Exploring the role of GHRH antagonist MIA-602 in overcoming Doxorubicin-resistance in acute myeloid leukemia.

Acute myeloid leukemia (AML) is characterized by the rapid proliferation of mutagenic hematopoietic progenitors in the bone marrow. Conventional therapies include chemotherapy and bone marrow stem cell transplantation; however, they are often associated with poor prognosis. Notably, growth hormone-releasing hormone (GHRH) receptor antagonist MIA-602 has been shown to impede the growth of various human cancer cell lines, including AML. This investigation examined the impact of MIA-602 as monotherapy and in combination with Doxorubicin on three Doxorubicin-resistant AML cell lines, KG-1A, U-937, and K-562. The in vitro results revealed a significant reduction in cell viability for all treated wild-type cells. Doxorubicin-resistant clones were similarly susceptible to MIA-602 as the wild-type counterpart. Our in vivo experiment of xenografted nude mice with Doxorubicin-resistant K-562 revealed a reduction in tumor volume with MIA-602 treatment compared to control. Our study demonstrates that these three AML cell lines, and their Doxorubicin-resistant clones, are susceptible to GHRH antagonist MIA-602.

Cationic exchange SPE combined with triple quadrupole UHPLC-MS/MS for detection of GHRHs in urine samples.

The use of growth hormone-releasing hormones (GHRHs) is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). Considering the complexity of urine samples and the low concentrations at which these analytes should be detected, analyzing GHRHs is a challenging task. In most of the studies, GHRHs are analyzed using UHPLC-HRMS with an orbitrap. The present developed and validated method for some GHRHs (tesamorelin, CJC-1295, sermorelin (GRF 1-29), sermorelin (3-29)-NH2, somatorelin) is based on the triple quadrupole UHPLC/MS-MS method with solid phase extraction (SPE) with weak cation exchange and is able to detect concentrations as low as 0.2 ng/mL (LOD), a limit of quantification (LOQ) at 0.6 ng/mL, and linearity across the range of 0.1 ng/mL to 1.2 ng/mL. The present method developed by our doping control laboratory was validated according to WADA technical documents for selectivity, limit of detection (LOD), carryover, reliability of detection, stability and recovery. The results show that the method has adequate recoveries and sensitivity, hence, it can be employed for routine screening in anti-doping laboratories.

Online large volume sample staking preconcentration and separation of enantiomeric GHRH analogs by capillary electrophoresis.

A capillary electrophoresis method is proposed to analyze the four most well-known growth hormone-releasing hormone (GHRH) analogs that are misused by athletes. Dimethyl-ß-cyclodextrin used as a chiral selector allowed, for the first time, the separation of those basic peptide analogs, including enantiopeptides (sermorelin and CJC-1293) that differ by the chirality of only one amino acid. To increase the method sensitivity, electrokinetic preconcentration methods have been investigated. The large volume sample stacking with polarity switching (PS-LVSS) method with an injected sample volume corresponding to 80% of the capillary one was found superior to the sweeping in terms of signal enhancement factor (SEF). Acid and organic solvent addition to the sample (0.1 mM phosphoric acid with 30% methanol) led to a twofold signal improvement, when compared to water as a matrix. We increased capillary dimensions to provide a signal enhancement through the injection of a larger sample volume. Finally, using a combination of the optimized PS-LVSS preconcentration with the chiral capillary zone electrophoresis (CZE), the GHRH analogs were separated and limits of detection between 75 and 200 ng/mL were reached. This method was successfully applied to urine after a desalting step. An optimized C18 SPE was used for that purpose in order to provide low sample conductivity (<130 µS/cm) and preserve the efficiency of LVSS preconcentration. SEF of 640 was obtained with desalted urine spiked with sermorelin by comparison to the CZE (without preconcentration) method.

Tumorigenic transformation of human prostatic epithelial cell line RWPE-1 by growth hormone-releasing hormone (GHRH).

