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Introduction

Cancer encompasses a range of conditions marked by an unregulated cell proliferation that infiltrates into nearby tissues, and potentially metastasizes to distant areas of the body (1). Despite progress in cancer treatment, it remains a worldwide major health issue. In 2023, the United States reported 1,958,310 new cases of cancer and 609,820 mortalities (2). Jordan also faces a similar challenge, and reported 11,559 new cases and 6,190 mortalities in 2023. Furthermore, patients are mainly diagnosed with breast (20%), colorectal (11.6%) and lung cancer (7.4%) (2).

Cancer treatment predominantly relies on conventional chemotherapy, which uses a combination of drugs (such as 5-fluorouracil, leucovorin, oxaliplatin and irinotecan) alongside surgical procedures and radiation therapy (3). The development of resistance towards these conventional chemotherapy agents reduces their effectiveness, resulting in only minimal and temporary advantages (4). However, the process of developing novel drugs has considerable challenges, including high financial burdens, extensive clinical trials and rigorous regulatory demands (5).

Nanomedicine is an emerging discipline and focuses on using nano-sized materials as biomedical tools for both diagnosis and treatment purposes, especially within oncology (6). This approach is gaining interest as a preferred method over chemotherapy drugs due to its enhanced safety and efficacy (7). It facilitates the amalgamation of drugs with synergistic effects, potentially enhancing treatment efficacy, reducing adverse effects and mitigating the emergence of drug resistance (8,9). These multifaceted benefits render the use of nano-scaled carriers and drugs a progressively favored option over conventional therapies, particularly in addressing diseases such as cancer (10-17).

Silver nanoparticles (AgNPs) have gained attention within pharmaceutical and medical settings due to having antimicrobial properties and inducing cytotoxicity in diverse cancer cell lines such as colorectal and breast cancer cell lines (18,19). AgNPs exhibit multifaceted applications in the research and therapy of cancer. They are utilized primarily as effective drug delivery systems, encapsulating anticancer agents (such as methotrexate, imatinib, doxorubicin and gemcitabine) to enhance their targeting and efficacy against tumors while minimizing systematic side effects (20). AgNPs themselves possess intrinsic anticancer properties through mechanisms such as the induction of apoptosis and disruption of cellular signaling pathways in cancer cells (21). Furthermore, AgNPs are used in diagnostics by functionalizing their surfaces with specific biomolecules (such as aptamer or specific antibodies) that bind to cancer biomarkers [such as platelet-derived growth factors, human epidermal growth factor receptor (EGFR) 2 and prostate-specific antigen], resulting in sensitive detection methods (22). Additionally, AgNPs are radiosensitizers and increase the effectiveness of radiotherapy by enhancing the DNA damage in cancer cells (23). These diverse applications highlight the potential for AgNPs to advance cancer treatment modalities through targeted drug delivery, diagnostic and therapeutic interventions.

Bioactive components sourced from a variety of organisms, including plants and animals, offer anticancer and various other biological effects, presenting opportunities for the development of pharmaceutical products (24-27). Phytoestrogens and Rhizoma polygonati exhibit anticancer and antiaging properties (28,29). Tribulus terrestris indicates a potential for neuroprotection, and may be a treatment option for cognitive disorders such as Parkinson's and Alzheimer's disease (30), while camel milk whey protein hydrolysates provide anti-inflammatory and anticancer benefits (31). Furthermore, actinobacterial agents (such as indole-3-acetic acid, polyamines and 1-aminocyclopropane-1-carboxylic acid deaminase) from Salicornia bigelovii indicate a potential for enhancing agricultural productivity, such as enhancing the root biomass, increasing the yield of the seeds and increasing the tolerance of plants to saline soil (32). Additionally, sea sponges in particular have shown antioxidant and anti-inflammatory properties, which reduce the risk of cancer (33).

Stylissa carteri and Amphimedon chloros, marine sponges that inhabit the exclusive economic zone of Indonesia, have gained attention for their therapeutic properties, particularly their potential in the treatment of cancer (34,35). The present study aimed to investigate the cytotoxic effects of AgNPs with extracts of Stylissa carteri or Amphimedon chloros, collected from the Red Sea coastline, against various types of cancer and bacterial strains. To the best of our knowledge, the present study was the first to conduct an evaluation of the cytotoxicity of Stylissa carteri and Amphimedon chloros.

Materials and methods

Materials

L-glutamine-containing Dulbecco's Modified Eagle Medium (DMEM), penicillin/streptomycin and fetal bovine serum (FBS) were purchased from EuroClone SpA. Both phosphate buffer saline and trypsin were purchased from Thermo Fisher Scientific, Inc. Ethyl acetate was purchased from Sigma-Aldrich; Merck KGaA. The MTT reagent the stop solution [dimethyl sulfoxide (DMSO)] and trypan blue dye were provided by Promega Corporation. Flow tubes (BD Biosciences) were used for centrifugation. BD FACSDiva™ software (version 8.0; BD Biosciences) and StemPro™ Accutase™ Cell Dissociation Reagent (Gibco; Thermo Fisher Scientific, Inc.) were used in the present study.

Biosynthesis of AgNPs using Aspergillus flavus

In the present study, AgNPs were synthesized using Aspergillus flavus strain MG973280, which was obtain from American Type Culture Collection (ATCC). The fungal culture was prepared as previously described (36). Briefly, Aspergillus flavus spores were adjusted to a concentration of 2.0x106 and cultivated in a complex broth medium that included 10 g/l glucose, 10 g/l yeast extract and 5 g/l NaCl (pH 7). Subsequently, the culture was incubated for 72 h at 33˚C and cells were agitated using an orbital shaker at 0.10 x g. Whatman® grade 1 filter paper was used to filter the culture at the end of the incubation period. The resultant mycelia, or biomass, were then collected and washed with deionized distilled water.

To produce AgNPs using the fungal biomass, the method reported in the study by Jadidev and Narasimha (37) was followed with minor adjustments. Briefly, 10 g of the first crude biomass were agitated using an orbital shaker at 0.10 x g for 72 h while submerged in 100 ml of sterile deionized water at 33˚C and pH 7.0. After the crude biomass was filtered using Whatman® grade 1 filter paper, the resulting suspension containing the fungal filtrate without biomass was collected. Subsequently, 100 ml of this biomass-free fungal filtrate was combined with 1 mM of silver nitrate to produce AgNPs. The mixtures were then continuously stirred in the dark at 27˚C for 72 h using a magnetic stirrer.

Morphology and particle size analysis

NPs were lyophilized at -54˚C and 0.2 mbar for 24 h using a Lyovapor L-200 freeze dryer (BUCHI UK Ltd.). Scanning electron microscope (SEM) images were obtained at 200 kV using a JEM-2010 microscope (JEOL, Ltd.) in order to analyze the surface morphology of the AgNPs. The powdered samples were coated with gold using sputter coater and a carbon thread coater (Leica Biosystems). The resulting particle size was measured using the Zetasizer Nano ZSP (Malvern Panalytical, Ltd.).

