Cytotoxic Potential of Alternaria tenuissima AUMC14342 Mycoendophyte Extract: A Study Combined with LC-MS/MS Metabolic Profiling and Molecular Docking Simulation

Breast, cervical, and ovarian cancers are among the most serious cancers and the main causes of mortality in females worldwide, necessitating urgent efforts to find newer sources of safe anticancer drugs. The present study aimed to evaluate the anticancer potency of mycoendophytic Alternaria tenuissima AUMC14342 ethyl acetate extract on HeLa (cervical cancer), SKOV-3 (ovarian cancer), and MCF-7 (breast adenocarcinoma) cell lines. The extract showed potent effect on MCF-7 cells with an IC50 value of 55.53 μg/mL. Cell cycle distribution analysis of treated MCF-7 cells revealed a cell cycle arrest at the S phase with a significant increase in the cell population (25.53%). When compared to control cells, no significant signs of necrotic or apoptotic cell death were observed. LC-MS/MS analysis of Alternaria tenuissima extract afforded the identification of 20 secondary metabolites, including 7-dehydrobrefeldin A, which exhibited the highest interaction score (−8.0156 kcal/mol) in molecular docking analysis against human aromatase. Regarding ADME pharmacokinetics and drug-likeness properties, 7-dehydrobrefeldin A, 4’-epialtenuene, and atransfusarin had good GIT absorption and water solubility without any violation of drug-likeness rules. These findings support the anticancer activity of bioactive metabolites derived from endophytic fungi and provide drug scaffolds and substitute sources for the future development of safe chemotherapy.


Introduction
Human health is at constant risk due to the occurrence of different types of noncommunicable chronic diseases [1]. Among the major non-communicable chronic diseases, cancer is considered the second principal cause of mortality in the world [2]. In 2020, an estimated 19.3 million cancer cases developed and around 10.0 million cancer deaths occurred worldwide [3]. Breast cancer was diagnosed in 2.3 million new cases (11.7%), followed by 11.4% for lung, 10.0% for colorectal, 7.3% for prostate, and 5.6% for stomach cancers [3]. Cancer incidence could be initiated by both extrinsic and intrinsic factors that trigger the activation or inactivation of certain genes, subsequently leading to abnormal growth of cells [4]. culture were inoculated into sterilized rice medium in 1 L Erlenmeyer flasks and incubated for 30 days at 28 ± 2 • C. The fermented culture was extracted twice by using ethyl acetate (EtOAc) [34], filtrated, and evaporated by a rotary evaporator at 45 • C to produce the dry extract that was kept for further investigations.

Cell Culture and Cytotoxicity Assay
The cancer cell lines were obtained from Nawah Scientific Inc. (Mokatam, Cairo, Egypt). HeLa (cervical cancer) and SKOV-3 (ovarian cancer) cells were cultured in RPMI medium amended with 100 units/mL penicillin, 100 mg/mL streptomycin, and 10% heatinactivated fetal bovine serum and incubated in a humidified 5% CO 2 atmosphere at 37 • C, while MCF-7 (breast adenocarcinoma) cells were grown in DMEM medium [31]. The Sulforhodamine B (SRB) assay was employed to estimate cancer cell viability, and a BMG LABTECH FLUOstar Omega microplate reader (Ortenberg, Germany) was utilized to measure absorbance at 540 nm [35,36].

Effect of A. tenuissima Extract on Cell Cycle Distribution of Breast Cancer Cells
After treatment with test compounds for 24 h, cells were analyzed for DNA content using a flow cytometry analysis protocol [37] with the FL2 (λex/em 535/617 nm) signal detector (ACEA Novocyte flow cytometer, ACEA Biosciences Inc., San Diego, CA, USA). Twelve thousand events were obtained for each tested sample. ACEA NovoExpress software (ACEA Biosciences Inc., San Diego, CA, USA) was utilized to calculate cell cycle distribution [38].

Assessment of Apoptotic Effect of A. tenuissima Extract
An Annexin-V/FITC kit (Abcam Inc., Cambridge Science Park, Cambridge, UK) for apoptosis detection combined with 2-fluorescent-channel flow cytometry was used to determine apoptotic and necrotic cell populations as previously described [35,37].

