Epidermal Mucus of Anabas testudineus as a Promising Source of Antibacterial and Anticancer Agents

Sains Malaysiana 51(5)(2022): 1363-1372 http://doi.org/10.17576/jsm-2022-5105-08

ABSTRACT Anabas testudineus is a sturdy freshwater fish that can live in a polluted environment due to the epidermal mucus (EM) that protects the fish from pathogens or germs. This study explored the functional properties of the EM as a potential antimicrobial and anticancer agent. Inactive Pseudomonas aeruginosa was introduced into fish tanks to stimulate the production of EM. This stimulus significantly increased EM production by more than 100% after 10 days of stimulation, indicating that EM production was influenced by environmental biotic stress. In vitro antibacterial activity tests showed that EM has significant antibacterial activity against Escherichia coli (12 ± 0.23 mm) and P. aeruginosa (10 ± 0.13 mm) at the tested concentration of 1000 μg/mL. Further characterisation against cells showed that EM has a cytotoxic effect against human breast cancer (MCF7) and human melanoma (A375.S2) producing an IC50 value of 4.97 ± 0.25 and 6.27 ± 0.17 mg/mL, respectively. In contrast, no cytotoxicity against normal fibroblast skin cells (HS27) was observed. In addition, apoptosis analysis showed that EM could cause DNA fragmentation of cancer cells, while no effect on normal cells was observed. These findings indicated that EM from A. testudineus could be further studied and explored as an anticancer agent.

INTRODUCTION Anabas testudineus, the climbing perch, is a native species found in Asia belonging to the Anabantide family. It inhabits brackish water and freshwaters of Asia, including Malaysia, Pakistan, Bangladesh, the Philippines, Myanmar, Thailand, Pakistan, India, Sri Lanka and China (Sarkar et al. 2005). It is highly adaptive as it can live in various environments and conditions (temperatures, reduced dissolved oxygen, and pH) and live without water for 6 to 10 h (Rahman 2005). One of the main contributing factors for fish well-being and survivability is the epidermal mucus (EM), which functions as a protective layer from both environmental biotic and abiotic factors (Al-Rasheed et al. 2018). It can also be characterised as a slimy, oily viscous colloid known as mucins composed of mucopolysaccharides, antibacterial enzymes, proteins and water (Dash et al. 2018) that coat the epithelial surfaces and act as a stable physical and chemical shield against invading pathogens (Arockiaraj et al. 2014). In addition, this mucus also contains various essential proteins such as proteases, antimicrobial peptides (AMPs), lectins, lysozymes, immunoglobulins, c-reactive protein (CRPs), transferrin, amphipathic lipid packing sensor (ALPs), and numerous other antibacterial proteins that play a significant role in fish immunity (Arockiaraj et al. 2013) the recombinant MrCrs gene was constructed and expressed in Escherichia coli BL21 (DE3. For example, Al-Rasheed et al. (2018) reported that the skin mucus of A. testudineus has the ability to inhibit the growth of several bacteria. Various studies have reported the antibacterial activity of EM against various bacterial species. For example, epidermal mucus from Atlantic flatfish has demonstrated antibacterial activities against several bacterial strains (Patrzykat et al. 2003). Moreover, antibacterial activities against Bacillus megaterium, Escherichia coli, and Candida albicans have also been reported for Atlantic cod (Gadus morhua) (Bergsson et al. 2005). Furthermore, antibacterial activity against the Gram-negative pathogenic bacteria, Pseudomonas aeruginosa, has been reported for snakehead fish (Channa striata) epidermal mucus (Wei et al. 2010). On top of this, Kumari et al. (2019) found that the EM of freshwater fish such as Hippelates nobilis, Ctenopharyngodon idella, and Cyprinus carpio showed antibacterial activity against various types of pathogens compared to snakehead fish (C. striata). In addition to antimicrobial activity, there is some scientific evidence showing that the epidermal mucus (EM) of freshwater fish and marine fish is cytotoxic against some tumour cells such as human lung cancer cells (Arulvasu et al. 2012), human breast cancer cells (Alijani Ardeshir et al. 2020; Teerasak et al. 2016) and colon cancer cells (Buhari et al. 2015). The cytotoxicity effect against human breast cancer cells by EM obtained from goby (Neogobius melanostomus) and common carp (C. carpio) fish have also been reported (Alijani Ardeshir et al. 2020). Not long ago, Fuochi et al. (2017) has found that the Dasyatis pastinaca (Linnaeus 1758) skin mucus blocked the proliferation of toxic leukaemia cells (HL60) while not affecting healthy neuroblastoma cells (SH-SY5Y) and multiple myeloma cell lines (MM1, U266). As part of EM, in recent years, AMPs have gained the attention of researchers due to their ability to inhibit both microbes and neoplasm (Jin & Weinberg 2019). The number of AMPs shown to have anticancer properties has steadily increased. An example of such AMP is the Aurein 1.2 (GLFDIKKIAESF), a peptide extracted from frog (Litoria aurea). The Aurein 1.2 was reportedly a potential toxin against different cancer cell lines in in vitro experiments. Conversely, Aurein 1.2 did not show any adverse effect against normal cell lines, but remarkably suppressed drug-resistant breast cancer cells both in in vitro and in vivo (Dennison et al. 2007; Rozek et al. 2000). More interestingly, these AMPs are not toxic to fibroblasts or erythrocytes (Hilchie et al. 2011). The physicochemical parameters that contribute to the AMP activity against cancers are mostly unknown, although the properties of AMP and anticancer peptides are very similar. Despite decades of medical and scientific research on most aquatic animals, A. testudineus is still under studied. Currently, A. testudineus could be one of the promising elected species for aquaculture activities and scientific research due to its robust characteristics mentioned previously (Al-Rasheed et al. 2018; Ndobe et al. 2019). Therefore, this study aimed to characterise the functional properties of pseudo-pathogenically induced A. testudineus EM extract and observe their antibacterial and anticancer activities.

Sains Malaysiana 51(5)(2022): 1363-1372 http://doi.org/10.17576/jsm-2022-5105-08