Macrophage Migration Inhibitory Factor (MIF) and D-Dopachrome Tautomerase (DDT): Pathways to Tumorigenesis and Therapeutic Opportunities.

Discovered as inflammatory cytokines, MIF and DDT exhibit widespread expression and have emerged as critical mediators in the response to infection, inflammation, and more recently, in cancer. In this comprehensive review, we provide details on their structures, binding partners, regulatory mechanisms, and roles in cancer. We also elaborate on their significant impact in driving tumorigenesis across various cancer types, supported by extensive in vitro, in vivo, bioinformatic, and clinical studies. To date, only a limited number of clinical trials have explored MIF as a therapeutic target in cancer patients, and DDT has not been evaluated. The ongoing pursuit of optimal strategies for targeting MIF and DDT highlights their potential as promising antitumor candidates. Dual inhibition of MIF and DDT may allow for the most effective suppression of canonical and non-canonical signaling pathways, warranting further investigations and clinical exploration.

MIF inhibition attenuates intervertebral disc degeneration by reducing nucleus pulposus cell apoptosis and inflammation.

Nucleus pulposus cells (NPCs) apoptosis and inflammation are the extremely critical factors of intervertebral disc degeneration (IVDD). Nevertheless, the underlying procedure remains mysterious. Macrophage migration inhibitory factor (MIF) is a cytokine that promotes inflammation and has been demonstrated to have a significant impact on apoptosis and inflammation. For this research, we employed a model of NPCs degeneration stimulated by lipopolysaccharides (LPS) and a rat acupuncture IVDD model to examine the role of MIF in vitro and in vivo, respectively. Initially, we verified that there was a significant rise of MIF expression in the NP tissues of individuals with IVDD, as well as in rat models of IVDD. Furthermore, this augmented expression of MIF was similarly evident in degenerated NPCs. Afterwards, it was discovered that ISO-1, a MIF inhibitor, effectively decreased the quantity of cells undergoing apoptosis and inhibited the release of inflammatory molecules (TNF-α, IL-1ß, IL-6). Furthermore, it has been shown that the PI3K/Akt pathway plays a vital part in the regulation of NPCs degeneration by MIF. Ultimately, we showcased that the IVDD process was impacted by the MIF inhibitor in the rat model. In summary, our experimental results substantiate the significant involvement of MIF in the degeneration of NPCs, and inhibiting MIF activity can effectively mitigate IVDD.

MIF-Modulated Spinal Proteins Associated with Persistent Bladder Pain: A Proteomics Study.

Bladder pain is a prominent symptom in Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS). We studied spinal mechanisms of bladder pain in mice using a model where repeated activation of intravesical Protease Activated Receptor-4 (PAR4) results in persistent bladder hyperalgesia (BHA) with little or no bladder inflammation. Persistent BHA is mediated by spinal macrophage migration inhibitory factor (MIF), and is associated with changes in lumbosacral proteomics. We investigated the contribution of individual spinal MIF receptors to persistent bladder pain as well as the spinal proteomics changes associated with relief of persistent BHA by spinal MIF antagonism. Female mice with persistent BHA received either intrathecal (i.t.) MIF monoclonal antibodies (mAb) or mouse IgG1 (isotype control antibody). MIF antagonism temporarily reversed persistent BHA (peak effect: 2 h), while control IgG1 had no effect. Moreover, i.t. antagonism of the MIF receptors CD74 and C-X-C chemokine receptor type 4 (CXCR4) partially reversed persistent BHA. For proteomics experiments, four separate groups of mice received either repeated intravesical scrambled peptide and sham i.t. injection (control, no pain group) or repeated intravesical PAR4 and: sham i.t.; isotype IgG1 i.t. (15 µg); or MIF mAb (15 µg). L6-S1 spinal segments were excised 2 h post-injection and examined for proteomics changes using LC-MS/MS. Unbiased proteomics analysis identified and relatively quantified 6739 proteins. We selected proteins that showed significant changes compared to control (no pain group) after intravesical PAR4 (sham or IgG i.t. treatment) and showed no significant change after i.t. MIF antagonism. Six proteins decreased during persistent BHA (V-set transmembrane domain-containing protein 2-like confirmed by immunohistochemistry), while two proteins increased. Spinal MIF antagonism reversed protein changes. Therefore, spinal MIF and MIF receptors mediate persistent BHA and changes in specific spinal proteins. These novel MIF-modulated spinal proteins represent possible new targets to disrupt spinal mechanisms that mediate persistent bladder pain.

Thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione Derivative Inhibits d-Dopachrome Tautomerase Activity and Suppresses the Proliferation of Non-Small Cell Lung Cancer Cells.

The homologous cytokines macrophage migration inhibitory factor (MIF) and d-dopachrome tautomerase (d-DT or MIF2) play key roles in cancers. Molecules binding to the MIF tautomerase active site interfere with its biological activity. In contrast, the lack of potent MIF2 inhibitors hinders the exploration of MIF2 as a drug target. In this work, screening of a focused compound collection enabled the identification of a MIF2 tautomerase inhibitor R110. Subsequent optimization provided inhibitor 5d with an IC50 of 1.0 µM for MIF2 tautomerase activity and a high selectivity over MIF. 5d suppressed the proliferation of non-small cell lung cancer cells in two-dimensional (2D) and three-dimensional (3D) cell cultures, which can be explained by the induction of cell cycle arrest via deactivation of the mitogen-activated protein kinase (MAPK) pathway. Thus, we discovered and characterized MIF2 inhibitors (5d) with improved antiproliferative activity in cellular models systems, which indicates the potential of targeting MIF2 in cancer treatment.

Repurposing old drugs as novel inhibitors of human MIF from structural and functional analysis.

Human macrophage migration inhibitory factor (MIF) is an important pro-inflammatory cytokine that plays multiple pleiotropic functions. It is considered as a promising therapeutic target for the infectious, autoimmune, and cardiovascular diseases and cancers. The development of MIF inhibitors has not been translated into clinical success despite decades of research. Given the time and cost of developing new drugs, existing drugs with clarified safety and pharmacokinetics are explored for their potential as novel MIF inhibitors. This study identified five known drugs that could inhibit MIF's tautomerase activity and MIF-mediated cell chemotaxis in RAW264.7 cells. It was found that compounds D2 (histamine), D5 (metaraminol), and D8 (nebivolol) exhibited micromolar-range inhibition potency close to the positive control ISO-1. Kinetics and the mechanism for inhibition were subsequently determined. Moreover, the detailed inhibitor-binding patterns were investigated by X-ray crystallography, computational molecular docking, and structure-based analysis. Therefore, this study elucidates the molecular mechanism of repurposed drugs acting on MIF and provides a structural foundation for lead optimization to promote the clinical development of MIF-targeted drugs.

HDAC/MIF dual inhibitor inhibits NSCLC cell survival and proliferation by blocking the AKT pathway.

Non-small-cell lung carcinoma (NSCLC) is one of the most common forms of lung cancer, and a leading cause of cancer death among human beings. There is an urgent demand for novel therapeutics for the treatment of NSCLC to enhance the efficacy of the currently applied Tyrosine kinase inhibitors (TKIs) therapy and to overcome therapy-resistance. Here, we report a novel small-molecule inhibitor that simultaneously targets histone deacetylase (HDAC) and macrophage migration inhibitory factor (MIF). The HDAC/MIF dual inhibitor proved to be toxic for EGFR mutated (H1650, TKI-resistant) or knock out (A549 EGFR-/-) NSCLC cell lines. Further experiments showed that HDAC inhibition inhibits cell survival and proliferation, while MIF inhibition downregulates pAKT or AKT expression level, which both interfere with cell survival. Furthermore, the combination treatment of TKI and HDAC/MIF dual inhibitor showed that the dual inhibitor enhanced TKI inhibitory efficacy, highlighting the advantages of HDAC/MIF dual inhibitor for more effective treatment of NSCLC.

