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Molecular Human Reproduction, Vol. 10, No. 1, pp. 43-48, 2004
© European Society of Human Reproduction and Embryology 2004

Molecular mechanisms involved in GnRH analogue-related apoptosis for uterine leiomyomas

G. Bifulco1,3, C. Miele2, M. Pellicano1, A. Trencia1, M. Ferraioli1, F. Paturzo2, G.A. Tommaselli1, F. Beguinot2 and C. Nappi1

1Dipartimento di Scienze Ostetrico–Ginecologiche, Urologiche e Medicina della Riproduzione and 2Dipartimento di Biologia e Patologia Cellulare e Molecolare ‘L. Califano’ & Istituto di Endocrinologia ed Oncologia Sperimentale del C.N.R. ‘G. Salvatore’, University of Naples ‘Federico II’, Italy

3 To whom correspondence should be addressed at: Dipartimento di Scienze Ostetrico–Ginecologiche, Urologiche e Medicina della Riproduzione, Via Pansini, 5, 80131 Napoli, Italy. e-mail: giuseppebifulco{at}hotmail.com


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and methods
 Results
 Discussion
 REFERENCES
 
GnRH agonist therapy is known to reduce uterine leiomyoma volume, although the molecular mechanisms responsible for this effect remain poorly understood. In this study, we have investigated the molecular mechanisms involved in the anti-proliferative effect of a GnRH agonist, leuprolide acetate (LA), in uterine leiomyomas obtained from six patients treated with LA for 3 months before surgery (group B), compared with tumours from six untreated patients (group A). To this end, we have evaluated the expression and the activity of molecules involved in the regulation of cell survival and proliferation. In group B, the total activity of PI3K was reduced by 60% compared with control samples. Furthermore, LA caused a reduction of PKB activation of ~50%, measured as serine 473 phosphorylation. In parallel with PKB reduction in LA samples, we observed a 60% reduction in the phosphorylation of its substrate BAD. While Bcl-xL/BAD association was not significantly modified in LA-treated leiomyomas, BAD/14.3.3 interaction was reduced, due to a 50% decreased 14.3.3 expression. In addition, LA was able to reduce the expression of the antiapoptotic proteins FLIP and PED/PEA15 by 70 and 50% respectively, compared with control samples. We next evaluated the activation of MAP kinases in leiomyomas. Activation of p42 and p44 MAP kinase isoforms was increased by 30% in group B. However, the phosphorylation of the transcription factor Elk1 was not increased in a similar fashion in LA-treated leiomyomas compared with group A. Thus, these data suggest that LA reduction of leiomyoma volume is mediated at least in part by a decreased activation of the PI3K/PKB survival pathway and by the suppression of antiapoptotic factors.

Key words: Key words: apoptosis/GnRH analogue/leuprolide acetate/myoma/proliferation


   Introduction
Top
 ABSTRACT
 Introduction
Materials and methods
 Results
 Discussion
 REFERENCES
 
Uterine leiomyomas are benign tumours originating from uterine smooth muscle cells during the reproductive period. These tumours have estrogen and progesterone receptors, and their growth and maintenance are sensitive to estrogen and/or progesterone (Rein et al., 1992, 1995). The depletion of these steroids induces a significant regression of leiomyomas (Friedman, 1993). GnRH is released from the hypothalamus and binds to specific receptors on pituitary gonadotrophs, resulting in stimulation of gonadotrophin biosynthesis, which leads to subsequent modulation of ovarian activities (Conn et al., 1991). GnRH agonist decreases estrogen and progesterone secretion from the ovary (Conn et al., 1991; Krey, 1993) and has been used in the therapy of some sex hormone-dependent cancers, including breast (Manni et al., 1986), prostatic (Beland et al., 1990), pancreatic (Gonzales-Barcena et al., 1989), endometrial (Gallagher et al., 1991) and ovarian (Emons et al., 1992) cancers. Although this effect may be mediated by an indirect mechanism based on the reduction in sex hormone secretion, there is evidence that GnRH agonist suppresses in vitro the growth of the cancer (Eidne et al., 1987; Kleinman et al., 1994) and specific binding sites for GnRH have been demonstrated in several tumours responsive to GnRH agonist (Eidne et al., 1987; Emons et al., 1993). Therefore, therapy of leiomyomas with GnRH agonist has been tried and has been shown to reduce leiomyoma volume (Filicori et al., 1983; Andreyko et al., 1988; Friedman, 1993; Zullo et al., 1998). The reduction of tumour volume is associated with a decrease in cell proliferation and with an increase in cell loss by apoptosis (Mizutani et al., 1998), but the molecular mechanisms involved have not been fully clarified.

