No abstract

The expression of GnRH and its receptor as a part of an autocrine regulatory system of cell proliferation has been demonstrated in a number of human malignant tumors, including cancers of the breast, ovary and endometrium. Dose-dependent antiproliferative effects of GnRH agonists in cell lines derived from these cancers have been observed by various investigators. GnRH antagonists also have marked antiproliferative activity in most breast, ovarian and endometrial cancer cell lines tested, indicating that the dichotomy of GnRH agonists and antagonists might not apply to the GnRH system in cancer cells. The classical GnRH receptor signal-transduction mechanisms, known to operate in the pituitary, are not involved in the mediation of antiproliferative effects of GnRH analogs in cancer cells. Rather, the GnRH receptor interacts with the mitogenic signal transduction of growth factor receptors and related oncogene products associated with tyrosine kinase activity, via activation of a phosphotyrosine phosphatase, resulting in downregulation of cancer cell proliferation. In addition, GnRH activates nuclear factor kB and protects the cancer cells from apoptosis. Furthermore, GnRH induces activation of the c-Jun N-terminal kinase/activator protein-1 (AP-1) pathway independent of the known AP-1 activators, protein kinase or mitogen activated protein kinase.

The signaling pathway through which LHRH acts in endometrial and ovarian cancers is distinct from that in the anterior pituitary. The LHRH receptor interacts with the mitogenic signal transduction of growth factor receptors, resulting in down-regulation of expression of c-fos and proliferation. Only limited data are available on the cross-talk between LHRH receptor signaling and inhibition of mitogenic signal transduction. The present experiments were performed to analyze in endometrial and ovarian cancer cells: 1) whether mutations or splice variants of the LHRH receptor are responsible for differences in LHRH signaling, 2) the coupling of G protein subtypes to LHRH receptor, 3) the phosphotyrosine phosphatase (PTP) activation counteracting growth factor receptor tyrosine kinase activity. For these studies, the well characterized human Ishikawa and Hec-1A endometrial cancer cell lines and human EFO-21 and EFO-27 ovarian cancer cell lines were used, which express LHRH and its receptor. 1) Sequencing of the complementary DNA of the LHRH receptor from position 31 to position 1204, covering the complete coding region (position 56 to position 1042) showed that there are neither mutations nor splice variants of the LHRH receptor transcript in Ishikawa and Hec-1A endometrial cancer cells or in EFO-21 and EFO-27 ovarian cancer cells. 2) All analyzed cell lines except for the ovarian cancer cell line EFO-27 expressed both G proteins, alpha(i) and alpha(q), as shown by RT-PCR and Western blotting. In the EFO-27 cell line only G protein alpha(i), not G protein alpha(q), expression was found. Cross-linking experiments using disuccinimidyl suberate revealed that in the cell lines expressing G protein alpha(i) and G protein alpha(q), both G proteins coupled to the LHRH receptor. Inhibition of epidermal growth factor (EGF)-induced c-fos expression by LHRH, however, was mediated through pertussis toxin (PTX)-sensitive G protein alpha(i). Moreover, LHRH substantially antagonized the PTX-catalyzed ADP-ribosylation of G protein alpha(i). 3) Using a phosphotyrosine phosphatase assay based on molybdate-malachite green, treatment of quiescent EFO-21 and EFO-27 ovarian cancer cells and quiescent Ishikawa and Hec-1A endometrial cancer cells with 100 nM of the LHRH agonist triptorelin resulted in a 4-fold increase in PTP activity (P < 0.001). This effect was completely blocked by simultaneous treatment with PTX, supporting the concept of mediation through G protein alpha(i). As shown by quantitative Western blotting, EGF-induced tyrosine autophosphorylation of EGF receptors was reduced 45-63% after LHRH (100 nM) treatment (P < 0.001). This effect was completely blocked using the PTP inhibitor vanadate (P < 0.001). These results demonstrate that mutations or splice variants of the LHRH receptor in human endometrial and ovarian cancer cells are not responsible for the different signal transduction compared with that in pituitary gonadotrophs. We provide evidence that the tumor LHRH receptor couples to multiple G proteins, but the antiprol...

Background: The majority of human endometrial and ovarian cancer cell lines express receptors for GnRH. Their proliferation is time-and dose-dependently reduced by GnRH-I and its superagonistic analogues. Recently, we have demonstrated that, in human endometrial and ovarian cancer cell lines except for the ovarian cancer cell line EFO-27, the GnRH-I antagonist cetrorelix has antiproliferative effects comparable to those of GnRH-I agonists, indicating that the dichotomy between GnRH-I agonists and antagonists might not apply to the GnRH system in cancer cells. We were also able to show that the proliferation of human endometrial and ovarian cancer cells was doseand time-dependently reduced by GnRH-II to a greater extent than by GnRH-I agonists. Objective: In this study we have assessed whether or not the antiproliferative effects of the GnRH-I antagonist cetrorelix in endometrial and ovarian cancer cells are mediated through the GnRH-I receptor. Methods: We analysed the antiproliferative effects of the GnRH-I agonist triptorelin, the GnRH-I antagonist cetrorelix and GnRH-II in a panel of endometrial and ovarian cancer cell lines expressing GnRH-I receptors, in the SK-OV-3 ovarian cancer cell line that does not express GnRH-I receptors, and in four GnRH-I receptor positive GnRH-I receptor knockout cell lines. Results: We found that, after knockout of the GnRH-I receptor, the antiproliferative effects of the GnRH-I agonist triptorelin were abrogated, whereas those of the GnRH-I antagonist cetrorelix and of GnRH-II persisted. Conclusions: These data suggest that, in endometrial and ovarian cancer cells, the antiproliferative effects of cetrorelix and of GnRH-II are not mediated through the GnRH-I receptor.

Recently it was shown that a second GnRH system exists in primates. This study was conducted to investigate whether or not the receptor specific for GnRH type II is expressed in human endometrial and ovarian cancer cells and whether or not GnRH-II has effects on tumor cell proliferation. Expression of GnRH-II receptor mRNA in endometrial and ovarian cancer cell lines was demonstrated using RT-PCR and Southern blot analysis. The proliferation of these cell lines was dose- and time-dependently reduced by native GnRH-II. These effects were significantly more potent than the anitproliferative effects of equimolar doses of GnRH-I agonist Triptorelin (p<0.001). In the GnRH-II receptor positive but GnRH-I receptor negative ovarian cancer cell line SK-OV-3 native GnRH-II but not GnRH-I agonist Triptorelin had antiproliferative effects.