Simple sugar intake and hepatocellular carcinoma: epidemiological and mechanistic insight

Sugar intake has dramatically increased during the last few decades. Specifically, there has been a clear trend towards higher consumption of fructose and high fructose corn syrup, which are the most common added sugars in processed food, soft drinks and other sweetened beverages. Although still controversial, this rising trend in simple sugar consumption has been positively associated with weight gain and obesity, insulin resistance and type 2 diabetes mellitus and non-alcoholic fatty liver disease. Interestingly, all of these metabolic alterations have also been related to the development of hepatocellular carcinoma. The purpose of this review is to discuss the evidence coming from epidemiological studies and data from animal models relating the consumption of simple sugars, and specifically fructose, with an increased risk of hepatocellular carcinoma and to gain insight into the putative molecular mechanisms involved.

Diagnosis, staging and treatment of hepatocellular carcinoma

Hepatocellular carcinomas are aggressive tumors with a high dissemination power. An early diagnosis of these tumors is of great importance in order to offer the possibility of curative treatment. For an early diagnosis, abdominal ultrasound and serum alpha-fetoprotein determinations at 6-month intervals are suggested for all patients with cirrhosis of the liver, since this disease is considered to be the main risk factor for the development of the neoplasia. Helicoidal computed tomography, magnetic resonance and/or hepatic arteriography are suggested for diagnostic confirmation and tumor staging. The need to obtain a fragment of the focal lesion for cytology and/or histology for a diagnosis of hepatocellular carcinoma depends on the inability of imaging methods to diagnose the lesion. Several classifications are currently available for tumor staging in order to determine patient prognosis. All take into consideration not only the stage of the tumor but also the degree of hepatocellular dysfunction, which is known to be the main factor related to patient survival. Classifications, however, fail to correlate treatment with prognosis and cannot suggest the ideal treatment for each tumor stage. The Barcelona Classification (BCLC) attempts to correlate tumor stage with treatment but requires prospective studies for validation. For single tumors smaller than 5 cm or up to three nodules smaller than 3 cm, surgical resection, liver transplantation and percutaneous treatment may offer good anti-tumoral results, as well as improved patient survival. Embolization or chemoembolization are therapeutic alternatives for patients who do not benefit from curative therapies.

Low occurrence of occult hepatitis B virus infection and high frequency of hepatitis C virus genotype 3 in hepatocellular carcinoma in Brazil

Occult hepatitis B virus (HBV) infection has been reported among patients with hepatitis C virus (HCV) infection and hepatocellular carcinoma (HCC). Our aim was to evaluate the presence of occult HBV infection in patients with HCV-related liver cirrhosis (LC) with or without HCC in São Paulo, Brazil. Serum and liver tissue samples from 50 hepatitis B surface antigen-negative patients with HCV-related LC who underwent liver transplantation at the University of São Paulo School of Medicine Hospital from 1993 to 2004 were divided into groups with LC only (N = 33) and with LC plus HCC (N = 17). HBV DNA was assayed for serum and paraffin-embedded liver tissue (tumoral and non-tumoral) using real time PCR and only 1 case with HCC had HBV DNA-positive serum. All liver samples were negative. HCV genotype 3 was detected in 17/39 (43.7%) cases. In conclusion, using a sensitive real time PCR directed to detect HBV variants circulating in Brazil, occult hepatitis B infection was not found among HCV-positive cirrhotic patients and was rarely found among HCV-positive HCC patients. These results are probably related to the low prevalence of HBV infection in our population. Furthermore, we have also shown that HCV genotype 3 is frequently found in Brazilian cirrhotic patients, particularly when they also have HCC. More studies involving a large number of cases should be carried out to confirm these data and to further characterize Brazilian HCV genotype isolates to elucidate genetic features that might be related to its carcinogenic potential.

Does hepatocellular carcinoma in non-alcoholic steatohepatitis exist in cirrhotic and non-cirrhotic patients?