BACKGROUND: Growth hormone-releasing hormone (GHRH) and its receptors have been implicated in the progression of various tumors. In this study, we analyzed the carcinogenetic potential of exposure to GHRH of a nontumor human prostate epithelial cell line (RWPE-1) as well as its transforming effect in a xenograft model. METHODS: We performed cell viability, cell proliferation, adhesion and migration assays. In addition, metalloprotease (MMP)-2 activity by means gelatin zymography, GHRH-R subcellular location using confocal immunofluorescence microscopy and vascular endothelial growth factor (VEGF) levels by enzyme-linked immunoassay were assessed. Besides, we developed an in vivo model in order vivo model to determine the role of GHRH on tumorigenic transformation of RWPE-1 cells. RESULTS: In cell cultures, we observed development of a migratory phenotype consistent with the gelatinolytic activity of MMP-2, expression of VEGF, as well as E-cadherin-mediated cell-cell adhesion and increased cell motility. Treatment with 0.1 µM GHRH for 24 h significantly increased cell viability and cell proliferation. Similar effects of GHRH were seen in RWPE-1 tumors developed by subcutaneous injection of GHRH-treated cells in athymic nude mice, 49 days after inoculation. CONCLUSIONS: Thus, GHRH appears to act as a cytokine in the transformation of RWPE-1 cells by mechanisms that likely involve epithelial-mesenchymal transition, thus reinforcing the role of GHRH in tumorigenesis of prostate.

An antibody-free, ultrafiltration-based assay for the detection of growth hormone-releasing hormones in urine at low pg/mL concentrations using nanoLC-HRMS/MS.

This work presents an ultrafiltration-based, validated method for the screening and confirmation of prohibited growth hormone-releasing hormone (GHRH) analogues (sermorelin/CJC-1293, sermorelin metabolite, CJC-1295 and tesamorelin) in urine by nanoLC-HRMS/MS. Sample preparation avoids the use of laborious antibody-based extraction approaches and consists solely of preconcentration by ultrafiltration. Even in the absence of immuno-affinity purification steps, high sensitivity was still ensured as limits of detection between 5 and 25 pg/mL and limits of identification between 25 and 50 pg/mL were established. The robustness of the miniaturized chromatographic setup was evaluated through the injection of 200 + preconcentrated urinary extracts. In a comparison with immuno-affinity purification, enhanced recoveries (59 - 115%) and similar sensitivity were achieved, yet at lower operational costs. Stability experiments showed the importance of the proper handling of urine samples to avoid degradation of these peptide hormones, especially for sermorelin and its metabolite which were found to rapidly degrade at temperatures > 4 °C and pH values < 7 in accordance with earlier studies. Without the need for specific antibodies, this method may be expanded to cover emerging peptide drugs (≥ ~3 kDa), as well as their metabolites in the future to facilitate coverage for this class of prohibited substances.

Impact of growth hormone-releasing hormone antagonist on decidual stromal cell growth and apoptosis in vitro†.

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.

Synthesis of potent antagonists of receptors for growth hormone-releasing hormone with antitumor and anti-inflammatory activity.

The syntheses and biological evaluation of GHRH antagonists of AVR series with high anticancer and anti-inflammatory activities are described. Compared to our previously reported GHRH antagonist 602 of MIAMI series, AVR analogs contain additional modifications at positions 0, 6, 8, 10, 11, 12, 20, 21, 29 and 30, which induce greater antitumor activities. Five of nineteen tested AVR analogs presented binding affinities to the membrane GHRH receptors on human pituitary, 2-4-fold better than MIA-602. The antineoplastic properties of these analogs were evaluated in vitro using proliferation assays and in vivo in nude mice xenografted with various human cancer cell lines including lung (NSCLC-ADC HCC827 and NSCLC H460), gastric (NCI-N87), pancreatic (PANC-1 and CFPAC-1), colorectal (HT-29), breast (MX-1), glioblastoma (U87), ovarian (SK-OV-3 and OVCAR-3) and prostatic (PC3) cancers. In vitro AVR analogs showed inhibition of cell viability equal to or greater than MIA-602. After subcutaneous administration at 5 µg/day doses, some AVR antagonists demonstrated better inhibition of tumor growth in nude mice bearing various human cancers, with analog AVR-353 inducing stronger suppression than MIA-602 in lung, gastric, pancreatic and colorectal cancers and AVR-352 in ovarian cancers and glioblastoma. Both antagonists induced greater inhibition of GH release than MIA-602 in vitro in cultured rat pituitary cells and in vivo in rats. AVR-352 also demonstrated stronger anti-inflammatory effects in lung granulomas from mice with lung inflammation. Our studies demonstrate the merit of further investigation of AVR GHRH antagonists and support their potential use for clinical therapy of human cancers and other diseases.

