Growth Hormone-Releasing Hormone Antagonist JV-1-36 Suppresses Reactive Oxygen Species Generation in A549 Lung Cancer Cells

Growth hormone-releasing hormone (GHRH) and its receptors are expressed in a variety of human cancers, and have been involved in malignancies. GHRH antagonists (GHRHAnt) were developed to suppress tumor progression and metastasis. Previous studies demonstrate the involvement of reactive oxygen species (ROS) in cancer progression. Herein, we investigate the effect of a commercially available GHRH antagonist, namely JV-1–36, in the redox status of the A549 human cancer cell line. Our results suggest that this peptide significantly reduces ROS production in those cells in a time-dependent manner and counteracts H2O2-induced ROS. Our study supports the anti-oxidative effects of JV-1–36 and contributes in our knowledge towards the in vitro effects of GHRHAnt in cancers.


Introduction
Growth hormone-releasing hormone (GHRH) is a hypothalamic hormone which regulates the release of growth hormone (GH) from the anterior pituitary gland. It consists of 44 amino acids and has been involved in mitogenic processes of a diverse variety of human malignancies including breast, lung, ovary, and prostate cancers [1]. It can directly bind to the pituitary-type GHRH receptor (GHRH-R), a member of the class B G-protein-coupled This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). receptor (GPCR) family [2]. That binding activates signaling cascades which promote cancer aggression and inflammation, at least in part due to insulin-like growth factor 1 (IGF1) release [1].
GHRH-R shares homology with the vasoactive intestinal peptide (VIP), pituitary adenyl cyclase activating polypeptide, and calcitonin receptors. The NH 2 -terminal 29 amino-acid sequence of GHRH preserves its biological activity [3]. GHRH-R contains seven hydrophobic transmembrane domains and regulates cell cycle progression and differentiation [4]. The proliferation rate of breast, prostate, and lung cancer cells is suppressed when intrinsic GHRH production is silenced due to small interfering RNA specifically designed for this neuropeptide [5]. Moreover, active receptors for GHRH were detected in surgical specimens of human prostate, ovarian, endometrial, adrenal, and pancreatic cancers [6]. Considering that GHRH is ectopically expressed, it was suggested that it can act as an autocrine/paracrine growth factor in tumor development [7].
Splice variants (SVs) of GHRH-R were detected in a diverse variety of human and animal tissues, including the lungs [8]. Although several SVs were characterized, SV1 is the most abundantly expressed and possesses ligand-dependent and -independent activities [9]. Pancreatic, colon, and gastric cancer cells expressed mRNA for both GHRH and SV1 [10]. That discovery shed light onto the mechanisms mediating the anti-cancer activities of GHRHAnt [1].

Western Blot Analysis
Cell proteins were isolated with RIPA buffer. Equal protein amounts were separated by sodium dodecyl sulfate (SDS-PAGE) Tris-HCl gel electrophoresis. Wet transfer was used to pass the proteins onto nitrocellulose membranes, which were incubated for 60 min at room temperature in 5% nonfat dry milk, so to cover non-specific binding sites. Those membranes were then exposed overnight (4 °C) to appropriate primary antibodies (1:1000). The signal for the immunoreactive proteins was developed by using the corresponding secondary antibodies (1:2000). Chemiluminescent substrate (VWR, Radnor, PA, USA) was used to develop the signal, visualized in a ChemiDoc Touch Imaging System from Bio-Rad (Hercules, CA, USA). The β-actin antibody was used as a loading control.

Cell Culture
Human lung cancer cells A549 and cervical cancer cells HeLa were obtained from ATCC (Manassas, VA, USA). They were cultured in DMEM (VWRL0101-0500) medium supplemented with 10% fetal bovine serum and 1X penicillin/streptomycin. Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO 2 -95% air. All reagents were purchased from VWR (Radnor, PA, USA).

Measurement of Cell Viability
A549 and HeLa cells were seeded onto 96-well culture plates (10,000 cells/well) in complete growth media, and were treated with GHRHAnt (0.01-15 μM) and H 2 O 2 (0.001-2 mM) for 8 hours (h). After that time-point, the media was replaced with serum-free media containing 5 mg/mL MTT, and incubated for 3.5 h. DMSO (100 μL/well) was added to dissolve MTT crystals, and 15 min later, absorbance was measured (570 nm) utilizing the SPECTROstar Nano ® Absorbance Plate Reader by BMG LABTECH.

