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.

CONCLUSION:

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: Andreas R Günthert, Carsten Gründker, Till Bongertz, Thilo Schlott, Attila Nagy, Andrew V Schally, Günter Emons
• Journal: American journal of obstetrics and gynecology (Am J Obstet Gynecol) Vol. 191 Issue 4 Pg. 1164-72 (Oct 2004) ISSN: 0002-9378 [Print] United States
• PMID: 15507937 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibiotics, Antineoplastic
• Receptors, LH
• LHRH, lysine(6)-doxorubicin
• Gonadotropin-Releasing Hormone
• Doxorubicin
• Phosphoric Triester Hydrolases
• diisopropyl-fluorophosphatase

Topics

• Antibiotics, Antineoplastic (pharmacology)
• Apoptosis (drug effects)
• Dose-Response Relationship, Drug
• Doxorubicin (analogs & derivatives, pharmacology)
• Endometrial Neoplasms (metabolism)
• Female
• Gene Expression Regulation, Neoplastic (drug effects)
• Genes, MDR (drug effects)
• Gonadotropin-Releasing Hormone (analogs & derivatives, pharmacology)
• Humans
• Microscopy, Confocal
• Ovarian Neoplasms (metabolism)
• Phosphoric Triester Hydrolases (pharmacology)
• Receptors, LH (drug effects, physiology)
• Tumor Cells, Cultured (drug effects)