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Endometrial stromal cells remodeling is critical during human pregnancy. Growth hormone-releasing hormone and its functional receptor have been shown to be expressed in gynecological cancer cells and eutopic endometrial stromal cells. Recent studies have demonstrated the potential clinical uses of antagonists of growth hormone-releasing hormone as effective antitumor agents because of its directly antagonistic effect on the locally produced growth hormone-releasing hormone in gynecological tumors. However, the impact of growth hormone-releasing hormone antagonists on normal endometrial stromal cell growth remained to be elucidated. The aim of this study was to investigate the effect of a growth hormone-releasing hormone antagonist (JMR-132) on cell proliferation and apoptosis of human decidual stromal cells and the underlying molecular mechanisms. Our results showed that growth hormone-releasing hormone and the splice variant 1 of growth hormone-releasing hormone receptor are expressed in human decidual stromal cells isolated from the decidual tissues of early pregnant women receiving surgical abortion. In addition, treatment of stroma cells with JMR-132 induced cell apoptosis with increasing cleaved caspase-3 and caspase-9 activities and decrease cell viability in a time- and dose-dependent manner. Using a dual inhibition approach (pharmacological inhibitors and siRNA-mediated knockdown), we showed that JMR-132-induced activation of apoptotic signals are mediated by the activation of ERK1/2 and JNK signaling pathways and the subsequent upregulation of GADD45alpha. Taken together, JMR-132 suppresses cell survival of decidual stromal cells by inducing apoptosis through the activation of ERK1/2- and JNK-mediated upregulation of GADD45alpha in human endometrial stromal cells. Our findings provide new insights into the potential impact of growth hormone-releasing hormone antagonist on the decidual programming in humans.

Anxiety and depression have been suggested to increase the risk for post-traumatic stress disorders (PTSD). A link between all these mental illnesses, inflammation and oxidative stress is also well established. Recent behavior studies by our group clearly demonstrate a powerful anxiolytic and antidepressant-like effects of a novel growth hormone releasing hormone (GHRH) antagonist of MIAMI class, MIA-690, probably related to modulatory effects on the inflammatory and oxidative status. In the present work we investigated the potential beneficial effects of MIA-602, another recently developed GHRH antagonist, in mood disorders, as anxiety and depression, and the possible brain pathways involved in its protective activity, in adult mice. MIA-602 exhibited antinflammatory and antioxidant effects in ex vivo and in vivo experimental models, inducing anxiolytic and antidepressant-like behavior in mice subcutaneously treated for 4 weeks. The beneficial effect of MIA-602 on inflammatory and oxidative status and synaptogenesis resulting in anxiolytic and antidepressant-like effects could be related by increases of nuclear factor erythroid 2-related factor 2 (Nrf2) and of brain-derived neurotrophic factor (BDNF) signaling pathways in the hippocampus and prefrontal cortex. These results strongly suggest that GHRH analogs should be tried clinically for the treatment of mood disorders including PTSD.

Current therapeutic approaches to Alzheimer disease (AD) remain disappointing and, hence, there is an urgent need for effective treatments. Here, we provide a perspective review on the emerging role of “metabolic inflammation” and stress as a key factor in the pathogenesis of AD and propose a novel rationale for correction of metabolic inflammation, increase resilience and potentially slow-down or halt the progression of the neurodegenerative process. Based on recent evidence and observations of an early pilot trial, we posit a potential use of extracorporeal apheresis in the prevention and treatment of AD. Apolipoprotein E, lipoprotein(a), oxidized LDL (low density lipoprotein)'s and large LDL particles, as well as other proinflammatory lipids and stress hormones such as cortisol, have been recognized as key factors in amyloid plaque formation and aggravation of AD. Extracorporeal lipoprotein apheresis systems employ well-established, powerful methods to provide an acute, reliable 60–80% reduction in the circulating concentration of these lipid classes and reduce acute cortisol levels. Following a double-membrane extracorporeal apheresis in patients with AD, there was a significant reduction of proinflammatory lipids, circulating cytokines, immune complexes, proinflammatory metals and toxic chaperones in patients with AD. On the basis of the above, we suggest designing clinical trials to assess the promising potential of such “cerebropheresis” treatment in patients with AD and, possibly, other neurodegenerative diseases.

In this article, we briefly review the identification of GHRH, provide an abridged overview of GHRH antagonists, and focus on studies with GHRH agonists. Potent GHRH agonists of JI and MR class were synthesized and evaluated biologically. Besides the induction of the release of pituitary GH, GHRH analogs promote cell proliferation and exert stimulatory effects on various tissues, which express GHRH receptors (GHRH-Rs). A large body of work shows that GHRH agonists, such as MR-409, improve pancreatic β-cell proliferation and metabolic functions and facilitate engraftment of islets after transplantation in rodents. Accordingly, GHRH agonists offer a new therapeutic approach to treating diabetes. Various studies demonstrate that GHRH agonists promote repair of cardiac tissue, producing improvement of ejection fraction and reduction of infarct size in rats, reduction of infarct scar in swine, and attenuation of cardiac hypertrophy in mice, suggesting clinical applications. The presence of GHRH-Rs in ocular tissues and neuroprotective effects of GHRH analogs in experimental diabetic retinopathy indicates their possible therapeutic applications for eye diseases. Other effects of GHRH agonists, include acceleration of wound healing, activation of immune cells, and action on the central nervous system. As GHRH might function as a growth factor, we examined effects of GHRH agonists on tumors. In vitro, GHRH agonists stimulate growth of human cancer cells and upregulate GHRH-Rs. However, in vivo, GHRH agonists inhibit growth of human cancers xenografted into nude mice and downregulate pituitary and tumoral GHRH-Rs. Therapeutic applications of GHRH analogs are discussed. The development of GHRH analogs should lead to their clinical use.

The phase II trial of AEZS-108 examined the role of the hybrid molecule as salvage chemotherapy in pretreated patients with disease progressing during standard therapies including multiple lines of hormonal agents and taxane-based chemotherapies. The compound showed promising activity in this cohort of patients with 13 of 25 (52%) patients achieving clinical benefit as well as radiographic (56%) and prostate-specific antigen stabilization (84%).

