Abstract
Cancer-fighting needs more effective and targeted drugs, desirably with least side-effects and from natural bases. The assessment and synergistic actions of multiple anticancer drug candidates were investigated. Fucoidan (Fu) was extracted from Sargassum cinereum, which was attained from the Saudi Red-Sea coast. The extract of Ganoderma lucidum or Reishi mushroom (Re) was achieved, intermixed with Fu solution, and they were used for reducing/decorating/capping selenium nanoparticles (SeNPs). The infrared analysis of produced/synthesized agents emphasized their biochemical structures and interactions. The decorated Fu/Re/SeNPs had negative (−30.6 mV) zeta potential and the SeNPs mean particle size was 6.5 nm. The transmission imaging of SeNPs indicated their spherical profiles and well-dispersion within Fu/Re composite. The anticancer potentiality of Fu/Re and Fu/Re/SeNPs against human colorectal adenocarcinoma cells was investigated using MTT, 4′,6-diamidino-2-phenylindole staining and comet assay. Both composites exhibited potent bioactivities toward adenocarcinoma cells; the reduced cells survival was detected with IC50 of 57.82 and 16.43 µg·mL−1 for Fu/Re and Fu/Re/SeNPs, respectively. The constrained apoptosis was notably observed from treated cells with the composites; substantial molecular damages were also verified via the comet assay, especially in Fu/Re/SeNPs treated cells. The innovative innocuous composite from Fu/Re/SeNPs is highly recommended to deactivate/destruct colorectal adenocarcinoma cells.
1 Introduction
Ganoderma lucidum (syn. Reishi or Lingzhi) belongs to the family of medicinal edible mushrooms that were described in early history (e.g., 2700 BCE) and validated to possess numerous bioactive characteristics, enabling their applications in the treatment of several diseases [1,2]. The G. lucidum or Reishi extract (Re) could have treasures of bioactive constituents, for example, polysaccharides (including β-1,3-glucans and polysaccharide peptides like peptidoglycan), triterpenoids (over 120 triterpenoids types), nucleotide bases (including thymine, guanosine, uridine, inosine, and adenosine), bioactive proteins (such as Lingzhi-8, ganodermin A, and hexameric lectin), sterols (such as ergosterol), fatty acids (nonadecenoic and cis-9-nonadecenoic acid), vitamins (including riboflavin and vitamin C), minerals, enzymes, and antioxidants [3–6]. These bioactive constituents possess super immune stimulation potentialities that could protect the whole body/organs [3]. Besides the Re immunity-boosting potentialities, G. lucidum components exhibited influential anti-hepatotoxic, immune-modulatory, anti-diabetic, anti-atherosclerotic, anti-nociceptive, cardiovascular, anti-aging, respiratory, anti-inflammatory, anti-oxidative, antifungal, and antitumor potentialities [4–6].
The anticancerous powers of Re and its components were documented through both cellular and molecular actions using in vitro protocols (against tumor cell lines) or in vivo (in experimental animals); the molecular studies indicated the Re capability to suppress cancerous genes and metabolic pathways [7–10].
The algal-derived polysaccharides that include laminarin, carrageenans, agarose, alginate, ulvan, and fucoidan (Fu), are expressively containing plentiful from valuable groups of bioactive compounds, which greatly inspire their therapeutic/biomedical applications (e.g., antimicrobial, anti-inflammatory, immunostimulants, antiviral, antioxidant, and anticancer agents) [11,12].
Fu belongs to sulfated algal polysaccharides that could be extracted from several brown macroalgal species; Fu attained extra interests due to its treasurable bioactivities in biomedical fields [13]. The nature of Fu types is defined as water-soluble, hetero-, and homo-polysaccharides that consist of l-fucose along with sulfate ester groups [13–15,51].
The elevated Fu biocompatibility, biosafety, and bioactivities (e.g., anticancer, immunomodulatory, antioxidant, antimicrobial, anti-inflammatory, antiviral, and antiallergic potentialities) advocated its biomedical usage for treating numerous human diseases [11,14–16]. Fu anticancer competencies were documented and confirmed, mainly attributing to Fu’s capabilities in inflammation suppression, oxidation protection, and apoptosis induction in cancerous cells [15,16]. The Fu conjugation/coating with nanometals and/or other bioactive biomolecules (e.g., manganese dioxide, silver oxide, sulfides, rutin, and chitosan) could reinforce their combined action as anticancer composites through increased tumor shrinkage, apoptosis induction, and molecular cancerous pathways suppression, leading to cancerous cells’ eradication and death [17–21].
