Cancer continues to pose a significant worldwide health issue, accounting for countless fatalities each year. In spite of notable progress in traditional cancer treatments like chemotherapy, radiotherapy, and targeted therapies, these methods frequently entail serious side effects and the potential for drug resistance (Newman & Cragg, 2020). Consequently, the investigation of natural bioactive substances has garnered significant attention within the realm of oncology research. Within the diverse array of natural origins, both marine and freshwater life forms, especially fish, have surfaced as a noteworthy source of bioactive substances that exhibit considerable anticancer properties (Mayer et al., 2019). Bioactive compounds sourced from marine environments, such as peptides, polysaccharides, and alkaloids, have shown significant cytotoxic properties against a range of cancer cell lines. These substances demonstrate a variety of mechanisms through which they operate, including the promotion of apoptosis, the suppression of angiogenesis, and the alteration of immune responses (Zhou et al., 2021). Antimicrobial peptides (AMPs) derived from fish, for example, specifically hone in on cancer cells by compromising their membranes, positioning them as intriguing prospects for alternative cancer therapies (Wang et al., 2022). In a similar vein, fucoidans and polysaccharides derived from algae associated with fish demonstrate the ability to impede tumour development by disrupting the adhesion, invasion, and immune evasion tactics employed by cancer cells (Apostolidis et al., 2018).

In the past few years, alkaloids sourced from microbes associated with fish have garnered significant interest due to their powerful anticancer properties. These substances disrupt the DNA replication of cancer cells and hinder the progression of the cell cycle, ultimately inhibiting tumour growth (Hamed et al., 2019). Moreover, omega-3 fatty acids along with chitin derivatives sourced from fish have demonstrated the ability to prevent cancer, thereby enhancing the possibilities of utilising bioactive compounds derived from fish in cancer treatment (Rahman et al., 2018; Li et al., 2021). From a molecular standpoint, compounds derived from fish demonstrate their anticancer properties via multiple pathways. They amplify apoptotic signalling through the upregulation of pro-apoptotic genes like Bax and caspase-3, consequently initiating programmed cell death in cancerous cells (Nguyen et al., 2022). Additionally, they impede angiogenesis through the downregulation of vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9), consequently limiting the development of new blood vessels that are crucial for tumour proliferation and metastasis (Jin et al., 2021). Moreover, bioactive compounds derived from fish have been discovered to boost the functionality of the immune system, resulting in enhanced detection and eradication of cancer cells (Singh et al., 2020). In spite of these encouraging results, the practical use of bioactive compounds sourced from fish encounters numerous obstacles. The large-scale extraction and purification of these compounds necessitate sophisticated biotechnological methods, and a more thorough assessment of their pharmacokinetics and bioavailability in humans is essential (Kim et al., 2021). Nonetheless, as the focus on natural and targeted treatments in cancer care continues to expand, bioactive compounds sourced from fish present significant opportunities for the advancement of future pharmaceuticals.

Bioactive Compounds from Fish and Their Anticancer Properties

The oceanic ecosystem has been acknowledged for an extended period as a rich reservoir of biologically potent substances that hold considerable promise for therapeutic applications. Fish and their related microbial populations have demonstrated the ability to generate a range of bioactive compounds that display potent anticancer effects. These substances fall into various classifications, such as antimicrobial peptides, polysaccharides, and alkaloids, with each category demonstrating distinct cytotoxic impacts on cancerous cells.

Antimicrobial peptides (AMPs) sourced from fish have emerged as intriguing prospects in the realm of cancer treatment, owing to their capacity to specifically hone in on malignant cells while leaving healthy tissues unharmed. These peptides operate by compromising the structural stability of cancer cell membranes, resulting in heightened permeability and subsequent cell lysis (Wang et al., 2020). Research has shown that antimicrobial peptides sourced from fish display strong cytotoxic effects on a range of cancer cell lines, such as those found in breast, lung, and colon cancers, while causing little harm to healthy cells (Zhang et al., 2022). The discerning characteristics of these peptides render them appealing for the advancement of precise cancer treatment strategies. A different category of bioactive substances exhibiting anticancer properties includes fucoidans and polysaccharides, which are typically derived from algae associated with fish and various other marine origins. These substances have demonstrated the ability to suppress tumour development by altering immune responses and disrupting the  adhesion  and invasion  abilities of cancer  cells  (Apostolidis et al., 2018).

