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Endometriosis is a benign gynecologic disorder causing dysmenorrhea, pelvic pain, and subfertility. Receptors for the growth hormone-releasing hormone (GHRH) were found in endometriotic tissues. Antagonists of GHRH have been used to inhibit the growth of endometriotic endometrial stromal cells. In this study, the GHRH receptor splice variant (SV) 1 was detected in human endometrial tissue samples by Western blots and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The highest messenger RNA (mRNA) and protein levels of SV1 were found in eutopic endometrium from patients with endometriosis compared to ectopic endometriotic tissues and endometrium from normal patients. The highest expression for GHRH mRNA was found by qRT-PCR in ectopic endometriosis lesions. In an in vivo mouse model with human endometrial explants from patients with endometriosis, 10 μg MIA-602 per day resulted in significantly smaller human endometrial xenotransplants after 4 weeks compared to mice treated with vehicle. The endometrial tissues expressed SV1 before and after xenotransplantation. The proliferation of endometrial stromal cells as well as the endometriosis cell lines 12-Z and 49-Z was decreased by exposure to 1 μM MIA-602 after 72 hours. The protein levels of epithelial growth factor receptors in 12-Z and 49-Z cell lines were reduced 48 and 72 hours after the administration of 1 μM MIA-602. MIA-602 decreased the activation of the MAP-kinases ERK-1/2. Our study demonstrates the presence of SV1 receptor as a target for treatment with GHRH antagonist in endometriosis. Endometrial tissues respond to MIA-602 with inhibition of proliferation in vitro and in vivo. The use of MIA-602 could be an effective supplement to the treatment strategies in endometriosis.

Receptors for LHRH (luteinizing hormone-releasing hormone) are expressed in about 80% of human endometrial, ovarian and prostate cancers and are also found in more than 50% of breast cancers including triple negative breast cancers. In the human body, LHRH receptors are found at significant levels in the pituitary and reproductive organs. Other benign tissues or hematopoietic stem cells express only low levels of receptors for LHRH or no receptors. Thus LHRH receptors are promising targets for a receptor- mediated chemotherapy with cytotoxic hybrid molecules. Cytotoxic analogs of LHRH consist of a LHRH agonist, which is used as a carrier peptide and DOX or its derivatives. Cytotoxic analogs of LHRH, AEZS-108 (formerly known as AN-152) and AN-207, exhibit anti-cancer activity in various in vitro and in vivo models of LHRH-receptor positive cancers. In AEZS-108 (zoptarelin DOX) DOX is covalently linked to the LHRH agonist [D-Lys(6)]LHRH. Results of phase I and II clinical studies in patients with breast, endometrial and ovarian cancers demonstrated good anticancer activity with moderate toxic side effects and without any sign of cardiotoxicity so far. AEZS-108 is also being evaluated in phase I/II studies in castration resistant prostate cancer and metastatic bladder cancer. Because of the very promising phase II results in endometrial cancer, a multinational, multicenter phase III study of this malignancy has been initiated and is currently recruiting patients.

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 vitro fertilization 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.

Intraabdominal adhesions are the most frequent complication of abdominal surgery and may represent one of the greatest unsolved problems in medicine today. They severely diminish quality of life of many patients who are affected by bowel obstruction, infertility and chronic abdominal pain. Hence, adhesions and their consequences are associated with high morbidity, a considerable risk of mortality as well as an increasing burden of medicolegal claims and socioeconomic expenses. This chapter summarizes key steps in anticipating, diagnosing and treating intraabdominal adhesions. Additionally, strategies to reduce adhesions formation are summarized. Up to 90 % of previously operated patients may have adhesions, most are asymptomatic. There is no standard imaging modality available to safely identify or rule out adhesions. In patients with clinically expected intraabdominal adhesions, adequate surgical steps (patient consent, adjustment of abdominal entry as an example) should be followed. There is strong evidence to lyse potentially symptomatic adhesions, whereas adhesiolysis in asymptomatic patients may not have a benefit. Patients with an increased individual risk of adhesion formation could be treated with anti-adhesion products. Although some of these products proofed successfully to reduce adhesion formation in several clinical trials, their usage is limited due to financial considerations. Further, it is highly recommended to clarify the adhesion formation risk and related complications during written consent before every surgery.

