High-mobility Group Box 1 siRNA Research Articles

HMGB1-activated tumor-associated macrophages promote migration and invasion via NF-κB/IL-6 signaling in oral squamous cell carcinoma

Tumor-associated macrophages (TAMs) are a highly abundant cell population within the tumor microenvironment of oral squamous cell carcinomas (OSCC). Recent studies have identified an intricate cross-talk between cancer cells and macrophages in the tumor microenvironment. However, the underlying mechanism remains unclear. High-mobility group box 1 (HMGB1) was linked to metastasis and an unfavorable prognosis in head and neck squamous cell carcinoma. Furthermore, it was significantly upregulated in moderately differentiated OSCC tissues and the OSCC cell lines CAL27 and SCC9. HMGB1 knockdown impedes the ability of TAMs to induce invasion and migration of OSCC cells. Phenotypic changes in macrophages were measured after incubation of supernatant from OSCC cells transfected with HMGB1 siRNA or supplemented with recombinant HMGB1. HMGB1 induced M1 polarization of macrophages and the secretion of IL-6 via the NF-κB pathway, contributing to the OSCC malignant migration. HMGB1 originating from OSCC cells, along with its downstream signaling pathways, holds promise as a potential therapeutic target for mitigating metastasis and improving the survival rate of OSCC.

Release of HMGB1 in Podocytes Exacerbates Lipopolysaccharide-Induced Acute Kidney Injury.

Objective Acute kidney injury (AKI) usually occurs during sepsis. Inflammation factors, such as high-mobility group box 1 (HMGB1), are dramatically upregulated under septic conditions. In our current work, the functions of HMGB1 in AKI were explored. Methods An AKI model was induced by the lipopolysaccharide (LPS) challenge in C57 mice. Podocytes were challenged by LPS for different durations. Subsequently, podocytes transfected with HMGB1 siRNA were exposed to LPS for 24 h. The expressions of supernatant HMGB1 and cellular active caspase-3 were examined by Western blotting analysis. To explore the effect of HMGB1 on tubular epithelial cells (TECs), HK-2 cells were exposed to HMGB1 at various concentrations for 24 h. Epithelial-mesenchymal transition (EMT) of HK-2 cells was evaluated by Western blotting analysis. Mitochondrial division and apoptosis of HK-2 cells were assessed by MitoTracker Red and Western blotting analysis, respectively. Results Compared with the sham control group, the expression of HMGB1 was increased in the kidney of AKI mice. Moreover, the expression of supernatant HMGB1 was increased in LPS-challenged podocytes compared with the control group. Knockdown of HMGB1 attenuated LPS-induced podocyte injury. Besides, EMT in TECs was triggered by HMGB1. Mitochondrial damage and apoptosis of HK-2 cells exposed to HMGB1 were markedly elevated compared with the control group. Conclusions Collectively, HMGB1 release in podocytes was induced by LPS, subsequently leading to exacerbated AKI.

Intrinsic Activation of the NLRP3 Inflammasome in Primary Human ALL Cells Is Regulated By Endogenous HMGB1 and Bone Marrow Macrophages

Inflammasomes are cytosolic signaling complexes that assemble in response to damage-associated molecular patterns (DAMPs). Intrinsic expression of the NLRP3 has been identified in acute lymphoblastic leukemia (ALL) cell lines and implicated in glucocorticoid resistance (Paugh W et al., 2015). The endogenous function of NLRP3 in ALL remains unknown. In this study, we use primary human ALL cells to demonstrate that NLRP3 is activated in primary ALL cells in response to chemotherapy stress, is regulated by the endogenously expressed “danger protein” HMGB1 and can be silenced through direct contact with bone marrow macrophages.To determine whether chemotherapy treatment of primary ALL cells induces NLRP3 expression, we treated CD19-selected cells from n=3 pediatric patients with ALL and lymphocytes from healthy donors with either 2nM doxorubicin or vehicle and compared NLRP3 expression by qPCR and Western blot at 24 hours. NLRP3 mRNA expression was increased by 4.5 + 0.8- fold over baseline in doxorubicin-treated ALL cells compared to no change in vehicle-treated ALL and undetectable levels in healthy lymphocytes. This corresponded to a 2-fold increase in NLRP3 protein expression was measured by Western Blot. Neither doxorubicin-treated nor vehicle-treated ALL expressed other NOD-family inflammasome proteins, including NLRP1 and NLRC4.In healthy monocytes, NLRP3 activates caspase-1 to induce transcription of inflammatory cytokines. To determine whether NLRP3 up-regulation has a similar function in primary ALL cells, we measured pro- and cleaved-caspase-1 in doxorubicin-treated primary ALL, vehicle-treated ALL and healthy lymphocytes. Primary ALL cells treated with doxorubicin expressed the transcriptionally active p20 subunit of caspase-1 by Western blot; vehicle-treated ALL cells and healthy lymphocytes did not. This corresponded to a 3.7 + 0.61-fold increase in global caspase-1 transcription by mRNA in doxorubicin-treated ALL cells and a significant increase in soluble IL1B production by ELISA (p<0.02). Treatment of primary ALL with staurosporin (an autophagy-inducer) and serum starvation up-regulated NLRP3 but not caspase 1/p20. Taken together, these data established proof-of-principle that primary ALL cells activate the NLRP3 inflammasome in response to clinically relevant cellular stress.The activating signal for NLRP3 assembly is unknown in leukemia. We have previously observed that DNA-binding DAMP high mobility group-box 1 (HMGB1) is endogenously expressed in ALL cells. To determine whether HMGB1 activates NLRP in leukemia, we knocked down HMGB1 by siRNA in primary ALL cells (n=3 patients) and measured NLRP3 and caspase-1/p20 at baseline and after treatment with doxorubicin vs. vehicle. HMGB1 siRNA ALL cells displayed at least a 2-fold decrease in NLRP3 protein expression 48 hours after knockdown compared to control siRNA cells that could not be rescued with doxorubicin treatment. Co-immunoprecipitation studies indicate that HMGB1 forms a binding complex in primary ALL cells.To determine whether NLRP3 expression in primary ALL is impacted by cells in the nonmalignant field, we co-cultured primary ALL cells with bone marrow-derived monocyte/macrophage, either directly or separated by 0.6 uM transwell. Unexpectedly, NLRP3 expression was completely abrogated in primary ALL cells when they were placed in direct, but not transwell, co-culture with macrophages. Direct co-culture of ALL with peripheral monocytes, however, did not abrogate NLRP3. These data indicate that direct interactions of ALL with macrophages in the nonmalignant bone marrow microenvironment have a negative regulatory effect on NLRP3 inflammasome activation.Taken together these data show, for the first time in primary pediatric ALL cells, that NLRP3 inflammasome is activated in response to chemotherapy cell stress that is regulated by endogenous HMGB1 expression and direct interactions with bone marrow macrophages. Studies are underway to define these interactions and their physiologic consequences for ALL cell survival in tne marrow niche. DisclosuresNo relevant conflicts of interest to declare.

