Heparin and Liver Heparan Sulfate Can Rescue Hepatoma Cells from Topotecan Action

Topotecan (TpT) is a major inhibitory compound of topoisomerase (topo) I that plays important roles in gene transcription and cell division. We have previously reported that heparin and heparan sulfate (HS) might be transported to the cell nucleus and they can interact with topoisomerase I. We hypothesized that heparin and HS might interfere with the action of TpT. To test this hypothesis we isolated topoisomerase I containing cell nuclear protein fractions from normal liver, liver cancer tissues, and hepatoma cell lines. The enzymatic activity of these extracts was measured in the presence of heparin, liver HS, and liver cancer HS. In addition, topo I activity, cell viability, and apoptosis of HepG2 and Hep3B cells were investigated after heparin and TpT treatments. Liver cancer HS inhibited topo I activity in vitro. Heparin treatment abrogated topo I enzyme activity in Hep3B cells, but not in HepG2 cells, where the basal activity was higher. Heparin protected the two hepatoma cell lines from TpT actions and decreased the rate of TpT induced S phase block and cell death. These results suggest that heparin and HS might interfere with the function of TpT in liver and liver cancer

Elevated miR-33a and miR-224 in steatotic chronic hepatitis C liver biopsies

AIM: To assess the expression of selected microRNAs (miRNA) in hepatitis C, steatotic hepatitis C, noninfected steatotic and normal liver tissues. METHODS: The relative expression levels of miR-21, miR-33a, miR-96, miR-122, miR-125b, miR-221 and miR-224 were determined in 76 RNA samples isolated from 18 non-steatotic and 28 steatotic chronic hepatitis C (CHC and CHC-Steatosis, respectively) cases, 18 non-infected, steatotic liver biopsies of metabolic origin (Steatosis) and 12 normal formalin-fixed paraffin-embedded liver tissues using TaqMan MicroRNA Assays. All CHC biopsy samples were obtained prior to initiating therapy. Patients’ serum biochemical values, which included glucose, triglyceride, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl-transferase (GGT), alkaline phosphatase (AP), were obtained and correlated with relative miRNA expression. RESULTS: When compared with control non-infected liver samples, miR-122 and miR-221 levels were reduced in CHC-Steatosis (P < 0.03) and in CHC, CHC-Steatosis and Steatosis (P < 0.01). Alternatively, the expression of miR-33a and miR-224 were elevated in CHC-Steatosis and Steatosis in comparison to control tissue (P < 0.01). The levels of miR-33a and miR-224 in CHC-Steatosis (P < 0.02) and miR-224 in Steatosis (P < 0.001) were increased in comparison to CHC samples. By contrast, the expression of miR-21 did not differ statistically between diseased and normal liver samples. Levels of miR-33a correlated negatively with serum AST and AP levels in Steatosis as well as with necroinflammatory grade in CHC, whereas miR-21 correlated positively with AST in Steatosis and displayed negative correlation with triglyceride level in CHC-Steatosis. In contrast, miRNA levels were not correlated with ALT, GGT, cholesterol levels or fibrosis stage. CONCLUSION: Differences in miRNA expression were observed between CHC and steatotic CHC, CHC and steatotic liver, but not between steatotic CHC and steatotic liver of metabolic origin

Elevated miR-33a and miR-224 in steatotic chronic hepatitis C liver biopsies.

