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1.  Bax Inhibitor-1 down-regulation in the progression of chronic liver diseases 
BMC Gastroenterology  2010;10:35.
Background
Bax inhibitor-1 (BI-1) is an evolutionary conserved endoplasmic reticulum protein that, when overexpressed in mammalian cells, suppresses the apoptosis induced by Bax, a pro-apoptotic member of the Bcl-2 family. The aims of this study were: (1) to clarify the role of intrinsic anti- and pro-apoptotic mediators, evaluating Bax and BI-1 mRNA and protein expressions in liver tissues from patients with different degrees of liver damage; (2) to determine whether HCV and HBV infections modulate said expression.
Methods
We examined 62 patients: 39 with chronic hepatitis (CH) (31 HCV-related and 8 HBV-related); 7 with cirrhosis (6 HCV-related and 1 HBV-related); 13 with hepatocellular carcinoma (HCC) [7 in viral cirrhosis (6 HCV- and 1 HBV-related), 6 in non-viral cirrhosis]; and 3 controls. Bax and BI-1 mRNAs were quantified by real-time PCR, and BI-1 protein expression by Western blot.
Results
CH tissues expressed significantly higher BI-1 mRNA levels than cirrhotic tissues surrounding HCC (P < 0.0001) or HCC (P < 0.0001). Significantly higher Bax transcripts were observed in HCV-genotype-1-related than in HCV-genotype-3-related CH (P = 0.033). A positive correlation emerged between BI-1 and Bax transcripts in CH tissues, even when HCV-related CH and HCV-genotype-1-related CH were considered alone (P = 0.0007, P = 0.0005 and P = 0.0017, respectively).
Conclusions
BI-1 expression is down-regulated as liver damage progresses. The high BI-1 mRNAs levels observed in early liver disease may protect virus-infected cells against apoptosis, while their progressive downregulation may facilitate hepatocellular carcinogenesis. HCV genotype seems to have a relevant role in Bax transcript expression.
doi:10.1186/1471-230X-10-35
PMCID: PMC2873598  PMID: 20359348
2.  The expression of beclin-1, an autophagic gene, in hepatocellular carcinoma associated with clinical pathological and prognostic significance 
BMC Cancer  2014;14:327.
Background
A role for autophagy, a conserved cellular response to stress, has recently been demonstrated in human cancers. Aberrant expression of Beclin-1, an important autophagic gene, has been reported in various human cancers. In the present study, we investigated the significance and relationship between Beclin-1 expression and cell proliferation, apoptosis, microvessel density (MVD) and clinical pathological changes or prognosis in human hepatocellular carcinoma (HCC).
Methods
A total of 103 primary HCC patients were involved in the study. Expression of Beclin-1, PCNA, NET-1, Bcl-2, Bax, Survivin in cancer cells and CD34 in stromal microvessels were evaluated immunohistochemically in tissue microarrays comprising 103 cases of HCC and 57 matched adjacent nontumor liver tissues. Correlations between clinicopathological characteristics and survival of HCC patients were explored.
Results
The positive rate of Beclin-1 was significantly lower in HCC tissues than adjacent tissues (72.8 vs. 89.5%, χ2 = 6.085, P = 0.015). In HCC, Beclin-1 expression was negatively correlated with cirrhosis background (r = −0.216, P = 0.029), Edmondson grade (r = −0.249, P = 0.011), vascular invasion (r = −0.246, P = 0.012), PCNA (r = −0.242, P = 0.014), NET-1 (r = −0.245, P = 0.013), anti-apoptosis protein Bcl-2 (r = −0.245, P = 0.013) and MVD (r = −0.292, P = 0.003), and positively correlated with pro-apoptosis protein Bax (r = 0.242, P = 0.014).
Significant differences in the 5-year survival rates were seen among patients with Beclin-1 strong positive (++) (59.1%, 13/22), moderate positive (+) (28.3%, 15/53) and weak negative expression (−) (14.6%, 7/28) (P = 0.043). Significant differences were detected between Beclin-1 (++) and either Beclin-1 (+) (P = 0.036) or Beclin-1 (−) groups (P = 0.008), but no significant difference between Beclin-1 (+) and Beclin-1 (−) groups (P = 0.281) was observed.
Survival rates were positively related to high Beclin-1 co-expressed with low PCNA, NET-1, or Bcl-2, lower MVD, and high Bax. Univariate and multivariate Cox regression analysis revealed that Beclin-1 expression was an independent indicator for overall survival in HCC patients (P < 0.05).
Conclusions
The pathogenesis and progression of HCC are associated with reduced autophagy. The expression of Beclin-1 and Bax in HCC tissues may provide a synergistic effect towards inhibiting HCC proliferation, infiltration, metastasis and angiogenesis. Beclin-1 expression may be a valuable prognostic marker of HCC.
doi:10.1186/1471-2407-14-327
PMCID: PMC4020872  PMID: 24885292
Beclin-1; Autophagy; HCC; Prognosis proliferation, Apoptosis; Microvessels
3.  Oxidative DNA damage correlates with cell immortalization and mir-92 expression in hepatocellular carcinoma 
BMC Cancer  2012;12:177.
Background
MicroRNAs expression has been extensively studied in hepatocellular carcinoma but little is known regarding the relationship, if any, with inflammation, production of reactive oxygen species (ROS), host’s repair mechanisms and cell immortalization. This study aimed at assessing the extent of oxidative DNA damage (8-hydroxydeoxyguanosine - 8-OHdG) in different phases of the carcinogenetic process, in relation to DNA repair gene polymorphism, telomeric dysfunction and to the expression of several microRNAs, non-coding genes involved in post-transcriptional regulation, cell proliferation, differentiation and death.
Methods
Tissue samples obtained either at surgery, [neoplastic (HCC) and adjacent non-cancerous cirrhotic tissues (NCCT)] at percutaneous or laparoscopic biopsy (patients with HCV or HBV-related hepatitis or patients undergoing cholecystectomy) were analysed for 8-OHdG (HPLC-ED), OGG1 (a DNA repair gene) polymorphism (PCR-RFLP), telomerase activity, telomere length (T/S, by RT-PCR), Taqman microRNA assay and Bad/Bax mRNA (RT-PCR). Fifty-eight samples from 29 HCC patients (obtained in both neoplastic and peritumoral tissues), 22 from chronic hepatitis (CH) and 10 controls (cholecystectomy patients - CON) were examined.
Results
Eight-OHdG levels were significantly higher in HCC and NCCT than in CH and CON (p=0.001). Telomerase activity was significantly higher in HCC than in the remaining subgroups (p=0.002); conversely T/S was significantly lower in HCC (p=0.05). MiR-199a-b, -195, -122, -92a and −145 were down-regulated in the majority of HCCs while miR-222 was up-regulated. A positive correlation was observed among 8-OHdG levels, disease stage, telomerase activity, OGG1 polymorphisms and ALT/GGT levels. In HCC, miR-92 expression correlated positively with telomerase activity, 8-OHdG levels and Bad/Bax mRNA.