Non-alcoholic steatohepatitis (NASH) has been associated with hepatocellular carcinoma (HCC) often arising in histologically advanced disease when steatohepatitis is not active (cryptogenic cirrhosis). Our objective was to characterize patients with HCC and active, histologically defined steatohepatitis. Among 394 patients with HCC detected by ultrasound imaging over 8 years and staged by the Barcelona Clinic Liver Cancer (BCLC) criteria, we identified 7 cases (1.7%) with HCC occurring in the setting of active biopsy-proven NASH. All were negative for other liver diseases such as hepatitis C, hepatitis B, autoimmune hepatitis, Wilson disease, and hemochromatosis. The patients (4 males and 3 females, age 63 ± 13 years) were either overweight (4) or obese (3); 57% were diabetic and 28.5% had dyslipidemia. Cirrhosis was present in 6 of 7 patients, but 1 patient had well-differentiated HCC in the setting of NASH without cirrhosis (fibrosis stage 1) based on repeated liver biopsies, the absence of portal hypertension by clinical and radiographic evaluations and by direct surgical inspection. Among the cirrhotic patients, 71.4% were clinically staged as Child A and 14.2% as Child B. Tumor size ranged from 1.0 to 5.2 cm and 5 of 7 patients were classified as early stage; 46% of all nodules were hyper-echoic and 57% were <3 cm. HCC was well differentiated in 1/6 and moderately differentiated in 5/6. Alpha-fetoprotein was <100 ng/mL in all patients. HCC in patients with active steatohepatitis is often multifocal, may precede clinically advanced disease and occurs without diagnostic levels of alpha-fetoprotein. Importantly, HCC may occur in NASH in the absence of cirrhosis. More aggressive screening of NASH patients may be warranted.

Retinoic acid and cAMP inhibit rat hepatocellular carcinoma cell proliferation and enhance cell differentiation

Hepatocellular carcinoma (HCC) is the third highest cause of cancer death worldwide. In general, the disease is diagnosed at an advanced stage when potentially curative therapies are no longer feasible. For this reason, it is very important to develop new therapeutic approaches. Retinoic acid (RA) is a natural derivative of vitamin A that regulates important biological processes including cell proliferation and differentiation. In vitro studies have shown that RA is effective in inhibiting growth of HCC cells; however, responsiveness to treatment varies among different HCC cell lines. The objective of the present study was to determine if the combined use of RA (0.1 µM) and cAMP (1 mM), an important second messenger, improves the responsiveness of HCC cells to RA treatment. We evaluated the proliferative behavior of an HCC cell line (HTC) and the expression profile of genes related to cancer signaling pathway (ERK and GSK-3β) and liver differentiation (E-cadherin, connexin 26 (Cx26), and Cx32). RA and cAMP were effective in inhibiting the proliferation of HTC cells independently of combined use. However, when a mixture of RA and cAMP was used, the signals concerning the degree of cell differentiation were increased. As demonstrated by Western blot, the treatment increased E-cadherin, Cx26, Cx32 and Ser9-GSK-3β (inactive form) expression while the expression of Cx43, Tyr216-GSK-3β (active form) and phosphorylated ERK decreased. Furthermore, telomerase activity was inhibited along treatment. Taken together, the results showed that the combined use of RA and cAMP is more effective in inducing differentiation of HTC cells.

IL-6 upregulation contributes to the reduction of miR-26a expression in hepatocellular carcinoma cells

A recent study showed that miR-26a is downregulated in hepatocellular carcinoma tissues and that this downregulation is an independent predictor of survival. Interestingly, the same study also reported that miR-26a downregulation causes a concomitant elevation of IL-6 expression. Because miR-26a expression was found to be transcriptionally downregulated by oncogene c-Myc in various cancers, and the expression of c-Myc was increased by IL-6 stimulation, we hypothesized that IL-6 contributes to reduction of miR-26a in hepatocellular carcinoma. Serum IL-6 was measured by ELISA and miR-26a was detected by qRT-PCR. The data of 30 patients with hepatocellular carcinoma who had undergone surgical tumor resection revealed that serum IL-6 could be considered to be a predictor of survival up to 5 years for hepatocellular carcinoma patients (log-rank test, P < 0.05). We observed that the serum IL-6 concentration was inversely correlated with miR-26a expression in cancerous tissues (Pearson correlation test, r = -0.651, P < 0.01). Furthermore, by in vitro experiments with HepG2 cells, we showed that IL-6 stimulation can lead to miR-26a suppression via c-Myc activation, whereas in normal hepatocyte LO2 cells incubation with IL-6 had no significant effect on miR-26a expression. Taken together, these results indicate that miR-26a reduction in hepatocellular carcinoma might be due to IL-6 upregulation.

