Macroalgae have been used for millennia as food sources in different countries. Its nutritional interest is based on high levels of dietary fibers, polyunsaturated fatty acids, vitamins, and minerals. Due to the great biodiversity in the oceans, marine organisms, including macroalgae, are a valuable source of minerals, as well as a reservoir of new biologically active compounds [1] [2]. Antarctic macroalgae are adapted to an extreme environment, presenting a diversity of metabolites and, therefore, being considered promising sources of compounds with anticancer activity [3] [4].

Due to side effects promoted by many cancer therapies and also tumor cell resistance to conventional drugs, the search for compounds of natural origin is still being investigated [5] [6]. Anticancer chemotherapeutic drugs are currently developed to inhibit the growth of tumor cells, but chemotherapy may fail due to the development of drug resistance promoted by apoptosis inhibition mechanisms. Apoptosis is a regulated cell death program that plays an essential role in the development and maintenance of tissue homeostasis by eliminating unnecessary or mutated cells. As regulation of apoptosis relies on multiple cell signaling mechanisms, cancer cells use number of different strategies to suppress a protective apoptotic response. Phosphatidylserine exposure on the cellular surface occurring at the initial or early apoptosis stages promotes membrane blebbing, nuclear fragmentation, decreased cellular volume and a formation of apoptotic bodies [7] [8] [9]. It can be further defined as cell death accompanied by the activation of caspase proteases [10]. Impairment of this mechanism generates abnormal cellular proliferation and the accumulation of genetic alterations that may contribute to drug resistance [11].

Identifying the type of cell death after treatment may contribute to selecting the best therapy modality. Therefore, a better understanding of the cell death signaling pathways can improve the efficacy of cancer therapy and bypass resistance [11] [12] [13]. Bioactive compounds that induce apoptosis in cancer cells can be used as agents for cancer chemoprevention and/or chemotherapy [14]. Accumulating evidence suggests that bioactive compounds extracted from algae produce anticancer effects through multiple mechanisms of action, including inhibition of cancer cell growth, invasion, and metastasis, and through the induction of apoptosis [15] [16].

Until present, there is no literature demonstrating reduction of cellular viability of colorectal cancer cells after treatment exposition to extract fractions of macroalgae D. anceps and the mechanism of cell death after de exposition of crude extract. However, some isolated compounds taken from brown Antarctic macroalgae such as fucoidan and fucosterol have been investigated with promising antitumor activity [17] [18].

In this context, the aim of this study was to evaluate mechanisms of cell death induced after treatment of colorectal cancer cell line HCT 116 with chloroform crude extract of D. anceps. In this study, the extract was fractioned and the chemical characterization of the crude extract and its fractions were further investigated.

Seaweeds are used as functional food and medicinal herbs, and have a long history of applications in Asian countries. Since, crude extracts or partially purified or isolated compounds from many species of brown, green, and red algae have been tested for their antitumor activities. These studies have indicated that marine algae constitute a promising source of novel compounds with potential as human therapeutic agents. In particular, macroalgae have been considered as a source of new bioactive compounds [16].

In this study, GC identified many compounds in the chloroform extract and fractions of the Antarctic macroalgae D. anceps. Fucosterol is a sterol commonly seen in algae and has been attributed to several biological activities such as anti-inflammatory, antioxidant, hepatoprotective and anticancer [18] [20]. Khanavi et al. [21] isolated fucosterol from brown alga Sargassumangustifolium and showed an anticancer activity against HT29 colorectal and T47D breast cancer lines. Among them, fucosterol has been investigated for its anticancer properties [22]. Therefore, other unidentified compounds that may act in isolate or through synergism mechanism may be involved in the anticancer potential of D. anceps. Recently, in our studies, we identified a series of steroids in Antarctic macroalgae using liquid chromatography coupled with tandem mass spectrometry [22]. The chemical composition of extract may vary according to extraction technique and methodology.

Bicyclo [3.1.1] heptane, 2,6,6-trimethyl (pinane) is a compound from the terpene class and was identified by Santana et al. [23] as a constituent of the essential oil of S. terebinthifolius, showing cytotoxic properties against human melanoma (A2058), breast adenocarcinoma (MCF7), leukemia (HL-60) and cervical carcinoma (HeLa) cell lines. Boyom et al. [24] identified the presence of alpha-pinene in the essential oil composition of Cleistopholispatens and Uvariastrumpierreanum, both showing antimalarial effect. Maruthupandian & Mohan [25] identified the presence of acid n-hexadecanoic with biological activities such as antioxidant, nematicide, antiinflammatory, hypocholesterolemic, cannabinoid. Members of the vitamin E family are hydrophobic fat-soluble compounds found in a variety of food sources such as corn oil, peanuts, vegetable oils, fruits and vegetables consumed through diet. The health benefit of consuming vitamin E, present in present in F2, through diet or supplementation is believed to be for its antioxidant properties. Vitamin E protects cells from cell damage caused by free radicals that damage cell membranes through lipid oxidation, leading to DNA damage and cancer development [26].

