Human milk as a source of tumor killing molecules. From MAL to HAMLET

Sammanfattning: HAMLET (Human alphalactalbumin made lethal to tumor cells), a complex between the partially unfolded alphalactalbumin and oleic acid, was discovered by serendipity when anti-adhesive properties of human milk were examined. HAMLET kills tumor cells but not healthy differentiated cells. Native alphalactalbumin (HLA) can be conveted to HAMLET by a two-step process including: 1) calcium ion removal inducing a teritary change of the protein structure. 2) incorporation of oleic acid into the apo-protein by an ion-exchange chromatography. Alphalactalbumin from several species can be converted to HAMLET like complexes. The tumor killing complex is present in human casein after low pH preicipitation in contrast to animal-casein. The results suggest that the alphalactalbumin protein from different species fulfill the properties for HAMLET formation but oleic acid is not accessible in the animal caseins. HAMLET interaction with membranes was investigated as a mechanism for HAMLET uptake. HAMLET interacts and pertubes the structure of membrane vesicle and induce leakage of small molecules as well as morphological changes but there was no evidence of HAMLET uptake into the vesicles. The HAMLET activity was investigated in vivo. Patients with bladder cancer were subjected to daily intravesicle HAMLET instillations. Massive tumor cell shedding was detected into urine after each instillation and a decrease in tumor volume was detected at surgery. HAMLET had a tumor selective activity in human patients as there were no detectable signs of cell death in the healthy tissue of the bladder. The therapeutically value of HAMLET was further investigated in an orthotopic mouse model. Tumor area in HAMLET treated animals were significant reduced compare to controls. Whole body imaging showed retention of HAMLET in tumor bearing tissue as Alexa-labelled HAMLET was visualized in the bladder 4 hours after the instillation. In summery, we show that human milk is a source for tumor killing molecules. We described the composition and the conditions for HAMLET formation and we also shows evidence for therapeutically value in vivo.