Triple negative breast cancers (TNBC) are associated with an adverse outcome, although these tumors are sensitive to chemotherapy. In part, this phenomenon could be caused by tumor immune escape. The current study investigates immunogenicity of TNBC cells in vitro and the presence of immunosuppressive factors in the tumor microenvironment (pAKT and B7H1 expression, infiltration with regulatory T cells, [Tregs]). Natural killer (NK)-cell induced lysis was evaluated in estrogen receptor (ER) positive MCF 7 breast cancers, in MDA-MB231 and MDA-MB468 and in HCC-1937 (BRCA 1 mutated) and HCC-1806 TNBC cells. Expression of pAKT, B7H1 and infiltration with Tregs were determined by immunohistochemistry in human specimens of benign and malignant breast disease. NK-cell induced lysis was significantly increased (p

Lobaplatin as a single agent and in combination with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is investigated in in-vitro models of p53-negative triple-negative breast cancers (TNBCs) and compared with a model of oestrogen receptor-positive p53-positive breast cancer. In addition, the induction of programmed cell death by lobaplatin is further explored. By using cell viability assays and western blotting, the cytotoxic effects of lobaplatin alone and in combination with TRAIL are compared with cisplatin in HCC 1806, HCC 1937, and MCF 7 cells. The multicaspase inhibitor z-VAD-fmk and necrostatin, an inhibitor of necroptosis, are used to demonstrate the mechanism of cell death caused by lobaplatin. Lobaplatin displayed antitumour activity in all three cell lines, which increased time dependently. Cotreatment of lobaplatin and TRAIL induced an increase in cytotoxicity by 30-50% in the different cell lines. The pan-caspase inhibitor z-VAD-fmk as well as necrostatin could weaken but not abolish the cytotoxic effect of lobaplatin and cisplatin. Lobaplatin showed substantial cytotoxic effects in two in-vitro models of p53-mutated TNBC. Cotreatment with TRAIL and platinum agents resulted in increased antitumour activity in the TNBC cell lines investigated. Cell death subsequent to treatment with cisplatin and lobaplatin occurred because of apoptosis. However, caspase-independent mechanisms of programmed cell death were also involved. It was also demonstrated that platinum compounds could induce necroptosis, although to a minor extent.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that is clinically negative for the expression of estrogen and progesterone receptors (ER/PR) and human epidermal growth factor receptor-2 (HER2). Patients with TNBC have a worse clinical outcome, as measured by time to metastasis and median overall survival. Chemotherapy has been the mainstay of treatment of TNBC but responses are disappointing. A substantial proportion of TNBC expresses luteinizing hormone-releasing hormone (LHRH), receptors for LHRH, in addition to receptors for growth hormone-releasing hormone (GHRH). These receptors represent potential therapeutic targets. Potent antagonists of GHRH and LHRH receptors have been developed in recent years and these antagonists inhibit the growth, tumorigenicity and metastatic potential of various human experimental malignancies. These antagonists could be utilized for the treatment of TNBC. The targeted cytotoxic analog of LHRH, AN-152 (AEZS-108) containing doxorubicin, must also be strongly considered for therapy of TNBC. Experimental studies suggest the merit of clinical trials with LHRH antagonists and AEZS-108 in TNBC patients.

