Two Red Sea Sponge Extracts ( Negombata magniﬁca and Callyspongia siphonella ) Induced Anticancer and Antimicrobial Activity

: Bioactive compounds extracted from marine organisms showed several biological activities. The present study is an extension of our earlier studies where we assessed the antiproliferative and pro-apoptotic activities of ethanol, methylene chloride, ethyl acetate, acetone, and chloroform crude extracts of sponges: Negombata magniﬁca ( Nm E) and Callyspongia siphonella ( Cs E) against cancer cells. Herein, we are extending our previous ﬁndings on both sponge species depending on an alternative methanol extraction method with more advanced molecular biochemical insights as additional proof for anticancer and antimicrobial activity of N. magniﬁca and C. siphonella . Therefore, sponge specimens were collected during winter 2020 from the Dahab region at the Gulf of Aqaba. Each sponge was macerated with methanol to obtain the crude extracts; Nm E and Cs E. GC–MS analysis presented a total of 117 chemical compounds; 37 bioactive, 11 represented previously as constituents for a natural organism, and 69 had no biological activities. Nm E dose-dependently inhibited the growth of HepG2, MCF-7, and Caco-2 carcinoma cell lines compared to Cs E, which unfortunately has no antiproliferative activity against the same cancer cells. Nm E was found to induce G0/G1 cell cycle arrest in HepG2 cells with its inhibition for CDK6, Cyclins D1, and E1 in HepG2, MCF-7, and Caco-2 cells. Nm E also activated ROS production in HepG2 cells and induced apoptosis in HepG2, MCF-7, and Caco-2 cells via an increase in pro-apoptotic protein Bax, caspase-3, and cleavage PARP, and a decrease in anti-apoptotic protein BCL2. Unlike its anticancer potential, Cs E exhibited clear superior results as an antimicrobial agent with a wider range against six microbial strains, whereas Nm E showed a positive antibacterial activity against only two strains.


Introduction
Marine species have had a lot of success in the last several years as a source of novel bioactive chemicals for medication development [1]. As a result, since the middle of the they found all extracts inhibited the growth of HepG2, MCF-7, and Caco-2 cancer cells and induced apoptosis in the same cancer cell lines via Bax and caspase-3 increase, and BCL2 decrease. Herein, the present work is an extension of this work, but here we prepare methanolic extracts for same sponge species (NmE and CsE) and state their bioactive compounds and introduce their anticancer and antimicrobial activities.

Sampling and Identification of Sponge Specimens
The sponge specimens were collected from the Dahab coast, Gulf of Aqaba at 30 m depth by SCUBA diving during winter (February) 2020. The specimens were carefully identified based on Porifera morphological characters according to Systema Porifera [43] associated with the most recent update undertaken in the World Porifera Database [44].

Preparation of NmE and CsE
This procedure was carried out according to El-naggar and Hasaballah, 2018 [20] with some modifications. Firstly, 100 gm of sponge were macerated with 200 mL of 70% aqueous methanol. The mixture was continuously stirred by a rotary shaker (200 rpm) for one week in dark. Then, the mixtures were filtered through Whatman 542 filter paper, and the solvent was evaporated using a rotary evaporator at 40 • C (LARK, Model: VC-100A) and preserved at −20 • C for further analysis.

GC-MS Analysis of NmE and CsE
One microliter of each extract was examined for its contents of bioactive compounds using a Gas Chromatography-Mass Spectrometer (Hewlett Packard HP-5890 series II) equipped with a split/splitless injector and a capillary column (30 m, 0.25 mm, 0.25 mL) fused with phenyl polysilphenylene siloxane. The temperatures of injector and detector were set at 280 and 300 • C, respectively, and the oven temperature was kept at 80 • C for 1 min, and rose to 300 • C at 20 • C/min. The helium was used as carrier gas at a constant flow of 1.0 mL/min. A volume of 2 microliter was injected in the splitless mode and the purge time was 1 min. The MS (Hewlett-Packard 5889B MS Engine) with selected ion monitoring (SIM) was used. The mass spectrometer was operated at 70 eV, scanning fragments from 50 to 1000 m/z. Peak identification of crude extract was performed based on comparing the obtained mass spectra with those available in the NIST library [45].

