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. 2012 Jun;55(6):1863-75.
doi: 10.1002/hep.25572.

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

Wei Yan  1 , Ying Chang, Xiaoyan Liang, Jon S Cardinal, Hai Huang, Stephen H Thorne, Satdarshan P S Monga, David A Geller, Michael T Lotze, Allan Tsung
Affiliations

"VSports在线直播" High-mobility group box 1 activates caspase-1 and promotes hepatocellular carcinoma invasiveness and metastases

"VSports最新版本" Wei Yan et al. Hepatology. 2012 Jun.

"V体育ios版" Abstract

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 VSports手机版. 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. .

Conclusion: 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. V体育安卓版.

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VSports手机版 - Figures

Fig. 1
Fig. 1. Overexpression and nuclear and cytoplasmic localization of HMGB1 in HCC cells
(A) HMGB1 protein levels were measured with western blot in twenty paired HCC samples and their nontumor counterparts. Protein expression results were normalized to internal control β-actin. ***P<0.001. N, nontumorous liver; T, tumor. (B) Expressions of HMGB1 in two primary hepatocytes (mouse and human hepatocytes) and five HCC cell lines were detected by western blot analysis. (C) The expression of HMGB1 in cytoplasm was measured with in HCC samples and their nontumor counterparts. (D) The expression of HMGB1 in normal and HCC serums were detected by ELISA. **P <0.01. (E) The nontumor and HCC tissues were stained for HMGB1. Red, HMGB1; blue, nuclei. (F) Hepa1-6 and Huh7 cells were analyzed in normoxia or hypoxia by immunostaining. Green, HMGB1; blue, nuclei; red, F-actin. Imaging shown is representative of three experiments with similar results.
Fig. 2
Fig. 2. Hypoxia induces translocation and release of HMGB1
(A) The expression of HMGB1 was determined by western blot in Hepa1-6 and Huh7 cells subjected to 24 h of hypoxia (1% O2). Cell supernatants and cytoplasmic extracts from Hepa1-6 cells (B) or Huh7 cells (C) that were subjected to a time course of hypoxia were collected. HMGB1 was determined by Western Blot. The blot shown is representative of three experiments with similar results. (D) Viability was determined by crystal violet assay on Hepa1-6 and Huh7 cells subjected to 24 h of hypoxia.
Fig. 3
Fig. 3. Hypoxia induces caspase-1 activation in HCC cells
Caspase-1 expression was determined by western blot in Hepa1-6 cells (A) or Huh7 cells (B) subjected to a time course of hypoxia. Blot shown is representative of three experiments with similar results. (C) Whole cell lysates were extracted from Hepa1-6 and Huh7 cells. Caspase-1 activity was determined by colorimetric assay.*P <0.05. Assay shown is representative of three experiments with similar results.
Fig. 4
Fig. 4. HMGB1 is necessary for hypoxia-induced activation of caspase-1 and induces activation of caspase-1 in normoxia
(A) Western blot analysis for caspase-1 from Hepa1-6 cells treated with 10 mM ethyl pyruvate (EP) or 1 μg/ml anti-HMGB1 neutralizing antibody (a-HMGB1) in hypoxia. The blot shown is representative of three experiments with similar results. (B) Caspase-1 activity was determined by colorimetric assay after various treatments. *P < 0.05 versus Hepa1-6 cells that were subjected to hypoxia. (C) Western blot analysis of caspase-1 in Hepa 1-6 cells treated with various dose rhHMGB1 for 24h. (D) Western blot analysis of caspase-1 in Hepa1-6 cells treated with 1 μg/ml rhHMGB1 for various times. (E) Ectopic overexpression of HMGB1-GFP fusion protein in Hepa1-6 stable clones. GFP and HMGB1-GFP fusion expression were confirmed via western blot analysis with GFP antibody. (F) Cleaved caspase-1 was significantly increased in HMGB1 stable transfectants as compared with vector controls via western blot analysis.
Fig. 5
Fig. 5. Signaling pathways downstream of HMGB1 are involved in hypoxia-induced activation of caspase-1
(A) Expression of TLR4 from Hepa1-6 cells subjected to hypoxia for various time points. (B) Western blot analysis for caspase-1 from hypoxic Hepa1-6 cells transfected with scrambled siRNA or TLR4 siRNA or treated with 5 μg/ml anti-TLR4 neutralizing antibody (a-TLR4). (C) Expression of RAGE from Hepa1-6 cells subjected to hypoxia for various time points. (D) Western blot analysis for caspase-1 from hypoxic Hepa1-6 cells transfected with scrambled siRNA or RAGE siRNA or treated with 5 μg/ml sRAGE. (E) Western blot analysis for caspase-1 from Hepa1-6 cells treated with a-TLR4 or/and sRAGE. The blot shown is representative of three experiments with similar results.
Fig. 6
Fig. 6. NLRP3-dependent caspase-1 activation promotes cleavage of IL-1β, IL-18 in hypoxia
(A) Western blot analysis for caspase-1 from Hepa1-6 cells treated with NLRP3 siRNA in hypoxia. (B) Hepa1-6 cells were treated with a caspase-1 inhibitor Z-YVAD-FMK (10 μM) and then analyzed by western blot for caspase-1. (C) Western blot analysis for IL-1β after treatment with Z-YVAD-FMK. (D) Expression of IL-18 from Hepa1-6 cells was determined by western blot.
Fig. 7
Fig. 7. HMGB1-induced caspase-1 activation is involved in hypoxia-induced cell invasion
(A) Transwell chamber studies were performed for 24 h in 21% O2 or 1.0% O2. Migration and invasion are expressed relative to the levels observed with normoxic controls. (mean ± SEM; n = 6). **P < 0.01. (B) Hepa1-6 cells were treated with rhHMGB1 (1 μg/ml) or Z-YVAD-FMK (10 μM) in normoxia or a-HMGB1 (1 μg/ml) or Z-YVAD-FMK in hypoxia. Cells were allowed to invade matrigel for 24 h. (C) The number of invasive cells was quantified. Invasion is expressed relative to the levels observed with normoxic controls. *P < 0.05; **P < 0.01. (D) Huh7 cells were treated with a-HMGB1 or Z-YVAD-FMK in hypoxia. Cells were allowed to invade matrigel for 24 h. (E) The number of invasive cells was quantified. Invasion is expressed relative to the levels observed with normoxic controls. *P < 0.05.
Fig. 8
Fig. 8. HMGB1 promotes HCC cells invasion and metastasis in vitro and in vivo
Stable HMGB1 overexpressing (A) or knockdown cells (B) were seeded onto the upper chamber of the transwell and allowed to invade matrigel for 24 h. The number of invasive cells was quantified. Invasion is expressed relative to the levels observed with vector controls. **P < 0.01. (C) A luciferase signal was generated from luciferase-labeled Hepa1-6 cells. The signal was observed in the lungs of the C57BL/6 mice. A stronger signal was observed in the vector control group. (D) The luciferase signal was determined once a week in both the vector control group and the shRNA group. Each group contained five mice. (E) The HMGB1 in serum was measured by ELISA in different groups. (F) Metastatic nodules on the surface of lung are shown (indicated by arrows). The numbers of tumor nodules were quantified.
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