Cholangiocarcinoma (CCA) is an aggressive cancer associated with a very poor prognosis and low survival rates, primarily due to late-stage diagnosis and low response rates to conventional chemotherapy. Therefore, there is an urgent need to identify effective therapeutic strategies that can improve patient outcomes. Flavonoids, such as quercetin and kaempferol, are naturally occurring compounds that have attracted significant attention for their potential in cancer therapy by targeting multiple genes. In this study, we employed network pharmacology and bioinformatic analysis to identify potential targets of quercetin and kaempferol. The results revealed that the target genes of these flavonoids were enriched in G2/M-related genes, and higher expression of G2/M signature genes was significantly associated with shorter survival in CCA patients. Furthermore, in vitro experiments using CCA cells demonstrated that quercetin or kaempferol induced cell-cycle arrest in the G2/M phase. Additionally, when combined with a Smac mimetic LCL-161, an IAP antagonist, quercetin or kaempferol synergistically induced RIPK1/RIPK3/MLKL-mediated necroptosis in CCA cells while sparing non-tumor cholangiocyte cells. These findings shed light on an innovative therapeutic combination of flavonoids, particularly quercetin and kaempferol, with Smac mimetics, suggesting great promise as a necroptosis-based approach for treating CCA and potentially other types of cancer. Cholangiocarcinoma (CCA) originates from cholangiocytes lining the biliary tree, both intrahepatic- and extrahepatic-bile ducts. Although the incidence of CCA in Thailand is almost 100 times higher than in other regions , the incidence rate has globally increased in recent decades ,3. Even We have previously reported that Smac mimetics sensitize gemcitabine to induce necroptosis in human CCA cells by degrading the cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2) . This stFlavonoids are naturally occurring compounds found in vegetables and fruits. Quercetin and kaempferol are two noteworthy compounds among the flavonoids. Quercetin is abundantly found in foods such as onions, asparagus, and berries. On the other hand, kaempferol is primarily present in green leafy vegetables such as spinach and kale, as well as herbs such as dill, chives, and tarragon . FlavonoTherefore, this current study aimed to comprehensively analyze the potential molecular targets of both quercetin and kaempferol related to cell-cycle regulation and DNA topoisomerase II using network pharmacology. Additionally, we investigated their associations with the prognosis of CCA patients using public databases and integrated bioinformatics tools. In vitro validation related to the effect of cell-cycle and proliferation inhibition was carried out in two representative CCA cells. Finally, we explored the novel therapeutic combination of quercetin or kaempferol with Smac mimetics to induce necroptosis in CCA cells.The crystallographic structure of DNA topoisomerase II (PDB ID: 5GWK) was obtained from the RCSB Protein Data Bank. To prepare the protein for docking, a previously established procedure was followed. In summary, all non-protein molecules were removed from the structure, and missing hydrogen atoms and Kollman charges were added using AutoDockTools-1.5.7 software . The prepared protein structures were saved in a PDBQT format to be used in subsequent molecular docking studies.https://pubchem.ncbi.nlm.nih.gov, accessed on 24 March 2023) in SDF format. The ligand structures were converted to a PDB format using BIOVIA Discovery Studio 2020  and then further converted to a PDBQT format using the AutoDockTools-1.5.7 program.The ligand structures, including etoposide (PubChem CID: 36462), quercetin (PubChem CID: 5280343), and kaempferol (PubChem CID: 5280863), were retrieved from the PubChem online database . The conformation with the lowest binding energy was selected, and the protein–ligand interaction was evaluated using BIOVIA Discovery Studio 2021 .http://www.swisstargetprediction.ch/, accessed on 29 March 2023) [https://www.bindingdb.org/rwd/bind/index.jsp, accessed on 3 April 2023) [http://lilab.ecust.edu.cn/pharmmapper/, accessed on 24 March 2023) [https://prediction.charite.de/, accessed on 29 March 2023) [https://pubchem.ncbi.nlm.nih.gov/, accessed on 24 March 2023) for use in SwissTargetPrediction and BindingDB. The structures of quercetin and kaempferol files (PubChem CID: 5280343 and 5280863) were uploaded into PharmMapper to predict possible targets. The names of quercetin and kaempferol were used to search for targets in Super-PRED. Additionally, a total of 157 cell cycle-related genes were obtained from the Kyoto Encyclopedia of Genes and Genomes . Finally, a Venn diagram  was used to identify the target genes shared by quercetin or kaempferol and cell cycle-related genes.The potential target genes were collected from various sources, including SwissTargetPrediction (ch 2023) , Bindingil 2023) , PharmMach 2023) , and Supch 2023) . The SMIhttps://www.string-db.org/, accessed on 2 May 2023) [A protein–protein interaction (PPI) network analysis was performed on the target genes using the STRING database (ay 2023) . In addihttps://www.ncbi.nlm.nih.gov/geo/, accessed on 8 May 2023). A comparative analysis of the 18 overlapping genes expressions was performed on 91 pairs of tumor and non-tumor tissues from the GSE76297 dataset using a paired-sample t-test presented in a box plot. A Kaplan–Meier and log-rank test were used to compare patient survival between G2/M groups using R statistical software (version 4.1.0)  with R’s “survminer” package. Survival data were obtained from the GSE89749 dataset. The G2/M signature group, which includes CCNA2, CCNB1, CCNB2, CDC25B, CDC25C, CDK1, ABL1, AURKB, CHEK1, and PLK1, was used to stratify patients into two groups. Each gene was initially divided into low and high groups based on the median as the cutoff, resulting in a score of 0 and 1, respectively. Subsequently, the scores of all genes in the G2/M signature were summed, and patients were stratified into low (scores ranging from 0 to 4) and high (scores ranging from 5 to 10) groups.The GSE76297 and GSE89749 datasets were downloaded from the Gene Expression Omnibus (GEO) database . Pan-caspase inhibitor, zVAD-fmk , GSK’782, and necrosulfonamide (NSA) were purchased from Calbiochem . Quercetin, kaempferol, and necrostatin-1 were purchased from Sigma-Aldrich  and dissolved in dimethyl sulfoxide (DMSO) following the manufacturer’s instructions to prepare stock solutions. Working solutions were subsequently prepared by diluting the stock solutions and ensuring that they contained a non-toxic concentration of DMSO, less than 0.15%.2. Furthermore, rigorous testing confirmed that there was no mycoplasma contamination in any of the cell lines.The HuCCT-1 cell line, derived from human Cholangiocarcinoma (CCA), was obtained from the Japanese Collection of Research Bioresources (JCRB) Cell Bank in Osaka, Japan. The RMCCA-1 cell line, also a human CCA cell line, was developed from Thai CCA patients . Both HuCCA cells were treated with quercetin or kaempferol as specified concentrations for 48 h. Following the treatment, cell-cycle analysis was conducted by employing propidium iodide (PI) staining to assess DNA content. Briefly, the cells were fixed with 70% ethanol and subsequently washed with Phosphate Buffered Saline . The cells were then resuspended in PBS containing 0.25% Triton X, along with RNase A (100 μg/mL) and PI (50 μg/mL) and incubated for 30 min. Finally, the stained cells were analyzed using flow cytometry for further examination. Data analysis was then performed using the FlowJo™ version 10 software (Becton Dickinson and Company (BD) Franklin Lakes, NJ, USA).For the treatment conditions involving Smac mimetic LCL-161 (5 μM for RMCCA-1 and 25 μM for HuCCT-1) and zVAD-fmk (20 μM), the inhibitors were pre-treated for a minimum of 2 h followed by treatment with quercetin or kaempferol (concentrations as indicated). Cell death was assessed through Annexin V-FITC and PI staining, followed by flow cytometry analysis. In brief, cells were washed and then suspended in Annexin V binding buffer containing recombinant Annexin V-FITC  and PI . The stained cells were examined using a flow cytometer . A total of ten thousand events were collected for each sample, and the data were analyzed using Navios software. The combination index (CI) was calculated based on the Chou-Talalay method, where CI values of 1, <1, and >1 indicate an additive effect, synergism, and antagonism, respectively .Lentiviral plasmids carrying shRNA constructs were obtained from Sigma . The shRNA sequences targeted human MLKL (NM_152649.4) at the 3′ untranslated regions 2025–2045 (shMLKL#1) and 1907–1927 (shMLKL#2). For CRISPR experiments, plasmids targeting human RIPK1 (NM_003804) and RIPK3 (NM_006871) were generated following Zhang’s protocol [The cells were washed twice with ice-cold PBS and lysed in RIPA buffer  supplemented with a proteinase inhibitor cocktail  on ice for 30 min. The total protein concentrations were determined using the Bradford assay . Total proteins (20–50 μg) were separated by 10–20% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with a 5% blotting-grade blocker  at room temperature for 1 h, followed by overnight incubation with primary antibodies at 4 °C. The