Advancing cancer therapeutics: a mini-review of the challenges and opportunities in exploring dinoflagellate-derived bioactive molecules through human cancer cell models

Avances en la terapia contra el cáncer: una mini revisión sobre los retos y las oportunidades en la exploración de moléculas bioactivas derivadas de dinoflagelados a través de modelos de cultivo celular con células de cáncer humano

María del Carmen Osorio-Ramírez¹ & Lorena M. Durán-Riveroll²

¹ Departamento de Biotecnología Marina, Centro de Investigación Científica y de Educación Superior de Ensenada, Baja California, Ensenada, México. ORCID 0000-0002-3467-5941
²SECIHTI-Departamento de Biotecnología Marina, Centro de Investigación Científica y de Educación Superior de Ensenada, Baja California, Ensenada, México. ORCID 0000-0001-8805-7228

Email: lduran@secihti.mx

Osorio-Ramírez, M.A. & L.M. Durán-Riveroll. 2025. Advancing cancer therapeutics: a mini-review of the challenges and opportunities in exploring dinoflagellate-derived bioactive molecules through human cell culture models. Cymbella 11 (2-3): 138-142
DOI: https://doi.org/10.22201/fc.24488100e.2025.11.

INTRODUCTION

Dinoflagellates (Dinophyceae) are a taxonomic group of photosynthetic organisms that play different roles in freshwater and marine ecosystems as primary producers and some build symbiotic relationships with other organisms (De Vera et al. 2018; Orefice et al. 2023). These organisms are well-known producers of toxins such as the saxitoxins, amphidinols, ciguatoxins, brevetoxins, and cooliatoxin. However, these microalgae have been reported as producers of other secondary metabolites with different biological activities and pharmacological potential, because these microorganisms are found in various environmental conditions. Hence, these compounds are produced through several metabolic pathways; therefore, they have diverse molecular targets for illnesses like cancer (Assunção et al. 2017; Cousseau et al. 2020).

These compounds acquire a significant relevance in today’s world due to their cytotoxic, antineoplastic, and antitumor activities, given that cancer is a growing problem of global concern (Assunção et al. 2017). Despite advances in treatments, the incidence of new cases has increased considerably in recent decades, and this group of diseases remains one of the leading causes of death worldwide. One of the most substantial challenges in its treatment is the resistance of cells to standard chemotherapy drugs. This need has driven research to find new molecules with cytotoxic activity, and marine dinoflagellates are a promising source of bioactive compounds (Abd El- Hack et al. 2019). In fact, despite massive research identifying over 300 marine algal toxins, the gaps remain in understanding their mechanisms of action (Gao et al. 2024).

OBTAINING BIOACTIVE COMPOUNDS FROM DINOFLAGELLATES

The search for bioactive compounds in marine and limnological organisms, such as dinoflagellates, represents a complex challenge. This process involves culturing, harvesting, and processing biomass (preferably by freeze-drying), and the use of purified solvents according to the potential components in the mix. The most common solvents are ethanol, methanol, acetone, and water. The reason for using these solvents is that they are polar solvents and they can extract a variety of non-polar and polar compounds with high efficiency. However, the best or optimal solvent depends on the compounds’ polarity in the crude extract (Lee et al. 2024). Once extracts are dry, to ensure the complete removal of any solvent residues, bioassay-guided fractionation is a common approach to discovering natural products. This method involves a series of steps requiring extract fractionation and purification based on biological activity (Kildgaard et al. 2017; Reverter et al. 2020).