Conclusions
The above findings confirm the accumulation, in the progression of chronic liver damage to HCC, of a ROS-mediated oxidative DNA damage, and suggest that this correlates with induction of telomerase activity and, as a novel finding, with over-expression of miR-92, a microRNA that plays a role in both the apoptotic process and in cellular proliferation pathways.
doi:10.1186/1471-2407-12-177
PMCID: PMC3420318  PMID: 22587342
Hepatocellular carcinoma; 8-hydroxydeoxyguanosine; miR-92; Telomeric dysfunction; OGG1 gene
4.  microRNA-122 as a regulator of mitochondrial metabolic gene network in hepatocellular carcinoma 
A moderate loss of miR-122 function correlates with up-regulation of seed-matched genes and down-regulation of mitochondrially localized genes in both human hepatocellular carcinoma and in normal mice treated with anti-miR-122 antagomir.Putative direct targets up-regulated with loss of miR-122 and secondary targets down-regulated with loss of miR-122 are conserved between human beings and mice and are rapidly regulated in vitro in response to miR-122 over- and under-expression.Loss of miR-122 secondary target expression in either tumorous or adjacent non-tumorous tissue predicts poor survival of heptatocellular carcinoma patients.
Hepatocellular carcinoma (HCC) is one of the most aggressive human malignancies, common in Asia, Africa, and in areas with endemic infections of hepatitis-B or -C viruses (HBV or HCV) (But et al, 2008). Globally, the 5-year survival rate of HCC is <5% and about 600 000 HCC patients die each year. The high mortality associated with this disease is mainly attributed to the failure to diagnose HCC patients at an early stage and a lack of effective therapies for patients with advanced stage HCC. Understanding the relationships between phenotypic and molecular changes in HCC is, therefore, of paramount importance for the development of improved HCC diagnosis and treatment methods.
In this study, we examined mRNA and microRNA (miRNA)-expression profiles of tumor and adjacent non-tumor liver tissue from HCC patients. The patient population was selected from a region of endemic HBV infection, and HBV infection appears to contribute to the etiology of HCC in these patients. A total of 96 HCC patients were included in the study, of which about 88% tested positive for HBV antigen; patients testing positive for HCV antigen were excluded. Among the 220 miRNAs profiled, miR-122 was the most highly expressed miRNA in liver, and its expression was decreased almost two-fold in HCC tissue relative to adjacent non-tumor tissue, confirming earlier observations (Lagos-Quintana et al, 2002; Kutay et al, 2006; Budhu et al, 2008).
Over 1000 transcripts were correlated and over 1000 transcripts were anti-correlated with miR-122 expression. Consistent with the idea that transcripts anti-correlated with miR-122 are potential miR-122 targets, the most highly anti-correlated transcripts were highly enriched for the presence of the miR-122 central seed hexamer, CACTCC, in the 3′UTR. Although the complete set of negatively correlated genes was enriched for cell-cycle genes, the subset of seed-matched genes had no significant KEGG Pathway annotation, suggesting that miR-122 is unlikely to directly regulate the cell cycle in these patients. In contrast, transcripts positively correlated with miR-122 were not enriched for 3′UTR seed matches to miR-122. Interestingly, these 1042 transcripts were enriched for genes coding for mitochondrially localized proteins and for metabolic functions.
To analyze the impact of loss of miR-122 in vivo, silencing of miR-122 was performed by antisense inhibition (anti-miR-122) in wild-type mice (Figure 3). As with the genes negatively correlated with miR-122 in HCC patients, no significant biological annotation was associated with the seed-matched genes up-regulated by anti-miR-122 in mouse livers. The most significantly enriched biological annotation for anti-miR-122 down-regulated genes, as for positively correlated genes in HCC, was mitochondrial localization; the down-regulated mitochondrial genes were enriched for metabolic functions. Putative direct and downstream targets with orthologs on both the human and mouse microarrays showed significant overlap for regulations in the same direction. These overlaps defined sets of putative miR-122 primary and secondary targets. The results were further extended in the analysis of a separate dataset from 180 HCC, 40 cirrhotic, and 6 normal liver tissue samples (Figure 4), showing anti-correlation of proposed primary and secondary targets in non-healthy tissues.
To validate the direct correlation between miR-122 and some of the primary and secondary targets, we determined the expression of putative targets after transfection of miR-122 mimetic into PLC/PRF/5 HCC cells, including the putative direct targets SMARCD1 and MAP3K3 (MEKK3), a target described in the literature, CAT-1 (SLC7A1), and three putative secondary targets, PPARGC1A (PGC-1α) and succinate dehydrogenase subunits A and B. As expected, the putative direct targets showed reduced expression, whereas the putative secondary target genes showed increased expression in cells over-expressing miR-122 (Figure 4).
Functional classification of genes using the total ancestry method (Yu et al, 2007) identified PPARGC1A (PGC-1α) as the most connected secondary target. PPARGC1A has been proposed to function as a master regulator of mitochondrial biogenesis (Ventura-Clapier et al, 2008), suggesting that loss of PPARGC1A expression may contribute to the loss of mitochondrial gene expression correlated with loss of miR-122 expression. To further validate the link of miR-122 and PGC-1α protein, we transfected PLC/PRF/5 cells with miR-122-expression vector, and observed an increase in PGC-1α protein levels. Importantly, transfection of both miR-122 mimetic and miR-122-expression vector significantly reduced the lactate content of PLC/PRF/5 cells, whereas anti-miR-122 treatment increased lactate production. Together, the data support the function of miR-122 in mitochondrial metabolic functions.
Patient survival was not directly associated with miR-122-expression levels. However, miR-122 secondary targets were expressed at significantly higher levels in both tumor and adjacent non-tumor tissues among survivors as compared with deceased patients, providing supporting evidence for the potential relevance of loss of miR-122 function in HCC patient morbidity and mortality.
Overall, our findings reveal potentially new biological functions for miR-122 in liver physiology. We observed decreased expression of miR-122, a liver-specific miRNA, in HBV-associated HCC, and loss of miR-122 seemed to correlate with the decrease of mitochondrion-related metabolic pathway gene expression in HCC and in non-tumor liver tissues, a result that is consistent with the outcome of treatment of mice with anti-miR-122 and is of prognostic significance for HCC patients. Further investigation will be conducted to dissect the regulatory function of miR-122 on mitochondrial metabolism in HCC and to test whether increasing miR-122 expression can improve mitochondrial function in liver and perhaps in liver tumor tissues. Moreover, these results support the idea that primary targets of a given miRNA may be distributed over a variety of functional categories while resulting in a coordinated secondary response, potentially through synergistic action (Linsley et al, 2007).
Tumorigenesis involves multistep genetic alterations. To elucidate the microRNA (miRNA)–gene interaction network in carcinogenesis, we examined their genome-wide expression profiles in 96 pairs of tumor/non-tumor tissues from hepatocellular carcinoma (HCC). Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-122 is under-expressed in HCC and that increased expression of miR-122 seed-matched genes leads to a loss of mitochondrial metabolic function. Furthermore, the miR-122 secondary targets, which decrease in expression, are good prognostic markers for HCC. Transcriptome profiling data from additional 180 HCC and 40 liver cirrhotic patients in the same cohort were used to confirm the anti-correlation of miR-122 primary and secondary target gene sets. The HCC findings can be recapitulated in mouse liver by silencing miR-122 with antagomir treatment followed by gene-expression microarray analysis. In vitro miR-122 data further provided a direct link between induction of miR-122-controlled genes and impairment of mitochondrial metabolism. In conclusion, miR-122 regulates mitochondrial metabolism and its loss may be detrimental to sustaining critical liver function and contribute to morbidity and mortality of liver cancer patients.
doi:10.1038/msb.2010.58
PMCID: PMC2950084  PMID: 20739924
hepatocellular carcinoma; microarray; miR-122; mitochondrial; survival
5.  Hepatocyte-specific deletion of the anti-apoptotic protein Mcl-1 triggers proliferation and hepatocarcinogenesis in mice 
Hepatology (Baltimore, Md.)  2010;51(4):1226-1236.