Anticancer Assay 2.4.1. Cell Culture
HepG2, MCF-7, and Caco-2 cancer cell lines were obtained from American Type Culture Collection (Manassas, VA, USA) and (VACsERA Co., Cairo, Egypt). Those cancer cells were cultured in DMEM (Corning Thomas Scientific, Swedesboro, NJ, USA). DMSO was purchased from (Sigma-Aldrich, Saint Louis, MO, USA), while FBS was purchased from Hyclone (Pittsburgh, PA, USA) and PSA was obtained from Mediatech Inc. (Herndon, VA, USA). Unless otherwise indicated, cancer cells were cultured in DMEM supplemented with 5% heat-inactivated FBS and 1% PSA at 37 • C in a 5% CO 2 incubator, and the untreated growth media containing vehicle DMSO (0.01%) were used as negative controls for all assays [46].

Measurement of Cytotoxicity by MTT Assay
The cytotoxicity of NmE and CsE against HepG2, Mcf-7, and Caco-2 was measured by Cell Proliferation Kit I MTT (Sigma-Aldrich, Saint Louis, MO, USA). Accordingly, the cells were cultivated in 96 well culture plates at 5000/well for 24 h. Then, cells were provided with different concentrations of NmE (0, 6.25, 12.5, and 25 µg/mL) and CsE (0, 156.2, 312.5, 625, 1250, and 2500 µg/mL) individually for 48 h in 37 • C and humidity 5% in CO 2 incubator. Treated and untreated Cells were incubated with 10 µL of the MTT powder (final concentration, 0.5 mg/mL) for 2 h after until violet crystals were formed at different color hues indicating cell metabolic activity. The insoluble formazan crystals are dissolved using a solubilization solution (Phosphate buffered saline). Colorimetric absorbance was measured at 620 nm (A620) and 570 nm (A570) by Synergy™ 2 Multi-Mode Microplate Reader (BioTek Inc., Winooski, VT, USA). The cell viability was calculated using the following equation [47].

Percentage of viability
The IC 50 was calculated by the IC 50 calculator as previously described by Luparello et al. [48]. The protocols of treatment and analysis were performed at least three times.
Due to the NmE potentiality as an antiproliferative active agent, it was chosen for the current investigations.

Protein Extraction and Western Blotting
The medium was discarded 24 h after HepG2, MCF-7, and Caco-2 cells treatment with NmE. Then, cells were washed with cold PBS (10 mmol/L, pH 7.4) followed by incubation on an ice-cold lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EGTA, 1 mmol/L EDTA, 20 mmol/L NaF, 100 mmol/L Na 3 VO 4 , 0.5% NP40, 1% Triton X-100, 1 mmol/L PMSF (pH 7.4)) with a freshly added inhibitor cocktail (Inhibitor Cocktail Set III, Calbiochem, La Jolla, CA, USA) over ice for 30 min [49]. The cells were scraped, and the suspension was passed many times through a 21.5-gauge needle up and down in a microfuge tube to split up any cell aggregates [50]. Then, centrifugation for lysate at 14,000× g for 25 min at 4 • C, and the total cell lysate (supernatant) was stored at 80 • C for further analysis [49].
Bax, BCl2, Caspase-3, CDK6, Cyclin D1, cyclin E1, PARP, HRP anti-mouse, and antirabbit secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA), whereas GAPDH, β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz Co., Santa Cruz, CA, USA). Mini-protean precast Tris-Glycine gels were from BioRad (Hercules, CA, USA). ECL detection system was from GE Healthcare (Buckinghamshire, UK). A 2% (w/v) Aqueous Solution of Gentian Violet was from Ricca Chemical Company (Arlington, TX, USA). Invitrogen Novex precast Tris-Glycine gels were from Corning. Briefly, 25-40 µg of protein were resolved over 8%-12% polyacrylamide gels and transferred to a nitrocellulose membrane. The blot was blocked in blocking buffer (7% nonfat dry milk/1% Tween 20; in 20 mmol/L TBS (pH 7.6)) for 1 h at room temperature, incubated with the appropriate monoclonal or polyclonal primary antibody in blocking buffer for 2 h at room temperature or overnight at 4 • C, followed by incubation with an appropriate secondary antibody HRP conjugate. The blots were exposed to enhanced chemiluminescence (Thermo Scientific Pierce, Rockford, IL, USA) and subjected to autoradiography using a BioRad (Hercules, CA, USA) imaging system. The digitalized scientific software program Quantity One (BioRad) was used for the analysis of the Western blot to assess the densitometric measurements of the bands, and treatment protocol was carried out at least three times, as well as each protein expression, was analyzed three times with analogous results [49,50].

