Although many cancers arise from chronic inflammation, the relationships between carcinogenesis, cancer promotion, and its molecular characteristics remain poorly understood. Hepatocellular carcinoma (HCC), which typifies an inflammation-related tumor, is one of the most common malignancies in the world, especially in Asia and Africa. Japan has a high incidence of chronic viral hepatitis, cirrhosis, and HCC. The resolution of inflammatory activity at the molecular level may correlate with prevention of hepatocarcinogenesis and cancer promotion. 1 2 3 5 6 5 7 8 9 10 11 12 13 14 15 15 16 17 MATERIAL AND METHODS Human samples 18 19 20 21 1 TABLE 1. Background of patients Gender   Male 30 (83%)   Female 6 (17%) Mean age 67.2 years Virus type   B 7 (19.4%)   C 21 (58.3%)   None 8 (22.2%) Background of livers   Chronic hepatitis 24 (60.7%)   Liver cirrhosis 4 (11.1%)   Normal liver 8 (22.2%)   Mean tumor size  49.7mm Histological grade   Well differentiated 4 (11.1%)   Moderately differentiated 28 (77.8%)   Poorly differentiated 4 (11.1%) Postoperative tumor recurrence 11 (30.6%). Immunohistochemistry 2 2 Cell and cell culture 2 Gene silencing of RAGE with specific siRNA 5  TM MTT assay Cell viability was monitored after incubation for 24, 36, and 48 hours by MTT assay. Briefly, 0.5 mg/mL 3-[4,5]-2,5-diphenyltetrazolium bromide (MTT) in fresh medium was added to each well and the cells were incubated for an additional 3 hours. Afterwards, the blue formazan crystals were dissolved in 1 mL isopropanol and measured spectrophotometrically at 570 nm. Immunoblot analysis Whole cell lysates were prepared as per the Santa Cruz protocol. One milliliter of Radio-Immunoprecipitation Assay (RIPA) buffer was added to a 100 mm cell culture plate. The plates were gently rocked for 15 min at 4°C. Adherent cells were scrpaed with a cell scraper, followed by incubation for 30–60 min on ice. The cell lysate was microcentrifuged at 10,000g for 10 min at 4°C. The supernatant fluid was the total cell lysate. The supernatant was transferred to a new microfuge tube and the pellet was discarded. Twelve microgram lysates were subjected to immunoblot analysis using a 12.5% sodium dodecyl sulfate (SDS) -polyacrylamide gel followed by electrotransfer onto nitrocellulose filters. The filters were immunoreacted with anti-RAGE antibody (a gift from TORAY Research Institute, Sagamihara, Japan) or with anti-HMGB1 antibody (BD Biosciences, Tokyo, Japan) and then incubated with peroxidase-conjugated anti-goat IgG (Medical and Biological Laboratories, Nagoya, Japan). The immune complex was visualized using the Enhanced Chemiluminescence (ECL) Western blot detection system (PIERCE, Rockford, IL, USA). The amount of B-actin as an internal control was also examined using a specific antibody (Cytoskelton Inc., Denver, CO, USA and Santa Cruz, CA, USA). At least three independent experiments were performed. Quantitative RT-PCR 2 R R Statistical analysis t t U χ 2  p RESULTS RAGE and HMGB1 expression in normal liver, hepatitis, and HCC 1 FIG. 1. (a) RAGE expression by Western blotting (A) and RT-PCR (B). (b) HMGB1 expression by Western blotting (A) and RT-PCR (B). Abbreviations: normal, normal liver; CH, chronic hepatitis.  Quantitative RAGE mRNA expression in HCC and noncancerous lesions p 2 p p 2 FIG. 2. n n  Relationship between RAGE mRNA expression and clinicopathological features 2 23 p p TABLE 2. Relationship between tumor RAGE expression and clinicopathological features Factors Tumor RAGE mRNA expression p n Mean ± SD Gender   Male 30 2.267 ± 0.341 0.03   female 6 2.777 ± 0.619  Age   ≥65 years 26 2.468 ± 0.568 0.03   <65 years 10 2.052 ± 0.638  Tumor size (mm)   ≥30 11 2.395 ± 0.613 0.39   <30 25 2.333 ± 0.620  Portal invasion   Absent 22 2.358 ± 0.655 0.52   Present 14 2.348 ± 0.594  Venous invasion   Absent 26 2.339 ± 0.577 0.47   Present 10 2.357 ± 0.633  Vascular invasion   Absent 19 2.362 ± 0.633 0.46   Present 17 2.340 ± 0.601  Intrahepatic metastasis   Absent 27 2.378 ± 0.662 0.33   Present 9 2.273 ± 0.435  Gross classification   Localized type 21 2.448 ± 0.636 0.13   Invasive type 15 2.217 ± 0.564  Differentiation   Well 4 2.365 ± 0.566     0.43   Moderately 28 2.420 ± 0.580     0.08   Poorly 4 1.860 ± 0.783  Stage   I,II 16 2.367 ± 0.643 0.55   III,IV 20 2.340 ± 0.597  PIVKA‡U   Normal 9 2.704 ± 0.385    High 25 2.186 ± 0.630 0.03 AFP   Normal 12 2.557 ± 0.586 0.16   High 24 2.250 ± 0.607  Virus   B 7 2.237 ± 0.606    C 21 2.382 ± 0.569    None 8 2.374 ± 0.779  Recurrence   Absent 25 2.485 ± 0.628 0.04   Present 11 2.050 ± 0.456  RAGE expression and tumor differentiation in HCC 3 3 FIG. 3. RAGE expression by immunohistochemical staining: (a) well-differentiated HCC, (b) moderately (mod.) differentiated HCC, (c) poorly differentiated HCC, (e) the number of RAGE positive or negative cases according to tumor differentiation.  