Association of MDR1 gene polymorphisms with the risk of hepatocellular carcinoma in the Chinese Han population

The multidrug resistance 1 gene (MDR1) is an important candidate gene for influencing susceptibility to hepatocellular carcinoma (HCC). The objective of the present study was to evaluate the association ofMDR1 polymorphisms with the risk of HCC in the Chinese Han population. A total of 353 HCC patients and 335 healthy subjects were enrolled in the study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), created restriction site-PCR (CRS-PCR) and DNA sequencing methods were used to identify MDR1 gene polymorphisms. Two allelic variants (c.335T>C and c.3073A>C) were detected. The CC genotype of the c.335T>C polymorphism was associated with an increased risk of developing HCC compared to the TT genotype (OR = 2.161, 95%CI = 1.350-3.459, χ2 = 10.55, P = 0.0011). The risk of HCC was significantly higher for the CC genotype in the c.3073A>C polymorphism compared to the AA genotype in the studied populations (CCvs AA: OR = 2.575, 95%CI = 1.646-4.028, χ2 = 17.64, P < 0.0001). The C allele of the c.335T>C and c.3073A>C variants may contribute to the risk of HCC (Cvs T of c.335T>C: OR = 1.512, 95%CI = 1.208-1.893, χ2 = 13.07, P = 0.0003, and Cvs A of c.3073A>C: OR = 1.646, 95%CI = 1.322-2.049, χ2 = 20.03, P < 0.0001). The c.335T>C and c.3073A>C polymorphisms of the MDR1 gene were associated with the risk of occurrence of HCC in the Chinese Han population. Further investigations are needed to confirm these results in larger different populations.

Hepatitis B virus subgenotype C2- and B2-associated mutation patterns may be responsible for liver cirrhosis and hepatocellular carcinoma, respectively

The objective of this study was to examine hepatitis B virus (HBV) subgenotypes and mutations in enhancer II, basal core promoter, and precore regions of HBV in relation to risks of liver cirrhosis (LC) and hepatocellular carcinoma (HCC) in Southeast China. A case-control study was performed, including chronic hepatitis B (CHB; n=125), LC (n=120), and HCC (n=136). HBV was genotyped by multiplex polymerase chain reaction and subgenotyped by restriction fragment length polymorphism. HBV mutations were measured by DNA sequencing. HBV genotype C (68.2%) predominated and genotype B (30.2%) was the second most common. Of these, C2 (67.5%) was the most prevalent subgenotype, and B2 (30.2%) ranked second. Thirteen mutations with a frequency >5% were detected. Seven mutation patterns (C1653T, G1719T, G1730C, T1753C, A1762T, G1764A, and G1799C) were associated with C2, and four patterns (C1810T, A1846T, G1862T, and G1896A) were associated with B2. Six patterns (C1653T, G1730C, T1753C, A1762T, G1764A, and G1799C) were obviously associated with LC, and 10 patterns (C1653T, G1730C, T1753C, A1762T, G1764A, G1799C, C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with CHB. Four patterns (C1810T, A1846T, G1862T, and G1896A) were significantly associated with HCC compared with LC. Multivariate regression analyses showed that HBV subgenotype C2 and C2-associated mutation patterns (C1653T, T1753C, A1762T, and G1764A) were independent risk factors for LC when CHB was the control, and that B2-associated mutation patterns (C1810T, A1846T, G1862T, and G1896A) were independent risk factors for HCC when LC was the control.