Sponge collection and identification

Sponge samples were gathered from several locations in the Gulf of Aqaba (29˚27' N, 34˚58' E) by specialists from the Marine Science Station at the University of Jordan (Aqaba, Jordan). The sponges were found by divers between the depths of 1-18 meters. After being cleared of debris, the samples were frozen at -80˚C, and then sent in sealed sterile polyethylene containers (which were submerged in seawater in order to maintain a moist environment during transport) to Aqaba International Laboratories-BEN HAYYAN for freeze-drying and extraction. Using specified morphological traits listed in the Systema Porifera and World Porifera Database (35,38-42), the sponges, Stylissa carteri and Amphimedon chloros, were identified.

Extraction of sponge extracts

Sponge fragments were weighed, freeze-dried for 48 h at -40˚C and then ground into a powder. The powder (weighing 45.0-350.0 g) was soaked at room temperature for 48 h in a 1:1 mixture of methanol and dichloromethane. Subsequently, a lyophilizer was used to filter and dehydrate the solution. The final pure extract was divided into non-polar, semi-polar and polar components by dispersing it in distilled water and using n-hexane and ethyl acetate as partitioning agents. The pure extract from each solvent was labeled and kept frozen at -20˚C (43,44).

Cancer cell lines culture

In the present study, to investigate the anticancer activity of the candidate mixtures, four cancer cell lines were used: i) A human lung cancer cell line (A549; cat. no. CCL-185; ATCC); ii) a human colorectal cancer cell line (HT-29; cat. no. HTB-38; ATCC); iii) a human breast cancer cell line (MCF7; cat. no. HTB-22; ATCC); and iv) a pancreatic cancer cell line (PANC-1; cat. no. CRL-1469; ATCC). For selective purposes, a normal human umbilical vein endothelial cell line (HUVEC; cat. no. CRL-1730; ATCC) was used. All of the cell lines, were cultured in DMEM containing 10% FBS, 10 mM HEPES buffer, 100 µg/ml L-glutamine, 50 µg/ml gentamicin, 100 µg/ml penicillin and 100 mg/ml streptomycin. ATCC provided authenticated cell lines, which ensured that the cell lines were correctly identified and contamination-free. Additionally, mycoplasma testing was performed on all of the cell lines used in the present study, which confirmed the absence of mycoplasma contamination.

Colorimetric MTT assay

A colorimetric MTT assay was used to evaluate the viability of the cells. Each type of cell line was seeded in 96-well plates at a density of 1x104 cells per well and cultured for 24 h at 37˚C (45). Subsequently, the cells were treated with: i) The extracts of each of the sponge species, Stylissa carteri and Amphimedon chloros, separately at concentrations ranging from 6-200 µg/ml; ii) AgNPs at concentrations ranging from 0.75-200 µg/ml; or iii) mixtures of 0.75 µg/ml AgNPs with extracts of Stylissa carteri or Amphimedon chloros at concentrations ranging from 6-200 µg/ml. Following a 72-h incubation period at 37˚C (46), 15 µl of MTT solution was added to each well, and then the plates were incubated at 37˚C for 4 h. Subsequently, 100 µl of DMSO was added to each well to dissolve the formed formazan crystal. The BioTek ELx800™ microplate reader was then used to measure the optical density at 590/630 nm to assess cell growth.

Screening and identifying compounds from the sponge extracts using liquid chromatography-mass spectrometry (LC-MS)

LC-MS is an analytical method that combines MS and LC. LC separates components of a mixture by passing them through a chromatographic column. Even when LC is unable to positively identify these separated components, MS can be used to identify both known and unknown chemicals and provide information on their structures (47).

A mobile phase comprising solvents A and B in a gradient was used in the LC-MS analysis. Solvent A consisted of formic acid dissolved in water at a concentration of 0.1% (v/v), and solvent B consisted of formic acid dissolved in acetonitrile at the same concentration. The experimental parameters used included an Agilent Zorbax Eclipse XDB-C18 column (Agilent Technologies, Inc.) measuring 2.1x150 mm x3.5 µm, a temperature maintained at 25˚C, the sponge extract dissolved in methanol at a concentration of 18 mg/ml and an injection volume of 1 µl. The sample was injectedinto the LC-MS system, which included the following components: A mass detector using a SIL-30AC autosampler with a cooler, a Shimadzu CBM-20A system controller, an LC-30AD pump, a CTO-30 column oven and an electrospray ion-mass spectrometer with a skimmer voltage of 65 V and a fragmentor voltage of 125 V, all components were of the Shimadzu LC-MS 8030 (Shimadzu Corporation). Nitrogen gas with 99.99% purity and a flow rate of 10 l/min served as the drying gas during operation in positive ion mode. Additionally, a nebulizer operating at a pressure of 45 psi and a capillary temperature of 350˚C were used. Subsequently, the mass/number of ions of the eluent was scanned from positions 100-1,000. Authentic standard substances were used for result validation.

Molecular docking

Molecular docking simulations were performed using tyrosine kinase receptor A [TrkA; Protein Data Bank (PDB) ID, 7VKO; https://www.rcsb.org/structure/7VKO] (48) and an EGFR kinase domain (PDB ID, 4I23; https://www.rcsb.org/structure/4i23) (49) in order to investigate the binding affinities of the major components in Stylissa carteri or Amphimedon chloros. AutoDock (version 4.2.6) (50,51) was used according the methods described in the studies by Saqallah et al (52) and Shtaiwi et al (53) with slight modifications. Briefly, all protein structures were prepared using BIOVIA® Discovery Studio® (version 16.1) (54) by removing water molecules (if applicable) and complexed co-structures. Complexed inhibitors (dacomitinib and repotrectinib in the EGFR kinase domain and TrkA structures, respectively) were separated from the crystal structures to be used as control ligands. Using AutoDockTools (version 1.5.6) (50), Kollman charges and polar hydrogen atoms were assigned to the proteins. Additionally, the 3D conformers of the compounds identified in the sponge extracts were downloaded from the NCBI PubChem database (pubchem.ncbi.nlm.nih.gov) and Gasteiger charges were assigned accordingly. A grid box with the size of 153 Å was set with the coordinates of -0.697, -52.750, -23.233 as x, y, z, respectively, for the EGFR kinase domain protein, and with the same size at -18.081, -43.125, -13.177 as x, y, z, respectively, for the TrkA protein. Simulations were carried out using 100 Lamarckian Genetic Algorithm runs with default parameters. Conformations with the lowest free energy of binding and the most populated cluster were selected for further analysis. Analyses of the interactions were carried out using BIOVIA® Discovery Studio® (version 16.1).