A critical role in the regulation of cell growth and differentiation is played by the MAP kinase (ERK1/2) signalling pathway. Upon growth factor stimulation, a protein kinase cascade is initiated leading to the activation of Erk1/2, which in turn phosphorylates the nuclear transcription factor Elk-1 (Marshall, 1995) and induces the expression of mitogenic genes.

On the other hand, apoptosis, or programmed cell death, is a regulated physiological process leading to cell death and is characterized by cell shrinkage, membrane blebbing and DNA fragmentation. Antiapoptotic ligands, including growth factors and cytokines, activate the S/T kinase PKB/Akt which in turn inhibits the proapoptotic member of the Bcl2 family BAD, thereby preventing cytochrome c release (Filippa et al., 1999). PKB/Akt plays a critical role in controlling the balance between survival and apoptosis (Franke et al., 1997). This protein kinase is activated by phosphorylation within the C-terminus at Ser473 by insulin and various growth and survival factors, and functions in a wortmannin-sensitive pathway involving PI3 kinase (Vanhaesebroeck et al., 2000). PKB/Akt functions to promote cell survival by inhibiting apoptosis by means of its ability to phosphorylate and inactivate several targets, including BAD (Cardone et al., 1998). BAD is a proapoptotic member of the Bcl-2 family that can displace Bax from binding to Bcl-2 and Bcl-xL, resulting in cell death (Yang et al., 1995; Zha et al., 1996). Survival factors can inhibit the apoptotic activity of BAD by activating intracellular signalling pathways that result in the phosphorylation of BAD at Ser112 and Ser136 (Zha et al., 1996). Phosphorylation of BAD at both serine residues results in the binding of Bad to 14.3.3 proteins and the inhibition of BAD binding to Bcl-xL and Bcl-2 (Zha et al., 1996).

cFLIP is a potent inhibitor of death receptor-induced apoptosis in many cell types. cFLIP binds to the adaptor molecule Fas-associated death domain and prevents the formation of the death-inducing signalling complex (DISC). Furthermore, we have recently shown that the protein PED/PEA-15, by a similar mechanism, protects cells overexpressing it from apoptosis (Condorelli et al., 1999).

In this study we have investigated the molecular mechanisms involved in the anti-proliferative effect of a GnRH agonist, leuprolide acetate (LA), in uterine leiomyomas obtained from six patients treated with LA for 3 months before surgery, compared with tumours from six untreated patients. To this end we have investigated the effect of LA on the different pathways involved in the balance between regulation of cell proliferation and apoptosis.


   Materials and methods
Top
 ABSTRACT
 Introduction
 Materials and methods
Results
 Discussion
 REFERENCES
 
General
Electrophoresis and Western blot reagents were purchased from Bio-Rad, and protein A beads (Trisacryl) from Pierce. Unless specified otherwise, all the other chemicals were from Sigma.

Antibodies
Phospho-specific PKB (Ser473) and anti-phospho MAP kinase were from New England Biolabs (USA). All the other antibodies were purchased from either Upstate Biotechnology Inc. (USA) or Santa Cruz Biotechnology (USA).

Patients
Twelve pre-menopausal women with symptomatic uterine leiomyomata were enrolled in the study. The patients’ characteristics are reported in Table I.