Esculetin, a coumarin derivative, exerts in vitro and in vivo antiproliferative activity against hepatocellular carcinoma by initiating a mitochondrial-dependent apoptosis pathway

This study investigated the in vitro and in vivo antiproliferative activity of esculetin against hepatocellular carcinoma, and clarified its potential molecular mechanisms. Cell viability was determined by the MTT (tetrazolium) colorimetric assay. In vivoantitumor activity of esculetin was evaluated in a hepatocellular carcinoma mouse model. Seventy-five C57BL/6J mice were implanted with Hepa1-6 cells and randomized into five groups (n=15 each) given daily intraperitoneal injections of vehicle (physiological saline), esculetin (200, 400, or 700 mg·kg-1·day-1), or 5-Fu (200 mg·kg-1·day-1) for 15 days. Esculetin significantly decreased tumor growth in mice bearing Hepa1-6 cells. Tumor weight was decreased by 20.33, 40.37, and 55.42% with increasing doses of esculetin. Esculetin significantly inhibited proliferation of HCC cells in a concentration- and time-dependent manner and with an IC50 value of 2.24 mM. It blocked the cell cycle at S phase and induced apoptosis in SMMC-7721 cells with significant elevation of caspase-3 and caspase-9 activity, but did not affect caspase-8 activity. Moreover, esculetin treatment resulted in the collapse of mitochondrial membrane potential in vitro and in vivo accompanied by increased Bax expression and decreased Bcl-2 expression at both transcriptional and translational levels. Thus, esculetin exerted in vitro and in vivo antiproliferative activity in hepatocellular carcinoma, and its mechanisms involved initiation of a mitochondrial-mediated, caspase-dependent apoptosis pathway.

An exploratory study for the discovery of non-invasive hepatocellular carcinoma biomarkers among high-risk hepatitis C virus infected patients