Isolation of the bacterial strains utilized in antibacterial studies

To study the antibacterial activity of the sponge-AgNPs mixtures, bacterial cells were obtained from hydatid cyst fluid sourced from various affected anatomical sites, such as the liver and lung, and five different isolated bacterial species were obtained in a previous study by Al Qaisi et al (55). The bacterial species used in the present study were Staphylococcus xylosus, Klebsiella oxytoca, Enterobacter aerogenes, Micrococcus spp. and Escherichia coli. Pseudomonas aeruginosa (cat. no. 27853; ATCC) was used as a control. The isolates were preserved on nutrient agar slants at 4˚C for up to 1 month.

Antibacterial agar disc diffusion assay

Isolated bacteria were first cultured in Muller Hinton Broth at 37˚C for 24 h for activation before carrying out the antibacterial tests. The antibacterial activity of the sponge extracts and AgNPs were measured using Muller Hinton agar and the disc diffusion method. Inhibitory zone diameters were calculated in mm (56). Briefly, 20 µg of sponge extract (either from Stylissa carteri or Amphimedon chloros) or AgNPs were used to impregnate sterile filter paper discs, which were then put on inoculated Petri dishes containing 0.1 ml of a bacterial solution containing 1.5x108 colony forming units/ml. Discs pre-dosed with 50 g/ml chloramphenicol and 50 g/ml ampicillin were used as positive controls, while discs containing 5% DMSO served as negative controls. After 24 h of incubation at 37˚C, the diameters of the inhibition zones around the extract-impregnated discs were measured and compared with those of the controls. To identify active and inactive sponge preparations, the inhibition zone diameter was used as a metric. Each sample was tested three times. To test for sponge extract-AgNP synergy, two different concentrations were used, which were 10 µg sponge extract and 10 µg AgNPs/disc, and 10 µg sponge extract and 5 µg AgNPs/disc (57).

Statistical analysis

Results from three or four independent experiments are presented as the means ± standard deviation. Statistical variances between the control group and various treatment groups were evaluated using GraphPad Prism version 10 (Dotmatics). Results were analyzed using one-way ANOVA followed by the Dunnett's test, or using an unpaired t-test. P<0.05 was considered to indicate a statistically significant difference.

Results

Lyophilization alters the size, charge and stability of AgNPs

A significant difference in charge, but not in size and polydispersity index (PDI) between the pre-and post-lyophilization states are presented in Fig. 1. Before lyophilization, the mean charge was-25.30±0.28 mV, the mean size was 108.65±3.45 nm and the PDI was 0.27±0.07. After the process of lyophilization, the mean size increased to 171.60±13.01 nm, the mean charge increased to -15.05±0.01 mV and the PDI increased to 0.34±0.01. Although all measurements for nanoparticle size (ideal range, 50-200 nm), charge (ideal range, -10 to -30 mV) and PDI (ideal range, ≤0.5) remained within the ideal ranges, these differences indicated that the lyophilization process did not significantly affect the properties of the AgNPs.

Figure 1 Size, PDI and surface charge of AgNPs before and after lyophilization. While there was not a significant difference in the size and PDI of the AgNPs before and after lypophilization, a significant difference in the surface charge was revealed after lyophilization. The results are presented as means ± SD (n=3). *P<0.05. AgNPs, silver nanoparticles; PDI, polydispersity index; ns, not significant.

SEM analysis reveals uniform distribution and nanoscale size of the AgNPs with minimal aggregation. To investigate the produced AgNPs, SEM was used (Fig. 2). The AgNPs had a uniform distribution and an approximately spherical shape, with negligible aggregation, which indicated a stable production process. Based on the 200 nm scale bar, the majority of the particles had a diameter of <100 nm, which indicated that the size distribution was in the nanoscale zone. Particle aggregation occurs frequently in nanoparticle samples (58); however, in the present study, only minimal aggregation was observed, which indicated high dispersion stability. The AgNPs appeared to have a smooth surface, which is consistent with the nature of nanoparticles made using chemical reduction techniques (59,60). These features implied that these AgNPs were suitable for various applications including anticancer and antimicrobial activity.

Figure 2 SEM micrograph of AgNPs. A high-resolution SEM image of the morphology and distribution of AgNPs, which were synthesized using 1.0 mM silver nitrate and the fungus Aspergillus flavus. SEM, scanning electron microscopy; AgNPs, silver nanoparticles; HV, high voltage (accelerating voltage in kV applied to the microscope); spot, spot size (beam diameter for optimal resolution); pA, picoamperes; curr, current (electron beam current in pA); det, detector (type of detector used to capture the image); STEM II, scanning transmission electron microscopy secondary image; BF, bright field (imaging mode providing high-contrast details in transmission images); WD, working distance (distance between sample surface and objective lens); mag, magnification; HFW, horizontal field width; CTC JU, Cells Therapy Center, Jordan University.

AgNPs with sponge extracts reveal selective cytotoxicity when using the HUVEC normal cell line

To investigate the selective cytotoxicity of AgNPs on the HUVEC normal cell line, an MTT assay was carried out. Cytotoxicity was observed across concentrations ranging from 0.75-200 µg/ml of AgNPs (Fig. 3A). There was a significant difference in the cell cytotoxicity between the 0.75 and 3 µg/ml AgNPs (Fig. 3A), which indicated that 3 µg/ml AgNPs had a lower cytotoxicity compared with 0.75 µg/ml AgNPs in the HUVEC normal cell line. However, 0.75 µg/ml AgNPs were used in the subsequent experiments based on several factors, such as to prevent drug resistance. An advantage of combination therapy is the prevention of drug resistance, which often occurs when high doses of a single agent are used repeatedly (61); therefore, using a lower concentration (0.75 µg/ml) of AgNPs avoided a high-dose exposure, which could otherwise lead to resistance. Additionally, lower doses of nanoparticles may be preferable for long-term use in order to minimize potential side effects while still retaining efficacy (62). Therefore, the subsequent combination experiments used Stylissa carteri or Amphimedon chloros extracts with 0.75 µg/ml AgNPs to minimize the cytotoxic effect of the AgNPs on the HUVEC normal cell line while maximizing the cytotoxic potential against various cancer cell lines. Fig. 3B presents the cytotoxic effects of Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs. Fig. 3C presents the cytotoxicity of Amphimedon chloros extract alone and in combination with 0.75 µg/ml AgNPs. The results indicated a significant reduction in HUVEC cytotoxicity when the Amphimedon chloros extract was combined with AgNPs compared with the Amphimedon chloros extract alone.

