Jörg B. Engel

Antagonists of growth-hormone-releasing hormone: an emerging new therapy for cancer

This article reviews the potential clinical uses of antagonists of growth-hormone-releasing hormone (GHRH) for tumor therapy. GHRH antagonists suppress the growth of various human cancer lines xenografted into nude mice; such tumors include breast, ovarian, endometrial and prostate cancers, lung cancers (small-cell lung carcinomas and non-small-cell lung carcinomas), renal, pancreatic, gastric and colorectal carcinomas, brain tumors (malignant gliomas), osteogenic sarcomas and non-Hodgkins lymphomas. The antitumor effects of GHRH antagonists are exerted in part indirectly through the inhibition of the secretion of GH from the pituitary and the resulting reduction in the levels of hepatic insulin-like growth factor I (IGF-I). The main effects of the GHRH antagonists are, however, exerted directly on tumors. GHRH ligand is present in various human cancers and might function as an autocrine and/or paracrine growth factor. Pituitary-type GHRH receptors and their splice variants are also found in many human cancers. The inhibitory effects of GHRH antagonists seem to be due to the blockade of action of tumoral GHRH. Antagonists of GHRH can also suppress cancer growth by blocking production of IGF-I and/or IGF-II by the tumor. Further development of GHRH antagonists that are still-more potent should lead to potential therapeutic agents for various cancers.

Drug Insight: clinical use of agonists and antagonists of luteinizing-hormone-releasing hormone

This article reviews the clinical uses of agonists and antagonists of luteinizing-hormone-releasing hormone (LHRH), also known as gonadotropin-releasing hormone. In particular, the state of the art treatment of breast, ovarian and prostate cancer, reproductive disorders, uterine leiomyoma, endometriosis and benign prostatic hypertrophy is reported. Clinical applications of LHRH agonists are based on gradual downregulation of pituitary receptors for LHRH, which leads to inhibition of the secretion of gonadotropins and sex steroids. LHRH antagonists immediately block pituitary LHRH receptors and, therefore, achieve rapid therapeutic effects. LHRH agonists and antagonists can be used to treat uterine leiomyoma and endometriosis; furthermore, both types of LHRH analogs are used to block the secretion of endogenous gonadotropins in ovarian-stimulation programs for assisted reproduction. The preferred primary treatment of patients with advanced, androgen-dependent prostate cancer is based on the periodic administration of depot preparations of LHRH agonists; these agonists can be likewise used to treat estrogen-sensitive breast cancer in premenopausal women. LHRH antagonists have been successfully used to treat prostate cancer and benign prostatic hypertrophy. Since receptors for LHRH are present on a variety of human tumors, (notably breast, prostate, ovarian, endometrial and renal cancers), cytotoxic therapy that targets these tumors with hybrid molecules of LHRH might be possible in the near future. Analogs of LHRH are now a well-established means of treating sex-steroid-dependent, benign and malignant disorders.

Ectonucleotidases CD39 and CD73 on OvCA cells are potent adenosine-generating enzymes responsible for adenosine receptor 2A-dependent suppression of T cell function and NK cell cytotoxicity

The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A2A adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (Treg) that are associated with increased mortality in OvCA. Immunohistochemical staining of human OvCA tissue specimens revealed further aberrant expression of CD39 in 29/36 OvCA samples, whereas only 1/9 benign ovaries showed weak stromal CD39 expression. CD73 could be detected on 31/34 OvCA samples. While 8/9 benign ovaries also showed CD73 immunoreactivity, expression levels were lower than in tumour specimens. Infiltration by CD4+ and CD8+ T cells was enhanced in tumour specimens and significantly correlated with CD39 and CD73 levels on stromal, but not on tumour cells. In vitro, human OvCA cell lines SK-OV-3 and OaW42 as well as 11/15 ascites-derived primary OvCA cell cultures expressed both functional CD39 and CD73 leading to more efficient depletion of extracellular ATP and enhanced generation of adenosine as compared to activated Treg. Functional assays using siRNAs against CD39 and CD73 or pharmacological inhibitors of CD39, CD73 and ADORA2A revealed that tumour-derived adenosine inhibits the proliferation of allogeneic human CD4+ T cells in co-culture with OvCA cells as well as cytotoxic T cell priming and NK cell cytotoxicity against SK-OV3 or OAW42 cells. Thus, both the ectonucleotidases CD39 and CD73 and ADORA2A appear as possible targets for novel treatments in OvCA, which may not only affect the function of Treg but also relieve intrinsic immunosuppressive properties of tumour and stromal cells.