Hepatocellular Carcinoma (HCC) is a major healthcare problem, representing the third most common cause of cancer-related mortality worldwide. Chronic infections with Hepatitis B virus (HBV) and/or Hepatitis C virus (HCV) are the major risk factors for the development of HCC. The incidence of HBV -associated HCC is in decline as a result of an effective HBV vaccine; however, since an equally effective HCV vaccine has not yet been developed, there are 130 million HCV infected patients worldwide who are at a high-risk for developing HCC. Because reliable parameters and/or tools for the early detection of HCC among high-risk individuals are severely lacking, HCC patients are always diagnosed at a late stage where surgical solutions or effective treatment are not possible. Using urine as a non-invasive sample source, two different approaches (proteomic-based and genomic-based approaches) were pursued with the common goal of discovering potential biomarker candidates for the early detection of HCC among high-risk chronic HCV infected patients. Urine was collected from 106 HCV infected Egyptian patients, 32 of whom had already developed HCC and 74 patients who were diagnosed as HCC-free at the time of initial sample collection. In addition to these patients, urine samples were also collected from 12 healthy control individuals. Total urinary proteins, Trans-renal nucleic acid (Tr-NA) and microRNA (miRNA) were isolated from urine using novel methodologies and silicon carbide-loaded spin columns. In the first, "proteomic-based", approach, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to identify potential candidates from pooled urine samples. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR (qRT-PCR). This approach revealed that significant over-expression of three proteins: DJ-1, Chromatin Assembly Factor-1 (CAF-1) and 11 Moemen Abdalla HCC Biomarkers Heat Shock Protein 60 (HSP60), were characteristic events among HCC-post HCV infected patients. As a single-based HCC biomarker, CAF-1 over-expression identified HCC among HCV infected patients with a specificity of 90%, sensitivity of 66% and with an overall diagnostic accuracy of 78%. Moreover, the CAF-lIHSP60 tandem identified HCC among HCV infected patients with a specificity of 92%, sensitivity of 61 % and with an overall diagnostic accuracy of 77%. In the second genomic-based approach, two different approaches were processed. The first approach was the miRNA-based approach. The expression levels of miRNAs isolated from urine were studied using the Illumina MicroRNA Expression Profiling Assay. This was followed by qRT-PCR-based validation of deregulated expression of identified miRNA candidates among all the patients. This approach shed the light on the deregulated expression of a number of miRNAs, which may have a role in either the development of HCC among HCV infected patients (i.e. miR-640, miR-765, miR-200a, miR-521 and miR-520) or may allow for a better understanding of the viral-host interaction (miR-152, miR-486, miR-219, miR452, miR-425, miR-154 and miR-31). Moreover, the deregulated expression of both miR-618 and miR-650 appeared to be a common event among HCC-post HCV infected patients. The results of the search for putative targets of these two miRNA suggested that miR-618 may be a potent oncogene, as it targets the tumor-suppressor gene Low density lipoprotein-related protein 12 (LPR12), while miR-650 may be a potent tumor-suppressor gene, as it is supposed to downregulate the TNF receptor-associated factor-4 (TRAF4) oncogene. The specificity of miR-618 and miR-650 deregulated expression patterns for the early detection of HCC among HCV infected patients was 68% and 58%, respectively, whereas the sensitivity was 64% and 72%, respectively. When the deregulated expression of both miRNAs was combined as a tandem biomarker, the specificity and the sensitivity were 75% and 58% respectively. 111 Moemen Abdalla HCC Biomarkers In the second, "Trans-renal nucleic acid-based", approach, the urinary apoptotic nucleic acid (uaNA) levels of 70ng/mL or more were found to be a good predictor of HCC among chronic HCV infected patients. The specificity and the sensitivity of this diagnostic approach were 76% and 86%, respectively, with an overall diagnostic value of 81 %. The uaNA levels positively correlated to HCC disease progression as monitored by epigenetic changes of a panel of eight tumor-suppressor genes (TSGs) using methylation-sensitive PCR. Moreover, the pairing of high uaNA levels (:::: 70 ng/mL) and CAF-1 over-expreSSIOn produced a highly specific (l 00%) multiple-based HCC biomarker with an acceptable sensitivity of 64%, and with a diagnostic accuracy of 82%. In comparison to the previous pairing, the uaNA levels (:::: 70 ng/mL) in tandem with HSP60 over-expression was less specific (89%) but highly sensitive (72%), resulting in a diagnostic accuracy of 64%. The specificities of miR-650 deregulated expression in combination with either high uaNA content or HSP 60 over-expression were 82% and 79%, respectively, whereas, the sensitivities of these combinations were 64% and 58%, respectively. The potential biomarkers identified in this study compare favorably with the diagnostic accuracy of the a-fetoprotein levels test, which has a specificity of 75%, sensitivity of 68% and an overall diagnostic accuracy of 70%. Here we present an intriguing study which shows the significance of using urine as a noninvasive sample source for the identification of promising HCC biomarkers. We have also introduced new techniques for the isolation of different urinary macromolecules, especially miRNA, from urine. Furthermore, we strongly recommend the potential biomarkers indentified in this study as focal points of any future research on HCC diagnosis. A larger testing pool will determine if their use is practical for mass population screening. This explorative study identified potential targets that merit further investigation for the development of diagnostically accurate biomarkers isolated from 1-2 mL urine samples that were acquired in a non-invasive manner.

Wavelet-based algorithm to the evaluation of contrasted hepatocellular carcinoma in CT-images after transarterial chemoembolization

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Sorafenib after arterial chemoembolization in child-pugh A and B cirrhotic patients with intermediate hepatocellular carcinoma: a retrospective analysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cyto and genotoxicity of gold nanoparticles in human hepatocellular carcinoma and peripheral blood mononuclear cells