Discovery of a Highly Potent and Selective Degrader Targeting Hematopoietic Prostaglandin D Synthase via In Silico Design.

Targeted protein degradation by proteolysis-targeting chimera (PROTAC) is one of the exciting modalities for drug discovery and biological discovery. It is important to select an appropriate linker, an E3 ligase ligand, and a target protein ligand in the development; however, it is necessary to synthesize a large number of PROTACs through trial and error. Herein, using a docking simulation of the ternary complex of a hematopoietic prostaglandin D synthase (H-PGDS) degrader, H-PGDS, and cereblon, we have succeeded in developing PROTAC(H-PGDS)-7 (6), which showed potent and selective degradation activity (DC50 = 17.3 pM) and potent suppression of prostaglandin D2 production in KU812 cells. Additionally, in a Duchenne muscular dystrophy model using mdx mice with cardiac hypertrophy, compound 6 showed better inhibition of inflammatory cytokines than a potent H-PGDS inhibitor TFC-007. Thus, our results demonstrated that in silico simulation would be useful for the rational development of PROTACs.

D-dopachrome tautomerase activates COX2/PGE2 pathway of astrocytes to mediate inflammation following spinal cord injury.

BACKGROUND: Astrocytes are the predominant glial cell type in the central nervous system (CNS) that can secrete various cytokines and chemokines mediating neuropathology in response to danger signals. D-dopachrome tautomerase (D-DT), a newly described cytokine and a close homolog of macrophage migration inhibitory factor (MIF) protein, has been revealed to share an overlapping function with MIF in some ways. However, its cellular distribution pattern and mediated astrocyte neuropathological function in the CNS remain unclear. METHODS: A contusion model of the rat spinal cord was established. The protein levels of D-DT and PGE2 synthesis-related proteinase were assayed by Western blot and immunohistochemistry. Primary astrocytes were stimulated by different concentrations of D-DT in the presence or absence of various inhibitors to examine relevant signal pathways. The post-injury locomotor functions were assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. RESULTS: D-DT was inducibly expressed within astrocytes and neurons, rather than in microglia following spinal cord contusion. D-DT was able to activate the COX2/PGE2 signal pathway of astrocytes through CD74 receptor, and the intracellular activation of mitogen-activated protein kinases (MAPKs) was involved in the regulation of D-DT action. The selective inhibitor of D-DT was efficient in attenuating D-DT-induced astrocyte production of PGE2 following spinal cord injury, which contributed to the improvement of locomotor functions. CONCLUSION: Collectively, these data reveal a novel inflammatory activator of astrocytes following spinal cord injury, which might be beneficial for the development of anti-inflammation drug in neuropathological CNS.

A knowledge-based, structural-aided discovery of a novel class of 2-phenylimidazo[1,2-a]pyridine-6-carboxamide H-PGDS inhibitors.

Through an internal virtual screen at GlaxoSmithKline a distinct class of 2-phenylimidazo[1,2-a]pyridine-6-carboxamide H-PGDS inhibitors were discovered. Careful evaluation of crystal structures and SAR led to a novel, potent, and orally active imidazopyridine inhibitor of H-PGDS, 20b. Herein, describes the identification of 2 classes of inhibitors, their syntheses, and their challenges.

Inhibition of macrophage migration inhibitory factor (MIF) as a therapeutic target in bleomycin-induced pulmonary fibrosis rats.