Regulation of hepatocellular apoptosis is crucial for liver homeostasis. Increased sensitivity of hepatocytes towards apoptosis results in chronic liver injury, whereas apoptosis resistance is linked to hepatocarcinogenesis and non-responsiveness to therapy-induced cell death. Recently, we have demonstrated an essential role of the anti-apoptotic Bcl-2 family member Myeloid cell leukemia-1 (Mcl-1) in hepatocyte survival. In mice lacking Mcl-1 specifically in hepatocytes (Mcl-1Δhep) spontaneous apoptosis caused severe liver damage. Here, we demonstrate that chronically increased apoptosis of hepatocytes coincides with strong hepatocyte proliferation resulting in hepatocellular carcinoma (HCC). Liver cell tumor formation was observed in >50% of Mcl-1Δhep mice already by the age of 8 months, whereas 12 month-old wild-type and heterozygous Mcl-1flox/wt mice lacked tumors. Tumors revealed a heterogenous spectrum ranging from small dysplastic nodules to HCC. The neoplastic nature of the tumors was confirmed by histology, expression of the HCC marker glutamine synthetase and chromosomal aberrations. Liver carcinogenesis in Mcl-1Δhep mice was paralleled by markedly increased levels of survivin, an important regulator of mitosis which is selectively overexpressed in common human cancers.
Conclusion
The present study provides in vivo evidence that increased apoptosis of hepatocytes not only impairs liver homeostasis but is also accompanied by hepatocyte proliferation and hepatocarcinogenesis. Our findings might have implications for understanding apoptosis-related human liver diseases.
The survival of multicellular organisms depends on the maintenance of tissue homeostasis. Under physiological conditions apoptosis contributes to liver homeostasis by removing damaged hepatocytes. Proliferation, growth and programmed hepatocyte cell death are highly coordinated and tightly controlled events in the normal liver (1).
On the one hand, increased apoptosis sensitivity contributes to liver injury. On the other hand, defective apoptosis was demonstrated to lead to excessive hepatocellular survival and has emerged as a major mechanism by which pre-malignant hepatocytes obtain a competitive advantage over normal liver cells (2). Various molecular alterations have been characterized causing an imbalance in the regulation of apoptosis. Among these are alterations in p53 signalling, expression of death receptors, growth factors and mitochondrial integrity (3). Decreased activity of pro-apoptotic signalling as well as increased activity of anti-apoptotic events are associated with HCC development and progression (4).
Among the main cellular changes that trigger apoptosis of hepatocytes is the permeabilization of the outer mitochondrial membrane followed by the release of pro-apoptotic factors (5). The Bcl-2 protein family plays a pivotal role for mitochondrial integrity and the selective interactions between pro- and anti-apoptotic family members regulate mitochondrial activation (6). Bcl-2 family members are similar within the Bcl-2 homology regions (BH1-BH4) and can be divided in pro- and anti-apoptotic Bcl-2 proteins.
Pro-apoptotic Bcl-2 proteins comprise (1) multi-domain members, which lack the BH4 domain (e.g. Bax, Bak), and (2) BH3-only proteins, which lack BH1, 2 and 4 domains (e.g. Bid, Noxa, Puma). BH3-only proteins initiate the mitochondrial signalling cascade by sensing cellular damage (7). After activation, BH3-only proteins are released to neutralise anti-apoptotic Bcl-2 proteins. Subsequently, Bax and Bak trigger mitochondrial membrane leakage and the release of mitochondrial proteins, including cytochrome c, Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP-binding protein with low pI) and apoptosis-inducing factor (AIF). Smac/DIABLO proteins inactivate the IAP (inhibitors of apoptosis proteins) family, which consists of IAP1/2, BRUCE, NAIP, ILP2, ML-IAP, survivin and XIAP. XIAP is a direct caspase inhibitor. Other IAPs including survivin have several functions apart from caspase inhibition, eg, triggering of ubiquitination processes (8). Anti-apoptotic Bcl-2 family members (eg, Bcl-2, Bcl-xL and Mcl-1), interact with Bax and Bak to inhibit the activation of mitochondria (7).
Both Bcl-xL and Mcl-1 have been identified as major anti-apoptotic Bcl-2 proteins in the liver (9-11). Liver homeostasis is severely disturbed in Mcl-1Δhep mice (10, 11). Spontaneous hepatocyte apoptosis was observed in livers of Mcl-1Δhep mice in profound liver cell damage and increased susceptibility of hepatocytes towards pro-apoptotic stimuli (10). In addition, Mcl-1 has been shown to be highly expressed in a subset of human HCC, contributing to apoptosis resistance of cancer cells (12, 13). Thus, abrogation of the pro-survival function of Mcl-1 (1) either by diminishing its levels or (2) by inactivating its function, have shown promising results with regards to treatment of HCC (12, 13).
In this study, we show that liver-specific depletion of Mcl-1 increases hepatocyte apoptosis, induces hepatocellular proliferation and causes HCC in the absence of overt inflammation.
doi:10.1002/hep.23479
PMCID: PMC2936921  PMID: 20099303
liver; hepatocellular carcinoma; apoptosis; Bcl-2 proteins; survivin
6.  Induction of Bcl-2 Expression by Hepatitis B Virus Pre-S2 Mutant Large Surface Protein Resistance to 5-Fluorouracil Treatment in Huh-7 Cells 
PLoS ONE  2011;6(12):e28977.
Background
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Our previous studies have indicated that expression of Hepatitis B virus pre-S2 large mutant surface antigen (HBV pre-S2Δ) is associated with a significant risk of developing HCC. However, the relationship between HBV pre-S2Δ protein and the resistance of chemotherapeutic drug treatment is still unclear.
Methodology/Principal Findings
Here, we show that the expression of HBV pre-S2Δ mutant surface protein in Huh-7 cell significantly promoted cell growth and colony formation. Furthermore, HBV pre-S2Δ protein increased both mRNA (2.7±0.5-fold vs. vehicle, p = 0.05) and protein (3.2±0.3-fold vs. vehicle, p = 0.01) levels of Bcl-2 in Huh-7 cells. HBV pre-S2Δ protein also enhances Bcl-2 family, Bcl-xL and Mcl-1, expression in Huh-7 cells. Meanwhile, induction of NF-κB p65, ERK, and Akt phosphorylation, and GRP78 expression, an unfolded protein response chaperone, were observed in HBV pre-S2Δ and HBV pre-S-expressing cells. Induction of Bcl-2 expression by HBV pre-S2Δ protein resulted in resistance to 5-fluorouracil treatment in colony formation, caspase-3 assay, and cell apoptosis, and can enhance cell death by co-incubation with Bcl-2 inhibitor. Similarly, transgenic mice showed higher expression of Bcl-2 in liver tissue expressing HBV pre-S2Δ large surface protein in vivo.