AIM: To assess the expression of selected microRNAs (miRNA) in hepatitis C, steatotic hepatitis C, noninfected steatotic and normal liver tissues. METHODS: The relative expression levels of miR-21, miR-33a, miR-96, miR-122, miR-125b, miR-221 and miR-224 were determined in 76 RNA samples isolated from 18 non-steatotic and 28 steatotic chronic hepatitis C (CHC and CHC-Steatosis, respectively) cases, 18 non-infected, steatotic liver biopsies of metabolic origin (Steatosis) and 12 normal formalin-fixed paraffin-embedded liver tissues using TaqMan MicroRNA Assays. All CHC biopsy samples were obtained prior to initiating therapy. Patients' serum biochemical values, which included glucose, triglyceride, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl-transferase (GGT), alkaline phosphatase (AP), were obtained and correlated with relative miRNA expression. RESULTS: When compared with control non-infected liver samples, miR-122 and miR-221 levels were reduced in CHC-Steatosis (P < 0.03) and in CHC, CHC-Steatosis and Steatosis (P < 0.01). Alternatively, the expression of miR-33a and miR-224 were elevated in CHC-Steatosis and Steatosis in comparison to control tissue (P < 0.01). The levels of miR-33a and miR-224 in CHC-Steatosis (P < 0.02) and miR-224 in Steatosis (P < 0.001) were increased in comparison to CHC samples. By contrast, the expression of miR-21 did not differ statistically between diseased and normal liver samples. Levels of miR-33a correlated negatively with serum AST and AP levels in Steatosis as well as with necroinflammatory grade in CHC, whereas miR-21 correlated positively with AST in Steatosis and displayed negative correlation with triglyceride level in CHC-Steatosis. In contrast, miRNA levels were not correlated with ALT, GGT, cholesterol levels or fibrosis stage. CONCLUSION: Differences in miRNA expression were observed between CHC and steatotic CHC, CHC and steatotic liver, but not between steatotic CHC and steatotic liver of metabolic origin

Elevated miR-33a and miR-224 in steatotic chronic hepatitis C liver biopsies

AIM: To assess the expression of selected microRNAs (miRNA) in hepatitis C, steatotic hepatitis C, noninfected steatotic and normal liver tissues. METHODS: The relative expression levels of miR-21, miR-33a, miR-96, miR-122, miR-125b, miR-221 and miR-224 were determined in 76 RNA samples isolated from 18 non-steatotic and 28 steatotic chronic hepatitis C (CHC and CHC-Steatosis, respectively) cases, 18 non-infected, steatotic liver biopsies of metabolic origin (Steatosis) and 12 normal formalin-fixed paraffin-embedded liver tissues using TaqMan MicroRNA Assays. All CHC biopsy samples were obtained prior to initiating therapy. Patients' serum biochemical values, which included glucose, triglyceride, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl-transferase (GGT), alkaline phosphatase (AP), were obtained and correlated with relative miRNA expression. RESULTS: When compared with control non-infected liver samples, miR-122 and miR-221 levels were reduced in CHC-Steatosis (P < 0.03) and in CHC, CHC-Steatosis and Steatosis (P < 0.01). Alternatively, the expression of miR-33a and miR-224 were elevated in CHC-Steatosis and Steatosis in comparison to control tissue (P < 0.01). The levels of miR-33a and miR-224 in CHC-Steatosis (P < 0.02) and miR-224 in Steatosis (P < 0.001) were increased in comparison to CHC samples. By contrast, the expression of miR-21 did not differ statistically between diseased and normal liver samples. Levels of miR-33a correlated negatively with serum AST and AP levels in Steatosis as well as with necroinflammatory grade in CHC, whereas miR-21 correlated positively with AST in Steatosis and displayed negative correlation with triglyceride level in CHC-Steatosis. In contrast, miRNA levels were not correlated with ALT, GGT, cholesterol levels or fibrosis stage. CONCLUSION: Differences in miRNA expression were observed between CHC and steatotic CHC, CHC and steatotic liver, but not between steatotic CHC and steatotic liver of metabolic origin

Evaluation of 9-cis retinoic acid and mitotane as antitumoral agents in an adrenocortical xenograft model

The available drug treatment options for adrenocortical carcinoma (ACC) are limited. In our previous studies, the in vitro activity of 9-cis retinoic acid (9-cisRA) on adrenocortical NCI-H295R cells was shown along with its antitumoral effects in a small pilot xenograft study. Our aim was to dissect the antitumoral effects of 9-cisRA on ACC in a large-scale xenograft study involving mitotane, 9-cisRA and their combination. 43 male SCID mice inoculated with NCI-H295R cells were treated in four groups (i. control, ii. 9-cisRA, iii. mitotane, iv. 9-cisRA + mitotane) for 28 days. Tumor size follow-up, histological and immunohistochemical (Ki-67) analysis, tissue gene expression microarray, quantitative real-time-PCR for the validation of microarray results and to detect circulating microRNAs were performed. Protein expression was studied by proteomics and Western-blot validation. Only mitotane alone and the combination of 9-cisRA and mitotane resulted in significant tumor size reduction. The Ki-67 index was significantly reduced in both 9-cisRA and 9-cisRA+mitotane groups. Only modest changes at the mRNA level were found: the 9-cisRA-induced overexpression of apolipoprotein A4 and down-regulation of phosphodiesterase 4A was validated. The expression of circulating hsa-miR-483-5p was significantly reduced in the combined treatment group. The SET protein was validated as being significantly down-regulated in the combined mitotane+9-cisRA group. 9-cisRA might be a helpful additive agent in the treatment of ACC in combination with mitotane. Circulating hsa-miR-483-5p could be utilized for monitoring the treatment efficacy in ACC patients, and the treatment-induced reduction in protein SET expression might raise its relevance in ACC biology