However, there are numerous challenges in obtaining bioactive compounds. One of the main obstacles is the amount of available biological material, as the cultivation times are often long, and yields are often low. The efficiency of cell disruption methods is critical to ensure the release of all target compounds (López Rodríguez et al. 2020). In addition, other technical factors can hinder the process, for example, co-extraction of other compounds, use of large volumes of one or more solvents, or poor selection of these (Getachew et al. 2020). Once a pure (or fairly purified) compound is obtained, advanced spectroscopic techniques, such as mass spectrometry (MS), infrared spectroscopy (IRS), and nuclear magnetic resonance (NMR), are used for structural identification. For instance, NMR is a sensitive technique for determining chemical structures, as it is effective with small quantities such as micrograms or even nanograms for analysis. However, it has a significant limitation: the compound must be pure and available for its correct identification (Reverter et al. 2020).

A promising strategy to overcome these challenges involves the adoption of high-throughput technologies capable of detecting molecular targets within minimal sample volumes. Notably, the development of omics approaches — Including genomics, transcriptomics, and metabolomics — has revolutionized the study of secondary metabolites. These techniques facilitate not only the comprehensive analysis of low molecular weight compounds (<2 000 Da) but also enable the mapping of vast chemical diversity through metabolomic and proteomic profiling. Simultaneously, genomic and transcriptomic analyses elucidate the underlying biosynthetic pathways and regulatory mechanisms governing metabolite production (Reverter et al. 2020; Rochfort 2005).

PRINCIPLES OF HUMAN CANCER CELL CULTURE AS RESEARCH MODELS

Human cell culture is one of the most important techniques for cellular and molecular biology research. The process consists of the growth of cells under controlled conditions (in vitro), e.g., temperature, pH, and nutrients. The use of cultured cells is linked to creating model systems of several diseases that have led to studying basic cell biology, mechanisms of action, investigating the toxicity of novel drug compounds (Segeritz & Vallier 2017), and to screen novel chemicals, cosmetics, and other compounds for efficacy and to assess drug cytotoxicity (Gilmor et al. 2023).

In this review we are focused on the research on cancer cells, because this group of illnesses is a leading cause of incidence and death in the world, and the search for new molecules continues to be a challenge (Gilmor et al. 2023). For this reason, exploring bioactive compounds of dinoflagellates can be used to determine the potential of those that possess biological activity against cancer (Pradhan & Ki 2022).

In this context, we will expand on important aspects of human cell cultures and cancer to evaluate secondary metabolites of dinoflagellates as potential treatments. Depending on the cell type (i.e., the cancer type), some of the discussed parameters may vary. This is particularly true for protocols involving activation, maintenance, and cryopreservation. In this context, cell viability is considered the most critical factor, as experimental results and their reproducibility, the number of passages, and the activation process post-thaw all depend on cells being in optimal condition (Geraghty et al. 2014).

IMPLEMENTING BEST PRACTICES FOR OPTIMAL HUMAN CELL CULTURE

Good practices in the cell culture laboratory are necessary to evaluate different biological activities. These include: 1) safe handling of human cell lines; 2) safe and reliable experimental designs and procedures; 3) aseptic technique in the handling of cells, an organized work environment, and sufficient reagents and media for cell culture; 4) the correct selection of cell lines to be used; and 5) the proper conditions under which cells are maintained, and the know-how of subculture maintenance and cell cryopreservation processes (Dubovi & Rankin 2023; Gilmor et al. 2023; Segeritz & Vallier 2017; Weiskirchen et al. 2023; Zhao 2023).

In addition to these considerations, it is important to record the number of passages performed from the start of the culture. The consequence of not having these records is that some cell lines are unsuitable for experimental work beyond a certain number of passages, such as a high number of passages and a prolonged time between subcultures, which can lead to mutations, duplications, and epigenetic changes. For this reason, it is important to certify the cells from the start of the experiment, as these changes can alter the morphology and proliferation rate and increase or decrease the cell divisions over time, affecting cell behavior and overall cell health. Consequently, these changes could affect reproducibility in experiments and results (Geraghty et al. 2014). Another problem is cell misidentification or contamination. This includes the use of illegible labels and mislabeling of cell culture vessels during manipulation. The misidentification of cell lines has resulted in erroneous results, interpretations and publications (Babic et al. 2019).