Introduction: Overexpression of MAPK has been detected in 34 % of TNBC and has been found to be associated with anthracycline resistance (1). AEZS-131 is a highly selective orally active ERK1/2-inhibitor, which has shown in vivo activity (first in class) in colon cancer. The following study explores mode of action and efficacy in models of TNBC. Study design: AEZS-131 was tested in a kinase panel of 36 STK, 26 TK and 7 PIK to check for selectivity. Inhbition of Rsk-phosphorylation (cellular substrate of ERK) was evaluated by western blot analysis. Mode of action was explored by cell cycle FACS-analysis. Cleavage of PARP was explored by Western blot. Cytotoxic efficacy was evaluated in 5 TNBC, of which 2 had mutations in the MAPK signal transduction pathway lines, by MTT assay. Additionally it was explored if inhibition of classical apoptosis could abrogate the effect of AEZS-131. Results: AEZS-131 selectively inhibited ERK with an IC50 Conclusions: AZS-131 was shown to selectively inhibit ERK at low nM concentrations and to induce G1-arrest. Accordingly, the cytotoxic effect most pronounced in TNBC cell lines with mutations in the MAPK pathway. If classical apoptosis is inhibited, cytotoxic effects remain unchanged, suggesting that AEZS-131 can also induce nonclassical forms of programmed cell death. AEZS-131 should be further explored in TNBCs with overexpression of MAPK or mutations in the MAPK-pathway. *Æterna Zentaris GmbH, Frankfurt/M, Germany. (1): MAPK overexpression is associated with anthracycline resistance and increased risk for recurrence in patients with triple-negative breast cancer. Eralp Y, Derin D, Ozluk Y, Yavuz E, Guney N, Saip P, Muslumanoglu M, Igci A, Kücücük S, Dincer M, Aydiner A, Topuz E. Ann Oncol. 2008 Apr;19(4):669–74. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P3-18-06.

A case of a papillary squamotransitional cell carcinoma (PSTCC) of the vagina with a follow-up of 3 years is presented here. The characteristics of this case support a squamous rather than urothelial origin of this rare entity. Unlike its counterparts in the cervix uteri, the clinical behavior of vaginal PSTCC is more favorable than squamous cell carcinoma. Histological and clinical features are compared to those of previously described cases of vaginal and cervical PSTCC.

Although trastuzumab (Herceptin) has substantially improved the overall survival of patients with mammary carcinomas, even initially well-responding tumors often become resistant. Considering that clinical efficacy of trastuzumab correlates with the infiltration of natural killer (NK) cells into the tumor site, we established a cell-culture-system to select for ovarian cancer and mammary carcinoma cells that survive a challenge by trastuzumab and NK cells. Under these conditions, NK cells can eliminate tumor cells either via antibody-dependent-cell-mediatedcytotoxicity (ADCC) or by direct recognition of tumor-associated ligands for NKG2D or DNAM-1. The most striking phenotypic alteration observed in immunoselected ovarian cancer cells was a down-regulation of HER2 expression, leading to an ADCC-resistant phenotype. All breast cancer cells tested (MCF7, SK-BR-3, MDA-MB231, BT474), however, failed to develop resistance in vitro. Instead, treatment with trastuzumab and polyclonal NK-cells resulted in the preferential survival of individual sphere-forming cells that displayed a CD44highCD24low ‘'cancer-stem-celllike” phenotype (CSC) and expressed significantly less HER2 compared with non-stem cells. Moreover, the molecular determinants for the direct recognition of transformed cells, most notably the NKG2D ligands MICA, MICB, ULBP1-4 and the DNAM-1 ligands CD112 and CD155, were virtually absent from CSC. When immunoseleced breast cancer cells were then re-expanded, they mostly lost the observed phenotype and regenerated a tumor cell culture that displayed initial HER2 surface expression and ADCC susceptibility, but was enriched in CD44highCD24low CSC. This translated into increased clonogenicity in vitro and tumorigenicity in vivo. We provide evidence that recruition of NK-cells and induction of ADCC by trastuzumab may spare actual tumor-initiating-cells, which could explain clinical relapse. Moreover, our observation that “relapsed” in vitro cultures show identical HER2 surface expression and susceptibility toward ADCC suggests that administration of trastuzumab beyond relapse might be considered, especially when combined with proteasome inhibition which increase the expression of NKG2D ligands. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-05.