Glycyrrhizin improved autophagy flux via HMGB1-dependent Akt/mTOR signaling pathway to prevent Doxorubicin-induced cardiotoxicity

Doxorubicin (DOX) is one of the most effective and irreplaceable chemotherapeutic agents but its clinical use is limited due to its cardiotoxicity. Glycyrrhizin(GL) has been applied to liver disorders for long. However, little is known that if GL could be meaningful in attenuating cardiotoxicity. The aim of this study is to investigate the cardioprotective effects of GL in DOX-induced cardiotoxicity (DIC) and the underlying mechanism. Here, H9c2 cardiomyoblasts, Neonatal rat cardiomyocytes (NRCMs), and Rats were introduced as test models. A single dose of 20 mg/kg DOX (i.p.) was applied to induce acute cardiotoxicity in vivo, as reflected by growth inhibition, increased levels of AST and CK-MB, and reduction of SOD activity, while GL (25 or 50 mg/kg/d, 14 d, i.p.) could counteract these effects. Moreover, pre-incubation with GL (0.8 mM for 12 h) in H9c2 cells protected against DOX-induced cytotoxicity, oxidative stress and depolarization of mitochondrial membrane potential (MMP). Besides, Western blot analysis showed that DOX upregulated the expression of LC3 II and p62 whereas GL reversed that both in vitro and in vivo and improved the obstructed autophagy flux in DOX-treated H9c2 cells with an autophagy inhibitor Bafilomycin A1 (Baf A1, 50 nM, 2 h). It has been previously documented that High-mobility group box 1 (HMGB1) was involved in DIC. In our work, knockdown of HMGB1 significantly increased cell viability and LC3 II level in H9c2, suggesting HMGB1 was crucial in DOX-induced autophagy-triggering cell death. Intriguingly, GL is a direct inhibitor of HMGB1. We found that GL downregulated Akt/mTOR autophagy signaling pathway in DOX-treated H9c2 cells. More importantly, in non-silencing H9c2 cells (transfected with negative control siRNA) cells, the expression of phospho-Akt, phospho-mTOR, p62, and LC3 II was significantly decreased with GL pretreament compared to DOX alone. However, in H9c2/HMGB1－(transfected with HMGB1 siRNA) cells exposed to DOX, the expression of p-Akt, p-mTOR, p62, LC3 II had no statistical difference with or without GL, revealing that HMGB1 mediated the cardioprotective action of GL in DIC.Taken together, our findings demonstrate that improved autophagy flux via HMGB1-dependent Akt/mTOR signaling pathway might contribute to attenuate DIC and go a novel insight into the underlying mechanisms of GL’s cardioprotective action. GL could be a potential candidate for the prevention of DIC.

Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats.

