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

Abstract

High-grade gliomas represent the most common group of infiltrative primary brain tumors in adults associated with high invasiveness, agressivity, and resistance to therapy, which highlights the need to develop potent drugs with novel mechanisms of action. The aim of this study is to reveal changes in proteome profiles under stressful conditions to identify prognostic biomarkers and altered apoptogenic pathways involved in the anticancer action of human isocitrate dehydrogenase (IDH) mutant high-grade gliomas. Our protocol consists first of a 3D in vitro developing neurospheroid model and then treatment by a pesticide mixture at relevant concentrations. Furthermore, we adopted an untargeted proteomic-based approach with high-resolution mass spectrometry for a comparative analysis of the differentially expressed proteins between treated and nontreated spheroids. Our analysis revealed that the majority of altered proteins were key members in glioma pathogenesis, implicated in the cellular metabolism, biological regulation, binding, and catalytic and structural activity and linked to many cascading regulatory pathways. Our finding revealed that grade-IV astrocytomas promote the downstream of the mitogen-activated-protein-kinases/extracellular-signal-regulated kinase (MAPK1/ERK2) pathway involving massive calcium influx. The gonadotrophin-releasing-hormone signaling enhances MAKP activity and may serve as a negative feedback compensating regulator. Thus, our study can pave the way for effective new therapeutic and diagnostic strategies to improve the overall survival.

Keywords: 3D neurospheroids; PPI networks; apoptogenic pathways; bioinformatic analysis; gene ontology; grade-IV astrocytomas IDH mutant; nanoflow UHPLC; neurotoxicity; proteomics; tandem mass spectrometry (MS/MS).

Figure 1

(A) Histopathological diagnosis of high-grade gliomas (WHO grade-IV astrocytomas): high cellularity diffuse proliferation with hyperplastic endothelial cells (H&E × 200). (B) Immunoreactivity of tumor cells with the isoform IDH1, confirming the diagnosis of the IDH mutant WHO grade-IV astrocytomas (IHS × 200). H&E, Hematoxylin & Eosin; IHS, immunohistochemical staining.

Figure 2

3D-spheroid culture model of glioma stem-like cells before pesticide treatment. Representative images of 3D-spheroid growth by an inverted phase contrast microscope MOTIC AE21 (Motic Incorporation Ltd. Hong Kong, China), equipped with an AXIOCAM CAMERA 208 (Sony CMOS image sensor color, Rolling Shutter; square pixels of 1.85 μM side length; 3840 × 2160 pixel resolution; ultra-HD (4K); 3 × 8-bits/pixel) (Zeiss, France). Selected images were performed with magnifications of 100 and 150 μm. By nonenzymatic protocol: Glioma stem-like cells gave rise to homogeneous cells having a fusiform format and arranged in multidirectional bundles, called primary spheres (A, left panels). After two additional days, primary spheres gave rise to neurospheres (A, middle panels) with self-renewal features such that they continuously formed secondary spheres or subspheres on day 10 (B). By enzymatic cell dissociation protocol: Neurospheres derived straight from fresh tumors have a less developed and robust morphology than those isolated from a tumor primary culture (A, right panels). After 15 days, both types of developed 3D neurospheroids could form astrocytoma daughter adherent cells, which display extensive neurite outgrowth and begin to extend thick bundles of dendrites outward to have spindle-form or polygonal to amorphous shapes (C).

Figure 3

(A) Representative microphotographs of histopathology shown on (H&E) staining obtained from tumor 3D-spheroid sections before and after culture medium. 1: Moderate cell density proliferation on day 0 without culture medium (×100); 2: very high cell density proliferation after 4 days of culture (×100). (B) H&D of 3D-spheroid growth after 24 h of pesticide treatment at three different concentrations (×100). C1: Moderate cell density proliferation; C2: weak cell density proliferation with a light appearance of apoptotic and necrotic cells after a moderate dose of treatment; C3: very weak cell density proliferation with diffuse appearance of apoptotic and necrotic cells after a high dose of treatment.