Background: Screening is an unsolved problem for ovarian cancer (OvCA). As late detection is equivalent to poor prognosis, we analysed whether OvCA patients show diagnostically meaningful microRNA (miRNA) patterns in blood cells. Methods: Blood-borne whole miRNome profiles from 24 patients with OvCA and 15 age- and sex-matched healthy controls were biostatistically evaluated. Results: Student's t-test revealed 147 significantly deregulated miRNAs before and 4 after Benjamini–Hochberg adjustment. Although these included miRNAs already linked to OvCA (e.g., miR-16, miR-155), others had never before been connected to specific diseases. A bioinformatically calculated miRNA profile allowed for discrimination between blood samples of OvCA patients and healthy controls with an accuracy of >76%. When only cancers of the serous subtype were considered and compared with an extended control group (n=39), accuracy, specificity and sensitivity all increased to >85%. Conclusion: Our proof-of-principle study strengthens the hypothesis that neoplastic diseases generate characteristic miRNA fingerprints in blood cells. Still, the obtained OvCA-associated miRNA pattern is not yet sensitive and specific enough to permit the monitoring of disease progression or even preventive screening. Microarray-based miRNA profiling from peripheral blood could thus be combined with other markers to improve the notoriously difficult but important screening for OvCA.

We analyzed the anti-tumor effect and the mechanism of action of perifosine, an orally active alkylphospholipid AKT inhibitor using in vitro models of human ovarian cancer.Ovarian cancer cells OAW42, PA-1, SKOV3, and A2780 as well as platinum resistant A2780cis cells were incubated with increasing concentrations of perifosine, with and without multi-caspase inhibitor zVAD-FMK. The effect of a combined treatment with cisplatin and perifosine was investigated in OAW42, SKOV3, A2780 and A2780cis cells. Cytotoxic effects of perifosine were analyzed using crystal violet staining, FACS analysis of DNA content as well as Annexin V/propidium iodide-double staining. The effect of perifosine on AKT phosphorylation was determined by Western blotting.Perifosine displayed anti-tumor activity in all five cell lines, which increased time-dependently. While IC(50) values at 24 h were40 μM, IC(50) values after 72 h decreased to 10 μM in OAW42 and 25 μM in PA-1 and 30 μm in SKOV3 cells. In platinum resistant A2780cis cells perifosine showed good antiproliferative activity (IC(50) = 3 μm). At adequate doses, perifosine increased cytotoxic effects of cisplatin in OAW42, A2780 and A2780cis cell. Anti-tumor activity of perifosine was not confined to a specific phase of the cell cycle and could not be decreased by the pan-caspase inhibitor zVAD-FMK. AnnexinV/propidium iodide-double staining after treatment with perifosine was not indicative of classical apoptosis. AKT phosphorylation was dose-dependently inhibited by perifosine.Perifosine showed substantial cytotoxic effects in various in vitro models of ovarian cancer. Since anti-tumor effects were not confined to platinum-sensitive cells perifosine seems to be a good candidate for clinical studies in patients especially with platinum resistant ovarian cancer.

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 10 μM and 40 μM 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 24 h, 48 h and 72 h 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 24 h were >40 μM, IC 50 values after 48 h were approximately 7 μM in Ishikawa and 25 μM in HEC 1A cells. After 72 h, the IC 50 was below 1.25 μM for Ishikawa and about 6 μM 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.

The objective was to assess the efficacy and safety of different dosing schedules of cetrorelix acetate as a short term treatment for 4 weeks prior to surgery in patients with uterine fibroids. A Randomized double-blind placebo-controlled study was used and the patients were 109 premenopausal women with at least one uterine fibroid more than 4 cm in diameter. Groups 1-3 received placebo 5 and 10 mg of cetrorelix on days 1 8 15 and 22 respectively group 4 received 10 mg of cetrorelix on days 1 and 15. MRI scan was performed at screening and on day 29. The main outcome measure was the reduction of uterine volume on day 29 and response defined as >30% size reduction. Mean (+or- S.D.) reduction of uterine volume on day 29 (MRI scan) was 5.1 +or- 32.1% with placebo 15.6 +or- 20.2% with 4 x 5 mg 15.4 +or- 34.6% with 4 x 10 mg and 0.6 +or- 30.6% with 2 x 10 mg cetrorelix. Significant response versus placebo ( p < 0.05) occurred in the 4 x 10 mg group (42.3% versus 11.1%) Best objective response after 4 weeks of treatment was achieved after therapy with 4 x 10 mg of cetrorelix acetate. Short term presurgical treatment with the LHRH-antagonist cetrorelix is a flexible treatment protocol without any major side effects. (authors)