Cell Treatment and ROS Measurement
Cells were seeded onto a 96-well plate at a density of 1 × 10 4 cells/well, and were exposed to vehicle (0.1% DMSO) or GHRHAnt (1 μM) for 4, 8, 16, or 24 h. In another set of experiments, cells were treated with vehicle (0.1% DMSO) or GHRHAnt (1 μM) for 8 h, before their exposure to vehicle (PBS) or H 2 O 2 (0.1 mM) for 8 h. After those treatments, the cells were incubated with 25 μM of DCFDA for 45 min. The fluorescence intensity was measured by the fluorescence plate reader (Synergy H1 Hybrid Multi-Mode Reader from Biotek) using the excitation wavelength of 485 nm and emission wavelength of 535 nm.

Expression of GHRH-R in Cancer Cells
The expression of GHRH-R in A549 human lung and HeLa cervical cancer cells was analyzed by Western blot analysis. The HeLa cell line was used as a negative control, due to the absence of GHRH-R [15]. Our results suggest that A549 express GHRH-R ( Figure 1A).

Effects of GHRHAnt on Cell Viability in A549 Cells
The lung cancer cells were seeded onto 96-well culture plates (10,000 cells/well) in complete growth media and were treated with either vehicle (0.1% DMSO) or JV-1-36 (0.01-15 μM). After 8 h, the media was replaced with fresh media containing 5 mg/mL MTT. After 3.5 h of incubation, DMSO was added to dissolve the MTT crystals, and absorbance was measured. Our results suggest that moderate concentrations of GHRHAnt (0.01-2 μM) do not exhibit toxicity in A549 cells. However, JV-1-36 (5-15 μM) reduced cell viability at higher concentrations ( Figure 1B).

Effects of H 2 O 2 on A549 Cell Viability
A549 cells were treated with either vehicle (PBS) or H 2 O 2 (0.001-2 mM) for 8 h. As shown in Figure 1D, H 2 O 2 at 0.001-0.5 mM did not significantly affect A549. Those groups exposed to higher concentrations (1-2 mM) of that ROS generator depicted low viability levels.

Effects of H 2 O 2 on HeLa Cell Viability
HeLa cells were exposed to vehicle (PBS) or H 2 O 2 (0.001-2 mM) (8 h). At the concentrations of 0.001-0.1 mM, H 2 O 2 did not affect cell viability, in contrast to the higher doses (0.5-2 mM) ( Figure 1E).

Effects of GHRHAnt on ROS Generation in A549 and HeLa Cells
Cells were exposed to either vehicle (0.1% DMSO) or GHRHAnt (1 μM) for 4, 8, 16, and 24 h. Our results demonstrate that GHRHAnt JV-1-36 inhibits ROS production in A549 cells (Figure 2A), while HeLa cells were unaffected ( Figure 2B). The latter cell line does not express GHRH-R.

Effects of GHRHAnt on H 2 O 2 -Induced ROS Generation in A549 and HeLa Cells
To evaluate the effects of GHRHAnt JV-1-36 on H 2 O 2 -induced ROS generation, the cells were exposed to vehicle (0.1% DMSO) or JV-1-36 (1 μM Figure 2C). That antagonist did not affect the HeLa cells ( Figure 2D).