Polysaccharides sourced from fish not only inhibit tumour advancement but also bolster the body's inherent immune responses by activating immune cell function (Singh et al., 2020). The combined mechanism—exerting direct cytotoxic impacts on cancerous cells while simultaneously bolstering the immune system—further underscores the significance of polysaccharides sourced from fish in cancer-related applications.

Alkaloids, a distinct category of bioactive compounds sourced from fish, hold considerable importance in cancer treatment by influencing DNA replication and the progression of the cell cycle. A significant number of these alkaloids are generated by microbial populations linked to marine fish, and they have been documented to demonstrate potent inhibitory effects on cancerous cells (Hamed et al., 2019). These compounds disrupt DNA replication, trigger oxidative stress, and facilitate programmed cell death in a range of cancer cell varieties (Rahman et al., 2018). The capacity of alkaloids sourced from fish to specifically interfere with the functions of cancerous cells, while sparing healthy cells to a significant extent, highlights their promise as agents for chemotherapy. Alongside these significant categories of bioactive compounds, the exploration of fish lipids and omega-3 fatty acids has also been undertaken regarding their potential in cancer prevention and therapy. A multitude of research has shown that omega-3 fatty acids sourced from fish oil exhibit both anti-inflammatory and pro-apoptotic characteristics, playing a significant role in their anticancer properties (Gupta et al., 2021). These lipids influence the metabolic processes of cancer cells, diminish tumour formation driven by inflammation, and improve the effectiveness of standard cancer treatments when administered together (Kim et al., 2021). The variety of bioactive substances found in fish highlights their significant promise in cancer treatment. These organic compounds present a viable substitute for traditional chemotherapy drugs, potentially minimising adverse effects and addressing drug resistance in the treatment of cancer. The upcoming segment delves into the intricate molecular processes by which these substances demonstrate their anticancer properties.

Mechanisms of Action of Fish-Derived Bioactive Molecules

Bioactive compounds sourced from fish demonstrate their anticancer properties via numerous molecular pathways, such as triggering apoptosis, suppressing angiogenesis, and influencing immune system functions. These processes are essential for hindering cancer advancement and enhancing the effectiveness of treatments. A key mechanism by which bioactive compounds derived from fish fight cancer is by triggering apoptosis, the process of programmed cell death that effectively removes damaged and malignant cells. A variety of peptides and alkaloids sourced from fish stimulate both intrinsic and extrinsic apoptotic pathways through the upregulation of pro-apoptotic genes, including Bax and caspase-3 (Nguyen et al., 2022). These substances compromise mitochondrial stability, resulting in the release of cytochrome c and the subsequent initiation of caspase cascades, which ultimately activate apoptosis in cancerous cells (Fernando et al., 2019). The capacity to specifically trigger programmed cell death in malignant cells while preserving healthy tissues renders compounds sourced from fish exceptionally appealing for cancer treatment strategies.

A vital anticancer strategy encompasses the suppression of angiogenesis, which is the mechanism through which tumours generate new blood vessels to support their expansion and spread. Bioactive compounds sourced from fish have been discovered to inhibit the expression

of angiogenic indicators, notably vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9), both of which are crucial in the process of blood vessel development (Jin et al., 2021). Through the downregulation of these angiogenic elements, compounds sourced from fish proficiently deprive tumours of their essential blood supply, resulting in inhibited growth and ultimately, tumour regression (Moreno et al., 2022). This system underscores the promise of fish-derived bioactive compounds in the realm of anti- angiogenic treatments for cancer. Alongside the inhibition of apoptosis and the suppression of angiogenesis, bioactive substances sourced from fish also influence immune system functions, boosting the body's inherent capacity to identify and eradicate cancerous cells. Fish-derived polysaccharides and peptides have been shown to enhance the generation of immune cells, including natural killer (NK) cells and cytotoxic T lymphocytes, which are essential in identifying and eliminating cancerous cells (Singh et al., 2020). Certain molecules sourced from fish have been discovered to inhibit immune checkpoint proteins, which in turn hinders the ability of cancer cells to evade the immune system and enhances the efficacy of immunotherapeutic strategies (Hassan et al., 2020).Additionally, recent studies have uncovered the significance of compounds sourced from fish in the realm of epigenetic regulation, demonstrating their ability to affect gene expression without modifying the DNA sequences themselves. Certain marine-derived peptides and polysaccharides have demonstrated the ability to alter histone acetylation profiles, resulting in the activation of tumour suppressor genes while simultaneously repressing oncogenes (Rodrigues et al., 2018). This system offers an extra dimension of treatment possibilities, given that epigenetic alterations are capable of being reversed and can be aimed at reinstating typical cellular operations in malignant tissues. Although the cancer-fighting attributes of bioactive compounds sourced from fish are thoroughly established, numerous obstacles persist in converting these discoveries into practical clinical uses. The retrieval and extensive manufacturing of these compounds necessitate sophisticated biotechnological methods, and their bioavailability along with pharmacokinetics must be fine-tuned for human intake (Kim et al., 2021). Nevertheless, the intricate molecular variety and specific mechanisms of action exhibited by these compounds position them as highly promising prospects for the advancement of cancer therapeutics in the future. Through the incorporation of bioactive compounds sourced from fish into cancer treatment protocols, innovative therapeutic approaches can be formulated to improve effectiveness and reduce the negative impacts linked to traditional chemotherapy. Subsequent investigations ought to concentrate on refining techniques for compound extraction, undertaking both preclinical and clinical studies, and examining combination therapies to completely leverage the capabilities of these natural anticancer substances (Yu et al., 2020).