Figure 3 Cytotoxicity of HUVEC cells to AgNPs, Stylissa carteri and Amphimedon chloros. (A) Cytotoxicity of different concentrations of AgNPs on the HUVEC cell line (analyzed using one-way ANOVA followed by the Dunnett's test). Cytotoxicity of (B) the Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs and (C) the Amphimedon chloros extract alone and in combination with 0.75 µg/ml AgNPs on the endothelial HUVEC cell line (analyzed using unpaired t-tests). The results are presented as means ± SD (n=3). *P<0.05, ***P<0.001 and ****P<0.0001. AgNPs, silver nanoparticles; HUVEC, human umbilical vein endothelial cell line.

Enhancement of the cytotoxicity of AgNPs with sponge extracts when using the A549 cell line

Fig. 4A demonstrates the cytotoxic impact of the AgNPs on the A549 cell line. The results revealed a significant increase in the cytotoxicity within the concentration range of 1.5-200 µg/ml AgNPs compared with the concentration of 0.75 µg/ml AgNPs. Fig. 4B presents the cytotoxicity of the Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs. Fig. 4C presents the cytotoxic effects of the Amphimedon chloros extract alone and in combination with 0.75 µg/ml AgNPs. The results revealed a significant augmentation in the A549 cell cytotoxicity when the Stylissa carteri extract was co-administered with 0.75 µg/ml AgNPs compared with the cytotoxic effects of the Stylissa carteri extract alone.

Figure 4 Cytotoxicity of A549 cells to AgNPs, Stylissa carteri and Amphimedon chloros. (A) Cytotoxicity of different concentrations of AgNPs on the lung cancer A549 cell line (analyzed using one-way ANOVA followed by the Dunnett's test). Cytotoxicity of (B) the Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs and (C) the Amphimedon chloros extract alone and in combination with 0.75 µg/ml AgNPs on the lung cancer A549 cell line (analyzed using unpaired t-tests). The results are presented as means ± SD (n=3). *P<0.05 and ****P<0.0001. AgNPs, silver nanoparticles.

Neither of the sponge extracts significantly increase the cytotoxicity of the AgNPs when using the MCF7 cell line

Fig. 5A demonstrates the cytotoxic effects of the AgNPs on the MCF7 cell line. The results revealed a significant increase in the cytotoxicity within the concentration range of 1.5-200 µg/ml AgNPs compared with the concentration of 0.75 µg/ml AgNPs. Fig. 5B presents the cytotoxicity of the Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs, and Fig. 5C presents the cytotoxicity of the Amphimedon chloros extract alone and in combination with 0.75 µg/ml AgNPs, when using the MCF7 cell line. However, the cytotoxic effects of the Stylissa carteri or the Amphimedon chloros extract with 0.75 µg/ml AgNPs were not significantly different using the MCF7 cell line compared with the cytotoxic effects of the Stylissa carteri or the Amphimedon chloros extracts alone, respectively.

Figure 5 Cytotoxicity of MCF7 cells to AgNPs, Stylissa carteri and Amphimedon chloros. (A) Cytotoxicity of different concentrations of AgNPs on the breast cancer MCF7 cell line (analyzed using one-way ANOVA followed by the Dunnett's test). Cytotoxicity of (B) the Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs and (C) the Amphimedon chloros extract alone and in combination with 0.75 µg/ml AgNPs on the breast cancer MCF7 cell line (analyzed using unpaired t-tests). The results are presented as means ± SD (n=3). ****P<0.0001. AgNPs, silver nanoparticles.

Neither of the sponge extracts significantly increase the cytotoxicity of the AgNPs when using the PANC-1 cell line

Using the PANC-1 cell line, there was a significant increase in the cytotoxicity within the concentration range of 1.5-200 µg/ml AgNPs compared with the concentration of 0.75 µg/ml AgNPs (Fig. 6A). Fig. 6B and C presents the cytotoxic effects of the Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs, along with the cytotoxicity of the Amphimedon chloros extract alone and with 0.75 µg/ml AgNPs, when using the PANC-1 cell line. However, the cytotoxic effects of the Stylissa carteri or the Amphimedon chloros extract with 0.75 µg/ml AgNPs were not significantly different using the PANC-1 cell line compared with the cytotoxic effects of the Stylissa carteri or the Amphimedon chloros extracts alone, respectively.

Figure 6 Cytotoxicity of PANC-1 cells to AgNPs, Stylissa carteri and Amphimedon chloros. (A) Cytotoxicity of different concentrations of AgNPs on the pancreatic cancer PANC-1 cell line (analyzed using one-way ANOVA followed by the Dunnett's test). Cytotoxicity of (B) the Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs and (C) the Amphimedon chloros extract alone and in combination with 0.75 µg/ml AgNPs on the pancreatic cancer PANC-1 cell line (analyzed using unpaired t-tests). The results are presented as means ± SD (n=3). ****P<0.0001. AgNPs, silver nanoparticles.

Neither of the sponge extracts significantly increase the cytotoxicity of the AgNPs when using the HT-29 cell line

Fig. 7A presents the cytotoxic effects of the AgNPs on the HT-29 cell line across a concentration range of 0.75-200 µg/ml. The results revealed a significant increase in the cytotoxicity within the concentration range of 1.5-200 µg/ml AgNPs compared with the concentration of 0.75 µg/ml AgNPs. Furthermore, a notable increase in the cell cytotoxicity was observed at AgNP concentrations of 12-200 µg/ml. Additionally, Fig. 7B presents the cytotoxicity of the Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs, and Fig. 7C presents the cytotoxicity of the Amphimedon chloros extract alone and in combination with 0.75 µg/ml AgNPs, when using the HT-29 cell line. However, the cytotoxic effects of the Stylissa carteri or the Amphimedon chloros extract with 0.75 µg/ml AgNPs were not significantly different using the HT-29 cell line compared with the cytotoxic effects of the Stylissa carteri or the Amphimedon chloros extracts alone, respectively.

Figure 7 Cytooxicity of HT-29 cells to AgNPs, Stylissa carteri and Amphimedon chloros. (A) Cytotoxicity of different concentrations of AgNPs on the colorectal cancer HT-29 cell line (analyzed using one-way ANOVA followed by the Dunnett's test). Cytotoxicity of (B) the Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs and (C) the Amphimedon chloros extract alone and in combination with 0.75 µg/ml AgNPs on the colorectal cancer HT-29 cell line (analyzed using unpaired t-tests). The results are presented as means ± SD (n=3). **P<0.01 and ****P<0.0001. AgNPs, silver nanoparticles.

LC-MS reveals key bioactive compounds in the sponge extracts, indicating their potential mechanisms of action

LC-MS analysis revealed that there were different chemical components contained in the Stylissa carteri and Amphimedon chloros extracts (Tables I and II, respectively). Based on the LC-MS analysis, manzacidine A, debromohymenialdisine and hymenialdisine were identified as the major components in the Stylissa carteri extract (Fig. 8). Whereas in the Amphimedon chloros extract, methoxyhexadecanoic acid, keramaphidin B and hydroxytricosanoic acid were identified as the major components (Fig. 9). The chemical structures of the predicated components contained in the Stylissa carteri and the Amphimedon chloros extracts are presented in Fig. 10.