RAGE expression in hepatoma cell lines 4 FIG. 4. (a) RAGE expression in hepatoma cell lines by Western blotting. (b) RAGE expression in hepatoma cell lines under hypoxic conditions by Western blotting. N, normoxic conditions; A, anaerobic conditions.  Enhanced RAGE expression under hypoxic conditions 15 23 24 4 Enhancement of cell survival in anaerobic conditions following RAGE transfection 5 FIG. 5. Comparison of cell survival of RAGE-transfected Cos7 and mock-transfected Cos7 cells under anaerobic conditions. Cell survival of both groups was estimated from six dishes at each time point using the MTT assay. *p < 0.01 by Student’s t-test.  Decline of cell survival in hypoxic conditions following RAGE reduced by siRNA 6 FIG. 6. RAGE level in RAGE-transfected Cos7 cells were reduced by siRNA RAGE-transfected Cos7 cells reduced RAGE level by siRNA decreased more than the cells mixed with only transfection reagent. *p < 0.01 by Student’s t-test.  DISCUSSION 19 25 25 26 27 28 The present study demonstrated that increased RAGE expression was highly associated with the status of pathological “differentiation” in HCC, which played a significant role in acquisition of the hypoxia-resistant phenotype of tumor cells. This conclusion is supported by several lines of experimental evidence. First, the level of RAGE expression was higher in well- and moderately differentiated HCC, while it diminished as the tumors dedifferentiated to poorly differentiated HCC. This was consistent with the evidence that a negative correlation was observed between the level of RAGE mRNA expression and either the level of PIVKA II or the incidence of postoperative recurrence. Second, the analysis of five HCC lines revealed that three of these (Li7, Hep3B, and HuH7) that are resistant to hypoxic stress characteristically showed higher levels of RAGE expression compared to the two hypoxia-intolerant cell lines HepG2 and HT17. Third, sublethal hypoxia exposure induced significantly increased RAGE expression in hypoxia-resistant HCC lines. In the analysis of the association between the level of RAGE expression and the “differentiation status” of HCC lines, the level of RAGE expression was higher in “possibly differentiated” lines (i.e., HCC with low Alpha-Fet protein (AFP) production), consistent with the results from clinical samples. Finally, cells overexpressing RAGE exogenously showed prolonged survival under hypoxic conditions compared to control mock-transfected cells, and siRNA experiments demonstrated similar results. 7 15 29 30 31 33 9 11 14 34 FIG. 7 Scheme of the change of RAGE expression according to the sequential change of liver tissue: normal → chronic hepatitis (CH) → cirrhosis → HCC. The value indicates the quantitative RAGE mRNA expression.  Some reports have shown that RAGE-expressed cells have invasion and migration potential. Our data from clinical samples did no’ relate to the potential. In vivo, invasive and metastatic potential are reflected by many factors, which may have caused our results. Our results raise many questions concerning mechanistic and practical processes. It is important to know whether RAGE can bind HMGB1 secreted from activated macrophages in hepatic inflammation, or if occupancy of RAGE by inhibitors would obviate binding of the stimulatory ligands. The functional role of downstream signaling of the RAGE would also be important to determine the cytoprotective mechanism under hypoxia. Our findings have provided the first evidence of the clinical relevance and function of RAGE in HCC, namely differentiation-associated RAGE expression that confers a hypoxia-resistant phenotype. Although other mechanisms may also be important, our data also introduce the concept that RAGE and its functions may be possible candidates for therapeutic targets in the treatment of HCC.