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Endocrinology Vol. 144, No. 4 1456-1463
Copyright © 2003 by The Endocrine Society

ARTICLE

Cytotoxic Activity of Gonadotropin-Releasing Hormone (GnRH)-Pokeweed Antiviral Protein Conjugates in Cell Lines Expressing GnRH Receptors

Wei-Hsiung Yang, Maciej Wieczorck, Matt C. Allen and Terry M. Nett

Animal Reproduction and Biotechnology Laboratory, Department of Physiology, Colorado State University, Fort Collins, Colorado 80523

Address all correspondence and requests for reprints to: Terry M. Nett, Animal Reproduction and Biotechnology Laboratory, Department of Physiology, Colorado State University, Fort Collins, Colorado 80523. E-mail: terry.nett{at}colostate.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pokeweed antiviral protein (PAP), a 29-kDa ribosome-inactivating protein isolated from the leaves of Phytolacca americana, has potent cytotoxic activity once it enters the cytoplasm of a cell. It is incapable of entering cells by itself. Therefore, our objective was to determine whether a GnRH analog could be used to deliver PAP specifically to cells expressing GnRH receptors. D-Lys6-GnRH-Pro9-ethylamide was conjugated to PAP (GnRH-PAP). Chinese hamster ovary cells stably transfected with cDNA for the murine GnRH receptor and a mouse gonadotroph tumor cell line that expresses endogenous GnRH receptors ({alpha}T3-1 cells) were used to evaluate the cytotoxic effects of GnRH-PAP. We also examined cytotoxicity of GnRH-PAP using human endometrial, breast, and prostate cancer cell lines. Treatment of GnRH receptor-positive cells with GnRH-PAP resulted in dose-dependent cytotoxicity. Cytotoxicity of GnRH-PAP was dependent on number of GnRH receptors (r2 = 0.871, P < 0.05) and duration of exposure of GnRH-PAP to the cells. In contrast, GnRH-PAP was not cytotoxic to Chinese hamster ovary cells not harboring GnRH receptors. Moreover, the cytotoxic activity of GnRH-PAP could be inhibited by addition of excess GnRH analog. Neither PAP nor GnRH analog alone was cytotoxic. These results suggest that GnRH analogs can be used to specifically deliver toxin molecules to cells that express GnRH receptors. Thus, a new class of biomedicines that act as hormonotoxins against cells expressing GnRH receptors provides a novel approach for inhibiting reproduction and treating cancers that are dependent on reproductive hormones.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IMMUNOTOXINS AND HORMONOTOXINS are cytotoxic agents designed to selectively destroy targeted populations of cells that display specific surface antigens or receptors. Most immunotoxins and hormonotoxins have been developed for cancer treatment (1, 2). Immunotoxins and hormonotoxins contain a ligand such as a hormone (or growth factor), monoclonal antibody, or fragment of an antibody linked to a toxic protein from bacteria or plants. After the ligand binds to the surface of the target cell, the complex molecule (ligand, toxin, and receptor) is internalized and the toxin kills the cell. Bacterial toxins that have been targeted to cancer cells include Pseudomonas exotoxin and diphtheria toxin (3, 4, 5). However, bacterial-derived toxins are very immunogenic (6). Moreover, these toxins often display some degree of nonspecific toxicity because they are able to penetrate living cells via their cell recognition domain (6, 7).

Many plant species synthesize toxic peptides called ribosome-inactivating proteins (RIPs). Pokeweed antiviral protein (PAP) produced by Phytolacca americana belongs to this peptide family (8). This enzyme is an RNA N-glycosidase that specifically removes an adenine residue from a highly conserved and exposed surface region in the large rRNA of eukaryotic and prokaryotic ribosomes (9, 10, 11, 12), inducing a conformational change in the subunit. This irreversibly inactivates the ribosomal subunit and prevents the GTP-dependent binding of elongation factor-2 to the affected ribosome (13), thus inhibiting translation and blocking protein synthesis; this in turn leads to cell death. Recent evidence indicates that L3, a highly conserved ribosomal protein at the peptidyltransferase center, may provide a binding site for PAP, allowing depurination of the target adenine in its RNA subunit (14). PAP is a single-chain RIP (type I). In contrast, type II RIPs are composed of two peptides (15), one of them, designated the A chain, containing the N-glycosidase activity, and the other, designated the B chain, containing the binding moiety, or cell recognition domain. Unlike type II RIPs (e.g. ricin) or bacterial toxins, which are able to penetrate living cells through their cell recognition domain, PAP alone, which does not contain the cell-binding domain, is not able to penetrate living cells. PAP, which contains four cysteine residues involved in two intramolecular disulfide bonds (Cys-34 to Cys-258 and Cys-84 to Cys-105), is not glycosylated. It has been shown that glycosylated proteins (i.e. ricin) increase lectin-dependent cytotoxic activity (16, 17). Thus, PAP alone should not be cytotoxic. In fact, most type I RIPs do not have a cell recognition domain and therefore are much less likely to enter nontarget cells.

