Cisplatin-induced tumor cell death signaling and potentiation by energy metabolism inhibitors

Abstract

Treatment of cancer is associated with challenges including the tumor development of resistance. By elucidating intracellular signal transduction pathways elicited by therapy it is possible to identify structures and processes crucial for induction of cell death. The aim is to exploit this knowledge to improve treatment strategies.

In this thesis, evidence is presented that cisplatin treatment of tumor cell lines induces separate signal transduction events. Upon cisplatin treatment, calpain, a cytosolic protease, is shown to become activated. Activated calpain is demonstrated to cleave the BH3 only protein Bid between residues Gly70 and Arg71, generating a fragment with the ability to induce release of cytochrome c from the mitochondria. Cisplatin induces signals via MEKK1, however, the activation of calpain is shown to be independent of overexpression of the dominante negative form of MEKK1 (dnMEKK1). The dominant positive form of MEKK1 (Dp MEKK1) induces a conformational change in the proapoptotic protein Bak. This conformational activation was revealed not to directly lead to apoptosis. However, cisplatin induces both conformational change of Bak as well as Bak oligomerization. Bak complexes of sizes between 80 kDa and 170 kDa are shown to be associated with apoptosis formation indicating that in addition to activation of MEKK1 another signal is needed. This signal is constituted by JNK, shown as a JNK inhibitor blocked cisplatin-induced apoptosis and the formation of Bak complexes in the critical size range. These results are further confirmed using cells deficient for JNK.

In addition to the development of tumor resistance to treatment, another problem in tumor therapy is the toxicity to normal tissues unaffected by disease. An approach to overcome these processes is the use of therapy sensitization, i.e. using low dose of a standard treatment in combination with a drug that specifically targets crucial processes in the tumor cell to increase the effects of the other drug. In this thesis, the effects of sensitizing tumor cells to cisplatin by inhibition of their energy production are investigated. Both an inhibitor of glycolysis, 2-deoxyglucose (DG) as well as an inhibitor of fatty acid â oxidation, etomoxir, were shown to potentiate the effects of cisplatininduced cell death in several tumor cell lines. These results are further confirmed in treatment of xenografted mouse tumors.