Normal tissues are continuously maintained and renewed by a balance between mass growth and autophagic degradation at the cellular level, and between cell division and apoptotic cell death at the tissue level. The uncontrolled growth of malignant tumours reflects the propensity of cancer cells to up-regulate mass growth and cell division as well as to down-regulate autophagy and apoptosis. Our project studies the molecular mechanisms involved in the regulation of the latter two processes, attempting to identify features that distinguish malignant cells from their normal counterparts.

Autophagy is a process by which cells, under conditions of stress or starvation, digest their own cytoplasm to obtain small molecules (like amino acids) needed for maintenance and renewal of essential cell functions. Specialized intracellular membrane cisternae, called phagophores, excise and enwrap pieces of cytoplasm, forming cytoplasm-filled vacuoles called autophagosomes. The autophagosomes subsequently fuse with endosomes (vacuoles that contain endocytosed material of extracellular origin) to form amphisomes, which eventually deliver their contents of cytoplasm and exogenous material to lysosomes (the digestive vacuoles of the cell) for degradation.

Our project has developed a method for purification of autophagosomes from isolated rat liver cells (Strømhaug et al. 1998, Biochem. J. 335, 217-224), and characterized these organelles biochemically as well as structurally (Fengsrud et al. 2000, Eur. J. Cell Biol. 79, 871-882). By the use of proteomics (protein separation by two-dimensional gel electrophoresis and column chromatography, combined with protein identification by mass spectrometry), we have been able to identify about 40 proteins that are selectively associated with autophagosomal membranes, including many novel enzyme forms (Fengsrud et al. 2000, Biochem. J. 352, 773-781). Several of these enzymes are functionally related, suggesting the participation of specific biochemical processes in the regulation of autophagic activity.

The use of proteomic methods has, furtermore, enabled our project to identify a signalling pathway that shuts down autophagy in liver cells during toxic stress. This pathway, which includes the AMP-activated protein kinase (AMPK), the stress-activated kinases SEK1 and JNK, and possibly S6-kinase (Ruud Larsen et al. 2002, J.Biol.Chem. 277, 34826-34835), also appears to regulate cytoskeletal organization and apoptotic cell death. Algal toxins like okadaic acid and microcystin induce a rapid activating phosphorylation of AMPK, and of cytoskeletal proteins like plectin and keratin, along with a disruption of the intracellular network of keratin intermediate filaments. This collapse of an important cytoskeletal element could well be a common cause of both the suppression of autophagy and of the apoptotic cell death that ensues several hours later.

A grapefuit flavonoid, naringin, prevents the AMPK phosphorylation, the keratin cytoskeletal collapse, the autophagy suppression and the apoptotic death elicited by algal toxins, probably reflecting an inhibitory effect of the flavonoid on an AMPK-activating protein kinase. Interestingly, naringin does not exert any such protective effect on liver cancer (hepatoma) cells of rat or human origin (Blankson et al. 2000, Cell Death Differ. 7, 739-746). The latter observation suggests the possibility of developing a novel type of cancer therapy, based on the use of a nonspecific cytotoxin (such as, e.g., okadaic acid) in combination with a specific cytoprotectant of normal cells (such as, e.g., naringin) to achieve a selective killing of cancer cells.