Michael A. Kern

Cyclooxygenase-2 inhibition induces apoptosis signaling via death receptors and mitochondria in hepatocellular carcinoma.

Inhibition of cyclooxygenase (COX)-2 elicits chemopreventive and therapeutic effects in solid tumors that are coupled with the induction of apoptosis in tumor cells. We investigated the mechanisms by which COX-2 inhibition induces apoptosis in hepatocellular carcinoma (HCC) cells. COX-2 inhibition triggered expression of the CD95, tumor necrosis factor (TNF)-R, and TNF-related apoptosis-inducing ligand (TRAIL)-R1 and TRAIL-R2 death receptors. Addition of the respective specific ligands further increased apoptosis, indicating that COX-2 inhibition induced the expression of functional death receptors. Overexpression of a dominant-negative Fas-associated death domain mutant reduced COX-2 inhibitor-mediated apoptosis. Furthermore, our findings showed a link between COX-2 inhibition and the mitochondrial apoptosis pathway. COX-2 inhibition led to a rapid down-regulation of myeloid cell leukemia-1 (Mcl-1), an antiapoptotic member of the Bcl-2 family, followed by translocation of Bax to mitochondria and cytochrome c release from mitochondria. Consequently, overexpression of Mcl-1 led to inhibition of COX-2 inhibitor-mediated apoptosis. Furthermore, blocking endogenous Mcl-1 function using a small-interfering RNA approach enhanced COX-2 inhibitor-mediated apoptosis. It is of clinical importance that celecoxib acted synergistically with chemotherapeutic drugs in the induction of apoptosis in HCC cells. The clinical relevance of these results is further substantiated by the finding that COX-2 inhibitors did not sensitize primary human hepatocytes toward chemotherapy-induced apoptosis. In conclusion, COX-2 inhibition engages different apoptosis pathways in HCC cells stimulating death receptor signaling, activation of caspases, and apoptosis originating from mitochondria.

Expression of c-Met in developing rat hippocampus: evidence for HGF as a neurotrophic factor for calbindin D-expressing neurons.

Hepatocyte growth factor‐scatter factor (HGF) is expressed in different parts of the nervous system, and has been shown to exhibit neurotrophic activity. Here we show that c‐Met, the receptor for HGF, is expressed in developing rat hippocampus, with the highest levels during the first postnatal weeks. To study the function of HGF, hippocampal neurons were prepared from embryonic rats and treated with different HGF concentrations. In these cultures, HGF increased the number of neurons expressing the 28‐kDa calcium‐binding protein (calbindin D) in a dose‐dependent manner. The effect of HGF was larger than that observed with either brain‐derived neurotrophic factor (BDNF) or neurotrophin‐3 (NT‐3), and cotreatment of the cultures with HGF and the neurotrophins was additive with respect to calbindin D neurons. Besides affecting the number of neurons, HGF significantly increased the degree of sprouting of calbindin D‐positive neurons, suggesting an influence on neuronal maturation. BDNF and NT‐3 stimulated neurite outgrowth of calbindin D neurons to a much smaller degree. In contrast to calbindin D neurons, HGF did not significantly increase the number of neurons immunoreactive with the neurotransmitter γ‐aminobutyric acid (GABA) in the hippocampal cultures. Immunohistochemical studies showed that c‐Met‐, calbindin D‐ and HGF‐immunoreactive cells are all present in the dentate gyrus and partly colocalize within neurons. These results show that HGF acts on calbindin D‐containing hippocampal neurons and increases their neurite outgrowth, suggesting that HGF plays an important role for the maturation and function of these neurons in the hippocampus.