The correct selection of cell lines is important for experimental design. Understanding the fundamental classification of cell types is essential, as cells are broadly categorized into three groups:

a) stem cells are considered undifferentiated cells capable of self-renewal and multilineage differentiation, e.g. embryonic stem cells (Zumwalt & Reddy 2020);

b) primary cells or fine cell lines: these cells are directly taken from a body or tissue. As the name implies, they can only be cultured for a finite number of passages, e.g., fibroblasts (Godbey 2014);

c) immortalized cells or continuous cell lines: these are primary cells that have been genetically modified for infinite culture, e.g., human cancer cell lines (Rahman et al. 2016).

CELL VIABILITY AND PROGRAMMED CELLULAR DEATH DETECTION ASSAYS

In vitro studies are essential steps in the search for new therapies against cancer, and specific mechanisms of action have been found. Therefore, depending on the biological activity of interest, such as cytotoxic, antineoplastic, or antitumor, different assays are employed to determine whether these compounds are bioactive. However, it is necessary to first define some concepts:

Viability, which is the capacity to perform the essential metabolic processes to maintain structural and functional integrity, is represented as the percentage of healthy cells in the population.

Proliferation is a process that leads to an increased number of cells through cell division.

Cell death refers to irreversible conditions or processes in non-viable cells, including apoptosis, autophagy, and necrosis.

Cytotoxicity is a cellular injury that may be reversible or progress to cell death (Adan et al. 2016).

Cell death includes the measurement of dead cells (cytotoxicity assay), quantification of live cells (viability assay), and finally, determination of the accurate mechanisms of death (Cousseau et al. 2020). For this, various assays can be used, such as dye exclusion, one of the most common being trypan blue dye exclusion. Colorimetric assays, including lactate dehydrogenase, 3-(4,5-dimethylthiazol-2-yl)-5-3(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and luminometric assays, such as the ATP assay, are used to determine the viability of cells and proliferation assays (Aslantürk 2018). According to the many proteins involved in the apoptosis signaling cascade, there are different apoptosis assays to assess cell death. Two or more assays should be used to detect apoptosis accurately since many overlapping features exist between apoptosis and necrosis. There are six major groups of available assays to detect apoptosis: membrane alteration, mitochondrial assays, cytomorphological alterations, DNA fragmentation, detection of caspases, cleaved substrate, inhibitors and regulators, and detection of apoptosis. Among those assays, early apoptosis could be detected through annexin V or acridine orange and ethidium bromide (AO/EB), which is based on the loss of cellular membrane integrity. Also, many assays can detect the midphase of apoptosis using caspase activation and molecular processing, including the key enzyme poly (ADP-ribose) polymerase (PARP). The late phase of apoptosis could be detected with DNA fragmentation assays. Combinations of these assays allow us to identify the mechanisms of apoptosis induction after a specific stimulus (Alshiraihi & Kato 2023; Azqueta et al. 2022).

FINAL CONSIDERATIONS

The exploration of secondary metabolites from dinoflagellates offers innovative opportunities for discovering novel cancer therapeutics. However, the success of such research relies heavily on the chemical identification of the compounds of interest. As emphasized in this work, this process requires obtaining purified compounds from cell cultures, coupled with the rigorous application of human cell culture techniques to enable reliable evaluation of their biological activities and mechanisms of action. Therefore, integrating appropriate practices in metabolite extraction, handling both cancerous and non-cancerous cell lines, and employing specific assays to assess viability, proliferation, and cell death is essential to ensure reproducible results and accurately propose mechanisms of action for dinoflagellate-derived metabolites. Ultimately, cell culture serves as a critical bridge connecting marine biodiversity to the development of future pharmacology strategies based on natural products.

REFERENCES

Sometido: 28 de abril de 2025
Revisado: 28 de mayo de 2025
Corregido: 17 de septiembre de 2025
Aceptado: 19 de septiembre de 2025