Conclusion/Significance
Our result demonstrates that HBV pre-S2Δ increased Bcl-2 expression which plays an important role in resistance to 5-fluorouracil-caused cell death. Therefore, these data provide an important chemotherapeutic strategy in HBV pre-S2Δ-associated tumor.
doi:10.1371/journal.pone.0028977
PMCID: PMC3245229  PMID: 22216150
7.  Major HGF-mediated regenerative pathways are similarly affected in human and canine cirrhosis 
Background
The availability of non-rodent animal models for human cirrhosis is limited. We investigated whether privately-owned dogs (Canis familiaris) are potential model animals for liver disease focusing on regenerative pathways. Several forms of canine hepatitis were examined: Acute Hepatitis (AH), Chronic Hepatitis (CH), Lobular Dissecting Hepatitis (LDH, a specific form of micronodulair cirrhosis), and Cirrhosis (CIRR). Canine cirrhotic samples were compared to human liver samples from cirrhotic stages of alcoholic liver disease (hALC) and chronic hepatitis C infection (hHC).
Results
Canine specific mRNA expression of the regenerative hepatocyte growth factor (HGF) signaling pathway and relevant down-stream pathways were measured by semi-quantitative PCR and Western blot (STAT3, PKB, ERK1/2, and p38-MAPK). In all canine groups, levels of c-MET mRNA (proto-oncogenic receptor for HGF) were significantly decreased (p < 0.05). Surprisingly, ERK1/2 and p38-MAPK were increased in CH and LDH. In the human liver samples Western blotting indicated a high homology of down-stream pathways between different etiologies (hALC and hHC). Similarly activated pathways were found in CIRR, hALC, and hHC.
Conclusion
In canine hepatitis and cirrhosis the major regenerative downstream pathways were activated. Signaling pathways are similarly activated in human cirrhotic liver samples, irrespective of the differences in etiology in the human samples (alcohol abuse and HCV-infection). Therefore, canine hepatitis and cirrhosis could be an important clinical model to evaluate novel interventions prior to human clinical trials.
doi:10.1186/1476-5926-6-8
PMCID: PMC1971050  PMID: 17672890
8.  Characterization of chronic HCV infection-induced apoptosis 
Background
To understand the complex and largely not well-understood apoptotic pathway and immune system evasion mechanisms in hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) and HCV associated chronic hepatitis (CH), we studied the expression patterns of a number of pro-apoptotic and anti-apoptotic genes (Fas, FasL, Bcl-2, Bcl-xL and Bak) in HepG2 cell line harboring HCV- genotype-4 replication. For confirmation, we also assessed the expression levels of the same group of genes in clinical samples obtained from 35 HCC and 34 CH patients.
Methods
Viral replication was assessed in the tissue culture medium by RT-PCR, quantitative Real-Time PCR (qRT-PCR); detection of HCV core protein by western blot and inhibition of HCV replication with siRNA. The expression level of Fas, FasL, Bcl-2, Bcl-xL and Bak was assessed by immunohistochemistry and RT-PCR whereas caspases 3, 8 and 9 were assessed by colorimetric assay kits up to 135 days post infection.
Results
There was a consistent increase in apoptotic activity for the first 4 weeks post-CV infection followed by a consistent decrease up to the end of the experiment. The concordance between the changes in the expression levels of Fas, FasL, Bcl-2, Bcl-xL and Bak in vitro and in situ was statistically significant (p < 0.05). Fas was highly expressed at early stages of infection in cell lines and in normal control liver tissues followed by a dramatic reduction post-HCV infection and an increase in the expression level of FasL post HCV infection. The effect of HCV infection on other apoptotic proteins started very early post-infection, suggesting that hepatitis C modulating apoptosis by modulating intracellular pro-apoptotic signals.
Conclusions
Chronic HCV infection differently modulates the apoptotic machinery during the course of infection, where the virus induces apoptosis early in the course of infection, and as the disease progresses apoptosis is modulated. This study could open a new opportunity for understanding the various signaling of apoptosis and in the developing a targeted therapy to inhibit viral persistence and HCC development.
doi:10.1186/1476-5926-10-4
PMCID: PMC3160349  PMID: 21781333
9.  Global Gene Expression Profiling Reveals SPINK1 as a Potential Hepatocellular Carcinoma Marker 
PLoS ONE  2013;8(3):e59459.
Background
Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) but the role of liver disease aetiology in cancer development remains under-explored. We investigated global gene expression profiles from HCC arising in different liver diseases to test whether HCC development is driven by expression of common or different genes, which could provide new diagnostic markers or therapeutic targets.
Methodology and Principal Findings
Global gene expression profiling was performed for 4 normal (control) livers as well as 8 background liver and 7 HCC from 3 patients with hereditary haemochromatosis (HH) undergoing surgery. In order to investigate different disease phenotypes causing HCC, the data were compared with public microarray repositories for gene expression in normal liver, hepatitis C virus (HCV) cirrhosis, HCV-related HCC (HCV-HCC), hepatitis B virus (HBV) cirrhosis and HBV-related HCC (HBV-HCC). Principal component analysis and differential gene expression analysis were carried out using R Bioconductor. Liver disease-specific and shared gene lists were created and genes identified as highly expressed in hereditary haemochromatosis HCC (HH-HCC) were validated using quantitative RT-PCR. Selected genes were investigated further using immunohistochemistry in 86 HCC arising in liver disorders with varied aetiology. Using a 2-fold cut-off, 9 genes were highly expressed in all HCC, 11 in HH-HCC, 270 in HBV-HCC and 9 in HCV-HCC. Six genes identified by microarray as highly expressed in HH-HCC were confirmed by RT qPCR. Serine peptidase inhibitor, Kazal type 1 (SPINK1) mRNA was very highly expressed in HH-HCC (median fold change 2291, p = 0.0072) and was detected by immunohistochemistry in 91% of HH-HCC, 0% of HH-related cirrhotic or dysplastic nodules and 79% of mixed-aetiology HCC.
Conclusion
HCC, arising from diverse backgrounds, uniformly over-express a small set of genes. SPINK1, a secretory trypsin inhibitor, demonstrated potential as a diagnostic HCC marker and should be evaluated in future studies.
doi:10.1371/journal.pone.0059459
PMCID: PMC3601070  PMID: 23527199
10.  Autophagy in rat annulus fibrosus cells: evidence and possible implications 
Arthritis Research & Therapy  2011;13(4):R132.
Introduction
Programmed cell death of intervertebral disc (IVD) cells plays an important role in IVD degeneration, but the role of autophagy, a closely related cell death event, in IVD cells has not been documented. The current study was designed to investigate the effect of interleukin (IL)-1β on the occurrence of autophagy of rat annulus fibrosus (AF) cells and the interrelationship between autophagy and apoptosis.
Methods
Rat AF cells were isolated and exposed, in tissue cultures with or without serum, to IL-1β in different concentrations for 24 hours. Ultrastructural analysis, flow cytometry and lysosomal activity assessment were performed after the in vitro treatment to determine the presence and levels of autophagy. The mRNA expression of autophagy-related proteins (Beclin-1, Bcl-2 and microtubule associated protein 1 light chain 3 (LC3)) were evaluated using real-time PCR. 3-methyladenine (3-MA), a PI3K inhibitor, was used to determine the interaction between autophagy and apoptosis via the suppression of autophagy.
Results
Autophagy was detected in rat AF cells under serum starvation condition by transmission electron microscopy. PCR and flow cytometry results showed that IL-1β enhanced the autophagy-induction effect of serum deprivation in a dose-dependent manner. However, IL-1β alone failed to induce autophagy in AF cells cultured without serum starvation. When autophagy was suppressed by 3-MA, the apoptosis incidence was increased. Serum supplement also partly reversed the autophagy incidence without affecting the apoptosis incidence in the same cells.
