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. 2021 Jun 14:12:650177.
doi: 10.3389/fmicb.2021.650177. eCollection 2021.

Diversity of Marine Macro-Algicolous Endophytic Fungi and Cytotoxic Potential of Biscogniauxia petrensis Metabolites Against Cancer Cell Lines

Subhadarsini Sahoo  1 , Kamalraj Subban  1 , Jayabaskaran Chelliah  1
Affiliations

Diversity of Marine Macro-Algicolous Endophytic Fungi and Cytotoxic Potential of Biscogniauxia petrensis Metabolites Against Cancer Cell Lines

Subhadarsini Sahoo et al. Front Microbiol. .

Abstract

Hypersaline environments are known to support diverse fungal species from various orders. The production of secondary metabolites is one of the strategies that fungi adopt to thrive under such extreme environments, bringing up the stress tolerance response. Some such unique secondary metabolites also exhibit clinical significance. The increasing prevalence of drug resistance in cancer therapy demands further exploration of these novel bioactive compounds as cancer therapeutics. In the present study, a total of 31 endophytic fungi harboring inside red, green, and brown marine algae have been isolated and identified. The maximum likelihood analysis and diversity indices of fungal endophytes revealed the phylogenetic relationship and species richness. The genus Aspergillus was found to be the dominating fungus, followed by Cladosporium spp. All the isolated endophytic fungal extracts were tested for their cytotoxicity against HeLa and A431 cancer cell lines. Nine isolates were further analyzed for their cytotoxic activity from the culture filtrate and mycelia extract. Among these isolates, Biscogniauxia petrensis showed potential cytotoxicity with CC50 values of 18.04 and 24.85 μg/ml against HeLa and A431 cells, respectively. Furthermore, the media and solvent extraction optimization revealed the highest cytotoxic active compounds in ethyl acetate extract from the potato dextrose yeast extract broth medium. The compound-induced cell death via apoptosis was 50-60 and 45% when assayed using propidium iodide-live/dead and loss of mitochondrial membrane potential assay, respectively, in HeLa cells. Four bioactive fractions (bioassay-based) were obtained and analyzed using chromatography and spectroscopy. This study reports, for the first time, the cytotoxic activity of an endophytic fungal community that was isolated from marine macro-algae in the Rameswaram coastal region of Tamil Nadu, India. In addition, B. petrensis is a prominent apoptotic agent, which can be used in pharmaceutical applications as a therapeutic.

Keywords: Biscogniauxia petrensis; algicolous fungi; biodiversity; cytotoxicity; secondary metabolites.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Different endophytic fungi (morpho species) isolated from marine brown, green, and red algae.
FIGURE 2
FIGURE 2
(A,B) In vitro cytotoxicity of fungal ethyl acetate extracts on human cancer cells HeLa and A431.
FIGURE 3
FIGURE 3
(A,B) In vitro cytotoxicity of selected fungal culture filtrates and mycelia extracts against A431 (A) and HeLa (B) cancer cell line.
FIGURE 4
FIGURE 4
Morphological observation of Biscogniauxia petrensis. The colonies at 7 days of inoculation on potato dextrose agar (PDA) plate front (A) and rear (B) view, respectively. (C) Appearance of red droplets at 10–14 days of growth on PDA plate. (D) Growth of B. petrensis in PDYEB at 21 days of culture. (E–G) Light microscopy image of the B. petrensis spores (×40 magnification) and microscopy image of chlamydospore.
FIGURE 5
FIGURE 5
Cytotoxic effects of B. petrensis culture extracts grown in different media on HeLa (A) and A431 (B) at 25 μg/ml.
FIGURE 6
FIGURE 6
Cytotoxic effect of Bp culture extracts on HeLa cells and estimated using PI staining by FACS analysis. (A) Untreated (B) culture filtrate extract (25 μg/ml), (C) culture filtrate extract (50 μg/ml), (D) mycelial extract (25 μg/ml), (E) mycelial extract (50 μg/ml), (F) paclitaxel (12 nM), and (G) bar diagram representing the distribution of cell death. The data are results from three independent experiments.
FIGURE 7
FIGURE 7
Determination of apoptosis through loss of mitochondrial membrane potential in HeLa cells induced by Bp culture extract and quantified by JC-1 monomer percentage. (A) Untreated (B) culture filtrate extract (25 μg/ml), (C) culture filtrate extract (50 μg/ml), (D) mycelial extract (25 μg/ml), (E) mycelial extract (50 μg/ml), (F) 2,4-DNP, and (G) statistical analysis of loss of mitochondrial membrane potential acquired in a flow cytometer. The experiments were conducted three times, and results are obtained from mean ± SD.
FIGURE 8
FIGURE 8
Cell cycle analysis of HeLa cells treated with Bp culture extract estimated using propidium iodide staining by flow cytometry analysis. (A) Untreated (B) culture filtrate extract (25 μg/ml), (C) culture filtrate extract (50 μg/ml), (D) mycelial extract (25 μg/ml), and (E) mycelial extract (50 μg/ml). (F) Statistical analysis showing the percentage of cell cycle in each phase. The experiments were conducted three times, and results are obtained from mean ± SD.
FIGURE 9
FIGURE 9
Cytotoxic effects of purified bioactive fractions from B. petrensis culture extracts. The cytotoxic effect was determined against HeLa, A431, MCF-7, and HepG2 cancer cell lines with different concentrations of bioactive fractions: (A) C2, (B) C5, (C) M3, and (D) M4.
FIGURE 10
FIGURE 10
UV–vis absorbance spectra of purified bioactive fractions from B. petrensis culture extracts. The UV–visible spectrum of bioactive fraction showed two main absorption peaks at λ282 and λ375 for C2 (A), λ269 and λ302 for C5 (B), λ273 and λ394 for M3 (C), and λ266 and λ392 for M4 (D).
FIGURE 11
FIGURE 11
Liquid chromatogram profile from culture filtrate extract of B. petrensis (A), liquid chromatogram profile of purified C2 fraction (B), and C5 fraction (C). The LC peak at Rt 33.31 and 10.13 displaying the purified bioactive fractions C2 and C5 corresponding to fungal culture filtrate extract Rt 33.31 and 10.14, respectively (A–C). The mass spectrum displaying the feature m/z 212.02, 185.11 [M+H]+ of compounds 2-(1,3-benzothiazol-2-ylsulfanyl)ethanol and 5-cyclohexyl-1-oxido-triazolidin-4-one obtained a similar characteristic feature of molecular mass in CFE with a positive ion mode (D–G).
FIGURE 12
FIGURE 12
Liquid chromatogram profile from culture filtrate extract of B. petrensis (A) and liquid chromatogram profile of purified M3 fraction (B) and M4 fraction (C). The LC peak at Rt 27.9 and 13.4 displaying the purified bioactive fractions M3 and M4 corresponding to fungal culture filtrate extract Rt 33.31 and 13.7, respectively (A,B,E,F). The mass spectrum displaying the feature m/z 229.08, 185.11 [M+H]+ of compounds 3-hydroxy-7-propyl-naphthalene-2-carboxylic acid and 2,2-bis(azidomethyl)butan-1-ol obtained a similar characteristic feature of molecular mass in ME with a positive ion mode (C,D,G,H).
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