Molecular pathogenesis of human hepatocellular carcinoma

Publisher Summary Hepatocellular carcinoma (HCC) is one of the most frequent human cancers. This chapter focuses on the compilation of molecular and functional changes identified in human HCCs and their interrelation and impact on tumor formation and therapy. The chapter explores the experimental data obtained in animal models, such as transgenic mouse lines, chemical rodent hepatocarcinogenesis, and models of virus-induced liver cancer. Human hepatocarcinogenesis usually develops on the histological background of a chronic liver disease, such as chronic hepatitis, chronic alcoholic liver disease, and hemochromatosis. The biology and prognosis of hepatocellular carcinoma significantly correlates with tumor stage and grade, but currently there is no evidence that tumor behavior is predictable from the macroscopic growth pattern, microscopic subtype, or any of the available immuno-histological markers. Only fibrolamellar HCC that is a rare, stroma-rich, oncocytic variant occurring predominantly in younger patients seems to have a better prognosis. Interference with growth factor signaling pathways by small molecules is an attractive therapeutic scheme with potential application in HCCs. All therapeutic approaches have to respect that in the majority of HCC patients, the surrounding liver is severely damaged, often showing complete cirrhosis. This imposes restrictions on therapeutic intervention and is often life-limiting in a manner comparable to the HCC itself.

Severe familial left ventricular non-compaction cardiomyopathy due to a novel troponin T (TNNT2) mutation

AIMS Left ventricular non-compaction (LVNC) is caused by mutations in multiple genes. It is still unclear whether LVNC is the primary determinant of cardiomyopathy or rather a secondary phenomenon with intrinsic cardiomyocyte dysfunction being the actual cause of the disease. Here, we describe a family with LVNC due to a novel missense mutation, pE96K, in the cardiac troponin T gene (TNNT2). METHODS AND RESULTS The novel mutation was identified in the index patient and all affected relatives, but not in 430 healthy control individuals. Mutations in known LVNC-associated genes were excluded. To investigate the pathophysiological implications of the mutation, we generated transgenic mice expressing human wild-type cTNT (hcTNT) or a human troponin T harbouring the pE96K mutation (mut cTNT). Animals were characterized by echocardiography, histology, and gene expression analysis. Mut cTNT mice displayed an impaired left ventricular function and induction of marker genes of heart failure. Remarkably, left ventricular non-compaction was not observed. CONCLUSION Familial co-segregation and the cardiomyopathy phenotype of mut cTNT mice strongly support a causal relationship of the pE96K mutation and disease in our index patient. In addition, our data suggest that a non-compaction phenotype is not required for the development of cardiomyopathy in this specific TNNT2 mutation leading to LVNC.

Targeting heat shock protein 90 with non-quinone inhibitors: a novel chemotherapeutic approach in human hepatocellular carcinoma.

The inhibition of heat shock protein 90 (Hsp90) has emerged as a promising antineoplastic strategy in diverse human malignancies. Hsp90 has been predicted to be involved in hepatocellular carcinoma (HCC) development; however, its role in hepatocarcinogenesis remains elusive. Using chemically distinctive Hsp90 inhibitors, we show that Hsp90 capacitates the aberrant expression and activity of crucial hepatocarcinogenesis‐driving factors (e.g., insulin‐like growth factor receptor 1, hepatocyte growth factor receptor, protein kinase B, v‐raf‐1 murine leukemia viral oncogene homolog 1, and cyclin‐dependent kinase 4). In vitro, Hsp90 inhibition with both geldanamycin analogs (17‐allylamino‐17‐desmethoxygeldanamycin (17‐AAG) and 17‐dimethylaminoethylamino‐17‐desmethoxygeldanamycin (17‐DMAG)) and the non‐quinone compound 8‐(6‐iodobenzo[d][1,3]dioxol‐5‐ylthio)‐9‐(3‐(isopropylamino)propyl)‐9H‐purin‐6‐amine (PU‐H71) reduced the viability of various HCC cell lines, induced the simultaneous degradation of numerous hepatocarcinogenic factors, and caused substantial cell cycle arrest and apoptosis. In contrast, nontumorigenic hepatocytes were less susceptible to Hsp90 inhibition. Because conventional geldanamycin‐derivate Hsp90 inhibitors induce dose‐limiting liver toxicity, we tested whether novel Hsp90 inhibitors lacking the benzoquinone moiety, which has been deemed responsible for hepatotoxicity, can elicit antineoplastic activity without causing significant liver damage. In HCC xenograft mouse models, PU‐H71 was retained in tumors at pharmacologically relevant concentrations while being rapidly cleared from nontumorous liver. PU‐H71 showed potent and prolonged in vivo Hsp90 inhibitory activity and reduced tumor growth without causing toxicity. Conclusion: Hsp90 constitutes a promising therapeutic target in HCC. Non‐quinone Hsp90 inhibitors exhibit tumor‐specific accumulation and exert potent antineoplastic activity without causing significant hepatotoxicity. (HEPATOLOGY 2009.)

