Management of castration-resistant prostate cancer (CRPC) is challenging due to lack of efficacious therapy. Luteinizing hormone-releasing hormone analogs appear to act directly on cells based on the LHRH receptors on human prostate adenocarcinoma cells. We explored anticancer activity of a cytotoxic analog of LHRH, AEZS-108 consisting of LHRH agonist linked to doxorubicin. Nude mice bearing DU-145 tumors were used to compare antitumor effects of AEZS-108 with its individual constituents or their unconjugated combination. The tumor growth inhibition of conjugate was greatest among treatment groups (90.5% inhibition vs. 41% by [D-Lys(6)]LHRH+DOX). The presence of LHRH receptors on DU-145 cells was confirmed by immunocytochemistry. In vitro, AEZS-108 significantly inhibited cell proliferation (61.2% inhibition) and elevated apoptosis rates (by 46%). By the detection of the inherent doxorubicin fluorescence, unconjugated doxorubicin was seen in the nucleus; the conjugate was perinuclear and at cell membrane. Autophagy, visualized by GFP-tagged p62 reporter, was increased by AEZS-108 (7.9-fold vs. 5.3-fold by DOX+[D-Lys(6)]LHRH. AEZS-108 more effectively increased reactive oxygen species (ROS, 2-fold vs. 1.4-fold by DOX+[D-Lys(6)]LHRH) and levels of the apoptotic regulator p21 in vivo and in vitro. We demonstrate robust inhibitory effects of the targeted cytotoxic LHRH analog AEZS-108 on LHRHR positive castration-resistant prostate cancer cells.

Pancreatic carcinoma is one of the cancers with the worse prognosis, thus any therapeutic improvement is imperative. Cytotoxic LH-RH analog, AN-152 (proprietary designation, AEZS-108), consisting of doxorubicin (DOX) conjugated to D-Lys⁶LH-RH, is now in clinical trials for targeted therapy of several sex hormone-dependent tumors that express LH-RH receptors. We investigated LH-RH receptors in human pancreatic carcinoma and the effects of AN-152 (AEZS-108) on experimental pancreatic cancers. We determined LH-RH receptor presence in human pancreatic cancer samples by immunohistochemistry and, in three human pancreatic cancer lines (SW-1990, Panc-1 and CFPAC-1), by binding assays and Western blotting. The effects of the cytotoxic LH-RH analog were investigated on growth of these same cancer lines xenografted into nude mice. We also analyzed differences between the antitumor effects of the cytotoxic analog and its cytotoxic radical alone, doxorubicin (DOX), on the expression of cancer-related genes by PCR arrays. LH-RH receptors were expressed in two randomly selected surgically removed human pancreatic cancer samples and in all three cancer lines. Cytotoxic LH-RH analogs powerfully inhibited growth of all three tumor lines in nude mice; AN-152 was significantly stronger than DOX on Panc-1 and CFPAC-1 cancers. PCR array showed that cytotoxic LH-RH analog AN-152 affected the expression of genes associated with cellular migration, invasion, metastasis and angiogenesis more favorably than DOX, however the changes in gene expression varied considerably among the three cancer lines. Cytotoxic LH-RH analog, AEZS-108, may be a useful agent for the treatment of LH-RH receptor positive advanced pancreatic carcinoma.