Cell Cycle Assay with PI
The effect of NmE on HepG2 cell cycle was handled as described by Kim and Sederstrom, 2015 [51]. Briefly, cells were seeded in 6-well plates (3 × 10 5 cells per well) and allowed to attach and grow for 24 h in the mean culture conditions. The cells were treated with NmE (0, 6.25, 12.5, and 25 µg/mL) for 24 h. At the time of detection, cells were harvested, and both attached and floating cells were collected. The cells were fixed by slowly dropping the 70% ice-cold ethanol and stored at 4 • C for at least 30 min. Later, the cells were washed twice with ice-cold PBS at 2000 rpm, then incubated in PBS with RNase A (10 mg/mL) (QIAGEN, Hilden, Germany) and PI (50 µg/mL) at room temperature for 10 min. Cell cycles were analyzed using a BD FACSCalibur™ Cell analyzer equipped with Cell Quest software at an emission of >575 nm (FL3). The area occupied by all the cell cycle phases (Sub G, G0/G1, S, and G2/M phase), expressed in resulting histograms, were measured and graphed together.

ROS Assay
The probably oxidative stress effect induced by NmE through the ROS production was tested by the DCFDA/H2DCFDA-Cellular ROS Assay Kit (ABCAM, Cambridge, UK) and flow cytometer according to the manufacturer instructions, as described before by Degl'Innocenti et al., 2019 [53]. HepG2 cancer cells were seeded in 6-well plates (3 × 105 cells per well) and allowed to attach for 24 h as described above. The cells were treated with NmE (0, 6.25, 12.5, and 25 µg/mL) for 24 h. At the detection time, cells were trypsinized, and all attached and floating cells were collected. The cells were, then, treated by DCFDA, and stored at 37 • C for 30 min. Before analysis, the cells were washed twice with ice-cold PBS, analyzed using a flow cytometer at an emission of 530 nm (FL1 channel) and 150 mV.

Microbial Indicator Strains
The bacterial indicators were provided by the regional center for mycology and biotechnology, Al-Azhar University. Ten test microorganisms were selected to cover all categories either filamentous fungi, yeasts, or Gram-positive and Gram-negative bacteria as follows: Aspergillus flavus, Penicillium expansum IMI28169, Candida lipolytica, Cryptococcus neoformas, Micrococcus sp., Streptococcus mutants ATCC25175, Methicillin-resistant Staphylococcus aureus, Salmonella typhimurium ATCC 14028, Escherichia coli ATCC 25955, and Klebsiella pneumonia ATCC 13883.

Media and Inocula
Mueller-Hinton agar medium was used for bacterial growth assay. It is composed of g/L; beef dehydrated infusion 300, casein hydrolysate 17.5, starch 1.5, and agar 17; this medium is ready to use when it has been prepared by weighing 38 g and up to 1 L distilled water. Furthermore, fungi and yeasts were cultivated onto malt extract agar MEA medium, which is composed of (g/L); malt extract 30, mycological peptone 5, and agar 20; all ingredients were properly combined up to 1 L with distilled water and well-cooked. The inoculum of every test microorganism was prepared according to Microbiology, 2000 [54].