Analgesic tolerance and hyperalgesia hinder the long-term utility of opioids. We examined whether spinal high mobility group box 1 (HMGB1) is involved in morphine tolerance and its underlying mechanisms by using a model of repeated intrathecal (i.t.) injections of morphine. The results showed that chronic i.t. morphine exposure led to increased expression of HMGB1, Toll-like receptor 4 (TLR4), and receptor for advanced glycation end products (RAGE) and their mRNAs in the dorsal horn. Morphine challenge also promoted HMGB1 expression and release in cultured spinal neurons, but these effects were inhibited by TAK-242, naloxone (antagonists of TLR4), and TLR4 siRNA. Intrathecal coadministration of morphine with TAK-242 or PDTC (inhibitor of NF-κB activation) also reduced HMGB1 expression in the spinal cord. Repeated i.t. coinjections of morphine with glycyrrhizin (GL, an HMGB1 inhibitor) or HMGB1 siRNA prevented reduction of the maximal possible analgesic effect (MPAE) of morphine and alleviated morphine withdrawal-induced hyperalgesia. The established morphine tolerance and hyperalgesia were partially reversed when i.t. injections of GL or HMGB1 antibody started at day 7 of morphine injection. Repeated i.t. injections of morphine with HMGB1 siRNA inhibited the activation of NF-κB, but not that of JNK and p38. A single i.t. injection of HMGB1 in naïve rats caused pain-related hypersensitivity and reduction in MPAE. Moreover, phosphorylated NF-κB p65, TNF-α, and IL-1β levels in the dorsal horn were upregulated following this treatment, but this upregulation was prevented by coinjection with TAK-242. Together, these results suggest that morphine-mediated upregulation of spinal HMGB1 contributes to analgesic tolerance and hyperalgesia via activation of TLR4/NF-κB signaling, and the HMGB1 inhibitor might be a promising adjuvant to morphine in the treatment of intractable pain in the clinic.

HMGB1 upregulates NF-kB by inhibiting IKB-α and associates with diabetic retinopathy

AimsDiabetic retinopathy (DR) is the main cause of blindness in adults and investigating new therapeutic targets for DR is necessary. This study aimed to investigate the effect of high-mobility group box 1 (HMGB1) protein and its mechanism in diabetic retinopathy (DR) were investigated. Main methodsHuman retinal endothelial cells (HREC) were uesd for chip-seq. Sprague Dawley (SD) rats were randomly divided into control group, HMGB1 group, diabetes mellitus (DM) combined with HMGB1 siRNA group, and DM group. Next, eyeballs were removed and retinas were detached for western blot. The DM model of cell was built by increasing the glucose concentration in cell culture medium. The regulation of HMGB1 was achieved by short hairpin (sh)-HMGB1 transfection, then, the transfected cells were harvested for luciferase assay, western blot and qRT-PCR analyses as well as proliferation and apoptosis detection. Key findingsChip-seq and luciferase assay showed the possible transcription factor functions of HMGB1 and IKB-α was one of the HMGB1 binding sites. In vivo and in vitro results indicated high expression of HMGB1 and NF-kB and low expression of IKB-α in DR and the expression of IKB-α and NF-kB was regulated by HMGB1. Moreover, cell assays showed that HMGB1 inhibited cell proliferation and promoted apoptosis. SignificanceThe results from the present study showed that HMGB1 may be involved in the pathogenesis of DR as a transcription factor through NF-kB pathway. Therefore, blockade of HMGB1 may be a new method for the treatment of DR.

Delivery of High Mobility Group Box-1 siRNA Using Brain-Targeting Exosomes for Ischemic Stroke Therapy.

Ischemic strokes are caused by decreased blood flow into the brain, due to narrowed cerebral arteries. In the ischemic brain, high-mobility group box 1 (HMGB1) is released into extracellular spaces and induces inflammatory reactions. In this study, HMGB1 small interfering RNA (siRNA) was delivered into ischemic brains by intravenous administration using rabies virus glycoprotein (RVG) peptide-decorated exosomes. A fusion protein of RVG and Lamp2b was expressed in 293T cells. Since Lamp2b is an exosome membrane-integral protein, RVG-Lamp2b is integrated into the exosomes, producing RVG-decorated exosomes (RVG-Exo). HMGB1-siRNA was loaded into RVG-Exo and unmodified exosomes (Unmod-Exo) by electroporation. The exosomes were homogenous with a size of less than 50 nm and a negative surface charge. In vitro delivery assays showed that RVG-Exo showed higher efficiency to Neuro2A cells than Unmod-Exo. Also, HMGB1 levels were reduced more effectively by RVG-Exo/HMGB1-siRNA. In vivo delivery efficiency and therapeutic effects of RVG-Exo/HMGB1-siRNA were evaluated in a middle cerebral artery occlusion (MCAO) model. RVG-Exo/HMGB1-siRNA, Unmod-Exo/HMGB1-siRNA, and PEI25k/HMGB1-siRNA were administrated into the MCAO model intravenously through the tail vein. The results showed that HMGB1, tumor necrosis factor-α (TNF-α), and apoptosis levels in the brain were reduced in the RVG-Exo/HMGB1-siRNA group more efficiently than the other groups. In addition, the infarct size was decreased in the RVG-Exo/HMGB1 group more effectively than the other groups. These results suggest that RVG-Exo with HMGB1-siRNA may have potential as a therapeutic system for the treatment of ischemic strokes.

Inhibition of high mobility group box 1 (HMGB1) attenuates podocyte apoptosis and epithelial-mesenchymal transition by regulating autophagy flux.