Macrophage migration inhibitory factor (MIF) inhibition can attenuate pulmonary fibrosis, but the antifibrotic mechanism is unclear. Here we investigated the antifibrotic effect of MIF knockdown in rats with bleomycin (BLM)-induced pulmonary fibrosis. The results showed that MIF inhibition attenuated lung injury and extracellular matrix deposition; significantly reduced the levels of cytokines including transforming growth factor-ß1 (TGF-ß1), tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), hydroxyproline (hyp), fibroblast growth factor 23 (FGF23), and secreted phosphoprotein 1 (Spp1); and inhibited the expression of CD68, F4/80, and α-smooth muscle actin (α-SMA) protein. MIF inhibition is associated with reduction of proinflammatory mediators and macrophage infiltration in lungs. In addition, MIF knockdown in the day 14 group was significantly better than MIF knockdown in day 1 group in terms of the above mentioned cytokines TGF-ß1, IL-17, TNF-α. MIF knockdown in day 14 group showed a better trend than MIF knockdown in day 1 group in inhibition of hyp and α-SMA formation. Furthermore, MIF inhibition downregulated the FGF23, Spp1, anti-integrin alpha 10 (Itga10), laminin subunit alpha 1 (Lama1), thrombospondin 2 (THBS2), and Serpin family B member 5 (SERPINB5) mRNA levels and the p-Smad2/3 protein level. MIF knockdown may inhibit fibrosis through the TGF-ß1/Smads signaling pathway. In addition, MIF inhibition protects against vascular remodeling via Thbs2 and Serpinb5 signaling. In summary, our study showed that knockdown of MIF can significantly inhibit lung inflammation and fibrosis in rats with BLM-induced pulmonary fibrosis. The future development of inhibitors targeting MIF may contribute to the treatment of pulmonary fibrosis.

MIF inhibitor ISO-1 alleviates severe acute pancreatitis-associated acute kidney injury by suppressing the NLRP3 inflammasome signaling pathway.

BACKGROUND: Acute kidney injury (AKI) is an important complication of severe acute pancreatitis (SAP) with a poor prognosis. The methyl ester of (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (ISO-1), an inhibitor of macrophage migration inhibitory factor (MIF), has protective effects against many diseases. Our previous study confirmed MIF inhibition alleviated SAP. Here, we explored the effects of ISO-1 in an experimental mouse model of SAP-associated AKI induced by l-arginine. METHODS: Mice were randomly divided into four treatment groups (n = 6 each): control (CON), SAP, SAP + ISO-1, and ISO-1. Histopathologic examination was used to observe damage in pancreatic and renal tissues. Biochemical and enzyme-linked immunosorbent assays (ELISA) kits were used to measure the serologic indicators amylase, lipase, creatinine, uric acid, interleukin (IL)-6, and tumor necrosis factor (TNF)-α. Immunohistochemistry was used to detect protein expression of NLRP3, ASC and caspase-1, and the infiltration of myeloperoxidase (MPO)-positive neutrophils in kidney tissue. Western blotting was used to detect NLRP3, ASC and caspase-1 and IL-1ß protein expression, and real-time PCR was used to measure MIF, IL-6, TNF-α, IL-1ß and IL-18 mRNA levels in kidney tissue. RESULTS: ISO-1 treatment alleviated pathological damage in pancreatic and renal tissues, and reduced the serum levels of amylase, lipase, creatinine, uric acid, IL-6 and TNF-α. ISO-1 also reduced protein expression of NLRP3, ASC, caspase-1 and IL-1ß, mRNA expression of MIF, IL-6, TNF-α, IL-1ß and IL-18, and the infiltration of MPO-positive neutrophils in kidney tissue. CONCLUSION: ISO-1 has a protective effect against experimental SAP-associated AKI. And the mechanism may be associated with ISO-1 inhibiting NLRP3 inflammasome signaling pathway.

Selective inhibition of prostaglandin D2 biosynthesis in human mast cells to overcome need for multiple receptor antagonists: Biochemical consequences.