Use of Analogs of Peptide Hormones Conjugated to Cytotoxic Radicals for Chemotherapy Targeted to Receptors on Tumors

Specific receptors for luteinizing hormone-releasing hormone (LH-RH), somatostatin, bombesin, and other peptides are found on various cancers. We review the development of cytotoxic analogs of LH-RH, somatostatin, and bombesin/gastrin releasing peptide (GRP) designed for targeting chemotherapy to peptide receptors on various cancers. Cytotoxic analogs of LH-RH, AN-152 and AN-207, containing doxorubicin (DOX) or 2-pyrrolino-DOX (AN-201), respectively, target LH-RH receptors and may be used for the treatment of prostatic and urinary bladder (urothelial), breast, ovarian and endometrial cancers, non-Hodgkins lymphomas, melanomas, and renal cell carcinomas. DOX and AN-201 have also been incorporated into the cytotoxic analogs of somatostatin, AN-162 and AN-238, respectively, which are targeted to receptors for somatostatin in prostatic, mammary, ovarian, gastric, renal, colorectal and pancreatic cancers, non-Hodgkins lymphomas, as well as glioblastomas and lung cancers. They are found to suppress the growth of these tumors and their metastases. A cytotoxic analog of bombesin/GRP, AN-215, containing 2-pyrrolino-Dox, has also been synthesized and shown to inhibit growth of various human cancer lines expressing receptors for bombesin/GRP. The toxicity, pharmacokinetics and maximum tolerated doses of AN-152 were assessed in a phase I clinical trial in women with ovarian or endometrial cancer. Disease stabilization and objective responses were found. Analog AN-152 is now in phase II clinical trials. Phase I/II studies with AN-152 in men with hormone-independent relapsed prostate cancer and patients with pancreatic and bladder cancers are pending. Targeted cytotoxic peptide analogs could provide a more efficacious and less toxic therapy for various cancers.

Therapy of ovarian cancers with targeted cytotoxic analogs of bombesin, somatostatin, and luteinizing hormone-releasing hormone and their combinations

The aim of this study was to investigate the effect of treatment of experimental ovarian cancers with targeted cytotoxic analogs as single compounds and in combination. Targeted cytotoxic analogs of bombesin (AN-215), somatostatin (AN-238), and luteinizing hormone-releasing hormone (AN-207) consisted of 2-pyrrolinodoxorubicin (AN-201) linked to the respective peptide carrier. AN-238 at 200 nmol/kg significantly inhibited growth of UCI-107, ES-2 and OV-1063 ovarian cancers. AN-215 alone at 200 nmol/kg and its combination with AN-238 at one-half of the dose were also able to inhibit the growth of UCI-107 tumors. A combination of AN-238 with AN-207at 50% of the dose strongly suppressed the proliferation of ES-2 and OV-1063 ovarian tumors. Cytotoxic radical AN-201 was toxic and had no significant effect on tumor growth. In contrast, the toxicity of the conjugated peptide analogs was low. Because ovarian cancers tend to acquire chemoresistance, we used real-time PCR to measure the mRNA expression of multidrug resistance protein 1, multidrug resistance-related protein 1, and breast cancer resistance protein after treatment. Low or no induction of multidrug resistance protein 1, multidrug resistance-related protein, and breast cancer resistance protein occurred after treatment with AN-238, AN-215, and the combination of AN-238 with AN-207 or AN-215. These results demonstrate that a therapy with cytotoxic analogs such as single agents and combinations is effective and nontoxic. Our work suggests that cytotoxic peptide analogs of luteinizing hormone-releasing hormone, somatostatin, and bombesin could be used for the therapy of ovarian cancers, considering the lack of induction of chemoresistance.