Advances in the detection of growth hormone releasing hormone synthetic analogs.

The administration of growth hormone releasing hormone (GHRH) and its synthetic analogs is prohibited by the World Anti-Doping Agency (WADA). Although there is evidence of their use, based on admissions and intelligence, they do not appear to have been found in anti-doping samples by WADA accredited laboratories. This might be due to their small concentration in urine and limited knowledge about their metabolism, especially for unapproved synthetic analogs. This study investigates the in vitro metabolism and detection of four of the larger GHRH synthetic analogs (sermorelin, tesamorelin, CJC-1295, and CJC-1295 with drug affinity complex) in fortified urine. Nineteen major in vitro metabolites were identified, selected for synthesis, purified, and characterized in house. These were used as reference materials to spike into urine together with commercially available parent peptides and a metabolite of sermorelin (sermorelin(3-29)-NH2 ) to develop a sensitive liquid chromatography-tandem mass spectrometry method for their detection to help prove GHRH administration. Limits of detection of the target peptides were generally 1 ng/ml (WADA required performance limit) or less.

Effects of growth hormone-releasing hormone agonistic analog MR-409 on insulin-secreting cells under cyclopiazonic acid-induced endoplasmic reticulum stress.

The endoplasmic reticulum (ER) stress is one of the mechanisms related to decreased insulin secretion and beta cell death, contributing to the progress of type 2 diabetes mellitus (T2D). Thus, investigating agents that can influence this process would help prevent the development of T2D. Recently, the growth-hormone-releasing hormone (GHRH) action has been demonstrated in INS-1E cells, in which it increases cell proliferation and insulin secretion. As the effects of GHRH and its agonists have not been fully elucidated in the beta cell, we proposed to investigate them by evaluating the role of the GHRH agonist, MR-409, in cells under ER stress. Our results show that the agonist was unable to ameliorate or prevent ER stress. However, cells exposed to the agonist showed less oxidative stress and greater survival even under ER stress. The mechanisms by which GHRH agonist, MR-409, leads to these outcomes require further investigation.

Effects of growth hormone-releasing hormone receptor antagonist MIA-602 in mice with emotional disorders: a potential treatment for PTSD.

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.

Agonist of growth hormone-releasing hormone enhances retinal ganglion cell protection induced by macrophages after optic nerve injury.

Optic neuropathies are leading causes of irreversible visual impairment and blindness, currently affecting more than 100 million people worldwide. Glaucoma is a group of optic neuropathies attributed to progressive degeneration of retinal ganglion cells (RGCs). We have previously demonstrated an increase in survival of RGCs by the activation of macrophages, whereas the inhibition of macrophages was involved in the alleviation on endotoxin-induced inflammation by antagonist of growth hormone-releasing hormone (GHRH). Herein, we hypothesized that GHRH receptor (GHRH-R) signaling could be involved in the survival of RGCs mediated by inflammation. We found the expression of GHRH-R in RGCs of adult rat retina. After optic nerve crush, subcutaneous application of GHRH agonist MR-409 or antagonist MIA-602 promoted the survival of RGCs. Both the GHRH agonist and antagonist increased the phosphorylation of Akt in the retina, but only agonist MR-409 promoted microglia activation in the retina. The antagonist MIA-602 reduced significantly the expression of inflammation-related genes Il1b, Il6, and Tnf Moreover, agonist MR-409 further enhanced the promotion of RGC survival by lens injury or zymosan-induced macrophage activation, whereas antagonist MIA-602 attenuated the enhancement in RGC survival. Our findings reveal the protective effect of agonistic analogs of GHRH on RGCs in rats after optic nerve injury and its additive effect to macrophage activation, indicating a therapeutic potential of GHRH agonists for the protection of RGCs against optic neuropathies especially in glaucoma.

Growth hormone-releasing hormone antagonistic analog MIA-690 stimulates food intake in mice.