Because of these characteristics, use of a member of the RIPs may reduce problems associated with nonspecific toxicity of targeted cytotoxins (18, 19, 20). Recently it was found that PAP was nontoxic to human sperm and epithelial cells in the female genital tract even at a concentration 2000 times higher than its IC50 value against HIV-1 (10). Consequently, PAP represents an excellent candidate for the toxic moiety of an immunotoxin or hormonotoxin. In fact, PAP has been chemically linked to monoclonal antibodies to make immunotoxins because of its toxic activity and because the protein lacks carbohydrate residues, thus reducing nonspecific interactions (19). When targeted to human B cells, PAP eradicates leukemic progenitor cells obtained from patients with common-B-lineage acute lymphoblastic leukemia (21, 22, 23). Thus, PAP may be useful when linked to a targeting protein, such as an antibody or hormone, to kill cancerous cells expressing unique antigens on their surface or destroy cells harboring receptors for a particular hormone.

GnRH has long been recognized as the central regulator of the reproductive axis. Potent GnRH agonists and antagonists have been used to treat sex steroid hormone-dependent cancers of the breast, endometrium, ovary, and prostate (24, 25, 26, 27). Suppression of gonadal steroid secretion by hormonal therapy with these agonists and antagonists reduces the growth of these hormone-dependent tumors. Moreover, many human breast, endometrial, ovarian, and prostate cancers and/or cell lines have GnRH receptors on their surface, even after they have lost their dependency on sex steroid hormones for growth (28, 29, 30, 31, 32, 33). This suggests that targeted chemotherapy may provide an alternative treatment for steroid-resistant cancers in these tissues. Recently highly potent GnRH analog conjugates showed selective cytotoxicity against human breast and prostate cancer cell lines in vitro (5, 34). Here we describe the cytotoxic activity of a GnRH-PAP conjugate in several cell lines expressing GnRH receptors.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Conjugates
PAP was purified from Pokeweed (P. americana) leaves as described previously (35) with minor modifications. Briefly, Pokeweed leaves were homogenized in 5 mM sodium phosphate, pH 6.5, in a blender (Waring, Torrington, CT). The extract-containing PAP was filtered through a strainer and centrifuged at 10,000 x g. PAP was then purified by ammonium sulfate precipitation (40–100% saturation), Fractogel EMD-CM (EM Science, Gibbston, NJ) ion-exchange chromatography and DEAE Sepharose CL-6B (Sigma, St. Louis, MO) chromatography. The purified protein was dialyzed against water and lyophilized. Purity of PAP was assessed by SDS-PAGE (12% reducing gel).

Conjugates of D-Lys6-GnRH with PAP (GnRH-PAP) were prepared basically as below: 1) Thiolation of D-Lys6-GnRH: D-Lys6-GnRH (14 µmol) was reacted with 2-iminothiolane (17 µmol) to produce SH-GnRH. The yield of the SH-GnRH analog was 60–70%. The progress of the reaction was monitored by C18-HPLC. The final product was analyzed by mass spectroscopy; 2) introduction of a maleimidobutyryl group into PAP. PAP (4.8 µmol) was reacted with sulfo-GMBS (14.7 µmol; Pierce Chemical Co., Rockford, IL) for 60 min at room temperature; and 3) reaction of thiolated D-Lys6-GnRH (14 µmol) with maleimidobutyryl-PAP (4.8 µmol). After incubation for 40 min at room temperature, Cys-SH was added to block any residual maleimide groups. Therefore, PAP was conjugated to D-Lys6-GnRH with a maleimidobutyryl cross-linker. SDS-PAGE (12% reducing gel) analysis and mass spectrometry showed that the final product (as expected) was heterogenous and contained three major fractions: PAP with one GnRH molecule attached, PAP with two GnRH molecules attached, and unconjugated PAP. Unconjugated PAP in the final product was estimated to be in the range of 25–35%.