SPOCK1 Promotes the Development of Hepatocellular Carcinoma

The extracellular matrix proteoglycan SPOCK1 is increasingly recognized as a contributor to the development and progression of cancers. Here, we study how SPOCK1, which is present in non-tumorous hepatocytes at low concentrations, promotes the development and progression of malignant hepatocellular tumors. Although SPOCK1 is an extracellular matrix proteoglycan, its concentration increases in the cytoplasm of hepatocytes starting with very low expression in the normal cells and then appearing in much higher quantities in cells of cirrhotic human liver and hepatocellular carcinoma. This observation is similar to that observed after diethylnitrosamine induction of mouse hepatocarcinogenesis. Furthermore, syndecan-1, the major proteoglycan of the liver, and SPOCK1 are in inverse correlation in the course of these events. In hepatoma cell lines, the cytoplasmic SPOCK1 colocalized with mitochondrial markers, such as MitoTracker and TOMM20, a characteristic protein of the outer membrane of the mitochondrion and could be detected in the cell nucleus. SPOCK1 downregulation of hepatoma cell lines by siRNA inhibited cell proliferation, upregulated p21 and p27, and interfered with pAkt and CDK4 expression. A tyrosine kinase array revealed that inhibition of SPOCK1 in the liver cancer cells altered MAPK signaling and downregulated several members of the Sarc family, all related to the aggressivity of the hepatoma cell lines. These studies support the idea that SPOCK1 enhancement in the liver is an active contributor to human and rodent hepatocarcinogenesis and cancer progression. However, its mitochondrial localization raises the possibility that it has a currently unidentified physiological function in normal hepatocytes

Decorin deficiency promotes hepatic carcinogenesis

Hepatocellular carcinoma represents one of the most-rapidly spreading cancers in the world. In the majority of cases, an inflammation-driven fibrosis or cirrhosis precedes the development of the tumor. During malignant transformation, the tumor microenvironment undergoes qualitative and quantitative changes that modulate the behavior of the malignant cells. A key constituent for the hepatic microenvironment is the small leucine-rich proteoglycan decorin, known to interfere with cellular events of tumorigenesis mainly by blocking various receptor tyrosine kinases (RTK) such as EGFR, Met, IGF-IR, PDGFR and VEGFR2. In this study, we characterized cell signaling events evoked by decorin deficiency in two experimental models of hepatocarcinogenesis using thioacetamide or diethyl nitrosamine as carcinogens. Genetic ablation of decorin led to enhanced tumor occurrence as compared to wild-type animals. These findings correlated with decreased levels of the cyclin-dependent kinase inhibitor p21WAF1/CIP1 and a concurrent elevation in retinoblastoma protein phosphorylation via cyclin dependent kinase 4. Decreased steady state p21Waf1/Cip1 levels correlated with enhanced expression of transcription factor AP4, a known transcriptional repressor of p21Waf1/Cip1, and enhanced c-Myc protein levels. In addition, translocation of beta-catenin was a typical event in diethyl nitrosamine-evoked tumors. In parallel, decreased phosphorylation of both c-Myc and beta-catenin was observed in Dcn-/- livers likely due to the hindered GSK3beta-mediated targeting of these proteins to proteasomal degradation. We discovered that in a genetic background lacking decorin, four RTKs were constitutively activated (phosphorylated), including three known targets of decorin such as PDGFRalpha, EGFR, IGF-IR, and a novel RTK MSPR/RON. Our findings provide powerful genetic evidence for a crucial in vivo role of decorin during hepatocarcinogenesis as lack of decorin in the liver and hepatic stroma facilitates experimental carcinogenesis by providing an environment devoid of this potent pan-RTK inhibitor. Thus, our results support future utilization of decorin as an antitumor agent in liver cancer