primary antibodies used in this study were anti-RIPK1 (610459) from BD Biosciences , anti-MLKL (ab184718) and anti-phosphorylated MLKL (ab187091) from Abcam , and anti-RIPK3 (8457) and anti-β-Actin (4970) from Cell Signaling . After incubation with primary antibodies, the blots were washed three times with TBS-T  buffer and incubated with horseradish peroxidase-conjugated secondary antibodies  at room temperature for 1 h. The proteins were visualized using enhanced chemiluminescence according to the manufacturer’s instructions  using Amersham ImageQuant 800 Western blot imaging systems. All the Western blots shown are representative of at least three independent experiments.t-tests to assess group differences. The log-rank test was utilized to examine variations in survival between G2/M signature groups, with Kaplan-Meier curves used to visualize patient survival. Pearson’s correlation coefficient (r) was employed for all correlation analyses. For in vitro experiments, the results were presented as the mean ± standard deviation (S.D.) of at least three independent experiments. Student’s t-test was conducted to compare two groups. Statistical significance was defined as a p-value < 0.05, indicated by asterisks .All data were analyzed using either R statistical software (version 4.1.0) or SPSS . Bioinformatics analysis involved the use of Wilcoxon or Student’s Previous research has demonstrated that flavonoids, including quercetin and kaempferol, with multiple phenolic rings can interact and inhibit DNA topoisomerase II similar to the chemotherapeutic drug etoposide, which can induce DNA double-strand breaks . The indABL1, AURKB, CCNA2, CCNB1, CCNB2, CCNB3, CDC25B, CDC25C, CDK1, CDK2, CDK6, CDK7, CHEK1, GSK3B, HDAC2, HDAC8, PLK1, and TGFB2)  A,B. To sd TGFB2) C. In thed TGFB2) A. Phosphd TGFB2) B. Moreovd TGFB2) C. These CCNA2, CCNB1, CCNB2, CDC25B, CDC25C, and CDK1), in addition to four other G2/M hallmark genes from the MSigDB database . Our results revealed a significant association between high expression levels of the G2/M signature gene set and poorer survival in CCA patients   B. These Our bioinformatics analysis identified genes in G2/M cell-cycle associated pathways as targets of quercetin and kaempferol. To further investigate whether quercetin and kaempferol can induce G2/M cell-cycle arrest, we conducted in vitro experiments using CCA cell models, RMCCA-1 and HuCCT-1. The effects of quercetin and kaempferol on the cell cycle in CCA cells were examined. We analyzed the cell-cycle perturbations induced by quercetin and kaempferol, as depicted in the representative histograms A,B. The In our previous studies, we demonstrated the potential of Smac mimetics to synergize with different inducers and induce necroptosis in CCA cells ,47, partRIPK1 or RIPK3 in CCA cells significantly suppressed cell death induced by the combination treatment , a specific marker of necroptosis, using Western blot analysis. The results demonstrated that the combination treatment of quercetin A or kaemreatment E,F. Simi LCL-161 G,H. CollSeveral studies have demonstrated the improvement of Smac mimetics as a monotherapy treatment by exploring the rationale combination of cytotoxic drugs and DNA-damaging agents in the context of chemotherapy with Smac mimetics. This study is the first to explore the novel potential combination of flavonoids, specifically quercetin and kaempferol, with Smac mimetics. Several key findings were identified in the present study. First, we identified the molecular targets related to G2/M cell-cycle regulators of quercetin and kaempferol. These target genes are associated with shorter overall survival of CCA patients, suggesting that G2/M cell-cycle regulators could be potential therapeutic targets for CCA therapy. Second, using an in silico molecular docking approach, we observed potential interactions between quercetin and kaempferol and DNA topoisomerase II. Third, we have shown that both quercetin and kaempferol can effectively arrest CCA cells in the G2/M cell-cycle phase. This effect is likely due to the ability of quercetin and kaempferol to target multiple G2/M cell-cycle regulators and DNA topoisomerase II. Fourth, we have demonstrated that the Smac mimetic LCL-161 sensitizes CCA cells to necroptosis when caspases are inhibited in an RIPK1/RIPK3/MLKL-dependent manner. This effect was demonstrated through experiments utilizing both pharmacological inhibitors and genetic approaches. Notably, the combination effect was observed at relatively lower cytotoxic doses of quercetin and kaempferol. Lastly, the synergistic combination of flavonoids (quercetin and kaempferol) and Smac mimetics specifically induced necroptosis in CCA cell lines, while the non-tumor cholangiocyte remained resistant. Our findings provide a novel therapeutic combination between flavonoids, specifically quercetin and kaempferol, with Smac mimetics. This innovative combination holds potential for the development of a necroptosis-based therapeutic approach, not only for CCA but also for other cancer types.Necroptosis can be considered as immunogenic cell death (ICD) due to its dual beneficial roles in killing tumor cells and inducing antitumor immunity . StudiesCDC25C and CDC25B, were enriched in the Gene Ontology (GO) pathway enrichment analysis; CDK1, CCNA2, CCNB1, and CCNB2 were enriched in the reactome pathway enrichment analysis; and finally, CDK1, CCNB1, and CCNB2 were enriched in the local network cluster (STRING) enrichment analysis. This study is the first to utilize network pharmacology to identify potential target genes of flavonoids (quercetin and kaempferol) in the G2/M cell cycle-related pathways. Although, we did not validate the expression changes of these target genes following quercetin and kaempferol treatment, in vitro functional experiments confirmed that both quercetin and kaempferol dramatically arrested CCA cells in the G2/M cell-cycle phase. This effect may be attributed to their targeting of multiple genes related to the G2/M cell-cycle phase. Our findings are consistent with previous studies that have demonstrated the ability of both quercetin and kaempferol to induce G2/M cell-cycle arrest, leading to apoptosis in cancer cells [Natural compounds with functional properties similar to chemotherapeutic drugs, such as DNA-damaging effects, targeting cell-cycle or DNA repair pathways, or inhibiting cell proliferation are promising candidates. Initially, we identified these natural compounds through a literature search. Among them, natural flavonoids, particularly quercetin and kaempferol, have been extensively studied and gained importance as cytotoxic and cytostatic agents, exhibiting the aforementioned functional properties. However, the combination of flavonoids (quercetin and kaempferol) with Smac mimetics has not yet been evaluated both in CCA or other cancers. To identify potential targets related to the cell cycle regulators of quercetin and kaempferol, we collected a list of potential target genes of quercetin and kaempferol and overlapped them with a set of 157 cell cycle-related genes. A total of 18 overlapping genes were identified. Furthermore, the protein–protein interaction (PPI) network and functional pathway enrichment analysis of these 18 overlapping target genes revealed that most of the overlapping target genes were enriched in the G2/M cell cycle-associated pathways. Specifically, the following G2/M cell cycle-related genes including er cells ,31,32,34In contrast to previous studies, we selected concentrations of quercetin and kaempferol that exhibit cell proliferation inhibition while inducing minimal cell death for their combination with the Smac mimetic. Additionally, in this study, we utilized the Smac mimetic LCL-161, which has entered clinical trials for both monotherapy and combination therapy in the treatment of certain cancers . InteresTo examine whether the combination treatment is specific to CCA cells, we performed the same experiment setting in a non-tumor cholangiocyte, MMNK-1, as a representative of non-tumor cells. The combination treatment did not trigger cell death when caspases were inhibited; however, treatment with quercetin or kaempferol alone significantly induced cell death, most likely apoptosis, in a dose-dependent manner. The non-tumor cholangiocyte was more sensitive to a single treatment with quercetin than kaempferol. In contrast to CCA cell lines (RMCCA-1 and HuCCT-1) which express key necroptotic proteins, including RIPK1, RIPK3, and MLKL , MMNK-1 CCNA2, CCNB1, CCNB2, CDC25B, CDC25C, CDK1, ABL1, AURKB, CHEK1, and PLK1, we used this G2/M gene signature to stratify patients into two groups. Interestingly, CCA patients with high expression of this G2/M gene signature were associated with unfavorable survival outcomes (p = 0.017), indicating that these genes might be druggable targets for CCA therapy. Although, when analyzing the expression of each gene with patients’ survival, only PLK1 turned out to be significantly associated with patients’ survival (p = 0.026)  . This fiCA cells ,57,58. WCA cells , PLK1 inCA cells . These rThis is the first study to explore the novel potential combination of flavonoids, specifically quercetin and kaempferol, with Smac mimetics. This novel combination shows promise for the development of a necroptosis-based therapeutic approach, not only for CCA but also for other cancer types.