Engineered nanomaterials have been extensively applied as active materials for technological applications. Since the impact of these nanomaterials on health and environment remains undefined, research on their possible toxic effects has attracted considerable attention. It is known that in humans, for example, the primary site of gold nanoparticles (AuNps) accumulation is the liver. The latter has motivated research regarding the use of AuNps for cancer therapy, since specific organs can be target upon appropriate functionalization of specific nanoparticles. In this study, we investigate the geno and cytotoxicity of two types of AuNps against human hepatocellular carcinoma cells (HepG2) and peripheral blood mononuclear cells (PBMC) from healthy human volunteers. The cells were incubated in the presence of different concentrations of AuNps capped with either sodium citrate or polyamidoamine dendrimers (PAMAM). Our results suggest that both types of AuNps interact with HepG2 cells and PBMC and may exhibit in vitro geno and cytotoxicity even at very low concentrations. In addition, the PBMC were less sensitive to DNA damage toxicity effects than cancer HepG2 cells upon exposure to AuNps. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Transforming growth factor alpha immunoreactivity. A study in hepatocellular carcinoma and in non-neoplastic liver tissue

Background. Transforming growth factor alpha (TGF alpha) is an important mitogen that binds to epidermal growth factor receptor and is associated with the development of several tumors. Aims. Assessment of the immunoexpression of TGF alpha in hepatocellular carcinoma (HCC) and in non-neoplastic liver tissue and its relationship to morphological patterns of HCC. Material and methods. The immunohistochemical expression of TGF alpha was studied in 47 cases of HCC (27 multinodular, 20 nodular lesions). Five lesions measured up to 5 cm and 15 lesions above 5 cm. Thirty-two cases were graded as I or II and 15 as III or IV. The non-neoplastic tissue was examined in 40 cases, of which 22 had cirrhosis. HBsAg and anti-HCV were positive in 5/38 and 15/37 patients, respectively. The statistical analysis for possible association of immunostaining of TGF alpha and pathological features was performed through chi-square test. Results. TGF alpha was detected in 31.9% of the HCC and in 42.5% of the non-neoplastic. There was a statistically significant association between the expression of TGF alpha and cirrhosis (OR = 8.75, 95% CI = [1.93, 39.75]). The TGF alpha was detected more frequently in patients anti-HCV(+) than in those HBsAg(+). The immunoexpression of TGF alpha was not found related to tumor size or differentiation. In conclusion the TGF alpha is present in hepatocarcinogenesis in HBV negative patients. Further analysis is needed to examine the involvement of TGF alpha in the carcinogenesis associated with HCV and other possible agents. In addition, TGF alpha has an higher expression in hepatocyte regeneration and proliferation in cirrhotic livers than in HCC.

Retinoic acid and cAMP inhibit rat hepatocellular carcinoma cell proliferation and enhance cell differentiation

Hepatocellular carcinoma (HCC) is the third highest cause of cancer death worldwide. In general, the disease is diagnosed at an advanced stage when potentially curative therapies are no longer feasible. For this reason, it is very important to develop new therapeutic approaches. Retinoic acid (RA) is a natural derivative of vitamin A that regulates important biological processes including cell proliferation and differentiation. In vitro studies have shown that RA is effective in inhibiting growth of HCC cells; however, responsiveness to treatment varies among different HCC cell lines. The objective of the present study was to determine if the combined use of RA (0.1 µM) and cAMP (1 mM), an important second messenger, improves the responsiveness of HCC cells to RA treatment. We evaluated the proliferative behavior of an HCC cell line (HTC) and the expression profile of genes related to cancer signaling pathway (ERK and GSK-3β) and liver differentiation (E-cadherin, connexin 26 (Cx26), and Cx32). RA and cAMP were effective in inhibiting the proliferation of HTC cells independently of combined use. However, when a mixture of RA and cAMP was used, the signals concerning the degree of cell differentiation were increased. As demonstrated by Western blot, the treatment increased E-cadherin, Cx26, Cx32 and Ser9-GSK-3β (inactive form) expression while the expression of Cx43, Tyr216-GSK-3β (active form) and phosphorylated ERK decreased. Furthermore, telomerase activity was inhibited along treatment. Taken together, the results showed that the combined use of RA and cAMP is more effective in inducing differentiation of HTC cells.