Melatonin protects from hepatic reperfusion injury through inhibition of IKK and JNK pathways and modification of cell proliferation.

Abstract:  Reactive oxygen species (ROS) are involved in pathophysiology of ischemia/reperfusion injury. Melatonin is a potent scavenger of ROS. Thus, this study was designed to elucidate its effects in a combined hepatic warm ischemia and resection model. The right lateral and caudate lobes (32% of liver volume) of Sprague–Dawley rats underwent warm ischemia for 30 min followed by reperfusion and subsequent resection of the nonischemic liver tissue. Some rats were gavaged with 50 mg/kg melatonin 2 hr before the onset of experiments. Controls received the same volume of microcrystalline cellulose. Survival, transaminases, histology, flow cytometry, inducible nitric oxide synthase (iNOS) expression, and activation of signal transduction pathways [c‐Jun N‐terminal kinase (JNK), cJUN, IκB kinase α (IKKα), proliferating cell nuclear antigen (PCNA), and Ki67] were assessed for hepatic injury, oxidative stress, and cell proliferation. Melatonin significantly improved animal survival and decreased transaminase levels, the indices for necrosis, liver damage, leukocyte infiltration, and iNOS expression. In parallel, the expression of IKKα, JNK1, and cJUN decreased by 35–50% after melatonin (P < 0.05). At the same time, melatonin reduced the expression of both PCNA and Ki67 in liver (P < 0.05). Melatonin is hepatoprotective most likely via mechanisms including inhibition of IKK and JNK pathways and regulation of cell proliferation.

Down‐regulation of tumor suppressor a kinase anchor protein 12 in human hepatocarcinogenesis by epigenetic mechanisms

The A kinase anchor protein 12 (AKAP12) is a central mediator of protein kinase A and protein kinase C signaling. Although AKAP12 has been described to act as a tumor suppressor and its expression is frequently down‐regulated in several human malignancies, the underlying molecular mechanisms responsible for the AKAP12 reduction are poorly understood. We therefore analyzed the expression of AKAP12 and its genetic and epigenetic regulatory mechanisms in human hepatocarcinogenesis. Based on tissue microarray analyses (n = 388) and western immunoblotting, we observed a significant reduction of AKAP12 in cirrhotic liver (CL), premalignant lesions (DN), and hepatocellular carcinomas (HCCs) compared to histologically normal liver specimens (NL). Analyses of array comparative genomic hybridization data (aCGH) from human HCCs revealed chromosomal losses of AKAP12 in 36% of cases but suggested additional mechanisms underlying the observed reduction of AKAP12 expression in hepatocarcinogenesis. Quantitative methylation analysis by MassARRAY of NL, CL, DN, and HCC tissues, as well as of various tumorigenic and nontumorigenic liver cell lines revealed specific hypermethylation of the AKAP12α promoter but not of the AKAP12β promoter in HCC specimens and in HCC cell lines. Consequently, restoration experiments performed with 5‐aza‐2′deoxycytidine drastically increased AKAP12α mRNA levels in a HCC cell line (AKN1) paralleled by AKAP12α promoter demethylation. As hypermethylation is not observed in CL and DN, we investigated microRNA‐mediated posttranscriptional regulation as an additional mechanism to explain reduced AKAP12 expression. We found that miR‐183 and miR‐186 are up‐regulated in CL and DN and are able to target AKAP12. Conclusion: In addition to genetic alterations, epigenetic mechanisms are responsible for the reduction of the tumor suppressor gene AKAP12 in human hepatocarcinogenesis. (HEPATOLOGY 2010;.)

Expression pattern and functional relevance of epidermal growth factor receptor and cyclooxygenase-2: novel chemotherapeutic targets in pancreatic endocrine tumors?

