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were treated with LHRH agonist Triptorelin or with cytotoxic agent. Doxorubicin in the absence or presence of Triptorelin. Apoptotic cells were quantified by flow ...
0021-972X/00/$03.00/0 The Journal of Clinical Endocrinology & Metabolism Copyright © 2000 by The Endocrine Society

Vol. 85, No. 10 Printed in U.S.A.

Luteinizing Hormone-Releasing Hormone Induces Nuclear Factor ␬B-Activation and Inhibits Apoptosis in Ovarian Cancer Cells ¨ NDKER, KATJA SCHULZ, ANDREAS R. GU ¨ NTHERT, CARSTEN GRU ¨ GUNTER EMONS

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Department of Gynecology and Obstetrics, Georg-August University, D-37070 Go¨ttingen, Germany ABSTRACT More than 80% of human ovarian cancers express LHRH and its receptor as part of a negative autocrine mechanism of growth control. This study was conducted to investigate whether LHRH affects apoptosis in ovarian cancer. EFO-21 and EFO-27 ovarian cancer cells were treated with LHRH agonist Triptorelin or with cytotoxic agent Doxorubicin in the absence or presence of Triptorelin. Apoptotic cells were quantified by flow cytometry. Expression of nuclear factor kappa B (NF␬B) was assessed by RT-PCR and immunoblotting. For determination of Triptorelin-induced NF␬B activation, cells were transfected with a NF␬B-secreted alkaline phosphatase reporter gene plasmid (pNF␬B-SEAP) and cultured for 96 h with or without Triptorelin. The causal relation between Triptorelin-induced NF␬B activation and Triptorelin-induced protection against apoptosis was investigated using SN50, an inhibitor for nuclear translocation of activated NF␬B. Apoptosis induction by Triptorelin was never observed. Treatment with Doxorubicin (1 nmol/L) for 72 h increased the percentage of apoptotic cells in EFO-21 and EFO-27 ovarian cancer cell lines to 31%

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N A SERIES of recent studies, it could be demonstrated that ovarian, endometrial, and breast cancer cell lines and primary tumors of these organs express LHRH immuno- and bioactivity, as well as the messenger RNA (mRNA) for LHRH (1– 4). In addition, specific high-affinity binding sites for LHRH and the expression of the mRNA for the pituitary LHRH receptor have been detected in ovarian, endometrial, and breast cancer cell lines and in over 80% (ovarian and endometrial cancers) or 50% (breast cancer) of biopsy specimens of these cancers, suggesting the existence of some components essential for a local regulatory system based on LHRH (5). Several groups provided evidence that the in vitro proliferation of a variety of human ovarian, endometrial, and breast cancer cell lines can be inhibited by agonistic and/or antagonistic analogs of LHRH in a dose- and time-dependent manner (2, 6 –9). Comparable data were obtained in human prostate cancer (10). The function of the expression of LHRH and its receptor in human cancers has been the subject of intensive research. In view of the apparent similarity of LHRH receptors in ovarian cancers to those in the pituitary, it seemed reasonable to speculate that LHRH signal trans-

Received December 30, 1999. Revision received May 23, 2000. Accepted July 7, 2000. Address correspondence and requests for reprints to: Gu¨nter Emons, M.D., Georg-August University, Department of Gynecology and Obstetrics, Robert-Koch Street 40, D-37075 Go¨ttingen, Germany. E-mail: [email protected].