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Malignant mixed Müllerian tumors (MMMTs) are aggressive malignant neoplasms with a high recurrence rate and poor prognosis. Despite advances in adjuvant therapies in recent years, the prognosis of these tumors has not improved. Growth hormone-releasing hormone (GHRH) is produced by a variety of malignant tumors and acts as a growth factor in an autocrine/paracrine manner. Its function requires the presence of its receptors to exert its effects on neoplastic cells. In this study, we evaluated the expression of GHRH receptors (GHRH-R) in a group of MMMTs. Thirty-one examples of MMMTs from endometrium, ovary, uterine tube, and pelvic peritoneum were retrieved from the files of Department of Pathology at the University of Miami, Jackson Memorial Hospital. Immunohistochemistry for GHRH-R was performed on paraffin sections and the staining results were evaluated separately in both epithelial and mesenchymal components of each tumor. The presence of pituitary type growth hormone-releasing hormone receptor mRNA and that of its biologically active splice variant were also evaluated by RT-PCR in 6 of the tumors. Positive immunohistochemical reaction for GHRH-R was detected in 30 tumors (96%). The epithelial and sarcomatous components were positive in 30 (96%), whereas one endometrial tumor was negative in both components. The mRNA for GHRH-R and its splice variant was found in all 6 tested tumors. This study shows that GHRH-R is expressed by the majority of MMMTs in both epithelial and mesenchymal components. This finding could potentially serve as a basis for therapeutic approaches using synthetic peptide antagonists of GHRH-R that have shown significant efficacy with minimal side effects in experimental models.

Introduction:Increasing evidence suggests that prostate cancer cells undergo unique metabolic reprogramming during transformation. A master regulator of cellular homeostasis, 5′-AMP-activated protein kinase (AMPK), directs metabolic adaptation that supports the growth demands of rapidly dividing cancer cells. The utilization of AMPK as a therapeutic target may therefore provide an effective strategy in the treatment of prostate cancer.Areas covered:Our review describes the regulation of AMPK by androgens and upstream kinases including the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) in prostate cancer. Oncogenic, AMPK-regulated pathways that direct various metabolic processes are also addressed. Furthermore, we discuss the role of AMPK in growth arrest and autophagy as a potential survival pathway for cancer cells. In addition, by regulating non-metabolic pathways, AMPK may stimulate migration and mitosis. Finally, this review summarizes efforts to treat prostate cancer with pharmacological agents capable of modulating AMPK signaling.Expert opinion:Current research is primarily focused on developing drugs that activate AMPK as a treatment for prostate cancer. However, oncogenic aspects of AMPK signaling calls for caution about employing such therapies. We think that inhibitors of CaMKK2 or AMPK, or perhaps the modulation of downstream targets of AMPK, will gain importance in the clinical management of prostate cancer.

Growth hormone releasing hormone (GHRH) from hypothalamus nominatively stimulates growth hormone release from adenohypophysis. GHRH is also produced by cancers, acting as an autocrine/paracrine growth factor. This growth factor function is seen in lymphoma, melanoma, colorectal, liver, lung, breast, prostate, kidney, bladder cancers. Pituitary type GHRH receptors and their splice variants are also expressed in these malignancies. Synthetic antagonists of the GHRH receptor inhibit proliferation of cancers. Besides direct inhibitory effects on tumors, GHRH antagonists also enhance cytotoxic chemotherapy. GHRH antagonists potentiate docetaxel effects on growth of H460 non-small cell lung cancer (NSCLC) and MX-1 breast cancer plus suppressive action of doxorubicin on MX-1 and HCC1806 breast cancer. We investigated mechanisms of antagonists on tumor growth, inflammatory signaling, doxorubicin response, expression of drug resistance genes, and efflux pump function. Triple negative breast cancer cell xenografted into nude mice were treated with GHRH antagonist, doxorubicin, or their combination. The combination reduced tumor growth, inflammatory gene expression, drug-resistance gene expression, cancer stem-cell marker expression, and efflux-pump function. Thus, antagonists increased the efficacy of doxorubicin in HCC1806 and MX-1 tumors. Growth inhibition of H460 NSCLC by GHRH antagonists induced marked downregulation in expression of prosurvival proteins K-Ras, COX-2, and pAKT. In HT-29, HCT-116 and HCT-15 colorectal cancer lines, GHRH antagonist treatment caused cellular arrest in S-phase of cell cycle, potentiated inhibition of in vitro proliferation and in vivo growth produced by S-phase specific cytotoxic agents, 5-FU, irinotecan and cisplatin. This enhancement of cytotoxic therapy by GHRH antagonists should have clinical applications.

Agonists of luteinizing hormone-releasing hormone (LHRH) induce a reversible hypoestrogenic state through the down-regulation of LHRH receptors and desensitization of the pituitary. Since endometrial implants are estrogen sensitive, LHRH agonists have frequently been used for medical treatment of endometriosis. Nowadays, LHRH agonists can be considered in general as a second-line medical treatment for endometriosis-related symptoms, as oral therapy with dienogest is as effective and has fewer side effects. However, therapy with LHRH agonists for 3-6 months prior to in vitrofertilization remains the treatment of choice in patients with endometriosis, as it significantly increases pregnancy rates.LHRH agonists are used prior to surgery and as an adjuvant after an operation to prevent recurrence or prolong disease-free intervals. Adverse effects of LHRH agonists are due to hypoestrogenism and include hot flushes, vaginal dryness, loss of libido, sleep disturbances and a diminished bone density which limits the duration of their administration to 6 months. For long-term treatment, add-back of estrogen and/or progestin,/or progestin only with or without bisphosphonates, can be used, but existing studies only cover a 12-month period of treatment.LHRH antagonists competitively block the pituitary receptors for LHRH. Consequently, a partial pharmacological hypophysectomy with a reduction of the estrogen levels to a desired level is possible if LHRH antagonists are adequately dosed. As endometriotic implants require relatively high levels of estrogen, partially lower plasma levels of estrogens are sufficient to prevent the loss of bone density. A long-term treatment without add-back therapy is also possible.