UHPLC-ESI-MS/MS Profiling
The UHPLC-ESI-MS/MS analysis of the ethyl acetate extract of A. tenuissima was performed on an ExionLC AC system coupled with a SCIEX Triple Quad 5500 + MS/MS system equipped with an electrospray ionization (ESI) system. An Ascentis C18 Column (4.6 × 150 mm, 3 µm) was employed as the stationary phase, and the sample was eluted with mobile phases consisting of eluent A (0.1% formic acid) and eluent B (acetonitrile, LC grade) with following mobile phase gradient: 10% B at 0-1 min, 10%-90% B at 1-21 min, 90% B at 21-25 min, and 10% at 25.01-28 min. The flow rate was 0.5 mL/min, and the injection volume was 10 µL. MS/MS analysis used positive and negative ionization modes with a scan (EMS-IDA-EPI) [35]. The compounds were identified by using MS-DIAL version 4.70, Natural Products Atlas, and CFM-ID version 4.0 software [39][40][41][42].

Molecular Docking Simulation
The binding affinity of the identified metabolites was evaluated by molecular docking analysis in comparison with a standard reference inhibitor using the "Molecular Operating Environment (MOE 2014.09) [35]. The compounds were imported to MOE and subjected to 3D protonation and Merck molecular force field (MMFF94x) energy minimization, and they were partially charged. A stochastic conformational search was conducted, the minimum dE conformers were selected, and a virtual ligand database was constructed [43]. The structure of human placental aromatase cytochrome P450 (CYP19A1) (PDB ID: 3S79) was acquired from the Protein Data Bank [33]. All the hetero atoms and unbound water molecules were removed from the target proteins, and their structures were optimized for docking simulation. The parameters of scoring were Triangle Matcher, scoring was set at London dG and rescoring at GBVI/WSA dG, and the docking poses were set at 30 poses for the initial energy score and 10 for refinement [43]. The docking pose score (the process of evaluating a particular pose by counting the number of favorable intermolecular interactions such as hydrogen bonds and hydrophobic contacts and computed by summing all the applicable scores of any interacting surface points between cavity and ligand) [44], root mean square deviation (RMSD) (RMSD measures the difference in conformation and position between two poses of a molecule), and ligand-receptor complexes were tested for interaction analysis. The 3D images were created using the MOE visualizing tool [33,35]. The protocol was validated after protein preparation by running redocking of the complexed inhibitor to the active site, and the RSMD value was 0.18 Å. Complexed ligand and redocked ligand overlays are shown in the Supplementary Materials (Supplementary Figure S1).

Drug-like Properties and ADME Prediction of High-Affinity Compounds
The Molinspiration web server was used to determine the molecular properties [45,46], and the SwissADME website server was employed to calculate the drug-likeness, ADME, and pharmacokinetic parameters of the identified metabolites [33,42].

Cytotoxic Activity of A. tenuissima Ethyl Acetate Extract
In the current study, the ethyl acetate extract of A. tenuissima was tested for its effect on the cell viability of three cancer cell lines, namely HeLa, SKOV-3, and MCF-7, by SRB assay, and the IC 50 values were determined from dose-response curves of different concentrations ( Figure 1).
The results showed that the extract exhibited potential cytotoxic activity against the tested cell lines and had a prominent influence against the MCF-7 cell line, with the IC 50 value of 55.53 µg/mL (Table 1), in comparison with doxorubicin as a standard anticancer drug. The histopathology study of the extract at a concentration of 100 µg/mL showed consistency with the dose-response curve (Figures 1 and 2) and IC 50 results (Table 1). Figure 2 shows the effect of the extract on different cell lines in comparison with the control and doxorubicin treatment. The observation of the optical microscope staining image changes of cancer cells showed that the control cells had normal morphology and attachment, while the cells treated with A. tenuissima EtOAc extract showed a significant reduction in cells at 100 µg/mL, confirming the cytotoxicity of the tested extract.

Effect of A. Tenuissima Extract on Cell Cycle Distribution of MCF-7 Cells
Breast cancer cells (MCF-7) were manipulated for 24 h with the predetermined IC 50 (55.53 µg/mL) of the A. tenuissima EtOAc extract, and the DNA content was measured using flow cytometry to determine the effect of the tested sample on MCF-7 cell cycle distribution ( Figure 3).    With no discernible effects on other phases, the tested extract caused S-phase arrest and increased this cell population significantly, by 25.53%, from 16.80% to 22.56% (Table 2, Figures 3 and 4), suggesting that the effect on the DNA synthesis step of replication may be the cause of the extract's cytotoxic effect.