Preparation of bovine pituitary membranes and GnRH receptor-binding assay
Bovine pituitary membranes were prepared as described previously (36). D-Ala6-desGly10-GnRH-Pro9-ethylamide (D-Ala6-GnRH) was radioiodinated using a glucose-oxidase procedure, and reaction products were separated on QAE Sephadex (36). Freshly prepared [125I]D-Ala6-GnRH (0.044 ng) in 50 µl ice-cold assay buffer was added to each tube in the presence of varying concentrations of unlabeled D-Lys6-GnRH (between 0.09 nM and 9 nM), GnRH-PAP (between 0.11 nM and 333.33 nM), or PAP (between 0.1 and 330 nM). Reactions were incubated on ice for 4 h and then centrifuged (10,000 x g, 15 min, 4 C). Radioactivity in the pellet was quantified using an Apex automatic {gamma}-spectrometer (Micromedic Systems, Inc., Horsham, PA).

Quantification of GnRH receptors in cell lines
Approximately 500,000 cells/tube from the {alpha}T3-1, Chinese hamster ovary (CHO)-GnRHR, LßT2, DU145, Ishikawa, LNCaP, MCF-7, PC3, and ppC1 cell lines were placed in plastic assay tubes. Concentrations of freshly prepared [125I]D-Ala6-GnRH between 20 pM and 4.5 nM in 200 µl ice-cold complete medium were then added to each tube in the presence or absence of 450 nM unlabeled D-Ala6-GnRH. Cells were incubated on ice for 4–5 h. Then 3 ml ice-cold binding assay buffer was added to each tube before centrifugation (10,000 x g, 20 min, 4 C). Radioactivity in the pellet was quantified using an Apex automatic {gamma}-spectrometer (Micromedic Systems, Inc.). At least three independent experiments were conducted, and the pooled data were analyzed by a nonlinear regression using GraphPad prism Software (GraphPad Software, Inc., San Diego, CA). A one-site model provided the best fit of the data.

Inhibition of in vitro translation
Varying concentrations of GnRH-PAP (0.2–400 pM) or PAP (0.2–400 pM) (2 µl) were added to the translation mixture. The latter contained 35 µl rabbit reticulocyte lysate (Promega Corp., Madison, WI), 0.2 µl of a mixture of amino acids at a concentration of 1 mM, 1.4 µl of 2.5 M KCl, 1 µl ribonuclease inhibitor (40 U/µl), and 10.6 µl H2O. The reaction was started by the addition of 1 µl luciferase control RNA (1 mg/ml). After incubation (90 min at 30 C), protein synthesis was determined by analysis of luciferase (Promega Corp.) according to the manufacture’s instructions.

Cell culture
CHO cells were transfected with cDNA for the murine GnRH receptor fused to green fluorescent protein and yellow fluorescent protein to create a cell line expressing high levels of GnRH receptor (CHO-GnRHR) (37). Functional characteristics of the GnRH/green and yellow fluorescent protein receptors appeared to be identical with native receptors (38). These CHO-GnRHR cells were a generous gift from Dr. Colin Clay (Animal Reproduction and Biotechnology Laboratory, CSU). CHO-GnRHR cells were maintained in DMEM (Sigma) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 10% fetal bovine serum (FBS; Gemini Biological Products, Calabasas, CA), and 1% nonessential amino acids (Life Technologies, Inc., Grand Island, NY).

{alpha}T3-1 cells (a mouse gonadotrope tumor cell line that expresses endogenous GnRH receptors; Ref. 39) were maintained in DMEM (Sigma) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 5% FBS, and 5% horse serum. LßT2 cells (a mouse clonal gonadotrope tumor cell line that expresses endogenous GnRH receptors; Ref. 39) were maintained in DMEM (Sigma) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FBS. Both of these cell lines were provided kindly by Dr. Pamela Mellon of the University of California (San Diego, CA).

Du145 human prostate adenocarcinoma (androgen-insensitive and moderately metastatic) cells, Ishikawa human endometrial adenocarcinoma cells, LNCaP human prostate adenocarcinoma (androgen-sensitive and nonmetastatic) cells, MCF-7 human breast adenocarcinoma cells, PC3 human prostate adenocarcinoma (androgen-insensitive and highly metastatic) cells, and ppC1 human prostate adenocarcinoma cells were a generous gift from Dr. Mike Glode (University of Colorado Health Science Center, Denver, CO). These cells were maintained in RPMI 1640 medium (Sigma) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FBS.

