| IntroductionHepatocellular carcinoma (HCC) is the fifth commonest malignancy and the third leading cause of cancer death in the world, being responsible for 80% of the primary malignant liver tumors in adults. The five-year relative survival rate is about 7% and causes more than 600,000 deaths annually worldwide. Although the prevelance is highest in Africa and Asia, the incidence in western countries is rising, mainly due to increasing rates of alcoholic liver diseases and hepatitis C infection. HCC has several interesting epidemiologic features including dynamic temporal trends; marked variations among geographic regions, racial and ethnic groups, and between men and women; and the presence of several well-documented environmental potentially preventable risk factors.Global gene expression profiling of HCC is a promising new technology that has already refined the diagnosis and prognostic prediction of HCC patients. As expected, prediction models based on gene expression profiling discriminate HCC from non-tumor livers with high accuracy successfully. A great deal of studies have identified differentially expressed genes in HCC that differ according to etiological factors, mutations of tumor suppressor genes, rate of recurrence, intrahepatic metastasis, and different stages. However, most of these studies have identified genes associated with limited aspects of tumor pathogenesis, but do not necessarily reflect the underlying biological properties likely driving tumor behavior. This is because the level of mRNA do not always reflect the accurately the levels of corresponding proteins nor do they reveal changes in epigenetic posttranscriptional modulation of proteins or changes in proteins degradation rates. Therefore, it is important to analyze difference in protein levels or modification between tumor cells and normal cells to complement studies on mRNA expression. The Pathway Array Technique is an innovative proteomic assay that allows screen globally the changes in protein expression. The focus of pathway array is to determine the signaling network that related to cancer development (initiation, promotion, invasion). The proteins selected from those highly expressed in cancer cells are functionally linked to the angiogenesis, apoptosis, cell cycle regulation, DNA repair, migration, proliferation, signaling, stem cell association and transcription activity. In our study, we will mainly use this technique to determine changes of protein levels and signaling networks in HCC.Human hepatocellular carcinomas (HCCs) usually develop as late complications of chronic liver injury, and are characterized by mononuclear cell infiltration of the parenchyma at the margin of liver lobules and apoptotic disappearance of contiguous hepatocytes. Failure of apoptosis is considered to be important for the survival of hepatocytes that are prone to undergo genetic damage and cellular transformation by an increased proliferative rate due to hepatocyte regeneration. In fact, most HCC cells show strong resistance to various stimuli that induce apoptosis. However, the anti-apoptotic mechanisms of HCC cells have not been well elucidated.Recently, it was demonstrated that the inhibitors of apoptosis (IAP) are crucial regulators in the molecular mechanism of apoptosis. To date,6 human IAP proteins, X-linked IAP (XIAP), c-IAP1, c-IAP2, NAIP, survivin, and Apollon have been cloned. They are characterized by the presence of 1-3 copies of a 70-amino acid motif, the BIR domain, which bears homology to sequences found in the baculovirus IAP proteins and plays an important role in binding and inhibition of caspase, particularly caspase 3 and 7. In addition, c-IAP1 and c-IAP2 were shown to interact with tumor necrosis factor (TNF) receptor-associated factor(TRAF)-1 and (TRAF)-2 in the yeast 2-hybrid system. These results suggested that c-IAPl and c-IAP2 are involved in regulation of TNF-induced NF-κB activation via TRAF. The XIAP protein consists of 3 BIR domains and 1 RING finger domain and appears to be a more potent inhibitor of caspase 3 and 7 than c-IAP1 and c-IAP2. XIAP mRNA was observed in all adult and fetal tissues examined except peripheral blood leukocytes, indicating that it is a ubiquitously expressed member of the IAP family.Therefore, in our study, we examined the level of expression and phosphorylation of important proteins involved in cell proliferation, apoptosis and cell cycle progression in HCC using our newly developed Protein Pathway Array technology. We demonstrated the significant crosstalk between cell cycle progression and apoptosis in HCC via a central regulatory protein, XIAP. This finding will certainly add in future development of effective anti-HCC therapy.Methods1. We collected 12-paired liver resection tissue samples including tumor tissues and normal tissues, extracted proteins to run pathway array analysis, in order to determine their expression levels.2. After embelin, as XIAP specific