Figure 4

Volcano plot illustrating a total of 510 proteins with increased and decreased expressions following pesticide treatment. The horizontal coordinate is the difference multiple (logarithmic transformation at the base of 2), and the vertical coordinate is the significant difference in P value (logarithmic transformation at the base of 10). Data sets were analyzed by setting the cutoff criterion as log2 FC (up: ≥ 0, down: < 0) and a false discovery rate (FDR) ≤ 0.05 as thresholds. The heatmap was plotted by https://www.bioinformatics.com.cn/en, a free online platform for data analysis and visualization.

Figure 5

Gene ontology (GO) term enrichment analysis of the differently expressed proteins at upregulation (A), downregulation (B), and only yielded in the treated samples (C) based on three subontologies, namely, biological process (BP), molecular function (MF), and cellular component (CC). The heatmap was plotted by https://www.bioinformatics.com.cn/en, a free online platform for data analysis and visualization.

Figure 6

Pathways enrichment analysis with METASCAPE of the differently expressed proteins at upregulation (A), downregulation (B), and only yieded in the treated samples (C).

Figure 7

Inter-relational pathway enrichment analysis visualized by the ClueGO & CluePedia Cytoscape plug-in module: (A) for proteins only yielded in the treated neurospheroids; (B) for the upregulated proteins; and (C) for the downregulated proteins.

Figure 8

PPI network analysis of the differentially expressed genes at upregulation (A) and only seen in the treated samples (B). The line color indicates the type of interaction evidence, and the line thickness indicates the strength of data support. The node color indicates enrichment of the reactome pathway of proteins. Edges represent meaningful protein–protein associations.

