of the cytotoxic Alternaria sp. KTDL7’s crude extract. After analyzing LC-QTOF-MS/MS spectrum data for Alternaria sp. KDTL7’s crude extract for previously characterized compounds, seven secondary metabolites (Compounds 1 to 6) were identified from compound libraries.
Compound
1 (1,8-dihydroxynaphthalene) is a key intermediate in the synthesis of dihydroxynaphtalene (DHN)-melanin, commonly found in fungi and is synthesized via the polyketide pathway [
58]. Fungi within the
Alternaria genus studied up to date have been shown to be DHN-melanin producers, including
A. alternata 15A [
59], and
A. infectoria CBS 137.90 [
60]. DHN-melanin was tested for antifungal activity on clinical isolates and was found to have a half-minimum inhibitory concentration (MIC
50) of 128 μg/mL for
Aspergillus flavus, 64 μg/mL for
A. niger, 256 μg/mL for
A. fumigatus and 512 μg/mL for
A. tamarii [
61].
Compound
2 (anserinone) is a polyketide that has been previously isolated from the cophrophilous
Podospora anserine, where it was found to reduce radial growth of
Sordaria fimicola and
Ascobolus furfuraceus by 50 and 37% respectively [
62,
63]. In that same study, anserinone B was found to be moderately cytotoxic with an average IC
50 of 4.4 μg/mL after being tested on the National Cancer Institute’s 60 human tumor cell line panel [
62,
64].
Compound
3 (phelligridin B) is a styrylpyrone derivative which is synthesized within the shikimate and acetate pathways [
65]. This secondary metabolite has been found in ethanolic extracts of
Phellinus linteus (Sang Huang) and has been shown to exhibit cytotoxic activity against Bel-7402 cells at an IC
50 of 0.050 μM [
66].
Compound
4 (metacytofilin) has been previously identified from the culture filtrate of
Metarhizium sp. TA2759 and possess immunosuppressive properties [
67]. It is a two-residue depsipeptide synthesized by non-ribosomal peptide synthases in combination with polyketide synthase [
68].
Compound
5 (phomopsidin) is an interesting polyketide which has been previously isolated from a marine derived
Phomopsis sp. TUF95F47 [
69]. This secondary metabolite showed inhibition of microtubule assembly at an IC
50 of 5.7 μM in the in vitro assembly analysis of porcine brain tubulin assay [
70,
71].
Compound
6 (vermixocin) is a diphenyl ether derivative, previously isolated from a marine fungus,
Talaromyces sp. LF458 [
71]. Vermixocins were previously found to inhibit RNA synthesis as they interfered with incorporation of labeled uridine in a murine P388 leukemia cell line [
72].
Commonly known secondary metabolites which have been previously identified in extracts of fungi from the
Alternaria genus include alternariol, alternariol monomethyl ether, tentoxin, altesertin, alteichin, stemphyltoxin, altersolanol, altenusin and tenuazenoic acid were not detected in this study [
73]. A possible explanation for this occurrence is that different fungal strains in the same genus have the biosynthetic capability of producing a wide variety of chemically diverse secondary metabolites [
74]. The type of method and solvent used in the extraction process may also significantly affect the nature and quantity of secondary metabolites recovered [
75]. Some researchers have reported the use of acidified extracting organic solvents or acidified filtrate broth to increase the solubility of fungal secondary metabolites in organic solvents [
76].
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.