What began on a deadline, lives on by choice: Discovering the anticancer potential of Galaxaura rugosa

What was initially thought to be a simple special project for one of my courses turned into a truly exciting discovery. We were given excess frozen seaweed samples stored in our lab to conduct a practice experiment and apply our learnings from the Marine Natural Products course. Our team investigated Galaxaura rugosa, a red macroalga, for its potential cytotoxic activity against HCT116 human colorectal adenocarcinoma cells using a tetrazolium-based assay.

During the initial screening of fractions from the crude extract, one fraction demonstrated notably strong cytotoxic activity. At a concentration of 10 µg/mL, it resulted in 88.6% cell death, and even at a lower concentration of 1 µg/mL, it maintained 82.1% cytotoxicity. These results were interesting enough to warrant further investigation. We proceeded with detailed spectral analyses, curious about the chemical constituents responsible for such effects. What we discovered was a complex mixture, rich in alkaloids, terpenoids, and both linear and aromatic polyketides—compounds well known for their diverse biological activities and potential therapeutic relevance.

Due to these compelling results, what began as a course requirement has since evolved into a long-term side project that we continue to pursue today.

Lighting the way forward: The promising potential of Philippine seaweeds as neuroactive agents

As one of the youngest members of the lab, I was fortunate to have seniors who took turns teaching me various assays and experimental techniques. Among them, the one that captivated me the most was the Dorsal Root Ganglion (DRG) assay.

This is a two-day procedure. On the first day, DRG neurones were isolated from ICR mice aged 14-60 days postnatal and were incubated for 16-18 hours. On the second day, the cultured neurones were loaded with the calcium-sensitive dye Fura-2-AM. Intracellular [Ca²⁺] influx in response to KCl stimulation was then monitored through ratiometric imaging at 340- and 380-nm light excitation. Lastly, perturbations in calcium signalling patterns were analysed to assess neuroactive responses [1].

Figure 1. Day 2 of DRG Assay (Calcium Imaging) (Photo from UP MSI)

Initially, I practiced the assay using available samples in the lab. But things took a turn when I began testing seaweed extracts from one of our lab’s projects, “Phytochemical Characterisation of Macroalgae for Food and High-Value Products (PhycoPRO).” The results were surprisingly striking—so much so that I was motivated to test every remaining sample from the project and ultimately make it the focus of my master’s thesis.

In the end, I screened a total of 57 seaweed species collected from across the Philippines—an unexpected journey that turned a simple learning exercise into a meaningful contribution to our research.

Among the 57 seaweed extracts tested, 38 induced amplification of [Ca²⁺] influx in DRG neurones, 5 inhibited [Ca²⁺] influx, and 8 showed both amplification and inhibition—highlighting their potential neuroactivity. One particularly promising species was Tydemania expeditionis, which exhibited chemical features consistent with fatty acid amides, compounds known for their neuroactive properties. A specific fraction from T. expeditionis caused irreversible inhibition of [Ca²⁺] influx in 59 neurones (30.6%), amplification in 11 neurones (5.7%), and no observable activity in 123 neurones (63.7%). These results suggest its potential role as an anti-nociceptive or pain-modulating agent, warranting further investigation and isolation of its active components.

This is particularly exciting because the DRG assay platform has a proven track record of success in natural products and drug discovery research. It has been used alongside other bioassay systems for the bioassay-guided purification of conopeptides from Conus venoms [1], forming part of the pioneering work led by renowned Filipino scientists Dr. Baldomero Olivera and Dr. Lourdes Cruz. The DRG assay was originally developed at the University of Utah, and the capability was transferred to University of the Philippines Marine Science Institute (UP MSI) by Dr. Olivera through the “Philippine Mollusc Symbiont International Cooperative Biodiversity Group (PMS-ICBG)” project. The beauty of the assay lies in the heterogeneity of the DRG neuronal population. Unlike conventional assays that use a single immortalised cell line, the mammalian dorsal root ganglion (DRG) comprises up to 30 distinct neuronal subtypes, each responsible for transmitting different sensory modalities—including pain, touch, temperature, and itch. These specialised neurones can be functionally distinguished through the application of selective pharmacological agents that target receptors unique to each subtype. The resulting calcium response patterns offer valuable insights into the identity and function of the responding neurones [1].

In the Philippine setup, a single experiment typically monitors around 400 DRG neurones simultaneously. When three pharmacological agents are applied sequentially, this effectively results in 1,200 individual cellular assays within a single run (400 neurones × 3 pharmacological challenges). This high-throughput capability, combined with the assay’s cellular diversity, makes it a powerful tool for screening complex natural products such as seaweed extracts for neuroactive properties.

Both PMS-ICBG and PhycoPRO are UP’s collaborative efforts with local and international institutions—examples of how interdisciplinary and cross-institutional partnerships can drive innovation. Collaboration plays a crucial role in this field by bringing together diverse expertise in chemistry, biology, and pharmacology and by enabling access to unique biological resources and advanced technologies. By leveraging the outputs of such collaborations, we can expand our capabilities and pursue more ambitious scientific goals. It is through these collective efforts that groundbreaking discoveries, such as ziconotide, become possible.

Beyond the data

With over 1,065 seaweed taxa [2], the Philippines is home to an extraordinary wealth of marine macroalgae—yet this biodiversity remains a vast and largely untapped reservoir of potential bioactive compounds for use in medicine and biotechnology.

While global research has been shedding light on the neurotherapeutic promise of marine macroalgae, Philippine seaweeds have remained largely unexplored in this domain. Our study breaks new ground as the first comprehensive neuroactivity screening of Philippine seaweeds, utilising primary cultured dorsal root ganglion (DRG) sensory neurones via calcium imaging. This work not only fills a critical research gap but also opens the door to discovering novel marine-derived compounds for neurological applications.

Our findings underscore the urgent need for continued pursuit to uncover both cytotoxic and neuroactive compounds from native Philippine seaweed species. Just as important are the downstream efforts to isolate, characterise, and elucidate the structures of these bioactive natural products, which hold promise for therapeutic development.

Crucially, the establishment of curated chemical libraries would serve as a cornerstone for future drug discovery and biomedical research. Such libraries allow for efficient re-screening and the sustainable use of marine resources. 

For the Philippines, this represents a strategic investment in scientific advancement, biotechnological innovation, and the conservation of its marine biodiversity. It continues the development of this biodiversity in the food, cosmetic, pharmaceutical, and nutraceutical industries. Most encouragingly, it upholds a tradition of using Philippine seaweeds for remedies and medicine, a practice that has persisted for many centuries [3].

With continued exploration, the Philippines is poised to unlock novel marine-derived compounds from its rich seaweed biodiversity, potentially fuelling future breakthroughs in both cancer and neurological therapies.

Figure 2. Marine Pharmacognosy Laboratory Members (Marine Science Institute, University of the Philippines Diliman)

References

[1] Teichert, R. W.; Schmidt, E. W.; Olivera, B. M. Constellation Pharmacology: a new paradigm for drug discovery. The Annual Review of Pharmacology and Toxicology 2015, 55 (1), 573–589. https://doi.org/10.1146/annurev-pharmtox-010814-124551.

[2] Lastimoso, J. M.; Santiañez, W. J. Updated checklist of the Benthic Marine Macroalgae of the Philippines. The Philippine Journal of Science 2021, 150 (S1). https://doi.org/10.56899/150.s1.04.

[3] Dumilag, R.; Javier, R. Ethnobotany of medicinal seaweeds of Ilocos Norte, Philippines. The Philippine Journal of Science 2022, 151 (3). https://doi.org/10.56899/151.03.28.