Podocyte injury, characterized by podocyte hypertrophy, apoptosis, and epithelial-mesenchymal transition (EMT), is the major causative factor of diabetic nephropathy (DN). Autophagy dysfunction is regarded as the major risk factor for podocyte injury including EMT and apoptosis. High mobility group box 1 (HMGB1) is involved in the progression of DN through the induction of autophagy. However, the underlying mechanism remains unknown. Plasma HMGB1 concentrations were determined in DN patients using ELISA. Apoptosis of DN serum-treated podocytes was evaluated by flow cytometry. Podocyte autophagy flux was measured using immunofluorescence. Western blotting analysis was used to investigate HMGB1 expression, EMT, and autophagy-related signaling pathways. Upregulation of HMGB1 was found in DN patients and DN serum-treated podocytes. Removal of HMGB1 inhibited DN serum-mediated podocyte apoptosis by inhibiting autophagy and activating AKT/mammalian target of rapamycin (mTOR) signaling. In addition, HMGB1 depletion repressed the progression of podocyte EMT by inhibiting transforming growth factor (TGF)-β/smad1 signaling in vitro and in vivo. The combination of HMGB1 short interference (si) RNA and the autophagy activator rapamycin protected against podocyte apoptosis and EMT progression by inhibiting the AKT/mTOR and TGF-β/smad signaling pathway, respectively. Although HMGB1 siRNA and rapamycin treatment had opposite effects on autophagy and AKT/mTOR signaling, there was no contradiction about the role of HMGB1 siRNA and rapamycin on AKT/mTOR pathway because autophagy and AKT/mTOR signaling play dual roles in intracellular biological processes. Based on the findings of this study, we may assume that HMGB1-initiated autophagy is harmful, whereas rapamycin is beneficial to podocyte survival. Possibly combined treatment with HMGB1 siRNA and rapamycin improved podocyte damage and EMT by regulating autophagy homeostasis.

DNA binding protein HMGB1 secreted by activated microglia promotes the apoptosis of hippocampal neurons in diabetes complicated with OSA

Type 2 diabetes mellitus (T2DM) complicated with obstructive sleep apnea (OSA) may cause neuronal apoptosis and cognitive deficits, but the underlying mechanisms remain unclear. We aimed to determine the relationship between the activation of microglia and the apoptosis of hippocampal neurons, specifically in terms of high mobility group box-1 (HMGB1), after high glucose (HG) and intermittent hypoxia (IH) exposure. Diabetic KK-Ay mice and non-diabetic C57BL/6J mice (C57 mice) underwent IH or normoxia (control) exposure for 4 weeks. Cognitive function, microglial activation and hippocampal neuronal apoptosis were assessed after IH or normoxia exposure. Compared with C57 control mice, KK-Ay control mice exhibited increased cognitive dysfunction, microglial activation and hippocampal neuronal apoptosis. There were no differences between untreated KK-Ay control mice and C57 mice that had been exposed to IH. The abovementioned responses were aggravated in IH-exposed KK-Ay mice compared with control KK-Ay mice. In vitro, a cellular co-culture experiment showed that HG combined with IH could activate BV2 microglia, leading to the release of neuroinflammatory factors (ROS, TNF-α, IL-1β) and mediating the apoptosis of HT22 cells via the PI3K/Akt/GSK-3β signaling pathway. Meanwhile, HMGB1 was actively secreted into the extracellular environment from activated BV2 microglia. As a proinflammatory factor, it was able to sustain microglial activation by directly acting on those cells. The activation promoted positive feedback and aggravated neuronal damage further. In a cellular monoculture or co-culture system, HMGB1 siRNA was able to alleviate the activation of BV2 cells and the apoptosis of HT22 cells induced by HG combined with IH. Our object is to show that inhibition of HMGB1 may break the vicious cycle to prevent or treat neuroinflammation and hippocampal neuronal apoptosis caused by T2DM complicated with OSA.

PM2.5 Induced the Expression of Fibrogenic Mediators via HMGB1-RAGE Signaling in Human Airway Epithelial Cells.

Background The aim of the present study was to test whether fine particulate matter (PM2.5) induces the expression of platelet-derived growth factor-AB (PDGF-AB), PDGF-BB, and transforming growth factor-β1 (TGF-β1) in human bronchial epithelial cells (HBECs) in vitro via high-mobility group box 1 (HMGB1) receptor for advanced glycation end products (RAGE) signaling. Methods Sprague-Dawley rats were exposed to motor vehicle exhaust (MVE) or clean air. HBECs were either transfected with a small interfering RNA (siRNA) targeting HMGB1 or incubated with anti-RAGE antibodies and subsequently stimulated with PM2.5. Results The expression of HMGB1 and RAGE was elevated in MVE-treated rats compared with untreated rats, and PM2.5 increased the secretion of HMGB1 and upregulated RAGE expression and the translocation of nuclear factor κB (NF-κB) into the nucleus of HBECs. This activation was accompanied by an increase in the expression of PDGF-AB, PDGF-BB, and TGF-β1. The HMGB1 siRNA prevented these effects. Anti-RAGE antibodies attenuated the activation of NF-κB and decreased the secretion of TGF-β1, PDGF-AB, and PDGF-BB from HBECs. Conclusion PM2.5 induces the expression of TGF-β1, PDGF-AB, and PDGF-BB in vitro via HMGB1-RAGE signaling, suggesting that this pathway may contribute to the airway remodeling observed in patients with COPD.