All cell lines were cultured in a humidified environment with 5% CO2 at 37 C, and medium was replaced every 2–3 d.

Cytotoxicity studies
There are several different assays that can be used to assess cytotoxicity. For our studies, we chose two different assays, a clonogenic assay and a cell proliferation assay. In the clonogenic assay, cytotoxicity was assessed by the inhibition of formation of colonies of cells from individual cells attached to culture plates. The cell proliferation assay was a colorimetric method to determine cell viability. Details of these assays are described below.

Clonogenic assay
Cytotoxicity was assessed by colony formation as described previously (40). Briefly, 800 cells were placed in 100-mm diameter Petri dishes in 6 ml medium. Two days later, varying amounts of the test compounds were each added to the dishes that were then maintained for 6–7 d in a CO2 incubator. Cells were fixed with methanol-acetic acid (3:1) solution and then stained with crystal violet. The number of colonies, containing a minimum of approximately 50 cells, was counted using a dissection microscope. The number and area of colonies in treated cultures were expressed as a percentage of those in control cultures and taken as measure of cytotoxicity of GnRH-PAP.

Two different experiments were performed to demonstrate that the cytotoxicity was due to the binding of GnRH-PAP conjugates to GnRH receptors. First, the cytotoxic activity of GnRH-PAP against CHO-GnRHR cells was investigated in the presence of D-Lys6-GnRH. CHO-GnRHR cells were seeded in 100-mm diameter Petri dishes and maintained in complete medium at 37 C for 2 d. Then 1 µM GnRH-PAP was added in the presence of varying concentrations of D-Lys6-GnRH (1 µM-100 µM). After 6 d of incubation, cell viability was estimated with crystal violet staining. The number of colonies in treated cultures was expressed as a percentage of those in control cultures. Second, we investigated the effect of GnRH-PAP conjugates on CHO cells that did not express GnRH receptors on their surface. CHO-GnRHR and CHO cells were seeded in 100-mm diameter Petri dishes and maintained in complete medium at 37 C for 2 d. Then various concentrations of GnRH-PAP were added. After 6 d of incubation, the number of colonies was determined after staining with crystal violet.

The time of exposure of CHO-GnRHR and {alpha}T3-1 cells to GnRH-toxin conjugates needed for complete cytotoxicity was investigated as follows. Cells were placed in a 100-mm Petri dish at 800 cells/dish and incubated for 2 d (CHO-GnRHR cells) or 7 d ({alpha}T3-1 cells). The cells were grown for an additional 1, 6, 24, 48, 72, or 96 h in the presence of 1 µM GnRH-PAP conjugate. After removing the medium containing the GnRH-PAP conjugates at each time point, the cells were incubated until 7 d from the first addition of GnRH-PAP with normal culture medium. The cells were fixed with methanol-acetic acid (3:1) solution and then stained with crystal violet. The number of colonies, containing a minimum of 50 cells, was counted using a dissection microscope. The number and area of colonies in treated cultures was expressed as a percentage of those in control cultures.

Cell proliferation assay
Each well of a cell culture plate (96-well, Nunc, Rochester, NY) was seeded with approximately 2000 cells. After incubation at 37 C for 2 d, 100 µl of varying amounts of GnRH-PAP, D-Lys6-GnRH, or PAP in culture medium were added to each well. After incubation for an additional 2 d, viability was assessed using the CellTiter 96 Aqueous One solution cell proliferation assay kit (Promega Corp.) according to the manufacture’s instructions. The cell proliferation assay is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity studies. Therefore, the percentage of cell proliferation was expressed as an index of cytotoxicity. Each experiment was performed in triplicate.

Statistical analysis
All values are presented as mean ± SEM. Each experiment was performed at least in triplicate. Data were analyzed by the general linear model procedures using SAS (SAS Institute, Cary, NC). Where significant treatment effects were noted, differences among means were separated by Tukey’s test (41). Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GnRH receptor-binding assay
To determine whether GnRH-PAP conjugates or PAP alone bound to GnRH receptors, bovine pituitary membranes were incubated with varying amounts of GnRH-PAP, PAP, or D-Lys6-GnRH in the presence of [125I]D-Ala6-GnRH. As shown in Fig. 1Go, GnRH-PAP conjugates were able to displace binding of [125I]D-Ala6-GnRH from GnRH receptors in a bovine pituitary membrane preparation, albeit with slightly less efficiency than D-Lys6-GnRH alone (Kd for D-Lys6-GnRH was 0.4 nM, whereas that for GnRH-PAP was 1.0 nM). However, PAP alone did not displace binding of [125I]D-Ala6-GnRH to GnRH receptors at concentrations up to 330 nM.



