Topoisomerase inhibitor camptothecin sensitizes mouse hepatocytes in vitro and in vivo to TNF-mediated apoptosis.
Topoisomerases are nuclear enzymes that maintain and modulate DNA structure. Inhibitors of topoisomerases like camptothecin (CPT), etoposide, and others are widely used antitumor drugs that interfere with transcription, induce DNA strand breaks, and trigger apoptosis preferentially in dividing cells. Because transcription inhibitors (actinomycin D, galactosamine, alpha-amanitin) sensitize primary hepatocytes to the cytotoxic action of tumor necrosis factor (TNF), we reasoned whether topoisomerase inhibitors would act similarly. CPT alone was not toxic to primary cultured murine hepatocytes. When incubated with CPT, murine hepatocytes displayed an inhibition of protein synthesis and were thereby rendered sensitive to apoptosis induction by TNF. Apoptosis was characterized by morphology (condensed/fragmented nuclei, membrane blebbing), caspase-3-like protease activity, fragmentation of nuclear DNA, and late cytolysis. Hepatocytes derived from TNF receptor-1 knockout mice were resistant to CPT/TNF-induced apoptosis. CPT treatment completely abrogated the TNF-induced NF-kappa B activation, and mRNA expression of the antiapoptotic factors TNF-receptor associated factor 2, FLICE-inhibitory protein, and X-linked inhibitor of apoptosis protein was also inhibited by CPT. The caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-(OMe)-fluoromethylketone (zVAD-fmk) and benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-chloromethylketone (zDEVD-fmk), as well as depletion of intracellular ATP by fructose prevented CPT/TNF-induced apoptosis. In vivo, CPT treatment sensitized mice to TNF-induced liver damage. In conclusion, the combination of topoisomerase inhibition and TNF blocks survival signaling and elicits a type of hepatocyte death similar to actinomycin D/TNF or galactosamine/TNF. During antitumor treatment with topoisomerase inhibitors, an impaired immune function often results in opportunistic infections, a situation where the systemic presence of TNF might be critical for the hepatotoxicity reported in clinical topoisomerase inhibitor studies.
Hentze
Hannes
H
Biochemical Pharmacology, University of Konstanz, Konstanz, Germany. hhentze@escellinternational.com
Latta
Markus
M
Künstle
Gerald
G
Dhakshinamoorthy
Saravanakumar
S
Ng
Poh Yong
PY
Porter
Alan G
AG
Wendel
Albrecht
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002166:Camptothecin / Q000494:pharmacology*; D002352:Carrier Proteins / Q000235:genetics; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D015536:Down-Regulation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005047:Etoposide / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism; D011506:Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics* / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I; D013047:Specific Pathogen-Free Organisms; D047992:TNF Receptor-Associated Factor 2; D059003:Topoisomerase Inhibitors; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D051636:X-Linked Inhibitor of Apoptosis Protein
D015703:Antigens, CD; D000970:Antineoplastic Agents; D000972:Antineoplastic Agents, Phytogenic; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002352:Carrier Proteins; D061945:Caspase Inhibitors; C506387:Cflar protein, mouse; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D011506:Proteins; D012333:RNA, Messenger; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D047992:TNF Receptor-Associated Factor 2; D059003:Topoisomerase Inhibitors; D014409:Tumor Necrosis Factor-alpha; D051636:X-Linked Inhibitor of Apoptosis Protein; D005047:Etoposide; D000255:Adenosine Triphosphate; D020169:Caspases; D002166:Camptothecin