Discussion
Reactive oxygen species (ROS) are partially reduced metabolites of oxygen, characterized by strong oxidizing capabilities. The majority of those radicals are produced by mitochondria as a result of oxidative phosphorylation. The electron transport chain is a mitochondrial pathway that encompasses five multimeric complexes. Complex I and III generate superoxide toward the matrix and intermembrane space [16]. ROS contribute to tumor development by enhancing cell survival, proliferation, protein synthesis, and glucose metabolism [12,17]. Moreover, these oxygen radicals are associated with inflammation progression. Superoxide anion (O 2− ) and hydrogen peroxide (H 2 O 2 ) are second messengers capable of releasing growth factors, chemokines, and cytokines [18]. H 2 O 2 is derived from membrane and mitochondrial sources, and it reversibly oxidizes cysteine residues (e.g., protein tyrosine phosphatases, protein tyrosine kinases, receptor tyrosine kinases) [19]. This ROS inducer is produced by epithelial cells during wound healing, and triggers the conversion of fibroblasts into myofibroblasts [20]. However, H 2 O 2 production can impair DNA and cell metabolism, partially due to the Fenton reactioninduced hydroxyl radical (OH) [21]. At low concentrations, it mediates cell growth via mitogenic oxidase Nox1, a homologue of gp91phox [22]. In higher concentrations, H 2 O 2 triggers pro-inflammatory mediators, including tumor necrosis factor (TNF-α) [23].
GHRHAnt JV-1-36 significantly reduced ROS production in lung cancer cells (Figure 2A), whereas ROS generation in HeLa cells was unaffected ( Figure 2B). HeLa cells do not respond to GHRH, since they lack the corresponding receptors ( Figure 1A) [15]. Figure 2C demonstrates the significant induction of ROS by H 2 O 2 in A549 cells and demonstrates that JV-1-36 exerted anti-oxidative effects in those cells. However, no changes were observed in HeLa cells ( Figure 1D). Those outcomes align with previous observations on the antioxidative effects of another GHRHAnt, namely JMR132, in prostate cancers. It was reported that cyclooxygenase 2 (COX-2) and cytochrome c oxidase IV (COX-IV) were involved in those effects, since GHRHAnt suppressed their expression [17]. The anti-oxidative effects of GHRHAnt are not limited to malignancies. Indeed, the GHRHAnt MZ-5-156 was shown to reduce oxidative stress in aging mice brain, as reflected in the glutathione (GSH) and glutathione peroxidase (GPx) expression levels [29].
P53 coordinates a complex framework to maintain cell homeostasis and genome stability [37]. In addition, it regulates redox homeostasis through transcriptional and nontranscriptional activities. P53 induction suppresses oxidative stress in the lung endothelium, as indicated by the reduced expression of the lipid peroxidation marker malondialdehyde (MDA) [24,30,31]. Moreover, NIMA-related kinases, or never-in-mitosis-A-related kinases (NEKs), are involved in the regulation of P53. NEK2, NEK3, NEK4, NEK7, and NEK9 upregulation has been observed in the lungs of septic mice [38]. NEK2 phosphorylates P53 at Ser315 and decreases its stability [39]. Suppression of this transcription factor by a high glucose (HG) levels is associated with HG-induced oxidative stress in endothelial cells, leading to endothelial damage and tissue injury [40]. GHRHAnt can induce P53 expression levels in both lung endothelial cells [15,41] and cancers [17], indicating that the antioxidant effects of those peptides may be mediated by P53, a UPR downstream target. However, additional studies are needed to interrogate the molecular cascades involved in those events.
UPR consists of protein kinase RNA-like ER kinase (PERK), inositol requiring enzyme 1α (IRE1α), and activating transcription factor 6 (ATF6) [42,43]. ATF6 was reported to participate in adaptive responses, so to ameliorate severe inflammatory disorders [44,45]. Perturbation of protein folding promotes the dissociation of binding immunoglobulin protein (BiP) or 78 kDa glucose-regulated protein (GRP-78) from the luminal domain of those sensors, which in turn activates UPR [44]. UPR can also mediate the protective effects of GHRHAnt in barrier dysregulation, a hallmark of the potentially lethal ARDS. GHRHAnt counteracted the kifunensine (UPR suppressor)-induced lung endothelial barrier dysfunction [31]. ARDS is responsible for the death of millions worldwide due to COVID-19; and targeted medicine for that disorder does not exist, so far. UPR can also positively regulate P53, and its modulation regulates barrier function [46]. Hence, GHRHAnt/UPR/P53 may form an autoregulatory loop to protect against barrier disorders. Further research will shed light on the role of UPR in the anti-oxidative activities of GHRHAnt in the P53 context. To do so, we will utilize genetically modified mice which express more or less of P53, and elevated UPR levels, as performed before [44,47,48].
Our observations reveal that JV-1-36 exerts anti-oxidative effects in A549 lung cancer cells. Considering the anti-inflammatory effects of GHRHAnt in normal lung endothelial cells, and their anti-oxidative activities in the inflamed endothelium [31], we conclude that GHRHAnt represent an exciting therapeutic possibility for the treatment of lung inflammatory diseases. Future studies will address the exact interrelations of GHRHAnt, UPR, and P53 in cancers, utilizing transgenic mice and genetically modified cancer cells.  (8 h). MTT assay was used to assess cell viability. n = 3 per group. Means ± SEM. (C) HeLa cells were seeded onto a 96-well plate (10,000 cells/well) and were incubated with VEH (0.1% DMSO) or GHRHAnt (0.01-15 μM); and (E) VEH (PBS) or H 2 O 2 (0.001-2 mM) (8 h). GHRHAnt at 0.01-1 μM, and H 2 O 2 at 0.001-0.1 mM did not affect HeLa viability. However, viability was reduced in cells exposed to higher concentrations of GHRHAnt (2-15 μM) or H 2 O 2 (0.5-2 mM). The graphs represent three independent experiments. * p < 0.05 vs. VEH. Means ± SEM.