Selenium (Se), for decades, was identified as a crucial nutritional element involved in organisms’ physiology through numerous selenoproteins, which play significant preventive roles toward degenerative conditions, for example, inflammation, aging, cancer, diseases, infertility, neurological and infectious diseases, by certain cellular pathways [22]. Nanoscaled materials (including Se) have very minute sizes (frequently at 1–100 nm range) and astonishing surface areas, which provide them with numerous exceptional characteristics, including their augmented bioactivities and potentialities for applications biomedicine [23]. The biosynthesis (green synthesis) of metals nanoparticles (NPs) involved the application of biological organisms/derivatives (e.g., plants, biopolymers, microorganisms, algae, polysaccharides, and proteins) for reduction, stabilizing, and capping of metals NPs and their usages, mainly as antimicrobial and anticancerous agents [24–30]. The biosynthesis protocols possess elevated biosafety and do not generate hazardous residues (as the chemical methods for NPs synthesis), and are simple, low energy consuming, and cost-effective compared to physical methods [27–32]. Selenium nanoparticles (SeNPs) gathered great researchers’ attention due to their low toxicity and outstanding bioactivities [33,34]. The biosynthesized/stabilized SeNPs with biomolecules were employed in numerous medicinal and biological applications toward disease treatment, with minimized toxicity and side effects [35]. In addition, the conjugation/capping of SeNPs with biopolymers and polysaccharides (e.g., algal extract, Fu, Re, and microbial metabolites) was supposed to fortify their combined bioactivities as antimicrobial, immunomodulatory, anti-inflammatory, and anticancerous agents [22,27,31,32].
Colorectal cancers are the second type of vigorous tumor that causes cancer deaths among men and women [36]. The utmost frequent treatment of colorectal cancer is surgery, where tumors and their lymph nodes are removed, then chemotherapy is used for restraining cancer regeneration. However, the employment of biomolecules (e.g., Fu, Re, and NPs biosystems) attained success in inhibiting colorectal cancer’s growth and reducing the side-effects of chemotherapy [37,38].
Therefore, this study aimed to extract Fu from Sargassum cinereum brown macroalgae, attain Re from G. lucidum fruits, and innovatively use their composites in synthesizing/capping SeNPs. The innovative appraisal of Fu/Re/SeNPs anticancer potentialities against human colorectal adenocarcinoma (HT-29) cells was in vitro conducted.
2 Materials and methods
2.1 Materials and chemicals
The entire employed reagents, chemicals, and media in experiments, for example, NaOH, CaCl2, Na2SeO3 (≥98%), KOH, HCl, chloroform, ethanol (95%), methanol (99%), Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum, l-glutamine, penicillin, streptomycin, 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT), dimethylsulfoxide (DMSO), phosphate buffer solution (PBS), and 4′,6-diamidino-2-phenylindole (DAPI), were in analytical grades; they were acquired by an accredited supplier, Sigma Aldrich Inc., St Louis, MO.
2.2 Algae sampling and processing
The brown algae (S. cinereum) were manually collected from the Saudi western coast (near Jeddah, at the Red Sea), within 38°65′E–39°08′E and 17°23′N–24°75′N. Algal specimens were morphologically recognized by expert marine biologists at the King Abdulaziz University, KSA. The seaweed specimens were intensively cleansed via deionized water (DW), drained, dried by hot air at 45 ± 2°C, and finely pulverized.
2.3 Fu extraction
The extraction of Fu from S. cinereum powder involved hot water modified protocols [39]. Algal dried powder (12 g) was soaked in 120 mL of extraction mixture (2:4:1 of chloroform:methanol:DW) and kept for 10–12 h at room temperature (RT; 25 ± 1°C) with regular stirring to mostly discard algal pigments, lipids, and proteins. After filtration, the treated seaweed biomass was washed with DW and immersed in 360 mL of HCl solution (0.1 M) for ∼130 min at 80 ± 5°C, then the filtration and DW washing were repeated, and the resulted biomass was rewashed with acetone and dried overnight. Next, seaweed biomass was soaked in 350 mL of 1% CaCl2 solution with constant gentle stirring (110 × g) at RT for ∼12 h to trigger alginate precipitation; this precipitate was subsequently removed through centrifugation, Sigma 2–16 KL centrifuge, GmbH, Germany, at 8,650 × g for 35 min at 10°C. The remaining Fu in resulted supernatant was attained by precipitation with cold ethanol (3 folds, v/v) and holding overnight at 4 ± 1°C. Centrifugation (9,500 × g, 35 min) was used for recovering precipitated Fu, which was then washed (with DW and ethanol), frozen and lyophilized.