Conclusions
IL-1β up-regulates serum deprivation-induced autophagy of AF cells in a dose-dependent manner. Autophagy may represent a protective mechanism against apoptosis in AF cells and IVD degeneration.
doi:10.1186/ar3443
PMCID: PMC3239374  PMID: 21846367
Intervertebral disc; Autophagy; Apoptosis; Interleukin-1β; Serum deprivation
11.  Increased Autophagy in Placentas of Intrauterine Growth-Restricted Pregnancies 
PLoS ONE  2012;7(7):e40957.
Background
Unexplained intrauterine growth restriction (IUGR) may be a consequence of placental insufficiency; however, its etiology is not fully understood. We surmised that defective placentation in IUGR dysregulates cellular bioenergic homeostasis, leading to increased autophagy in the villous trophoblast. The aims of this work were (1) to compare the differences in autophagy, p53 expression, and apoptosis between placentas of women with normal or IUGR pregnancies; (2) to study the effects of hypoxia and the role of p53 in regulating trophoblast autophagy; and (3) to investigate the relationship between autophagy and apoptosis in hypoxic trophoblasts.
Methodology/Principal Findings
Compared with normal pregnant women, women with IUGR had higher placental levels of autophagy-related proteins LC3B-II, beclin-1, and damage-regulated autophagy modulator (DRAM), with increased p53 and caspase-cleaved cytokeratin 18 (M30). Furthermore, cytotrophoblasts cultured under hypoxia (2% oxygen) in the presence or absence of nutlin-3 (a p53 activity stimulator) had higher levels of LC3B-II, DRAM, and M30 proteins and increased Bax mRNA expression compared with controls cultured under standard conditions. In contrast, administration of pifithrin-α (a p53 activity inhibitor) during hypoxia resulted in protein levels that were similar to those of the control groups. Moreover, cytotrophoblasts transfected with LC3B, beclin-1, or DRAM siRNA had higher levels of M30 compared with the controls under hypoxia. However, transfection with Bcl-2 or Bax siRNA did not cause any significant change in the levels of LC3B-II in hypoxic cytotrophoblasts.
Conclusions/Significance
Together, these results suggest that there is a crosstalk between autophagy and apoptosis in IUGR and that p53 plays a pivotal and complex role in regulating trophoblast cell turnover in response to hypoxic stress.
doi:10.1371/journal.pone.0040957
PMCID: PMC3397998  PMID: 22815878
12.  Distinct expression patterns in hepatitis B virus- and hepatitis C virus-infected hepatocellular carcinoma 
AIM: To identify biomarkers indicating virus-specific hepatocarcinogenic process, differential mRNA expression in 32 patients with hepatitis B virus (HBV)-/hepatitis C virus (HCV)-associated hepatocellular carcinoma (HCC) were investigated by means of cDNA microarrays comprising of 886 genes.
METHODS: Thirty two HCC patients were divided into two groups based on viral markers: hepatitis B virus positive and HCV positive. The expression profiles of 32 pairs of specimens (tumorous and surrounding non-tumorous liver tissues), consisting of 886 genes were analyzed.
RESULTS: Seven up-regulated genes in HBV-associated HCC comprised genes involved in protein synthesis (RPS5), cytoskeletal organization (KRT8), apoptosis related genes (CFLAR), transport (ATP5F1), cell membrane receptor related genes (IGFBP2), signal transduction or transcription related genes (MAP3K5), and metastasis-related genes (MMP9). The up-regulated genes in HCV-infected group included 4 genes: VIM (cell structure), ACTB (cell structure), GAPD (glycolysis) and CD58 (cell adhesion). The expression patterns of the 11 genes, identified by cDNA microarray, were confirmed by quantitative RT-PCR in 32 specimens.
CONCLUSION: The patterns of all identified genes were classified based on the viral factor involved in HBV- and HCV-associated HCC. Our results strongly suggest that the pattern of gene expression in HCC is closely associated with the etiologic factor. The present study indicates that HBV and HCV cause hepatocarcinogenesis by different mechanisms, and provide novel tools for the diagnosis and treatment of HBV- and HCV-associated HCC.
doi:10.3748/wjg.14.6072
PMCID: PMC2760187  PMID: 18932288
Hepatocellular carcinoma; Hepatitis B virus; Hepatitis C virus-infected; cDNA microarray; Expression profiling
13.  Inhibition of autophagy significantly enhances combination therapy with sorafenib and HDAC inhibitors for human hepatoma cells 
AIM: To clarify whether histone deacetylase inhibitors histone deacetylase inhibitors (HDACIs) can sensitize hepatocellular carcinoma (HCC) cells to sorafenib treatment.
METHODS: Bax, Bcl-2, ATG5-ATG12, p21, and p27 protein levels in Hep3B, HepG2, and PLC/PRF/5 cells were examined by Western blot. CCK8 and a fluorometric caspase-3 assay were used to examine cellular viability and apoptosis levels. The effect of Beclin-1 on sensitization of HCC cells to sorafenib was examined by transfecting Beclin-1 siRNA into Hep3B, HepG2, and PLC/PRF/5 cells.
RESULTS: Autophagy inhibition enhances the inhibitory effects of vorinostat and sorafenib alone or in combination on HCC cell growth. Vorinostat and sorafenib synergistically induced apoptosis and cell cycle alterations. Western blot data indicated that HDACIs and Beclin-1 knockdown increased the p53 acetylation level. The knockdown of Beclin-1 enhanced the synergistic effect of the combination of vorinostat with sorafenib.
CONCLUSION: HDACIs can sensitize HCC cells to sorafenib treatment by regulating the acetylation level of Beclin-1.
doi:10.3748/wjg.v20.i17.4953
PMCID: PMC4009527  PMID: 24833845
Hepatocellular carcinoma; Histone deacetylase inhibitors; Autophagy; Sorafenib; Chemoresistance
14.  microRNA-122 Abundance in Hepatocellular Carcinoma and Non-Tumor Liver Tissue from Japanese Patients with Persistent HCV versus HBV Infection 
PLoS ONE  2013;8(10):e76867.
Mechanisms of hepatic carcinogenesis in chronic hepatitis B and hepatitis C are incompletely defined but often assumed to be similar and related to immune-mediated inflammation. Despite this, several studies hint at differences in expression of miR-122, a liver-specific microRNA with tumor suppressor properties, in hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) versus hepatitis C virus (HCV) infection. Differences in the expression of miR-122 in these cancers would be of interest, as miR-122 is an essential host factor for HCV but not HBV replication. To determine whether the abundance of miR-122 in cancer tissue is influenced by the nature of the underlying virus infection, we measured miR-122 by qRT-PCR in paired tumor and non-tumor tissues from cohorts of HBV- and HCV-infected Japanese patients. miR-122 abundance was significantly reduced from normal in HBV-associated HCC, but not in liver cancer associated with HCV infection. This difference was independent of the degree of differentiation of the liver cancer. Surprisingly, we also found significant differences in miR-122 expression in non-tumor tissue, with miR-122 abundance reduced from normal in HCV- but not HBV-infected liver. Similar differences were observed in HCV- vs. HBV-infected chimpanzees. Among HCV-infected Japanese subjects, reductions in miR-122 abundance in non-tumor tissue were associated with a single nucleotide polymorphism near the IL28B gene that predicts poor response to interferon-based therapy (TG vs. TT genotype at rs8099917), and correlated negatively with the abundance of multiple interferon-stimulated gene transcripts. Reduced levels of miR-122 in chronic hepatitis C thus appear to be associated with endogenous interferon responses to the virus, while differences in miR-122 expression in HCV- versus HBV-associated HCC likely reflect virus-specific mechanisms contributing to carcinogenesis. The continued expression of miR-122 in HCV-associated HCC may signify an important role for HCV replication late in the progression to malignancy.
doi:10.1371/journal.pone.0076867
PMCID: PMC3793926  PMID: 24130799
15.  Knockdown of autophagy enhances innate immune response in hepatitis C virus infected hepatocytes 
Hepatology (Baltimore, Md.)  2011;53(2):406-414.