Malignant melanoma is the deadliest form of skin cancer; the treatment of advanced and recurrent forms remains a challenge. It has recently been reported that growth hormone-releasing hormone (GHRH) receptor is involved in the pathogenesis of melanoma. Therefore, we investigated the effects of our new GHRH antagonists on a human melanoma cancer cell line. Antiproliferative effects of GHRH antagonists, MIA-602, MIA-606 and MIA-690, on the human melanoma cell line, A-375, were studied in vitrousing the MTS assay. The effect of MIA-690 (5 μg/day 28 d) was further evaluated in vivoin nude mice bearing xenografts of A-375. Subcellular localization of p27 was detected with Western blot and immunofluorescent staining. MIA-690 inhibited the proliferation of A-375 cells in a dose-dependent manner (33% at 10 μM, and 19.2% at 5 μM, P< 0 .05 vs. control), and suppressed the growth of xenografted tumors by 70.45% (P< 0.05). Flow cytometric analysis of cell cycle effects following the administration of MIA-690 revealed a decrease in the number of cells in G2/M phase (from 19.7% to 12.9%, P< 0.001). Additionally, Western blot and immunofluorescent studies showed that exposure of A-375 cells to MIA-690 triggered the nuclear accumulation of p27. MIA-690 inhibited tumor growth in vitroand in vivo, and increased the translocation of p27 into the nucleus thus inhibiting progression of the cell cycle. Our findings indicate that patients with malignant melanoma could benefit from treatment regimens, which combine existing chemotherapy agents and novel GHRH-antagonists.

Introduction:Androgen deprivation therapy (ADT) has been the first-line standard of care for treating patients with hormone-sensitive advanced prostate cancer (PCa) for many decades. The agonists of luteinizing hormone-releasing hormone (LHRH), also called gonadotropin-releasing hormone, are still the most frequently used form of medical ADT.Areas covered:This article reviews the available data and most recent information concerning the use of LHRH agonists in advanced PCa. This article also reviews the discovery and development of LHRH agonists and summarizes the clinical evidence for their efficacy in PCa.Expert opinion:The introduction and application of agonists of LHRH has modernized and improved the treatment of advanced PCa. The life-saving benefits of LHRH agonists are well established, yet underestimated. Despite their efficacy, agonists of LHRH have several disadvantages or drawbacks including disease flare. The approach to ADT has been recently further refined with the development of the LHRH antagonist degarelix. Degarelix, a highly clinically effective third-generation LHRH antagonist, is currently available in most countries for therapy of advanced PCa. This new drug offers attractive alternatives to LHRH agonists for treatment of advanced PCa. A therapy for castration-resistant PCa based on a targeted cytotoxic analog of LHRH, AEZS-108, is also emerging.

Previously, we have shown that the targeted cytotoxic somatostatin (sst) analogue AN-162 AZSE-124 inhibits the growth of MDA-MB-231 human breast cancers xenografted into nude mice. In this study, we examined the trafficking of AN-162 into the cell, the expression of the somatostatin receptors (sstr) in specimens of human triple-negative breast cancers (TNBC), and the effect of AN-162 on HCC 1806 human TNBC xenografts. The expression of sstr in TNBC tumor samples was investigated by immunohistochemical staining. The expression of sstr in HCC 1806 was evaluated by reverse transcription PCR. Internalization studies with 125I-labeled AN-162 were carried out and the autofluorescence sign of doxorubicin moiety in the cell nucleus after incubation with AN-162 was measured using a fluorescence assay. The effects of AN-162 on the growth of HCC 1806 xenografted into nude mice were studied. A fluorescence microscopy cytotoxicity assay in vitroto detect cell death after treatment with AN-162 was also carried out. About 28 of TNBC tumor specimens showed a positive staining for sstr subtype 2a. HCC 1806 expresses all five subtypes of sstr. In the fluorescence cytotoxicity assay, dead HCC 1806 cells were found 24 h after incubation with AN-162. The growth of HCC 1806 tumors in nude mice was significantly inhibited by treatment with AN-162. AN-162 was internalized into the HCC 1806 cells and doxorubicin moiety was detected in the cell nuclei. This study is the first to show that the trafficking of the cytotoxic sst analogue AN-162 into the cell is mediated by sstr. Our work shows that the growth of xenografted HCC 1806 TNBCs can be effectively inhibited in vivowith AN-162. This investigation provides information on the mechanism of action and efficacy of this new targeted cytotoxic sst analogue and identifies in this relation the sstr as a favorable therapeutic target in TNBC.

We provide new viewpoints of hormonal control of benign prostatic hyperplasia (BPH). The latest treatment findings with 5-alpha reductase inhibitors (5-ARIs) finasteride and dutasteride, refined indications, efficacy, and safety are discussed and compared. We also discuss potential new 5-ARIs and other hormonal treatments.

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 μM) combined with 5-FU (1.25 μM), irinotecan (1.25 μM) or cisplatin (1.25 μM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G1fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40–55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56–85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.

Hepatic carcinoma is a major health problem worldwide. Its incidence is increasing in Western countries and there is currently no effective systemic therapy against it. Targeted treatment modalities developed in the past few years have provided very limited success. Development of new treatment strategies is therefore essential. We investigated the effects of bombesingastrin-releasing peptide (BNGRP) antagonist RC-3940-II on experimental human liver cancers in nude mice. SK-Hep-1 and Hep-G2 cancers transplanted subcutaneously into nude mice were treated daily with 10 or 20 µg of RC-3940-II. Tumor growth was monitored for 50–184 days in five experiments. Tumor gene expression was analyzed with PCR array and protein expression by immunoblotting. Characteristics of BNGRP receptors in the tumors were analyzed by binding assays. Effects of RC-3940-II on cell proliferation were investigated in vitro. RC-3940-II inhibited the growth of SK-Hep-1 cancers in nude mice by 65–98, with total regression in 9 of 36 tumors in three experiments. The BNGRP antagonist inhibited the growth of Hep-G2 cancers as well by 73–82 in two experiments, being effective even on originally large tumors. Gene expression analysis showed an increase in several angiogenesis inhibitors and decrease in proangiogenic genes after RC-3940-II treatment. Receptor assays demonstrated high-affinity binding sites for BNGRP in both tumor lines. BNGRP antagonist RC-3940-II powerfully inhibits growth of SK-Hep-1 and Hep-G2 cancers in nude mice. Its effect may be linked to changes in expression of those cancer genes important in angiogenesis, invasion, and metastasis. RC-3940-II may be considered for further investigations in treatment of liver cancers.

