High Mobility Group Box 1 Knockdown Research Articles

Expression of the Inflammatory Molecule HMGB1 in Human Osteosarcoma and its Clinical Relevance

High mobility group box 1 (HMGB1), known as a pro-inflammatory cytokine and chromatin-binding molecule, plays an important role in the carcinogenesis and metastasis of various tumors. The present study aimed to investigate the expression of HMGB1 in human osteosarcoma and its clinical relevance. At first, human osteosarcom tissues and their corresponding adjacent non-cancerous tissues (ANCT) from forty consecutive cases were collected. The expression of HMGB1 was detected by immunohistochemical assay through tissue microarray procedure and the correlation of HMGB1 expression with clinicopathologic factors was evaluated. Secondly, through small hairpin RNA(shRNA)-mediated HMGB1 knockdown in MG-63 osteosarcoma cells, we observed the changes of the biological behaviors of the osteosarcoma cells. As a consequence, the rate of positive expression of HMGB1 was significantly higher in osteosarcoma tissues than in the ANCT (60% vs 15%, P < 0.01). HMGB1 expression had significant positive correlation with Ennecking staging ( P = 0.034) and distant metastases ( P = 0.003), but had no correlation with the factors including age and gender of the patients, or histology and location of the tumor (each P > 0.05). Knockdown of HMGB1 down-regulated the expression of p-AKT, p-PI3K, PCNA, MMP-9 and CyclinD1, while it up-regulated the expression of cleaved caspase-3. More importantly, HMGB1 knockdown inhibited the proliferative activities and invasive potential, and induced apoptosis and cycle arrest in MG63 osteosarcoma cells. Taken together, our results indicate that HMGB1 was highly expressed in human osteosarcoma tissues, and the patients with higher HMGB1 expression in osteosarcoma tissues were more likely to have progression and metastasis of the disease. Knockdown of HMGB1 could inhibit the proliferation and invasion of osteosarcoma cells and induce its apoptosis through down-regulation of PI3K/AKT signaling pathway. HMGB1 could be a potential therapeutic target for osteosarcoma.

Expression and clinical significance of HMGB1 in human liver cancer: Knockdown inhibits tumor growth and metastasis in vitro and in vivo

The high-mobility group box 1 (HMGB1) signaling pathway plays a crucial role in tumorigenesis and progression of many malignant cancers. The present study aimed to investigate the expression and clinical significance of HMGB1 in human primary liver cancer, and further explore the molecular mechanisms of HMGB1 in tumor growth and metastasis. Forty cases of human liver cancer and normal liver tissues were collected. The expression of HMGB1 was assessed using RT-PCR and western blot assays in biopsy samples. The HMGB1 pathway invitro was blocked using transfection of the recombinant small hairpin RNA adenovirus vector rAd5-HMGB1 into the human liver cancer cell line SMMC-7721. The expression of HMGB1, phosphorylated AKT (p-AKT), Ki-67 and matrix metallopeptidase-2 (MMP-2) was detected by Real-PCR and western blot assays. Cell proliferative activities and metastatic capability were determined by MTT and Transwell assays. Cell cycle distribution and apoptosis were detected by flow cytometry. A subcutaneous xenograft tumor model was established, validating the effects of rAd5-HMGB1 on tumor growth invivo. As a consequence, HMGB1 was found to be highly expressed in liver cancer compared with normal tissues, and was positively associated with pathological grade and distant metastases of liver cancer. Knockdown of HMGB1 downregulated the expression of p-AKT, Ki-67 and MMP-2, inhibited the proliferative activities and metastatic potential of SMMC-7721 cells, induced cell cycle arrest and apoptosis, and slowed the growth of xenograft tumors. Altogether, the expression of HMGB1 is closely correlated with pathological grade and distant metastases of liver cancer, and knockdown of HMGB1 inhibits liver cancer growth and metastasis, suggesting that HMGB1 may be involved in liver cancer development and progression through AKT-mediated regulation of Ki-67 and MMP-2 expression, and represent a potential therapeutic target for this aggressive malignancy.