Bioactive Compound Isolation

The isolation and purification of fish-derived bioactive compounds were carried out using a combination of chromatographic and spectroscopic techniques to ensure high purity and bioactivity. Marine fish species were selected based on their known potential for producing bioactive peptides, alkaloids, and polysaccharides. The extraction process involved an initial homogenization of fish tissues in an aqueous buffer, followed by centrifugation to separate the supernatant from cellular debris. For peptide extraction, enzymatic hydrolysis was performed using proteases such as trypsin and pepsin, which facilitated the breakdown of fish proteins into bioactive peptide fragments (Yu et al., 2020). The resulting peptide-rich extract was then subjected to high-performance liquid chromatography (HPLC) to separate individual peptides based on their hydrophobicity and molecular weight. The purified peptides were further characterized using mass spectrometry (MS) to determine their exact molecular composition and structure (Fernando et al., 2019).

Polysaccharides were extracted from fish tissues and associated marine algae using a hot water extraction method, followed by ethanol precipitation to isolate high-molecular-weight polysaccharides. The purity and composition of these polysaccharides were confirmed using gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy (Rodrigues et al., 2018). For alkaloid isolation, fish-derived microbial cultures were grown in nutrient-rich media to enhance secondary metabolite production. The alkaloids were extracted using organic solvents such as methanol and chloroform, followed by purification through thin-layer chromatography (TLC) and liquid chromatography-mass spectrometry (LC-MS) techniques (Hamed et al., 2019).

Once isolated, all bioactive compounds were tested for purity and stability before being used in downstream assays. Their concentrations were measured using spectrophotometric techniques, and their biological activity was confirmed through preliminary cytotoxicity screening.

Figure 1: Fish-Derived Bioactive Compounds Methodology Flowchart

Cancer Cell Line Assays

To evaluate the anticancer potential of fish-derived bioactive compounds, in vitro cytotoxicity assays were performed using established human cancer cell lines. The cell lines selected for this study included:

MCF-7 (breast cancer)

A549 (lung cancer)

 HT-29 (colon cancer)

These cancer cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) or RPMI-1640 medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were incubated at 37°C in a humidified 5% CO₂ atmosphere, ensuring optimal growth conditions before treatment.

The cytotoxic effects of the fish-derived compounds were assessed using the MTT assay, a widely used colorimetric method for measuring cell viability. Cancer cells were seeded into 96-well plates at a density of 5 × 10⁴ cells per well and allowed to attach overnight. The following day, cells were treated with different concentrations of the purified bioactive compounds, ranging from 1 µg/mL to 50 µg/mL, for 24 to 48 hours.

After treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well, and cells were incubated for an additional 4 hours to allow mitochondrial enzymes to convert MTT into formazan crystals. The formazan was then dissolved in dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm using a microplate reader (Jin et al., 2021). The percentage of cell viability was calculated relative to untreated control cells, and IC₅₀ values (the concentration required to inhibit 50% of cell viability) were determined for each compound.

For further confirmation, colony formation assays were conducted to assess the long-term effects of these compounds on cancer cell proliferation. Treated cells were plated in six-well plates and allowed to grow for 10–14 days, followed by staining with crystal violet to visualize colony formation (Singh et al., 2020). A reduction in colony formation indicated significant antiproliferative activity of the fish-derived bioactives.

Gene Expression Analysis

To elucidate the molecular mechanisms underlying the cytotoxic effects of fish-derived bioactive compounds, quantitative real-time PCR (qPCR) was performed to measure changes in gene expression related to apoptosis and angiogenesis.