The role of autophagy in disease pathogenesis following viral infection is beginning to be elucidated. We have previously reported that hepatitis C virus (HCV) infection in hepatocytes induces autophagy. However, the biological significance of HCV induced autophagy has not been clarified. Autophagy has recently been identified as a novel component of innate immune system against viral infection. In the present study, we have shown that knockdown of autophagy related protein Beclin1 or ATG7 in immortalized human hepatocytes (IHH) inhibited HCV growth. Beclin1 or ATG7 knockdown IHH when infected with HCV exhibited an increased expression of IFN-β, OAS-1, IFN-α and IFI27 mRNAs of the interferon signaling pathways as compared to infection of control IHH. Subsequent study demonstrated that HCV infection in autophagy impaired IHH displayed caspase activation, PARP cleavage and apoptotic cell death.
Conclusion
The disruption of autophagy machinery in HCV infected hepatocytes activated IFN signaling pathway, and induced apoptosis. Together, these results suggest that HCV induced autophagy impairs innate immune response.
doi:10.1002/hep.24073
PMCID: PMC3335751  PMID: 21274862
16.  Potential Prognostic Biomarkers for Bone Metastasis from Hepatocellular Carcinoma 
The Oncologist  2011;16(7):1028-1039.
Whether or not variations in gene and protein expression in hepatocellular carcinoma present at the time of surgery can enable the identification of patients at high risk for developing bone metastasis was examined.
Background.
Hepatocellular carcinoma (HCC) most commonly develops in patients who have a viral infection, especially in the case of hepatitis B virus (HBV), and in patients with a chronic liver disease. HCC patients with bone metastasis (BM) suffer from pain and other symptoms that significantly reduce their quality of life. Identification of patients who are at high risk for BM after undergoing potentially curative treatment for HCC remains challenging. Here, we aimed to identify HCC BM-related genes and proteins to establish prediction biomarkers.
Methods.
RNA was extracted from 48 pairs of intratumoral and peritumoral formalin-fixed, paraffin-embedded tissue from HCC patients with and without BM. A cDNA-mediated annealing, selection, extension and ligation assay containing 502 cancer-related genes was used to identify novel BM-associated genes. An additional independent study with 350 HCC patients who had undergone hepatectomy was conducted to evaluate the expression of candidate genes at the protein level using immunohistochemistry on tissue microarrays (TMAs). Of the 350 patients, 273 (78.0%) were infected with HBV.
Results.
Seven intratumoral genes and 17 peritumoral genes were overexpressed in patients with BM, whereas 15 intratumoral genes and 28 peritumoral genes were underexpressed in patients with BM. We selected the following four genes for further analysis because they were differentially expressed in the cancer gene-specific microarray and were previously reported to be associated with BM: connective tissue growth factor (CTGF), matrix metalloproteinase-1 (MMP-1), transforming growth factor β1 (TGF-β1), and interleukin-11 (IL-11). We assessed the protein expression of these selected genes using immunohistochemistry on TMAs including 350 HCC patient specimens. We determined that expression of intratumoral CTGF, intratumoral IL-11, and peritumoral MMP-1 were independent prognostic factors for developing BM in HCC patients. Combining intratumoral CTGF and IL-11 expression was also an independent risk factor for BM development.
Conclusions.
Sixty-seven genes were differentially expressed in HCC patients with and without BM. High intratumoral CTGF, positive IL-11, and high peritumoral MMP-1 expression were associated with BM after hepatectomy. Intratumoral CTGF expression combined with IL-11 expression may serve as a useful predictive biomarker for HCC BM.
doi:10.1634/theoncologist.2010-0358
PMCID: PMC3228144  PMID: 21665914
Gene expression; Tissue microarray; Hepatocellular carcinoma; Bone metastases
17.  Overexpression of DNA methyltransferase 1 and its biological significance in primary hepatocellular carcinoma 
AIM: To explore the relationship between DNA methyltransferase 1 (DNMT1) and hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) and its biological significance in primary HCC.
METHODS: We carried out an immunohistochemical examination of DNMT1 in both HCC and paired non-neoplastic liver tissues from Chinese subjects. DNMT1 mRNA was further examined in HCC cell lines by real-time PCR. We inhibited DNMT1 using siRNA and detected the effect of depletion of DNMT1 on cell proliferation ability and cell apoptosis in the HCC cell line SMMC-7721.
RESULTS: DNMT1 protein expression was increased in HCCs compared to histologically normal non-neoplastic liver tissues and the incidence of DNMT1 immunoreactivity in HCCs correlated significantly with poor tumor differentiation (P = 0.014). There were more cases with DNMT1 overexpression in HCC with HBV (42.85%) than in HCC without HBV (28.57%). However, no significant difference in DNMT1 expression was found in HBV-positive and HBV-negative cases in the Chinese HCC group. There was a trend that DNMT1 RNA expression increased more in HCC cell lines than in pericarcinoma cell lines and normal liver cell lines. In addition, we inhibited DNMT1 using siRNA in the SMMC-7721 HCC cell line and found depletion of DNMT1 suppressed cells growth independent of expression of proliferating cell nuclear antigen (PCNA), even in HCC cell lines where DNMT1 was stably decreased.
CONCLUSION: The findings implied that DNMT1 plays a key role in HBV-related hepatocellular tumorigenesis. Depletion of DNMT1 mediates growth suppression in SMMC-7721 cells.
doi:10.3748/wjg.15.2020
PMCID: PMC2675095  PMID: 19399937
DNA methyltransferase 1; Hepatitis B virus-related hepatocellular carcinoma; RNAi; Cell proliferation; Apoptosis
18.  Expression characteristics and diagnostic value of annexin A2 in hepatocellular carcinoma 
AIM: To investigate the characteristics and diagnostic value of annexin A2 (ANXA2) expression in cancerous tissues and sera of patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC).
METHODS: Levels of liver ANXA2 gene transcription or protein expression were analyzed in HCC-, their self-controlled precancerous-, and distant cancerous- tissues from 30 HCC. Serum levels of ANXA2 expression in 115 patients with HCC, 25 with metastatic liver cancer, 35 with chronic hepatitis, 28 with acute hepatitis, 38 with cirrhosis, and 30 healthy controls were determined. Clinicopathological characteristics of circulating ANXA2 expression were analyzed, and its diagnostic efficiency and clinical values in HCC were evaluated.