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  		Table I. Patients’ characteristics
 
Inclusion criteria were: history of infertility; recurrent abortion, symptoms of increased vaginal bleeding, pelvic pressure and pain, intramural leiomyoma with an ultrasonographic diameter of 5–8 cm. Women with liver disease, ischaemic heart disease, dyslipidaemia, diabetes, acute or recent vascular thrombosis, or carcinoma of the breast or endometrium were excluded.

Other exclusion criteria were the presence of submucosal fibroids as screened by hysteroscopy, a pattern of hyperplasia with cytological atypia in the endometrial biopsy performed because of menometrorrhagia, an abnormal result on Papanicolaou smear, a positive result on a urine pregnancy test, or ultrasonographically documented calcification.

The study was approved by our Institutional Review Board and written informed consent was obtained from each patients.

Group A consisted of six patients who received only iron tablets (2 oral tablets daily) (Isairon; Pfizer, Italy). Group B consisted of six patients who received i.m. LA (Enantone 3.75; Takeda, Italy), 3.75 mg every 28 days; iron tablets (2 oral tablets daily) (Isairon; Pfizer). The treatment was administered for three cycles. Laparoscopic myomectomy was performed during the fourth week, after the third injection of LA.

During surgery, operative samples were taken for all patients and kept at –80°C.

All the procedures and assays were performed by investigators blinded to the treatment used.

Immunoblot analysis
Leiomyoma samples were homogenized with a Polytron in a buffer containing 50 mmol/l HEPES (pH 7.6), 150 mmol/l NaCl, 10 mmol/l EDTA, 10 mmol/l Na4P2O7, 2 mmol/l sodium orthovanadate, 100 mmol/l NaF, 1 mmol/l phenylmethylsulphonyl fluoride (PMSF), 100 IU/ml aprotinin, 20 µmol/l leupeptin and 1% (v/v) Triton X-100 (TAT buffer) at 4°C. Aliquots (200 µg) of the lysates were clarified by centrifugation at 5000 g for 15 min at 4°C. Samples were either immunoprecipitated with specific antibodies or directly resolved by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore Corp., USA). Membranes were blocked for 1 h in TBS (10 nmol/l Tris–HCl, 140 mmol/l NaCl, pH 7.4) containing 4% w/v bovine serum albumin and then incubated with the appropriate antibody, as indicated. After extensive washing in TBS containing 1% v/v Triton X-100, detection was performed with horseradish peroxidase-conjugated anti-mouse antibody and ECL western blotting detection reagents (Amersham Pharmacia Biotech, UK) according to the manufacturer’s instructions.

Determination of PI 3-kinase activity
Cells were solubilized for 40 min at 4°C in 50 mmol/l HEPES, pH 7.5, 150 mmol/l NaCl, 10% glycerol, 1% Nonidet P-40, 10 mmol/l EDTA, 10 mmol/l Na4P2O7, 2 mmol/l sodium orthovanadate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 mmol/l NaF, 1 mmol/l PMSF. Aliquots (200 µl) of the lysates were precipitated with an anti-phosphotyrosine antibody coupled to protein A Sepharose for 2 h at 4°C. PI3K activity was determined in the immunoprecipitates as described in Filippa et al. (1999).

Statistical analysis
Differences between the two groups in optical density of western blots regarding proteins evaluated were assessed with Student’s t-test for unpaired data using Statistica for Windows (StatSoft; Italy). Statistical significance was defined as P < 0.05.


   Results
Top
 ABSTRACT
 Introduction
 Materials and methods
 Results
Discussion
 REFERENCES
 
In LA-treated myomas (group B), phosphotyrosine-associated PI3K activity, was reduced by 60% compared with control samples (group A) (P < 0.01) (Figure 1A). Furthermore, LA caused a reduction of PKB activation of ~50%, measured as serine 473 phosphorylation in treated leiomyomas compared with control samples (P < 0.05) (Figure 1B). In parallel with PKB reduction in LA samples, we observed a 60% reduction in the phosphorylation of its substrate BAD (P < 0.01) (Figure 2). The reduction in PKB and BAD phosphorylation was not due to modification of the total amount of the two proteins, as shown in Figures 1 and 2.