or 34%, respectively. In cultures treated simultaneously with Triptorelin (100 nmol/L), the percentage of apoptotic cells was reduced significantly, to 17% or 18%, respectively (P ⬍ 0.001). RT-PCR and immunoblotting experiments showed that NF␬B subunits p50 and p65 were expressed by ovarian cancer cell lines EFO-21 and EFO-27. When EFO-21 or EFO-27 cells were transfected with pNF␬B-SEAP and subsequently treated with Triptorelin (100 nmol/L), NF␬Binduced SEAP expression increased 5.3-fold or 4.7-fold, respectively (P ⬍ 0.001). Triptorelin-induced reduction of Doxorubicin-induced apoptosis was blocked by SN50-mediated inhibition of NF␬B translocation into the nucleus. We conclude that LHRH induces activation of NF␬B and thus reduces Doxorubicin-induced apoptosis in human ovarian cancer cells. This possibility to protect ovarian cancer cells from programmed cell death is an important feature in LHRH signaling in ovarian tumors, apart from the inhibitory interference with the mitogenic pathway. (J Clin Endocrinol Metab 85: 3815–3820, 2000)

duction pathways in tumors might be identical to those operating in pituitary gonadotrophs (11). Our findings, however, suggest that the classical LHRH receptor signal transduction pathways, known from pituitary gonadotrophs, such as phospholipase C or protein kinase C (11), are not involved in the mediation of antiproliferative effects of LHRH analogs in ovarian and endometrial cancer cells (12). LHRH analogs, rather, interfere with the mitogenic signal transduction pathway of growth factor receptors and related oncogene products associated with tyrosine kinase activity (5, 12). The mechanism of action is probably an LHRH-induced activation of a tyrosine phosphatase, counteracting the effects of receptor protein tyrosine kinase (5, 12). A comparable mechanism was detected in human prostate cancer cells (13). Imai et al. (14) suggested that LHRH-induced antiproliferative activity might be additionally mediated by stimulation of apoptotic cell death. However, in pilot experiments, we could not detect any signs of apoptosis induction by LHRH agonists or antagonists. In contrast to that, we found an antiapoptotic influence of LHRH agonists in control experiments, using cytotoxic agent Doxorubicin, a well established inductor of apoptosis. Looking for possible mechanisms that might be able to mediate this antiapoptotic effect, we regarded nuclear factor ␬B (NF␬B) to be an interesting candidate. In the present study, we checked whether the LHRH ag-

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onist Triptorelin is able to reduce Doxorubicin-induced apoptosis. In addition, we assessed whether the putative antiapoptotic effects of LHRH-agonists might be mediated through an increased activation of NF␬B. Subjects and Methods Cell lines and culture conditions The human ovarian cancer cell lines used were derived from a poorly differentiated serous adenocarcinoma (EFO-21) and a mucinous papillary adenocarcinoma of intermediate differentiation (EFO-27) (15). The cells were cultured as described in detail previously (8). To show that LHRH agonist Triptorelin ([D-Trp6]-LHRH, Ferring Pharmaceuticals Ltd., Kiel, Germany) does not induce apoptosis, cells were cultured for 72 h in the absence or presence of LHRH agonist Triptorelin (1 nmol/L–10 ␮mol/L). In a positive control experiment, cells were cultured in the presence of 1 nmol/L cytotoxic agent Doxorubicin (Sigma, Deisenhofen, Germany). To analyze whether LHRH agonist Triptorelin reduces Doxorubicin-induced apoptosis, the cells were cultured for 72 h in the presence of 1 nmol/L Doxorubicin, with or without 100 nmol/L Triptorelin. In a second set of experiments, cells were cultured for 1 h, in the presence of 100 nmol/L Doxorubicin, with or without pretreatment with Triptorelin (100 nmol/L), for 3 h. In a third set of experiments, the cells were cultured for 72 h in the presence of 1 nmol/L Doxorubicin, with or without 100 nmol/L Triptorelin, with or without 18 ␮mol/L SN50 (Calbiochem, Bad Soden, Germany), a cell membrane-permeable inhibitor peptide that blocks the nuclear translocation of the activated NF␬B complex into the nucleus (16). In control experiments, we used 18 ␮mol/L SN50 M (Calbiochem), an inactive control peptide for SN50, instead of SN50 (16). After Doxorubicin treatment, the cells were cultured in the absence of Triptorelin and Doxorubicin for 72 h. Attached and floating cells both were collected by gentle centrifugation and washed twice with PBS. To assess Triptorelin-induced NF␬B activation, cells were transfected with pNF␬B-SEAP (see below). Subsequently, these cells were cultured for 96 h in the absence of FCS and phenol red, or in the presence of 1% FCS with or without 100 nmol/L Triptorelin. Every 24 h, 100 ␮L of the media were collected and analyzed for SEAP activity (see below).