Antimicrobial Activity Assessment for CsE and NmE
The agar well diffusion technique was used to investigate the ability of the crude extracts of sponges to inhibit the growth of indicator bacteria, fungi, and yeasts [55], whereas gentamycin was used as a positive antibacterial standard while, ketoconazole was a positive antifungal standard. In addition, methanol was applied as a negative control. One hundred microliters of the tested crude extracts were transferred into each inoculated well after sterilizing by ultra-filtration [21]. All plates were incubated at an appropriate temperature for 24-48 h. After the incubation period, the radius of the clear zone around each well (Y) and the radius of the well (X) were linearly measured in mm, where dividing Y2 over X2 determines an absolute unit (AU) for the clear zone [19]. The absolute unit of crude extract, which indicates a positive result, was calculated according to the equation [56]: AU = Y2π/X2π.

Statistical Analysis
All statistical analyses were performed with GraphPad Prism™ version 9.1.0. (Graph-Pad Software Inc., San Diego, CA, USA). Statistical significance was determined using one-way ANOVA for multiple groups, followed by Tukey's test for multiple comparisons. All results were reported as the mean ± standard deviation (SD) with significance set at p values ≤ 0.05.

Sponge Identification
The collected sponges were identified to two species (Figure 1), as follows: 1-Finger-sponge (Negombata magnifica): It is a reddish-brown, narrow, crooked branched sponge, and lives between coral reefs and rocks. 2-Tube-sponge (Callyspongia siphonella): It is a cluster of vertical tubes with a common base that lives on sheltered, hard substrate. It is smooth and has a pale purple or reddish-brown color.

GC-MS Analysis of CsE and NmE
A total of 117 compounds have been found in both CsE and NmE, 58 for each, and 1 (10-Octadecenoic acid, methyl ester) shared. According to biological activity, 48 compounds were supposed to have biological activity (37 bioactive, 11 reported previously as a constituent for a natural organism), and 69 have no bioactivity yet. NmE has 9 compounds with anticancer activity and 6 compounds with antimicrobial activity; similarly, CsE contains 15 compounds, which have anticancer activity, 10 compounds with antimicrobial (bacteria, virus) activity, and 1 has antifungal activity ( Table 1; Figures 2 and 3).    (Table 2). Due to the NmE potentiality as an antiproliferative active agent, it was chosen for further investigations.  Statistical differences from control cultures are shown as bar graphs with error bars representing the means ± SD; ** p < 0.01, *** p < 0.001 and **** p <0.0001 vs. control (DMSO-treated) cells. In HepG2, NmE in a dose-dependent manner induced significant early and late apoptosis causing complete cell death (Figures 6 and 7), indicated by the elevated percentage of PI stain that binds to the genetic material (DNA and RNA) inside the damaged dead cells. In addition, the NmE apoptotic effect is confirmed through the expression of some regulatory proteins that are involved in either the extrinsic or intrinsic apoptotic pathways. NmE increased the level of the pro-apoptotic protein Bax, cleavage PARP, and caspase-3, while it decreased the level of the anti-apoptotic protein BCl2 in HepG2, MCF-7, and Caco-2 cancer cell lines ( Figure 8).

NmE Induced G0/G1-Phase Cell Cycle Arrest and Decreased Cyclins D1 and E1 and CDK6 Expressions in Cancer Cells
The cell cycle profile analysis revealed significant dose-dependent increases in the number of cells in the G0/G1 phase of the cell cycle in NmE-exposed HepG2 cells compared to no-treatment HepG2 cells control (Figures 9 and 10). The G0/G1 phase distribution of HepG2 cells after treatment with NmE (0, 6.25, 12.5, and 25 µg/mL) was 45.8%, 49.83%, 59.67%, and 67.56%, respectively ( Figure 10). The increases in the G0/G1 phase cell population were accompanied by decreases in the G2/M and S phase cell populations of the HepG2 cell line except for a slight increase for the S phase at the 25 µg/mL dose of NmE. This slight increase in the S phase at this highest dose of NmE (25 µg/mL) is suggestive as evidence of toxicity. Since the current cell cycle analysis elucidated that NmE treatment of HepG2 cells resulted in a G0/G1-phase arrest, we next assessed the effect of NmE on cell cycle regulatory proteins that are effective in the G1 phase. Thus, we examined the effect of NmE on the expression of cyclins D1 and E1 and CDK6. NmE showed a dose-dependent decrease in the expression of cyclins D1 and E1 and CDK6 ( Figure 11) in HepG2, MCF-7, and Caco-2 cancer cell lines. To conclude, NmE-induced cell cycle arrest is mediated via inhibiting CDK6 along with cyclins D1 and E1.