This study evaluated the effects of a modern antagonistic analog of GHRH on tumor growth and on expression of inflammatory cytokine genes in two models of human triple negative breast cancers (TNBC). The TNBC subtype is refractory to the treatment options available for other hormone-independent breast cancers. Inflammatory cytokines play a major role in the cellular signaling associated with breast cancer pathogenesis and enhance epithelial-mesenchymal transitions (EMT), drug resistance, and metastatic potential. Growth hormone-releasing hormone (GHRH) is a hypothalamic neuropeptide which regulates the synthesis and release of growth hormone by the pituitary and is an autocrine/paracrine growth factor for multiple human cancers. The effects of analogs of GHRH on tumoral cytokine expression have not been previously investigated. Animals bearing xenografts of the human TNBC cell lines, HCC1806 and MX-1, were treated with MIA-602, an antagonistic analog of GHRH. Treatment with MIA-602 significantly reduced tumor growth. We quantified transcript levels of the genes for several inflammatory cytokines. Expression of INFγ, IL-1α, IL-4, IL-6, IL-8, IL-10, and TNFα, was significantly reduced by treatment with MIA-602. We conclude that treatment of TNBC with GHRH antagonists reduces tumor growth through an action mediated by tumoral GHRH receptors and produces a suppression of inflammatory cytokine signaling. Silencing of GHRH receptors in vitro with siRNA inhibited the expression of GHRH-R genes and inflammatory cytokine genes in HCC1806 and MX-1 cells. Further studies on GHRH antagonists may facilitate the development of new strategies for the treatment of resistant cancers.

Many bladder cancers progress to invasion with poor prognosis; new therapeutic methods are needed. We developed a cytotoxic LH-RH analog, AN-152 (AEZS-108) containing doxorubicin (DOX), for targeted therapy of cancers expressing LHRH receptors. We investigated the expression of LH-RH receptors in clinical bladder cancers and in HT-1376, J82, RT-4 and HT-1197 human bladder cancer lines. The effect of analog, AN-152, on growth of these tumor lines xenografted into nude mice was analyzed. Using molecular and functional assays, we also evaluated the differences between the effects of AN-152, and DOX alone. We demonstrated the expression of LH-RH receptors on 18 clinical bladder cancers by immunohistochemistry and on four human urinary bladder cancer lines HT-1376, J82, RT-4 and HT-1197 by Western blotting and binding assays. AN-152 powerfully inhibited growth of these bladder cancers in nude mice. AN-152 exerted greater effects than DOX and was less toxic. DOX activated strong multidrug resistance mechanisms in RT-4 and HT-1197 cancers, while AN-152 had no or less such effect. PCR assays and in vitro studies revealed differences in the action of AN-152 and DOX on the expression of genes involved in apoptosis. These results suggest that targeted cytotoxic LH-RH analog, AN-152 (AEZS- 108), should be examined for treatment of patients with LH-RH receptor positive invasive bladder cancers.

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.

We previously showed that growth hormone-releasing hormone (GHRH) agonists are cardioprotective following myocardial infarction (MI). Here, our aim was to evaluate the in vitro and in vivo activities of highly potent new GHRH agonists, and elucidate their mechanisms of action in promoting cardiac repair.

This study evaluated the effects of an antagonistic analog of growth hormone-releasing hormone, MIA-602, on tumor growth, response to doxorubicin, expression of drug resistance genes, and efflux pump function in human triple negative breast cancers.

Antibiotic treatment of patients infected with G(-) or G(+) bacteria promotes release of the toxins lipopolysaccharide (LPS) and pneumolysin (PLY) in their lungs. Growth Hormone-releasing Hormone (GHRH) agonist JI-34 protects human lung microvascular endothelial cells (HL-MVEC), expressing splice variant 1 (SV-1) of the receptor, from PLY-induced barrier dysfunction. We investigated whether JI-34 also blunts LPS-induced hyperpermeability. Since GHRH receptor (GHRH-R) signaling can potentially stimulate both cAMP-dependent barrier-protective pathways as well as barrier-disruptive protein kinase C pathways, we studied their interaction in GHRH agonist-treated HL-MVEC, in the presence of PLY, by means of siRNA-mediated protein kinase A (PKA) depletion.