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  		Figure 1. Receptor-binding activity of GnRH-PAP. Freshly prepared [125I]D-Ala6-GnRH (0.044 ng) in 50 µl ice-cold assay buffer was added to each tube in the presence of varying concentrations of unlabeled D-Lys6-GnRH (between 0.09 nM and 9 nM), GnRH-PAP (between 0.11 nM and 333.33 nM), or PAP (between 0.11 nM and 333.33 nM). Reactions were incubated on ice for 4 h and then centrifuged. Radioactivity in the pellet was quantified using an Apex automatic {gamma}-counter. Similar experiments were performed in duplicate four times with equivalent results.

 
Inhibition of in vitro translation
Because PAP inhibits protein synthesis in vitro, using the rabbit reticulocyte lysate assay, we determined that GnRH-PAP had a similar inhibitory activity [IC50 of GnRH-PAP was 7.2 ± 0.8 pM, whereas the IC50 for purified PAP was 8.9 ± 0.2 pM (P > 0.05)].

Cytotoxic activity of GnRH-PAP conjugates on CHO-GnRHR and {alpha}T3-1 cells
Clonogenic assay.
To evaluate the ability of GnRH-PAP to decrease viability of cells that express GnRH receptors on their surface, CHO-GnRHR cells were treated with varying amounts of GnRH-PAP. Representative results with CHO-GnRHR cells in the clonogenic assay are depicted in Fig. 2Go. GnRH-PAP conjugate was cytotoxic (i.e. able to prevent clones of CHO-GnRHR cells from developing), whereas PAP alone did not influence the formation of clones. As shown in Fig. 3Go, GnRH-PAP conjugates decreased viability of CHO-GnRHR cells in a concentration-dependent manner, and the concentration of conjugate giving a 50% inhibition of cell viability was approximately 0.3 µM. However, neither GnRH nor PAP alone altered viability of CHO-GnRHR cells.



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  		Figure 2. Effects of GnRH-PAP on CHO-GnRHR cells in a representative experiment. Cells were untreated (A) or were treated with PAP at 10-6 M (B), GnRH-PAP at 10-7 M (C), or GnRH-PAP at 10-6 M (D) for 6 d. Similar experiments were performed in triplicate more than six times with equivalent results.

 


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  		Figure 3. Cytotoxicity of GnRH-PAP against CHO-GnRHR cells in the clonogenic assay. CHO-GnRHR cells were seeded in 100-mm-diameter Petri dishes and maintained in complete medium at 37 C for 2 d. Then various concentrations of either GnRH-PAP, D-Lys6-GnRH or PAP were added. After 6 d of incubation, cell viability was estimated with crystal violet staining. The experiment was performed in triplicate and replicated three times. The data are presented as mean ± SEM for a representative experiment.

 
Two different experiments were performed to demonstrate that the cytotoxicity was due to the binding of GnRH-PAP conjugates to GnRH receptors. First, the cytotoxic activity of GnRH-PAP against CHO-GnRHR cells was investigated in the presence of varying concentrations of D-Lys6-GnRH. Excess D-Lys6-GnRH effectively reduced the cytotoxicity of GnRH-PAP conjugates against CHO-GnRHR cells (Fig. 4Go). Second, we examined the effect of GnRH-PAP conjugates on CHO cells that did not express GnRH receptors on their surface. The conjugates had no cytotoxic activity against GnRH-receptor-negative CHO cells (Fig. 5Go).



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  		Figure 4. Inhibition of the cytotoxic effects of GnRH-PAP by increasing doses of D-Lys6-GnRH. CHO-GnRHR cells were seeded in 100-mm-diameter Petri dishes and maintained in complete medium at 37 C for 2 d. Then 1 µM GnRH-PAP was added in the presence of varying concentrations of D-Lys6-GnRH. After 6 d of incubation, cell viability was estimated with crystal violet staining. The number of colonies in treated cultures was expressed as a percentage of those in control cultures. The experiment was performed in triplicate and replicated four times. The data are presented as mean ± SEM. Means with the same letter are not significantly different.