Potentiation of mammary cancer inhibition by combination of antagonists of growth hormone-releasing hormone with docetaxel

Antagonists of growth hormone-releasing hormone (GHRH) are being developed for the treatment of various cancers. In this study, we investigated the effectiveness of treatment with GHRH antagonist JMR-132 alone and in combination with docetaxel chemotherapy in nude mice bearing MX-1 human breast cancers. Specific high-affinity binding sites for GHRH were found on MX-1 tumor membranes using ligand competition assays with 125I-labeled GHRH antagonist JV-1-42. JMR-132 displaced radiolabeled JV-1-42 with an IC50 of 0.14 nM, indicating a high affinity of JMR-132 to GHRH receptors. Treatment of nude mice bearing xenografts of MX-1 with JMR-132 at 10 μg per day s.c. for 22 days significantly (P < 0.05) inhibited tumor volume by 62.9% and tumor weight by 47.8%. Docetaxel given twice at a dose of 20 mg/kg i.p. significantly reduced tumor volume and weight by 74.1% and 58.6%, respectively. Combination treatment with JMR-132 (10 μg/day) and docetaxel (20 mg/kg i.p.) led to growth arrest of most tumors as shown by an inhibition of tumor volume and weight by 97.7% and 95.6%, respectively (P < 0.001). Because no vital cancer cells were detected in some of the excised tumors, a total regression of the tumors was achieved in some cases. Treatment with JMR-132 also strongly reduced the concentration of EGF receptors in MX-1 tumors. Our results demonstrate that GHRH antagonists might provide a therapy for breast cancer and could be combined with docetaxel chemotherapy to enhance the efficacy of treatment.

Human malignant melanomas express receptors for luteinizing hormone releasing hormone allowing targeted therapy with cytotoxic luteinizing hormone releasing hormone analogue.

Cytotoxic analogue of luteinizing hormone releasing hormone (LHRH), AN-207, binds with high affinity to LHRH receptors and can be targeted to tumors expressing these receptors. We investigated the expression of LHRH receptors in surgical specimens of human malignant melanoma and evaluated the effects of AN-207 in models of human melanoma. Human melanoma specimens derived from primary tumors or metastases were examined for LHRH receptor expression by immunohistochemistry. Binding assays, Western immunoblotting, and reverse transcription-PCR analyses were used to investigate LHRH receptors in MRI-H255 and MRI-H187 transplantable human melanoma tumor lines. Antitumor effects of AN-207 and its components were evaluated in vivo in nude mice bearing xenografts of either melanoma tumor line. All 19 human melanoma specimens examined showed positive staining for LHRH receptors. The mRNA for LHRH receptors, receptor protein and binding sites for LHRH were detected in both transplantable melanoma tumor lines. AN-207 significantly inhibited the growth of MRI-H255 and MRI-H187 xenografts in vivo, reducing tumor volume by 59.9% to 79.2% and tumor weight by 61.0% to 76.9% (all P < 0.05). The components of AN-207 (LH-RH analogue carrier and cytotoxic radical AN-201 as single drugs or as an unconjugated mixture) had no significant effects. Blockade of LHRH receptors by an excess of LHRH agonist Decapeptyl suppressed the effects of AN-207. LHRH receptors are expressed in a very high percentage of human malignant melanoma specimens and can be used for targeted chemotherapy with cytotoxic LHRH analogue AN-207.

Receptors for Luteinizing Hormone Releasing Hormone Expressed on Human Renal Cell Carcinomas Can Be Used for Targeted Chemotherapy with Cytotoxic Luteinizing Hormone Releasing Hormone Analogues

