Apoptosis and tumor cell death pathways in response to HAMLET

Sammanfattning: HAMLET (Human alpha-lactalbumin made lethal to tumor cells) is a molecular complex of alpha-lactalbumin and oleic acid, that induces programmed cells death in tumor cells but leaves healthy differentiated cells unaffected. It has a broad anti-tumor activity as more than 40 cell lines from different origin are sensitive. The cell death activity of HAMLET is retained in vivo. HAMLET limits the progression of human glioblastomas in a rat xenograft model and reduces the lesion volume in skin papilloma patients. In HAMLET treated cells there are characteristic morphological changes of apoptosis. This thesis aimed to elucidate the cell death mechanism(s) activated by HAMLET. A low apoptotic response was observed in HAMLET treated tumor cells, defined as caspase activity and phosphatidyl serine exposure. Caspase inhibition did not rescue cells from dying but it altered the mode of death. HAMLET triggered caspase dependent chromatin condensation but when caspases were blocked, alternative apoptosis like condensation was observed. Bcl-2 overexpression and p53 deletions or gain of function mutations did not influence the sensitivity to HAMLET. In conclusion, HAMLET induces cell death independently of caspases, Bcl-2 and p53. In HAMLET treated tumor there is extensive vesicle formation typical for autophagy. Since autophagy has been described as a cell death mechanism we next investigated the relevance of autophagy in HAMLET induced cell death. Morphological changes characteristic of macro-autophagy was observed. There was as an increase of cytoplasmic vesicles enwrapped in double and multiple membranes. The vesicular content was shown to be acidic by MDC staining and the increase in acidic vesicle formation was reversed by 3MA, which prevents autophagy. LC3 was modified and translocated to autophagosomes in HAMLET treated cells and this response was reversed by 3MA. We identified damaged mitochondria that may be the trigger of this pathway. In parallel, there were indications of chaperon-mediated autophagy. An increase of HSC70 expression was observed and HAMLET colocalized with HSC70 in the cytoplasm. However, autophagy was not the cause of death as 3MA treatment did not rescue the HAMLET treated tumor cells from death. These results suggest that HAMLET triggers an autophagy in tumor cells, but this was not the mechanism of death. As HAMLET kills tumor cells but not differentiated healthy cells we next investigated the influence of HAMLET on the cell cycle of tumor cells. In lymphoma cells, HAMLET preferentially killed G0/G1 at the LD50 concentration, but at higher doses all cells were killed regardless of cell cycle phase. The G2/M cells were the most sensitive to HAMLET in carcinoma cells, but at a high dose all cells were killed. Furthermore, the sensitivity to HAMLET decreased with the degree of differentiation. These results suggest that HAMLET differs from many cancer drugs by killing tumor cells regardless of cell cycle phase but with a preference for undifferentiated cells. HAMLET consists of ?-lactalbumin in the apo state stabilized by the lipid cofactor oleic acid. The effect of the unfolded protein alone has not been tested. ?-lactalbumin rapidly reverts to the native state when exposed to physiologic solvent conditions, we therefore used ?-lactalbumin mutants that are retained in the apo state even at physiologic conditions. These proteins were not able to induce cell death, but could be converted to active complexes when they were combined with the fatty acid. These results show that both the protein and the fatty acid are required to form the active complex.