2.4 G. lucidum (Reishi) extraction
Fruit powder of identified G. lucidum (Reishi) was supplied from DSM Pharmaceutical SDN BHD, Malaysia. The Re preparation involved soaking of 10 g of fruit powder in 150 mL of ethanol (70%), upholding the extraction solution for 8 h under stirring (135 × g) at RT, filtration to remove mushroom residues, and evaporating the extraction solvent under vacuum at 42°C until dry. The resulted Re powder was dissolved in DW to have a 5% concentration, which was the stock solution for further experiments.
2.5 Nanoparticles synthesis and characterization
For fabricating SeNPs using Fu and Re as reducing/stabilizing agents, solutions (1% concentration) were prepared in DW from Fu and Re. Equal volumes (25 mL) from each solution were mixed using high-speed stirring (720 × g) for 200 min at RT. Fresh solution (1 mM) from Na2SeO3 in DW was then prepared and 25 mL from it was slowly dropped into the stirred Fu/Re mixture solution, with the subsequent dropping of a few drops of 1% (w/v) ascorbic acid into the mixture as a supplemental NPs reducing agent [32]. Mixture stirring was continued for further 120 min after dropping; the visualized brownish-orange color of mixture solution indicated the formation of SeNPs. Successively, the Fu/Re/SeNPs containing solution was centrifuged (12,500 × g for 24 min) and the harvested pellet was washed (DW), frozen and lyophilized. The systematic protocol involved for the synthesis of decorated Fu/Re/SeNPs is illustrated in Figure 1.
A systematic schematic protocol involved in the synthesis of decorated Fu/Re/SeNPs.
2.6 Characterizations
The biochemical/structural characteristics of Re, Fu, and their composites (either alone or with SeNPs) were appraised via Fourier-transform infrared (FTIR) spectroscopy, JASCO FTIR-360, Japan, within 450–4,000 cm−1 wavenumber range, after samples intermingled with KBr.
The UV-Vis spectrophotometry, UV-2450 Shimadzu, Japan, was employed for assessing the surface plasmon resonance (SPR) of Fu/Re synthesized SeNPs, at a 200–800 nm wavelength range.
The NP size (P s) distribution and the NPs zeta potentiality (ζ) were appraised through dynamic light scattering (DLS), Brookhaven ZetaPlus, USA.
Moreover, the ultrastructure of biosynthesized NPs was broadcasted by transmission electron microscopy (TEM), JEOL Ltd, JEM-100CX, Japan, operated at 80 kV acceleration.
2.7 Anticancerous assaying
The HT-29 cell line (ATCC-HTB-38) was employed for anticancer potentiality assessment. Cells were propagated in T-flasks at 37°C and 5% CO2 humidified atmosphere was created using supplemented DMEM medium with glucose (4.5 g·L−1), l-glutamine (8 mM), fetal bovine serum (10%), and 1% of an antibiotic mixture (streptomycin and penicillin).
2.7.1 Viability assessment via MTT protocol
Grown lively cells in DMEM were planted in 96-well microtiter plate (with ∼20,000 cells per well), in triplicates, with negative control (untreated cells with composites; incubated only in plain DMEM medium) and zero-adjustment (medium only). After 24 h of incubation, media were amended by gradual concentrations (1, 2, 5, 10, 25, 50, 125, and 250 µg·mL−1) from either Fu/Re or Fu/Re/SeNPs composites. After a further 24 h of treatment, media were detached and cancerous cells were afforded with 20 µL of MTT solution (5 mg·mL−1) and incubated at 37°C for extra 4 h. About 150 µL from DMSO were carefully added, gently agitated, and the optical density of mixture absorbance was measured (at 570 nm) via microplates reader.
