Introduction

Among the therapeutic targets for cancer, the abnormal expression of the epidermal growth factor receptor (EGFR) is strongly associated with various malignancies such as breast, ovarian, non-small-cell lung, prostate, and colon cancers. The EGFR has been confirmed to be closely related to tumor growth, progression, and metastasis. The poor prognosis of cancer patients prompts extensive studies on the EGFR signaling pathway (Allam et al. 2020). Vascular endothelial growth factor (VEGF) is secreted by tumors and induces a mitogenic response through its binding to one of three tyrosine kinase receptors (VEGFR-1 to -3) on nearby endothelial cells; inhibition of this signaling pathway should block angiogenesis and subsequent tumor growth (Dziobek et al. 2019). The Ochrophyta, Polycladia myrica (S.G.Gmelin) Draisma, Ballesteros, F.Rousseau & T.Thibaut 2010, belongs to the family Sargassaceae while Dictyopteris acrostichoides (J.Agardh) Bornet, 1885, belongs to family Dictyotaceae. Caulerpa racemosa (Forsskål) J.Agardh, 1873, is a Chlorophyta that belongs to the family Caulerpaceae, and Halimeda opuntia (Linnaeus) J.V.Lamouroux, 1816 belongs to the family Halimedaceae (Guiry and Guiry 2023). Dictyotaceae is considered one of the most important brown algae in the treatment of tumors. The alga Dictyopteris sp. is an important group of marine seaweeds and is excessively distributed, known by their identified ocean smell due to its secondary metabolites including C11-hydrocarbons and sulfur compounds. Dictyopteris has a broad variety of phytoconstituents, which were reported to have antitumor, antimicrobial, alpha-amylase inhibitors, and anti-inflammatory activities; Dictyopteris acrostichoides has shown a wide range of biological activities, but its phytoconstituents are still elusive (Rushdi et al. 2022a). Thus, the current study characterizes the cytotoxic potential of ethanol extract of four algae including Caulerpa racemosa, Dictyopteris acrostichoides, Halimeda opuntia, and Polycladia myrica against HepG2 (human hepatoma), MCF-7 (human breast adenocarcinoma), and Caco-2 (human colon adenocarcinoma) cells assisted by both LC-HR-ESI–MS spectrometry metabolomics and molecular docking analyses to detect the chemical metabolites as well as their possible mode of action.

Materials and Methods

All used chemicals were of analytical grade and purchased from Sigma (USA), Merck (Germany), and SD Fine Chemicals (India). Caulerpa racemosa (Forsskål) J.Agardh, 1873, Caulerpaceae; Dictyopteris acrostichoides (J.Agardh) Bornet, 1885, Dictyotaceae; Halimeda opuntia (Linnaeus) J.V.Lamouroux, 1816, Halimedaceae; and Polycladia myrica (S.G.Gmelin) Draima, Ballesteros, F.Rousseau & T.Thibaut, 2010, Sargassaceae, samples were collected in August 2021 from various locations along the coastal region of Hurghada City coast line, Red Sea (27° 17′ 01.0″N and 33° 46′ 21.0″E), Egypt. The voucher numbers (Da-08/2021, Cr-08/2021, Ho-08/2021, and Pm-08/2021) were deposited in the herbarium unit at the Department of Pharmacognosy, Faculty of Pharmacy, South Valley University, Qena, Egypt. The samples were collected in sterilized polyethylene bags, and kept in an icebox, for transportation to the laboratory. Samples were rinsed with distilled water to remove any associated debris and allowed to air dry before being ground to a fine powder. The air-dried powdered algal materials (200 g) were separately extracted with 70% ethanol to get crude extracts from C. racemose (16 g), D. acrostichoides (23 g), H. opuntia (9 g), and P. myrica (28 g). The obtained solvent-free residues were stored at 4 °C for subsequent analysis. Cytotoxicity of the ethanol extracts was evaluated in cell lines manipulating the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to Cheng et al. (2017) against MCF-7 (human breast adenocarcinoma), Caco2 (human colon adenocarcinoma), and HepG2 (human hepatoma) cell lines. EGFR assay was performed according Huang et al (2021), while VEGFR assay was performed according Elrazaz et al (2021). Molecular modeling and visualization processes were performed within EGFR and VEGFR using Molecular Operating Environment (MOE 2019.0102, 2020; Chemical Computing Group, Montreal, QC, Canada). Metabolomic profiling of the ethanol extracts of D. acrostichoides was performed using analytical techniques of liquid chromatography high-resolution electrospray ionization mass spectrometry (Attia et al. 2022). The co-crystal structure was retrieved from the RCSB Protein Data Bank (PDB code 3UG2 for EGFR and 3CJG for VEGFR). The resulting docking poses were visually inspected, and the pose of the lowest binding free energy value was considered (Abdel-Rahman et al. 2022).