In addition to its metabolic and endocrine effects, growth hormone-releasing hormone (GHRH) was found to modulate feeding behavior in mammals. However, the role of recently synthetized GHRH antagonist MIA-690 and MR-409, a GHRH agonist, on feeding regulation remains to be evaluated. We investigated the effects of chronic subcutaneous administration of MIA-690 and MR-409 on feeding behavior and energy metabolism, in mice. Compared to vehicle, MIA-690 increased food intake and body weight, while MR-409 had no effect. Both analogs did not modify locomotor activity, as well as subcutaneous, visceral and brown adipose tissue (BAT) mass. A significant increase of hypothalamic agouti-related peptide (AgRP) gene expression and norepinephrine (NE) levels, along with a reduction of serotonin (5-HT) levels were found after MIA-690 treatment. MIA-690 was also found able to decrease gene expression of leptin in visceral adipose tissue. By contrast, MR-409 had no effect on the investigated markers. Concluding, chronic peripheral administration of MIA-690 could play an orexigenic role, paralleled by an increase in body weight. The stimulation of feeding could be mediated, albeit partially, by elevation of AgRP gene expression and NE levels and decreased 5-HT levels in the hypothalamus, along with reduced leptin gene expression, in the visceral adipose tissue.

Growth hormone-releasing hormone agonists ameliorate chronic kidney disease-induced heart failure with preserved ejection fraction.

Therapies for heart failure with preserved ejection fraction (HFpEF) are lacking. Growth hormone-releasing hormone agonists (GHRH-As) have salutary effects in ischemic and nonischemic heart failure animal models. Accordingly, we hypothesized that GHRH-A treatment ameliorates chronic kidney disease (CKD)-induced HFpEF in a large-animal model. Female Yorkshire pigs (n = 16) underwent 5/6 nephrectomy via renal artery embolization and 12 wk later were randomized to receive daily subcutaneous injections of GHRH-A (MR-409; n = 8; 30 µg/kg) or placebo (n = 8) for 4 to 6 wk. Renal and cardiac structure and function were serially assessed postembolization. Animals with 5/6 nephrectomy exhibited CKD (elevated blood urea nitrogen [BUN] and creatinine) and faithfully recapitulated the hemodynamic features of HFpEF. HFpEF was demonstrated at 12 wk by maintenance of ejection fraction associated with increased left ventricular mass, relative wall thickness, end-diastolic pressure (EDP), end-diastolic pressure/end-diastolic volume (EDP/EDV) ratio, and tau, the time constant of isovolumic diastolic relaxation. After 4 to 6 wk of treatment, the GHRH-A group exhibited normalization of EDP (P = 0.03), reduced EDP/EDV ratio (P = 0.018), and a reduction in myocardial pro-brain natriuretic peptide protein abundance. GHRH-A increased cardiomyocyte [Ca2+] transient amplitude (P = 0.009). Improvement of the diastolic function was also evidenced by increased abundance of titin isoforms and their ratio (P = 0.0022). GHRH-A exerted a beneficial effect on diastolic function in a CKD large-animal model as demonstrated by improving hemodynamic, structural, and molecular characteristics of HFpEF. These findings have important therapeutic implications for the HFpEF syndrome.

GHRH Antagonists Protect Against Hydrogen Peroxide-Induced Breakdown of Brain Microvascular Endothelium Integrity.

Growth hormone releasing hormone is a hypothalamic neuropeptide, which regulates the release of growth hormone from the anterior pituitary gland. Growth hormone releasing hormone antagonists are anticancer agents, associated with strong anti-inflammatory activities. In the present study, we investigated the effects of the GHRH antagonist MIA-602 in the integrity of the brain microvascular endothelium in vitro. Our observations suggest that MIA-602 protects against the H2O2-induced breakdown of the brain endothelium and enhances its integrity by inducing P53, deactivating cofilin, and suppressing the RhoA inflammatory pathway. Thus, GHRH antagonists may offer an exciting possibility for the treatment of pathologies related to the blood brain barrier dysfunction, including the Parkinson's and Alzheimer's diseases.

A glimpse at growth hormone-releasing hormone cosmos.

Growth hormone-releasing hormone is a hypothalamic neuropeptide, which regulates the secretion of growth hormone by the anterior pituitary gland. Recent evidence suggest that it exerts growth factor activities in a diverse variety of in vivo and in vitro experimental malignancies, which are counteracted by growth hormone-releasing hormone antagonists. Those peptides support lung endothelial barrier integrity by suppressing major inflammatory pathways and by inducing the endothelial defender P53. The present effort provides information regarding the effects of growth hormone-releasing hormone in the regulation of P53 and the unfolded protein response. Furthermore, it suggests the possible application of growth hormone-releasing hormone antagonists towards the management of acute lung injury, including the lethal acute respiratory distress syndrome.