2.7.2 DAPI staining
For detecting potential HT-29 cells’ apoptosis, sterilized coverslip slides are sited into a six-well culture plate, then grown HT-29 cells suspension (3 mL per well, ∼12,000 cells·mL−1) were appended to wells and incubated under optimal conditions (e.g., 37°C and humidified 5% CO2 atmosphere). After ∼50% confluence, untreated cells and amended wells with 2 × IC50, from Fu/Re and Fu/Re/SeNPs composites were incubated for further 24 h. Treated cells were gently washed with pre-warmed media, which were subsequently replaced with fixative solution carefully (pre-warmed DMEM containing 4% formaldehyde) and incubated for 5 min. After fixation, cells were washed by PBS twice, then permeabilized using permeabilization solution (PBS contained 0.1% Triton X-100) and 5 min incubation. Permeabilized cells were afterward stained by DAPI (100 µL per well, 250 ng·mL−1 concentration) and kept at RT for 3 min. Lastly, slides were PBS washed and inspected via fluorescent microscope (Olympus BX61, Japan) with U-MWU2 fluorescence filter.
2.7.3 Comet assay – single cell gel electrophoresis
The comet assaying protocol was conducted in alkaline conditions following the slightly modified procedure of Singh et al. [40]. The slides were processed after cell treatments (as above method), neutralized by 0.4 M cold Tris (pH 7.5), and analyzed using Komet 5.5 imaging system (Olympus, Japan), attached with a fluorescent microscope. The average tail lengths (µm) were the indicators for DNA damage after treatments with inspected anticancerous composites.
3 Results and discussion
The Fu extraction was efficaciously accomplished from S. cinereum with an average yield of 1.83%, which is comparable to formerly reported yields from relevant Sargassum spp. [13,27,39,41].
3.1 Compositional/biochemical structures analysis
The compositional/biochemical structures of produced biomolecules/composites (i.e., Re, Fu, Fu/Re, and Fu/Re/SeNPs) were inspected via FTIR spectroscopy (Figure 2), to appraise their potential compositional groups and biochemical interactions.
FTIR spectra of Re, Fu, composited Re/Fu, and composited Re/Fu with SeNPs (Re/Fu/Se).
For purified Re alone (Re in Figure 2), strong stretching peaks at 3,448 and 3,422 cm−1 are believingly ascribed to O‒H stretched vibration, while the sharp peak at about 2,926 and 1,658 cm−1 are associated with C‒H and C═O stretched vibrations, respectively [12]. The band at 1,713 cm−1 appointed the C═O stretch of carbonyl, whereas the characteristic peak at 1,458 cm−1 indicated the C‒H deformation vibration, and C═C stretching. The peaks at 1,035 and 1,075 cm−1 indicated the presence of carboxylic acid C‒OH group and hydroxyl/ether (C‒O‒C) stretched vibration, respectively. The peak at 637.47 cm−1 designated the alkyl halide presence [42,43].
The band at 1,249 cm−1 indicated the P═O bonds in phosphate-containing molecules (e.g., nucleic acids), and in addition, the appeared peaks in of 900–400 cm–1 region indicated the carbohydrates components in Re, especially β-glucans [22,43,44]. The appeared stretch in the 1,630–1,680 cm–1 wavelength could be frequently indicative of the amides and ketone compounds in the extract [1].
The spectrum of Fu (Fu in Figure 2) demonstrated the existence of key characteristic bonds that confirm algal Fu extraction; the peak around 3,400 cm−1 indicated the –OH presence (hydroxyl group); the band at 811 cm−1 indicated the axial/equatorial sulfate position; and the bands in 1,600–1,650 cm−1 region indicate the associated functional carbonyl groups to uronic acid [27,45]. The infrared spectrum of Fu also displayed additional characteristic bands at 1,671 cm−1 (C═O vibrated stretching of O‒ in acetyl group); at 1,421 cm−1 (the CH2 in xylose, galactose) and at 1,092 cm−1 (the symmetric strong stretching of O═S═O vibration in sulfate esters); the above bands are characteristics in sulfated polysaccharides [46]. As the sulfation level is associated with the peak intensity of sulfate groups [39], the strong band at 541 cm−1 (C‒O‒S) can indicate axial secondary sulfate presence at C-4 of fucopyranose.
The spectrum of Re/Fu composite had the main characteristic bands from both agents (Re/Fu in Figure 2); many distinctive peaks from Re and Fu spectra were overlapped, and others were changed in their intensities, which strongly indicate the biochemical interactions between Re and the sulfated polymer [47]. This innovative Re/Fu composition could be the start for numerous biomedical applications using the produced bioactive complex.