We investigated the efficacy of a powerful antagonist of bombesin/gastrin-releasing peptide (BN/GRP) RC-3940-II administered as a single agent or in combination with cytotoxic agents on the growth of HT-29, HCT-116 and HCT-15 human colon cancer in vitro and in vivo. GRP-receptor mRNA and protein were found in all three cell lines tested. Exposure of HT-29 cells to 10 μM RC-3940-II led to an increase in the number of cells blocked in S phase and G2/M and cells with lower G0/G1DNA content. Similar changes on the cell cycle traverse of HT-29 cells could also be seen at lower concentrations of RC-3940-II (1 μM) after pretreatment with 100 nM GRP (14–27), indicating a dose-dependent mechanism of action based on the blockage of BN/GRP induced proliferation of tumor cells at lower concentrations.Daily in vivo treatment with BN/GRP antagonist RC-3940-II decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice by 25 to 67% (p < 0.005). Combined treatment with RC-3940-II and chemotherapeutic agents 5-FU and irinotecan resulted in a synergistic tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts by 43% to 78%. In HT-29 and HCT-116 xenografts the inhibition for the combinations of RC-3940-II and irinotecan vs. single substances (p < 0.05) was significantly greater.These findings support the use of RC-3940-II as an anticancer agent and may help to design clinical trials using RC-3940-II in combinations with cytotoxic agents.

Introduction:Receptors for the luteinizing hormone-releasing hormone [LHRH, also known as gonadotropin-releasing hormone (GnRH)] can be regarded as an ideal target for a personalized medicine approach in cancer therapy. LHRH receptors are expressed in about 80% of human endometrial and ovarian cancers, 86% of prostate cancer, and about 50% of breast cancers including triple-negative breast cancer, as well as bladder, colorectal, and pancreatic cancers, sarcomas, lymphomas, melanomas, and renal cell carcinomas. Apart from the pituitary and reproductive organs, other organs and hematopoietic stem cells express LHRH receptors. Thus, a targeted cytotoxic LHRH analog such as AEZS-108(formerly known as AN-152), in which doxorubin is linked to the LHRH agonist [D-Lys6]LHRH, appears to be a suitable drug for targeted chemotherapy of cancers expressing receptors for LHRH, which would be more efficacious and less toxic than standard systemic chemotherapy.Areas covered:This review discusses the development of AEZS-108, its targeting mechanism, preclinical studies, and clinical trials in patients with endometrial, ovarian, prostatic, and bladder cancers. We emphasize its development as a personalized medicine approach. The studies reviewed demonstrate the effects of the cytotoxic LHRH analog, AEZS-108, mediated by LHRH receptors, in in vivomodels of LHRH-receptor-positive human endometrial, ovarian, breast, prostatic, colorectal, pancreatic, and bladder cancers xenografted into nude mice. Intravenous administration of AEZS 108 inhibits the growth of LHRH-receptor-positive tumors better than equimolar doses of the cytotoxic agent doxorubicin and is far less toxic. AEZS 108 has no antitumor activity in cancers negative to LHRH receptor. This strongly supports the concept of targeting cytotoxic chemotherapy to tumor cells expressing LHRH receptors. Early clinical trials have demonstrated the efficacy of AEZS-108. A Phase I trial assessed the maximum tolerated dose and pharmacokinetics and pharmacodynamics of AEZS-108 given once every 3 weeks in patients with gynecological cancers. Two Phase II studies in heavily pretreated ovarian and recurrent endometrial cancers showed good clinical activity after a maximum of six courses of AEZS-108 as a single agent. Ongoing clinical studies with AEZS-108 in men with castration-resistant prostate cancer and patients with chemotherapy refractory bladder cancer had shown early signs of clinical efficacy. Side effects are moderate and easily manageable. In particular, no pituitary or cardiac toxicity is observed.Expert opinion:AEZS-108 is a cytotoxic analog designed for receptor-mediated targeted chemotherapy and consists of an LHRH carrier linked to doxorubicin. Preclinical studies demonstrate that the uptake of AEZS-108 is achieved by receptor-mediated endocytosis. Results of Phase I and II clinical trials in patients with gynecological cancers demonstrated anticancer activity without cardiotoxicity even in highly pretreated patients. Phase I/II studies in castration-resistant prostate cancer and chemotherapy refractory bladder cancer are in progress. Targeted chemotherapy with a cytotoxic analog of LHRH, such as AEZS-108, is therefore being considered for Phase III studies in advanced endometrial cancers positive for LHRH receptor. LHRH receptors are also present in human colon cancers, melanomas, lymphomas, and sarcomas, and treatment of these cancers with AEZS-108 should also be undertaken. Before such treatment with AEZS-108 is begun, the status of tumoral LHRH receptors of patients must be determined.

AMP-activated protein kinase (AMPK) regulates cellular proliferation, growth and metabolism. Targeted activation of AMPK is considered an important therapeutic strategy for cancer treatment. To evaluate the effect of growth hormone-releasing hormone (GHRH) and its antagonist MZ-5-156 on the phosphorylation of AMPK and other related regulatory intracellular proteins we employed human non-small cell lung cancer cell line A549, which expresses GHRH receptors. Treatment of A549 cells with GHRH antagonist decreased cell proliferation and activated AMPK as well as glycogen synthase kinase (GSK)3β. Furthermore, MZ-5-156 inhibited Akt, the mammalian target of rapamycin (mTOR) and its downstream target eIF4E which controls protein synthesis and cell growth. GHRH(1-29)NH2 counteracted all these effects. HeLa human endometrial cancer cells which do not express any GHRH receptors were used as a negative control and GHRH did not induce the AMPK activation in these cells. Our results demonstrate for the first time that GHRH antagonists can regulate the AMPK metabolic pathway, which is crucial for the growth of non-small cell lung cancer and other major cancers.

