Synergistic interaction between the EGFR tyrosine kinase inhibitor gefitinib ("Iressa") and the DNA topoisomerase I inhibitor CPT-11 (irinotecan) in human colorectal cancer cells.
Epidermal growth factor receptor [EGFR (HER1, erbB1)] is a receptor with associated tyrosine kinase activity, and is expressed in colorectal cancers and many other solid tumors. We examined the effect of the selective EGFR tyrosine kinase inhibitor (EGFR-TKI) gefitinib ("Iressa") in combination with the DNA topoisomerase I inhibitor CPT-11 (irinotecan) on human colorectal cancer cells. EGFR mRNA and protein expression were detected by RT-PCR and immunoblotting in all 7 colorectal cancer cell lines studied. Gefitinib inhibited the cell growth of the cancer cell lines in vitro with an IC(50) range of 1.2-160 microM by 3,(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Lovo cells exhibited the highest level of protein and autophosphorylation of EGFR and were the most sensitive to gefitinib. The combination of gefitinib and CPT-11 induced supra-additive inhibitory effects in COLO320DM, WiDR and Lovo cells, assessed by an in vitro MTT assay. Administration of gefitinib and CPT-11 had a supra-additive inhibitory effect on WiDR cells and tumor shrinkage was observed in Lovo cell xenografts established in nude mice, whereas no additive effect of combination therapy was observed in COLO320DM cells. To elucidate the mechanisms of synergistic effects, the effect of CPT-11-exposure on phosphorylation of EGFR was examined by immunoprecipitation. CPT-11 increased phosphorylation of EGFR in Lovo and WiDR cells in time- and dose-dependent manners. This EGFR activation was completely inhibited by 5 microM gefitinib and gefitinib-induced apoptosis was enhanced by combination with CPT-11, measured by PARP activation although no PARP activation was induced by 5 microM CPT-11 alone. These results suggested that these modification of EGFR by CPT-11, in Lovo cells, is a possible mechanism for the synergistic effect of CPT-11 and gefitinib. These findings imply that the EGFR-TKI gefitinib and CPT-11 will be effective against colorectal tumor cells that express high levels of EGFR, and support clinical evaluation of gefitinib in combination with CPT-11, in the treatment of colorectal cancers.
Koizumi
Fumiaki
F
Support Facility of Project Ward, National Cancer Center Hospital, Tokyo, Japan.
Kanzawa
Fumihiko
F
Ueda
Yutaka
Y
Koh
Yasuhiro
Y
Tsukiyama
Shoji
S
Taguchi
Fumiko
F
Tamura
Tomohide
T
Saijo
Nagahiro
N
Nishio
Kazuto
K
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000627:therapeutic use*; D002455:Cell Division / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism; D004347:Drug Interactions; D004357:Drug Synergism; D004359:Drug Therapy, Combination; D004791:Enzyme Inhibitors / Q000627:therapeutic use*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005260:Female; D000077156:Gefitinib; D006801:Humans; D000077146:Irinotecan; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011799:Quinazolines / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D059004:Topoisomerase I Inhibitors; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
D002166:Camptothecin; D004264:DNA Topoisomerases, Type I; D004791:Enzyme Inhibitors; D066246:ErbB Receptors; D000077156:Gefitinib; D000077146:Irinotecan; D011065:Poly(ADP-ribose) Polymerases; D011799:Quinazolines; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; D059004:Topoisomerase I Inhibitors