BACKGROUND: The major mast cell prostanoid PGD2 is targeted for therapy of asthma and other diseases, because the biological actions include bronchoconstriction, vasodilation and regulation of immune cells mediated by three different receptors. It is not known if the alternative to selectively inhibit the biosynthesis of PGD2 affects release of other prostanoids in human mast cells. OBJECTIVES: To determine the biochemical consequences of inhibition of the hematopoietic prostaglandin D synthase (hPGDS) PGD2 in human mast cells. METHODS: Four human mast cell models, LAD2, cord blood derived mast cells (CBMC), peripheral blood derived mast cells (PBMC) and human lung mast cells (HLMC), were activated by anti-IgE or ionophore A23187. Prostanoids were measured by UPLC-MS/MS. RESULTS: All mast cells almost exclusively released PGD2 when activated by anti-IgE or A23187. The biosynthesis was in all four cell types entirely initiated by COX-1. When pharmacologic inhibition of hPGDS abolished formation of PGD2 , PGE2 was detected and release of TXA2 increased. Conversely, when the thromboxane synthase was inhibited, levels of PGD2 increased. Adding exogenous PGH2 confirmed predominant conversion to PGD2 under control conditions, and increased levels of TXB2 and PGE2 when hPGDS was inhibited. However, PGE2 was formed by non-enzymatic degradation. CONCLUSIONS: Inhibition of hPGDS effectively blocks mast cell dependent PGD2 formation. The inhibition was associated with redirected use of the intermediate PGH2 and shunting into biosynthesis of TXA2 . However, the levels of TXA2 did not reach those of PGD2 in naïve cells. It remains to determine if this diversion occurs in vivo and has clinical relevance.

Novel amide and imidazole compounds as potent hematopoietic prostaglandin D2 synthase inhibitors.

In seeking novel and potent small molecule hematopoietic prostaglandin D2 synthase (H-PGDS) inhibitors as potential therapies for PGD2-mediated diseases and conditions, we explored a series comprising multiple aryl/heteroaryl rings attached in a linear arrangement. Each compound incorporates an amide or imidazole "linker" between the pyrimidine or pyridine "core" ring and the "tail" ring system. We synthesized and screened twenty analogs by fluorescence polarization binding assay, thermal shift assay, glutathione S-transferase inhibition assay, and a cell-based assay measuring suppression of LPS-induced PGD2 stimulation. Amide analogs show ten-fold greater shift in the thermal shift assay in the presence of glutathione (GSH) versus the same assay run in the absence of GSH. The imidazole analogs did not produce a significant change in thermal shift between the two assay conditions, suggesting a possible stabilization effect of the amide linker in the synthase-GSH-inhibitor complex. Imidazole analog 23, (KMN-010034) demonstrates superior potency across the in vitro assays and good in vitro metabolic stability in both human and guinea pig liver microsomes.

Protective Mechanism of MIF Inhibitor ISO-1 on Intrahepatic Bile Duct Cells in Rats with Severe Acute Pancreatitis.

AIMS: This study aimed to explore the protection mechanism of ISO-1 on severe acute pancreatitis-associated intrahepatic bile duct (IBD) injury in rats. METHODS: Forty-eight specific-pathogen-free male Wistar rats were randomly divided into four groups (N = 12): a sham operation group (SO group), a severe acute pancreatitis model group (SAP group), a ISO-1 treatment group (ISO-1 + SAP group), and a ISO-1 control group (ISO-1 + SO group). All rats were killed after 12 h of being made models. Immunohistochemistry was used to detect the expression of MIF and P38 in IBD cells. MIF mRNA expression in IBD cells was observed using real-time fluorescent quantitative polymerase chain reaction (real-time PCR). In addition, Western blotting was performed to detect the protein expression of P38, phosphorylated P38 (P-P38), nuclear factor-κB (NF-κB p65), and tumor necrosis factor alpha (TNF-α). Enzyme-linked immunosorbent assays were used to analyze the levels of TNF-α, IL-1ß, and IL-6 in the IBD of rats. RESULTS: Compared with SAP, after treatment with ISO-1, the pathological injuries of pancreas, liver, and IBD cells in ISO-1 treatment group remarkably relieved. The expression of MIF in the IBD cells was significantly downregulated both at mRNA and at protein levels in ISO-1 treatment group. Besides, the protein expression levels of P38, P-P38, NF-κBp65, TNF-α, IL-1ß, and IL-6 in the IBD in rats were also significantly decreased in ISO-1 treatment group (all P < 0.05). CONCLUSION: ISO-1 may protect the IBD cells, reduce pathological injuries, and reduce the inflammatory response in SAP rats. Its mechanisms may be via inhibiting the expression of MIF and then blocking the activation of p38-MAPK and NF-κB signaling pathway.

