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. 2023 Dec 23;13(1):66.
doi: 10.3390/foods13010066.

Flammutoxin, a Degradation Product of Transepithelial Electrical Resistance-Decreasing Protein, Induces Reactive Oxygen Species and Apoptosis in HepG2 Cells

Jianguo Wu  1 , Yu Nong  1 , Bingzhi Chen  2 , Yuji Jiang  2 , Yuanhao Chen  1 , Chuanzheng Wei  1 , Yongxin Tao  1   3 , Baogui Xie  1
Affiliations

Flammutoxin, a Degradation Product of Transepithelial Electrical Resistance-Decreasing Protein, Induces Reactive Oxygen Species and Apoptosis in HepG2 Cells

Jianguo Wu et al. Foods. .

Abstract

Proteins from Flammulina filiformis were prepared by sodium chloride extraction and fractionated by ammonium sulfate precipitation with increasing saturation degrees to obtain the protein fractions Ffsp-30, Ffsp-50, Ffsp-70, Ffsp-90, and Ffp-90. Among these protein fractions, Ffsp-50 possessed the most significant cytotoxic effect against three human gastrointestinal cancer cell lines, viz. HT-29, SGC-7901, and HepG2. SDS-PAGE and MALDI-TOF/TOF MS/MS analyses revealed that flammutoxin (FTX) was present as a dominating protein in Ffsp-50, which was further evidenced by HPLC-MS/MS determination. Furthermore, native FTX was purified from Ffsp-50 with a molecular weight of 26.78 kDa, exhibiting notable cytotoxicity against gastrointestinal cancer cell lines. Both Ffsp-50 and FTX exposure could enhance intercellular reactive oxygen species (ROS) generation and induce significant apoptosis in HepG2 cells. FTX was identified to be relatively conserved in basidiomycetes according to phylogenetic analysis, and its expression was highly upregulated in the primordium as well as the pileus of the fruiting body from the elongation and maturation stages, as compared with that in mycelium. Taken together, FTX could remarkably inhibit cell growth and induce ROS and apoptosis in HepG2 cells, potentially participating in the growth and development of the fruiting body. These findings from our investigation provided insight into the antigastrointestinal cancer activity of FTX, which could serve as a biological source of health-promoting and biomedical applications.

Keywords: Flammulina filiformis; apoptosis; flammutoxin; gastrointestinal cancer; reactive oxygen species.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
SDS-PAGE analysis of protein fractions from the fruiting bodies of F. filiformis. Samples were electrophoresed on 12% gel and stained with Coomassie brilliant blue. Column M represents a pre-stained protein marker with Mw range of 15–170 kDa; columns a, b, c, d, and e represent Ffsp-30, Ffsp-50, Ffsp-70, Ffsp-90, and Ffp-90, respectively.
Figure 2
Figure 2
Purification and analysis of FTX protein. (A) Purification of native FTX from Ffsp-50 on a Cellufine Q-500 strong anion-exchange column. FTX was obtained from section (a) of eluents of 0.2 M NaCl in 0.02 M Tris-HCl buffer solution (pH 8.0). (B) SDS-PAGE analysis of purified FTX from the fruiting body of F. filiformis. Column M represents the pre-stained protein marker with Mw range of 10–170 kDa; column F represents purified FTX. (C) Chromatogram of purified FTX determined by HPGPC method.
Figure 3
Figure 3
AA sequence and molecular weight of FTX determined by MALDI-TOF/TOF MS/MS. (A) The deduced AA sequence of FTX precursor (GenBank accession no. BAA76510.1). The red parts indicate the peptide fragments (sequence coverage: 62%) detected by MALDI-TOF/TOF MS/MS; the region highlighted in luminous yellow denotes the presumed AA sequence of purified FTX lacking the initial Met (M) and C-terminal 30 AA residues (241 AA residues, Mw 26.78 kDa). (B) FTX molecular weight detected by MALDI-TOF/TOF MS/MS.
Figure 4
Figure 4
Growth-inhibiting effects on three human gastrointestinal cancer cell lines. Using a standard MTT assay, the relative cell viabilities of HT-29, SGC-7901, and HepG2 cells were determined after treatment with indicated concentrations of (A–C) Ffsp-30, Ffsp-50, Ffsp-70, Ffsp-90, and Ffp-90 for 24 h or (D–F) FTX for 24, 48, and 72 h. Values are represented as the percentage of viable cells, with vehicle-treated cells regarded as 100% viable. Data are presented as mean ± S.D. of three independent experiments. * p < 0.05, ** p < 0.01 compared with vehicle-treated controls.
Figure 5
Figure 5
Morphological changes in HepG2 cells examined by DAPI nuclear staining under a fluorescence microscope after challenge with indicated concentrations of (A) Ffsp-50 (0, 25, 50, and 100 μg/mL) and (B) FTX (0, 12.5, 25, and 50 μg/mL) for 24 h. Images are representative of three independent experiments (magnification ×200). Apoptosis-inducing effects of Ffsp-50 (C,E) and FTX (D,F) in HepG2 cells. HepG2 cells were treated with indicated concentrations of Ffsp-50 (0, 25, 50, and 100 μg/mL) and FTX (0, 12.5, 25, and 50 μg/mL) for 24 h and analyzed by flow cytometry with Annexin V/PI double staining. Representative FACS analysis scattergrams of Annexin V/PI show the four different cell populations, which are described as follows: double-negative stained cells (Annexin V−/PI−, lower left) indicating the live cell population; Annexin V positive/PI negative stained cells (Annexin V+/PI−, lower right) and double−positive (Annexin V+/PI+, upper right) stained cells showing early and late apoptotic cells, respectively; Annexin V negative/PI positive stained cells (Annexin V−/PI+, upper left) denoting dead cells. Data shown are presented as mean ± SD of three independent experiments. ** p < 0.01 compared with vehicle−treated controls.
Figure 6
Figure 6
Intracellular ROS generation in HepG2 cells detected by DCFH-DA staining using fluorescence microscope after treatment with indicated concentrations of (A) Ffsp-50 (0, 25, 50, and 100 μg/mL) and (B) FTX (0, 12.5, 25, and 50 μg/mL) for 24 h. Images are representative of three independent experiments (magnification ×200).
Figure 7
Figure 7
Phylogenetic tree of FTX protein in F. filiformis and other fungi.
See this image and copyright information in PMC

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