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AGG CTC AAA GTT CTC CAC CA 3⬘; p65: forward primer: 5⬘ TCA ATG GCT ACA CAG GAC CA 3⬘, backward primer: 5⬘ CAC TGT CAC CTG GAA GCA GA 3⬘), and 1.25 U Taq polymerase (Boehringer) in a PerkinElmer Corp. (Weiterstadt, Germany) DNA thermal cycler 2400. Thirty cycles of amplification were carried out: denaturation at 94 C for 30 sec, annealing at 56 C for 30 sec, followed by extension at 72 C for 60 sec. The PCR products amplified with the p50 or the p65 primers had a total length of 399 bp or 308 bp, respectively. PCR products were separated by gel electrophoresis in 1.5% agarose, and bands were visualized by ethidium bromide staining on a ultraviolet transilluminator.

Antibodies and immunoblotting For immunoblots, polyclonal rabbit antihuman p50 and rabbit antihuman p65 (Serotec, Oxford, UK) were used in an 1:1000 dilution, followed by a peroxidase-conjugated antirabbit IgG (Amersham Pharmacia Biotech, Buckinghamshire, UK) in an 1:1000 dilution. Proteins were detected with ECL reagents (Amersham Pharmacia Biotech).

Plasmids and transfection pNF␬B-SEAP (CLONTECH Laboratories, Inc., Palo Alto, CA) is designed to monitor the activation of NF␬B and NF␬B-mediated signal transduction pathways. pNF␬B-SEAP contains four tandem copies of the ␬B4 enhancer fused to the HSV-TK promotor. pTK-SEAP (CLONTECH Laboratories, Inc.) was used as a negative control to determine the background signals associated with the culture medium. The enhancerless pTK-SEAP contains HSV-TK upstream of the SEAP coding sequence. Cells were grown to approximately 50% confluence on 30-mm plates. Transfections were done using the Superfect liposome reagents and following the manufacturer’s instructions (QIAGEN), and cells were treated as described above.

Chemiluminescence Chemiluminescence detection of SEAP activity was performed according to the manufacturer’s instructions (CLONTECH Laboratories, Inc.) using a plate fluorometer (Berthold, Bad Wildbach, Germany).

Flow cytometry

Statistical analysis

To quantify cells with advanced DNA degradation, we used a procedure similar to that described by Nicoletti et al. (17). A pellet containing 1 ⫻ 106 cells (see above) was gently resuspended in 500 ␮L of hypotonic fluorochrome solution containing 0.1% Triton X-100 (Sigma), 0.1% sodium-citrate (Sigma), and 50 ␮g/mL propidium-iodide (Sigma). Cell suspensions were kept at 4 C in the dark, overnight, before flow cytometry analysis of cellular DNA content was performed using a FACScan (Becton Dickinson and Co., Mountain View, CA).

All experiments were reproduced four times in different passages of the EFO-21 and EFO-27 cell lines. Data were tested for significant differences using a Mann-Whitney U-Test.

Isolation of RNA and complementary DNA (cDNA) synthesis Total RNA was prepared, from cells grown in monolayer, using the RNeasy protocol (QIAGEN, Hilden, Germany). The concentration of RNA in each sample was determined by photospectroscopy. First-strand cDNA was generated by RT of 4 ␮g of total RNA using p(dT)15 primers (Boehringer, Mannheim, Germany) with Moloney murine leukemia virusreverse transcriptase, according to the instructions of the suppliers (Life Technologies, Karlsruhe, Germany). After determining the concentration of the cDNAs, the samples were used for semiquantitative PCR analysis. The integrity of the samples was tested by RT-PCR of the house keeping gene GAPDH (forward primer: 5⬘ CAT CAC CAT CTT CCA GGA GCG AGA 3⬘, backward primer: 5⬘ GTC TTC TGG GTG GCA GTG ATG G 3⬘).