Targeting HMGB1 inhibits ovarian cancer growth and metastasis by lentivirus‐mediated RNA interference

High-mobility group box 1 (HMGB1), a nuclear and extracellular protein, is implicated in the development and progression of some types of cancers. However, no information is available to date regarding the function of HMGB1 in ovarian cancer. In this study, we performed cDNA microarray analysis and identified HMGB1 as a gene dramatically elevated in the highly invasive subclone S1 compared with the low invasive subclone S21 derived from the same cell line SKOV3. Then lentivirus vector with HMGB1 shRNA was constructed and infected the highly invasive cell line S1, A1, and HO8910PM. Real-time RT-PCR, Western blot, and IHC results confirmed the down-regulation of HMGB1 expression by its shRNA was about 80-90% at both the mRNA and protein levels. Knockdown of HMGB1 significantly suppressed ovarian cancer cell proliferation and induced cell cycle arrest at the G1/G0 phase, which was accompanied by decreased expressions of cyclin D1 and PCNA. Furthermore, knockdown of HMGB1 induced ovarian cancer cell apoptosis, which was mediated by increased expression of Bax and decreased expression of Bcl-2. Finally, knockdown of HMGB1 significantly inhibited ovarian cancer cell invasion and metastasis, which was regulated by decreased expressions of MMP2 and MMP9. Serum HMGB1 levels in patients with epithelial ovarian cancer were significantly higher than that in patients with benign ovarian tumor and healthy controls. These results indicate that HMGB1 is a newly identified gene associated with ovarian cancer growth and metastasis. HMGB1 may serve as a new therapeutic target for the treatment of ovarian cancer in the future.

High-mobility group box 1 activates caspase-1 and promotes hepatocellular carcinoma invasiveness and metastases

Hypoxia is often found in solid tumors and is associated with tumor progression and poor clinical outcomes. The exact mechanisms related to hypoxia-induced invasion and metastasis remain unclear. We elucidated the mechanism by which the nuclear-damage-associated molecular pattern molecule, high-mobility group box 1 (HMGB1), released under hypoxic stress, can induce an inflammatory response to promote invasion and metastasis in hepatocellular carcinoma (HCC) cells. Caspase-1 activation was found to occur in hypoxic HCC cells in a process that was dependent on the extracellular release of HMGB1 and subsequent activation of both Toll-like receptor 4 (TLR4)- and receptor for advanced glycation endproducts (RAGE)-signaling pathways. Downstream from hypoxia-induced caspase-1 activation, cleavage and release of proinflammatory cytokines interleukin (IL)-1β and -18 occurred. We further demonstrate that overexpression of HMGB1 or treatment with recombinant HMGB1 enhanced the invasiveness of HCC cells, whereas stable knockdown of HMGB1 remarkably reduced HCC invasion. Moreover, in a murine model of HCC pulmonary metastasis, stable knockdown of HMGB1 suppressed HCC invasion and metastasis. These results suggest that in hypoxic HCC cells, HMGB1 activates TLR4- and RAGE-signaling pathways to induce caspase-1 activation with the subsequent production of multiple inflammatory mediators, which, in turn, promote cancer invasion and metastasis.

Glycyrrhizic acid affords robust neuroprotection in the postischemic brain via anti-inflammatory effect by inhibiting HMGB1 phosphorylation and secretion

High mobility group box 1 (HMGB1) is an endogenous danger signal molecule. In a previous report, we showed that HMGB1 is massively released during NMDA-induced acute damaging process in the postischemic brain and triggers inflammatory processes, like microglial activation. siRNA-mediated HMGB1 knockdown markedly reduced infarct volumes, confirming the crucial role played by HMGB1 in the postischemic brain. In the present study, we showed neuroprotective effects of glycyrrhizin (GL) in the postischemic rat brain after middle cerebral artery occlusion (MCAO). GL, a triterpene present in the roots and rhizomes of licorice, Glycyrrhiza glabra, has been shown to have anti-inflammatory and anti-viral effects. It has been reported that GL binds directly to HMGB1, and inhibits its chemoattractant and mitogenic activities. The administration of GL (10mg/kg) intravenously at 3 or 6h after MCAO reduced infarct volumes to 12.9±4.2% and 46.2±9.9%, respectively, of untreated control. This neuroprotective effect was accompanied by improvements in motor impairment and neurological deficits and suppressions of microglia activation and proinflammatory cytokine induction. Interestingly, GL almost completely blocked HMGB1 secretion in the postischemic brain and in lipopolysaccharide (LPS)-treated microglia cells. Furthermore, HMGB1 phosphorylation, which is the initial step for HMGB1 secretion, and the interaction between HMGB1 and protein kinase C (PKC) or calcium/calmodulin-dependent protein kinase IV (CaMKIV) were suppressed dose-dependently by GL. Here, we hypothesized that the blockage for the putative phosphorylation sites in HMGB1 by GL might be attributed to this suppression. In addition to the anti-inflammatory effects, we found that GL has anti-excitotoxic and anti-oxidative effects in neurons. Together these results indicate that GL has neuroprotective efficacy in the postischemic brain via its anti-inflammatory, anti-excitotoxic, and anti-oxidative effects and in particular, it exerts anti-inflammatory effect, at least in part, by inhibiting HMGB1 secretion.

