HAMLET: Functional Properties and Therapeutic Potential

James Ho CS; Anna Rydström; Maria Trulsson; Johannes Bålfors; Petter Storm; Manoj Puthia; Aftab Nadeem; Catharina Svanborg


Future Oncol. 2012;8(10):1301-1313. 

In This Article

Fatty Acids are Essential for the Formation & Function of the HAMLET Complex

Early studies identified oleic acid as a cofactor required for the formation of an active HAMLET complex. NMR spectroscopy suggested that oleic acid is integrated into the complex, as the signal was broader than for oleic acid alone,[10] and the lipid cofactor has been proposed to stabilize the partially unfolded state of α-lactalbumin, preventing the protein from reverting to the native state at physiologic solvent conditions.[10]

To address whether HAMLET complex formation is specific for oleic acid, a fatty acid screening study was performed. Ion exchange matrices were preconditioned with fatty acids differing in chain length, saturation and configuration of the double bond(s). Interestingly, oleic acid (C18:1:9 cis) and vaccenic acid (C18:1:11 cis) were identified as the most efficient cofactors in HAMLET complex formation. In addition, C16 or C20 cis unsaturated fatty acids readily formed complexes with apo α-lactalbumin,[41] but trans fatty acids of similar carbon chain length were unable to form complexes, as were saturated fatty acids. In addition, deprotonated oleic acid (oleate) has been used successfully as a cofactor in HAMLET or BAMLET (the bovine counterpart of HAMLET), as expected from the alkaline pH conditions used in HAMLET formation.[42] The results suggest that specific fatty acid may be suitable for complex formation, by binding to domains or epitopes in the partially unfolded protein.

C18 cis fatty acid complexes showed enhanced tumoricidal activity, compared with other complexes, suggesting that fatty acids also participate in the interaction of target cells with HAMLET. However, whether the fatty acids act as direct, independent tumor cell agonists has been debated. Studies of complexes with high lipid content have recently suggested that unfolded proteins may function solely as 'lipid carriers', and that the lipids constitute the tumoricidal entity.[43] At the concentrations present in HAMLET, oleic acid/oleate cause cellular responses including a change in morphology, although there is no evidence that oleic acid or oleate alone trigger the entire tumoricidal response seen with HAMLET.[44]