PCR The cDNAs (2 ng) were amplified in a 50-␮L reaction vol containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L potassium chloride, 1.5 mmol/L magnesium chloride, 200 ␮mol/L of each of the deoxynucleotide triphosphates, 1 ␮mol/L of the appropriate primers (p50: forward primer: 5⬘ CAC CTA GCT GCC AAA GAA GG 3⬘, backward primer: 5⬘

Results

Ovarian cancer cell lines EFO-21 and EFO-27, treated either with authentic LHRH or LHRH agonist Triptorelin (concentrations of 1 nmol/L–10 ␮mol/L), showed no morphologic signs of programmed cell death (not shown). The absence of apoptosis induction by LHRH agonist Triptorelin was also assessed by flow cytometry (Fig. 1). The percentage of living and apoptotic cells in the LHRH agonist-treated groups (94.0% and 5.9%, respectively; Fig. 1A) were in the same range as in the untreated controls (93.0% and 6.2%, respectively; Fig. 1B). In positive controls treated with Doxorubicin (1 nmol/L), the percentage of apoptotic cells was increased to 31% (Fig. 2A). When EFO-21 cells were treated with the cytotoxic agent Doxorubicin (1 nmol/L) for 72 h, 31% of the cells showed all morphologic signs of apoptosis, including DNA fragmentation. Vacuoles in the cytoplasm, rounding-up of cells, apoptotic bodies, and bleb formation were the most characteristic signs of the apoptotic process (not shown). The percentages of living cells and apoptotic cells, assessed by flow cytometry, were 64.0% and 31.0%, respectively (Fig. 2A). If the cells were simultaneously treated with LHRH

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FIG. 1. Flow cytometric analysis of ovarian cancer cell line EFO-21. After 72 h of treatment with 100 nmol/L of LHRH agonist Triptorelin, only marginal DNA degradation (5.9%) is detectable (A). After 72 h of incubation in the absence of LHRH agonist, comparably low amounts of DNA degradations (6.2%) are detectable (B). Experiments using ovarian cancer cell line EFO-27 gave results comparable with those of ovarian cancer cell line EFO-21.

FIG. 2. Flow cytometric analysis of ovarian cancer cell line EFO-21. After 72 h of treatment with 1 nmol/L of cytotoxic agent Doxorubicin, characteristic apoptotic DNA degradation (31.0%) is observed (A). After 72 h of treatment with 1 nmol/L of cytotoxic agent Doxorubicin, in the presence of 100 nmol/L of LHRH agonist Triptorelin, a significantly lower amount of characteristic apoptotic DNA degradation (17.0%) is observed (P ⬍ 0.001) (B). Experiments using ovarian cancer cell line EFO-27 gave results comparable with those of ovarian cancer cell line EFO-21 (cf. Fig. 5B).