The FTIR spectrum of synthesized/decorated SeNPs with Re/Fu composite (Re/Fu/Se in Figure 2) indicated the appearance of many novel peaks, especially in the wavelength range of 500–900 cm−1, which indicates the role of carbohydrates (e.g., β-glucans in Re) and C–O–S sulfate groups (from Fu) in the capping/stabilizing of SeNPs [48]. In addition, the emerged bands in Re/Fu/SeNPs spectrum, at 1,183, 1,419, and 3,752 cm−1 indicated the interactions between SeNPs and the biochemical groups of Re/Fu polysaccharides and proteins; this could effectively augment the NPs stability through their capping with the composite components [12,48].
3.2 Structural attributes of Re–Fu-mediated SeNPs
The optical analysis of Fu–Re-mediated SeNPs, via UV-vis analysis and direct visual examination, confirmed SeNPs formation after interactions Fu–Re (Figure 3a). The visual observation of Fu/Re/SeNPs solution indicated ongoing transformation of its color from clear to deep reddish-orange within 30 min of reaction (Figure 3a – upper photos), with no extra color-changing afterward. The highest absorption value in UV-vis curve (λ max) NPs peaks was recorded at 263 nm (Figure 3a – curve), which was in accordance with the attained λ max values in recent studies involving SeNPs synthesis/capping with other polysaccharides and biopolymers [30,32]. The color revolutions of NPs synthesizing solutions are predominantly associated with the excitation of SPR in synthesized NPs; more color depth of NPs solutions indicated smaller sizes of synthesized NPs [29]. Fu was stated individually to possess remarkable reducing powers that enabled it to generate metals NPs via direct interaction [49]. Although the capability of Fu for reducing/capping metals NPs (Ag) was recently documented [49], this study could be leading in Fu biopolymer application for reducing/decorating SeNPs and augmenting their bioactivities.
Structural attributes of Re–Fu-mediated SeNPs, including their UV-absorbance spectrum (a) and ultrastructure via TEM imaging (b).
The morphological features of synthesized/decorated SeNPs within Fu–Re matrix are elucidated via TEM imaging (Figure 3b); the NPs were homogenously distributed and appeared mostly with spherical and semispherical shapes, embedded within the biomolecule matrix. The estimated P s range of SeNPs, from the TEM image, was 3.74–34.65 nm, with an estimated mean P s diameter of 6.61 nm; this NPs size range and morphology were stated to be effectual in practical application for cancer management [12,27,38]. The metal NPs synthesis/capping with other natural polysaccharides and biopolymers were proved to provide an effectual approach for generating diminished size NPs with elevated efficiency [49]. The SeNPs ultrastructure analysis validated the elevated potentiality of Fu and its composite with Re to reduce/stabilize metal NPs [50,51]; which was innovatively achieved toward SeNPs.
The DLS analysis of extracted/composited materials indicated high negativity ζ potentials (charges) on their surfaces, for example, −33.8, −34.7, −31.1, and −30.6 mV for Fu, Re, Fu–Re, and Fu/Re/SeNPs, respectively. The average sizes of Fu/Re/SeNPs nanocomposite ranged from 3.12 to 38.28 nm, with a mean diameter of 6.47 nm, which was in accordance with sizes appraised from TEM analysis (Figure 3b).
The ζ potential determination can frequently indicate the carried charge onto individual molecules’ surface; NPs having ζ potentialities of ≤−30 mV or ≥+30 mV commonly exhibit advanced stability in solutions [52], triggered by interparticle electrostatic repulsion [53]. This means that the elevated values of ζ potentialities, in particular, NPs give their nanosuspensions good physical colloidal stability and prevent particle flocculation and aggregation due to the Van der Waals attraction forces. The slight decrement in ζ potential negativity after conjugation with further molecules/NPs is assumingly associated with the formation of novel electrochemical bonds among molecules and the occupation of free bonds with the conjugated particles [50,51].
3.3 Anticancerous potentiality assaying
3.3.1 MTT assay
The preliminary assessment of cytotoxicity and anticancer potentialities of Fu/Re and Fu/Re/SeNPs against HT-29 cells, using MTT protocol, revealed that both composites had remarkable activity toward decreasing cancerous cells’ viability (Figure 4). The Fu/Re/SeNPs anticancer effect was significantly stronger than Fu/Re effect (without SeNPs); the entire cells completely lost their viability after treatment with Fu/Re/SeNPs at 125 µg·mL−1 concentration. The calculated IC50 of composites toward HT-29 cells were 57.82 ± 1.25 µg·mL−1 (for Fu/Re) and 16.43 ± 0.21 µg·mL−1 (for Fu/Re/SeNPs). The antitumor potentialities of Fu extracted from various seaweeds were documented [37]; these actions were augmented via Fu conjugation with further bioactive molecules, NPs (e.g., gold, copper, manganese, and chitosan), or with accustomed anticancer drugs [54,55].
