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Investigation into the mechanisms of the synergistic interactions between tumor necrosis factor and the topoisomerase II inhibitors VP-16/VM-26

by
Weijia Zhu
Pathology and Laboratory Medicine

B.M.D., 2000, Fudan University, P.R. China

Thesis Advisor: Frederick D. Coffman, Ph.D.
Assistant Professor
Department: Pathology and Laboratory Medicine

Dental School
Room B721

Thursday, June 12, 2008
10:00 a.m.


Abstract

The ability of tumor necrosis factor (TNF) to induce apoptosis in many cancer cell lines has attracted considerable attention. However, elevated systemic levels of TNF cause septic shock, thus severely limiting the amount of TNF that can be used for therapeutic purposes. Our laboratory was one of the first to discover that certain topoisomerase II-directed chemotherapeutic agents, specifically the epipodophyllotoxins etoposide (VP-16) and teniposide (VM-26), synergistically enhanced TNF-mediated apoptosis in ME-180 human cervical carcinoma cells. Since TNF activates multiple cellular signaling pathways, it was our initial hypothesis that the observed cytotoxic synergy is due to the inhibition of one or more of the cell survival pathways and/or the enhancement of one or more of the cell death pathways. Treatment of ME-180 cells with TNF, either topoisomerase II inhibitor, or the combination of TNF and VP-16 did not significantly change NF-B protein levels or IB protein and message levels, nor did the addition of VM-26 markedly inhibit TNF-mediated NF-B nuclear translocation. Wortmannin, a specific and potent PI(3) kinase inhibitor, enhanced both TNF- and VP-16-mediated cytotoxicities to a degree, but did not affect the synergistic interaction between TNF and VP-16. Thus neither the NF-B nor the PI(3)K/Akt anti-apoptotic pathways contribute significantly to the cytotoxic synergy in ME-180 cells. The combination of TNF and VP-16 induced a biphasic activation of JNK, which was not observed in either TNF or VP-16 alone. However, VM-26 did not markedly enhance TNF-mediated p-JNK nuclear translocation. Also, levels of the anti-apoptotic c-FLIP protein, a key target of JNK-mediated apoptotic pathway, were not significantly altered under any treatment conditions. Most importantly, blocking the JNK pathway with JNK inhibitor 1 had no effect on the synergistic interactions between TNF and VP-16/VM-26. Therefore, while JNK activity is modulated during the process of cytotoxic synergy, it does not participate in the mechanism of synergy. Treatment of ME-180 cells with TNF and VP-16 increased caspase-2 and caspase-3 activities in a time-dependent manner, beginning at 6h, as compared to either TNF or VP-16 alone. The activation of caspase-3 was always significantly greater than the activation of caspase-2. Over the initial 10h, neither caspase-8 nor caspase-9 activity was markedly enhanced under any conditions. Thus the synergistic interaction in these cells appears directly correlated with caspase-2 and caspase-3 activation. ME-180 cells express the pro-apoptotic caspase-2L isoform, not the anti-apoptotic caspase-2S isoform, regardless of treatment conditions, and changes in caspase-2 transcription do not contribute to the cytotoxic synergy between TNF and VP-16.

In summary, these studies demonstrate that the synergistic interactions between tumor necrosis factor and the topoisomerase II inhibitors VP-16/VM-26 do not depend upon either the downregulation of the anti-apoptotic NF-B and PI(3)K/Akt pathways or the upregulation of the pro-apoptotic JNK pathway as originally hypothesized. Rather, the cytotoxic synergy in ME-180 cells appears to be correlated to the enhanced activation of caspase-2 and caspase-3.


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