agonist Triptorelin (100 nmol/L) for 72 h, the percentage of apoptotic cells was reduced significantly, to 17.0% (P ⬍ 0.001; Fig. 2B). When EFO-21 cells were treated for 1 h with Doxorubicin (100 nmol/L), previous incubation with LHRH agonist Triptorelin (3 h, 100 nmol/L) reduced the percentage of apoptotic cells from 83% (no LHRH pretreatment) to 52% (LHRH pretreatment; P ⬍ 0.001; not shown). Experiments using ovarian cancer cell line EFO-27 gave results comparable with those of ovarian cancer cell line EFO-21 (see Fig. 5). To check whether ovarian cancer cell lines EFO-21 and EFO-27 express NF␬B, we have assessed the presence of NF␬B subunits p50 and p65 mRNA and immunoreactivity. Both NF␬B subunits were expressed in both ovarian cancer cell lines (Fig. 3, A and B). To examine whether NF␬B plays a role in LHRH-mediated protection against apoptosis, we transiently transfected EFO-21 and EFO-27 ovarian cancer cells with a reporter vector containing a ␬B4 cis-acting DNA sequence (response element) and the SEAP reporter gene or the pTK-SEAP vector as a negative control. SEAP activity was detected using a chemiluminescence assay. During culture of the transfected EFO-21 or EFO-27 cells, under serum- and phenol-red-free conditions, LHRH agonist Triptorelin treatment resulted in a 3.4-fold (Fig. 4A) or in a 2.9-fold (data not shown) increase of NF␬B-induced SEAP expression, respectively (P ⬍ 0.001).

Under the same conditions, including 1% FCS, the LHRH agonist Triptorelin treatment resulted in a 5.3-fold (Fig. 4B) or in a 4.7-fold (not shown) increase of NF␬B-induced SEAP expression, respectively (P ⬍ 0.001). To study the causal relationship between Triptorelininduced NF␬B activation and Triptorelin-induced protection against Doxorubicin-induced apoptosis, we used SN50, an inhibitor of nuclear translocation of activated NF␬B. When EFO-21 (Fig. 5A) or EFO-27 (Fig. 5B) cells were treated with Doxorubicin (1 nmol/L) for 72 h, the percentages of apoptotic cells, assessed by flow cytometry, were 31.0% or 34.0%, respectively. If the cells were simultaneously treated with LHRH agonist Triptorelin (100 nmol/L) for 72 h, the percentage of apoptotic cells was reduced significantly, to 17.0% or 18.0%, respectively (P ⬍ 0.001). When the cells were treated with Doxorubicin (1 nmol/L) and simultaneously with LHRH agonist Triptorelin (100 nmol/L) and with NF␬B nuclear translocation inhibitor SN50 for 72 h, the percentage of apoptotic cells was increased to 28.0% or 31.0%, respectively (P ⬍ 0.001). The inactive control peptide SN50 M had no effects. Discussion

Looking for apoptosis induction by authentic LHRH or LHRH agonists, we found no signs for an increase of pro-

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FIG. 3. Expression of p50 and p65 NF␬B subunits in ovarian cancer cell line EFO21. RT-PCR using specific primers for human p50 (left bands) and p65 (right bands), respectively (A). Immunoblot using polyclonal antihuman p50 (left), antihuman p65 (middle), or normal serum (right), respectively (B). Experiments using ovarian cancer cell line EFO-27 gave identical results as ovarian cancer cell line EFO-21.

grammed cell death, as compared with controls. Flow cytometry showed that the percentage of living and apoptotic cells in LHRH-treated groups were always in the same range as in untreated controls. In contrast to these findings, Imai et al.(14) recently have shown that LHRH analogs are able to induce Fas-ligand production in LHRH receptor positive ovarian and endometrial cancer cells and that LHRH treatment can directly inhibit the growth of Fas positive endometrial cancer cells by increasing expression of Fas ligand. They concluded that the Fas/Fas-ligand system, linked to LHRH receptor activation, may be one of the candidates to mediate the antiproliferative action of LHRH analogs by increasing apoptotic cell death within the tumor (14). However, we were unable to show that authentic LHRH or LHRH agonist Triptorelin induce apoptosis in different ovarian cancer cell lines such as EFO-21 or EFO-27. We were unable to detect Fas or Fas-ligand expression in these cell lines using the same PCR primers as used by Imai (data not shown). It is probable that the LHRH systems in cancer cells are not uniform and that differences exist between individual cancer cell lines. Doxorubicin-treated EFO-21 and EFO-27 cells showed all signs of apoptosis, including DNA fragmentation. If the cells were treated simultaneously or pretreated with LHRH agonist Triptorelin, the percentage of apoptotic cells was reduced significantly. These experiments clearly demonstrate, for the first time, that LHRH agonist Triptorelin is able to partly inhibit Doxorubicin-induced apoptosis. Taking into account the expression of high-affinity LHRH receptors, as well as of bioactive LHRH in EFO-21 and EFO-27 ovarian cancer cell lines (4), it is reasonable to speculate on the existence of an autocrine/paracrine system based on LHRH preventing apoptosis. Looking for possible mechanisms that are able to mediate LHRH-induced protection against apoptosis, we regarded NF␬B to be an interesting candidate, because it was shown to suppress chemotherapy-induced apoptosis (18). NF␬B is an ubiquitous transcription factor that is activated by a variety of cytokines and mitogens and is thought to be a key regulator of genes involved in stress, immune, and inflammatory responses. NF␬B is a heterodimer of p50 and p65 subunits. The activity of NF␬B is strictly regulated by an inhibitor, I␬B, that forms a complex with NF␬B and keeps NF␬B in the cytoplasm. When cells receive signals that activate NF␬B, I␬B is phosphorylated and degraded through a ubiquitin/proteasome pathway. The degradation of I␬B triggers the translocation of NF␬B from the cytoplasm into the