Figure 8 Liquid chromatography-mass spectrometry chromatogram of the components in the Stylissa carteri extract. Chromatogram of the Stylissa carteri extract with peaks that correspond to individual compounds, which were identified based on their mass-to-charge ratio. Each peak provided detail on the chemical composition of the extract and the relative abundance of its components. The chromatogram revealed the molecular profile of Stylissa carteri and highlighted key compounds such as (a) debromohymenialdisine, (b) hymenialdisine, (c) agelongine, (d) calthramide, (e) manzacidine A, (f) 3-bromohymenialdisine, (g) spongiacidine and (h) ageliferin. The chromatogram corresponds to ions with an m/z of 193.00, which have a relative intensity of 81.82% compared with the total ion intensity across all detected m/z values. The maximum intensity range or full-scale limit of the detector was 1,000,000,000. m/z, mass-to-charge ratio.

Figure 9 Liquid chromatography-mass spectrometry chromatogram of the components in the Amphimedon chloros extract. Chromatogram of the Amphimedon chloros extract with peaks that correspond to individual compounds, which were identified based on their mass-to-charge ratio. Each peak provided detail on the chemical composition of the extract and the relative abundance of its components. The chromatogram revealed the molecular profile of Amphimedon chloros and highlighted key compounds such as (a) purine, (b) methoxyhexadecanoic acid, (c) hachijodine, (d) tricosenal, (e) kermaphidine B, (f) tricosenoic acid, (g) pentacosenal, (h) zamamidine, (i) karamamine, (j) pentacosenic acid, (k) ircinol, (l) pyrinodemin, (m) nakinadine and (n) hydroxytricosanoic acid. The chromatogram corresponds to ions with an m/z of 193.00, which have a relative intensity of 81.82% compared with the total ion intensity across all detected m/z values. The maximum intensity range or full-scale limit of the detector was 1,000,000,000. m/z, mass-to-charge ratio.

Figure 10 Chemical structures of the main components in the Stylissa carteri and Amphimedon chloros extracts. (A) Manzacidine A, (B) debromohymenialdisine and (C) hymenialdisine were the main components in the Stylissa carteri extract. (D) Hydroxytricosanoic acid, (E) kermaphidine B and (F) methoxyhexadecanoic acid were the main components in the Amphimedon chloros extract.

Table I Analysis of the Stylissa carteri extract components using liquid chromatography-mass spectrometry.

Table I

Analysis of the Stylissa carteri extract components using liquid chromatography-mass spectrometry.

Compound name	Molecular formula	M/Z	Composition (%)
Calthramide	C2H15BrN4O3	343.10	11.0
Agelongine	C13H11BrN2O4	339.10	9.6
Manzacidine A	C12H14BrN3O4	344.16	13.0
Debromohymenialdisine	C11H11N5O2	245.20	12.5
Spongiacidine	C11H11Br2N5O2	405.05	10.5
Hymenialdisine	C11H10BrN5O2	324.13	17.8
3-Bromohymenialdisine	C11H9Br2N5O2	403.03	9.1
Ageliferin	C22H24Br2N10O2	620.30	9.5

[i] M/Z, mass-to-charge ratio.

Table II Analysis of the Amphimedon chloros extract components using liquid chromatography-mass spectrometry.

Table II

Analysis of the Amphimedon chloros extract components using liquid chromatography-mass spectrometry.

Compound name	Molecular formula	M/Z	Composition (%)
Tricosenal	C23H44O	336.6	6.4
Tricosenoic acid	C23H44O2	352.6	4.1
Pentacosenal	C25H48O	364.6	3.5
Pentacosenic acid	C25H48O3	396.6	3.2
Methoxyhexadecanoic acid	C18H36O3	300.5	9.1
Hydroxytricosanoic acid	C23H46O3	370.6	11.4
Zamamidine	C49H60N6O	749.0	5.1
Keramamine	C23H33N3	380.0	8.7
Ircinol	C26H40N2O2	412.6	6.4
Keramaphidin B	C26H40N2	351.5	10.2
Purine	C5H4N4	120.1	7.1
Pyrinodemin	C37H57N3O	559.9	8.9
Nakinadine	C27H40N2O2	424.6	8.1
Hachijodine	C19H34N2O	306.5	3.4

[i] M/Z, mass-to-charge ratio.

Molecular docking reveals strong binding affinities of marine compounds with EGFR and TrkA kinases, highlighting the inhibitory potential of keramaphidin B

The present study investigated the docking interactions of six marine compounds (debromohymenialdisine, hydroxytricosanoic acid, hymenialdisine, keramaphidin B, manzacidine A and methoxyhexadecanoic acid) with the kinase domains of EGFR and TrkA, which have been previously revealed to be overexpressed in different cancer cells such as A549, HT-29, MCF7 and PANC-1 (63-68). The binding energies, specific amino acid interactions, presence of ionic bonds, hydrogen bonds and halogens were analyzed (Table III; Figs. 11 and 12).

Figure 11 Binding interactions of six marine-derived compounds with the epidermal growth factor receptor. Ball and stick representation of (A) debromohymenialdisine, (B) hydroxytricosanoic acid, (C) hymenialdisine, (D) keramaphidin B, (E) manzacidine A and (F) methoxyhexadecanoic acid docked against the epidermal growth factor receptor kinase domain (Protein Data Bank ID, 4I23; https://www.rcsb.org/structure/4i23). The figure was created using Biovia Discovery Studio Visualiser 2016® (Dassault Systèmes). H-bond, hydrogen bond.

Figure 12 Binding interactions of six marine-derived compounds with the tyrosine kinase receptor A. Ball and stick representation of (A) debromohymenialdisine, (B) hydroxytricosanoic acid, (C) hymenialdisine, (D) keramaphidin B, (E) manzacidine A and (F) methoxyhexadecanoic acid docked against tyrosine kinase receptor A (Protein Data Bank ID, 7VKO; https://www.rcsb.org/structure/7VKO). The figure was created using Biovia Discovery Studio Visualiser 2016® (Dassault Systèmes). H-bond, hydrogen bond.

Table III Binding interactions and affinity analysis of the selected marine components against the EGFR kinase domain and TrkA after molecular docking.

Table III

Binding interactions and affinity analysis of the selected marine components against the EGFR kinase domain and TrkA after molecular docking.