Signaling mechanisms of growth hormone-releasing hormone receptor in LPS-induced acute ocular inflammation.

Ocular inflammation is a major cause of visual impairment attributed to dysregulation of the immune system. Previously, we have shown that the receptor for growth-hormone-releasing hormone (GHRH-R) affects multiple inflammatory processes. To clarify the pathological roles of GHRH-R in acute ocular inflammation, we investigated the inflammatory cascades mediated by this receptor. In human ciliary epithelial cells, the NF-κB subunit p65 was phosphorylated in response to stimulation with lipopolysaccharide (LPS), resulting in transcriptional up-regulation of GHRH-R. Bioinformatics analysis and coimmunoprecipitation showed that GHRH-R had a direct interaction with JAK2. JAK2, but not JAK1, JAK3, and TYK2, was elevated in ciliary body and iris after treatment with LPS in a rat model of endotoxin-induced uveitis. This elevation augmented the phosphorylation of STAT3 and production of proinflammatory factors, including IL-6, IL-17A, COX2, and iNOS. In explants of iris and ciliary body, the GHRH-R antagonist, MIA-602, suppressed phosphorylation of STAT3 and attenuated expression of downstream proinflammatory factors after LPS treatment. A similar suppression of STAT3 phosphorylation was observed in human ciliary epithelial cells. In vivo studies showed that blocking of the GHRH-R/JAK2/STAT3 axis with the JAK inhibitor Ruxolitinib alleviated partially the LPS-induced acute ocular inflammation by reducing inflammatory cells and protein leakage in the aqueous humor and by repressing expression of STAT3 target genes in rat ciliary body and iris and in human ciliary epithelial cells. Our findings indicate a functional role of the GHRH-R/JAK2/STAT3-signaling axis in acute anterior uveitis and suggest a therapeutic strategy based on treatment with antagonists targeting this signaling pathway.

Splice variant of growth hormone-releasing hormone receptor drives esophageal squamous cell carcinoma conferring a therapeutic target.

The extrahypothalamic growth hormone-releasing hormone (GHRH) and its cognate receptors (GHRH-Rs) and splice variants are expressed in a variety of cancers. It has been shown that the pituitary type of GHRH-R (pGHRH-R) mediates the inhibition of tumor growth induced by GHRH-R antagonists. However, GHRH-R antagonists can also suppress some cancers that do not express pGHRH-R, yet the underlying mechanisms have not been determined. Here, using human esophageal squamous cell carcinoma (ESCC) as a model, we were able to reveal that SV1, a known splice variant of GHRH-R, is responsible for the inhibition induced by GHRH-R antagonist MIA-602. We demonstrated that GHRH-R splice variant 1 (SV1) is a hypoxia-driven promoter of tumor progression. Hypoxia-elevated SV1 activates a key glycolytic enzyme, muscle-type phosphofructokinase (PFKM), through the nuclear factor kappa B (NF-κB) pathway, which enhances glycolytic metabolism and promotes progression of ESCC. The malignant actions induced by the SV1-NF-κB-PFKM pathway could be reversed by MIA-602. Altogether, our studies demonstrate a mechanism by which GHRH-R antagonists target SV1. Our findings suggest that SV1 is a hypoxia-induced oncogenic promoter which can be an alternative target of GHRH-R antagonists.

Antinflammatory, antioxidant, and behavioral effects induced by administration of growth hormone-releasing hormone analogs in mice.