nucleus, where it regulates the transcription of NF␬Bresponsive genes by interacting with ␬B binding sites (19). The role of NF␬B in apoptosis seems to be complex, because it has been found to depend on the cell type. Some studies have implied that NF␬B promotes apoptosis in certain cells, such as neurons (20), Schwann cells (21), and embryonic kidney cells (22). In contrast, several recent reports provided convincing evidence that NF␬B is involved in apoptosis inhibition. Cells from transgenic mice deficient in NF␬B subunit p65 are highly susceptible to TNF-␣-induced apoptosis, and this susceptibility is reversed by transfection of the cells with the wild-type p65 gene (23). Inhibition of NF␬B induces apoptosis in murine B cells, a cell type expressing constitutively active NF␬B (24). Inhibition of NF␬B potentiates amyloid ␤-mediated neuronal apoptosis (25). In Chinese hamster ovary cells overexpressing wild-type insulin receptors, NF␬B plays an important role in the antiapoptotic function of insulin (26). As shown by RT-PCR and Western blotting, both NF␬B subunits were expressed in the ovarian cancer cell lines EFO-21 and EFO-27. Several other ovarian cancer cell lines were found to express NF␬B, including OVCAR-3 (27), CAOV-3 (28), and UT-OC-5 (29). One important pathway involved in initiating apoptosis is activated by stress inducers, including chemotherapeutic drugs (e.g. Doxorubicin) or ionizing radiation. These inducers damage mitochondria by an unknown mechanism, leading to the release of cytochrome c from mitochondria into the cytosol (30 –32). Cytochrome c and different other factors recruit and process procaspase 9 (31). The active caspase 9 activates the effector caspases, such as caspase 3, to induce apoptosis (31, 33, 34). The inducible transcription factor NF␬B plays an important role in inhibiting chemotherapyinduced apoptosis (23, 24, 35–37). NF␬B activation blocks caspase cleavage and cytochrome c release, indicating that NF␬B suppresses the earliest signaling components of the caspase cascade (38). Using a NF␬B-SEAP reporter gene construct, a significant increase in NF␬B activation was found in LHRH agonisttreated EFO-21 and EFO-27 ovarian cancer cells. To show the link between the inhibitory effect of Triptorelin on Doxorubicin-induced apoptosis and the Triptorelin-induced activation of NF␬B, we used a synthetic peptide (SN50), which inhibits the nuclear translocation of activated NF␬B (16). SN50 is a cell-permeable peptide constructed to control nuclear translocation of NF␬B in intact cells. If the cells were treated simultaneously with LHRH agonist Triptorelin and

