Institutionen för onkologi-patologi / Department of Oncology-Pathology
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