In this five-week experiment, seven groups of male albino rats were organized: G1 , G2 (SI), G3 (MnCl2), G4 (SI + MnCl2), G5 (SI + PUN), G6 (MnCl2 + PUN), and G7 (SI + PUN + MnCl2). The results revealed significant changes in behavior, biochemistry, and histopathology in rats exposed to SI and/or MnCl2, with the most pronounced effects detected in the SI rats concurrently exposed to MnCl2. These effects were associated with augmented oxidative stress biomarkers and reduced antioxidant activity of the Nrf2/HO-1 pathway. Additionally, inflammatory pathways (HMGB1/RAGE/TLR4/NF-ᴋB/NLRP3/Caspase-1 and JAK-2/STAT-3) were upregulated, while dysregulation of signaling pathways (PI3K/AKT/GSK-3β/CREB), sustained endoplasmic reticulum stress by activation PERK/CHOP/Bcl-2, and impaired autophagy (AMPK/SIRT-1/Beclin-1 axis) were observed. Apoptosis induction and a decrease in monoamine levels were also noted. Remarkably, treatment with PUN effectively alleviated behaviour, histopathological changes, and biochemical alterations induced by SI and/or MnCl2. These findings emphasize the role of SI in PD progress and propose PUN as a potential therapeutic intervention to mitigate PD. PUN’s mechanisms of action involve modulation of pathways such as HMGB1/RAGE/TLR4/NF-ᴋB/NLRP3/Caspase-1, JAK-2/STAT-3, PI3K/AKT/GSK-3β/CREB, AMPK/SIRT-1, Nrf2/HO-1, and PERK/CHOP/Bcl-2.Parkinson’s disease (PD) is a gradual deterioration of dopaminergic neurons, leading to motor impairments. Social isolation (SI), a recognized stressor, has recently gained attention as a potential influencing factor in the progress of neurodegenerative illnesses. We aimed to investigate the intricate relationship between SI and PD progression, both independently and in the presence of manganese chloride (MnCl Parkinson’s disease (PD) is a gradual neurodegenerative illness manifests as muscle rigidity, tremors, and bradykinesia . These fOxidative stress and neuroinflammation are interconnected processes that can mutually stimulate or repress each other . OxidatiNuclear factor-erythroid 2-related factor 2 (Nrf2) emerges as a central transcription factor associated with oxidative stress and neuroinflammation. Nrf2 effectively modulates the expression of genes encoding antioxidant enzymes and proteins, including heme-oxygenase-1 (HO-1). Nrf2/HO-1 pathway acts as a neuroprotective in various neurological conditions. Activation of this pathway has a significant impact on counteracting oxidative stress, impeding neurodegeneration, and attenuating neuroinflammation through the repression of NF-κB. Consequently, the regulation of Nrf2/ HO-1 holds a therapeutic potential for combating the deleterious consequences of oxidative stress and neuroinflammation in diverse neurological disorders .Moreover, the embarrassment of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway has been related to the aggravation of oxidative stress, neuroinflammation, and neuronal apoptosis in PD. PI3K, once activated, recruits, and activates AKT. Upon activation, AKT phosphorylates downstream targets, thereby deactivating glycogen synthase kinase 3β (GSK-3β) and triggering cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB). Furthermore, GSK-3β enhances neuroinflammation by activating the NF-κB pathway and promotes oxidative stress by impairing Nrf2 signaling . AdditioER is a principal organelle in proteostasis, where it controls appropriate protein folding and transport ,14. MisfThe balance between autophagy and apoptosis is disrupted during PD, with enhanced apoptosis and impaired autophagy, leading to the accumulation of abnormal misfolded α-syn protein, with progressive loss of dopaminergic neurons . UndeniaManganese (Mn) is a widely recognized environmental neurotoxicant . ElevateIt is noteworthy that social isolation (SI) serves as a prominent neurological stressor ,29. SI cPunica granatum L.). PUN has shown antioxidant, anti-inflammatory, and anti-apoptotic actions. It has gained significant attention for its potential health benefits, including cardioprotective, antibacterial, anticancer, anti-atherosclerotic, and neuroprotective properties [To date, available treatments only offer short-term relief from the symptoms of PD but do not effectively hinder the progression of the disease . This hioperties ,37. Prevoperties  and Alzhoperties , suggest2. The study aimed to elucidate the nature and pattern of this relationship by analysing behavioural, biochemical, and histopathological data and to explore the underlying mechanistic pathways that contribute to this interplay. Furthermore, the study examined the potential neuroprotective effects of PUN against the biochemical, behavioural, and histopathological alterations concomitant with SI and/or MnCl2 intoxication. Also, the investigation was designed to deliver mechanistic comprehensions into the role of various pathways, including HMGB1/RAGE/TLR4/NF-κB/NLRP3/Caspase-1, JAK-2/STAT-3, PI3K/AKT/GSK-3β/CREB, AMPK/SIRT-1, Nrf2/HO-1, and PERK/CHOP/Bcl-2, as potential contributors to these pathophysiological events.In recent times, there has been a growing focus on investigating the relationship between manganese exposure and the initiation of PD, as well as the subsequent neurotoxic outcomes. Mn is being explored as an inducer of PD in preclinical trials. This emerging field of research has provided valuable insights into the origins and advancement of Parkinson’s disease ,42,43,44Seventy adult male Sprague Dawley rats weighing between 300 and 320 g were acquired from The Nile Co. for Pharmaceuticals and Chemical Industries . Before beginning the experiment, the rats were provided a one-week acclimatization period. The rodents were housed in controlled conventional conditions at the Faculty of Pharmacy, Al-Azhar University’s animal house. These conditions entailed a 12-h light/dark cycle, 25 ± 1 °C temperature, and a humidity level of 50 ± 5%. The rats were given access to unlimited amounts of water and normal food pellets. All animal procedures and experimental protocols were ethically approved by the Animal Care and Use committee of the Faculty of Pharmacy, Al-Azhar University, with ethical approval number 395/2023. The handling of animals was according to the guidelines outlined in the “Guide for Care and Use of Laboratory Animals”, published by the National Institutes of Health .2.4H2O) and punicalagin were purchased from Sigma-Aldrich Chemical Co. . They were freshly dissolved in normal saline, as described [Manganese (Mn) chloride tetrahydrate (MnClescribed . All che2 at a dose of 10 mg/kg/day for 5 consecutive weeks to induce PD [2 for 5 weeks to induce depression concomitantly with PD (PDD). G V (SI + PUN group) received daily oral administration of PUN at a dose of 30 mg/kg in SI condition for 5 weeks, according to prior reports [2 and concurrent daily oral administration of PUN for 5 weeks. G VII (Mn + SI + PUN-group) consisted of SI rats treated with MnCl2 and concurrently receiving PUN for 5 weeks. The treatment was continued for five weeks [Seventy adult male albino rats were randomly assigned into 7 groups: 10 rats each. These groups (Gs) were as follows: G I (Control group) treated with 1 mL of normal saline orally and 0.2 mL of saline intraperitoneally (i.p.) in a group-housing condition . G II , which was then centrifuged at 4000 rpm for 15 min at 4 °C. The resulting supernatant was preserved at −20 °C for subsequent biochemical investigations [After euthanizing the rats, their brains were carefully extracted and then rinsed with saline. The rodents within each group were subsequently divided into 2 sets of rats. In the first set (n = 4). For histological investigation, the brains in the first set (n = 4) were preserved in 10% buffered formalin. The brains in the second set (n = 6) were each divided into two portions. The first portion of tissue underwent homogenization with phosphate buffer (pH = 7.4) to create a 10% homogenate  following the manufacturer’s instructions.The acetylcholinesterase (ACHE) activities in the brain tissue homogenate was estimated by a commercial kit , following the standard protocol instructions. Additionally, the measurements of the lipid peroxidation product, malondialdehyde (MDA) (Cat. No. MD 2528), the activity of superoxide dismutase (SOD) (Cat. No. SD 2520), and the levels of total antioxidant capacity (TAC) (Cat. No. TA 2512) in brain tissue homogenates were estimated by commercially available kits , norepinephrine (NE), and serotonin (5-HT), were measured without delay using fluorometric kits that were commercially available . As previously mentioned, the fluorometric method was employed to detect the monoamines present in the tissue samples, with specific excitation/emission wavelengths set at 320/480 nm for DA, 380/480 nm for NE, and 355/470 nm for 5-HT .ELISA technique was employed to quantify inflammatory markers in brain tissue homogenates by ELISA kits  according to the manufacturer’s recommendations. The levels of IL-1β (Cat.#MBS2023030), prostaglandin E2 (PGE-2) (Cat.#MBS262150), inducible nitric oxide synthase (iNOS) (Cat.#MBS723326), IL-6 (Cat.#MBS269892), cyclooxygenase-2 (COX-2) (Cat.#MBS725633) and TNF-α (Cat.#MBS175904). Moreover, the level of BDNF was also measured using a commercial kit (Cat.#MBS703433) provided by the same vendor.TM RT reagent Kit . The qRT-PCR were performed with SYBR Green reagents  on an Applied Biosystems instrument  with software version 3.1. The transcripts of B-cell lymphoma 2 protein (Bcl-2), Bcl-2-associated X protein (Bax), caspase-3, caspase-1, NLRP3, NF-κB, HO-1, Nrf2, TLR4, GSK-3β, PERK, CHOP, beclin-1, GRP78, PI3K, TrkB, AMPK, SIRT-1, AKT, CREB, HMGB1, RAGE, JAK-2 and STAT-3, were normalized to the expression level of the reference gene GAPDH. The relative expression of the target genes was evaluated by the Livak and Schmittgen method [−∆∆CT. The list of forward and reverse primer sequences for each gene used in qPCR amplification is provided in The total RNA was isolated from brain tissue samples by the standard protocol provided by Qiagen , per the manufacturer’s instructions. The concentration of mRNA was evaluated by a NanoDrop Spectrophotometer . Subsequently, 1 μg of mRNA was reverse transcribed into cDNA employing the PrimeScriptn method  with theFormalin-fixed paraffin-embedded blocks of brain tissues were cut into 5 m-thick slices, stained using the usual H&E method, allowed to air dry, then observed under a light microscope and photographed at a magnification of 40× ,65.p <0.05. Tukey’s multiple comparisons test was then performed. The statistical analysis and data visualization were done using GraphPad Prism® .Mean and SEM are used to describe the data. One-way ANOVA was used for the statistical analysis, and significance was determined at In our study, the influences of PUN were examined on a normal control group. However, it did not display any substantial differences from the control group regarding the measured parameters and histopathological findings. Therefore, these results were not presented to avoid complicating the data.2 groups exhibited a significant intensification in latency time  , along with a remarkable increase in rearing , grooming , and ambulation frequencies  when linked to their corresponding untreated groups, SI, MnCl2, or SI with MnCl2, correspondingly. Of note, the administration of PUN to the SI rats restored the latency time and grooming frequency to normal values.In ctively) A, accompctively) B, groomictively) C, and amctively) D, comparl2 alone . In cont2 resulted in a meaningful rise in the immobility score   and climbing scores , together with a notable increase in the swimming score  and percentage of spontaneous alteration  related to the matching untreated groups, SI, MnCl2, or SI with MnCl2, respectively (As displayed in ctively) A and clictively) B, along ctively) C and perctively) D, relateectively . Importa2 resulted in a significant reduction in brain monoamines; specifically, there