In 1999, the peptidic antagonist of gonadotropin-releasing hormone (GnRH), cetrorelix, was the first compound of this class to be marketed. Cetrorelix is a classic antagonist at the GnRH pituitary receptor. It is used to achieve reversible, dose-dependent suppression of the gonadotropins and the sex steroids. Cetrorelix is approved in over 80 countries including the USA and Japan for the prevention of the premature luteinizing hormone surge in controlled ovarian stimulation (COS) cycles. Using GnRH antagonists instead of GnRH agonists in COS cycles may facilitate infertility treatment. Pregnancy rates after both treatment regimens appear to be similar, while in GnRH-controlled COS cycles a lower rate of side effects such as ovarian hyperstimulation syndrome occur. Cetrorelix may also be useful in minimal stimulation cycles for in vitro fertilization or intrauterine insemination, although further and larger studies are required. Other indications for cetrorelix may be sex steroid-dependent benign and malig...

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 125 I-labeled GHRH antagonist JV-1-42. JMR-132 displaced radiolabeled JV-1-42 with an IC 50 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.

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

In this study we have investigated the efficacy and toxicity of targeted cytotoxic bombesin (BN) analog AN-215 and its effects on the expression of three multidrug resistance proteins in experimental human endometrial cancers. Nude mice bearing HEC-1A, RL-95-2 and AN3CA tumours were treated with AN-215 and its cytotoxic radical (AN-201). The BN receptor expression in tumours was followed by RT-PCR analysis and radioligand binding assays. Expression of drug resistance proteins MDR-1, MRP-1 and BCRP were measured by realtime PCR. AN-215 significantly (P0.05) inhibited the growth of HEC-1A, RL-95-2 and AN3CA tumours while AN-201 was ineffective. The expression of BN receptors was demonstrated in all three tumour models. AN-215 caused a lower induction of MDR-1 in HEC-1A and RL-95-2 cancers than AN-201. MRP-1 and BCRP were not induced by AN-215 or AN-201. Thus, targeted chemotherapy with AN-215 powerfully inhibits the growth of human BN receptor-positive endometrial cancers.

Antagonists of GHRH are being developed for the treatment of various cancers. In this study we investigated in vivo and in vitro the effects of the GHRH antagonist MZ-J-7-118 and its mechanism of action in HEC-1A human endometrial cancer. Treatment of nude mice bearing HEC-1A xenografts with 10 mug/d MZ-J-7-118 for 6 wk significantly inhibited the volume of HEC-1A tumors by 43%, tumor weight by 40% compared with controls and prolonged the tumor doubling time from 18.7 +/- 1.4 to 25.4 +/- 3.8 d. Administration of 20 mug MZ-J-7-118, sc, twice a day significantly (P0.05) decreased HEC-1A growth, as evidenced by a 57.9% decrease in tumor volume, a 50.7% reduction in tumor weight, and the extension of tumor doubling time from 17.5 +/- 2.8 to 36.4 +/- 6.5 d. Therapy with GHRH antagonists significantly decreased serum IGF-I levels in experiment 1, and significantly increased tumoral IGF-I levels in experiment 2 in treated mice. Levels of IGF-II and vascular endothelial growth factor-A in tumors were not changed. Specific high affinity binding sites for GHRH were found on HEC-1A tumor membranes using ligand competition assays with (125)I-labeled GHRH antagonist JV-1-42. MZ-J-7-118 displaced radiolabeled JV-1-42 with an IC(50) of 0.13 +/- 0.04 nm. The expression of mRNA for GHRH and splice variants of the GHRH receptor in HEC-1A tumors was demonstrated by real-time RT-PCR analysis. HEC-1A cells cultured in vitro secreted GHRH into the medium. The GHRH antagonist MZ-J-7-118 inhibited the growth of HEC-1A cells in vitro. Our results indicate that GHRH antagonists can reduce the growth of human endometrial cancer and could be used as an alternative adjuvant therapy for the management of endometrial cancer.