A, EGFR kinase domain
		Interacting amino acid per interaction type
Compound	LEB (kcal/mol)	Ionic	H-bond	Halogen	Other binding residues
Debromohymenialdisine	-6.68	None	Gln791, Met793, Arg841, Asn842 and Asp855	None	Leu 718, Ala 743, Lys 745, Thr 790, Leu792, Gly 796, Cys 797 and Thr 854
Hydroxytricosanoic acid	-3.77	None	Glu762 and Thr854	None	Leu718, Val726, Ala743, Lys 745, Met 766, Leu 788, Thr 790, Gln 791, Leu 792, Met 793,Pro 794, Gly 796, Cys 797,Arg 841,Asn 842, Leu 844, Asp 855 and Phe 856
Hymenialdisine	-6.65	None	Met 793, Arg 841, Asn 842 and Asp 855	None	Leu 718, Val 726, Ala 743, Lys 745, Thr 790, Gln791, Leu 792, Gly 796, Cys797, Leu 844 and Thr 854
Keramaphidin B	-7.15	None	None	None	Leu 718, Gly719, Ser 720, Val 726, Ala 743, Lys 745, Thr 790, Leu 792, Met 793, Gly 796, Cys 797, Asp 800, Arg 841, Leu 844, Thr 854 and Asp 855
Manzacidine A	-6.16	Asp 855	Glu 762 and Met 793	None	Leu 718, Val 726, Ala 743, Thr 754, Met 766, Cys 775, Leu 788, Thr 790, Leu 792, Pro 794, Gly 796 and Leu 844
Methoxyhexadecanoic acid	-4.62	None	Met 793	None	Leu 718, Val 726, Ala 743, Ile 744, Lys 745, Glu762, Met 766, Leu788, Thr790, Gln 791, Leu792, Gly796, Leu 844, Thr 854 and Asp 855
Dacomitinib (control)	-7.48	None	Thr 790 and Met 793	Glu 762	Leu718, Val726, Ala743, Lys745, Met766, Leu788, Gln791, Leu792, Pro794, Gly 796,Cys 797, Leu 844), Thr 854 and Asp 855
Repotrectinib (control)	-	-	-	-	-
B, TrkA
		Interacting amino acid per interaction type
Compound	LEB (kcal/mol)	Ionic	H-bond	Halogen	Other binding residues
Debromohymenialdisine	-6.71	None	Glu 590, Met 592 and Asp 668	None	Leu 516, Val 524, Ala542, Lys 544, Val 573, Phe 589, Tyr 591, Gly 595, Leu 657, Gly 667 and Arg 673
Hydroxytricosanoic acid	-4.60	None	Asp 596 and Arg 673	None	Leu 516, Gly 517, Val 524, Ala 542, Lys 544, Val 573, Phe 589, Gly 595, Arg 599, Arg 654, Asn 655, Cys 656, Leu 657, Gly 667 and Asp 668
Hymenialdisine	-6.95	None	Glu 590, Met 592 and Asp 668	None	Leu 516, Val 524, Ala 542, Lys 544, Val 573, Phe 589, Tyr 591, Gly 595, Asp 596, Leu 657, Gly 667 and Arg 673
Keramaphidin B	-7.08	None	None	None	Leu 516, Gly 517, Glu 518, Val 524, Lys 544, Asp 558, Gly 595, Asp 596, Arg 654, Asn 655, Cys 656, Leu 657, Gly 667 and Arg 673
Manzacidine A	-6.32	None	Glu 590, Met 592 and Arg 673	None	Leu 516, Gly 517, Val 524, Ala 542, Val 573, Phe 589, Tyr 591, Gly 595, Asp 596, Leu 657 and Gly 667
Methoxyhexadecanoic acid	-4.37	None	Asp 596 and Arg 673	None	Leu 516, Gly 517, Val 524, Ala 542, Lys 544, Val 573, Phe 589, Glu 590, Tyr 591, Met 592, Gly 595 and Leu 657
Dacomitinib (control)	-	-	-	-	-
Repotrectinib (control)	-9.23	None	Met 592 and Gly 667	Asn 655 and Cys 656	Leu 516, Gly 517, Val 524, Ala 542, Lys 544, Val 573, Phe 589, Glu 590, Tyr 591, Gly 595, Asp 596, Arg654, Leu 657, Asp 668 and Arg 673

[i] EGFR, epidermal growth factor receptor; TrkA, tyrosine kinase receptor A; LEB, lowest free energy of binding; H-bond, hydrogen bond.

All compounds except keramaphidin B formed hydrogen bonds with key amino acid residues such as Leu718, Lys745 or Met793 within EGFR. The results indicated that keramaphidin B had the most favorable binding energy (-7.15 kcal/mol), followed by debromohymenialdisine (-6.68 kcal/mol) and hymenialdisine (-6.65 kcal/mol). Dacomitinib, the control compound, indicated a binding energy of -7.48 kcal/mol and formed hydrogen bonds with Thr790 and Met793. However, it lacked several interactions observed in the marine compounds, such as interactions with Leu718 and Lys745.

Furthermore, keramaphidin B also demonstrated the strongest binding affinity (-7.08 kcal/mol), followed by hymenialdisine (-6.95 kcal/mol) and debromohymenialdisine (-6.71 kcal/mol), against TrkA. Similar to EGFR, all compounds except keramaphidin B formed hydrogen bonds with residues such as Leu516 and Val524.

The findings suggested that several marine compounds, particularly hymenialdisine and debromohymenialdisine, may potentially interact with and inhibit both EGFR and TrkA. These results indicated that these compounds interact with specific residues (Leu718 and Lys745 in EGFR; Leu516 and Val524 in TrkA), which was not observed with the control compounds. Therefore, this may indicate a unique binding mode.

Co-administering sponge extracts with AgNPs enhances the antibacterial activity

Sponge ethyl acetate extracts combined with AgNPs were tested against six bacterial species using the disc diffusion method (Table IV). In addition, 20 µg/disc AgNPs or sponge ethyl acetate extract alone was administered. The AgNPs and sponge ethyl acetate extract were combined in proportions of 5 or 10 µg/disc of AgNPs with 10 µg/disc of ethyl acetate extract for synergistic tests. Using 20 µg/disc AgNPs alone resulted in ~10 mm-diameter inhibition zones against the majority of bacteria, except for Enterobacter aerogenes, which had an increased susceptibility and an inhibition zone of 28.33 mm. All bacterial species, with inhibition zone diameters ranging from 21.43-13.58 mm, were suppressed by the 20 µg/disc Stylissa carteri ethyl acetate extract. However, the 20 µg/disc Amphimedon chloros extract had no effect on Enterobacter aerogenes or Klebsiella oxytoca, and the remaining bacterial species had inhibition zones that ranged in diameter from 7.34-15.15 mm. When using the extracts alone, the Stylissa carteri extract had an increased inhibitory effect against the six investigated bacterial species compared with the Amphimedon chloros extract. Additionally, when combining 10 µg/disc sponge extract with 5 or 10 µg/disc AgNPs, the majority of the bacterial species investigated had increased inhibition zones and increased fold area (IFA) values when using the Stylissa carteri extract compared with the Amphimedon chloros extract, except for E. coli in which the opposite results were demonstrated. The results indicated that the bacterial species exhibited different responses, which were caused either by the combined treatment of AgNPs with the sponge extract or by the effect of each treatment alone. These results suggest the possibility of using AgNPs in combination with a sponge ethyl acetate extract to treat bacterial infections in hydatid cysts.