Growth Hormone-Releasing Hormone (GHRH) is secreted by the hypothalamus and regulates the release of growth hormone from the anterior pituitary gland. In addition to its endocrine role, this peptide has been shown to act as a growth factor in diverse malignancies. Recent studies indicate that GHRH is also a regulator of several important physiological processes. These processes which include the regulation of the metabolism of the reactive oxygen and nitrogen species are similarly enhanced by GHRH agonists. In contrast, GHRH antagonists can counteract the growth factor effects of GHRH. GHRH and its agonists have been shown to contribute to the recovery of heart tissue after myocardial infarction and can promote the survival and proliferation of pancreatic islets after transplantation into diabetic animals.

Gastrin-releasing peptide (GRP), the mammalian bombesin (BN), appears to be involved in the growth of several neoplasms. BN/GRP receptors (BN/GRP-Rs) are expressed in a variety of cancer cells and have limited distribution in normal human tissue. Thus inhibition of BN/GRP-Rs represents an attractive target for pharmacological treatment of some human malignancies. This review will focus on intracellular signaling pathways, which have been characterized to mediate BN/GRP-dependent receptor biological effects as well as on various approaches to target BN/GRP-Rs for therapeutic and diagnostic interventions in human malignancies.

We investigated the mechanisms of inhibitory effect of growth hormone-releasing hormone (GHRH) antagonist JMR-132 on the growth of HT29, HCT-116 and HCT-15 human colon cancer cells in vitro and in vivo.  High-affinity binding sites for GHRH and mRNA for GHRH and splice variant-1 (SV1) of the GHRH receptor were found in all three cell lines tested. Proliferation of HT-29, HCT-116 and HCT-15 cells was significantly inhibited in vitro by JMR-132. Time course studies revealed that the treatment of human HCT-116 colon cancer cells with 10μM GHRH antagonist JMR-132 causes a significant DNA damage as shown by an increase in olive tail moment (OTM) and loss of inner mitochondrial membrane potential (∆Ψm). Western blotting demonstrated a time-dependent increase in protein levels of phospho-p53 (Ser46), Bax, cleaved caspase-9, -3, cleavage of poly(ADP-ribose)polymerase (PARP) and a decrease in Bcl-2 levels. An augmentation in cell cycle checkpoint protein p21Waf1/Cip1 was accompanied by a cell cycle arrest in S-phase. DNA fragmentation visualized by the comet assay and the number of apoptotic cells increased time dependently as determined by flow cytometric annexinV and PI staining assays. In vivo, JMR-132 decreased the volume of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic mice up to 75% (p

Growth-hormone-releasing hormone (GHRH) and its receptors are expressed in a wide variety of normal and malignant tissues. This article describes various GHRH antagonists, their mechanisms of action, andin vivostudies. These antagonists are a promising potential therapy for a wide range of cancers, and perhaps other diseases.

Both agonists and antagonists of luteinizing-hormone-releasing hormone (LHRH) are in clinical use for a wide range of cancers, benign prostatic hypertrophy, fibroids and reproductive disorders. This article describes the existing applications, those under investigation, and potential applications for this type of drug to target diseases such as Alzheimer's.

The development of antagonists of growth hormone (GH) - releasing hormone (GH-RH) is reviewed. GH-RH antagonists bind with a high affinity to pituitary receptors for GH-RH and inhibit the release of GH in vitro and in vivo. The main applications of GH-RH antagonists would be for tumor therapy. The antitumor effects of GH-RH antagonists are exerted in part indirectly through the inhibition of the secretion of pituitary GH and the reduction in the levels of hepatic insulin like growth factor (IGF-I). However, principal effects of the GH-RH antagonists are exerted directly on tumors. Antagonists of GH-RH inhibit the proliferation of various cancer cell lines in vitro and suppress in vivo the levels and the expression of mRNA for IGF-I and IGF-II in tumors. In many human cancers, the effects of GH-RH antagonists appear to be due to the blockade of the action of tumoral GH-RH. GH-RH ligand is present in various human cancers indicating that it may be an autocrine/paracrine growth factor. Splice variants (SVs) of GH-RH receptors and pituitary type of GH-RH receptors that might mediate effects of tumoral GH-RH and of GH-RH antagonists were demonstrated in many human cancers. This suggests the presence of a stimulatory loop based on GH-RH and SVs or pituitary type of GHRH receptors in diverse tumors. It was shown that GH-RH antagonists inhibited the growth of various human cancer lines xenografted into nude mice including mammary, ovarian, endometrial and prostate cancers, small cell lung carcinomas (SCLC) and non-SCLC, renal, pancreatic, gastric and colorectal carcinomas, malignant gliomas, osteosarcomas and Non- Hodgkin's lymphomas. Further development of GH-RH antagonists should lead to potential therapeutic agents for various cancers.

The cytotoxic analog of bombesin (BN)/gastrin releasing peptide (GRP) AN-215 consisting of 2-pyrrolinodoxorubicin (AN-201), a superactive derivative of doxorubicin linked to a bombesin analog carrier, displays a high affinity to BN/GRP receptors and can be targeted to tumors that express these receptors. We evaluated the antitumor effect and the toxicity of AN-215 in 5 human breast cancer cell lines xenografted into nude mice. In addition, we measured the mRNA expression of multi drug resistance protein 1 (MDR-1), multi drug resistance related protein 1 (MRP-1) and breast cancer resistance protein (BCRP) by real-time PCR analysis after treatment with AN-215. All five cell lines expressed BN/GRP receptors, and AN-215 significantly (P<0.05) inhibited tumor growth in all models, while its cytotoxic radical AN-201 had no significant effect in four models. In MX-1 tumors, AN-201 had a significantly weaker antitumor effect than AN-215. The effect of AN-215 was nullified by a blockade of BN/GRP receptors with a bombesin antagonist. Low or no induction of MDR-1, MRP-1 and BCRP occurred after treatment with AN-215. In conclusion, targeted chemotherapy with the cytotoxic BN/GRP analog AN-215 strongly inhibits breast cancers that express BN/GRP receptors and might provide a new treatment modality for mammary carcinoma.

