Quantitative proteomic analysis of GnRH agonist treated GBM cell line LN229 revealed regulatory proteins inhibiting cancer cell proliferation

Abstract

Background Gonadotropin-releasing hormone (GnRH) receptor, a rhodopsin-like G-protein coupled receptor (GPCR) family member involved in GnRH signaling, is reported to be expressed in several tumors including glioblastoma multiforme (GBM), one of the most malignant and aggressive forms of primary brain tumors. However, the molecular targets associated with GnRH receptor are not well studied in GBM or in other cancers. The present study aims at investigating the effect of GnRH agonist (Gosarelin acetate) on cell proliferation and associated signaling pathways in GBM cell line, LN229. Methods LN229 cells were treated with different concentrations of GnRH agonist (10−10 M to 10−5 M) and the effect on cell proliferation was analyzed by cell count method. Further, total protein was extracted from control and GnRH agonist treated cells (with maximum reduction in cell proliferation) followed by trypsin digestion, labeling with iTRAQ reagents and LC-MS/MS analysis to identify differentially expressed proteins. Bioinformatic analysis was performed for annotation of proteins for the associated molecular function, altered pathways and network analysis using STRING database. Results The treatment with different concentrations of GnRH agonist showed a reduction in cell proliferation with a maximum reduction of 48.2% observed at 10−6 M. Quantitative proteomic analysis after GnRH agonist treatment (10−6 M) led to the identification of a total of 29 differentially expressed proteins with 1.3-fold change (23 upregulated, such as, kininogen-1 (KNG1), alpha-2-HS-glycoprotein (AHSG), alpha-fetoprotein (AFP), and 6 downregulated, such as integrator complex subunit 11 (CPSF3L), protein FRG1 (FRG1). Some of them are known [KNG1, AHSG, AFP] while others such as inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2), ITIH4, and LIM domain-containing protein 1 (LIMD1) are novel to GnRH signaling pathway. Protein-protein interaction analysis showed a direct interaction of KNG1, a hub molecule, with GnRH, GnRH receptor, EGFR and other interactors including ITIH2, ITIH4 and AHSG. Overexpression of KNG1 after GnRH agonist treatment was validated using Western blot analysis, while a significant inhibition of EGFR was observed after GnRH agonist treatment. Conclusions The study suggests a possible link of GnRH signaling with EGFR signaling pathways likely via KNG1. KNG1 inhibitors may be investigated independently or in combination with GnRH agonist for therapeutic applications.

Characterization of Immune-Related Molecular Subtypes and a Prognostic Signature Correlating With the Response to Immunotherapy in Patients With Gastric Cancer

“…ITIH2 plays important roles in extracellular matrix stabilization and in the prevention of tumor metastasis ( 60 ). In addition, overexpression of the deregulated ITIH2 protein in glioma cells not only inhibits cancer cell invasion but also suppresses cell proliferation and promotes cell-cell adhesion ( 61 ). However, ITIH2 seemed to be a risk factor for patients with GC in our study, suggesting the need for more experiments.…”

Differential Proteome Profiling Analysis under Pesticide Stress by the Use of a Nano-UHPLC-MS/MS Untargeted Proteomic-Based Approach on a 3D-Developed Neurospheroid Model: Identification of Protein Interactions, Prognostic Biomarkers, and Potential Therapeutic Targets in Human IDH Mutant High-Grade Gliomas

“…et al (2022) has revealed an overexpression of a cysteine proteinase inhibitor, KNG1, that inhibits cell proliferation and angiogenesis in response to GnRH agonists in glioma cells. This inhibition was also related to the breakdown of EGFR/PI3K/AKT signaling, 111,112 assuming a likely strong link between GnRH and EGFR signaling pathways via KNG1. In contrast, in our study, both KNG1 and lactotransferrin isoform 2 (LTF), preventing cancer development and metastasis, were downregulated.…”

Quantitative tissue proteome profile reveals neutrophil degranulation and remodeling of extracellular matrix proteins in early stage gallbladder cancer

“…The blots were then incubated with secondary antibody (anti Rabbit-HRP, catalogue no. G-21234, 1/20,000) for 1 hr at RT and developed using the enhanced chemiluminescent (ECL) Kit (Millipore, USA) followed by image acquisition (24). The total density of the proteins in each lane was analyzed using densitometric analysis after SDS-PAGE analysis and was used for normalization (24).…”

“…G-21234, 1/20,000) for 1 hr at RT and developed using the enhanced chemiluminescent (ECL) Kit (Millipore, USA) followed by image acquisition (24). The total density of the proteins in each lane was analyzed using densitometric analysis after SDS-PAGE analysis and was used for normalization (24). For quantitative analysis, the maximum density among GSD cases was considered to define the fold change in expression in individual GBC cases.…”