was a decrease by 25.7% and 67.2% in DA levels  and HO-1 ; elevated SOD activity  and TAC levels , together with a significant diminution in brain MDA levels  and iNOS levels , related to the matching untreated groups, SI, MnCl2, or SI + MnCl2. It is worth noting that the administration of PUN to SI rats restored the brain iNOS level back to normal values.2 substantially raised the mRNA expression level of NF-ᴋB  , brain TNF-α , IL-1β , IL-6 , COX-2 , and PGE2  related to their untreated equivalent groups . It is worth noting that PUN treatment in SI rats restored the levels of brain IL-1β, IL-6, and COX-2 back to normal values.As displayed in ctively) A; the brctively) B–F. Furt2 meaningfully upregulated the mRNA expression levels of TLR4  , HMGB1 , RAGE , NLRP3 , caspase-1 , JAK-2 , and STAT-3  relative to their untreated corresponding groups . Importantly, PUN treatment in SI rats restored the mRNA expression levels of TLR4, JAK-2, and STAT-3 back to normal values.As depicted in ctively) A, HMGB1 ctively) B, RAGE (ctively) C, NLRP3 ctively) D, caspasctively) E, JAK-2 ctively) E, and STctively) F likened2 elicited substantial drop in the mRNA expression levels of PI3K  , AKT , CREB , TrKB , and BDNF , together with a marked reduction in mRNA expression levels of GSK-3β  when it was likened to their untreated corresponding groups . Remarkably, PUN treatment in SI rats restored the mRNA expression level of AKT back to normal values.As clarified in ctively) A, AKT (bctively) B, CREB (ctively) D, TrKB (ctively) E, and BDctively) F, along ctively) C linked 2 had shown mRNA upregulation of PERK  , GRP78 , CHOP , Bax  and caspase-3 , together with a substantial upregulation in mRNA expression level of Bcl-2 , matched to their untreated equivalent groups .As depicted in ctively) A, GRP78 ctively) B, CHOP (ctively) C, Bax (bctively) E and casctively) F, along ctively) D, compar2 resulted in a substantial diminution in mRNA expression levels of AMPK  , SIRT-1 , and beclin-1  matched to their untreated equivalent groups . It is worth noting that PUN in SI rats restored the mRNA expression level of AMPK back to its normal values.As illustrated in ctively) A, SIRT-1ctively) B, and bectively) C, compar2, or SI with MnCl2 ameliorated all the aforementioned histopathological alterations in the explored brain regions by varying degrees, compared to their untreated corresponding groups . It is worth noting that PUN treatment resulted in apparent normal histological architecture in all investigated brain areas in the SI + PUN group (Inserts e1–5), along with neurons of the cerebral cortex (Insert f1), hippocampus (subiculum and fascia dentate) (Inserts f2 and f3), and striatum (Insert f4) in the Mn + PUN group. Moreover, neurons of the cerebral cortex (Insert g1) and hippocampus (subiculum and fascia dentate) (Inserts g2 and g3) in the Mn Isolated + PUN group also exhibited normal histological architecture. However, there was still slight degeneration in some neurons of the substantia nigra in Mn + PUN group (Insert f5), along with a slight formation of focal fine plaques in the striatal area (Insert g4) and slight degeneration in some neurons of the substantia nigra noted in the Mn Isolated + PUN group (Insert g5).As illustrated in 2 via comparing PD-mediated biochemical, behavioural, and histopathological alterations, as well as outlining the possible molecular mechanisms involved in this relationship. Of particular significance, plant-derived phytochemicals with antioxidant and anti-inflammatory activities have gained much popularity as a potential therapeutic approach against neurodegenerative diseases. This approach targets multiple pathways, and one compound that has attracted considerable attention is PUN. PUN has been revealed to own various health benefits, including antioxidant, anti-inflammatory, and neuroprotective activities [2 in SI rats, focusing on HMGB1/RAGE/TLR4/ NF-ᴋB, NLRP3/Caspase-1, JAK-2/STAT-3, PI3K/AKT/GSK-3β/CREB, AMPK/SIRT-1, Nrf2/HO-1, and PERK/CHOP/Bcl-2 cues.PD is a debilitating neurological condition marked by progressive loss of dopaminergic neurons and accumulation of neurotoxic α-synuclein. This leads to a deficiency of dopamine (DA), which is responsible for motor symptoms . Notablytivities ,68. Cons2 intoxication exhibited more pronounced alterations in spatial working memory and motor activity. This was proven by a deterioration in cognitive function in the Y-maze test, increased latency in the OFT, decreased swimming score, and augmented immobility score in the FST. These observations suggest that the rats experienced negative mood, despair behaviour, bradykinesia, rigidity, motor deficits, and impaired learning and memory functions. Notably, these effects were more pronounced in the SI + MnCl2 group compared to the individual SI or MnCl2 groups. These findings suggested possible synergistic effects between SI and MnCl2 in worsening the behavioural outcomes of PD. The observed behavioural alterations in the SI + MnCl2 group can be attributed to significant depletion in the brain monoamines levels  and marked increment in ACHE activity, together with severe nuclear pyknosis and degeneration observed in the histopathological findings of the cerebral cortex and hippocampus in the SI + MnCl2 group, related to individual SI or PD groups. It is worth noting that these ongoing alterations could be partly attributed to enhanced oxidative stress, neuroinflammation, and neuronal damage in the brains of SI + MnCl2 rats. Disturbances in cholinergic neurotransmission, as indicated by the substantial rise in ACHE activity in the brains of the SI + MnCl2 group, may have a central role in the impaired learning, memory, and locomotor functions observed in this group, where maintaining normal cholinergic neurotransmission system is essential for normal brain functions, particularly locomotor and memory functions. Our results align with the findings of Abu-Elfotuh et al. [2 resulted in hippocampal neuronal cell death and a decline in cognitive, motor, and memory functions, as well as monoamine levels and ACHE activity in rats.Our results demonstrated that rats exposed to concurrent SI and MnClh et al. , where e2-intoxicated groups significantly ameliorated all the pervious alternations, compared to their corresponding control groups . These impacts could be endorsed to PUN’s antioxidant and anti-inflammatory actions [Similarly, the study by Famitafreshi et al. supported our findings, demonstrating the adverse effects of SI on the hippocampus and cerebral cortex, which have a key role in learning, memory, and cognitive functions, as well as dopamine levels ,71. On t actions ,48,49,502 intoxication experienced significant disruption in their redox balance. This was evident through the depletion of antioxidant defense mechanisms, including reduced expression of Nrf2 and HO-1 genes, decreased levels of SOD and TAC, and increased formation of ROS and reactive nitrogen species (RNS), as indicated by elevated iNOS levels and MDA compared to the individual SI or MnCl2 groups. These findings suggested an augmentation between SI and MnCl2 in promoting ROS production and impairing antioxidant defenses. The observed disturbances in redox balance can be attributed to the upregulation of GSK-3β expression in SI, MnCl2, or SI + MnCl2 groups. Enhanced GSK-3β expression can inhibit Nrf2 activation, leading to the downregulation of antioxidant genes including HO-1 and SOD [2 group compared to the respected individual groups. In contrast, treatment with PUN in SI and/or MnCl2-intoxicated groups effectively mitigated these redox imbalances compared to their respective control groups  because of antioxidant properties of PUN [2, or SI + MnCl2 groups. Previous studies further supported our findings which had also demonstrated the notable antioxidant effects of PUN in various diseases [It is interesting to note that PD development involves multiple interconnected processes, for instance, oxidative stress, neuroinflammation, protein aggregation, ER stress, dysregulated autophagy, and apoptosis of dopaminergic neurons ,72,73,74 and SOD . This ins of PUN . Moreovediseases ,77.2, and SI + MnCl2 groups. The initiation of NLRP3 further triggers caspase-1 activation, resulting in the cleavage of pro-IL-1β and the formation of mature IL-1β, thus strengthening the inflammatory response and promoting neuronal cell death [2, and SI + MnCl2 groups. Additionally, the elevation of pro-inflammatory mediators, primarily IL-6, observed in the SI, MnCl2, and SI + MnCl2 groups may induce JAK-2/STAT-3 pathway that involved in neuroinflammatory processes [2, and SI + MnCl2 groups. Interestingly, rats exposed to both SI and MnCl2 intoxication exhibited a more pronounced inflammatory response, as evidenced by increased gene expression of HMGB1, RAGE, TLR4, NF-κB, NLRP3, caspase-1, JAK-2, and STAT-3, along with elevated levels of TNF-α, IL-1β, IL-6, iNOS, COX-2, and PGE-2 compared to the SI or MnCl2 groups alone. These findings highlight the exacerbating effect of SI on neuroinflammation in rats with PD induced by MnCl2.Another significant pathophysiological event implicated in the progression of PD is the triggering of inflammatory signaling pathways, with a particular focus on the HMGB1 pathway. Increased expression of HMGB1 has been connected with the pathogenesis of PD ,79. The ll death ,83, whicrocesses ,85. Uponrocesses ,87, whic2, and SI + MnCl2 groups compared to their respective control groups. These favourable consequences could be attributed to the anti-inflammatory properties of PUN [2, and SI + MnCl2, thereby inhibiting the activation of both RAGE and TLR4 receptors. Furthermore, PUN exhibited antioxidant actions and downregulated the expression of GSK-3β, resulting in the suppression of NF-κB activation. Consequently, NF-κB inhibition by PUN resulted in the suppression of NLRP3 release and the formation of pro-inflammatory mediators. This preventedNLRP3 activation and consequently blocked the activity of caspase-1, thereby reducing the maturation of IL-1β, in agreement with previous studies [On the contrary, the administration of PUN substantially diminished the abnormal activation of the aforementioned proinflammatory and inflammatory pathways in the SI, MnCls of PUN . Notably studies ,90. Addi studies ,92,93. I studies ,88,94.2 intoxication, or SI combined with MnCl2 exhibited a disruption in PI3K/AKT pathway. This disruption was characterized by reduced expressions of PI3K, AKT, CREB, and TrKB, as well as decreased BDNF levels, accompanied by an elevated expression of GSK-3β in these groups.Prior studies have established the association between the dysregulation of the PI3K/AKT signaling pathway and the development of PD ,95. In t2, or SI + MnCl2 groups led to increased expressions of PI3K, AKT, CREB, and TrKB, along with downregulation of GSK-3β expression, in comparison to their corresponding control groups. These influences could be endorsed to the capability of PUN to reverse the repression of the PI3K/AKT pathway induced by SI, MnCl2, or SI + MnCl2. Specifically, the elevation of PI3K phosphorylated AKT, which in turn phosphorylated both GSK-3β and CREB, resulting in the stimulation of CREB and inhibition of GSK-3β activity. Activated CREB, in turn, resulted in the upregulation of BDNF, which interacted with TrKB to promote neuronal plasticity, memory regulation and inhibit neuronal apoptosis [2, or SI + MnCl2 groups.Conversely, administration of PUN to the SI, MnClpoptosis ,96,97. Fpoptosis . It is npoptosis ,99,100. poptosis ,102. The2, or SI + MnCl2 intoxication exhibited sustained ER stress, as corroborated by an increase in ER stress markers, counting the gene expression of PERK, GRP78, and CHOP, relative to the normal control group. These observed changes can be attributed to the capability of SI, MnCl2, or SI + MnCl2 to induce the overproduction of ROS, which provoked the buildup of misfolded proteins, resulting in