Targeted chemotherapy is a modern approach aimed at increasing the efficacy of systemic chemotherapy and reducing its side effects. The peptide receptors expressed primarily on cancerous cells can serve as targets for a selective destruction of malignant tumors. Binding sites for LHRH (now known in genome and microarray databases as GNRH1), were found on 52% of human breast cancers, about 80% of human ovarian and endometrial cancers, and 86% of human prostatic carcinoma specimens. Because LHRH receptors are not expressed on most normal tissues, they represent a specific target for cancer chemotherapy with antineoplastic agents linked to an LHRH vector molecule. To test the efficacy of targeted chemotherapy based on LHRH analogs, we recently developed a cytotoxic analog of LHRH, designated AN-152, which consists of [d-Lys6]LHRH covalently linked to one of the most widely used chemotherapeutic agents, doxorubicin (DOX). In addition, we designed and synthesized a highly active derivative of DOX, 2-pyrrolino-DOX (AN-201), which is 500–1000 times more potent than DOX in vitro. AN-201 is active against tumors resistant to DOX, and noncardiotoxic. As in the case of DOX, AN-201 was coupled to carrier peptide [d-Lys6]LHRH to form a superactive targeted cytotoxic LHRH analog, AN-207. Both AN-152 and AN-207 can effectively inhibit the growth of LHRH receptor-positive human breast, ovarian, endometrial, and prostate cancers xenografted into nude mice. DOX-containing cytotoxic LHRH analog AN-152 is scheduled for clinical phase I/IIa trials in patients with advanced ovarian and breast cancers in 2005.

Multidrug resistance (MDR) mediated by membrane transporters, such as P‐glycoprotein (MDR‐1) and MDR‐associated protein (MRP), remains a challenge in the therapy of renal cell carcinoma (RCC). Chemotherapy targeted to hormone receptors may provide a new approach to overcome chemoresistance. The cytotoxic analogue of bombesin/gastrin‐releasing peptide (GRP), AN‐215, consists of a superactive derivative of doxorubicin, AN‐201, which is linked to a bombesin analogue carrier: RC‐3094.The authors examined the expression of bombesin/GRP receptors in 3 human RCC cell lines (A‐498, ACHN. and 786‐0) by using reverse‐transcriptase‐polymerase chain reaction (RT‐PCR) analysis and radioligand‐binding assays. They also evaluated the effects of AN‐215 and its cytotoxic radical AN‐201 in the same RCC models in vivo, and they studied the effects of AN‐215 and AN‐201 on the expression levels of MDR‐1 and subtype 1 of MRP (MRP‐1) by using real‐time PCR.A N‐215 significantly (P < 0.05) inhibited the growth of A‐498, ACHN, and 786‐0 RCC xenografted into nude mice by 59.2–67.6%, whereas the cytotoxic radical AN‐201 alone had no significant antitumor effects. The efficacy of AN‐215 was independent of the expression patterns of MDR‐1 and MRP‐1 in these RCC cell lines. The induction of MDR‐1 by AN‐215 was similar (Experiment 2) or weaker (Experiment 1) compared with AN‐201. Both AN‐215 and AN‐201 caused only a minor induction of MRP‐1.The current findings indicated that targeted chemotherapy with cytotoxic bombesin/GRP analogue AN‐215 can inhibit the growth of RCC, providing a new treatment modality for patients with advanced RCC. Cancer 2005. © 2005 American Cancer Society.

Targeting anticancer agents to receptors for peptide hormones such as bombesin/gastrin-releasing peptide (GRP) on tumor cells increases the efficacy and lowers the toxicity of cancer therapy. We studied the expression of bombesin/GRP receptors in 6 experimental pancreatic cancers and evaluated tumor inhibition in vivo produced by targeted chemotherapy with the cytotoxic bombesin analog AN-215.

Chemoresistance mediated by membrane transporters such as multidrug resistance (MDR‐1) glycoprotein remains a challenge in the chemotherapy treatment of advanced or recurrent endometrial carcinoma. Targeted chemotherapy might overcome this resistance. The cytotoxic somatostatin (SST) analog, AN‐238, consists of a superactive derivative of doxorubicin (DOX), 2‐pyrrolino‐DOX (AN‐201), linked to the SST analog carrier, RC‐121. This conjugate binds strongly to SST receptor subtypes (sst) 2a (sst2a) and 5 (sst5) and can be targeted to tumors that express these receptors.The presence of sst2a and sst5 was determined in 3 human endometrial carcinoma cell lines (HEC‐1A, RL‐95‐2, and AN3CA). Nude mice bearing xenografts of these cancers were treated with AN‐238 and its radical, AN‐201. The antitumor effects and toxicity were compared. The authors studied the effects of AN‐238 and AN‐201 on the expression levels of MDR‐1, multidrug resistance related protein (MRP‐1), and breast carcinoma resistance protein (BCRP) by real‐time polymerase chain reaction.The authors demonstrated the presence of mRNA and receptor protein for sst2a and sst5 on HEC‐1A, RL‐95‐2, and AN3CA tumors. AN‐238 significantly (P < 0.05) inhibited the growth of these tumors, whereas AN‐201 had no effect. Blockade of SST receptors nullified the effects of AN‐238. In all 3 endometrial carcinoma lines, AN‐238 caused a weaker induction of MDR‐1 than AN‐201. No major induction of MRP‐1 and BCRP occurred after treatment with AN‐238 or AN‐201.Targeted chemotherapy with the cytotoxic SST analog, AN‐238, inhibited powerfully the growth of endometrial carcinoma, which express SST receptors, regardless of their expression level of MDR‐1. Cancer 2005. © 2005 American Cancer Society.

The targeted cytotoxic somatostatin analogue AN-238, consisting of 2-pyrrolinodoxorubicin (AN-201) linked to carrier octapeptide RC-121, is scheduled for clinical trials. To extend previous findings we tested AN-238 on human androgen sensitive MDA-PCa-2b prostate cancers grown subcutaneously and androgen independent LNCaP derived C4-2 prostate cancers xenografted into the tibiae of nude mice.