Cancer cells’ (HT-29) viability using MTT assay after treatment with Fu/Re and Fu/Re/SeNPs composite.
The anticancerous and immunoregulatory activities of Re were also reported and enforced with nanometals or biopolymers; the Re-mediated Au NPs could powerfully promote dendritic cell activation, reduce acid phosphatase action and phagocytic capability, and upsurge cytokine transcription [12,56]. In addition, the decorated SeNPs with Re sulfated polysaccharides exhibited elevated synergistic immunomodulatory actions [22]. The biofabricated nanometals (e.g., Au) with Re were validated to possess elevated cytotoxic and anticancerous efficacy toward colon cancer cells, in dose-dependent manners; the minute NPs size and their capping with Re biomolecules had vital functions in their bioactivities [38]. Existing results were supported with the aforementioned investigations and innovatively indicate the synergistic actions of Fu, Re, and SeNPs to inhibit HT-29 cells in a dose-dependent approach.
3.3.2 Apoptosis and DNA damage assay
The cytotoxic/anticancer consequences of Fu/Re and Fu/Re/SeNPs composites, toward HT-29 cancerous cells, were further elucidated via fluorescent imaging (Figure 5). The potential apoptosis/nuclear degradation effects after HT-29 cells’ treatment with the composites (at 2 × IC50 concentrations) were screened via DAPI staining (D in Figure 5), whereas the comet assay, The single cell gel electrophoresis assay was additionally applied as a measurable, versatile and sensitive technique for assessing the DNA damage and genotoxicity in treated cancerous cells (C in Figure 5). The cells' exposure to Fu/Re and Fu/Re/SeNPs composites induced apparent morphological signs of apoptosis in HT-29 cells, for example, blebbing membranes, rounding, and shrinkage cells. The apoptosis indicators became much more vigorous and seriously increased to comprise most treated cells with the Fu/Re/SeNPs treatment, compared to Fu/Re treated and control cells (D in Figure 5). While no apparent apoptosis signs were detected in untreated cells, the DAPI-stained exposed cells exhibited numerous luminously fluoresced nuclei/fragments; the increments of apoptosis indicators and DNA condensation were intensely detected in Fu/Re/SeNPs-treated cells.
Fluorescent imaging of treated HT-29 cancerous cells with Fu/Re (2) and Fu/Re/SeNPs (3) composites, compared to untreated cells (1) using DAPI staining (D) and comet assay (C).
The DNA damage and genotoxicity indicators appraisal (using comet assay) emphasized matching observations (C in Figure 5); the treatments of HT-29 cells Fu/Re and Fu/Re/SeNPs composites triggered notable DNA damages, distinguished with apparent tails moment. Significant increments in DNA damages were detected in treated cells over the control group, and in addition, in Fu/Re/SeNPs-exposed cells over the Fu/Re-treated group. The average tail lengths were computed as 24.3 µm for Fu/Re/SeNPs-exposed cells, 16.9 µm for Fu/Re-treated cells, compared with 6.4 µm in untreated control cells (C in Figure 5).
The perceived cellular DNA damage assumingly resulted from two main factors – the “reactive oxygen species” generation from composited NPs, and the direct interactions of Fu, Re, SeNPs and their conjugates with intercellular DNA, which could trigger breakage in double-stranded DNA [57]. Also, the anticancerous potentialities NPs-based composites were suggested to predominantly base on NPs adjacent contact with components of cancer cells, which prompted the development of cells’ apoptosis and/or necrosis [58]. This could clarify the forceful effects of Fu/Re/SeNPs composite toward HT-29 cells than the consequence of Fu/Re treatment alone. Although the current microscopic examinations using fluorescent staining and imaging provided strong evidence for the composites’ anticancer potentialities, other techniques such as confocal microscopy examination for cell cycle analysis is suggested to give more validation to their action on the cancer cell.