The role of high-mobility group box 1 in magnetic stimulation-induced migration of astrocytes

Objective To examine the molecular mechanism involved in astrocyte migration induced by magnetic stimulation (MS), and the role of high mobility group box 1 (HMGB1) in migration. Methods Astrocytes were isolated from the cortical tissues of 2-3 day old Sprague-Dawley rats and divided into a stimulus group given MS and a control group without MS. The stimulus group was further divided into an experimental group and a control group, with the former pre-stimulated with 10 μmol/ml U0126 for 30 minutes and no pre-stimulation for the latter.The cells in the experimental group were also randomly divided into siRNA and HMGB1-siRNA transfection groups to examine the role of HMGB1 in astrocyte migration induced by MS. The SiRNA group was transfected with HMGB1 siRNA. Western blotting was used to detect any effect of MS on HMGB1 and extracellular signal-regulated kinase1/2 (ERK1/2) .The migration of astrocytes was detected using the scratch assay. Results MS (10 Hz) can promote the phosphorylation of ERK, increase the migration of astrocytes and the expression of HMGB1. After the U0126 treatment and transfection with HMGB1 siRNA, the effects of MS on expression of HMGB1 and migration of astrocytes decreased significantly. Conclusions Magnetic stimulation-mediated migration of astrocytes via activation of the ERK pathway phosphorylation and autocrine of HMGB1. Key words: Magnetic stimulation; Astrocytes; Box 1; Migration

High-mobility group box-1 contributes tumor angiogenesis under interleukin-8 mediation during gastric cancer progression.

Many soluble factors are involved in tumor angiogenesis. Thus, it is valuable to identify novel soluble factors for effective control of tumor angiogenesis in gastric cancer (GC). We investigated the role of extracellular high‐mobility group box‐1 (HMGB1) and its associated soluble factors in the tumor angiogenesis of GC. Clinically, we measured serum levels of HMGB1 and GC‐associated cytokines/chemokines using GC serum samples (n = 120), and calculated microvessel density (MVD) by CD34 immunostaining using human GC tissues (n = 27). Then we analyzed the correlation of serum HMGB1 levels with MVD or that with cytokine/chemokine levels by linear regression. As in vitro angiogenesis assay for HMGB1, HUVEC migration and capillary tube formation assay were carried out using different histological types of human GC cells (N87 and KATOIII). CD34‐positive microvessels were detected from early GC, but MVD increased according to GC stages, and were closely correlated with serum HMGB1 levels (R = 0.608, P = 0.01). The HUVECs cultured in conditioned media derived from rhHMGB1‐treated or HMGB1‐TF GC cells showed remarkably enhanced migration and tube formation activities. These effects were abrogated by anti‐HMGB1 antibody or HMGB1 siRNA in both N87 and KATOIII cells (all P < 0.05). Among tested cytokines/chemokines, interleukin‐8 (IL‐8) was the most remarkable cytokine correlated with serum HMGB1 (P < 0.001), and enhanced HUVEC migration and tube formation activities by rhHMGB1 or HMGB1‐TF were significantly reversed by IL‐8 inhibition. These results indicate overexpressed HMGB1 contributes to tumor angiogenesis through IL‐8 mediation, and combined targeting of HMGB1 and IL‐8 can control tumor angiogenesis in GC.

HMGB1-mediated autophagy attenuates gemcitabine-induced apoptosis in bladder cancer cells involving JNK and ERK activation

High-mobility group box 1 (HMGB1) has been found to mediate autophagy during chemotherapy in several cancers. However, whether HMGB1plays a role in autophagy and chemoresistance in bladder cancer is elusive. In this report, HMGB1 expression was found to be increased in 30 primary bladder cancer tissue specimens compared to their matched adjacent non-tumor tissues. While gemcitabine induced apoptotic cell death, it also induced HMGB1 expression and autophagy in bladder cancer T24 and BIU-87 cells. Suppressing HMGB1 expression with siRNA strongly potentiated gemcitabine-induced apoptosis. HMGB1 siRNA or autophagy inhibitors suppressed gemcitabine-induced autophagy. Further, gemcitabine activated c-Jun N-terminal kinase (JNK) and extracellular regulated protein kinase (ERK) and Bcl-2 phosphorylation, and blocking ERK and JNK inhibited autophagy and increased apoptosis in gemcitabine-treated cells. Interestingly, suppressing HMGB1 expression attenuated gemcitabine-induced ERK and JNK activation and Bcl-2 phosphorylation. Thus, our results suggest that while gemcitabine kills bladder cancer cells through apoptosis, a cytoprotective autophagy is also induced involving HMGB1-mediated JNK and ERK to counteract the cytotoxicity of gemcitabine, and intervention targeting this pathway may improve the anticancer efficacy of gemcitabine against bladder cancer.