BACKGROUNDNew modalities are necessary for the treatment of patients with unresectable gastric carcinoma. The authors investigated whether receptors for somatostatin and bombesin were present in human gastric carcinoma lines and tested the antitumor effects of cytotoxic somatostatin analog AN238 and cytotoxic bombesin conjugate AN215.METHODSNude mice bearing AGS, Hs 746T, and NCIN87 human gastric carcinomas were treated with AN238, AN215, or their cytotoxic moiety 2pyrrolinodoxorubicin AN201. Tumor growth reduction and tumor doubling times were calculated, and histologic characteristics of cell proliferation and apoptosis were examined. The expression of mRNA for somatostatin and bombesin receptors in tumors was investigated by reverse transcriptase–polymerase chain reaction. Subtypes 2 and 5 of somatostatin receptor proteins sst2 and sst5, respectively were analyzed using immunohistochemistry and immunoblotting. Binding assays were performed with radiolabeled somatostatin and bombesin analogs.RESULTSCytotoxic bombesin analog AN215 powerfully inhibited the growth of AGS carcinomas that expressed highaffinity subtype 1 bombesin receptors. All three carcinomas expressed highaffinity sst2 and sst5 receptors. Cytotoxic somatostatin analog AN238 exerted a strong inhibitory effect on NCIN87 and Hs 746T carcinomas, which exhibited high concentrations of somatostatin receptors, but had a weaker effect on AGS tumors, which expressed the lowest receptor levels. AN201 had only nonsignificant effects.CONCLUSIONSExperimental human gastric carcinomas that expressed highaffinity subtype 1 bombesin receptors were inhibited by cytotoxic bombesin analog AN215, and tumors with high concentrations of sst2 or sst5 somatostatin receptors were suppressed by cytotoxic somatostatin analog AN238. These findings suggest that this class of targeted compounds should be considered for the therapy of patients with advanced gastric carcinoma. Cancer 2003;98:1401–10. © 2003 American Cancer Society.DOI 10.1002cncr.11649

Growth hormone-releasing hormone (GHRH) regulates growth hormone release from the pituitary. However, in addition to this neuroendocrine action, much evidence implies an additional role for GHRH in carcinogenesis in non-pituitary tissues. This role of GHRH in cancer development appears to be due to the operation of several mechanisms, which involve the regulation of the growth hormone-dependent hepatic insulin-like growth factor I (IGFI) production, tumoural IGF-I and IGF-II secretion and direct action of GHRH on tumour cells by autocrine and/or paracrine pathways. This review summarises the available information regarding the role of GHRH in tumorigenesis with special emphasis on the direct action of GHRH in primary and experimental cancers.

Antagonists of growth hormone-releasing hormone (GH-RH) can inhibit the proliferation of various tumors either indirectly through the suppression of the pituitary growth hormone/hepatic insulin-like growth factor I (IGF-I) axis and the lowering of serum IGF-I concentration or directly by reducing the levels of IGF-I and IGF-II and their mRNA expression in tumors and blocking the effect of autocrine GH-RH. In this study, the authors investigated the effects of the GH-RH antagonist JV-1-38 on MNNG/HOS human osteosarcoma and SK-ES-1 human Ewing sarcoma cell lines. Male nude mice bearing subcutaneous xenografts of MNNG/HOS or SK-ES-1 tumors were treated subcutaneously with JV-1-38 at a dose of 20 μg twice daily for 4 weeks. The concentrations of IGF-I and IGF-II in serum and in tumor tissue were measured by radioimmunoassay. Tumor and liver levels of mRNA for IGF-I and IGF-II were determined by reverse transcriptase-polymerase chain reaction analysis. The effects of JV-1-38, IGF-I, and IGF-II on cell proliferation in vitro were evaluated. GH-RH antagonist significantly (P &lt; 0.05) inhibited the tumor volume and tumor weight of MNNG/HOS and SK-ES-1 tumors by > 50% after 4 weeks and increased tumor doubling time. JV-1-38 lowered the serum IGF-I level, decreased the expression of mRNA for IGF-I in the liver, and significantly (P &lt; 0.05–0.01) reduced the concentration of IGF-II and mRNA levels for IGF-II in both sarcomas. The concentration of IGF-I was lowered only in SK-ES-1 tumors. In vitro, the proliferation of SK-ES-1 and MNNG/HOS cells was inhibited by JV-1-38 and by antisera to IGF-I and IGF-II. The inhibition of MNNG/HOS osteosarcoma and SK-ES-1 Ewing sarcoma by GH-RH antagonists was linked to a suppression of IGF-II production in tumors. However, in SK-ES-1 tumors, the effects on IGF-I also may be involved. Cancer 2002;95:1735–45. © 2002 American Cancer Society. DOI 10.1002/cncr.10865

BACKGROUND: Hypothalamic GH-releasing hormone (GHRH) regulates GH release from the pituitary, but an ectopic production of GHRH has been detected in various non-hypothalamic tissues, especially cancers. OBJECTIVE: To investigate whether endometrial tumors produce GHRH. METHODS: Twenty-four endometrioid, three serous papillary (SP), three mixed type endometrioid/serous papillary adenocarcinomas and one malignant mixed Müllerian tumor (MMMT) were assessed for GHRH immunoreactivity by the polyclonal anti-rabbit antibody SV95 and for the expression of GHRH mRNA by in situ hybridization using an oligonucleotide probe. RESULTS: Increased GHRH immunoreactivity was detected in 15 out of 24 (63%) of the endometrioid tumors, including two out of three (66%) of the mixed type endometrioid/serous adenocarcinomas but not in the three SP or the MMMT tumor. Cytoplasmic staining was detected in all positive cases, while in three of them strong nuclear localization of GHRH was also revealed. In situ hybridization indicated the presence of GHRH mRNA in six cases, all characterized as positive for GHRH immunoreactivity. CONCLUSION: GHRH is expressed in a subset of endometrial tumors, of the endometrioid type in particular. A paracrine/autocrine role for GHRH in the development of the disease should be considered.

