Recombinant CPE fused to tumor necrosis factor targets human ovarian cancer cells expressing the claudin-3 and claudin-4 receptors.
Using gene expression profiling, others and we have recently found that claudin-3 (CLDN3) and claudin-4 (CLDN4) are two of the most highly and consistently up-regulated genes in ovarian carcinomas. Because these tight junction proteins are the naturally occurring receptors for Clostridium perfringens enterotoxin (CPE), in this study, we used the COOH-terminal 30 amino acids of the CPE (CPE(290-319)), a fragment that is known to retain full binding affinity but have no cytolytic effect, to target tumor necrosis factor (TNF) to ovarian cancers. We constructed a pET32-based vector that expressed the fusion protein, designated here as CPE(290-319)-TNF, in which CPE(290-319) was fused to TNF at its NH(2)-terminal end. Western blotting confirmed presence of both CPE(290-319) and TNF in the fusion protein. The TNF component in CPE(290-319)-TNF was 5-fold less potent than free TNF as determined by a standard L-929 TNF bioassay. However, the CPE(290-319)-TNF was >6.7-fold more cytotoxic than free TNF to 2008 human ovarian cancer cells, which express both CLDN3 and CLDN4 receptors. shRNAi-mediated knockdown of either CLDN3 or CLDN4 expression in 2008 markedly attenuated the cytotoxic effects of CPE(290-319)-TNF. The fusion construct was efficiently delivered into target cells and located in both cytosol and vesicular compartments as assessed by immunofluorescent staining. We conclude that CPE(290-319) effectively targeted TNF to ovarian cancer cells and is an attractive targeting moiety for development of CPE-based toxins for therapy of ovarian carcinomas that overexpress CLDN3 and CLDN4.
Yuan
Xiaoqin
X
Department of Medicine and the Rebecca and John Moores University of California San Diego Cancer Center, University of California San Diego, La Jolla, CA 92093, USA.
Lin
Xinjian
X
Manorek
Gerald
G
Kanatani
Isao
I
Cheung
Lawrence H
LH
Rosenblum
Michael G
MG
Howell
Stephen B
SB
D015153:Blotting, Western; D045744:Cell Line, Tumor; D062465:Claudin-3; D062506:Claudin-4; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004768:Enterotoxins / Q000235:genetics*; D005260:Female; D005976:Glutaral / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019108:Tight Junctions; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
C568122:CLDN3 protein, human; C568157:CLDN4 protein, human; D062465:Claudin-3; D062506:Claudin-4; D003432:Cross-Linking Reagents; D004768:Enterotoxins; D008565:Membrane Proteins; D010446:Peptide Fragments; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C035598:enterotoxin, Clostridium; D005976:Glutaral