excessive ER stress in these groups. Specifically, α-syn aggregates-mediated sustained ER stress overactivated UPR that stimulated key sensing proteins, particularly; PERK, through the detachment of GRP78, consequently this severe/prolonged ER stress led to an elevation in the pro-apoptotic factor, CHOP, which initiated the apoptotic cascades, via enhancing caspases cascade and decreasing Bcl-2 [2 intoxication elicited pronounced elevation in ER stress markers and apoptotic markers, compared to that in either SI group or MnCl2 intoxicated group separately.Another mechanistic factor implicated in PD and regulating apoptosis is ER stress. Numerous studies have reported that the buildup of misfolded proteins leading to ER stress is a significant contributor to the development of PD, where excessive or prolonged ER stress had been closely associated with the initiation of neuronal apoptosis ,104,105.ng Bcl-2 ,103,104.ng Bcl-2 ,106,107.ng Bcl-2 ,109. Thi2-induced oxidative stress, which may suppress ROS-mediated abnormal α-syn protein misfolding, resulting in abating SI and/or MnCl2-mediated excessive ER stress markers alternations. In this frame, the depicted abilities of PUN to abrogate SI and/or MnCl2-mediated enhancement in ER stress, oxidative stress, neuroinflammation, and GSK-3β expression collectively resulted in halting SI and/or MnCl2-induced neuronal apoptosis and diminish the apoptosis biomarkers. These results align with previous studies that reported decrement of ER stress markers and apoptotic markers upon treatment with PUN in various diseases [In contrast, PUN, by virtue of its depicted antioxidant properties, demonstrated the ability to mitigate SI and/or MnCldiseases ,111,112.2, or SI + MnCl2 groups, the observed exaggerated ER stress resulted in enhanced neuronal apoptosis and alternation in autophagy and autophagy biomarkers compared to the normal control group. Significantly, SI + MnCl2 group displayed more noticeable impairment in autophagic mechanisms, specifically the prominent suppression of AMPK/SIRT-1/Beclin-1 pathway, compared to either SI or MnCl2 alone groups. In contrast, the administration of PUN to SI, MnCl2, or SI + MnCl2 groups demonstrated a significant improvement in autophagy. These effects could be attributed to PUN’s capability to reverse the repressive effects of SI, MnCl2, or SI + MnCl2 on the AMPK/SIRT-1 pathway. AMPK activation leads to SIRT-1 activation by increasing NAD/NADH ratio, promoting autophagy and upregulated the expression of the autophagy-related gene beclin-1, leading to enhanced clearance of aggregated misfolded α-syn and subsequently suppressing neuronal apoptosis while promoting neuronal survival and improving locomotor function in PD [Apart from inducing neuronal apoptosis, sustained ER stress also contributed to impaired autophagy, leading to disruption of the balance between neuronal apoptosis and autophagy, which is crucial in maintaining homeostasis, and this imbalance, is principally convoluted in the pathogenesis of PD . Indeed,on in PD ,119,120 on in PD . Additioon in PD . It is won in PD ,124. Earon in PD ,119.2 intoxication, which revealed varying degrees of histopathological alterations. These changes included nuclear pyknosis and degeneration in most neurons of the cerebral cortex and fascia dentata in the SI, MnCl2, or SI + MnCl2 groups. The MnCl2-intoxicated group also showed degeneration of some neurons in the subiculum and substantia nigra, while the SI + MnCl2 group exhibited degeneration in all neurons of the substantia nigra and subiculum. Additionally, the striatal areas in the MnCl2 or SI + MnCl2 groups displayed the formation of large eosinophilic plaques, and in SI + MnCl2 group, there was a loss of neurons along with multiple large eosinophilic plaques.Furthermore, it is vital to mention that histopathological findings validated all the aforementioned biochemical results of our study. The investigation of H&E-stained sections from the cerebral cortex, subiculum, fascia dentata, and hilus in the hippocampus, striatum, and substantia nigra regions of rats exposed to SI and/or MnCl2-induced histopathological alterations in all examined brain regions to varying degrees. It also preserved the normal histological picture in the cerebral cortex, hippocampus (subiculum and fascia dentata), and striatum in the SI + PUN group, as well as in the neurons of the cerebral cortex, hippocampus, and striatum in Mn + PUN group. Of note, there were slight degenerative changes in some neurons of the substantia nigra in both Mn + PUN and Mn Isolated + PUN groups, along with the slight formation of focal fine plaques in the striatal area in the Mn Isolated + PUN group.On the other hand, PUN treatment markedly relieved the SI and/or MnClThis investigation studied the impact of SI on the progression of PD by establishing a model of PD with concurrent exposure to SI. The study explored the interconnection between SI and PD and examined its effects on behavioural, biochemical, and histopathological findings; together with outlining the possible molecular contributors underlying this relationship.2 revealed that SI, as a psychological stressor, along with its associated depression, accelerated the progression of PD and worsened its prognosis. The behavioural, neurochemical, and histopathological alterations were more pronounced in the group exposed to SI with MnCl2 compared to the group exposed to MnCl2 alone. SI exacerbated oxidative stress, neuroinflammation, ER stress, and neuronal apoptosis while reducing autophagy in PD rats, compared to either SI or PD solo groups.Co-exposure to SI and MnCl2 intoxication in rats. It exerted antioxidant, anti-inflammatory, and anti-apoptotic properties, improved behavioural disturbances, normalized neurotransmitter levels, and mitigated histopathological changes. Mechanistically, PUN alleviated the progression of PD in rats exposed to SI with MnCl2 primarily by modulating HMGB1/RAGE/TLR4/NF-κB/NLRP3/Caspase-1, JAK-2/STAT-3, PI3K/AKT/GSK-3β/CREB, AMPK/SIRT-1, Nrf2/HO-1, and PERK/CHOP/Bcl-2 pathways .It is worth noting that PUN exhibited promising neuroprotective effects against SI and/or MnClOur study provided new evidence supporting using PUN as a safe, natural, and multitarget neuroprotective approach for halting neuronal damage, neurodegeneration, and enhancing neuronal survival, improving motor functions, and prognosis of PD. However, more pharmacological studies are needed before initiating clinical trials.


Multiple myeloma (MM) is characterized by multiple relapse and, despite the introduction of novel therapies, the disease becomes ultimately drug-resistant. The tumor microenvironment (TME) within the bone marrow niche includes dendritic cells, T-cytotoxic, T-helper, reactive B-lymphoid cells and macrophages, with a complex cross-talk between these cells and the MM tumor cells. Tumor-associated macrophages (TAM) have an important role in the MM pathogenesis, since they could promote plasma cells proliferation and angiogenesis, further supporting MM immune evasion and progression. TAM are polarized towards M1  and M2  subtypes. Many studies demonstrated a correlation between TAM, disease progression, drug-resistance and reduced survival in lymphoproliferative neoplasms, including MM. MM plasma cells in vitro could favor an M2 TAM polarization. Moreover, a possible correlation between the pro-tumor effect of M2 TAM and a reduced sensitivity to proteasome inhibitors and immunomodulatory drugs was hypothesized. Several clinical studies confirmed CD68/CD163 double-positive M2 TAM were associated with increased microvessel density, chemoresistance and reduced survival, independently of the MM stage. This review provided an overview of the biology and clinical relevance of TAM in MM, as well as a comprehensive evaluation of a potential TAM-targeted immunotherapy. Multiple myeloma (MM) represents a frequent hematological disease, characterized by a significant survival improvement during last years, thanks to the availability of novel agents, including proteasome inhibitors , monoclonal antibodies  and immunomodulatory drugs  ,2,3,4. HThe multiple intrinsic and extrinsic mechanisms of drug resistance include the inhibition of drug entrance into neoplastic plasma cells, a drug-efflux pump that reduces drug activity, a mutation in the gene encoding the drug’s target protein and a drug inactivation due to bone marrow (BM) stromal cells which constitute the tumor microenvironment (TME), as illustrated in The TME in the BM includes hematopoietic stem cells, endothelial cells, osteoclasts, osteoblasts, extracellular matrix proteins, mesenchymal stem cells (MSC) and tumor-associated macrophages (TAM) ,25,26,27Macrophages can release pro-inflammatory cytokines, promote the recruitment of leukocytes to the site of inflammation and give a contribution to the tissue reparation and phagocytosis of foreign antigens, such as neoplastic antigens ,32,33. THowever, an elevated macrophage number, as frequently reported in hematologic malignancies, could also contribute to tumor progression by multiple mechanisms, including angiogenesis, the reduction in CD8 T-cell proliferation, the recruitment of T-regulatory cells (T-regs) and the inhibition of apoptosis ,39,40,41The so-called TAM, as “bad guys”, are characterized by a complex interaction with malignant cells for patients with non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) and MM ,55,56,57We suggest TAM represent a subpopulation of macrophages located in the tumor site, which is strongly influenced by cancer cells and TME. TAM originate from circulating monocytes after recruitment at tumor site by cancer cells and progressively acquire pro-tumor properties, making themselves similar to M2 macrophages that are present in the site of injury upon removal of damaged tissue  ,60.In this field, during tissue reparation, there is a transition between classically activated M1 and alternatively activated M2 macrophages, which, in turn, coordinate the proliferation of cell subtypes useful for wound healing, such as vascular endothelial cells and fibroblasts . Tumor cSpecifically, the M1 subtype is activated by granulocyte-monocyte colony-stimulating factor (GM-CSF), interferon (IFN)-γ and bacterial products, while M2 activation is triggered by interleukin (IL)-4, IL-10, IL-13 ,59,60,61However, the separation between M1/M2 subtypes was developed more than 20 years ago and it could represent an oversimplification of a broader spectrum, including at least five subsets: M1, M2a, M2b, M2c and M2d. M2a was induced by IL-4 and/or IL-13 and showed a primary anti-inflammatory and pro-wound healing function. M2b was induced by IL-1b and demonstrated an immuno-regulatory role. Conversely, M2c were induced by IL-10 and showed an increased expression of tissue remodeling and immune suppressive markers. Finally, M2d subtype could be induced by IL-6 and could express angiogenic markers .To exert their action, TAM have to be recruited within TME; the recruitment is mediated by chemokines, including CCL2, CCL5, CCL7 and CXCL1, vascular endothelial growth factor (VEGF) and M-CSF ,64,65,66This review illustrates the manuscripts associated with the influence of TAM, with a particular focus on M2 subtype, in MM pathophysiology, proliferation, disease progression and drug resistance. Finally, we will also provide a summary of the possible use of TAM as a therapeutic target.We performed a computerized search in MEDLINE to find publications as full-text, written in English, focused on the relationship between TAM and MM. The key terms of our search included “tumor-associated macrophages OR TAM OR M1 macrophages OR M2 macrophages OR CD68 OR CD163 OR CD204 OR CD206 AND multiple myeloma.”We also searched in the reference list of selected articles to perform a more comprehensive research. We excluded conference abstract and case reports, but we included retrospective studies. For each preclinical and clinical