Reduced High-Mobility Group Box 1 Expression Induced by RNA Interference Inhibits the Bioactivity of Hepatocellular Carcinoma Cell Line HCCLM3

Increased expression of high-mobility group box 1 (HMGB1) has been observed in many tumor types, but the role of HMGB1 in hepatocellular carcinoma (HCC) is unknown. To examine the effects of RNA interference HMGB1 on the bioactivity of HCC cell line HCCLM3. We synthesized three specific small interfering RNAs of HMGB1 (HMGB1-siRNAs) and transfected these into HCCLM3 cells by use of Lipofectamine 2000. RT-PCR and Western blot were performed to determine the effects of HMGB1-siRNAs on HMGB1 expression and to detect NF-κB/p65 and VEGF-C expression after transfection of HMGB1-siRNAs into HCCLM3. In vitro proliferation was assessed by MTT assay. Migration and invasive ability were determined by use of the Transwell assay. Apoptosis was demonstrated by flow cytometry. RT-PCR and Western blotting showed that all three specific HMGB1-siRNAs significantly inhibited HMGB1 expression, with inhibition by HMGB1-siRNA-1 being highest (70-80%). MTT assay demonstrated that the growth of cells transfected with HMGB1-siRNA-1 was significantly lower than that of control cells (P < 0.01). The Transwell assay showed that cell migration and invasion were significantly inhibited in HMGB1 knockdown cells compared with control cells (P < 0.01). FCM revealed that apoptosis was significantly increased in HMGB1-siRNA-1-transfected cells compared with control cells (P < 0.01). Expression of NF-κB/p65 and VEGF-C was inhibited in HCCLM3 cells transfected with HMGB1-siRNA-1 compared with control cells (P < 0.01). Downregulation of HMGB1 could obviously inhibit the growth of HCCLM3 cells, and their migration and invasion ability. HMGB1 may serve as a potential target for treatment of HCC.

Knockdown of HMGB1 in Tumor Cells Attenuates Their Ability To Induce Regulatory T Cells and Uncovers Naturally Acquired CD8 T Cell-Dependent Antitumor Immunity

Although high mobility group box 1 (HMGB1) in tumor cells is involved in many aspects of tumor progression, its role in tumor immune suppression remains elusive. Host cell-derived IL-10 suppressed a naturally acquired CD8 T cell-dependent antitumor response. The suppressive activity of tumor-associated Foxp3(+)CD4(+)CD25(+) regulatory T cells (Treg) was IL-10 dependent. Neutralizing HMGB1 impaired tumor cell-promoted IL-10 production by Treg. Short hairpin RNA-mediated knockdown of HMGB1 (HMGB1 KD) in tumor cells did not affect tumor cell growth but uncovered naturally acquired long-lasting tumor-specific IFN-γ- or TNF-α-producing CD8 T cell responses and attenuated their ability to induce Treg, leading to naturally acquired CD8 T cell- or IFN-γ-dependent tumor rejection. The data suggest that tumor cell-derived HMGB1 may suppress naturally acquired CD8 T cell-dependent antitumor immunity via enhancing Treg to produce IL-10, which is necessary for Treg-mediated immune suppression.

Extracellular HMGB1 Released by NMDA Treatment Confers Neuronal Apoptosis via RAGE-p38 MAPK/ERK Signaling Pathway

High mobility group box 1 (HMGB1) was originally identified as ubiquitously expressed nonhistone DNA-binding protein, but recently, it was found to act as an endogenous danger molecule, which signals danger and traumatic cell death. Previously, the authors showed that HMGB1 is massively released immediately after an ischemic insult and that it subsequently activates microglia and induces inflammation in the postischemic brain. Here, we showed the endogenous danger molecule-like function of HMGB1 in primary cortical cultures. HMGB1 was found to be accumulated in NMDA-treated primary cortical culture media, and media collected from these cultures were able to induce neuronal cell death when added to fresh primary cortical cultures. However, HMGB1-depleted NMDA-conditioned media produced by HMGB1 siRNA transfection or by preincubation with anti-HMGB1 antibody or with HMGB1 A box failed to induce neuronal cell death. Furthermore, siRNA-mediated HMGB1 knockdown substantially suppressed NMDA- or Zn(2+)-induced cell death. It was interesting to find that extracellular HMGB1-induced neuronal apoptosis, as evidenced by TUNEL staining and caspase 3 assay in combination with double immunofluorescence staining. A series of RAGE and HMGB1 co-immunoprecipitation experiments in the presence of SB203580 and PD98059 (p38 MAPK and ERK inhibitors, respectively) demonstrated that RAGE-p38 MAPK and RAGE-ERK pathway might underlie extracellular HMGB1-mediated neuronal apoptosis. These results together with our previous reports regarding microglial activation by extracellular HMGB1 indicate that HMGB1 functions as a novel danger signal, which aggravates brain damage via autocrine and paracrine manners.