 


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  		Figure 5. Viability of CHO cells without GnRH receptors are not affected by GnRH-PAP. CHO-GnRHR and CHO cells were seeded in 100-mm-diameter Petri dishes and maintained in complete medium at 37 C for 2 d. Then various concentrations of GnRH-PAP were added. After 6 d of incubation, cell viability was estimated with crystal violet staining. This experiment was performed in triplicate and replicated four times. The data are presented as mean ± SEM for a representative experiment.

 
Cell proliferation assay.
To determine whether GnRH-PAP conjugates can inhibit proliferation of cells expressing GnRH receptors on their surface (another measure of cytotoxicity), CHO-GnRHR cells and {alpha}T3-1 cells were treated with varying amounts of conjugate for 2 d. As shown in Fig. 6Go, the conjugates inhibited the proliferation of these cells in a concentration-dependent manner, and the concentrations of conjugates giving a 50% inhibition of cell proliferation were approximately 0.87 µM and 0.37 µM for CHO-GnRHR and {alpha}T3-1 cells, respectively. However, neither GnRH nor PAP alone affected proliferation of CHO-GnRHR cells at these concentrations. At concentration of 5 µM, both GnRH and PAP inhibited the proliferation of {alpha}T3-1 cells slightly.



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  		Figure 6. Cytotoxicity of GnRH-PAP against CHO-GnRHR (A) and {alpha}T3-1 (B) cells in the cell proliferation assay. CHO-GnRHR and {alpha}T3-1 cells were seeded in 96-well cell culture plate and maintained in complete medium at 37 C for 2 d. Then various concentrations of GnRH-PAP, D-Lys6-GnRH or PAP were added. After 2 d of incubation, cell proliferation was measured. The experiment was performed in triplicate and replicated more than six times. The data are presented as mean ± SEM for a representative experiment.

 
Time course
We evaluated the time course of the cytotoxic activity of GnRH-PAP conjugates against CHO-GnRHR and {alpha}T3-1 cells by growing the cells for 1, 6, 24, 48, 72, and 96 h in the presence of conjugates. Exposure of each cell type to GnRH-PAP for 72 h or longer was required to achieve complete inhibition of cell growth (Fig. 7Go).



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  		Figure 7. Effect of time of exposure of CHO-GnRHR (A) and {alpha}T3-1 (B) cells to GnRH-PAP on cell viability. Cells were placed in a 100-mm Petri dish at 800 cells/dish and incubated for 2 d (CHO-GnRHR cells) or 7 d ({alpha}T3-1 cells). The cells were grown for an additional 1, 6, 24, 48, 72, or 96 h in the presence of 1 µM GnRH-PAP. After removing the medium containing 1 µM GnRH-PAP at each time point, the cells were incubated until 7 d from the first addition of GnRH-PAP with normal culture medium. The experiment was performed in triplicate and replicated three times (CHO-GnRHR cells) or five times ({alpha}T3-1 cells). The data are presented as mean ± SEM for a representative experiment.

 
Cytotoxic activity of GnRH-toxin conjugates on human tumor cell lines
To determine whether the human cancer cells had GnRH receptors, binding studies were performed on six human cancer cell lines and CHO-GnRHR, {alpha}T3-1, and LßT2 cells. Scatchard analysis of GnRH receptors (Table 1Go) indicated that high-affinity binding sites for GnRH were found in {alpha}T3-1, CHO-GnRHR, and LßT2 cells and each of the cancer cell lines. However, the concentration of GnRH receptors in human cancer cell lines was lower (P < 0.05) than that in {alpha}T3-1, CHO-GnRHR, and LßT2 cells.


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  		Table 1. The relationship between cytotoxic activity of GnRH-PAP and number of GnRH receptors on cell lines

 
The relationship between cell viability as assessed by the cell proliferation assay and number of GnRH receptors on each of the cell lines is shown in Table 1Go and Fig. 8Go. There was a linear (r2 = 0.871) relationship between the cytotoxicity of GnRH-PAP and numbers of GnRH receptors in the cell lines (Fig. 8Go).