Background: Triple negative breast cancer (TNBC) is a distinct subtype of breast cancer burdened with a dismal prognosis due to the lack of effective therapeutic agents. Receptors for LHRH (luteinizing hormone-releasing hormone) can be successfully targeted with AEZS-108 [AN-152], an analog of LHRH conjugated to doxorubicin. Our study evaluates the presence of this target LHRH receptor in human specimens of TNBC and investigates the efficacy and toxicity of AEZS-108 in vivo. We also studied in vitro activity of AEZS-125, a new LHRH analog conjugated with the highly potent natural compound, Disorazol Z. Methods: 69 human surgical specimens of TNBC were investigated for LHRH-R expression by immunohistochemistry. Expression of LHRH-R in two TNBC cell lines was evaluated by real time RT-PCR. Cytotoxicity of AEZS-125 was evaluated by Cell Titer Blue cytoxicity assay. LHRH- receptor expression was silenced with an siRNA in both cell lines. For the in vivo experiments an athymic nude mice model xenotransplanted with the cell lines, MDA-MB-231 and HCC 1806, was used. The animals were randomised to three groups receiving solvent only (d 1, 7, 14, i.v.) for control, AEZS-108 (d 1, 7, 14, i.v.) or doxorubicin at an equimolar dose (d 1, 7, 14, i.v.). Results: In human clinical specimens of TNBC, expression of the LHRH-receptor was present in 49% (n = 69). HCC 1806 and MDA-MB-231 TNBC cells expressed mRNA for the LHRH-receptor. Silencing of the LHRH-receptor significantly decreased the cytotoxic effect of AEZS-108. MDA-MB-231 and HCC 1806 tumors xenografted into nude mice were significantly inhibited by treatment with AEZS-108; doxorubicin at equimolar doses was ineffective. As compared to AEZS 108, the Disorazol Z – LHRH conjugate, AEZS-125, demonstrated an increased cytotoxicity in vitro in HCC 1806 and MDA-MB-231 TNBC; this was diminished by receptor blockade with synthetic LHRH agonist (triptorelin) pretreatment. Conclusion: The current study confirms that LHRH-receptors are expressed by a significant proportion of TNBC and can be successfully used as homing sites for cytotoxic analogs of LHRH, such as AEZS-108 and AEZS-125.