The purpose of this study was to investigate the effects of a potent LHRH agonist, [D-Trp⁶]LHRH on the basal and EGF-induced cell proliferation and the metastasis-associated properties in A431 human epidermoid carcinoma. [D-Trp⁶]LHRH time-dependently inhibited the basal and EGF-stimulated growth of A431 cancer cells. It is assumed that phosphorylation/dephosphorylation of cellular proteins is highly related to cell growth. This study demonstrates that [D-Trp⁶]LHRH decreased the basal and EGF-induced total cellular kinase activity, particularly the tyrosine phosphorylation of several cellular proteins including the EGFR. In contrast, [D-Trp⁶]LHRH did not cause detectable changes in basal and EGF-stimulated serine/threonine phosphorylation of A431 cellular proteins. The inhibitory effect of [D-Trp⁶]LHRH on A431 cell proliferation was associated with apoptosis as evidenced by the cell morphology and DNA integrity (ladder pattern), the expression of interleukin 1β-converting enzyme (ICE) and activation of caspase. Furthermore, EGF could rescue the remaining attached A431 cells following [D-Trp⁶]LHRH treatment for 48 hr, which suggests that limited exposure to [D-Trp⁶]LHRH did not channel all cells to irreversible apoptotic process. We also determined the effects of [D-Trp⁶]LHRH on metastasis-associated properties in A431 cells. [D-Trp⁶]LHRH reduced both basal and EGF-stimulated secretion of MMP-9 and MMP-2. In addition, [D-Trp⁶]LHRH suppressed the basal and EGF-induced invasive activity of A431 cells based on an in vitro invasion assay. In conclusion, this study indicates that [D-Trp⁶]LHRH may act partly through activating tyrosine phosphatase activity to inhibit cell proliferation and the metastasis-associated properties of A431 cancer cells. Our work suggests that [D-Trp⁶]LHRH may be therapeutically useful in limiting the tumor growth and metastasis of some neoplasms. © 2002 Wiley-Liss, Inc.

Vasoactive intestinal peptide (VIP) stimulates the proliferation and invasiveness of malignant prostatic cells. Receptors for VIP and the closely related growth hormone-releasing hormone (GH-RH) show considerable homology and are found in prostatic and other carcinomas. Among various analogs of GH-RH synthesized, JV-1-52 is a non-selective VIP/GH-RH antagonist, whereas JV-1-53 is a VIP antagonist devoid of GH-RH antagonistic effect. In our study, nude mice bearing PC-3 human androgen-independent prostate carcinomas were treated with JV-1-52 or JV-1-53 (20 μg/day, s.c.) for 28 days. Both antagonists produced a similar reduction in tumor volume (62–67%, p &lt; 0.01) and tumor weight (59–62%; p &lt; 0.05) vs. controls and extended tumor doubling-time from 9.1 to about 16 days (p &lt; 0.05). To investigate the mechanisms involved, in another study we compared the effects of JV-1-53 with those of somatostatin analog RC-160. VIP antagonist JV-1-53 reduced tumor weight by 67% (p &lt; 0.01) and suppressed the expression of mRNA for c-fos and c-jun oncogenes by about 34% (p &lt; 0.05), without affecting serum levels of insulin-like growth factor-I (IGF-I). In contrast, RC-160 (50 μg/day) reduced serum IGF-I by 19% (p &lt; 0.05), but did not significantly decrease tumor weight. mRNA for VIP and high affinity receptors for VIP were detected on PC-3 tumors. Our results suggest that VIP/GH-RH antagonists can inhibit the growth of androgen-independent prostate cancer by abrogating the autocrine/paracrine mitogenic stimuli of VIP. The ability of GH-RH antagonists to block tumoral VIP receptors, in addition to GH-RH receptors, could be potentially beneficial for prostate cancer therapy. © 2002 Wiley-Liss, Inc.

The use of peptide analogs for the therapy of various cancers is reviewed. Inhibition of the pituitary–gonadal axis forms the basis for oncological applications of luteinizing hormone-releasing hormone (LH-RH) agonists and antagonists, but direct effects on tumors may also play a role. Analogs of somatostatin are likewise used for treatment of various tumors. Radiolabeled somatostatin analogs have been successfully applied for the localization of tumors expressing somatostatin receptors. Studies on the role of tumoral LH-RH, growth hormone-releasing hormone (GH-RH), and bombesin/GRP and their receptors in the proliferation of various tumors are summarized, but the complete elucidation of all the mechanisms involved will require much additional work. Human tumors producing hypothalamic hormones are also discussed. Treatment of many cancers remains a major challenge, but new therapeutic modalities are being developed based on antagonists of GH-RH and bombesin, which inhibit growth factors or their receptors. Other approaches consist of the use of cytotoxic analogs of LH-RH, bombesin, and somatostatin, which can be targeted to receptors for these peptides in various cancers and their metastases. These new classes of peptide analogs should lead to a more effective treatment for various cancers.

Cytotoxic analogs of somatostatin (SST), such as AN-238, which consists of 2-pyrrolinodoxorubicin (AN-201) linked to the SST carrier RC-121, can be targeted to tumors that express SST receptors. Because SST receptors are present in ovarian carcinoma cells, the authors evaluated the effect of AN-238 on the UCI-107 ovarian carcinoma cell line. An analysis of microsatellite alleles in cocultured SST receptor positive and receptor negative cells was used for the demonstration of in vitro targeting. The toxicity and antitumor effects of AN-238 in nude mice bearing UCI-107 human ovarian tumors were investigated with or without pharmacologic inhibition of serum carboxylesterases (CE). The expression of SST receptor subtypes was determined by reverse transcriptase-polymerase chain reaction analysis, and the binding affinity of AN-238 to SST receptors was determined by radioligand assays. The proliferation of SST receptor positive UCI-107 cells in vitro was inhibited preferentially by AN-238. AN-238 showed high-affinity binding to UCI-107 tumor membranes at a 50% inhibition concentration of 3.39 nM ± 0.74 nM. In vivo, the volume and weights of UCI-107 tumors treated with AN-238 were decreased by more than 60% (P &lt; 0.05) compared with controls. Cytotoxic radical AN-201 or the unconjugated mixture of AN-201 with carrier RC-121 had no significant effects on tumors and were toxic. In mice with inhibited serum CE activity, AN-201 at 400 nmol/kg was lethal, whereas AN-238 at a total dose of 800 nmol/kg caused only 22% mortality and reduced tumor weight by 69% and volume by 70% (P &lt; 0.05 vs. control). Targeted chemotherapy with the SST conjugate AN-238 inhibits SST receptor positive experimental ovarian tumors. AN-238 may provide a new treatment modality for patients with advanced ovarian carcinoma. Cancer 2001;92:1168–76. © 2001 American Cancer Society.