Figure 9

Schema outlines the potential signaling pathways affected by pesticide treatment with the differently identified expressed proteins involved in the anticancer action of IDH1-mutant high-grade gliomas. (1) Impaired energy metabolism and mitochondrial dysfunction by the EGFr/PI3K/AKT/SREBP-1 signaling pathway: Oncogene mutation of Ras/GTPases activates mTOR via the PI3K-AKT-mTOR signaling pathway, which indirectly affects other metabolic pathways by activating HIF-1. Consequently, proteins related to glycolysis, the TCA cycle, and lipid metabolism were found to be altered by ER oxidative stress induced by pesticide treatment. (2) Protein disfolding degradation by the heat shock response/ubiquitin/proteasomal pathway. (3) Structural dysfunction, impaired axonal transportation, neuritic abnormalities, DNA damage, and impaired neuronal communication by the MAPK/ERK signaling pathway: MAP3Ks, which are activated by MAP4Ks or GTPases, mediate the phosphorylation and activation of MAP2Ks, which in turn phosphorylate and activate MAPKs. Activated MAPKs phosphorylate various substrate proteins including transcription factors, resulting in regulation of a set of cellular activities, namely, cell proliferation, differentiation, migration, inflammatory responses, and death. The mammalian MAPK gene family converges signals from G protein coupled receptors (GPCRs) or growth factor receptor-tyrosine kinases (RTKs), then via an adaptor molecule GRB2, and a guanine nucleotide exchange factor, mSOS, to activate the small GTP-binding protein, Ras, followed by activation of the MAPK cascade. The MAPK family includes ERK, p38, and JNK. In the ERK signaling pathway, ERK1/2 is activated by MEK1/2, which is activated by Raf. This latter is activated by Ras GTPase. In p38 signaling, the TNF receptor-associated factor (TRAF) activates ASK1, TAK1, or MEKK1, which activates MKK3/6, and then MKK3/6 phosphorylates p38 isoforms. In JNK signaling, RAC1 activates MEKK1 or MEKK2/3 to activate MKK4/7, and then, MKK4/7 phosphorylates JNK1/2/3. The ASK1 in the p38 signaling also activates MKK4/7 to cross-talk with JNK signaling. Ca²⁺ is required for MAPK/ERK pathway activation by the downstream of PKC and PTK signalings. (4) Pesticides may also mediate KEAP1/NRF2/ARE activation as well as the NF-kB signaling pathway responsible for cell defense against oxidative stress, with molecular cross-talk to the apoptosis-regulatory machinery through activation of the ASK1 kinase by a KEAP1 binding partner, PGAM5. (5) Cell signaling dysfunction by altering TGF-β and GnRH signaling pathways: pesticides may induce inflammatory cytokines (IL-6, IL11, IL-1ß) and caspases inhibiting growth hormone (GH) leading to endocrine disruption and activation of JAK2/STAT1 signaling. Phosphorylation of the C terminal of STAT1 enhances the activity of other factors such as p53; together with the action of DNA topoisomerase II, these molecules can cause DNA damage and eventually apoptosis. (6) Excitotoxicity: Pesticides disrupted Ca²⁺ homeostasis by influx of extracellular Ca²⁺ through L-type voltage-gated calcium channels, release from ER via inhibition of Ca²⁺-ATPase, and activation of NMDA receptors of glutamine. Stimulation of calcium-dependent apotogenic signaling pathways ultimately results in an increase of the Ca²⁺ level, activation of calpain and caspases, and thus excitotoxicity and cell death. (7) Pesticide mixture increases the nitric oxide species (NOXs), which in turn increases RNS/ROS signaling, promoting oxidative stress in cells, which may induce lipid, protein, and DNA oxidation, resulting in mitochondrial dysfunction and apoptosis. Upregulation and downregulation of the differently expressed proteins identified in the treated neurospheroids are highlighted in yellow color. AKT, serine/threonine kinase; AP2, adaptor-related protein complexes; ARE-Nrf2-NF-kB, antioxidant-responsive element-nuclear factor-erythroid-2-related factor 2, nuclear factor-kappa light chain enhancer of activated B cells; ASK1, apoptosis signal-regulating kinase 1; DAG, diacylglycerol; FABP, fatty acid binding proteins; FASN, fatty acid synthase; EGFR, epidermal growth factor receptor; ER, endoplasmic reticulum; FA, fatty acid; G3P, glyceraldehyde-3-phosphate; G6P, glucose-6-phosphate; GnrH, gonadotropin-releasing hormone; GH, growth hormone; GRB2, growth factor receptor-bound protein 2; HIF, hypoxia-induced factor; IP3R, IP3 receptor; IP3, inisitol-3-phosphate; JAK-STAT, Janus kinase-signal transducer and activator of transcription; JNK, c-Jun N-terminal kinases; KEAP1, Kelch-like ECH-associated protein-1; LDH, lactate deshydrogenease; MAPK/ERK, mitogen-activated protein kinase/extracellular-signal-regulated kinase; mSOS, mammalin son of sevenless; mTOR, mammalin target of rapamycin kinase; NMDA, N-methyl-d-aspartate; PLC, phospholipase C; PKC, protein kinase C; PDGFR, platelet-derived growth factor receptor β; PPAR, peroxisome proliferator-activated receptor; PI3K, phosphatidylinositol-3-kinase; PKC, protein kinase C; PTK, protein tyrosin kinase; PTEN, phosphatase and Tensin homolog; PDH, pyruvate deshydrogenase; PGAM5, phosphoglycerate mutase 5; RE, reticulim endoplasmic; ROS, reactive oxygen species; RNS, reactive nitrogen species; RTK, receptor-tyrosine kinase; SREBP-1, sterol regulatory element-binding transcription factor 1; TNF, tumor necrosis factor; TG, triglyceride; TGF, transforming growth factor; tPA, tissue plasminogen activator; VEGFR, vascular endothelial growth factor receptor. Created by K. Louati.