NmE Triggered ROS Production in HepG2 Cell Line
The current flowcytometric analysis showed that NmE-treated HepG2 cells produced more ROS compared to untreated HepG2 cells ( Figure 12). NmE was able to increase the ROS production, even the NmE lower concentration (6.25 µg/mL) was significant to induce higher ROS production than HepG2 cells control. In summary, it seems that NmE produced marked induction of apoptosis and anti-proliferative activity on HepG2, MCF-7, and Caco-2 cancer cells via multiple pathways ( Figure 13).

Antimicrobial Activity of CsE and NmE
The results showed a noticeable antimicrobial activity against a wide range of the test organisms (Table 3). CsE was effectively more than NmE against Aspergillus flavus, Cryptococcus neoformans, Salmonella typhimurium, Klebsiella pneumonia, Streptococcus mutants, and Candida lipolytica, while NmE showed positive antibacterial activity against Escherichia coli and MRSA. The most susceptible test organism is Salmonella typhimurium followed by Streptococcus mutants. The most resistant strain is Penicillium expansum. While CsE could not affect the growth of Escherichia coli and MRSA, the NmE did.   Data expressed as AU ranged from 2.09 to 4.46 for the sponge extracts in corresponding to the AU resulted by positive control, which ranged from 2.42 to 11.11. * Positive standard All the values resulting from the sponge crude extracts are lower than those obtained by the positive standard; however, it was noticed that CsE had an antibacterial potential against Klebsiella pneumonia while, ketoconazole as a positive control, had no antibacterial activity against this strain so, this is a very promising result.
Entirely, with a careful look at Table 3, it is observed that CsE has a superior antifungal rather than antibacterial activity, and therefore might be classified as a moderate potent antifungal agent and as a mild to moderate antibacterial, and vice versa with NmE, which has only a limited antibacterial activity, and therefore might be classified as weak to a mild antibacterial agent.