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  		Figure 8. Relationship between cell viability and number of GnRH receptors in the cell lines. The number of GnRH receptors on eight cell lines ({alpha}T3-1, CHO-GnRHR, LßT2, Du145, Ishikawa, MCF-7, PC3, and ppC1 cells) was determined by Scatchard analysis. Cell viability of eight cell lines after exposure to 1 µM GnRH-PAP was evaluated by cell proliferation assay. The experiment was performed in triplicate and replicated at least three times.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cytotoxicity of GnRH-PAP was tested in two cell lines expressing high numbers of GnRH receptors and in cells lacking GnRH receptors. The GnRH-PAP showed significant cytotoxicity in GnRH-receptor-positive cell lines, whereas the conjugate had no effect on GnRH-receptor-negative cell lines (i.e. CHO cells). Likewise, PAP or GnRH alone was not cytotoxic in cell lines harboring GnRH receptors. Further indication that the response was specific was that the cytotoxic activity of GnRH-PAP could be reduced by addition of exogenous GnRH. Exogenous GnRH was less effective at inhibiting cytotoxic activity of GnRH-PAP than at inhibiting binding of GnRH-PAP to cell membranes. This likely is due to the fact that PAP is an enzyme and once internalized into a cell, a single molecule can deactivate many ribosomes. Thus, virtually all of the GnRH-PAP must be prevented from binding to cells to prevent the cytotoxic effect. These results clearly indicate that GnRH-PAP conjugates can specifically destroy cell types that express GnRH receptors.

The slightly lower degree of inhibition obtained in the cell proliferation experiments may be due to the shorter time period for that assay (2 d), compared with the clonogenic assay (6–7 d). The antigrowth effect is both dose and time dependent: The longer the exposure time, the higher the degree of inhibition.

When compared with a GnRH-PAP conjugate constructed by using recombinant DNA technology (42), the cytotoxicity of our GnRH-PAP conjugate appears lower. This is somewhat perplexing. Cytotoxicity of GnRH-PAP conjugates is highly dependent on the targeting moiety and number of ligand receptors on the cell surface. Evidence is mounting that both ends of the GnRH molecule are required for receptor binding (43, 44, 45, 46). Therefore, fusion toxins in which either of the terminal amino acids of the GnRH molecule are altered or attached to another moiety are very likely to have reduced receptor binding activity. Moreover, highly potent analogs of GnRH-containing D-amino acids in position 6 can be chemically conjugated to toxic moieties, and this should increase binding affinity to GnRH receptors. Thus, it is unclear why recombinant GnRH-toxin conjugates (or fusion toxins) retain binding and have cytotoxic activity on GnRH-receptor-positive cells. Based on these observations, a direct comparison of GnRH fusion toxins constructed by using recombinant DNA technology and GnRH-cytotoxins prepared by chemical conjugation is needed.

Internalization and rate of delivery of the toxin into the cytoplasm are also important factors for efficacy of a targeted cytotoxin. For a GnRH-toxin conjugate to cause death of cells expressing GnRH receptors, the conjugate must bind to receptor and be internalized by the cell. Fortunately, GnRH appears to be internalized when conjugated to small (47) or large molecules (48); however, the rate of internalization of GnRH-toxin conjugates, compared with that of GnRH alone, has not yet been determined.

Many investigators have shown that specific high-affinity binding sites for GnRH are present in about 50% of breast cancers (49), approximately 80% of endometrial and ovarian cancers (50), and nearly 85% of prostate cancers (31). Our results clearly indicate that GnRH-PAP conjugates can kill cell lines that express the GnRH receptors. Therefore, GnRH-PAP conjugates provide a potential treatment for these cancers. Moreover, GnRH-PAP conjugates may have a dual effect if cancers are sex steroid hormone dependent and GnRH-receptor positive. That is, GnRH-PAP would decrease LH-dependent steroid production by destroying gonadotropes as well as having a direct affect on the cancer cells. This approach, which remains to be tested clinically, could open a new approach to cancer therapy.

In conclusion, because GnRH-PAP appears to destroy cells harboring GnRH receptors, it may prove useful for destruction of gonadotropin-secreting cells in the pituitary gland and thereby prevent reproduction (i.e. chemical castration) and for destroying tumor cells that harbor GnRH receptors (i.e. treat breast, endometrial, ovarian, and prostate cancers).


   Footnotes
 
This work was supported by NIH Grant CA-75662.

Abbreviations: CHO, Chinese hamster ovary; D-Ala6-GnRH, D-Ala6-desGly10-GnRH-Pro9-ethylamide; FBS, fetal bovine serum; PAP, Pokeweed antiviral protein; RIP, ribosome-inactivating protein.

Received September 3, 2002.

Accepted for publication December 20, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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