The polymeric capping/carrying was additionally advocated to enhance the half-life circulation and deposition of capped drugs/NPs into diseased sites, with minimized extravasation to somatic/normal tissues [59]; this characteristic was from the key goals to employ Fu/Re/SeNPs as anticancer composite, as the biopolymers (Fu and Re polysaccharides) are assumed to augment the biosafety of SeNPs toward healthy cells.
The sole Fu was recurrently documented to possess remarkable anticancer potentialities [14,37,39,41,55,60–62]. The deacetylated Fu was proposed as an effective treatment for inhibiting HT-29 cells’ growth [41]; Fu could inhibit sphere formation, growth, and migration of cells via suppression of PI3K/Akt/mTOR pathway [60]. Other reports for anticancer applications of Fu to combat colon cancer indicated that injection (intraperitoneally) of Fu diminished tumor volume; this reduction accompanied apoptosis induction and lowered angiogenesis intermediated via Akt signaling [37,61]. Another attempt suggested that Fu anticancer potentiality depended on “caspases” activation through mitochondria and/or death receptor-mediated pathways of apoptotic, and these were the main Fu functions for growth inhibition and apoptosis induction in colon tumor cells [62]. Complementary Fu administration directed the arrest of sub-G1 phase in tumor cell cycle, inhibited cellular tumor growth, and enforced their apoptotic death [37]. Because of the human inability to enzymatically hydrolyze Fu in the intestine, the Fu administration is associated with elevated concentrations of luminal Fu in the large intestine [60]. Considering that Fu was supposed as a very promising agent/carrier for the treatment/prevention of colon cancers, especially with its potentialities for combating cancerous cells in vitro/in vivo [37,61].
The Fu conjugations with metals NPs (e.g., MnO₂, CuS, and Ag) or biomolecules (e.g., rutin and chitosan) were validated for augmenting their bioactivities toward cancer system’s inhibition and elimination [18–21]; the Fu-based conjugates possessed higher toxicity and apoptosis induction in numerous cancers type, with increased biosafety levels for normal cells.
The anticancer potentialities of Re and its polysaccharides were stated to involve diverse molecular and cellular mechanisms (including apoptosis induction, antiangiogenic, anti-proliferation, antimetastatic, telomerase inhibition, autophagy induction, and cells’ cycles arresting) [6,12,63]. Besides Re antitumor immunology, the intrinsic/extrinsic initiated apoptosis pathways involved tumor angiogenesis inhibition (via affecting of vascular endothelial growth factor pro-angiogenic stimulus), inhibition of adenosine triphosphate-dependent transmembranes, and P-glycoprotein in the tumor cells surfaces (especially multidrug-resistant tumors), which have a significant role in delivering anticancer drugs to the intracellular tumor cells [7–10].
The Re-mediated AgNPs possessed potent anticancerous actions toward breast cancer cells [1], which were strengthened via conjugation of the two agents in cells’ treatment; thus, this provides further validation of anticancer bioactivities synergism when Re is conjugated with metals NPs [63].
4 Conclusion
Fucoidan was effectually from Sargassum cinereum brown seaweeds and its composite with G. lucidum extract (Re) was innovatively employed for synthesizing/decorating SeNPs. The innovative formulation of Fu/Re and Fu/Re/SeNPs composites exhibited promising anticancerous potentialities against HT-29 cells; these bio-composites provided effectual formulations for potential eradication of colorectal cancers with minimized human toxicity.
Acknowledgement
This project was funded by the DSR (Deanship of Scientific Research at King Abdulaziz University, Jeddah, KSA) under grant number G:310-662-1442. The authors, therefore, acknowledge with thanks DSR for the technical and financial support.
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Funding information: This project was funded by the DSR (Deanship of Scientific Research at King Abdulaziz University, Jeddah, KSA) under grant number G:310-662-1442.
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Author contributions: Mohamed E. El-Hefnawy: conceptualization, methodology, investigation, data curation, resources, formal analysis, writing – original draft; Mohsen M. El-Sherbiny: visualization, methodology, formal analysis, data curation, formal analysis, writing – original draft; Mamdouh Al-Harbi: conceptualization, supervision, resources, validation, writing – review and editing; Ahmed A. Tayel: conceptualization, methodology, investigation, formal analysis, writing – original draft, writing – review and editing.
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Conflict of interest: Authors state no conflict of interest.
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Data availability statement: The data that support the findings of this study are available on request from the corresponding author.
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