Induction of apoptosis by emestrin from the plant endophytic fungus Emericella nidulans ATCC 38163 in Huh-7 human hepatocellular carcinoma cells

Abstract This research aimed to investigate the anticancer properties of emestrin, a major constituent of Emericella nidulans ATCC 38163 through the induction of apoptosis in Huh-7 human hepatocellular carcinoma cells. In this study, this fungus was isolated from the fresh leaves of Ruprechtia salicifolia (Cham. & Schltdl.) C.A. Mey, and identified by morphology and 18S rDNA followed by large-scale fermentation in liquid biomalt broth medium. Epidithiodioxopiperazine derivative emestrin along with ten known metabolites were isolated and identified from the fungal extract. The cytotoxic assay revealed that emestrin had the strongest cytotoxicity against Huh-7 and A-549 cells with IC50 values of 4.89 and 6.3 μM, respectively. Using annexin V-FITC assay, treatment of Huh-7 cells with 4.89 µM for 24 h resulted in a significant increase in the percentage of early and late apoptosis (3.16% and 22.84%, respectively) compared to untreated cells. Additionally, Bax and bcl-2 protein levels were regulated, which induced apoptosis in treated cells. These results indicate that emestrin induces mitochondrial pathway to stimulate apoptosis and inhibits cell proliferation in hepatocellular carcinoma. Graphical Abstract


Introduction
Apoptosis is a type of cellular suicide, generally characterized by a series of biochemical and distinct morphologic changes in cells, including cell shrinkage and membrane blebbing (Kerr et al. 1972).The induction of apoptosis in cancer cells offers a promising mechanism for cancer therapy.Therefore, several cytotoxic methodologies have been used, such as anticancer drugs, gamma-irradiations, and immunotherapy (Debatin 1997;Fulda and Debatin 2013).Several compounds have been used as anticancer agents through inducing apoptosis in cancer cells such as ciplastin, etoposide, mitomycin, and actinomysin D (Debatin 2004;Wong 2011) Identifying a new compound with anticancer properties will increase the promising anticancer therapeutic agents (Solary et al. 2000;Wong 2011).
A previous study showed that emestrin induces cytotoxic effects against different types of cancer, including breast, liver, colon, and cervix cancer.The study investigated the role of anticancer activity of emestrin in breast cancer cell only (T47D) through the induction of cell cycle arrest and apoptosis (Nursid et al. 2011).Our study is the first to demonstrate the anticancer properties of the cytotoxic compound emestrin on hepatocellular carcinoma cells (Huh-7) which is mediated by cell cycle arrest and apoptosis.In addition, we examined the proteins level of the mitochondrial pathway as well as p53 after treatment with emestrin.

