In Vitro Anticancer Activity of Five Marine Seaweeds Extract From Egypt Against Human Breast and Colon Cancer Cell Lines


 Ulva fasciata, U. lactuca, Amphiroa anceps, Corallina mediterranea, and Sargassum filipendula extract were screened for their in vitro anticancer activity against human breast adenocarcinoma cell line (MCF-7) and colorectal carcinoma cell line (HCT-116). Algal extracts exhibited significantly dose-dependent anticancer activity without significant cytotoxicity against normal human fibroblasts. The maximum inhibitory percentages against MCF-7 and HCT-116 cell lines were recorded by U. lactuca (88.5 ± 1.08 %) and A. anceps (86.1 ± 2.88 %) extracts. Algal extracts showed cytotoxic effect against MCF-7 cell line with fifty percent inhibitory concentration (IC50) values ranging from 3.54 ± 1.2 to 21.2 ± 1.1 µg mL-1. The seaweeds extract has a less cytotoxic effect against HCT-116. FTIR and GC-MS analyses of the extracts indicated that the anticancer potential of the tested seaweeds may be returned to the presence of various anticancer compounds such as palmitic acid, oleic acid, retinoic acid, dihydroactinidiolide, thiosemicarbazide, diisobutyl phthalate, and phytol. Thus, seaweed extracts may be a promising natural source of safe anticancer agents against human breast and colon cancers.

The dried seaweed sample (25 g) was extracted using 250 ml methanol: hexane (1:1) at 65°C in the Soxhlet apparatus. The extraction procedure was repeated until the extract was clear (most compounds were completely extracted). The extraction process of U. fasciata, U. lactuca, A. anceps, C. mediterranea, and S. lipendula has run for 22,24,16,18, and 16 hours, respectively. The liquid extracts were then cooled and concentrated using a rotary evaporator at 30-45°C. Extracts were stored in labeled sterile screw-capped bottles at 4°C until it was tested. Methanol and hexane were of analytical grade and were purchased from Sigma Aldrich, USA. (Sigma-Aldrich, USA). Both media were supplemented with antibiotic-free 10% Fetal Bovine Serum (FBS, Sigma., USA), 100 U mL − 1 penicillin, and 2 mg mL − 1 streptomycin. The cells were maintained at 37°C in 95% relative humidi ed atmosphere containing 5% CO 2 .

Anticancer activity of organic extract of seaweeds using Sulforhodamine B (SRB) assay
After seaweed extraction, we get rid of methanol: hexane solvent and dissolve it in DMSO for further analyses. Various concentrations (0, 12.5, 25, 50, and 100 µg mL − 1 ) of organic seaweeds extract were prepared for the following in vitro anticancer assay. 0.5% (v/v) DMSO was used as a control sample.
In vitro anticancer assay of the algal extracts against MCF-7, HCT-116, and broblast normal cells were performed using a SRB assay as it is a sensitive method for evaluating cytotoxic activity (Skehan et al., 1990). All determinations were replicated three times in a day and the mean values were recorded. IC 50 (the extract concentration that resulted in 50% of cell growth inhibition) was calculated using the Prism program (Graph Pad prim version 7.0 software at a 95% con dence limit.). Also, the percent of cell inhibition (death) was calculated using the following formula: Percent cell inhibition (%) = 100 -(Absorbance of sample/Absorbance of control × 100) 2.5. Identi cation of phytochemicals in the algal extracts

Fourier transforms infrared spectrometry (FTIR) analysis
The FTIR characterization of powdered seaweeds was carried out with a Mattson 5000 FTIR spectrometer. Two mg of seaweed powder was ground with approximately 100 mg of KBr in a mortar until the mixture was formed in ne particles and then pressed as a disc (10 mm) for transforming IR spectral measurements in the frequency range of 4000 − 400 cm − 1 at 25 o C.

GC-MS analysis
Gas Chromatography-Mass Spectrometry (GC-MS) analysis of methanol: hexane extracts of the tested seaweeds were performed using Varian Chrompack CP-3800 GC/MS/MS-2000 equipped with split-splitless injector and DB-5.625 GC column (30 m × 0.25 mm i.d., 0.25 µm lm thickness). An electron ionization system with ionization energy of 70 eV was used. The injector temperature was set at 275°C for 5 min with injection of 5 µl volumes of extracts. A linear temperature program was adapted to separate the different components as follows: initially, the column maintained at 40°C for 3.5 min, ramped at a rate of 10°C min − 1 to 250°C at which it was held isothermal for 10 min; a second ramp (20°C min − 1 ) was then applied to 280°C and held isothermal for 5 min, the total run time was 75.5 min. The temperatures of the transfer line and ion source were maintained at 250°C and 200°C, respectively. Identi cation of the compounds was performed based on the comparison of their relative retention time (Rt) and mass spectra with those of the National Institute of Standards and Technology (NIST) and Wiley MS spectra libraries data of the GC/MS system. Also, the percent relative peak area of the identi ed components was estimated.

