ISOLATION AND CHARACTERISATION OF MARINE BIOACTIVES FROM GALAXEA FASCICULARIS

Abstract

The mucus or the hard coral. Galaxea fascicularis, contains a system of multiple cytotoxins, with unique anticancer potentials. Primary screening of the partially enriched mucus supernatant (MS) revealed its transient cytotoxicity towards normal rat liver cells, BL8L which acquired gradual resistance on repeated inoculation of MS. However, transformed rat hepatoma JB1 cells succumbed to a single inoculation of MS at half the dosage. Secondary screening or MS revealed its efficacy against a multiple-drug-resistant leukaemia cell line, P388/VCR. MS contains multiple cytotoxins, some or which are heat-labile and Proteinaccous, while others are heat-resistant, non-proteinaccous and glycosylated. MS was fractionated into 15 peaks through CM Sepharose, or which 9 peaks were bioactive. Peaks P4, P5, P6 and P8b were most potent and thence were targetted for further studies. Peak P8b, showing highest cytotoxicity against BL8L cells, was further purified through HPLC silica column to yield S2, a 720 Da cytotoxin comprising a NeuNAc?(2-6)Gal?(1-4)GlcNAc sugar moeity. Secondary anticancer screening found S2 to be most effective against P388/VCR leukaemia, with an LD50 of 0.3 µg dry weight/ml. The cytotoxic components of MS expressed their cytotoxicity via different cellular pathways. P4 was an agonist, while P5 was an antagonist of PKC, leading to the inhibition and activation or the PKC enzyme respectively. Since studies showed that PKC activity was higher in cancer cells than normal cells, the PKC inhibitor P4 could express selective cytotoxicity against cancer cells like P388/VCR but not on normal cells. On the other hand P5 activates the PKC enzyme leading to altered intracellular signalling, culminating in cancer cell death. Peak P6 contained a novel DNase activity that probably induced accelerated apoptosis specifically in P388/VCR cells via the triggering of intrinsic endonuclease within the P388/VCR cells, leading to in situ DNA degradation. Peak P8b and its purified component, S2 inhibited the relaxation of supercoiled DNA catalysed by both topoisomerase l and II, thus impairing the enhancement of the forward rate of DNA cleavage and their ability to religate cleaved DNA in rapidly growing cancer cells. S2 also expresses its cytotoxicity via the stabilisation of topoisormerase I cleavable complex thus causing the collision between the replication machinery and the reversible cleavable complex, culminating in cell death. In summary, the G. fascicularis harbour unique anticancer properties which have potentials for future drug screening and development into novel chemotherapeutics.