Purpose: To determine the expression of luteinizing hormone releasing hormone (LHRH) receptors in specimens and cell lines of human renal cell carcinoma (RCC) and to evaluate the antitumor efficacy of targeted therapy with a cytotoxic analogue of LHRH, AN-207, in vivo. AN-207, consisting of [D-Lys6] LHRH linked to a cytotoxic radical, 2-pyrrolinodoxorubicin (AN-201), binds with high affinity to LHRH receptors and can be targeted to tumors expressing these receptors. Experimental Design: The expression of LHRH receptors was investigated in 28 surgically removed specimens of human renal cell carcinoma (RCC) by immunohistochemistry and in three human RCC cell lines A-498, ACHN, and 786-0 by radioreceptor assays, Western immunoblotting, and reverse transcription-PCR analysis. Antitumor efficacy of AN-207 was examined in experimental models of these cell lines. Results: Positive staining for LHRH receptors was found in all (28 of 28) of the examined human RCC specimens. mRNA for LHRH receptor, receptor protein, and LHRH binding sites were detected in all three cell lines. AN-207 significantly (P < 0.05) inhibited the growth of A-498, ACHN, and 786-0 xenografts in vivo producing a 67.8% to 73.8% decrease in tumor volume and a 62.2% to 77.3% reduction in tumor weight. Nontargeted cytotoxic radical AN-201 had no significant antitumor effects. Blockade of LHRH receptors by an excess of LHRH agonist Decapeptyl suppressed tumor inhibitory effects of AN-207. Conclusions: Our findings indicate that LHRH receptors are expressed in human RCC specimens and can be used for targeted chemotherapy with cytotoxic LHRH analogues.

Perifosine inhibits growth of human experimental endometrial cancers by blockade of AKT phosphorylation

OBJECTIVE Perifosine is an orally active alkylphospholipid analog, which has shown anti-tumor activity in a variety of cancers by inhibition of AKT phosphorylation. The objective of the current study was to evaluate its efficacy in in vitro models of human endometrial cancer. STUDY DESIGN The effect of 10microM and 40microM perifosine on AKT phophorylation in human endometrial cancer cell lines Ishikawa and HEC 1A was determined by Western blotting. To screen for a putative anti-tumor effect, HEC 1A and Ishikawa cells were incubated with increasing concentrations of perifosine for 24h, 48h and 72h and the number of viable cells was determined by crystal violet staining. Also the effect of a combined treatment with cisplatin and perifosine was investigated in Ishikawa cells. Flow cytometric analysis of DNA content was used to determine the effect of perifosine on the cell cycle distribution of HEC 1A and Ishikawa cells and to assess potential toxic side effects of perifosine on peripheral blood lymphocytes (PBL). RESULTS AKT phosphorylation was dose-dependently inhibited by perifosine. Concomitantly, perifosine displayed anti-tumor activity in both cell lines at concentrations that showed no effect on peripheral blood lymphocytes. Growth inhibitory effects became more pronounced with increasing treatment time. While IC 50 values at 24h were >40microM, IC 50 values after 48h were approximately 7microM in Ishikawa and 25microM in HEC 1A cells. After 72h, the IC 50 was below 1.25microM for Ishikawa and about 6microM for HEC 1A cells. Perifosine cotreatment substantially increased cytotoxic effects of cisplatin in human Ishikawa endometrial cancer cells. Of note, the anti-tumor activity of perifosine was not confined to a specific phase of the cell cycle. CONCLUSIONS The small molecule AKT inhibitor perifosine showed substantial anti-tumor activity in human endometrial cancer cell lines. Since these effects were increased with cisplatin, perifosine seems to be a good candidate for treatment combinations with classical cytostatic compounds. Thus, perifosine should be further evaluated in clinical studies in endometrial cancer.

GnRH agonists and antagonists in assisted reproduction: pregnancy rate

Although gonadotrophin-releasing hormone (GnRH) antagonists offer many advantages when used in ovarian stimulation, their popularity is lower than expected. GnRH protocols are suspected to yield lower pregnancy rates compared with the long agonist protocol. In the current study, subgroup analyses from the German IVF registry (DIR) were performed to evaluate the hypothesis that GnRH antagonists are often used as second-line medication in patients with difficult medical conditions, and thus pregnancy rates may be biased. Consequently, pregnancy rates in the first six stimulation cycles (low rank cycles) were more favourable in the group treated according to the long protocol, while in the patient group requiring more stimulation cycles (high rank cycles; 7, 9 and 10), numerically higher pregnancy rates were achieved with GnRH antagonist protocols. On the other hand, in a patient collective with equal demographic and clinical features (<35 years, tubal infertility), the long and the GnRH antagonist protocols resulted in similar pregnancy rates, supporting the hypothesis that both stimulation protocols lead to equal results.