1D) These findings suggest

that HMGB1 may, in fact, be a

1D). These findings suggest

that HMGB1 may, in fact, be actively released into the circulation of patients with HCC. Hypoxia is a hallmark of solid tumors, including HCC.8 Accordingly, we found hypoxia-inducible factor-1 alpha levels to be substantially increased in liver homogenates of HCC specimens, compared to nontumor tissue (Supporting Fig. 1). Immunofluorescence Sirolimus ic50 showed that HMGB1 mainly localized in the nucleus of hepatocytes in the nontumorous liver. However, in addition to nuclear HMGB1, cytoplasmic HMGB1 was also present at high levels in HCC cells (Fig. 1E). To determine whether hypoxia plays a role in inducing the expression or translocation of HMGB1 in HCC cells, Hepa1-6 and Huh7 cells were cultured under normoxic (21% O2) or hypoxic (1% O2) conditions. Under normoxia in both cell lines, HMGB1 remained located predominantly in the nucleus. After exposure

to hypoxia, the expression of HMGB1 increased in the cytoplasm Bortezomib chemical structure (Fig. 1F). These findings indicate that hypoxia leads to the nuclear to cytoplasmic translocation of HMGB1 in HCC cells. To determine whether hypoxia induces the expression or translocation of HMGB1 in HCC, western blotting analysis was performed. The expression of HMGB1 in whole cell lysates was not significantly changed during hypoxia at either the protein or mRNA level (Fig. 2A; Supporting Fig. 2). In contrast, hypoxia led to a time-dependent increase of HMGB1 translocation to the cytoplasm and HMGB1 release into the media in both cell lines (Fig. 2B,C). In addition, cell viability was not substantially different, when

comparing HCC cells exposed to hypoxia and normoxia, as assessed in a crystal violet viability assay (Fig. 2D). These findings demonstrate that hypoxia leads to the translocation and release of HMGB1 in HCC cells. Caspases play an important role in programmed cell death and inflammation.16, 19 Though apoptosis inducers caspase-3 and -9 were activated in our hypoxic HCC cells (data not shown), we focused on caspase-1, which can induce inflammation and affect tumor progression. To characterize caspase-1 activation during hypoxia, Hepa1-6 and Huh7 cells were learn more cultured under hypoxic conditions for various times. The 45-kDa procaspase-1 was cleaved into the active 20-kDa caspase-1 during hypoxia and was increased in a time-dependent manner during hypoxia (Fig. 3A,B). Using a colorimetric assay to assess caspase-1 activity during hypoxia, we found that caspase-1 activity was also significantly increased in both Hepa1-6 and Huh7 cells subjected to hypoxia, compared to normoxic controls (Fig. 3C). To confirm the localization of cleaved caspase-1, we performed immunofluorescent staining for cleaved caspase-1. After hypoxia, cleaved caspase-1 significantly increased in the cytoplasm diffusely in both cell lines (Supporting Fig. 3). These findings demonstrate that hypoxia induces caspase-1 activation in HCC cells.

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