Glioblastoma multiforme is the most frequent tumor of the central nervous system in adults and has a dismal clinical outcome, which necessitates the development of new therapeutic approaches. We investigated in vivo the action of the targeted cytotoxic analog of luteinizing hormone releasing hormone, AN-152 (AEZS-108) in nude mice (Ncr nu/nu strain) bearing xenotransplanted U-87 MG glioblastoma tumors. We evaluated in vitro the expression of LHRH receptors, proliferation, apoptosis and the release of oncogenic and tumor suppressor cytokines. Clinical and U-87 MG samples of glioblastoma tumors expressed LHRH receptors. Treatment of nude mice with AN-152, once a week at an intravenous dose of 413 nmol/20 g, for six weeks resulted in 76 % reduction in tumor growth. AN-152 nearly completely abolished tumor progression and elicited remarkable apoptosis in vitro. Genomic (RT-PCR) and proteomic (ELISA, Western blot) studies revealed that AN-152 activated apoptosis, as reflected by the changes in p53 and its regulators and substrates, inhibited cell growth, and elicited changes in intermediary filament pattern. AN-152 similarly reestablished contact regulation as demonstrated by expression of adhesion molecules and inhibited vascularization, as reflected by the transcription of angiogenic factors. Our findings suggest that targeted cytotoxic analog AN-152 (AEZS-108) should be considered for a treatment of glioblastomas.

Currently, various techniques are available to mark and selectively remove initially suspicious axillary lymph nodes (target lymph nodes, TLNs) in breast cancer patients receiving neoadjuvant chemotherapy (NACT). To date, limited data are available on whether the use of magnetic seeds (MS) is suitable for localizing TLNs. This study aimed to investigate the feasibility of MS in patients undergoing target lymph node biopsy (TLNB) or targeted axillary dissection (TAD) after NACT.

Molecular investigations are crucial for further developments in precision medicine. RNA sequencing, alone or in combination with further omic-analyses, resulted in new therapeutic strategies. In this context, biobanks represent infrastructures to store tissue samples and body fluids in combination with clinical data to promote research for new predictive and prognostic biomarkers as well as therapeutic candidate molecules. Until today, the optimal storage conditions are a matter of debate especially with view to the storage temperature. In this unique approach we compared parallel samples from the same tumour, one half stored at - 80 °C and one half in the vapor phase of liquid nitrogen, with almost identical pre-analytical conditions. We demonstrated that RNA isolated from breast cancer samples revealed significantly higher RINe-values after 10 years of storage in the vapor phase of liquid nitrogen compared to storage at - 80 °C. In contrast, no significant difference was found regarding the DIN-values after DNA isolation. Morphological changes of the nucleus and cytoplasm, especially in the samples stored at - 80 °C, gave insights to degenerative effects, most possibly due to the storage protocol and its respective peculiarities. In addition, our results indicate that exact point-to point documentation beginning at the sample preparation is mandatory.

Checkpoint blockade is particularly based on PD-1/PD-L1-inhibiting antibodies. However, an efficient immunological tumor defense can be blocked not only by PD-(L)1 but also by the presence of additional immune checkpoint molecules. Here, we investigated the co-expression of several immune checkpoint proteins and the soluble forms thereof (e.g., PD-1, TIM-3, LAG-3, PD-L1, PD-L2 and others) in humanized tumor mice (HTM) simultaneously harboring cell line-derived (JIMT-1, MDA-MB-231, MCF-7) or patient-derived breast cancer and a functional human immune system. We identified tumor-infiltrating T cells with a triple-positive PD-1, LAG-3 and TIM-3 phenotype. While PD-1 expression was increased in both the CD4 and CD8 T cells, TIM-3 was found to be upregulated particularly in the cytotoxic T cells in the MDA-MB-231-based HTM model. High levels of soluble TIM-3 and galectin-9 (a TIM-3 ligand) were detected in the serum. Surprisingly, soluble PD-L2, but only low levels of sPD-L1, were found in mice harboring PD-L1-positive tumors. Analysis of a dataset containing 3039 primary breast cancer samples on the R2 Genomics Analysis Platform revealed increased TIM-3, galectin-9 and LAG-3 expression, not only in triple-negative breast cancer but also in the HER2+ and hormone receptor-positive breast cancer subtypes. These data indicate that LAG-3 and TIM-3 represent additional key molecules within the breast cancer anti-immunity landscape.