Designed CXCR4 mimic acts as a soluble chemokine receptor that blocks atherogenic inflammation by agonist-specific targeting.

Targeting a specific chemokine/receptor axis in atherosclerosis remains challenging. Soluble receptor-based strategies are not established for chemokine receptors due to their discontinuous architecture. Macrophage migration-inhibitory factor (MIF) is an atypical chemokine that promotes atherosclerosis through CXC-motif chemokine receptor-4 (CXCR4). However, CXCR4/CXCL12 interactions also mediate atheroprotection. Here, we show that constrained 31-residue-peptides ('msR4Ms') designed to mimic the CXCR4-binding site to MIF, selectively bind MIF with nanomolar affinity and block MIF/CXCR4 without affecting CXCL12/CXCR4. We identify msR4M-L1, which blocks MIF- but not CXCL12-elicited CXCR4 vascular cell activities. Its potency compares well with established MIF inhibitors, whereas msR4M-L1 does not interfere with cardioprotective MIF/CD74 signaling. In vivo-administered msR4M-L1 enriches in atherosclerotic plaques, blocks arterial leukocyte adhesion, and inhibits atherosclerosis and inflammation in hyperlipidemic Apoe-/- mice in vivo. Finally, msR4M-L1 binds to MIF in plaques from human carotid-endarterectomy specimens. Together, we establish an engineered GPCR-ectodomain-based mimicry principle that differentiates between disease-exacerbating and -protective pathways and chemokine-selectively interferes with atherosclerosis.

The exploration of aza-quinolines as hematopoietic prostaglandin D synthase (H-PGDS) inhibitors with low brain exposure.

GlaxoSmithKline and Astex Pharmaceuticals recently disclosed the discovery of the potent H-PGDS inhibitor GSK2894631A 1a (IC50 = 9.9 nM) as part of a fragment-based drug discovery collaboration with Astex Pharmaceuticals. This molecule exhibited good murine pharmacokinetics, allowing it to be utilized to explore H-PGDS pharmacology in vivo. Yet, with prolonged dosing at higher concentrations, 1a induced CNS toxicity. Looking to attenuate brain penetration in this series, aza-quinolines, were prepared with the intent of increasing polar surface area. Nitrogen substitutions at the 6- and 8-positions of the quinoline were discovered to be tolerated by the enzyme. Subsequent structure activity studies in these aza-quinoline scaffolds led to the identification of 1,8-naphthyridine 1y (IC50 = 9.4 nM) as a potent peripherally restricted H-PGDS inhibitor. Compound 1y is efficacious in four in vivo inflammatory models and exhibits no CNS toxicity.

7-Hydroxycoumarins Are Affinity-Based Fluorescent Probes for Competitive Binding Studies of Macrophage Migration Inhibitory Factor.

Macrophage migration inhibitory factor (MIF) is a cytokine with key roles in inflammation and cancer, which qualifies it as a potential drug target. Apart from its cytokine activity, MIF also harbors enzyme activity for keto-enol tautomerization. MIF enzymatic activity has been used for identification of MIF binding molecules that also interfere with its biological activity. However, MIF tautomerase activity assays are troubled by irregularities, thus creating a need for alternative methods. In this study, we identified a 7-hydroxycoumarin fluorophore with high affinity for the MIF tautomerase active site (Ki = 18 ± 1 nM) that binds with concomitant quenching of its fluorescence. This property enabled development of a novel competition-based assay format to quantify MIF binding. We also demonstrated that the 7-hydroxycoumarin fluorophore interfered with the MIF-CD74 interaction and inhibited proliferation of A549 cells. Thus, we provide a high-affinity MIF binder as a novel tool to advance MIF-oriented research.