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FIG. 4. NF␬B-induced SEAP expression of ovarian cancer cells (EFO21) transfected with pNF␬B-SEAP with or without (100 nmol/L) LHRH agonist Triptorelin treatment. Cells were cultured for 96 h in the absence of FCS and phenol red (A) or in the presence of 1% FCS (B). After treatment with Triptorelin, a significant increase of SEAP expression is observed (P ⬍ 0.001). A, pTK-SEAP without Triptorelin treatment; B, pTK-SEAK with Triptorelin treatment; C, pNF␬BSEAP without Triptorelin treatment; D, pNF␬B-SEAP with Triptorelin treatment. Experiments using ovarian cancer cell line EFO-27 gave results comparable with those of ovarian cancer cell line EFO-21.

with SN50 peptide, the LHRH-induced reduction of apoptosis in Doxorubicin-treated cells was virtually blunted. These experiments clearly suggest that LHRH agonist Triptorelin inhibits Doxorubicin-induced apoptosis via activation of NF␬B. Because the antiapoptotic effects of Triptorelin (45%) are much greater than its antiproliferative effects (20%) (7), it is unlikely that the antiapoptotic activity of Triptorelin is just a direct shot off of its antiproliferative effects. In addition the experiments with SN50 clearly support the concept that the activation of NF␬B induced by Triptorelin is the crucial mechanism mediating its antiapoptotic activity. The LHRH agonist-induced activation of NF␬B is presumably mediated by G protein i, because it can be inhibited by pertussis toxin (unpublished data). However, this effect seems to be independent of the LHRH-induced activation of tyrosine-phosphatase, because the phosphatase inhibitor vanadate does not have any influence on the LHRH agonistinduced NF␬B activation (unpublished data). Therefore, the LHRH-induced activation of NF␬B seems to be independent from interaction between LHRH agonists and the signal transduction of the EGF-receptor. In ovarian cancer cells,

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FIG. 5. Flow cytometric analysis of ovarian cancer cell lines EFO-21 (A) and EFO-27 (B). After 72 h of treatment with 1 nmol/L of cytotoxic agent Doxorubicin, characteristic apoptotic DNA degradation (31% or 34%, respectively) is observed (A). After 72 h of treatment with 1 nmol/L of cytotoxic agent Doxorubicin in the presence of 100 nmol/L of LHRH agonist Triptorelin, a significantly lower amount of characteristic apoptotic DNA degradation (17% or 18%, respectively) is observed (P ⬍ 0.001) (B). After 72 h of treatment with 1 nmol/L of cytotoxic agent Doxorubicin and with 100 nmol/L of LHRH agonist Triptorelin, in the presence of 18 ␮mol/L of NF␬B nuclear translocation inhibitor SN50, the amount of cells showing characteristic apoptotic DNA degradation is 28% or 31%, respectively (C). Using the inactive SN50 M control peptide instead of SN50, a significantly lower amount of characteristic apoptotic DNA degradation (16% or 15%, respectively) is observed (P ⬍ 0.001) (D).

LHRH seems to have two opposite activities: 1) the antimitotic activity through inhibition of signal transduction of mitogens such as EGF (5); and 2) the inhibition of Doxorubicin-induced apoptosis via activation of NF␬B as shown here. NF␬B plays a negative role in chemotherapy-mediated apoptosis, and LHRH is able to induce NF␬B activation, as shown by reporter gene assay. Inhibition of nuclear translocation of activated NF␬B abolishes the antiapoptotic effect of the LHRH agonist. We therefore believe that the LHRHinduced mechanism reducing apoptosis is likely to be important in blocking cell death induced by Doxorubicin therapy. We here present data showing, for the first time, a mechanism based on LHRH that suppresses chemotherapeutic drug-induced apoptosis, possibly mediated through

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NF␬B activation. The knowledge about this antiapoptotic mechanism ought to be deepened by further research and might open new therapeutic options. Acknowledgment We are grateful to Ferring-Arzneimittel (Kiel, Germany) for supplying the LHRH agonist Triptorelin.

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