Discussion
Bioactive chemicals generated from marine sponges are well-known for their pharmacodynamic potential in the treatment of a variety of disorders. The current high anticancer effect of NmE may be attributable to the fact that it contains nine anticancer chemicals (see Table 1) that triggered apoptosis and suppressed proliferation in cancer cell lines HepG2, MCF-7, and Caco-2. According to the present findings, NmE inhibited the growth of HepG2, MCF-7, and Caco-2 cancer cells dose-dependently with IC50 values of 10.33, 12.48, and 10.52 g/mL, respectively, and induced apoptosis in HepG2, MCF-7, and Caco-2 cells by increasing pro-apoptotic proteins Bax, caspase-3, and cleavage PARP, and decreasing antiapoptotic protein BCL2. Rady and Bashar [1] confirmed the different extracts (methylene chloride, ethyl acetate, acetone, and chloroform) of Negombata magnifica have induced antiproliferation activity against HepG2 cancer cell line within IC50 values 9.14, 10.94, 8.78, and 7.23 µg/mL, respectively; MCF-7 cancer cell lines within IC50 values of 7.65, 6.18, 11.82, and 8.26 µg/mL, respectively; and Caco-2 cancer cells within IC50 values of 12. 67, 9.27, 8.37, and 10.68 µg/mL, respectively, as well as the same extracts induced apoptosis in HepG2, MCF-7, and Caco-2 cancer cell lines through Bax and caspase-3 increase and BCL2 decrease. The ethanolic extract of Negombata magnifica induced intense anticancer activities against Caco-2 and MCF-7 cancer cells with lower values of IC50 (1.09 and 0.37 g/mL, respectively) [19], while the differences in values of IC50 overall indicated that all N. magnifica extracts are highly potential for anticancer properties. However, more research into the effects of the ethanolic extract on the cancer cell cycle and cancer cell-induced apoptosis is needed.
Similarly, CsE present modest anticancer activity could be linked to its GC-Mass study, which revealed that it contains 15 anticancer chemicals (Table 1). In the same context, El-Hawary et al. [39] discovered that the ethanol crude extract of Callyspongia siphonella has antiproliferative action against the MM.1S, OVCAR-3, and HT-29 cancer cells. Moreover, dehydroisophonochalynol and Callyspongenols were extracted from the organic extract of Callyspongia fistularis and showed cytotoxic activities against HeLa and P388 cancer cells [41]. On the other side, Rady and Bashar [1] stated that the different extracts of C. siphonella potentially inhibited the growth of Caco-2, MCF-7, and HepG2 cells with IC 50 values lower than that recorded in the present methanolic extract, as well as inducing apoptosis in Caco-2, MCF-7, and HepG2 cells through an increase in Bax and caspase-3 along with a decrease in BCL2. Ibrahim et al. [21] found the ethanolic extract of C. siphonella was more efficient as an anticancer agent against MCF-7 and Caco-2 cancer cells, than the current methanolic one.
Although each of the current CsE and NmE concentrations was found to inhibit cell proliferation in Caco-2, MCF-7, and HepG2 cancer cells in vitro, only three NmE concentrations (6.25, 12,5, and 25 g/mL) were used in the treatment of Caco-2, MCF-7, and HepG2 cancer cells, whereas five CsE concentrations (156.2, 312.5, 625, 1250, and 2500 µg/mL) were used. As previously stated, the highest concentration of CsE was 100 times that of NmE, which may explain the significant variation in the values of IC 50 for the two extracts for their cell proliferation inhibition properties in HepG2, MCF-7, and Caco-2 cancer cells, where the values of IC50 for CsE (271. 48, 467.19, and 1570.82 g/mL), respectively, were significantly higher than that of NmE (10.33, 12.48, and 10.52 µg/mL), respectively. Therefore, in the current investigation, NmE was chosen to undertake further anticancer screening methods, which revealed more about NmE anticancer efficacy in HepG2, MCF-7, and Caco-2 cancer cells. In HepG2, MCF-7, and Caco-2 cancer cells; however, the in vitro anticancer effects of NmE were supported by a decrease in CDK6, BCL2, and cyclins E1 and D1, as well as an increase in Bax, cleaved PARP, and caspase-3; in addition, the anticancer effects of other previously researched bioactive chemicals of marine species resulted in BCL2, STAT3, and MMP-9 decreases in HepG2 cancer cell line, in addition to the stimulation of G0/G1 cell cycle arrest and ROS buildup [97]. Furthermore, a study using quantitative real-time PCR analysis revealed that the administration of cerebrosides from Asterias amurensis or Acaudina molpadioides decreased BCL2 and Bcl-xL expression while increasing caspase-3, Cyt c, Bax, and caspase-9 expression in the murine sarcoma cell line-180 ascites [98,99]; unlike another in vivo investigation that indicated philinopside E anti-angiogenic activity correlated with the downturn of VEGFR2 signaling in murine sarcoma cancer cells, the current NmE was studied in vitro where it suppressed the expression of BCL2 and increased the expression of cleaved PARP, Bax, and caspase-3 in MCF-7, HepG2, and Caco-2 cancer cell lines. Even though clinical study on the anticancer efficacy of bioactive components from marine sources is highly limited, there is one study in which TBL12 was given to 20 patients with high-risk asymptomatic multiple myeloma. TBL12 was well tolerated, and 9 (45%) of the patients are still on treatment with only one minimal response [100].