The compound 3-methyl-2-cyclopenten-1-one and oleic acid observed in fractions 3/4 and 6, respectively, were found in plant extracts ofFoeniculum vulgare andshowed antimicrobial activity [27]. Phytol, the open chain diterpene, presented antimycobacterium activity [28] was identified in F2, F7, F9/10, F11/12.

Based on popular knowledge, algae have been investigated for their biological activities. However, few studies have reported the chemical composition and biological activity of Antarctic phytoflora [29] [30]. Clinical tests, both in vivo and in vitro, are performed to confirm the effectiveness of natural compounds [31].

Several studies have reported that compounds extracted from seaweed may be effective anticancer agents [16] [32] [33]. The DACExt and its fractions presented cytotoxicity against the colorectal cancer cell line HCT 116 at the analyzed times. The effect of the treatment on IC₅₀ concentration (37.7.7 µg∙mL⁻¹) of the cell line after 24 hours of exposure to the extract was also analyzed by scanning electron microscopy and, in addition to the reduction in the number of cells, loss of cell adhesion, cytoplasm retraction was found. As the concentration of the extract increased (75.4 µg∙mL⁻¹), sparse colonic formation was observed. Cytotoxic activity of extracts of different Antarctic seaweeds, C. jacquinotii, I. cordata, H. grandifolius, and P. endiviifolia were observed from our research group [4] [34] [35]. The anticancer potential of several macroalgae species has been demonstrated against colorectal cancer cells lines such as HT 29 e HCT 116 [14] [30] [31], Caco-2, WiDr, HCT116, and DLD-1 [36] [37] [38] [39]. This was the first study to demonstrate the anticancer activity extract fractions of macroalgae D. anceps against colorectal cancer HCT 116 cell line. In this way, D. anceps was shown to be a promising source of compounds with anticancer activity and target for new studies.

The elucidation of the molecular mechanisms involved in the pathogenesis of colorectal cancer may reduce incidence, as well as the morbidity and mortality. Thus, the identification of markers and the mechanisms that inhibit apoptosis and stimulate cell proliferation of transformed cells is of fundamental importance to reduce the prevalence of this disease.

In this study we evaluated by flow cytometry the effects on cell cycle of exposing colorectal cancer cells to different concentrations of DACExt and the mechanism of cell death. We verified an increase in the number of multinucleated cells and phase M progression, acquiring extra copies of the genome without making cytokinesis, perhaps by checkpoint failure in phase M. Increased ploidy may be causing genetic instability, which may be responsible for the cell being referred for apoptosis.

Tumors are complex systems that include heterogeneous cancer cells with markedly different sizes and genomic contents [40]. The bulk of cancer cells within the majority of solid tumors (~90%) are aneuploidy. Such cells have an alteration (often gain) of chromosome number that is not a multiple of the diploid (2n) component. Cancer stem cells are often much smaller than the bulk of cells, whereas polyploid cells are larger than bulk cells by virtue of their increased ploidy (>4n). Not surprisingly, cultures of established solid tumor-derived cell lines are also heterogeneous [41]. Similar results were found and described by Lu et al. [42]. In this study, reversine, a small molecule that was originally identified to induce dedifferentiation of murine myoblasts into multipotent progenitor cells, demonstrated anticancer activity and generated an increase in the number of multinucleated cells, suggesting that tumor cells can escape from mitosis without cell division.

Giant multinucleated cell formation is a cell phenotype due to verification failure at cell cycle checkpoints, mainly in DNA structure verification and mitotic spindle assembly. Failure to interrupt the cycle to phase M leads to an attempt at aberrant chromosomal segregation, which triggers the activation of the apoptotic mechanism. Due to damage to the mitotic spindle, cells are able to escape the M-phase without segregation of sister chromatids and cytokinesis, a process known as “mitotic slippage”. Cells stop at G1 because they activate a p53-mediated checkpoint. In the presence of p53, p21 activates polyploidy, which generates cell death to prevent late mitosis and aneuploidy errors [41].