Growth hormone-releasing hormone (GHRH) antagonist MIA-690 and GHRH agonist MR-409, previously synthesized and developed by us have demonstrated potent antitumor effects. However, little is known about the effects of these analogs on brain functions. We investigated the potential antinflammatory and antioxidant effects of GHRH antagonist MIA-690 and GHRH agonist MR-409, on isolated mouse prefrontal cortex specimens treated with lipopolysaccharide (LPS). Additionally, we studied their effects on emotional behavior after chronic in vivo treatment. Ex vivo, MIA-690 and MR-409 inhibited LPS-induced inflammatory and pro-oxidative markers. In vivo, both MIA-690 and MR-409 induced anxiolytic and antidepressant-like effects, increased norepinephrine and serotonin levels and decreased nuclear factor-kB, tumor necrosis factor-α and interleukin-6 gene expression in prefrontal cortex. Increased nuclear factor erythroid 2-related factor 2 expression was also found in mice treated with MIA-690 and MR-409. MIA-690 showed higher efficacy in inhibiting all tested inflammatory and oxidative markers. In addition, MR-409 induced a down regulation of the gene and protein expression of pituitary-type GHRH-receptor in prefrontal cortex of mice after 4 weeks of treatment at 5 µg/day. In conclusion, our results demonstrate anxiolytic and antidepressant-like effects of GHRH analogs that could involve modulatory effects on monoaminergic signaling, inflammatory and oxidative status.

Exquisite sensitivity of adrenocortical carcinomas to induction of ferroptosis.

Adrenocortical carcinomas (ACCs) are rare and highly malignant cancers associated with poor survival of patients. Currently, mitotane, a nonspecific derivative of the pesticide DDT (1,1-(dichlorobiphenyl)-2,2-dichloroethane), is used as the standard treatment, but its mechanism of action in ACCs remains elusive. Here we demonstrate that the human ACC NCI-H295R cell line is remarkably sensitive to induction of ferroptosis, while mitotane does not induce this iron-dependent mode of regulated necrosis. Supplementation with insulin, transferrin, and selenium (ITS) is commonly used to keep NCI-H295R cells in cell culture. We show that this supplementation prevents spontaneous ferroptosis, especially when it contains polyunsaturated fatty acids (PUFAs), such as linoleic acid. Inhibitors of apoptosis (zVAD, emricasan) do not prevent the mitotane-induced cell death but morphologically prevent membrane blebbing. The expression of glutathione peroxidase 4 (GPX4) in H295R cells, however, is significantly higher when compared to HT1080 fibrosarcoma cells, suggesting a role for ferroptosis. Direct inhibition of GPX4 in H295R cells led to high necrotic populations compared to control, while cotreatment with ferrostatin-1 (Fer-1) completely reverted ferroptosis. Interestingly, the analysis of public databases revealed that several key players of the ferroptosis pathway are hypermethylated and/or mutated in human ACCs. Finally, we also detected that growth hormone-releasing hormone (GHRH) antagonists, such as MIA602, kill H295R cells in a nonapoptotic manner. In summary, we found elevated expression of GPX4 and higher sensitivity to ferroptosis in ACCs. We hypothesize that instead of treatment with mitotane, human adrenocortical carcinomas may be much more sensitive to induction of ferroptosis.

Growth Hormone-Releasing Hormone Receptor Antagonist Modulates Lung Inflammation and Fibrosis due to Bleomycin.

PURPOSE: Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that a GHRH receptor (GHRH-R) antagonist, MIA-602, would inhibit bleomycin-induced lung inflammation and/or fibrosis in C57Bl/6J mice. METHODS: We tested whether MIA-602 (5 µg or vehicle given subcutaneously [SC] on days 1-21) would decrease lung inflammation (at day 14) and/or fibrosis (at day 28) in mice treated with intraperitoneal (IP) bleomycin (0.8 units on days 1, 3, 7, 10, 14, and 21). Bleomycin resulted in inflammation and fibrosis around airways and vessels evident histologically at days 14 and 28. RESULTS: Inflammation (histopathologic scores assessed blindly) was visibly less evident in mice treated with MIA-602 for 14 days. After 28 days, lung hydroxyproline (HP) content increased significantly in mice treated with vehicle; in contrast, lung HP did not increase significantly compared to naïve controls in mice treated with GHRH-R antagonist. GHRH-R antagonist increased basal and maximal oxygen consumption of cultured lung fibroblasts. Multiple genes related to chemotaxis, IL-1, chemokines, regulation of inflammation, and extracellular signal-regulated kinases (ERK) were upregulated in lungs of mice treated with bleomycin and MIA-602. MIA-602 also prominently suppressed multiple genes related to the cellular immune response including those for T-cell differentiation, receptor signaling, activation, and cytokine production. CONCLUSIONS: MIA-602 reduced lung inflammation and fibrosis due to bleomycin. Multiple genes related to immune response and T-cell functions were downregulated, supporting the view that MIA-602 can modulate the cellular immune response to bleomycin lung injury.