study, we extrapolated: methods of TAM determination, TAM markers, patient number , treatment schedule and the relationship between TAM, drug resistance and disease outcome, especially PFS and OS.TAM play a key role in MM pathophysiology, since they represent up to 10% of the BM cells of MM cases and could support disease progression, immune evasion, angiogenesis and drug resistance with multiple mechanisms, as represented in Immunohistochemical (IHC) studies showed an inconsistent evidence that total TAM number was increased in the BM of MM-bearing mice, if compared to monoclonal gammopathy of uncertain significance (MGUS) . ConversMM cells are known to secrete chemotactic factors that could influence the monocytes migration into the BM ,79,80. IA cornerstone for MM progression is represented by the initial homing and subsequent establishment within the BM of malignant PC . In vitrChemotactic molecules produced by TAM, such as IL-8, CCL2, CCL3 and (IGF)-1, play a relevant role in PC migration and homing ,91,92,93The role of IL-6 as PC growth factor was confirmed in a study in which the presence of IL-6 blocking antibody could reduce the proliferation index of co-cultured MM cells; interestingly, this effect was more pronounced in the presence of MSC and/or TAM . Further+ pro-angiogenic macrophages was associated with increased angiogenesis and disease progression in the Vk*Myc murine MM model [Access to vascular structures by proximity is critical to guarantee to MM cells an adequate supply of oxygen and nutrients. A growing evidence suggests TAM, within BM TME, could play a pro-angiogenic role through vascular mimicry and VEGF production, synergizing with the angiogenic properties of malignant PC . In a moMM model ,103.In the MM nude mouse subcutaneous xenograft model, TAM removal from the TME by using clodronate-liposome hindered tumor growth . MoreoveIn several reports, during progression from MGUS to MM, M2 TAM could direct angiogenesis through its expression and production of VEGF, the main angiogenic player ,106,107.In another study, TAM showed the capability to secrete IL-10, that could induce both PC proliferation and angiogenesis .TAM synthesize several angiogenic factors other than VEGF, such as fibroblast growth factor (FGF)2, inducible nitric oxide synthase (iNOS), IL-8 and TNFα ,107. TheOverall, these findings indicate TAM as a relevant component of the MM-associated pro-angiogenic network.+ T cell response, further supporting the hypothesis that M2 TAM, conversely, play a relevant role in the decreased T cell activation and resulting immunosuppression reported in MM, ultimately leading to disease progression [In vivo, the administration of the anti CSF-1 receptor antibody CS7 significantly reduced the MM tumor burden, while the in vitro administration of low-dose CS7 could contribute to TAM polarization towards an M1 subtype . M1 TAM gression .Macrophages, as a member of the innate immune system, could directly influence the development of an immunosuppressive TME in MM ,33. TAM + PC of the immune check protein CD47, which inhibits the macrophages-mediated phagocytosis of neoplastic cells by binding to SIRPα , was very elevated and was associated with disease stage [+ T cells and programmed death receptor ligand 1 (PD-L1) expression on MM cells [Notably, the expression on MM CD138se stage ,74. In vse stage ,114. In MM cells ,110.An in vitro suppression of T cell proliferation was obtained when MM cells were co-cultured with macrophages, further confirming MM cells could cross-talk with macrophages and polarize TAM towards an M2 immunosuppressive phenotype . TAM couAnother recently discovered mechanism is represented by the production of IL-32 by MM cells, which, in turn, increased production by TAM of indoleamine 2,3-dioxygenase (IDO), a molecule with a well-known inhibitory effect on T cells .The tendency to develop drug resistance towards anti-MM drugs represents an unmet medical need for MM patients and a relevant role for TME, including TAM, was demonstrated during the last few years ,33,116.The resistance to the alkylating agent melphalan was mediated by the interaction between intracellular adhesion molecule-1 (ICAM-1) and P-selectin glycoprotein ligand-1 (PSGL-1) on MM cells and P-selectin and CD18 on TAM ,118. TheTAM could enable drug resistance towards bortezomib through the production of IL-1β, which in turn increased the number of MM-initiating cells . In addiTotal TAM, determined as CD68-positive macrophages, were shown to inhibit in a pre-clinical model drug-induced apo ptosis by caspase 3 and poly-ADP ribose polymerase (PARP) cleavage ,118. RecSeveral studies confirmed the prognostic relevance of TAM for MM patients, especially CD68/CD163 double-positive M2 TAM, as illustrated in The difference of macrophage involvement in MM cases with different prognosis was initially demonstrated using CD68 as a single marker ,123. In Subsequently, in 198 MM patients receiving bortezomib-based regimens, CD163 expression was assessed by IHC as macrophage marker with the used cut-off of >55/high power field . MM patiThe clinical impact of different TAM subtypes by IHC was illustrated in a study in which total TAM were considered as CD68-positive and subclassified as M1 TAM  or M2 TAM  . In thisp = 0.02), further confirming an adverse prognostic influence for CD163-positive TAM [These results were confirmed in another study, in which the authors reported low M1 TAM infiltration, assessed by the concurrent expression of CD68 and C-C chemokine receptor 2 (CCR2) by flow cytometry, was correlated with an unsatisfactory response to bortezomib . In additive TAM .As MM PC, but not normal PC, express CD47 to evade a macrophage-mediated phagocytosis, its expression was associated with disease progression from MGUS to MM ,74. The The above-mentioned clinical relevance of TAM for MM patients led to translational research with the aim to discover the underlying mechanisms, as illustrated in The close interaction between TAM and MM cells could represent the basis to design targeted therapies and to overcome the well-known TAM-mediated drug resistance ,149,150.An important pathway for TAM recruitment and differentiation is represented by the CSF-1R signaling . CSF-1R Controlling monocyte recruitment to the TME could represent a promising mechanism to reduce TAM infiltration and it is regulated by citokines and chemokines produced by tumor cells and stromal cells. Since monocytes trafficking is regulated by the CCL2-CCR2 signaling axis, its blockade could reduce tumor proliferation in solid malignancies . CCL2 shCXCL12-CXCR4 represents another mechanism to recruit macrophage towards the MM BM ,132. MM The administration of clodronate-liposome demonstrated the ability to cause a global macrophage depletion in solid and hematologic malignancies ,27,88. I+ T-cell response [In another in vivo study in a murine MM model, the therapeutic effects of CSF-1R blocking monoclonal antibodies were investigated. Specifically, CSF-1R blockade could inhibit the M2 TAM proliferation and differentiation and repolarize TAM towards an antitumor M1 subtype . Notablyresponse . Antitumresponse . As muriresponse . After tresponse ,134.In addition, due to the BMI1 protein, released by MM cells, could promote TAM proliferation, angiogenesis and drug resistance, its inhibitor PTC596 was investigated in a murine MM model. This agent was able to reduced MM tumor burden and improve mice survival by depleting M2 TAM .In our opinion, TAM depletion, especially by using biphosphonates, could represent a promising therapy for MM patients. In particular, biphosphonates are already used in clinical daily practice to prevent skeletal complications in MM cases . These mIn the above-mentioned study, the potential therapeutic effect of an anti-CSF-1R monoclonal antibody was investigated. In vivo, CSF-1R blockade could inhibit MM growth by both depleting and polarizing TAM towards the M1 subtype .In a preclinical study, the JAK1/2 inhibitor ruxolitinib demonstrated the ability to suppress M2 TAM through the reduction in the tribbles homolog 1 protein kinase expression . In addiAnother promising target is represented by CD40, a cell surface costimulatory protein expressed on antigen-presenting cells (APC) and necessary for their activation. Agonistic antibodies were able to stimulate innate and adaptive immune response in cancer patients. A preclinical study in MM observed a repolarizing effect on TAM after sequential CD40 activation together with Toll-like receptor (TLR) ligation .TAM reprogramming aims to reduce the immunosuppressive M2 subtype, while promoting the immunostimulatory M1 subtype and represents a promising research field for MM therapy . InteresIn a pivotal paper, M1 TAM and M2 TAM, when co-cultured with MM cells, could play an antitumor and a protumor role, respectively. A double treatment with GM-CSF, a pro-M1 cytokine, in association with an inhibitor of the pro-M2 cytokine macrophage migration inhibitory factor (MIF) was performed . This coFinally, a relevant role was discovered for the IL-10/IL-10R pathway. Due to IL-10 being released by MM cells and potentially polarizing TAM towards an M2 subtype, the inhibition of IL-10/IL-10R pathway by using an anti-IL-10R blocking antibody could reprogram TAM to lose the M2 phenotype . The finIn our opinion, TAM reprogramming represents an interesting research field for MM patients. Unfortunately, the anti-CD40 agonistic monoclonal antibody mitazalimab showed only a modest activity in patients with solid neoplasms with moderate toxicity in a phase I study . Convers+ T-cell response and increased the production of anti-inflammatory citokines. Another study used an IDO inhibitor, called D,L-1methyl-tryptophan, in patient MM cells and reported the reversal of T-regs proliferation and the improvement of Th1 immune response [Due to TAM significantly contributing to immunosuppression and reducing T-cell response, the restoration of an adequate immune response through targeting TAM represents an interesting strategy. Notably, the lack of effect of several drugs in MM could be due to their effect on TME ,27. Cladresponse .Immune checkpoints represent inhibitory mechanisms frequently used by tumor cells to escape from recognition and killing by cells of the host immune system. Anti PD-1/PD-L1 monoclonal antibodies demonstrated high efficacy in HL treatment, but treatment results were disappointing in MM ,144. TheSince hematological malignancies, including MM, express high CD47 levels, preclinical research demonstrated that an antibody-mediated blockade of CD47-SIRPα signaling could promote tumor cell death, phagocytosis and improve T-cell response ,127,128.In addition, miR-155 was expressed at low levels in drug resistant MM cells and could directly regulate CD47 through its 3′UTR. An overexpression of miR-155 could suppress CD47 expression on MM cells surface, leading to the phagocytosis of MM cells by macrophages and the induction of apoptosis through targeting TNF AIP8 in vitro and in vivo .As previously illustrated, the JAK2 inhibitor ruxolitinib was able to reduce M2 TAM polarization in MM both in vitro and in vivo . InteresAnother strategy to overcome the bortezomib resistance was to target BAFF by neutralizing antibodies. In a xenograft model, anti-BAFF antibody in association with bortezomib showed a significantly delayed tumor growth and progression if compared to bortezomib as single-agent .A growing evidence was reported to indicate that TAM could contribute to MM drug resistance to several agents, including bortezomib, melphalan and lenalidomide ,26,27. AICAM-1 expressed on MM cells interacts with CD18 on TAM surface and plays a relevant role to confer drug resistance to MM cells . BI-505,Overall, there are limited available data about the role of TME, including TAM, in the natural history of MM. The adverse ISS stage, together with high-risk cytogenetics, was associated with distinct