MP28-20 EXPRESSION OF HMGB1 IN PROSTATE CANCER: CLINICAL AND BIOLOGICAL CORRELATIONS

You have accessJournal of UrologyProstate Cancer: Markers I1 Apr 2017MP28-20 EXPRESSION OF HMGB1 IN PROSTATE CANCER: CLINICAL AND BIOLOGICAL CORRELATIONS Yong Hyun Park, Ae Ryang Jung, Jin Bong Choi, U-Syn Ha, Sung-Hoo Hong, Sae Woong Kim, and Ji Youl Lee Yong Hyun ParkYong Hyun Park More articles by this author , Ae Ryang JungAe Ryang Jung More articles by this author , Jin Bong ChoiJin Bong Choi More articles by this author , U-Syn HaU-Syn Ha More articles by this author , Sung-Hoo HongSung-Hoo Hong More articles by this author , Sae Woong KimSae Woong Kim More articles by this author , and Ji Youl LeeJi Youl Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.833AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The high mobility group box1 (HMGB1), which is the important nuclear factor, has been noted to play a critical role of biological processes such as DNA repair, transcription and extracellular response. Increased expression of HMGB1 has been observed in several solid cancers and known to be associated with poor prognosis. However, there have been little studies of the role of HMGB1 in prostate cancer (PCa) development and progression. Therefore, in this study, we aimed to investigate 1) the role of HMGB1 on cellular proliferation, apoptosis, migration, and invasion, 2) the underlying biological mechanisms of HMGB1 in PCa, and 3) the expression pattern of HMGB1 in PCa patients with different stage and grade and its prognostic importance. METHODS After transient transfection of PC3 and DU-145 cells with HMGB1 siRNA, diverse experiments were performed to evaluate the changes in proliferation, apoptosis, migration and invasion. To determine whether HMGB1 affects the NF-?B pathway, subcellular localizations of p65 and phosphorylated p65 were assessed by western blot analysis. Using the Cancer Genome Atlas (TCGA) datasets, we determined the impact of HMGB1 on overall survival in PCa. We further validated the prognostic importance of HMGB1 by immunofluorescence staining in 131 PCa patients from the Korean Prostate Bank. RESULTS Inhibition of HMGB1 expression significantly reduced cell proliferation and increased cell cycle arrest in the sub-Go phase of PC3 and DU-145 cells. It also inhibited the migration and invasive capacity of PCa cells. Western blot analysis showed that inhibition of HMGB1 reduced p65 and phosphorylated p65 protein levels in nuclear fractions of PCa cells. In The Cancer Genome Atlas data set (n = 498), HMGB1 was altered in 61 of 498 patients (12%). Overall survival was shorter in the high HMGB1 expression group (medians: 115.0 months vs. not reached; P = 0.0296). In the Korean Prostate Bank cohort, the positive areas of HMGB1 differed in patients with BPH, and low-, intermediate-, high-risk, and metastatic PCa (4.6, 11.9, 18.6, 19.7, and 23.4%, p < 0.001). During the median follow-up of 32 months, increased expression of HMGB1 was associated with a significant decrease in biochemical recurrence free survival on Kaplan-Meier analysis. CONCLUSIONS Our findings demonstrate an important role of HMGB1 and novel relationship between HMGB1 and NF-?B pathway in PCa. Therapy targeting HMGB-associated pathways may represent a novel therapeutic avenue for PCa. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e346 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Yong Hyun Park More articles by this author Ae Ryang Jung More articles by this author Jin Bong Choi More articles by this author U-Syn Ha More articles by this author Sung-Hoo Hong More articles by this author Sae Woong Kim More articles by this author Ji Youl Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

HMGB1 regulates P-glycoprotein expression in status epilepticus rat brains via the RAGE/NF-κB signaling pathway.

Overexpression of P-glycoprotein (P-gp) in the brain is an important mechanism involved in drug-resistant epilepsy (DRE). High-mobility group box 1 (HMGB1), an inflammatory cytokine, significantly increases following seizures and may be involved in upregulation of P-gp. However, the underlying mechanisms remain elusive. The aim of the present study was to evaluate the role of HMGB1 and its downstream signaling components, receptor for advanced glycation end-product (RAGE) and nuclear factor-κB (NF-κB), on P-gp expression in rat brains during status epilepticus (SE). Small interfering RNA (siRNA) was administered to rats prior to induction of SE by pilocarpine, to block transcription of the genes encoding HMGB1 and RAGE, respectively. An inhibitor of NF-κB, pyrrolidinedithiocarbamic acid (PDTC), was utilized to inhibit activation of NF-κB. The expression levels of HMGB1, RAGE, phosphorylated-NF-κB p65 (p-p65) and P-gp were detected by western blotting. The relative mRNA expression levels of the genes encoding these proteins were measured using reverse transcription-quantitative polymerase chain reaction and the cellular localization of the proteins was determined by immunofluorescence. Pre-treatment with HMGB1 siRNA reduced the expression levels of RAGE, p-p65 and P-gp. PDTC reduced the expression levels of P-gp. These findings suggested that overexpression of P-gp during seizures may be regulated by HMGB1 via the RAGE/NF-κB signaling pathway, and may be a novel target for treating DRE.

