Interferon-gamma-induced sensitization of colon carcinomas to ZD9331 targets caspases, downstream of Fas, independent of mitochondrial signaling and the inhibitor of apoptosis survivin.
We have demonstrated previously a Fas-dependent component in thymineless death of human colon carcinoma cells. Importantly, the cytotoxic effects of thymidine deprivation induced by 5-fluorouracil (FUra) combined with leucovorin (LV) was enhanced by IFN-gamma, and the synergism was shown to be dependent on Fas, FUra-induced DNA damage, and independent of p53. Subsequently we examined the potential for synergistic interactions between IFN-gamma and the specific thymidylate synthase inhibitor, ZD9331. IFN-gamma sensitized colon carcinomas to ZD9331-induced apoptosis and loss in clonogenic survival, also dependent on ZD9331-induced DNA damage, independent of p53. Synergism occurred in HCT116, demonstrating previously RNA-mediated FUra/LV cytotoxicity that could not be potentiated by IFN-gamma. Manipulation of the Fas death receptor pathway from the level of the receptor (Nok1/Nok2, Fas overexpression, and DN-FADD) to the mitochondria (Bcl-xL and Bcl-2) did not modulate ZD9331 +/- IFN-gamma-induced cytotoxicity in HT29, with the exception that Nok1/Nok2-blocking antibodies partially protected HT29 from the cytotoxic activity of ZD9331 alone. However, IFN-gamma alone (but not ZD9331) up-regulated the expression of caspases -3, -4, -7, and -8, and in combination with ZD9331 demonstrated enhanced caspase activation and cleavage of poly(ADP-ribose) polymerase that was not prevented by overexpression of Bcl-2. Additionally, IFN-gamma increased the activity of the proteasome in HT29, leading to selective down-regulation of the antiapoptotic protein survivin, whereas simultaneously increasing Fas expression. However, reduction in the survivin:Fas ratio by transfection of survivin small interfering RNA and/or overexpression of Fas did not affect sensitivity of HT29 to ZD9331 +/- IFN-gamma. Data demonstrate that IFN-gamma combined with ZD9331 is synergistic in additional cell lines that demonstrate RNA-mediated FUra/LV cytotoxicity, and that a major target of interaction is at the level of caspases, downstream of Fas, and independent of involvement of either the mitochondria or survivin.
Geller
James
J
Division of Molecular Therapeutics, Department of Hematology-Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Petak
Istvan
I
Szucs
Kinga Szekely
KS
Nagy
Katalin
K
Tillman
David M
DM
Houghton
Janet A
JA
D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D020169:Caspases / Q000096:biosynthesis / Q000378:metabolism*; D053471:Caspases, Initiator; D002460:Cell Line; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000188:drug therapy*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005472:Fluorouracil / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D051034:Inhibitor of Apoptosis Proteins; D007371:Interferon-gamma / Q000378:metabolism*; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D009363:Neoplasm Proteins; D020411:Oligonucleotide Array Sequence Analysis; D046988:Proteasome Endopeptidase Complex; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011799:Quinazolines / Q000627:therapeutic use*; D034741:RNA, Small Interfering / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction; D000077022:Survivin; D013997:Time Factors; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation; D051020:bcl-X Protein; D019014:fas Receptor / Q000378:metabolism*
D000970:Antineoplastic Agents; C494811:BCL2L1 protein, human; C107489:BIRC5 protein, human; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011799:Quinazolines; D034741:RNA, Small Interfering; D000077022:Survivin; D016159:Tumor Suppressor Protein p53; C094299:ZD 9331; D051020:bcl-X Protein; D019014:fas Receptor; D007371:Interferon-gamma; C505407:CASP3 protein, human; C093516:CASP4 protein, human; C505520:CASP7 protein, human; C505530:CASP8 protein, human; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D020169:Caspases; D053471:Caspases, Initiator; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D005472:Fluorouracil