Statistical analysis
Data were analyzed by two-way analysis of variance (ANOVA) using SPSS version 22. The difference was considered signi cant when p < 0.05. All determinations were carried out in triplicates and the results are expressed as a mean ± standard deviation (SD).

Cytotoxicity activity of seaweeds extracts
Different concentrations (12.5, 25, 50, and 100 µg mL − 1 ) of organic extract (methanol: hexane) of U. fasciata, U. lactuca, A. anceps, C. mediterranea, and S. lipendula were applied to SRB assay to test their anticancer potential. Also, IC 50 was calculated for all extracts against both MCF-7 and HCT-116 cell lines. To evaluate the cytotoxic effect of the tested extracts against both MCF-7 and HCT-116 cell lines, the activity of these extracts was also examined using human broblast cells. All the tested extracts did not affect the viability of normal broblast cells.

GC-MS analysis
The chemical constituents of the crude extracts of the tested seaweeds were analyzed using GC-MS, and the identi ed compounds with their molecular formula, peak area, and the chemical group were presented in Tables 4-8. A total of 20, 12, 12, 19, and 23 different compounds were identi ed in U. fasciata, U. lactuca, A. anceps, C. mediterranea, and S. lipendula, respectively. The identi ed compounds in the tested seaweed extracts belong to eleven main chemical groups (esters, hydrocarbons, terpenes, ketones, fatty acids, alcohols, steroids, aldehyde, amides, amines, and phenols). 8-Heptadecene, Diisobutyl phthalate, and Octadecanal, 2-bromo-were the major compounds in extracts of U. fasciata, U. lactuca, and S. lipendula, respectively. Whereas 1,2-Benzenedicarboxylic acid, diisooctyl ester was the major compound in extracts of both A. anceps, and C. mediterranea.     Bioactive compounds of seaweeds such as terpenes, and steroids were highly diverse compared to higher plants (Manilal et al., 2013). Terpenes were detected in all the tested seaweeds except in A. anceps extract while steroids were found in the extracts of U. fasciata, and A. anceps. Most of these compounds expressed important various biological activities such as anticancers, antivirals, antioxidants, and anti-in ammatories. Some of the identi ed compounds in the extracts of the tested seaweeds like palmitic acid, oleic acid, retinoic acid, dihydroactinidiolide, thiosemicarbazide, diisobutyl phthalate, and phytol having anticancer activities ( Retinoic acid was found in S. lipendula extract. Retinoic acid is an active metabolite of vitamin A that was found to reduce breast (MCF-7), liver (HepG2), lung, prostate, bladder, ovarian, oral, and skin cancers via cell cycle inhibiting protein (p27) and cell cycle regulator (Cdk5) with cyto-differentiating, antiproliferative, and apoptotic effects that are mediated by activation of the nuclear hormone retinoic acid receptors RARα, RARβ and RARγ (Garattini et  GC-MS analysis of the methanol: hexane extract of C. mediterranea revealed the presence of thiosemicarbazide. It has an anticancer potential similar to that of triapine and methisazone (anticancer drugs). This activity was related to its ability to inhibit ribonucleotide reductase that is involved in the rate-limiting This study sheds light on the anticancer activity of Ulva fasciata, Ulva lactuca, Amphiroa anceps, Corallina mediterranea, and Sargassum lipendula collected from Egyptian coasts against human breast adenocarcinoma cell line (MCF-7) and colorectal carcinoma cell line (HCT-116). The crude extract of U. lactuca and A. anceps showed the maximum activity against MCF-7 and HCT-116 cell lines, respectively. The tested seaweeds contain different anticancer compounds that will make the seaweeds a promising source for cheap and safe anticancer drugs in the future. Therefore, puri cation of active substances from these seaweeds and the mechanisms by which the seaweeds induce anti-proliferation activity needs to be considered in further studies to facilitate the future potential application of these novel natural anticancer agents.

Declarations
Funding The authors received no nancial support for the research, authorship, and/or publication of this article.
Compliance with ethical standards Con ict of interest The authors declare that they have no con ict of interest.
Ethical standards This article does not contain any studies with human or animal subjects.
Informed consent There is no patient care involved in this article. Authors Contributions JM contributed to the study design and to collect data. MD contributed to the study conception and design. AS contributed to prepare materials and collect data. FW contributed to collect and analyze data. All authors wrote, read, and approved the nal manuscript.
Availability of data and materials Data sharing is not applicable to this article as no datasets were generated during the current study. The data that support the ndings of this secondary study are available from primary studies which were all cited.