The DNA-Binding Protein High Mobility Group Box 1 (HMGB1) Interacts with RelB to Maintain Non-Canonical NF-κb Signaling, Cell Survival and Immune Evasion in Infant Acute Lymphoblastic Leukemia

Disordered NF-κB signaling is a hallmark of many hematologic malignancies but has not been well-studied in MLL-rearranged infant lymphoblastic leukemia (iMLL-ALL), a subtype of pediatric ALL with an extremely poor prognosis in need of new therapy. Given the stable genome and short latency of iMLL-ALL, targeting of oncoproteins that play a coordinated role in regulation of the leukemic transcriptome represent ideal targets. Here, we present one such potential target: a complex formed between the nucleosome-stabilizing protein high mobility group box 1 (HMGB1) and RelB, a transcriptionally active subunit of the noncanonical NF-κB pathway.Disruptions in the non-canonical NF-κB pathway, which normally functions to support lymphogenesis, are known to drive lymphoid malignancies. Specifically, changes in proteasomal processing of the p100 subunit modulate nuclear p52/RelB regulation of the NF-κB2 gene. In stable short-term culture, we observed that primary infant MLL-ALL cells (derived from the diagnostic pheresis specimen of a 9 month old with t(4;11)) express an endogenous NF-κB subunit profile consistent with constitutive non-canonical activation, including significantly increased levels of IKKa, RelB, and NF-κB2 by Western Blot and qPCR compared to healthy human B cells and cytogenetically normal ALL. First, we asked whether exogenous "drivers" of the canonical (p65/RelA) pathway could alter this expression profile by treating iMLL-ALL with TNFalpha and bacterial LPS. Exogenous cytokine treatment did not alter expression levels of IKKa, RelB or NF-κB2, nor did it increase p65RelA, likely due to the lack of pattern recognition receptors on precursor lymphoblastic cells.We have previously demonstrated that infant MLL-ALL express high intracellular levels of the nucleosome-stabilizing protein high mobility group box-1 (HMGB1). During infection, HMGB1 forms a transcription regulatory complex with RelB that, by epigenetic means, silences inflammatory cytokine promoters as a negative feedback. In this study, we hypothesized that HMGB1-RelB interactions stabilize and maintain a pro-leukemic NF-κB program. Because the function of HMGB1 is determined by post-translational modifications, we first verified the DNA-binding disulfide form of the protein in the nuclear fraction of iMLL-ALL cells by immunoprecipitation. Next, we proved a physiologically relevant interaction between HMGB1 and RelB by co-immunoprecipitation; both HMGB1 and RelB could be identified by immunoblot from iMLL-ALL extracts probed for either for RelB or HMGB1. Detection of the complex required minimal stabilization, suggesting a durable interaction. Preliminary studies suggest that the complex associates with the acetylated form of histone 3 (H3), rather than histone 1 as in sepsis models.We then determined the effect of HMGB1 siRNA knockdown on the NF-κB subunit expression profile, proliferation and survival of iMLL-ALL, nonleukemic control PBMC and 293T cells. HMGB1 siRNA produced 30-50% spontaneous apoptosis in MLL-ALL, but not nonleukemic control cells, measured by Annexin/PI. iMLL-ALL transfected with a nonsense siRNA sequence did not apoptose. HMGB1 siRNA also silenced expression of RelB and NF-κB 2, measured by Western Blot and qPCR. Unexpectedly, HMGB1 siRNA restored expression of critical canonical NF-κB subunits: p65/RelA, the TNF-associated factor TRAF3, and the proteasome regulator NEMO. Further, HMGB1 siRNA knockdown resulted in a 51.3% (+/- 3.47, p<0.0001) increase in engulfment by activated bone marrow macrophages in a dual-label phagocytosis assay, compared to nonsense siRNA and control iMLL-ALL- suggesting that HMGB1 expression plays a role in evasion of iMLL-ALL by the innate immune system.Taken together, these data indicate that HMGB1-RelB interactions represent an important regulatory component of the NF-κB signaling program in human infant MLL-ALL cells that support survival and leukemogenesis. Current studies are comparing p100 processing and proteasome activity in iMLL-ALL with competent and disrupted HMGB1-RelB interactions, as well as comparing the effectiveness of proteasome inhibition with bortezomib. Near-future studies will screen other high-risk ALL’s for HMGB1-Rel activity and elaborate the downstream effects of complex binding on the MLL-ALL transcriptome to establish it as a rational target for drug development. DisclosuresNo relevant conflicts of interest to declare.

Influence of HMGB1/MAPK/m-TOR signaling pathway on cell autophagy and chemotherapy resistance in K562 cells

To observe the effect of high-mobility group box 1 (HMGB1) on autophagy and chemotherapy resistance in human leukemiacell line (K562) cells, and to explore the underlying mechanisms. Methods: The K562 cells were cultured in vitro and divided into 6 groups: a chemotherapeutic group, a chemotherapeutic control group, a HMGB1 preconditioning group, a HMGB1 preconditioning control group, a HMGB1 siRNA group and a siRNA control group. The chemotherapeutic group was further divided into a vincristine (VCR) group, an etoposide (VP-16) group, a cytosine arabinoside (Ara-C) group, a adriamycin (ADM) group and a arsenic trioxide (As2O3) group. The cell activity was evaluated by cell counting kit-8. The protein levels of HMGB1, microtubule-associate protein1light chain3 (LC3), AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (m-TOR) were determined by Western blotting. The level of serum HMGB1 was evaluated by enzyme-linked immunosorbent assay (ELISA). The autophagy was examined by monodansylcadaverine staining and observed under transmission electron microscopy. Results: Compared with the control group, the cell activity was significantly decreased and the level of serum HMGB1 was significantly increased in the chemotherapeutic (VCR, VP-16, Ara-C, ADM and As2O3) groups (all P<0.05). Compared with the control group, the cell activity and the level of serum HMGB1 were significantly increased in the HMGB1 preconditioning group (both P<0.05). Compared with the siRNA control group, the cell activity and the level of serum HMGB1 were significantly decreased in the HMGB1 siRNA group (both P<0.05). Compared with the control group, the expression of LC3-II and the formation of autophagic bodies were increased in the HMGB1 preconditioning group (both P<0.05), the p-AMPK expression was increased and p-mTOR expression was decreased (both P<0.05). Conclusion: HMGB1 can increase the autophagy and promote chemotherapy resistance through the pathway of AMPK/m-TOR in K562 cells.