“Triple negative breast cancer” (TNBC) represents a subgroup of breast cancers (BC), which do not express receptors for either estrogen or progesterone and which do not overexpress HER-2 receptors. Although this subgroup is characterized by immunohistochemistry, there is a seventy percent overlap with basal-like breast cancers, defined by gene-expression profiling and thus these terms are frequently used as synonyms. Because of this lack of therapeutic targets, TNBC is burdened with a dismal prognosis as compared to other subgroups of breast cancer and thus, a taxane- and anthracycline-based chemotherapy are the only options for patients with TNBC [1]. Various clinical studies investigated the effect of neoadjuvant chemotherapy in TNBC, i.e. systemic treatment was administered prior to surgery (as a means of an in vivo sensitivity testing). Two subgroups of TNBC with a distinct prognosis can be distinguished, based on the response to this neoadjuvant chemotherapy. Tumors, which are sensitive to chemotherapy as mirrored by a pathologic complete response (PCR), i.e. no surviving tumor tissue is detected in the surgical specimen, and cancers which are chemoresistant, as reflected by residual disease being detected after neodjuvant chemotherapy. While the former patient group has a good prognosis, overall survival of patients with chemoresistant TNBC is dramatically decreased [2]. These clinical observations emphasize, that it is of paramount importance to identify ways to overcome the chemoresistance of TNBC. GHRH antagonists inhibit the growth of various experimental human cancers [3] including TNBC [4]. These peptide hormone analogs suppress tumor growth through both direct and indirect pathways. It was observed that GHRH-antagonists can inhibit the proliferation of diverse cancer lines by direct action in vitro, under conditions in which the contribution of the hypothalamic GHRH/pituitary GH/hepatic IGF-I axis is clearly excluded [5]. These studies led to the conclusion that the main mechanism responsible for tumor inhibition could be a direct effect of the antagonists on the tumor tissue. In more than 70 % of TNBC, mRNA for receptors for GHRH and its respective ligands was detected and accordingly tumor growth of TNBC cell lines could be powerfully decreased by antagonists of GHRH [4]. The indirect, endocrine mechanism operates through the suppression of the release of GH from the pituitary, and the resulting inhibition of the hepatic production of IGF-I [3]. The current publication by Perez et al. [6] further extends our knowledge on the therapeutic potential of GHRH antagonists. This experimental study was carried out by the group of Nobel laureate Dr. A.V. Schally, the leading world expert on agonistic an antagonistic analogs of GHRH. The current study demonstrates synergy of GHRH-antagonist MIA-602 with doxorubicin as shown in an in vivo model of TNBC. More importantly, the authors clearly document in vivo that the resistance to doxorubicin of human MX-1 TNBC can be overcome by the addition of treatment with MIA-602. Thus, treatment of doxorubicin-resistant MX-1 TNBC with MIA-602 can reactivate sensitivity to doxorubicin mirrored by the synergistic effect of combination treatment; doxorubicin administered as single agent has no effect on this tumor's growth. A possible explanation for this important finding, is the suppression of the expression of the multi-drug resistance factors, MDR-1 and NANOG, by MIA-602, which has been demonstrated in vivo. In addition, in order to demonstrate that the decrease of this expression is mechanistically relevant, it was shown that treatment with MIA-602 inhibits the function of the efflux pump coded by the MDR-1 gene. Thus, strong evidence is presented, that besides being a potent tumor inhibiting agent, which shows a synergistic effect with anthracycline treatment, GHRH antagonist MIA-602 is also an inhibitor of the MDR-1 drug efflux pump. Accordingly MIA-602 can overcome resistance to doxorubicin in cancer therapy. The results presented are clinically relevant as resistance to anthracycline is a major problem in the treatment of TNBC, The prognosis of chemoresistant TNBC is thus significantly decreased. Accordingly, we believe that phase I studies should be carried out investigating combined treatment of MIA-602 and doxorubicin in patients with locally advanced or metastatic anthracycline resistant TNBC.

The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A(2A) adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (T(reg)) 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 T(reg). 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 T(reg) but also relieve intrinsic immunosuppressive properties of tumour and stromal cells.

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-Hodgkin's 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-Hodgkin's 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.

The aim of the present study was to evaluate the expression of receptors for luteinizing hormone-releasing hormone (LHRH) in human specimens of triple-negative breast cancers (TNBC). In addition, we used in vitro and in vivo models of TNBC to investigate if these receptors are suitable targets for the treatment with the LHRH antagonist cetrorelix. Receptors for LHRH were expressed in all tumor samples and in the TNBC cell lines HCC1806 and HCC1937. The proliferation of both TNBC cell lines was significantly inhibited in vitro by 1 microM cetrorelix. Injections of 3 mg cetrorelix on day 1 and 21 resulted in a significant growth inhibition of HCC1806 tumors xenografted into nude mice. Tumors of mice treated with cetrorelix expressed less mRNA for EGFR and HER3 receptors than untreated tumors. After treatment of cells with Cetrorelix a flow cytometric analysis of the cell cycle revealed a decrease in S-phase. Given the low toxicity and clinical availability of cetrorelix, this peptide antagonist should be considered for phase II studies in patients with advanced TNBC.

AEZS 112 is an orally active small molecule anticancer drug which inhibits the polymerization of tubulin at low micromolar concentrations. The current study investigates the anti-tumor effect and the mechanism of action of AEZS 112 in in vitro models of human ovarian and endometrial cancers. Four human ovarian and 2 endometrial cancer cell lines were incubated with increasing concentrations of AEZS 112 with and without multi-caspase inhibitor zVAD-FMK for 72 hours. Cytotoxic effects of AEZS 112 were analyzed using crystal violet staining, FACS analysis of DNA content as well as Annexin V/propidium iodide-double staining. AEZS 112 displayed anti-tumor activity in all six cell lines. The EC50 determined after 72-h incubation for Ishikawa and HEC 1A was 0.0312 and 0.125 microm, respectively. The EC50 was 5 microm for SKOV 3 cells, 1 microm for 0.5 microm for OAW 42 cells, 0.125 microm for OvW 1 cells and 0.0312 microm for PA 1 cells. Cytotoxic effects of AEZS 112 could not be abrogated by caspase inhibition with pan-caspase inhibitor zVAD-fmk. Annexin V/propidium iodide-double staining after treatment with AEZS 112 was indicative of necrosis-like cell death. AEZS 112 dose-dependently increased non-vital hypodiploid cells and the cytotoxic effect was least pronounced in G2 phase of the cell cycle, indicating cell death during mitosis, as determined by FACS analysis. The orally active small molecule tubulin inhibitor AEZS 112 showed anti-tumor activity in human ovarian and endometrial cancer cell lines at low micromolar concentrations, which could not be abrogated by caspase inhibition and is therefore a good candidate for in vivo studies in these tumors.