Results and discussion
The plant endophytic fungus E. nidulans ATCC 38163 was identified by morphological characterization and authenticated by phylogenetic analyses based on internal transcribed spacer (ITS) regions of rDNA containing ITS1-5.8S-rRNA-ITS4 gene sequences.The fungus was grown in a static liquid biomalt medium.The culture broth was extracted with EtOAc, and the total extract was subjected to a combination of column chromatography (CC) including silica gel and Sephadex LH-20 to yield eleven metabolites 1-11 (Figure 1).The structural elucidation of the isolated compounds based on the spectral analyses (MS, 1D-and 2D-NMR), and comparison with literature data.Alkaloids 1-7 were detected as their violet to pink colour on silica gel TLC when spraying with Ehrlich's reagent (Stahl and Kaldewey 1961).The NMR spectra of these compounds revealed the macrocyclic epidithiodioxo-piperazine derivatives emestrin (1), emestrin B (2) as mycotoxins of Emericella (Nursid et al. 2015).Piperazines derivatives secoemestrin C (3), dethiosecoemestrin (4) and aurantioemestrin (5) together with violacetic acid (8) were also isolated, which seems to be biogenetically derived from emestrin.It is postulated that epitetrathiodioxopiperazine derivative 3 may be the key intermediate from emestrin and/or emestrin B to trioxopiperazine 4 (Ooike  et al. 1997).The later derivative 4 may be also the key intermediate from the dioxopiperazinethione 5 via emestrin (Kawai et al. 1987).Moreover, due to the long fungal fermentation, it seems likely that 4 biosynthesized from emestrin by desulfurization and oxidative degradation, and thus was easily hydrolyzed to give 8 (Seya et al. 1986).Indoloditerpene, emindol SA (6) (Nozawa et al. 1988), and a purine nucleoside, cordycepin (7) (Zhou et al. 2016) were also identified.Additionally, shamixanthone (9), emericellin (10) were identified as two hydroxyquinone derivatives (showing dark green color with ethanolic FeCl 3 and red color with KOH) (Inoue et al. 2018), and glycerol-1-O-palmiteolate (11) as a lipoidal metabolite (Sun et al. 2016).
The agar diffusion disc method (Cosentino et al. 1999) was used to screen the antibacterial activity of some isolated compounds against Gram-positive bacteria (Micrococcus luteus ATCC 10240 and Staphylococcus aureus ATCC 6538) and Gramnegative bacteria (Escherichia coli ATCC 25922).Emestrin (1) and cordycepin (7) showed significant antibacterial activity against M. luteus with inhibition zone diameters 17 and 15 mm, respectively, compared to gentamycin as a reference drug (20 mm).However, the MIC values of both active compounds were above 1000 mg/ml.Other compounds, 2 and 9-11 were inactive on all microbial strains.
The cytotoxic activity of compounds 1, 2, 7, 9-11 against three human cancer cell lines including A-549, Huh-7, and Caco2 was estimated using crystal violet assay (Mosmann 1983).Vinblastine sulphate, a broad-spectrum anticancer drug was used as a positive control (Table S1).The results showed that there were differences in the cytotoxic effect depending on the structure of tested compounds (Panggabean et al. 2022).Emestrin (1) showed the lowest IC 50 , indicating high potency against all cancer cell lines (Huh-7 ¼ 4.8 mM, A-549 ¼ 6.3 mM, Caco2 ¼ 9.28 mM), followed by cordycepin (7) with IC 50 values ranging from 18.84 to 29.2 mM.When dithio group of emestrin is replaced by a trithio group in emestrin B (2), the activity is reversed and emestrin B showed the weakest cytotoxic activity.In addition, glycerol-1-O-palmiteolate (11) slightly inhibited the proliferative of the different cancer cells with IC 50 values ranging from 50.9 to 83.9 mM, whilst compound 9 was inactive (IC 50 >100 lM).In order to assess the compounds' selectivity towards cancerous cells, the compounds were screened against normal human lung fibroblast cells, MRC-5.As shown in Table S2, emestrin and cordycepin demonstrated significant selectivity against all cell lines with SI values ranging from 8.8-16.7 and 4.34-6.75mM, respectively.However, according to the 'selectivity criteria' only emestrin could be considered a selective compound against Huh-7 cells.Therefore, emestrin has been assessed using protein expression studies of mitochondrial pathway, and cell cycle analysis by flow cytometry against human hepatoma cell line Huh-7.
During apoptosis, cells display numerous common morphological changes such as nuclear condensation, cell shrinkage, and DNA fragmentation.Externalization of phosphatidylserine (PS) of the plasma membrane is a unique biomarker of apoptotic cells (Kerr et al. 1972;Renehan et al. 2001).Annexin V is a cellular phospholipid binding protein with a high affinity to bind to PS in the presence of calcium, therefore can be used as a probe for early detection of apoptosis (Kerr et al. 1972;Crowley et al. 2016).In this study, hepatocellular carcinoma cell (Huh-7) was treated with emestrin to examine its ability to induce apoptosis using Annexin V-FITC assay.Interestingly, treated cells at an IC 50 concentration (4.89 mM) for 24 h resulted in a significant increase in the percentage of early and late apoptosis with 3.16 and 22.84%, respectively, (p < 0.05).Moreover, a remarkable increase in the percentage of the cells at the late apoptosis stage with 22.84% compared to necrotic cells after 24 h of treatment (Figure S1a) was detected (p < 0.05).These results indicate that emestrin has an antiproliferative effect on Huh-7 through inducing apoptosis cells.
One of the main biochemical hallmarks of apoptosis is the formation of DNA fragments.Indeed, the well characterized apoptotic morphology is internucleosomal DNA fragmentation.During the cell cycle, the first step in interphase is the first gap phase (G1) including synthesizing mRNA and proteins in preparation for mitosis.The following step is S phase in which DNA is replicated (Saraste and Pulkki 2000).To determine more the anticancer activity of emestrin against hepatocellular carcinoma cells (Huh-7), a cell cycle assay was used.The treated cells with emestrin resulted in G1 and S phase cell cycle arrest with 94.4% cells in both G1and S phases compared with 82.7% in control (Figure S1b).This result indicates the association between cell cycle arrest activity and emestrin through affecting the proliferation of Huh-7 cells by inhibiting the G1/S phase.As a result, this will delay the following cell cycle phase in the treated cells with a decrease in the percentage of treated cells with 5.5% in G2/M phase of the cell cycle from control.Interestingly, the percentage of pre-G1 was high with 37.5% (which represents the apoptotic cells) in treated cells compared to the control with 2.1%.These results indicate the cell cycle arrest and apoptosis induced in Huh-7 cells after emestrin treatment.
Next, the role of emestrin in inducing apoptosis through the mitochondrial pathway that is regulated by bcl family members was investigated.Bax/proapoptotic and bcl-2/antiapoptotic are vital regulators of apoptosis cell death (Wang and Youle 2009).Interestingly, an up-regulation of Bax protein expression and downregulation of bcl-2 in treated cells compared to the control was detected (Figure S1c).As a result, activation of caspase-9 was found in treated cells compared to the control.Moreover, up regulation of the tumor suppressor protein a (p53) level was found after treatment with emestrin as shown in Figure S1c.These results indicate that emestrin induces mitochondrial pathway to stimulate apoptosis and inhibits cell proliferation in hepatocellular carcinoma.

Experimental
See Supplementary Material.

Conclusions
Plant-endophytic E. nidulans ATCC 38163 produced eleven metabolites.Emestrin was identified as a cytotoxic compound that exhibited significant activity against Huh-7 and A-549 cells.Furthermore, the capability of emestrin to induce apoptosis in the hepatocellular carcinoma cell (Huh-7) was evaluated.The induction of apoptosis was exhibited through a significant increase in the percentage of early and late apoptosis.In addition, treating the cell with emestrin resulted in G1 and S phase cell cycle arrest.Also, emestrin regulates mitochondrial pathway to stimulate apoptosis in treated cells leading to suppression of cell proliferation.Together, the above study determines the cytotoxic effects of the emestrin on hepatocellular carcinoma cells as a potential anticancer agent.