As a result, the current potent CsE antibacterial activities can be attributed to its GC-Mass analysis, which contains five antimicrobial molecules. The Callyspongia siphonella methanol extract within its antibacterial components explains antimicrobial activities against Streptococcus mutants, Klebsiella pneumonia, and Salmonella typhimurium, in comparison to the antimicrobial activities of another ethanolic extract of the same sponge, including its two brominated oxindole alkaloids bioactive substances, which undoubtedly played a crucial part in robust antibacterial activity against Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, as well as moderate biofilm inhibitory action against Pseudomonas aeruginosa [39]. Furthermore, when compared to methanolic extract of Callyspongia reticutis, which showed less antibacterial activities in a specific fungicidal against Candida albicans, Penicillium notatum, and Aspergillus niger [101], the present CsE had a wider and more effective antimicrobial activity toward Cryptococcus neoformas, Klebsiella pneumonia, Salmonella typhimurium, Aspergillus flavus, Candida spp., and Streptococcus mutants. Like the antimicrobial activity of CsE, the sponges Ircinia strobilinia, Ircinia felix, and Acanthella carteri displayed wide-spectrum antimicrobial activity toward Candida tropicalis, C. albicans, Bacillus subtilis, Proteus vulgaris, Escherichia coli, and Staphylococcus aureus [102].
Emerging bacterial resistance leads to finding new advanced antibacterial agents [103] specifically those that belong to the natural origin starting from the microorganisms [104,105], plants [55], and other living forms. So, the present moderate NmE antibacterial properties can be attributed to the GC-Mass analysis of it, which contains only four antimicrobial substances as explained in Table 1. In addition, two compounds might have antimicrobial activity: β-Guaiene has been reported as an essential oil extract constituent [61]; and androstan-17-one, 3-ethyl-3-hydroxy-, (5α) has been reported as an Araucaria cunninghamii methanol extract constituent [66]. The current NmE induced positive antibacterial activity against Escherichia coli and MRSA, whereas the ethanol extract of Negombata magnifica [19] and the methanol extract of Negombata cortica [102] had no antimicrobial activity.
Some of the current GC-Mass compounds have additional bioactivity besides their anticancer or antimicrobial activity, for instance, fenretinide and ethyl iso-allocholate, which had anti-COVID-19 activity [11,12]. Although the current investigation assessed the anticancer and antimicrobial activity of NmE and CsE, there are about eighty-one chemical compounds in the present GC-Mass analysis for both NmE and CsE that have not yet revealed any anticancer or antimicrobial properties, and they will require more research to demonstrate their anticancer or antimicrobial activity. Sixty-nine of those chemical compounds have no reports for their biological activity, e.g., ethyl 9,12-hexadecadienoate and 2-heptadecanone, while the other 12 compounds are biologically active, such as desoximetasone, which has shown anti-psoriasis activity [106,107], and ergosta-5,22, which has demonstrated anti-inflammatory activity [108].
In the end, we conclude that because of the worrisome increase in the incidence of cancer and other emerging microbes or viruses, such as SARS-CoV-2, humanity needs to procedure many studies directed towards finding promising solutions to overcome these stubborn diseases. Though chemotherapy is an effective way to treat many types of cancer, chemotherapy treatment carries a risk of side effects. Some chemotherapy side effects are mild and treatable, while others can cause serious complications. Therefore, the trend now is to return to nature to discover more natural remedies that contain natural chemicals, which do not have, or have few, side effects. The marine ecosystem is rich in its components that are characterized as diverse in their species and benefits, which vary from the unseen microorganisms to huge macroorganisms [109][110][111][112].

Conclusions
To our knowledge, the present study represents the first assessment of NmE influence on the cancer cell (HepG2) cycle profile analysis, and ROS production, of the antimicrobial activity of both CsE and NmE. The data generated during the current study emphasize our hypothesis, namely, that NmE has anti-cancer properties that might be promising, and can be developed in the future as an anti-cancer drug for cancer therapy outcomes, and also that CsE acts as a vital source for antimicrobial drug discovery. Therefore, the findings in this study recommend increasing research in this field, which can have solutions in treating many intractable diseases.

Data Availability Statement:
The datasets generated during and/or analyzed through the current study are available from all authors.