Assessment of Apoptotic Effect of A. tenuissima Extract
After being exposed to the predetermined IC 50 's, cells were evaluated using Annexin-V/FITC staining in conjunction with flow cytometry to determine the impact of the extract on the mechanism of cell death. After 24 h, the evaluated extract showed no significant signs of necrotic or apoptotic cell death when compared to untreated control cells (Table 3, Figure 5). Table 3. Apoptosis/necrosis assessment in MCF7 cell line after exposure to A. tenuissima EtOAc extract and control for 24 h.

Metabolite Analysis of A. tenuissima EtOAc by UPLC-ESI-MS/MS
The analysis of the sample was performed using LC-ESI-MS/MS for the separation and detection of metabolites. Positive and negative ionization modes were employed to characterize the corresponding signals. The total ion current map of the sample was produced. The TIC of the A. tenuissima EtAOc extract is shown in Figure 6. Structural analysis was performed and the components were analyzed using two-stage mass spectrometry to obtain the mass, metabolite molecular formula, and characteristic fragment ions. Moreover, the previously isolated compounds of the Alternaria genus were used as a tool for the identification of detected compounds by comparison of the obtained molecular formula with the published data.
In total, 20 secondary metabolites were identified by analysis of the results (Table 4, Figure 7) based on their precursor ion and their MS 2 fragmentation patterns compared with the Competitive Fragmentation Modeling for Metabolite Identification online database; the retention time, high-resolution molecular ion mass, chemical formula, and MS 2 fragments of the identified chemical constituents are shown in Table 4 and Supplementary Figures S2-S21. The compounds are arranged according to their retention time. Analyzing the A. tenuissima extract revealed several compounds (Table 4)   fit to isocoumarin derivatives altenuene (4 -epialtenuene), alternariol, 3-O-demethylaltenuisol, and alternariol-9-methyl ether. In addition, (S)-alternariphent A, alternapyran, 4-methoxy-6-methyl-5-(3-oxobutyl)-2H-pyran-2-one, and resveratrodehyde C were also detected at t R 12.43, 12.84, 15.04, and 30.02, respectively.

Molecular Docking Study
The compounds' docking results revealed an affinity range of −8.0156 to −4.7618 kcal/mol. The top-scoring compounds were 4'-epialtenuene (4'), 7-dehydrobrefeldin A (9), and atransfusarin (19), which had a stronger affinity to the human placental aromatase cytochrome P450 (CYP19A1) active site relative to the complexed inhibitor ligand 4-androstene-3-17-dione (Table 5). Molecular docking analysis revealed that 7-dehydrobrefeldin A (9) showed the highest interaction score with human aromatase (posing score -8.0156 kcal/mol and RMSD value of 1.16 Å) as compared to 4-androstene-3-17-dione, standard inhibitor (−9.0037 kcal/mol). 7-dehydrobrefeldin A interacts through hydrogen bond formation with the Met 374 amino acid residue in the catalytic cleft of aromatase in addition to several hydrophobic interactions with the active site ( Figure 8). Moreover, the compound 4'-Epialtenuene showed a pose score of −7.2929 kcal/mol with an RMSD value of 1.30 Å and was found to bind with the receptor active site through the formation of hydrogen bonds with Leu 477 and Met 374 amino acid residues as Hdonors as well as through a pi-H bond with VAL 370 amino acid (Figure 9). Furthermore, atransfusarin (19) interacts with Met 374 amino acid residue as an Hdonor and with VAL 370 amino acid through a pi-H bond ( Figure 10) with a pose score of −7.2688 kcal/mol. It could be concluded that these compounds could be used as a scaffold for the development of bioactive treatment for breast cancer through inhibition of the selected target (human aromatase cytochrome P450) Figure 10. The 2D and 3D interactions of atransfusarin with human aromatase cytochrome P450 active site.

ADME Pharmacokinetics and Drug-Likeness Properties of High-Affinity Compounds
The compounds that showed the highest affinity to the human aromatase cytochrome P450 active site were screened for their drug-likeness and ADME pharmacokinetics using the website servers. All tested compounds had good GIT absorption and water solubility without any violation of drug-likeness rules. In addition, the compounds had promising bioavailability scores with a value of 0.55. 4'-epialtenuene and dehydrobrefeldin A showed promising lead-likeness and synthetic accessibility ( Table 6). Table 6. Detailed in silico assessment of molecular properties, drug-likeness, absorption, distribution, metabolism, and excretion of 4'-epialtenuene, 7-dehydrobrefeldin A, and atransfusarin.