immune profiles in a cohort of MM cases receiving bortezomib, lenalidomide and dexamethasone (VRD) . In thisLenalidomide demonstrated the ability to drive TAM towards an immunostimulatory M1 subtype through the cereblon-CRL4 E3 ligase to degrade by ubiquitination the transcription factor IKAROS family zinc finger 1 (IKZF1) . LenalidDue to preclinical studies demonstrated CD47 blockade could induce TAM activation, resulting in MM cells death, the anti CD47 magrolimab could be used as single-agent or in combination with other drugs in MM. Magrolimab in association with commonly used anti MM therapies is under investigation in an ongoing phase II study .In recent years, the introduction of anti CD38 monoclonal antibodies daratumumab and isatuximab dramatically changed MM treatment landscape ,156,157.Finally, TAM influence in the response to antibody-drug conjugates, chimeric antigen receptor T cells (CAR-T) and Bi specific T-cell engagers is currently under investigation. In a recently published preclinical paper , RO72970The treatment landscape of MM was revolutionized in recent years and the modulation of TME, including TAM, could represent a promising strategy to overcome MM drug resistance, in association with conventional and/or novel agents. We summarized the critical TAM influence in MM pathophysiology, disease progression, immunosuppression and drug resistance. Specifically, M2 TAM, which are characterized by immunosuppressive phenotype, were associated with reduced PFS and OS for MM patients. However, most of the available studies were preclinical and a great effort will be necessary to bring these findings from bench to bedside, with the aim to design safe and effective therapies for R/R MM cases.Interestingly, a TAM-targeted treatment strategy could be associated with checkpoint inhibitors, CAR-T cells and/or Bi specific T-cell engagers, with the aim to reduce TAM-mediated immunosuppression.In conclusion, TAM could represent an interesting target for future therapies, to counteract the frequently observed drug resistance in R/R MM cases, but many issues still need to be explored, including understanding the molecular mechanisms regulating cross talk between TAM, MM cells and other elements within TME. Finally, these promising results have to be validated in clinical studies, with the aim to give to each patients an increasingly personalized treatment strategy.


Background: Tocotrienol, a type of vitamin E, is well known for its anti-cancer and other biological activities. This systematic review aims to summarize the involvement of endoplasmic reticulum stress (ERS) and subsequent unfolded protein response (UPR) as the underlying molecular mechanisms for the anticancer properties of tocotrienol. Method: A comprehensive literature search was performed in March 2023 using the PubMed, Scopus, Web of Science, and EMBASE databases. In vitro, in vivo, and human studies were considered. Result: A total of 840 articles were retrieved during the initial search, and 11 articles that fit the selection criteria were included for qualitative analysis. The current mechanistic findings are based solely on in vitro studies. Tocotrienol induces cancer cell growth arrest, autophagy, and cell death primarily through apoptosis but also through paraptosis-like cell death. Tocotrienol-rich fractions, including α-, γ- and δ-tocotrienols, induce ERS, as evidenced by upregulation of UPR markers and/or ERS-related apoptosis markers. Early endoplasmic reticulum calcium ion release, increased ceramide level, proteasomal inhibition, and upregulation of microRNA-190b were suggested to be essential in modulating tocotrienol-mediated ERS/UPR transduction. Nevertheless, the upstream molecular mechanism of tocotrienol-induced ERS is largely unknown. Conclusion: ERS and UPR are essential in modulating tocotrienol-mediated anti-cancer effects. Further investigation is needed to elucidate the upstream molecular mechanism of tocotrienol-mediated ERS. The endoplasmic reticulum (ER) is one of the essential intracellular organelles. It can be divided into rough and smooth ER, depending on the presence of ribosomes on the outer surface. Rough ER synthesizes proteins and subjects them to folding and post-translational modification . ProteinThere are three major UPR downstream pathways, which involve protein kinase-like ER kinase (PERK), inositol-requiring element 1 , and activating transcription factor 6 α type (ATF6α) as ERS sensors or UPR markers  3]. In . In 3]. The activated PERK, on the other hand, phosphorylates downstream targets such as eukaryotic initiation factor 2 α subunit (eIF2α) to suppress global native protein synthesis . At the On the other hand, unbounded ATF6α is dissociated from ER and then translocated into the Golgi apparatus to be cleaved into a smaller cytosolic N-terminal ATF6 fragment (ATF6f) ,5,14. ATBioactive compounds from natural resources possess great potential in novel drug design and development. Tocotrienol and tocopherol are two natural vitamin E families. Structurally, tocopherol and tocotrienol consist of a chromanol ring and a carbon tail. The distinction is that tocopherol has a saturated hydrocarbon phytyl tail and tocotrienol has an unsaturated farnesyl tail with three carbon-carbon double bonds ,17,18. TTocotrienol demonstrates broad biological activities, including antioxidant, anti-inflammatory, cardioprotective, neuroprotective, anti-metabolic, anti-osteoporotic, anti-rheumatic, and anti-diabetic properties ,24,25,26https://doi.org/10.17605/OSF.IO/B4KAQ, registered on 15 March 2023). In addition, four databases, including PubMed, Scopus, Web of Science, and EMBASE, were used for literature searching in March 2023. The following search terms were used: (1) (“vitamin E” OR tocotrienol) and (2) (“endoplasmic reticulum stress” OR ERS OR “unfolded protein response” OR UPR). Additional records were identified manually from the reference list of included research articles or relevant review articles. The detailed search strategy is listed in This systematic review was conducted according to preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines  40]. Th. Th40]. The inclusion criteria for this systematic review were studies that reported tocotrienol-mediated ERS or UPR and the associated molecular mechanism. There is no restriction on the year of publication or study model . The exclusion criteria were: (1) articles not written in English; (2) articles without primary data; (3) studies using semi-synthesized or modified tocotrienol; and (4) combinational therapy, whereby the effects of tocotrienol treatment alone could not be delineated. The article identification and screening process was summarized in Two independent reviewers (K.-L.P. and C.-W.M.) extracted the data and performed the screening of the titles and abstracts, followed by the screening of the full-text articles. Inclusion and exclusion criteria were applied, and any discrepancy between reviewers was resolved by discussing it with the third author (K.-Y.C.). The extracted information was summarized in an evidence table , includiTwo independent reviewers (K.-L.P. and C.-W.M.) evaluated the quality of included articles by using the Office of Health Assessment and Translation (OHAT) risk of bias tool for non-human studies . Nine don = 2), lack of primary data (n = 43), and being irrelevant to the topic (n = 448). After assessing the eligibility of the full-text articles, 12 articles were excluded because 10 were not studying tocotrienol and 2 were using combination treatment, and the effects of tocotrienol treatment could not be determined. Two additional articles were included, and a final 11 articles were included for qualitative analysis in this systematic review.A total of 840 records  were identified. After removing the duplicates, 514 articles were screened for titles and abstracts. A total of 493 articles were excluded due to language   or tocotSeveral ERS inducers were used in previous studies as comparative controls, including tunicamycin ,46,50, tFrom the included articles, tocotrienols demonstrated superior selectivity on cancerous cells and were not toxic to several non-cancerous cells within the anticancer concentration range, including human mammary tissue MCF-10A ,49, humaCHOP and IRE1 mRNA and demonstrated the nuclear translocation of CHOP and ATF4 proteins [Tocotrienol-mediated ERS was demonstrated by activation of caspase-4  or caspaproteins .MAPK signaling, including JNK and p38 MAPK, was activated after tocotrienol-mediated ERS. Tocotrienol activated p38 MAPK ,49,52 anThe upstream molecular mechanism of tocotrienol-induced ERS is largely unknown. Comitato et al. demonstrated α-, δ- & γ-tocotrienols induced an early ER calcium ion release into the cytoplasm as early as 15 min, which serves as the early signal in triggering ERS . The relThe current finding summarizes the anticancer properties of tocotrienol by triggering ERS, UPR, and ERS-related apoptosis or autophagy on several cancerous cells. Interestingly, tocotrienol-mediated ERS and ERS-mediated apoptosis were not dependent on HMG CoA reductase inhibition ,48. ThisAdditionally, δ-tocotrienol also induced prostate tumor cell paraptosis with extensive cytoplasmic vacuolation . A similTocotrienol, especially γ-tocotrienol, specifically induced the extrinsic pathway of apoptosis, as evidenced by the upregulation of death receptors ,45,47. TFurthermore, tocotrienol was reported to activate some but not all ERS sensors or UPR markers. Wali et al. demonstrated that γ-tocotrienol  significantly increased the phosphorylated PERK and ATF6 degradation in +SA cells . A 24h γATF3, CHOP, PERK, and GRP78 [Several transcriptomic analyses revealed the involvement of ERS and/or UPR response pathways upon tocotrienol treatment ,46,66. γnd GRP78 . Similarnd GRP78 ,67. Addind GRP78 . ER diland GRP78 ,67. Somend GRP78 ,51,52 onMicroRNAs are a type of short noncoding RNAs (~22 nucleotides) important in regulating cellular response and function, including UPR ,70. SeveThe upstream molecular mechanism of tocotrienol-induced ERS, including the activation of PERK and IRE1, is largely unknown. All the included studies only demonstrated the activation of UPR markers; the accumulation of misfolded or unfolded protein upon tocotrienol treatment has not been determined. In addition, how tocotrienol treatment triggers the activation of UPR markers is not fully understood. Tocotrienol induces very early ER calcium ion release, which suggests that it serves as an early signal in triggering ERS . GlucoseOn the other hand, oxidative stress is known to cause ERS by directly causing oxidative damage to proteins, thereby compromising the ER protein folding process and causing the accumulation of unfolded or misfolded proteins ,85. In aOn the other hand, tocopherol ,55,91,92There are several limitations to this systematic review. The reproducibility of data is of concern, whereby some of the included studies did not disclose the tocotrienol stock solution preparation ,52 and dThe currently available evidence has consistently demonstrated ERS-inducing properties of tocotrienols via the upregulation or activation of ERS sensors or UPR markers. Tocotrienol-induced ERS was crucial in the subsequent ERS-mediated apoptosis. Nevertheless, the upstream molecular mechanisms of tocotrienols in triggering ERS are largely unknown. Several events, such as ER calcium release, ceramide and/or dihydroceramide upregulation, proteasomal inhibition, and miR-190b upregulation, were suggested to serve as the early events in modulating or triggering the ERS. However, the protein folding status and the precise mechanism of action of tocotrienol have not been determined. Further studies are required to elucidate the molecular mechanism and the upstream molecular targets in tocotrienol-mediated ERS, wherein these mechanistic findings will be essential in developing tocotrienol or chemical derivatives in personalized medicine.


