title: Internalization of cytotoxic analog AN-152 of luteinizing hormone-releasing hormone induces apoptosis in human endometrial and ovarian cancer cell lines independent of multidrug resistance-1 (MDR-1) system.
abstract: OBJECTIVE
  Eighty percent of human ovarian and endometrial cancers express receptors for luteinizing hormone-releasing hormone (LHRH-R). These receptors can be used for targeted chemotherapy with agents such as AN-152, in which doxorubicin is linked to analog [D-Lys(6)]-LHRH. Direct receptor-mediated antiproliferative effects of AN-152 have been shown in vitro and in vivo. In LHRH-R positive cell lines, AN-152 was more effective than doxorubicin at equimolar concentrations. This study was designed to investigate the mechanism of action of AN-512 in ovarian and endometrial cancer cells in vitro. Study design Three ovarian (SKOV-3, NIH:OVCAR-3, EFO-21) and 2 endometrial carcinoma cell lines (Ishikawa, HEC-1A) were evaluated for doxorubicin- or AN-152-induced apoptosis. Internalization and cytoplasmic release of AN-152 was monitored by confocal laser scanning microscopy and inhibited by chloroquine. Cleavage of doxorubicin from AN-152 was inhibited by carboxylesterase inhibitor, diisopropyl fluorophosphate (DFP). The surface expression of multidrug resistance-1 (MDR-1) gene product P-glycoprotein (Pgp) was measured by flow cytometry.
  
  
  RESULTS
  Induction of apoptosis by AN-152 in LHRH-R positive Ishikawa, HEC-1A, EFO-21, and NIH:OVCAR-3 cells was significantly higher than that induced by doxorubicin, whereas the percentage of apoptotic cells in LHRH-R negative SKOV-3 was higher after treatment with doxorubicin. In EFO-21 cells, apoptosis induced by AN-152 was inhibited by pretreatment with chloroquine. Pretreatment with DFP increased AN-152-induced apoptosis in LHRH-R positive cells and reduced apoptosis in LHRH-R negative SKOV-3. Both AN-152 and doxorubicin induced surface expression of MDR-1 gene product Pgp, but the effect of AN-152 was smaller than that of doxorubicin. Pgp surface expression induced by AN-152 was inhibited by pretreatment with DFP.
  
  
  CONCLUSIONS
  AN-152 is internalized through the LHRH-R and induces apoptosis in LHRH-R-positive human ovarian and endometrial cancer cell lines without activating the MDR-1 efflux pump system. The efficacy and specificity of AN-152 is inversely correlated with carboxylesterase activity.
authors.affiliation: Department of Gynecology and Obstetrics, Georg-August-University, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany.
authors.forename: Andreas R
authors.initials: AR
authors.lastname: Günthert
authors.forename: Carsten
authors.initials: C
authors.lastname: Gründker
authors.forename: Till
authors.initials: T
authors.lastname: Bongertz
authors.forename: Thilo
authors.initials: T
authors.lastname: Schlott
authors.forename: Attila
authors.initials: A
authors.lastname: Nagy
authors.forename: Andrew V
authors.initials: AV
authors.lastname: Schally
authors.forename: Günter
authors.initials: G
authors.lastname: Emons
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000031:analogs & derivatives* / Q000494:pharmacology*; D016889:Endometrial Neoplasms / Q000378:metabolism; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D019450:Genes, MDR / Q000187:drug effects*; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives* / Q000494:pharmacology*; D006801:Humans; D018613:Microscopy, Confocal; D010051:Ovarian Neoplasms / Q000378:metabolism; D044345:Phosphoric Triester Hydrolases / Q000494:pharmacology; D011974:Receptors, LH / Q000187:drug effects* / Q000502:physiology; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D000903:Antibiotics, Antineoplastic; D011974:Receptors, LH; C080883:LHRH, lysine(6)-doxorubicin; D007987:Gonadotropin-Releasing Hormone; D004317:Doxorubicin; D044345:Phosphoric Triester Hydrolases; C020061:diisopropyl-fluorophosphatase