Results and Discussion

Epidermal growth factor receptor stimulates tumor growth and progression through several mechanisms. The expression of transcription, mutation, and/or gene amplification may be the cause of EGFR activation in tumor cells. The increased protein and transcribed levels of EGFR will correspond to poor prognosis in several cancers such as lung cancer and colorectal cancer (Allam et al. 2020). The VEGFR has multiple immediate effects on cancer cells and is known to have a major contribution to angiogenesis that aims to abolish the nutrient and oxygen supply to the tumor cells through the decrease of the vascular network and the avoidance of new blood vessel formation (Dziobek et al. 2019). LC-HR-MS analysis (Fig. 1S) for dereplication purposes was adopted for the identification of metabolites from ethanol extract of Dictyopteris acrostichoides (Table 1S). The dereplication study of the metabolites against the DNP (Dictionary of Natural Products) (Buckingham 2014) and Marin Lit databases (Munro and Blunt 2023) resulted in the identification of 17 compounds. Ethanol extract of D. acrostichoides as a source of bioactive natural products resulted in the detection of α-dictyopterol (1), β-dictyopterol (2) (Etsuro et al. 1966), germacra-1(10),4(15)-11-trien-5S-ol (3) (Toshi et al. 1964), 4α,5β-dihydroxycubenol (4), cadinan-1,4,5-triol (5) (Qiao et al. 2009), (1R,3R,4S,11R)-3,4;7,8-bisepoxydolabellan-12(18)-ene (6) (Wright et al. 1990), chromenol (7) (Wang et al. 2018), dictyochromenol (8) (Li et al. 2022), isochromazonarol (9), chromazonarol (10), 2-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl] benzene-1,4-diol (11), zonaric acid (12), (Ishibashi et al. 2013), isozonarol (13), zonarol (14) (Fenical et al. 1973), and dictyvaric acid (15) (Song et al. 2005), (3-oxo-undecyldisulfanyl)-undecan-3-one (16) (Schnitzler et al. 1998), and di(3-acetoxy-5-undecenyl) disulfide (17) (Moore et al. 1972) from LC-HR-ESI–MS metabolic profiling of the ethanol extract of D. acrostichoides.

figure b

(1R,3R,4S,11R)-3,4;7,8-Bisepoxydolabellan-12(18)-ene (6) is a dolabellane diterpene, typical product of algae of the genus Dictyota (Rushdi et al. 2022b); this mixing (Dictyota and Dictyopteris) must have occurred during the collection process. The cytotoxic potential obtained results revealed that the ethanol extract of D. acrostichoides had promising cytotoxicity activities against HepG2, MCF-7, and Caco-2 cell lines with IC50 values of 11.65, 9.28, and 16.86 µg/ml, respectively, compared to doxorubicin (IC50 5.72, 5.17, and 5.81 µg/ml, respectively); thus, it was further subjected to other assays while other extracts of C. racemosa, H. opuntia, and P. myrica did not show cytotoxic activities against tested cell lines. Further screen of ethanol extract of D. acrostichoides displayed in vitro activity against EGFR (IC50 0.11 µg/ml) compared to lapatinib as a positive control (IC50 0.03 ± 0.002 µg/ml) and against VEGF (IC50 0.276 µg/ml) compared to sorafenib as a positive control (IC50 0.049 ± 0.003 µg/ml). Most compounds showed hydrogen bonding with the gatekeeper mutant Met790. Interestingly, compounds 17 and 16 showed higher docking scores than Gefitinib (Table 2S, Fig. 2S–4S). Compounds (2, 5, 8–15) were of good binding energies ranging from − 6.9734 to − 6.1317 kcal/mol while (1, 3–4, 6–7) were of fair binding energies less than 6 kcal/mol. The docking on VEGFR revealed that most compounds formed hydrogen bonding with Asp 1044 like that of the ligand KIM. Compound 17 has a binding score (− 8.2601 kcal/mol more than ligand KIM (− 7.8176 kcal/mol) while compounds 16 and 12 showed − 7.4368 and − 7.0233 kcal/mol, respectively (Table 3S, Fig. 4S–6S). Compounds 8–13 and 15 showed good binding energies ranging from − 6.8160 to − 6.3920 kcal/mol. Most compounds formed hydrogen bonding with Asp 1044 like that of the ligand KIM.

Conclusions

Dictyopteris acrostichoides ethanol extract displayed cytotoxicity against HepG2, MCF-7, and CACO-2 (IC50 11.65, 9.28, and 16.86 µg/ml, respectively) compared to doxorubicin as a positive control (IC50 5.72, 5.17, and 5.81 µg/ml, respectively). Furthermore, the ethanol extract of D. acrostichoides was tested against EGFR (IC50 0.11 µg/ml) compared to lapatinib as a positive control (IC50 0.03 µg/m) and against VEGF (IC50 0.276 µg/ml) compared to sorafenib as a positive control (IC50 0.049 µg/ml). Seventeen metabolites were identified from the ethanol extract of D. acrostichoides but were detected using LC-HR-ESI–MS for dereplication purposes. 1-(3-Oxo-undecyldisulfanyl)-undecan-3-one (16) confirmed considerable interaction energies and formed substantial interactions EGFR binding site in the cancer cell receptors. Interestingly, di(3-acetoxy-5-undecenyl) disulfide (17) confirmed considerable interaction energies and formed substantial interactions VEGFR binding site.