A higher EMP 1 H-score was observed in pheochromocytomas with a GAPP score ≥3 (p = 0.018). In univariate analysis, high levels of EMP 1 and EMP 3 expression in ACN were associated with shorter overall survival (p = 0.001). Differences were observed in the expression of EMPs between ACN and pheochromocytoma. EMPs are associated with malignant tumor biology in adrenal cortical neoplasm and pheochromocytoma, suggesting the role of a prognostic and/or predictive factor for EMPs in adrenal tumor.The purpose of this study is to investigate the expression of the epithelial membrane proteins (EMP) 1, 2, and 3 in adrenal gland neoplasm and to explore the broader implications of this. Tissue microarrays were constructed for 132 cases of adrenal cortical neoplasms (ACN) , and carcinoma (17 cases)) and 189 cases of pheochromocytoma. Immunohistochemical staining was performed to identify EMP 1, 2, and 3, and was compared with clinicopathological parameters. The H-score of EMP 3 ( Adrenal gland neoplasms primarily encompass adrenal cortical neoplasms (ACNs) and pheochromocytomas (PCCs), localized in the adrenal cortex and adrenal medulla, respectively. ACNs, comprising adrenal cortical adenomas (ACAs) and adrenal cortical carcinomas (ACCs), are relatively uncommon tumors, and distinguishing between them histologically presents challenges . ACCs, cPCC is commonly discovered incidentally through health screenings, accounting for approximately 20–60% of cases, and it represents about 4–8% of all adrenal incidentalomas ,16. PrevEpithelial membrane proteins (EMPs) 1, 2, and 3 are members of the myelin protein 22-kDa (PMP22) gene family. While they primarily function in the peripheral nervous system, their distinct roles in various tumors have been documented ,28. EMP p < 0.001). Additionally, ACC displayed features such as diffuse architecture, necrosis, and invasion of venous/sinusoidal structures and capsules. Although two ACC cases had Weiss scores of 4 or less, they were diagnosed as ACC due to the presence of distant metastasis at the time of diagnosis. Notably, tumor recurrence, distant metastasis, and patient mortality were exclusively observed in ACC cases. Regarding PCC, the GAPP score ranged from 0 to 2 in 138 cases (73.0%), 3 to 6 in 50 cases (26.5%), and 7 to 10 in 1 case (0.5%). Tumor recurrence occurred in 5 cases (2.6%), distant metastasis in 7 cases (3.7%), and patient death in 11 cases (5.8%).The basal characteristics of the patients included are presented in The immunohistochemistry results for EMP 1, 2, and 3, including H-scores, are shown in p < 0.001). Specifically, PCC exhibited significantly higher H-scores compared to ACN (p < 0.001) and EMP 3 (p < 0.001), with ACC showing higher H-scores than ACA (p < 0.001) and EMP 3 (p < 0.001). Notably, ACC exhibited a higher proportion of high expression compared to ACA , and the norepinephrine type was associated with high expression levels of EMP 1 (p < 0.001), atypical mitosis (p < 0.001), clear-cell proportion (p < 0.001), diffuse architecture proportion (p < 0.001), necrosis (p < 0.001), and the Weiss score (p < 0.001). High expression levels of EMP 1 and 3 were associated with higher levels of mitosis, atypical mitosis, lower clear-cell proportions, higher diffuse architecture proportions, an increase in necrosis, and higher Weiss scores when compared to that of low levels of EMP 1 and 3 expressions  was the only factor that showed a significant association with shorter OS  is a likely tumor-suppressor gene, because of its genomic deletion in the 19q13 chromosomal region in neural origin tumors, such as neuroblastoma and glial tumors ,75. FurtIn PCC, there was a significant increase in the expression of EMP 1 when the GAPP score was three or higher. EMP 1 expression is commonly detected in early and immature neurons, indicating its potential association with neurogenesis during the development of both the central and peripheral nervous systems . Since ap-Akt (Ser473), a molecule related to the PI3K/AKT pathway, than that of ACA and normal tissue [Our results suggest that EMP 1 and 3 may contribute to the oncogenic role in malignant tumors in ACN, as they showed significantly higher expression in ACC than in ACA. As a possible mechanism of action for EMP 1 and 3 in ACC, cross-talk with the ErbB family receptors , ErbB-2 (HER2), ErbB-3 (HER3), and ErbB-4 (HER4)) may exist. Previous studies have reported functional interactions between EM 3 and HER-2 in urothelial and breast cancer ,43,88, al tissue ,94. Sincl tissue ,32,33, tl tissue  and pedil tissue . Furtherl tissue , breast l tissue , gastricl tissue , and urol tissue , while tl tissue . TherefoThis study suggests that EMP may be a therapeutic target for adrenal neoplasms such as ACC and PCC. Previous studies have demonstrated the inhibitory effects of anti-EMP 2 recombinant bivalent antibody fragments (diabodies) on the proliferation and induction of apoptosis in uterine endometrial and ovarian cancer ,98. MoreIn conclusion, EMP expression levels were shown to be significantly different between ACN and PCC, and EMPs are associated with malignant tumor biology in adrenal cortical neoplasm and pheochromocytoma, suggesting the role of a prognostic and/or predictive factor for EMPs in adrenal tumor.The focus of this research was on individuals who underwent surgery for ACC and PCC between January 2000 and December 2012 at Severance Hospital. The study did not include individuals who underwent preoperative chemotherapy. The Institutional Review Board of Yonsei University Severance Hospital granted approval for the study (IRB number: 4-2021-0393). Endocrine pathologists (Koo JS) retrospectively reviewed all cases, conducting histology using hematoxylin and eosin (H&E)-stained slides. Clinicopathological data, such as age at diagnosis, disease recurrence, metastasis, current status, and length of follow-up, were extracted from the patients’ medical records.After careful selection of representative regions on H&E-stained slides, corresponding spots were identified on the surface of the corresponding paraffin block. Core biopsies measuring 5 mm were then obtained from the chosen regions and placed into a recipient block measuring 5 × 4. To reduce extraction bias, more than two tissue cores were taken from each case. Each tissue core was assigned a distinct microarray location number, which was linked to a comprehensive database containing additional clinicopathological information.In this research, immunohistochemistry (IHC) was carried out utilizing formalin-fixed, paraffin-embedded tissue sections. Tissue sections measuring 3 μm in thickness were prepared from paraffin blocks. These sections underwent a process of deparaffinization and rehydration using xylene and alcohol solutions. For antigen retrieval, cell conditioning 1 (CC1) buffer  was employed. IHC staining was conducted using the Ventana Discovery XT automated stainer , which included appropriate positive and negative controls. The antibodies employed for IHC in this study are listed in Immunohistochemical markers were visualized using light microscopy. The expression of these markers was assessed using the semi-quantitative H-score method and evaluated in tumor cells. The H-score system generates a comprehensive range from 0 to 300, which is determined by multiplying the dominant staining intensity score  by the percentage (0–100%) of positive cells . If the t-test and Fisher’s exact test were employed for continuous and categorical variables, respectively. In the case of analyzing data with multiple comparisons, a corrected p-value was calculated using the Bonferroni multiple comparison procedure. Statistical differences were considered significant if p < 0.05. Kaplan–Meier survival curves and log-rank statistics were employed to evaluate tumor recurrence time and overall survival (OS). Additionally, the Cox proportional hazards model was utilized to perform multivariate regression analysis.The Statistical Package for the Social Sciences (SPSS) for Windows, Version 24.0  was utilized for data analysis. To determine statistical significance, Student’s