RESULTS: ANXA2 expression was localized in both cell membrane and cytoplasm in HCC tissue, mainly in the cytoplasm of matched adjacent cancerous tissue, and there was almost no positive staining in matched distant cancerous tissue. Abnormal expression of liver ANXA2 was present in HCC tissues compared with self-controlled adjacent- and distant-cancerous tissues at protein or mRNA level. Circulating ANXA2 in HCC patients was significantly higher than that of other liver diseases (P < 0.01) except metastatic liver cancer. If the diagnostic cutoff value of ANXA2 level was more than 18 ng/mL, the incidence of serum ANXA2 was 86.96% in the HCC group, 80% in the metastatic liver cancer group, 31.58% in the liver cirrhosis group, none in the chronic hepatitis or acute hepatitis or normal control group, respectively. Serum ANXA2 expression in HCC patients was correlated with HBV infection (27.38 ± 5.67 ng/mL vs 18.58 ± 7.83 ng/mL, P < 0.01), extrahepatic metastasis (26.11 ± 5.43 ng/mL vs 22.79 ± 5.64 ng/mL, P < 0.01), and portal vein thrombus (26.03 ± 5.99 ng/mL vs 23.06 ± 5.03 ng/mL, P < 0.01), and was significantly higher (P < 0.01) in the moderately- (26.19 ± 5.34 ng/mL) or the poorly- differentiated group (27.05 ± 5.13 ng/mL) than in the well differentiated group (20.43 ± 4.97 ng/mL), and in the tumor node metastasis stages III-IV (P < 0.01) than in stages I-II. ANXA2 was not correlated with patient sex, age, size or α-fetoprotein (AFP) level. Area under the receiver operating characteristic curve for the whole range of sensitivities and specificities was 0.796 for ANXA2 and 0.782 for AFP. Combining detection of serum ANXA2 and AFP substantially improved the diagnostic efficiency (96.52%) and the negative predictive value (96.61%) for HCC.
CONCLUSION: The characteristics and distribution of ANXA2 expression has good diagnostic potential for HCC diagnosis.
doi:10.3748/wjg.v18.i41.5897
PMCID: PMC3491596  PMID: 23139605
Hepatocellular carcinoma; Annexin A2; Liver; Upregulation; Clinicopathological characteristics; Diagnosis; Expression; Biomarker
19.  Hepatitis B Virus Inhibits Apoptosis of Hepatoma Cells by Sponging the MicroRNA 15a/16 Cluster 
Journal of Virology  2013;87(24):13370-13378.
Hepatitis B virus (HBV) causes chronic hepatitis in hundreds of millions of people worldwide, which can eventually lead to hepatocellular carcinoma (HCC). The molecular mechanisms underlying HBV persistence are not well understood. In this study, we found that HBV inhibited the chemotherapy drug etoposide-induced apoptosis of hepatoma cells. Further analysis revealed that HBV mRNAs possess a microRNA 15a/16 (miR-15a/16)-complementary site (HBV nucleotides [nt] 1362 to 1383) that acts as a sponge to bind and sequester endogenous miR-15a/16. Consequently, Bcl-2, known as the target of miR-15a/16, was upregulated in HBV-infected cells. The data from HBV-transgenic mice further confirmed that HBV transcripts cause the reduction of miR-15a/16 and increase of Bcl-2. More importantly, we examined the levels of HBV transcripts and miR-15a/16 in HBV-infected HCC from patients and found that the amount of HBV mRNA and the level of miR-15a/16 were negatively correlated. Consistently, the level of Bcl-2 mRNA was upregulated in HBV-infected patients. In conclusion, we identified a novel HBV mRNA–miR-15a/16–Bcl-2 regulatory pathway that is involved in inhibiting etoposide-induced apoptosis of hepatoma cells, which may contribute to facilitating chronic HBV infection and hepatoma development.
doi:10.1128/JVI.02130-13
PMCID: PMC3838258  PMID: 24089558
20.  ShRNA-Targeted Centromere Protein A Inhibits Hepatocellular Carcinoma Growth 
PLoS ONE  2011;6(3):e17794.
Background
Centromere protein A (CENP-A) plays important roles in cell-cycle regulation and genetic stability. Herein, we aimed to investigate its expression pattern, clinical significance, and biological function in hepatocellular carcinoma (HCC).
Methodology/Principal Findings
CENP-A expression at the mRNA and protein levels was examined in 20 pairs of fresh HCCs and corresponding nontumor liver tissues. Immunohistochemistry for CENP-A was performed on 80 paraffin-embedded HCC specimens, and the clinical significance of its expression was analyzed. A human HCC cell line HepG2 with high abundance of CENP-A was used to study the effects of manipulating CENP-A on HCC growth. Quantitative real-time polymerase chain reaction arrays and Western blot analysis were employed to identify the cell-cycle control- and apoptosis-related genes regulated by CENP-A. The results showed that CENP-A was aberrantly overexpressed in HCCs relative to adjacent nontumor tissues. This overexpression was significantly associated with positive serum HBsAg status, increased histological grade, high Ki-67 index and P53 immunopositivity. Knockdown of CENP-A in HepG2 cells reduced cell proliferation, blocked cell cycle at the G1 phase, and increased apoptosis. The antiproliferative effects of CENP-A silencing were also observed in vivo. Conversely, CENP-A overexpression promoted HCC cell growth and reduced apoptosis. Furthermore, many genes implicated in cell-cycle regulation and apoptosis, including CHK2, P21waf1, P27 Kip1, SKP2, cyclin G1, MDM2, Bcl-2, and Bax, were deregulated by manipulating CENP-A.
Conclusions/Significance
Overexpression of CENP-A is frequently observed in HCC. Targeting CENP-A can inhibit HCC growth, likely through the regulation of a large number genes involved in cell-cycle progression and apoptosis, and thereby represents a potential therapeutic strategy for this malignancy.
doi:10.1371/journal.pone.0017794
PMCID: PMC3058037  PMID: 21423629
21.  Beclin 1 Expression is Closely Linked to Colorectal Carcinogenesis and Distant Metastasis of Colorectal Carcinoma 
Beclin 1 participates in development, autophagy, differentiation, anti- apoptosis, neurodegeneration, tumorigenesis and cancer progression. The roles of Beclin 1 in colorectal carcinogenesis and its subsequent progression are still unclear. Here, the mRNA and protein expression of Beclin 1 were determined in colorectal carcinoma and matched mucosa by Reverse transcriptase-polymerase chain reaction and Western blot. Immunohistochemistry and in situ hybridization (ISH) were performed on tissue microarryer with colorectal carcinoma, adenoma and mucosa. The expression of Beclin 1 mRNA and protein was found to be higher in colorectal carcinoma than matched mucosa by real-time PCR and Western blot (p < 0.05). According to the ISH data, Beclin 1 expression was lower in colorectal non-neoplastic mucosa (NNM) than adenoma and carcinoma (p < 0.05). Immunohistochemically, primary carcinoma showed stronger Beclin 1 expression than NNM and metastatic carcinoma in the liver (p < 0.05). Beclin 1 protein expression was negatively related to liver and distant metastasis (p < 0.05), but not correlated with age, sex, depth of invasion, lymphatic or venous invasion, lymph node metastasis, tumor-node-metastasis (TNM) staging, differentiation or serum carcinoembryonic antigen (CEA) concentration (p > 0.05). Survival analysis indicated that Beclin 1 expression was not linked to favorable prognosis of the patients with colorectal carcinoma (p > 0.05). Cox’s model indicated that depth of invasion and distant metastasis were independent prognostic factors for colorectal carcinomas (p < 0.05). It was suggested that Beclin 1 expression is closely linked to colorectal carcinogenesis and distant metastasis of colorectal carcinoma.
doi:10.3390/ijms150814372
PMCID: PMC4159856  PMID: 25196438
colorectal carcinoma; Beclin 1; tumorigenesis; pathogenesis; prognosis
22.  Hedgehog Signaling Pathway Regulates Autophagy in Human Hepatocellular Carcinoma Cells 
Hepatology (Baltimore, Md.)  2013;58(3):995-1010.