Discussion
Fungi are a valuable source of bioefficient natural products that can be employed to fabricate new analogs for cancer treatment [5]. Several metabolites from Alternaria spp. of various chemical classes and biological activities have been identified [23]. Alternaria extracts and secondary metabolites have been shown to have a wide range of biological activities and functions, including cytotoxic, antimicrobial, and herbicide effects. The promising bioactivities have attracted the interest of pharmaceutical researchers in the discovery of novel natural therapeutics [23,34].
In the present study, the IC 50 values of the A. tenuissima EtOAc extract on the viability of HeLa, SKOV-3, and MCF-7 cancer cell lines determined by SRB assay were 67.76, 74.60, and 55.53 µg/mL, respectively. Cell cycle distribution analysis of the treated MCF-7 cell line showed a cell cycle arrest at the S phase with a significant increase in the cell population (25.53%), while no significant signs of necrotic or apoptotic cell death were observed. It is noteworthy that the previous studies of A. tenuissima revealed the isolation of toxic metabolites such as alternariol and alternariol monomethyl ether which possessed strong cytotoxicity against KB and KBv200 tumor cell lines [47] and L5178Y mouse lymphoma cells [48]. Interestingly, Lehmann et al. reported the estrogenic potential of alternariol, as well as its cell proliferation inhibitory effect via interference with the cell cycle [49]. Furthermore, tenuazonic acid, a metabolite isolated from several endophytic Alternaria species, exhibited a broad spectrum of biological activity, such as antineoplastic, antiviral, and antibiotic effects [24,50,51]. Additionally, A. tenuissima TER995 was employed as a promising source for the submerged fermentation-based production of paclitaxel (taxol), the most valuable anticancer drug [52].
The cytotoxicity assay revealed that A. tenuissima has a potent impact on MCF-7 cells, with an IC 50 value of 55.53 µg/mL. In order to evaluate the possible affinity of identified compounds and understand their binding possibility as inhibitors for the aromatase enzyme as a selective target for breast cancer treatment, a molecular docking analysis was conducted [13,33]. Aromatase is responsible for estrogen biosynthesis by catalyzing the bioconversion of androgens to estrogens by the aromatization of androstenedione to estrone [27,70]. Aromatase inhibitors are classified into steroidal and non-steroidal types depending on their chemical structure [71]. Both steroidal and non-steroidal inhibitors of aromatase block the biosynthesis of estrogens by inhibiting aromatase; steroidal inhibitors inhibit aromatase in an irreversible manner, while non-steroidal inhibitors inhibit aromatase in a reversible (competitive) manner and are more effective [72]. The general mode of action of aromatase inhibitors has been suggested to be due to the coordination of the inhibitor with the iron atom of the catalytic heme group within aromatase [73]. The aromatase catalytic cleft contains the amino acids Met 374 from the b3 loop; Leu 477 and Ser 478 from the b8-b9 loop; Ile 133 and Phe 134 from the B-C loop; Val 370, Leu 372, and Val 373 from the K-helix-b3 loop; Ile 305, Ala 306, Asp 309, and Thr 310 from the I-helix; and Phe 221 and Trp 224 from the F-helix [33]. 7-dehydrobrefeldin A, 4'-epialtenuene, and atransfusarin, which exhibited the highest affinity to human aromatase cytochrome P450 active site, were screened for their drug-likeness and ADME pharmacokinetics using the website servers [43,74] and showed promising GIT absorption and water solubility.

Conclusions
The ethyl acetate extract from endophytic Alternaria tenuissima AUMC14342 showed anticancer potential against HeLa (cervical cancer), SKOV-3 (ovarian cancer), and MCF-7 (breast adenocarcinoma) cell lines in an SRB assay used to assess cancer cell viability. Cell cycle distribution analysis of treated MCF-7 cells revealed cell cycle arrest at the S phase with a significantly increased cell population. Bioactive secondary metabolites in the EtOAc extract were characterized using LC-MS/MS, and their molecular docking analysis against human placental aromatase exhibited a promising affinity to the aromatase active site. Moreover, ADME pharmacokinetics and drug-likeness properties of 7-dehydrobrefeldin A, 4'-epialtenuene, and atransfusarin revealed good GIT absorption and water solubility. The current research will enrich and enhance the bioprospecting of anticancer natural metabolites from endophytic fungi to achieve a sustainable supplement for safe chemotherapy.