Macrophage migration inhibitory factor inhibition as a novel therapeutic approach against triple-negative breast cancer.

Triple-negative breast cancer (TNBC), defined as loss of estrogen, progesterone, and Her2 receptors, is a subtype of highly aggressive breast cancer with worse prognosis and poor survival rate. Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory cytokine aberrantly expressed in many solid tumors and known to promote tumor progression and metastasis. However, its role in TNBC progression and metastasis is unexplored. Here we have shown that in TNBC patients, MIF expression was significantly enriched in the tumor compared to adjacent normal tissue. Using publically available patient datasets, we showed that MIF overexpression correlates with worse survival in TNBC compared to other hormonal status. Orthotopic implantation of TNBC cells into MIF knockout mice showed reduced tumor growth compared to wild-type mice. In addition, we have shown that MIF downregulation inhibits TNBC growth and progression in a syngeneic mouse model. We further showed that CPSI-1306, a small-molecule MIF inhibitor, inhibits the growth of TNBC cells in vitro. Mechanistic studies revealed that CPSI-1306 induces intrinsic apoptosis by alteration in mitochondrial membrane potential, cytochrome c (Cyt c) release, and activation of different caspases. In addition, CPSI-1306 inhibits the activation of cell survival and proliferation-related molecules. CPSI-1306 treatment also reduced the tumor growth and metastasis in orthotopic mouse models of mammary carcinoma. CPSI-1306 treatment of tumor-bearing mice significantly inhibited TNBC growth and pulmonary metastasis in a dose-dependent manner. Histological analysis of xenograft tumors revealed a higher number of apoptotic cells in CPSI-1306-treated tumors compared to vehicle controls. Our studies, for the first time, show that MIF overexpression in TNBC enhances growth and metastasis. Taken together, our results indicate that using small molecular weight MIF inhibitors could be a promising strategy to inhibit TNBC progression and metastasis.

Inhibition of Macrophage Migration Inhibitory Factor Protects against Inflammation through a Toll-like Receptor-Related Pathway after Diffuse Axonal Injury in Rats.

OBJECTIVE: We have previously demonstrated that inflammation induced by toll-like receptors (TLRs) 2/4 exert cerebral deleterious effects after diffuse axonal injury (DAI); however, the underlying mechanisms are not fully understood. Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine involved in inflammatory responses. The purpose of this study was to investigate the role of MIF in inflammation induced by TLRs in the cortices of DAI rats. METHODS: The rat DAI model was established by head rotational acceleration and confirmed by ß-APP, HE, and silver staining. MIF protein expression at 3 h, 6 h, 12 h, 1 d, and 3 d after DAI was measured by western blot. The localization of MIF was measured by immunofluorescence. MIF antagonist ISO-1 was intracerebroventricularly injected to inhibit MIF. Neuronal and axonal injury and glial responses were assessed by TUNEL, immunohistochemistry, and TEM. Expression of TLR2, TLR4, ERK, phospho-ERK, NF-κB, and phospho-NF-κB was examined by western blot. The level of IL-1ß, IL-6, and TNF-α was measured by ELISA. RESULTS: MIF expression was significantly increased, peaking at 1 day after DAI, and MIF was mainly localized in microglial cells and neurons. ISO-1 suppressed neuronal apoptosis, axonal injury, and glial responses and decreased the expression of downstream signaling molecules related to TLR2/4, including ERK, phospho-ERK, NF-κB, phospho-NF-κB, IL-1ß, IL-6, and TNF-α. CONCLUSION: MIF was involved in the neuronal and axonal damage through a TLR-related pathway following DAI.