Hedgehog (Hh) signaling plays an important role in embryonic development and in the regulation of a variety of cellular functions. Aberrant activation of Hh signaling has been implicated in several human cancers including hepatocellular carcinoma (HCC). In this study we examined the pathobiological functions and molecular mechanisms of Hh signaling pathway in HCC cells. Treatment of cultured human HCC cells (Huh7, Hep3B and HepG2) with the Hh signaling ligand (recombinant Shh) or agonist, SAG and purmorphamine, prevented the induction of autophagy. In contrast, GANT61 (a small molecule inhibitor of Gli1 and Gli2) induced autophagy, as determined by immunobloting for microtubule-associated protein light chain 3 (LC3) and p62, GFP-LC3 puncta, monodansylcadaverine (MDC) staining and transmission electron microscopy. Hh inhibition-induced autophagy was associated with upregulation of Bnip3, as determined by immunoblotting and real-time PCR assay. Knockdown of Bnip3 by RNAi impaired GANT61-induced autophagy. Additionally, Hh inhibition-induced autophagy was associated with Bnip3-mediated displacement of Bcl-2 from Beclin-1, as determined by immunoblotting and immunoprecipitation assays. Furthermore, inhibition of Hh signaling increased HCC cell apoptosis and decreased cell viability, as determined by caspase and WST-1 assays. Pharmacological or genetic inhibition of autophagy by 3-methyladenine (3-MA) or Beclin-1 siRNA partially suppressed GANT61-induced cell apoptosis and cytotoxicity. In a tumor xenograft model using SCID mice inoculated with Huh7 cells, administration of GANT61 inhibited tumor formation and decreased tumor volume; this effect was partially blocked by the autophagy inhibitor, 3-MA. These findings provide novel evidence that hedgehog inhibition induces autophagy through upregulation of Bnip3 and that this mechanism contributes to apoptosis. Therefore, the status of autophagy is a key factor that determines the therapeutic response to Hh-targeted therapies.
doi:10.1002/hep.26394
PMCID: PMC3706478  PMID: 23504944
Hedgehog signaling; GANT61; autophagy; hepatocellular carcinoma; apoptosis
23.  Identification of microRNAs Specifically Expressed in Hepatitis C Virus-Associated Hepatocellular Carcinoma 
Although several studies have investigated the association of miRNAs with hepatocellular carcinoma (HCC), the data published so far are not concordant. A reason for these discrepancies may be the fact that most studies used as a control the non-tumorous tissue surrounding the HCC lesion, which is almost invariably affected by cirrhosis or chronic hepatitis, as well as other pathological conditions such as hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. Moreover, HCC is often analyzed as a single group regardless of the different viral etiologies. In this study, we investigated miRNAs differentially expressed in HCV-related HCC by comparing the tumorous tissues to a wide range of liver specimens, including healthy livers obtained from liver donors and patients who underwent liver resection for angioma, in addition to tissues from various acute and chronic liver diseases, including HCV-related cirrhosis not associated with HCC, HCV-related cirrhosis associated with HCC, and HBV-associated acute liver failure (ALF). Our study examined the whole set of 2,226 human miRNAs, including 1,121 pre miRNAs and 1,105 mature miRNAs, available in a microarray platform.Stringent statistical methods were applied to reduce the risk of false discoveries to less than 1%. Our data identified 18 miRNAs exclusively expressed in HCV-associated HCC, characterized by high specificity and selectivity versus all other liver diseases and healthy conditions, and connected into a regulatory network pivoting on p53, phosphatase and tensin homolog (PTEN), and all-trans retinoic acid signaling.
doi:10.1002/ijc.28075
PMCID: PMC3830961  PMID: 23390000
Liver; hepatocellular carcinoma; hepatitis C virus; microRNA; pathogenesis
24.  CHOP mediates ASPP2-induced autophagic apoptosis in hepatoma cells by releasing Beclin-1 from Bcl-2 and inducing nuclear translocation of Bcl-2 
Liu, K | Shi, Y | Guo, X | Wang, S | Ouyang, Y | Hao, M | Liu, D | Qiao, L | Li, N | Zheng, J | Chen, D
Cell Death & Disease  2014;5(7):e1323-.
Apoptosis-stimulating protein of p53-2 (ASPP2) induces apoptosis by promoting the expression of pro-apoptotic genes via binding to p53 or p73; however, the exact mechanisms by which ASPP2 induces apoptotic death in hepatoma cells are still unclear. Here, we show that the transient overexpression of ASPP2 induces autophagic apoptosis in hepatoma cells by promoting p53- or p73-independent C/EBP homologous protein (CHOP) expression. CHOP expression decreases the expression of Bcl-2; this change releases Beclin-1 from cytoplasmic Bcl-2-Beclin-1 complexes and allows it to initiate autophagy. However, transient overexpression of Beclin-1 can induce autophagy but not apoptosis. Our results show that ASPP2 induces the expression of damage-regulated autophagy modulator (DRAM), another critical factor that cooperates with free Beclin-1 to induce autophagic apoptosis. The effect of CHOP on the translocation and sequestration of Bcl-2 in the nucleus, which requires the binding of Bcl-2 to ASPP2, is also critical for ASPP2-induced autophagic apoptosis. Although the role of nuclear ASPP2–Bcl-2 complexes is still unclear, our results suggest that nuclear ASPP2 can prevent the translocation of the remaining Bcl-2 to the cytoplasm by binding to Bcl-2 in a CHOP-dependent manner, and this effect also contributes to Beclin-1-initiated autophagy. Thus, CHOP is critical for mediating ASPP2-induced autophagic apoptosis by decreasing Bcl-2 expression and maintaining nuclear ASPP2–Bcl-2 complexes. Our results, which define a mechanism whereby ASPP2 overexpression induces autophagic apoptosis, open a new avenue for promoting autophagy in treatments to cure hepatocellular carcinoma.
doi:10.1038/cddis.2014.276
PMCID: PMC4123070  PMID: 25032846
25.  Progesterone Increases Apoptosis and Inversely Decreases Autophagy in Human Hepatoma HA22T/VGH Cells Treated with Epirubicin 
The Scientific World Journal  2014;2014:567148.
Hepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide. Epirubicin can induce intracellular reactive oxygen species and is widely used to treat unresectable HCC. Progesterone has been found to inhibit the proliferation of hepatoma cells. This study was designed to test the combined effects of epirubicin and progesterone on human hepatoma cell line, HA22T/VGH. These cells were treated with different concentrations of epirubicin with or without the coaddition of 30 μM progesterone and then analyzed for apoptosis, autophagy, and expressions of apoptotic-related proteins and multidrug-resistant gene. Epirubicin treatment dose-dependently inhibited the growth of HA22T/VGH cells. Addition of 30 μM progesterone, which was inactive alone, augmented the effect of epirubicin on the inhibition of growth of HA22T/VGH cells. Cotreatment with progesterone enhanced epirubicin-induced apoptosis, as evidenced by greater increase in caspase-3 activity and in the ratio of the apoptosis-regulating protein, Bax/Bcl-XL. The combination also caused a decrease in autophagy and in the expression of multidrug resistance-related protein 1 mRNA compared to epirubicin alone. This study shows the epirubicin/progesterone combination was more effective in increasing apoptosis and inversely decreasing autophagy on HA22T/VGH cells treated with epirubicin alone, suggesting that this combination can potentially be used to treat HCC.
doi:10.1155/2014/567148
PMCID: PMC4055367  PMID: 24971383

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