The mechanism of cell death identified was initial apoptosis. Cell death was evaluated by several studies using different compounds and extracts from algae in a wide variety of cell models. Laminarin from brown seaweed Laminariadigitata induced apoptosis in HT-29 colon cancer cells (5 mg∙mL⁻¹) in a dose-dependent manner [43]. The ethanolic extract of algae Undariapinnatifida induced the reduction of cell viability of the HCT 116 cell line (2% of cell extract reduces in 80% cell viability) by apoptotic mechanism [44]. Fucoindan, sulfated polysaccharides purified from the brown algae Fucusvesiculosus, induced apoptosis in the HT 29 adenocarcinoma line (5 - 20 µg∙mL⁻¹) [14]. Fucoxanthin, a carotenoid present in brown seaweeds, has shown to suppress the level of Bcl-2 protein and induce apoptosis in human colon cancer cells Caco-2, HT-29 and DLD-1. After exposure at concentration 15.2 µM, fucoxoxantin inhibited, respectively, the viability of the cells in 14.8%, 50.8% and 29.4%. In addition, fucoxanthinol, fucoxanthin metabolite, also had a stronger anti-proliferative effect than fucoxanthin on Caco-2 human colon cancer cells [45] [46].

Most reports studying cytotoxic effects of macroalgae present a range of IC₅₀ from 5 µg/mL to 869 µg∙mL⁻¹. Various therapeutic compounds from seaweed are able to induce apoptosis through different pathways and molecular mechanisms. Recent cancer preventive studies have been focused on designing anticancer drugs from natural sources because of fewer side effects as compared to synthetic anticancer drugs [16] [32] [17] [47].

The inhibition of apoptosis in colorectal cancer cells enhances tumor growth, promotes neoplastic progression, and confers resistance to cytotoxic anticancer agents. Thus, bioactive compounds that induce apoptosis in cancer cells can be usefull as agents for cancer chemoprevention and/or chemotherapy [17].

This study was the first to demonstrate the reduction of the cellular viability of colorectal cancer cells through the induction of apoptosis pathway after treatment with chloroform extract of the brown Antarctic macroalgae D. anceps, suggesting that this macroalgae is a promising candidate for the isolation of molecules with anticancer activity. Fractionation by bioassay-guided is an interesting tool to separate, isolate and purify natural compounds with anticancer activity. Some isolated compounds such as fucoidan and fucosterol have been investigated with promising antitumor activity. However, more studies are necessary in order to elucidate the mechanism of action of macroalgae and the pathways of induction of apoptosis.

The authors thank Brazilian Research Funding Program (CAPES), University of Caxias do Sul (UCS), Brazilian Algae Research Group (RedeAlgas), Antarctic Brazilian Program (PROANTAR) for financial support for the development of this work.

The authors Rafaele Frassini, Daniela Steffens, Sidnei Moura, Aline Paternostro Martins, Cesar Aguzzoli, Pio Colepicolo, Mutue Toyota Fujii, Nair S. Yokoya, Cláudio Martin Pereira de Pereira, Ana Cláudia Phillipus, Miriam de Barcellos Flakenberg, João Antonio Pegas Henriques and Mariana Roesch-Ely, declare to be responsible for drafting the manuscript entitled “Desmaretia anceps Montagne Against Colorectal Cancer Cells: Cytotoxic Activity and Proapopitotic Effects.”

The authors Rafaele Frassini, João Antonio Pegas Henriques and Mariana Roesch-Ely participated in project design, in the execution of all experiments and data collection. Daniela Steffens assisted in the cytometry, cell cycle assay and statistical analysis. Aline Paternostro, Mutue Toyota Fujii, Nair S. Yokoya and Pio Colepicolo were responsible for the collection, storage and botanical identification of seaweed Desmarestiaanceps. Cesar Aguzzoli contributed to the SEM analysis. Cláudio Martin Pereira de Pereira, Ana Cláudia Phillipus, Miriam de Barcellos Flakenberg helped in chemical characterization, vacuum liquid chromatography and nuclear magnetic resonance.

The authors participated in data analysis, discussion of results and writing of the manuscript. The manuscript was written, read and approved by all the authors. The material has not been published, in whole or in part, and it also is not under consideration for publication elsewhere.

The authors declare no conflicts of interest regarding the publication of this paper.

Frassini, R., Steffens, D., Moura, S., Aguzzoli, C., Martins, A.P., Colepicolo, P., Fujii, M.T., Yokoya, N.S., De Pereira, C.M.P., Phillipus, A.C., De Barcellos Falkenberg, M., Henriques, J.A.P. and Roesch-Ely, M. (2022) Desmarestiaanceps Montagne (Phaeophyceae) against Colorectal Cancer Cells: Cytotoxic Activity and Proapoptotic Effects. Advances in Biological Chemistry, 12, 228-245. https://doi.org/10.4236/abc.2022.126019