Table IV Antibacterial and synergistic effects of Stylissa carteri or Amphimedon chloros ethyl acetate extracts and AgNPs against bacteria isolated from echinococcal hydatid cyst fluid.

Table IV

Antibacterial and synergistic effects of Stylissa carteri or Amphimedon chloros ethyl acetate extracts and AgNPs against bacteria isolated from echinococcal hydatid cyst fluid.

A, Stylissa carteri
	Inhibition zones (mm)
Bacteria	AgNPs (20 µg/disc)	Sponge (20 µg/disc)	AgNPs (5 µg/disc) plus sponge (10 µg/disc)	AgNPs (10 µg/disc) plus sponge (10 µg/disc)	IFAa
Staphylococcus xylosus	10.40±0.15	21.43±0.17	16.00±0.12	20.00±0.18	2.70±0.13
Klebsiella oxytoca	10.65±0.14	15.92±0.16	12.77±0.13	16.54±0.19	1.40±0.08
Enterobacter aerogenes	28.33±0.18	13.58±0.15	17.14±0.14	30.79±0.16	0.18±0.02
Micrococcus spp.	9.75±0.13	21.00±0.18	13.60±0.12	20.52±0.17	3.43±0.14
Pseudomonas aeruginosa	9.40±0.16	16.09±0.15	13.38±0.11	14.96±0.13	1.53±0.10
Escherichia coli	10.17±0.14	13.93±0.17	12.44±0.16	16.71±0.18	1.70±0.09
B, Amphimedon chloros
	Inhibition zones (mm)
Bacteria	AgNPs (20 µg/disc)	Sponge (20 µg/disc)	AgNPs (5 µg/disc) plus sponge (10 µg/disc)	AgNPs (10 µg/disc) plus sponge (10 µg/disc)	IFAa
Staphylococcus xylosus	10.40±0.13	9.00±0.12	11.19±0.15	13.23±0.14	0.62±0.05
Klebsiella oxytoca	10.65±0.16	0.00±0.00	9.48±0.14	12.28±0.15	0.33±0.03
Enterobacter aerogenes	28.33±0.17	0.00±0.00	8.88±0.12	15.93±0.16	0.00±0.00
Micrococcus spp.	9.75±0.12	8.77±0.13	8.90±0.14	11.99±0.15	0.51±0.05
Pseudomonas aeruginosa	9.40±0.14	15.00±0.16	9.66±0.11	11.39±0.13	0.47±0.06
Escherichia coli	10.17±0.15	7.34±0.12	18.00±0.19	19.71±0.18	2.76±0.13

[i] ^aAn IFA of individual AgNPs, which was calculated using C=(B²-A²)/A²; where, A and B are the inhibition zones (mm) obtained for 20 µg/disc AgNPs alone and 10 µg/disc AgNPs with 10 µg/disc Stylissa carteri or Amphimedon chloros ethyl acetate extract, respectively. The results are presented as means ± SD (n=3). AgNPs, silver nanoparticles; IFA, increase in fold area.

Discussion

To the best of our knowledge, the present study was the first to investigate the combined effects of AgNPs with the extracts of two sponge species, Stylissa carteri and Amphimedon chloros, in order to evaluate their potential as antibacterial and anticancer agents. In the present study, four cancer cell lines (namely A549, PANC-1, HT-29 and MCF7 from lung, pancreatic, colorectal and breast cancer, respectively). These cancer types were selected due to their notable incidence rates, and across all types of cancer diagnoses globally, have incidence rates of 18.4% for lung cancer, 13.1% for breast cancer, 9.7% for pancreatic cancer and 11.2% for colorectal (colon) cancer. In terms of the global cancer-related mortality rates, lung cancer accounts for 32.8% of mortalities, followed by pancreatic and colorectal cancers at 7.4% each, and breast cancer at 4.0% (2,69). Furthermore, these tumors frequently have non-specific symptoms such as, fatigue, weight loss, pain and gastrointestinal disorders, which can interfere with the correct diagnosis as these symptoms are observed in a number of different types of cancer (69,70). In addition, they have genetic and epigenetic changes, such as mutations in the Myc and Ras oncogenes, which are considered notable targets for developing treatments (71-73). Treatment of these types of cancer cells is made more difficult by their metastatic propensity, which requires an alternative treatment approach (74). Therefore, investigating these cell lines is necessary in order to develop treatment approaches and reduce their impact on public health (75,76).

To investigate the anticancer activities of the Amphimedon chloros and Stylissa carteri marine sponge extracts, the cytotoxic effects were investigated using the extracts separately and in combination with AgNPs. Various cancer cell lines were used including A549, HT-29, MCF7 and PANC-1, as well as the HUVEC normal cell line. To investigate the antibacterial activity of the Amphimedon chloros and Stylissa carteri extracts alone or with AgNPs, pathogenic bacteria were used, which were isolated from hydatid cyst fluid from damaged anatomical sites including the lung and liver (55). Several previous studies investigate the anticancer and antimicrobial properties of naturally occurring AgNPs derived from fungal origins (77-86).

The present study used LC-MS analysis, which revealed the main active chemical components in both sponge species, and molecular docking, which indicated the potential processes that may underlie their bioactivity. Cytotoxicity was observed when Amphimedon chloros or Stylissa carteri were combined with AgNPs and applied to different cancer cell lines. However, the impact of AgNPs in combination with Stylissa carteri was only significant in the A549 cell line. This synergistic effect may be explained by the presence of the bioactive chemicals present in the sponge extract, which may have increased the efficiency of the AgNPs by facilitating their contact with or absorption by the cancer cells. The LC-MS experiment indicated that Amphimedon chloros and Stylissa carteri had different chemical components, which may be the reason for the differences in their synergistic effects. Substances such as debromohymenialdisine, hymenialdisine and keramaphidin B may act in combination with AgNPs to inhibit the growth of cancer cells (87-92). Recently, certain sponge components, such as stylissamide, 6-bromotrisindoline, N-(2-hydroxyphenyl)-acetamide, petrocidin A, 2,3-dihydroxybenzamid, 6-bromotrisindoline and geodiataurine, have been revealed to have anti-proliferative capabilities against cancer cells (93,94). Additionally, keramaphidin B, which is known to be an important constituent of Amphimedon chloros, has a cytotoxic impact on a number of cancer cell lines, including P388 murine leukemia cells and KB human epidermoid carcinoma cells (89). However, neither the mechanism nor the possible synergistic effects of keramaphidin B in combination with AgNPs have been investigated. Furthermore, hymenialdisine, which is identified as the main component of Stylissa carteri, inhibited the growth of ovarian cancer cell lines, and demonstrated an antiangiogenic activity by blocking NF-κB activity and angiogenic factors such as vascular endothelial growth factor and IL-8 (91,92). Furthermore, debromohymenialdisine, which is also indicated to be a component of Stylissa carteri, increases cell cycle arrest by inhibiting the G2 phase in the MCF7 cell line (87).