OBJECTIVES:Pancreatic endocrine tumors represent morphologically and biologically heterogeneous neoplasms. Well-differentiated endocrine tumors (benign or of uncertain behavior) can be distinguished from well-differentiated and poorly differentiated endocrine carcinomas. Although many well-differentiated endocrine carcinomas show rather low rates of tumor growth, more than two-thirds of pancreatic endocrine carcinomas display distant metastases at the time of diagnosis. As the currently applied therapies beyond surgery only achieve partial or complete response rates of approximately 15%, additional chemotherapeutic targets are needed, especially in the therapy of inoperable and progressive pancreatic endocrine carcinomas.METHODS:The expression of epidermal growth factor receptor (EGFR) and cyclooxygenase (COX)-2 were investigated in 110 clinically and pathomorphologically well-characterized pancreatic endocrine tumors, using immunohistochemistry and immunoblot analyses. Functional tests were performed using the human pancreas carcinoid cell line BON and the mouse insulinoma cell line β-TC-3.RESULTS:The expression of EGFR correlated significantly with the grade of malignancy, increasing from low rates of expression in benign tumors and tumors of uncertain behavior to high rates of expression in well- and poorly differentiated endocrine carcinomas. The expression of COX-2 was independent of the malignant potential, but was more frequently expressed in primary tumors than in metastases. The treatment of the human pancreas carcinoid cell line BON and the mouse insulinoma cell line β-TC-3 with EGFR and COX-2 inhibitors (monotherapy and combined therapy) resulted in a significant, dose-dependent reduction of cell viability coupled with increased apoptosis.CONCLUSIONS:Our results suggest that EGFR and COX-2 may represent useful additional chemotherapeutic targets in pancreatic endocrine tumors.

Cyclooxygenase-2 (COX-2) - A Therapeutic Target in Liver Cancer?

Targeting COX-2, a key-enzyme of the prostaglandin metabolism, for the treatment of cancer has been in the focus of researchers for about a decade. However, only recently has this topic been related to hepatocellular carcinoma (HCC). HCC is one of the most common cancers and a growing health problem worldwide. At present, only few promising treatment options are available, accentuating the urgent need for novel therapeutic approaches. Since the first report of COX-2 overexpression in HCC, several findings support the notion that selective COX-2 inhibition proves to be beneficial in this malignancy. This review focuses on recent discoveries regarding the pro-tumorigenic potential of COX-2 in HCC and the functional effects of COX-2 inhibition on molecular mechanisms of this malignancy. Of clinical interest, promising data from in vivo experiments and case studies suggest a beneficial effect of COX-2 inhibitors for HCC- therapy. Detailed analysis of COX-2- activated pathways and related mechanisms may enable the evaluation and design of even more specific and combinatorial treatment approaches in the future.

Establishment, characterization and drug sensitivity testing in primary cultures of human thymoma and thymic carcinoma

Thymomas and thymic carcinomas are peculiar epithelial tumors of the anterior mediastinum. They may show aggressive clinical behavior and are a paradigm for the interaction between the tumor and the immune system. So far, adequate functional studies enabling a better understanding of this malignancy have not been performed, since human thymoma/thymic carcinoma cell lines have not been available. Here, the authors describe the establishment, characterization and functional analyses of epithelial cell lines from a Type B1‐thymoma and a poorly differentiated thymic carcinoma. By Fluorescence‐activated cell sorting (FACS) analyses, both cell lines were aneuploid. The aneuploid cell fraction of the thymic carcinoma cell line was characterized by a high proliferation index of 55.9%, in contrast to a lower proliferation rate of the aneuploid cell fraction of the thymoma (19.7%). Array‐based comparative genomic hybridization (aCGH) and conventional cytogenetic analysis of the thymoma revealed only minor imbalances whereas the thymic carcinoma was characterized by a complex karyotype in the hyperdiploid range that was readily defined with multicolor FISH (mFISH). Application of a selective COX‐2 inhibitor reduced cell viability in both cell lines in a dose‐dependent manner. In conclusion, these first cell lines of a thymoma and a CD5‐positive thymic carcinoma are useful tools for further in vitro studies of cellular, molecular and genetic aspects of the disease and for functional tests to evaluate new therapeutic targets.