Methotrexate affects HMGB1 expression in rheumatoid arthritis, and the downregulation of HMGB1 prevents rheumatoid arthritis progression.

High-mobility group box 1 (HMGB1) is associated with the development of rheumatoid arthritis (RA). Recent studies have shown that methotrexate (MTX) may inhibit the expression of HMGB1. This study examined whether HMGB1 might be involved in the treatment of RA using MTX. Synovial tissues were collected from RA patients who were treated with MTX for at least 6months (RA-MTX group, 7 cases) and from those without MTX treatment (RA-noMTX group, 7 cases). Additionally, patients with osteoarthritis (OA group, 7 cases) were used as controls. The expression and locations of HMGB1 in the tissues were detected using real-time PCR, western blot, and immunohistochemistry. Additionally, OA-fibroblast-like synoviocytes (FLSs) and RA-FLSs were isolated and cultured, and the expression of HMGB1 was reduced in these cells by transfection with HMGB1 siRNA. Cell proliferation, migration, and invasion abilities were detected. Furthermore, the effects of HMGB1 on matrix metalloproteinase (MMP)-2 and MMP-13 were measured using western blot analysis. At the tissue level, HMGB1 expression in synovial membrane did not differ significantly between the OA and RA-MTX groups, but was significantly lower in these groups than in the RA-noMTX group. In cell experiments, the cell doubling time in the RA-FLS HMGB1 siRNA group was significantly extended compared with that in the RA-FLS negative control (NC)-siRNA group. The amount of cell migration and invasion in the RA-FLS HMGB1 siRNA group was significantly lower compared with that in the NC-siRNA group; the MMP-2 and MMP-13 expression levels were also lower. These results showed that MTX reduced HMGB1 expression in RA synovial tissues, and through the downregulation of HMGB1 expression in tissues, MTX may slow disease progression of RA.

Role of high-mobility group box 1 in methamphetamine-induced activation and migration of astrocytes.

BackgroundMounting evidence has indicated that high-mobility group box 1 (HMGB1) is involved in cell activation and migration. Our previous study demonstrated that methamphetamine mediates activation of astrocytes via sigma-1 receptor (σ-1R). However, the elements downstream of σ-1R in this process remain poorly understood. Thus, we examined the molecular mechanisms involved in astrocyte activation and migration induced by methamphetamine.MethodsThe expression of HMGB1, σ-1R, and glial fibrillary acidic protein (GFAP) was examined by western blot and immunofluorescent staining. The phosphorylation of cell signaling pathways was detected by western blot, and cell migration was examined using a wound-healing assay in rat C6 astroglia-like cells transfected with lentivirus containing red fluorescent protein (LV-RFP) as well as in primary human astrocytes. The role of HMGB1 in astrocyte activation and migration was validated using a siRNA approach.ResultsExposure of C6 cells to methamphetamine increased the expression of HMGB1 via the activation of σ-1R, Src, ERK mitogen-activated protein kinase, and downstream NF-κB p65 pathways. Moreover, methamphetamine treatment resulted in increased cell activation and migration in C6 cells and primary human astrocytes. Knockdown of HMGB1 in astrocytes transfected with HMGB1 siRNA attenuated the increased cell activation and migration induced by methamphetamine, thereby implicating the role of HMGB1 in the activation and migration of C6 cells and primary human astrocytes.ConclusionsThis study demonstrated that methamphetamine-mediated activation and migration of astrocytes involved HMGB1 up-regulation through an autocrine mechanism. Targeting HMGB1 could provide insights into the development of a potential therapeutic approach for alleviation of cell activation and migration of astrocytes induced by methamphetamine.

Downregulation of high mobility group box1 attenuates the severity of acute lung injury in endotoxemic mice.

High mobility group box1 (HMGB1) is a systemic inflammation‑associated cytokine mediator. The aim of the present study was to examine the effect of the downregulation of HMGB1 in a lipopolysaccharide (LPS)‑induced mouse model of acute lung injury (ALI). It was identified that serum levels of tumor necrosis factor‑α and interleukin‑1β, lung myeloperoxidase activity and malondialdehyde content, as well as lung wet/dry weight ratios were all increased following LPS challenge. However, LPS‑mediated increases in these parameters were significantly downregulated in HMGB1 small interfering (si)RNA‑treated mice versus the negative control siRNA‑treated mice. In addition, the administration of HMGB1 siRNA in LPS‑treated mice resulted in a decreased DNA binding activity of nuclear factor‑κB (NF‑κB) in the lung. It was demonstrated that downregulation of HMGB1 decreases inflammation and the severity of sepsis associated with ALI, possibly via inhibiting the NF‑κB DNA‑binding activity. The present data support HMGB1 as a contributor to the pathogenesis of LPS‑induced sepsis and ALI.