Due to their function in cancer cell signaling pathways, their overexpression or mutation in a variety of types of cancer (including lung, pancreatic, colorectal and breast cancers) and their potential as therapeutic targets for inhibiting tumor growth and progression, EGFR and TrkA were chosen for docking experiments (95). We hypothesize that the activity of the sponge components (such as debromohymenialdisine, hydroxytricosanoic acid, hymenialdisine, keramaphidin B, manzacidine A and methoxyhexadecanoic acid) originates from the possible interaction of these components with TrkA and EGFR, which in turn may inhibit various pathways that promote the growth of cancer, including the phoshoinositide-3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathways. This may accelerate the process of apoptosis, cause cell cycle arrest, oppose the metastatic stage and prevent cancer cells from proliferating. Furthermore, the present study also demonstrated the cytotoxicity of combining the sponge extracts with AgNPs. The combination of 6 µg/ml Stylissa carteri extract with 0.75 µg/ml AgNPs revealed a relatively low cytotoxicity in HUVEC cells, with a 25.97% cell cytotoxicity, suggesting minimal toxicity at this dose. However, this cytotoxic effect was not significantly different compared with the Stylissa carteri extract alone at the same concentration. By contrast, a significant reduction in the cytotoxicity of the HUVEC cells was revealed when the Amphimedon chloros extract was used across concentrations ranging from 6-200 µg/ml in combination with 0.75 µg/ml AgNPs compared with the Amphimedon chloros extract alone. This selectivity suggested that the combination of sponge extracts with AgNPs has a possible therapeutic potential as a low cytotoxicity against HUVEC normal cells is an important factor for the development of safer anticancer agents (96). The present study highlighted the potential of analyzing marine products, indicating that they may be used to find new drugs among the vast resources that are present in the water of the Aqaba Gulf and other seas located within the sub-tropical arid climate.

The combination of the Stylissa carteri extract with AgNPs demonstrated the strongest synergistic action against Micrococcus spp., with an IFA of 3.43. The catalytic reactivity of nanoparticles is primarily dependent on their surface area and increases as the surface energy increases (97). However, AgNPs can have harmful effects (such as DNA damage, inflammation and apoptosis) due to their ability to induce the production of reactive oxygen species (such as hydrogen peroxide and superoxide anions) (57,98). The antibacterial activities of the AgNPs, the sponge ethyl acetate extracts and the AgNPs combined with the sponge ethyl acetate extracts demonstrated inconsistencies between the different treatment groups, regardless of Gram status of the bacteria. Therefore, it was hypothesized that the structural variations of the membranes could not be the reason behind this variation. A number of compounds, such as debromohymenialdisine, hydroxytricosanoic acid, hymenialdisine, keramaphidin B, manzacidine A and methoxyhexadecanoic acid, extracted using ethyl acetate, which is a semi-polar solvent, have a high level of antibacterial activity (99-104). Using the Stylissa carteri extract alone produced inhibitory zones in the six bacterial species investigated, and had an increased antibacterial efficacy compared with the extract of Amphimedon chloros. Using marine natural components reveals synergistic effects with antimicrobial medicines (99,105). For example, several marine sources have produced natural compounds such as equisetin, D-mannose, cis-vaccenic acid, trans-13-octadecenoic acid, stigmasterol and retinoyl-β-glucuronide with antibiotic-resistant microorganism-fighting capabilities (94,106,107). A number of them have stronger antimicrobial activity compared with therapeutic antibacterial/antifungal drugs. For example, an epoxy sponge sterol, 9α,11α-epoxycholest-7-ene-3β,5α,6α,19-tetrol-6 acetate (ECTA) was the first marine natural substance to reverse the multidrug efflux pump-mediated fluconazole resistance of Candida albicans (108,109). Combining fluconazole with ECTA (3.8 µM) increases its antifungal efficacy by 35 times (105). Marine sponges are the animal kingdoms that create the largest quantities of bioactive chemicals (110). Currently, it is unclear if the combination of AgNPs and sponge ethyl acetate extracts will be additive or synergistic, as their ability to penetrate the bacterial envelope is still poorly understood.

Although the present study offered insights regarding the potential synergistic effects of AgNPs combined with the ethyl acetate extracts of marine sponges Amphimedon chloros or Stylissa carteri, a number of limitations should be addressed. Firstly, there was a lack of results on the combined effect of Stylissa carteri and Amphimedon chloros extracts with and without AgNPs, as well as a lack of an evaluation of these combinations on a control ‘normal’ human cell line. Additionally, in vivo validation is required to investigate the therapeutic potential and safety. Furthermore, the mechanisms proposed in the present study regarding TrkA and EGFR requires further comprehensive mechanistic investigation. Although important chemical components were identified using LC-MS analysis, the entire spectrum of bioactive chemicals was not fully investigated. In addition, the number of cancer cell lines and bacterial strains investigated in the presence of these compounds was limited in the present study. Therefore, the present study was not sufficient in demonstrating the therapeutic prospects of these components for cancer and bacterial infections. However, it provided a framework for future investigations and highlighted the possibility of combining AgNPs with marine sponge extracts as potential options for antibacterial and cancer therapy.

In conclusion, the observed cytotoxic effects of Amphimedon chloros and Stylissa carteri extracts combined with AgNPs against a variety of cancer cell lines and bacterial strains suggests that further investigation on the anticancer and antibacterial potential is warranted.

Future studies should focus on elucidating the precise mechanisms of the interaction between the sponge-derived compounds and AgNPs. Furthermore, the combination ratios between the sponge extracts (such as Stylissa carteri and Amphimedon chloros) and between the sponges extracts and AgNPs should be optimized for the maximum efficacy. Additionally, the therapeutic potential should be evaluated in vivo. The results of the present study indicated the importance of marine biodiversity as a source of novel therapeutic agents and highlighted the potential of combining natural products with nanotechnology in order to increase their anticancer and antibacterial properties.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

The data generated in the present study may be found in Figshare under accession number 26255297 or at the following URL: https://doi.org/10.6084/m9.figshare.26255297.

Authors' contributions

MA, KK, AAl-S, DA, YQ and AAls participated in the conception and design of the study. Material preparation and analysis were carried out by MA, KK, AAl-S, DA and AF. The docking experiment was carried out by BA and FS. The manuscript was written by MA, YQ and AAls. All authors read and approved the final version of the manuscript. MA and KK confirm the authenticity of all the raw data.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References