On the pro-apoptotic mechanisms of the antitumor drugs Doxorubicin and Interferon-alpha

Abstract: Anti-cancer drugs act primarily by inducing apoptosis. However, knowledge of how various substances induce apoptosis is still incomplete, and so is the basis for the great variation in cellular sensitivity to cytotoxic drugs. A detailed understanding of how anti-cancer agents induce cell death and how defects in cell death pathways promote resistance will change the way chemotherapeutic drugs are used and designed. The aim of this thesis was to investigate pro-apoptotic signaling induced by two commonly used anti-cancer drugs, Doxorubicin and Interferon-á. Doxorubicin (DXR), an anthracycline, is a major antitumor agent known to cause cellular damage via a number of mechanisms including free radical formation and inhibition of topoisomerase II. Interferon-á (IFN-á) is a pleiotropic cytokine and its ability to induce apoptosis has been proposed to be of major importance for its clinical anti-tumor activity. The results demonstrate that the mechanisms of induction of apoptosis by both drugs are strikingly similar with respect to the signaling involved. Clinically relevant concentrations of both agents induce the activation of the pro-apoptotic Bcl-2 family members Bak and Bax prior to apoptosis and anti-apoptotic Bcl-2 family members regulate this response. We could also demonstrate that Bak is activated prior to Bax by both agents. Upstream of Bak, Bax and the mitochondria, two kinases that are known to be activated by cellular stress, JNK and PKCä, are involved, both with respect to DXR and IFN-á. We demonstrated the requirement of Bak and Bax for the induction of apoptosis by DXR by using bax- as well as bak-deficient mouse embryo fibroblasts (MEFs). The BH3-only protein, Bik, which is induced in response to DXR, could be an activator of Bak and Bax. Upstream of the Bcl-2 family members, caspase-2 is activated and was found to be required for DXR-induced apoptosis in Jurkat cells. PKCä was found to be one of the critical downstream targets of caspase-2 following DXR treatment. By using chemical inhibitors against caspase-2, PKCä and JNK, our data suggest a signaling model involving caspase-2, PKCä and JNK. Survival signaling could mask the true potential of chemotherapeutic agents as demonstrated by co-incubation of a PI3K-inhibitor with DXR. Inhibition of PI3K potentiated the DXR-induced Bak, Bax activation and apoptosis in a Bcl-2 dependent but in a caspase-2, JNK and PKCä-independent manner. The upstream signaling in IFN-á-induced apoptosis was also addressed. Upstream of the mitochondria, IFN-a induces JNK phosphorylation/activation. Inhibition of JNK significantly blocked IFN-á-induced Bak and Bax activation and apoptosis, but did not affect the IFN-á-stimulated Jak/STAT signaling. This suggests that the canonical IFN-á induced pathway is not sufficient for this response. Inhibition of JNK was also found to influence the phosphorylation of the pro-apoptotic PKC family member, PKCä. Furthermore, PKCä inhibition blocked apoptosis and Bak activation induced by IFN-á. We conclude that IFN-á-induced apoptosis involves the mitochondrial pathway and the kinases JNK and PKCä. Furthermore this stress-related IFN-induced pathway is unrelated to the Jak/STAT signaling which is generally thought to mediate IFN-á's cellular responses.