inhibitor, treating HCC cells, we proceeded cell viability analysis, cell cycle analysis and cell apoptosis analysis to make sure whether embelin could effect HCC cell proliferation, cell cycle and cell apoptosis.3. After embelin treating HCC cells, we examined protein expression levels of XIAP, CDK4, CDK6 and cyclin D1 by western-blot. Subsequently, we confirmed XIAP, CDK4, CDK6 and cyclin D1 expression through transfection of XIAP siRNA and XIAP expression vector into Huh7 cells.4. Formalin-fixed, paraffin-embedded tissue samples from 59 primary human HCC were proceeded to immunohistochemical staining about target proteins.Results1. Twelve pairs of HCC and surrounding tissues were assessed for the expression of proteins and phosphorylation using Protein Pathway Array. Fifty four percent tumors had increased expression of XIAP and 46% had increased expression of cdk6. Interestingly,46% tumor tissues had increased expression of both XIAP and cdk6. Similarly,8% tumors had increased expression of XIAP, cyclinDl and cdk4.2. Our results showed that the number of viable cells decreased with increase of embelin, suggesting an effective inhibition of HCC cell growth. The 50% inhibition concentration (IC50) at 24 hours was 35 uM for HepG2/2.2.1 cells and 25 uM for Huh7 cells. Therefore, it was proved that XIAP could promote cell proliferation in HCC.3. Our results showed that embelin significantly blocked Huh7 cell growth at G1 phase (F=8.040, P=0.008) with concomitant decrease of S phases (F=7.104, P=0.012). It was confirmed that XIAP could induce cell entering to G1 phase, then leading to cell proliferation as the result in HCC.4. Our results showed that apoptotic cell number increased with increase of embelin concentration (F=3169.485, P=0.000). The percentage of apoptotic cells increased from 1.430% in control cells to 7.967% in treated cells. XIAP could actuate cell proliferation through inducing cell entering to G1 phase, and inhibiting cell apoptosis in HCC.5. Our results also showed embelin significantly inhibited the expression of XIAP, CDK4/6 and cyclin D1 in a dose-dependent manner. To further determine the regulatory role of XIAP on CDK4/6 and cyclin D1, siRNA specific to XIAP was transiently introduced into the Huh 7 cells. As a consequence, the protein level of XIAP, CDK 4/6 and cyclin D1 was also reduced. To further confirm this relationship, the expression vector containing XIAP gene was also introduced into Huh 7 cells. The results showed that the protein level of CDK4/6 and cyclin D1 was also increased.6. The patients with a high XIAP expression had a significantly lower overall survival rate as compared to those with low XIAP expression (x2=8.384, P=0.004). Similarly, lower overall survival rates were observed in patients with high level expression of CDK4 (x2=12.721, P =0.000)and cyclin D1(x2=20.894, P=0.000) as compared to low level expression groups. The 3-year-survival rates were 90.0% for group with low XIAP and low CDK4 expression (Group 1),66.7% for group with divergent XIAP and CDK4 expression (Group 3) and 12.9% for group with high XIAP and high CDK4 expression (Group 5). The prognosis of group with high XIAP and high CDK4 expression (Group 5) was much more poor than group with divergent XIAP and CDK4 expression (Group 3)(x2=9.833, P=0.002)and group with low XIAP and low CDK4 expression (Group 1) (x2=16.899, P=0.000). Similarly, the 3-year-survival rates were 83.3% for group with low XIAP and low cyclin D1 expression (Group 2), 54.5% for group with divergent XIAP and cyclin D1 expression (Group 4) and 12.0% for group with high XIAP and high cyclin D1 expression (Group 6). The prognosis of group with high XIAP and high cyclin D1 expression (Group 6) was much more poor than group with divergent XIAP and cyclin D1 expression (Group 4) (x 2=12.006, P =0.001) and group with low XIAP and low cyclin D1 expression (Group 2) (x 2=15.979, P=0.000). Finally, we performed Cox multivariate regression analysis to determine the independent prognostic factors among XIAP,CDK4,cyclin D1,XIAP+CDK4/cyclin D1,maximal tumor size. We found XIAP+CDK4,maximal tumor size could be used as independent factors for overall survival of HCC patiets.Conclusions 1. XIAP, CDK4, CDK6, cyclin D1 were significantly overexpressed in HCC tissues, suggesting significant roles of these proteins in HCC development. Furthermore, co-expression of XIAP with cdk4/cdk6/cyclinD1 suggests a regulatory relation between XIAP and cyclinDl/cdk4/cdk6.2. XIAP could promote cell proliferation through regulating G1 protein expression of CDK4, CDK6 and cyclin D1,leading cell entering to G1 cell cycle and reducing cell apoptosis in HCC cells.3. Co-expression of XIAP and cyclin D1/CDK4 correlated with a poor prognosis of the HCC patients, suggesting a regulatory link between cyclin D1/CDK4 and XIAP. XIAP+CDK4 could be used as independent factor for overall survival of HCC patients.
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