Cancer cells were treated with bioactive compounds at their respective IC₅₀ concentrations for 24 hours. After treatment, total RNA was extracted using the TRIzol reagent, and RNA purity and concentration were determined using a NanoDrop spectrophotometer. Complementary DNA (cDNA) synthesis was carried out using a reverse transcription kit, and qPCR analysis was conducted using SYBR Green chemistry.

The expression levels of key pro-apoptotic genes, such as Bax, caspase-3, and p53, were analyzed to determine the extent of apoptosis induction (Nguyen et al., 2022). Conversely, the expression of anti-apoptotic genes such as Bcl-2 was assessed to evaluate whether treatment suppressed survival pathways. Additionally, genes related to angiogenesis, including VEGF and MMP-9, were analyzed to determine whether the fish-derived compounds inhibited tumor- associated blood vessel formation (Kim et al., 2021). The ΔΔCt method was used to calculate relative gene expression, with GAPDH serving as the housekeeping gene for normalization.

To further confirm apoptotic induction, Western blot analysis was performed to detect the protein expression levels of Bax, caspase-3, and Bcl-2. Cells were lysed using RIPA buffer, and total protein extracts were separated via SDS-PAGE before being transferred onto PVDF membranes. Membranes were incubated with primary antibodies against the target proteins, followed by secondary antibodies conjugated to horseradish peroxidase (HRP). Protein bands were visualized using an enhanced chemiluminescence detection system (Rahman et al., 2018). Furthermore, to assess potential DNA damage, comet assays (single-cell gel electrophoresis) were performed, revealing whether fish-derived compounds caused significant DNA fragmentation, a hallmark of apoptosis (Hassan et al., 2020).

Statistical Analysis

All experimental data were analyzed using GraphPad Prism 9.0. Cytotoxicity data were expressed as mean ± standard deviation (SD) from at least three independent experiments, and statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered statistically significant. For gene expression analysis, qPCR data were analyzed using Student’s t-test, comparing treated samples to untreated controls. Western blot band intensities were quantified using ImageJ software, and results were normalized against β-actin. The reproducibility of the experiments was ensured by performing all assays in triplicates, and data trends were validated through biological replicates.

This section presents the results of bioactive compound isolation, cytotoxicity assays, gene expression analysis, protein expression, angiogenesis inhibition, DNA fragmentation analysis, and statistical evaluation of the findings.

Bioactive Compound Isolation

The extraction and characterization of bioactive compounds from different fish species were performed using enzymatic hydrolysis, solvent extraction, and chromatographic techniques. Peptides, alkaloids, and polysaccharides were quantified to determine their potential anticancer effects. The table below presents the concentration of these compounds and the respective extraction methods used.

Table 1: Fish-Derived Bioactive Compounds and Their Extraction Methods

Figure 2: Fish-Derived Bioactive Compounds and Their Extraction Methods

The results indicate that different fish species provide varying concentrations of bioactive compounds, with Mackerel and Salmon yielding the highest levels of peptides, suggesting their potential as rich sources of bioactive proteins. Cod and Sardine exhibited higher alkaloid concentrations, indicating their effectiveness in DNA-interacting anticancer mechanisms. The

presence of polysaccharides in all fish species further reinforces their significance in immune modulation and tumor suppression, with Mackerel demonstrating the highest polysaccharide content (30.2 mg/g). These findings support the potential use of fish-derived bioactive compounds in cancer therapy, emphasizing the need for further characterization and bioavailability studies.

Cancer Cell Line Assays

To assess the cytotoxic potential of fish-derived bioactive compounds, different cancer cell lines were cultured and maintained under optimal conditions. These included breast, lung, colon, cervical, and prostate cancer models. The table below presents the key characteristics of the selected cell lines.

Table 2: Cancer Cell Lines Used in the Study

Figure 3: Cancer Cell Lines Used in the Study

The results indicate that HT-29 colon cancer cells had the fastest doubling time of 20 hours, making them an aggressive model for studying cytotoxicity. A549 lung cancer cells also demonstrated rapid proliferation, with a doubling time of 22 hours, indicating their potential for assessing the early-stage effects of bioactive compounds. MCF-7 breast cancer cells, on

the other hand, exhibited a slower doubling time of 36 hours, making them an ideal model for studying long-term cytotoxic effects. The PC-3 prostate cancer cell line had a relatively slower proliferation rate (30 hours), reflecting its unique tumor biology. These findings provide a strong foundation for evaluating the differential responses of various cancer cell types to fish-derived bioactive compounds.