The glycoprotein macrophage migration inhibitory factor (MIF) is a cytokine that has been shown to promote tumor progression and tumor immune escape in ovarian cancer. The present study investigates MIF in uterine cervical cancer. Eighty surgical biopsies (32 cervical dysplasias, 23 in situ carcinomas and 25 invasive carcinomas) of uterine cervical tissue were evaluated immunohistochemically for MIF expression. In uterine cervical cancer cell lines SiHa and CaSki and their respective supernatants, MIF protein expression was analyzed by Western blotting, enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase polymerase chain reaction (RT-PCR). Immunohistochemical analysis shows that MIF is clearly overexpressed on the protein level in invasive cervical cancer compared to cervical dysplasias. MIF overexpression was confirmed by RT-PCR in surgical biopsies of invasive cervical cancer. Western blotting reveals that the MIF protein is overexpressed in SiHA und CaSki cervical cancer cell lines, whereas the ELISA reveals that cervical cancer cells secrete MIF. MIF has been shown to promote tumor immune escape mechanisms in other cancer entities, which makes it an interesting target for cancer therapy, given the known significance of immune mechanisms for uterine cervical cancer. The overexpression of MIF on the protein and mRNA level, as well as its secretion by cervical cancer cells points to a critical role of the protein for the pathogenesis of uterine cervical cancer.

Triple-negative breast cancers do not express receptors for estrogen or progesterone and do not overexpress HER2. These tumors have an unfavorable prognosis and at present chemotherapy is the only treatment option. Because the antagonists of growth hormone-releasing hormone (GHRH) have been shown to inhibit growth of a variety of cancers by endocrine and paracrine/autocrine mechanisms, we evaluated the expression of GHRH receptors in human specimens of triple-negative breast cancers and the response to GHRH by in vitro models. In samples of triple-negative breast cancers we found mRNA expression for the GHRH receptor and its functional splice variant SV1 in 25 and 70% of the cases, respectively and for GHRH in 80% of the samples. Immunoreaction of SV1 was detected in the human triple-negative breast cancer cell line HCC1806 while HCC1937 was negative. The growth of HCC1806 was stimulated by GHRH(1-44)NH(2) and inhibited by GHRH antagonist MZ-J-7-118. In addition, in HCC1806 MAP-kinases ERK-1/2 were activated by GHRH. Our findings suggest the existence of an autocrine loop consisting of GHRH and GHRH receptors in triple-negative breast cancers. Our in vitro studies demonstrate that targeting the GHRH receptor may be a therapeutic option which should be evaluated in studies in vivo.

Gynecological cancers such as breast, ovarian, and endometrial carcinoma express receptors for luteinizing hormone-releasing hormone (LHRH), bombesin/gastrin-releasing peptide (BN/GRP), and somatostatin (SST). These tumors are therefore suitable candidates for targeted therapy with cytotoxic hybrid molecules consisting of a cytotoxic radical and a peptide hormone analogue as a carrier. These compounds have been shown to be more active and less toxic in vivo than nontargeted chemotherapy in models of various human cancers which express the respective receptors. The current review summarizes experimental and clinical findings with cytotoxic peptide hormone analogues of LHRH (AN-152 [AEZS 108], AN-207), BN/GRP (AN-215), and SST (AN-238) in breast, ovarian, and endometrial cancers.

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-Hodgkin's 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.