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  		Figure 1. Effect of leuprolide acetate (LA) on PI 3-kinase (PI3K) activity in leiomyoma. Samples obtained from either control (group A) or LA-treated leiomyoma (group B) were lysed as reported in Materials and methods. Lysates were immunoprecipitated with an antibody to antiphosphotyrosine to measure PI3K activity (A, left histograms) or subjected to SDS–PAGE followed by western blot analysis with anti-p85PI3K (A, right histograms), antiphospho-specific PKB (Ser473) (B, left histograms) or anti-PKB (B, right histograms) antibodies. The blotted proteins were revealed by chemiluminescence as described in Material and methods. Relative quantifications of PI3K activity and p85PI3K total amounts and of the phosphorylated and total form of PKB are shown. Results are representative of at least three independent experiments.

 


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  		Figure 2. Effect of leuprolide acetate (LA) on BAD in leiomyoma. Samples obtained from either control (group A) or LA-treated leiomyoma (group B) were lysed as reported in Materials and methods. Lysates were subjected to SDS–PAGE followed by Western blot analysis with antibodies to phosphorylated BAD (A). Total amount of proteins was verified by reblotting with BAD antibody. The blotted proteins were revealed by chemiluminescence as described in Material and methods. Relative quantifications of phosphorylated and total form of BAD are reported in (B). Results are representative of at least three independent experiments.

 
No significant differences in the total amount of the Bcl-xL protein were detected in the two groups of samples (Figure 3A). On the contrary, 14.3.3 expression was reduced by 50% in the LA-treated samples (P < 0.05) (Figure 3B). Bcl-xL/BAD association was not significantly modified in treated leiomyomas compared with group A (Figure 3A), while the association between phosphorylated BAD and 14.3.3 was significantly reduced in myomas obtained from treated patients (P < 0.01) (Figure 3B). Moreover, LA was able to reduce the expression of the antiapoptotic proteins FLIP by 70% compared with control samples (P < 0.01) (Figure 4A and B). Similarly, PED protein levels were reduced by 50% in group B compared with group A (P < 0.05) (Figure 4A and B).



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  		Figure 3. Effect of leuprolide acetate (LA) on Bcl-xL and 14.3.3 proteins. Samples obtained from either control (group A) or LA-treated leiomyoma (group B) were lysed as reported in Materials and methods. Lysates were either subjected to SDS–PAGE followed by western blot analysis with antibodies to Bcl-xL (A, left histograms) or to 14.3.3 (B, left histograms) or immunoprecipitated with anti-Bcl-xL (A, right histograms) or 14.3.3 (B, right histograms) antibodies followed by immunoblotting with BAD and phospho-BAD (Ser136) antibodies. The blotted proteins were revealed by chemiluminescence as described in Materials and methods. Relative quantifications are reported. The blots are representative of at least three independent experiments.

 


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  		Figure 4. Effect of leuprolide acetate (LA) on c-FLIP and PED/PEA15. Samples obtained from either control (group A) or LA-treated leiomyoma (group B) were lysed as reported in Materials and methods. Lysates were subjected to SDS–PAGE followed by western blot analysis with antibodies to FLIP (A, upper blot) or to PED/PEA15 (A, middle blot). The blotted proteins were revealed by chemiluminescence as described in Materials and methods. Relative quantifications of PED and FLIP total proteins are reported in (B). The intensities for PED and FLIP were normalized by the intensities for ß-actin. The blots shown are representative of at least three independent experiments.

 
We next evaluated the activation of MAP kinases in leiomyomas. Activation of p42 and p44 MAP kinase isoforms, measured as the level of phosphorylation of the two isoforms with a specific antibody, was increased by 30% in group B (P < 0.05) (Figure 5A). However, the phosphorylation of the nuclear substrate Elk1 was not increased in treated leiomyomas compared with control samples (Figure 5B).



