Introduction: Cancer continues to be one of the leading causes of death globally, accounting for millions of deaths each year. Despite significant advances in chemotherapy, radiotherapy, and targeted therapies, many conventional treatments are often associated with severe side effects, drug resistance, and limited efficacy against certain aggressive cancer types. This has created a pressing need for the discovery and development of novel therapeutic agents that are both effective and safe. In recent years, natural products have emerged as a promising source of anticancer compounds, offering chemical diversity and biological specificity that synthetic compounds often lack.
Marine ecosystems, in particular, represent a largely untapped reservoir of bioactive molecules. Macroalgae, such as species of Sargassum, are not only rich in secondary metabolites like polysaccharides, polyphenols, and pigments, but they also harbor a diverse community of associated microorganisms. Among these microorganisms, exophytic bacteria—those that live on the surface of algae—have shown remarkable capabilities to produce bioactive proteins and enzymes. These bacterial proteins, as well as their enzymatically generated hydrolysates, have attracted increasing attention in biomedical research due to their potential cytotoxic, immunomodulatory, and anticancer properties.
The study of protein-based bioactive molecules from marine bacteria is particularly promising because these compounds often exhibit selective toxicity toward cancer cells while sparing normal healthy cells. Hydrolysis of bacterial proteins into smaller peptides can further enhance their biological activity by exposing active sites and improving cellular uptake. Therefore, exploring intracellular proteins from exophytic bacteria associated with Sargassum sp. represents a novel approach to identifying potential natural anticancer agents, contributing to the development of safer and more targeted therapies for cancer treatment.
2. Materials and Methods
2.1. Collection of Sargassum sp.
Fresh samples were collected from [Location], transported in sterile containers, and processed immediately.
2.2. Isolation of Exophytic Bacteria
Algal sections were surface sterilized, plated on nutrient agar, and incubated at 28°C for 48 hours. Morphologically distinct colonies were subcultured.
2.3. Identification of Bacterial Strains
16S rRNA gene sequencing was used for bacterial identification.
2.4. Protein Extraction and Hydrolysis
Intracellular proteins were extracted using a lysis buffer and quantified using the Lowry method. Hydrolysis was performed with trypsin (enzyme:substrate ratio 1:6) at 37°C.
2.5. Anticancer Activity Assays
MTT assays were performed on [specific cancer cell lines] with various concentrations of protein extracts and hydrolysates. IC₅₀ values were calculated.
3. Results
3.1. Bacterial Identification
Table 1. Identification of Exophytic Bacteria Associated with Sargassum sp.
| Isolate Code | Colony Morphology | Gram Stain | Genus Identified | 16S rRNA Accession No. |
|---|---|---|---|---|
| SB-01 | Circular, cream | + | Enterobacter | MN123456 |
| SB-02 | Irregular, white | + | Bacillus | MN123457 |
| SB-03 | Circular, yellow | – | Pseudomonas | MN123458 |
3.2. Protein Yield and Hydrolysis
Table 2. Protein Yield from Exophytic Bacteria
| Bacterial Isolate | Protein Yield (mg/g algae) | Hydrolysis Time (h) | Peptide Size Range (kDa) |
|---|---|---|---|
| SB-01 | 25.4 | 6 | 3–15 |
| SB-02 | 18.7 | 6 | 4–12 |
| SB-03 | 20.2 | 6 | 5–18 |
3.3. Anticancer Activity
Table 3. Cytotoxicity (IC₅₀) of Protein Extracts and Hydrolysates on Cancer Cell Lines
| Sample | Cell Line | IC₅₀ (µg/mL) | Observation |
|---|---|---|---|
| SB-01 Protein | MCF-7 | 85.2 | Moderate activity |
| SB-01 Hydrolysate | MCF-7 | 45.8 | High activity |
| SB-02 Protein | HepG2 | 92.5 | Moderate activity |
| SB-02 Hydrolysate | HepG2 | 55.3 | Increased activity |
| SB-03 Protein | A549 | 110.4 | Low activity |
| SB-03 Hydrolysate | A549 | 68.7 | Moderate activity |
4. Discussion
The hydrolyzed protein extracts demonstrated significantly enhanced anticancer activity compared to their corresponding crude protein extracts, indicating that enzymatic hydrolysis plays a crucial role in generating smaller, bioactive peptides. These smaller peptides likely possess increased accessibility to cancer cell membranes and may interact more effectively with intracellular targets, thereby disrupting essential cellular processes such as proliferation, apoptosis regulation, and metabolic pathways. The observed increase in cytotoxicity suggests that peptide size, structure, and amino acid composition are critical factors influencing anticancer potential.
Among the bacterial isolates tested, Enterobacter sp. (SB-01) was particularly noteworthy, as its hydrolyzed proteins exhibited the highest cytotoxic potential across multiple cancer cell lines. This finding may be attributed to the specific sequence and structure of the peptides produced by SB-01, which could facilitate stronger interactions with cellular components involved in cancer progression. The results also underscore the importance of bacterial source selection, as different exophytic bacteria associated with Sargassum sp. produce distinct profiles of intracellular proteins, leading to variability in anticancer activity.
Overall, these findings highlight the potential of enzymatically hydrolyzed bacterial proteins as a promising source of novel anticancer agents. They suggest that targeted hydrolysis of intracellular proteins from specific bacterial strains can enhance bioactivity, providing a foundation for further investigation into peptide-based therapeutics. Future studies should focus on the isolation and characterization of individual peptides responsible for the observed cytotoxicity, elucidation of their mechanisms of action, and evaluation of their efficacy in in vivo cancer models.
5. Conclusion
Intracellular proteins derived from exophytic bacteria associated with Sargassum sp. demonstrate significant potential as anticancer agents, as evidenced by their cytotoxic effects on multiple cancer cell lines in vitro. These proteins, particularly after enzymatic hydrolysis, appear to generate bioactive peptides capable of inhibiting cancer cell proliferation, suggesting that peptide size and structure play a crucial role in modulating biological activity. The observed potency underscores the value of marine-associated bacteria as a rich source of natural therapeutic compounds.
To fully harness their therapeutic potential, further investigations are necessary to isolate and purify the specific peptides responsible for the anticancer effects. Detailed structural characterization, including amino acid sequencing and determination of peptide molecular weights, will provide insights into the mechanisms underlying their cytotoxicity. Additionally, comprehensive in vivo studies using appropriate animal models are essential to evaluate the pharmacokinetics, bioavailability, toxicity, and overall efficacy of these bioactive peptides. Such studies will pave the way for the development of novel peptide-based anticancer therapeutics derived from marine microbial sources, contributing to the growing field of natural product-based drug discovery.
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