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  		Figure 5. Effect of leuprolide acetate (LA) on ERK1/2 and Elk-1 phosphorylation. Samples obtained from either control (group A) or LA-treated leiomyoma (group B) were subjected to SDS–PAGE followed by Western blot analysis with an antibody to phosphorylated ERK1/2 kinase (A) or to phospho-Elk1 (B). Total amount of protein was verified by reblotting with the respective antibodies. Results shown are representative of at least three independent experiments.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and methods
 Results
 Discussion
REFERENCES
 
In this study we attempted to clarify the effect of the GnRH analogue LA on different molecules involved in controlling the balance between cell death and survival.

The serine/threonine kinase Akt, also referred to as PKB, plays a critical role in controlling the balance between survival and apoptosis within a pathway involving PI3K (Franke et al., 1997; Vanhaesebroeck et al., 2000). Here we show that LA treatment induces a reduction of PI3K activity, having as a consequence a significant decrease in Akt phosphorylation. Akt has been shown to promote cell survival via its ability to phosphorylate BAD at Ser136 (Datta et al., 1997; del Peso et al., 1997). In LA-treated myoma, BAD phosphorylation at Ser136 is indeed reduced. Hence, BAD association with 14.3.3 proteins is significantly reduced, due also to the reduced amount of 14.3.3. Interestingly, phosphorylation of BADSer112 is also reduced by LA treatment, thus indicating that other kinases could be involved. Studies are in progress in our laboratory to identify those kinases.

Together with the inhibition of the PI3K/PKB pathway, in LA-treated myoma we observed a significant reduction in the total amount of the antiapoptotic molecules PED/PEA15 and FLIP. This suggests that LA, besides inhibiting activation of some proteins important for cell survival, reduces the expression of antiapoptotic proteins and the combination of these two events moves the cellular balance from life to death.

It has been shown that LA treatment induces ERK1/2 activation in different tissues (Kang et al., 2000). In our samples, ERK1/2 phosphorylation is induced by LA treatment. Nevertheless, we do not observe a parallel increase in the phosphorylation of the nuclear substrate of ERK, the transcription factor Elk1. This could indicate that ERK, in spite of its overactivation, is unable to increase nuclear signalling. This in turn prevents ERK proliferative effects in LA-treated myoma. Others have proposed that ERK can be sequestered into the cytoplasm by certain proteins (Formstecher et al., 2001). This could be possible in LA-treated myomas. However, the protein suggested to block ERK in the cytoplasm, PED, cannot be involved in our system, being reduced by LA treatment.

GnRH agonist therapy induces the reduction of gonadal estrogen, growth factors and blood flow, which are required for the growth of leiomyomas. Recently, Cheng et al. (2001) have reported that GnRH agonist determines DNA damage in leiomyoma cells through the indirect effect of reducing estrogen levels. On the other hand, the GnRH receptor or binding sites are present in endometrium, myometrium and leiomyoma (Wiznitzer et al., 1988; Chegini et al., 1996; Raga et al., 1998), suggesting also a direct effect of GnRH agonist in leiomyoma regression.

In conclusion, these data support the hypothesis that the GnRH agonist LA, in addition to inhibition of estrogen production, leads to the inhibition of the activation of proteins involved in cell survival and reduces the expression of antiapoptotic proteins. A proposed model is shown in Figure 6.




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  		Figure 6. Diagram of normal (A) and leuprolide acetate (LA)-modified (B) regulation of survival/apoptosis balance. LA treatment induces a reduction in PI3Kactivity determining a significant decrease in Akt phosphorylation. This event causes a reduction in BAD phosphorylation and an inhibition of cell survival. Although ERK1-2 phosphorylation is induced by LA treatment, no effect was observed on the Elk1 activation, indicating that after LA treatment ERK are unable to increase nuclear proliferation signalling. Together with these effects there is a significant reduction in the total amount of two antiapoptotic molecules (PED/PEA15, FLIP) resulting in an induction of apoptosis.

 


   REFERENCES
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 ABSTRACT
 Introduction
 Materials and methods
 Results
 Discussion
 REFERENCES
 
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Submitted on May 19, 2003; resubmitted on August 7, 2003; accepted on August 14, 2003.


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