Susanne Strand

Drug-induced apoptosis in hepatoma cells is mediated by the CD95 (APO-1/Fas) receptor/ligand system and involves activation of wild-type p53.

Chemotherapeutic drugs are cytotoxic by induction of apoptosis in drug-sensitive cells. We investigated the mechanism of bleomycin-induced cytotoxicity in hepatoma cells. At concentrations present in the sera of patients during therapy, bleomycin induced transient accumulation of nuclear wild-type (wt) p53 and upregulated expression of cell surface CD95 (APO-1/Fas) receptor in hepatoma cells carrying wt p53 (HepG2). Bleomycin did not increase CD95 in hepatoma cells with mutated p53 (Huh7) or in hepatoma cells which were p53-/- (Hep3B). In addition, sensitivity towards CD95-mediated apoptosis was also increased in wt p53 positive HepG2 cells. Microinjection of wt p53 cDNA into HepG2 cells had the same effect. In contrast, bleomycin did not enhance susceptibility towards CD95-mediated apoptosis in Huh7 and in Hep3B cells. Furthermore, bleomycin treatment of HepG2 cells increased CD95 ligand (CD95L) mRNA expression. Most notably, bleomycin-induced apoptosis in HepG2 cells was almost completely inhibited by antibodies which interfere with CD95 receptor/ligand interaction. These data suggest that apoptosis induced by bleomycin is mediated, at least in part, by p53-dependent stimulation of the CD95 receptor/ligand system. The same applies to other anti-cancer drugs such as cisplatin and methotrexate. These data may have major consequences for drug treatment of cancer and the explanation of drug sensitivity and resistance.

CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are immunosuppressive drugs that are used in organ transplantation and autoimmune and chronic inflammatory diseases such as Crohn disease. However, their molecular mechanism of action is unknown. In the present study, we have identified a unique and unexpected role for azathioprine and its metabolites in the control of T cell apoptosis by modulation of Rac1 activation upon CD28 costimulation. We found that azathioprine and its metabolites induced apoptosis of T cells from patients with Crohn disease and control patients. Apoptosis induction required costimulation with CD28 and was mediated by specific blockade of Rac1 activation through binding of azathioprine-generated 6-thioguanine triphosphate (6-Thio-GTP) to Rac1 instead of GTP. The activation of Rac1 target genes such as mitogen-activated protein kinase kinase (MEK), NF-kappaB, and bcl-x(L) was suppressed by azathioprine, leading to a mitochondrial pathway of apoptosis. Azathioprine thus converts a costimulatory signal into an apoptotic signal by modulating Rac1 activity. These findings explain the immunosuppressive effects of azathioprine and suggest that 6-Thio-GTP derivates may be useful as potent immunosuppressive agents in autoimmune diseases and organ transplantation.

Involvement of the CD95 (APO-1/Fas) Receptor and Ligand System in Helicobacter pylori -induced Gastric Epithelial Apoptosis

Helicobacter pylori infection is associated with chronic gastritis, peptic ulceration, and gastric carcinoma. The potential role of CD95-mediated apoptosis was investigated in a panel of gastric biopsies obtained from patients with H. pylori-associated chronic gastritis (n = 29) and with noninfected normal mucosa (n = 10). Immunohistochemistry revealed increased CD95 receptor expression in epithelial and lamina propria cells in chronic gastritis. By in situ hybridization, CD95 ligand mRNA was absent or low in normal mucosa but expressed at high levels in lamina propria lymphocytes and, unexpectedly, in epithelial cells in chronic gastritis. Apoptotic cells were rare in normal mucosa but were observed regularly in chronic gastritis in close proximity to CD95 ligand mRNA expression throughout the epithelial and lamina propria cells. In a functional analysis gastric epithelial cell lines were incubated with supernatants of H. pylori. Treatment with the cytotoxic isolate H. pylori 60190 but not with the noncytotoxic isolate Tx30a upregulated CD95 in up to 50% of gastric epithelial cells and induced apoptosis in these cells. H. pylori-induced apoptosis was partially prevented by blocking CD95, demonstrating the functional role of the CD95 system. These findings suggest that H. pylori-associated chronic gastritis involves apoptosis of gastric epithelial cells by activation of the CD95 receptor and ligand system.

Reactive Oxygen Intermediates Are Involved in the Induction of CD95 Ligand mRNA Expression by Cytostatic Drugs in Hepatoma Cells

Oxidative stress has been associated with the induction of programmed cell death. The CD95 ligand/receptor system is a specific mediator of apoptosis. We have used the model of drug-induced apoptosis to assess whether the CD95 ligand mRNA is induced by reactive oxygen intermediates. Treatment of HepG2 hepatoma cells with bleomycin induced the production of reactive oxygen intermediates and, as an additional parameter of oxidative stress, resulted in glutathione (GSH) depletion. In parallel, CD95 ligand mRNA expression was induced. In a similar fashion CD95 ligand mRNA expression increased after treatment with H2O2. Additional treatment with the antioxidant and GSH precursor N-acetylcysteine resulted in partial restoration of intracellular GSH levels and in reduced induction of CD95 ligand mRNA. Induction of CD95 ligand mRNA by bleomycin was further reduced by combined treatment withN-acetylcysteine and deferoxamine. These data suggest a direct role of oxygen radicals in the induction of the CD95 ligand.

Reduced expression of Hugl-1, the human homologue of Drosophila tumour suppressor gene lgl, contributes to progression of colorectal cancer.

The human gene, human giant larvae (Hugl-1/Llg1/Lgl1) has significant homology to the Drosophila tumour suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that binds Myosin II and is involved in maintaining cell polarity and epithelial integrity. The human protein, Hugl-1 contains several conserved functional domains found in Lgl, suggesting that these proteins may have closely related functions. Whether loss of Hugl expression plays a role in human tumorigenesis has so far not been extensively investigated. Thus, we evaluated tumour tissues from 94 patients undergoing surgery for colorectal cancer (CRC) for loss of Hugl-1 transcription and compared our findings with the clinical data from each of these patients. We found that Hugl-1 was lost in 75% of tumour samples and these losses were associated with advanced stage and particularly with lymph node metastases. Reduced Hugl-1 expression during the adenoma-carcinoma sequence occurring as early as in colorectal adenomas was detected by both immunohistochemical and reverse transcription–polymerase chain reaction analysis. Functional assays with ecdysone-inducible cell lines revealed that Hugl-1 expression increased cell adhesion and decreased cell migration. Our studies thus indicate that downregulation of Hugl-1 contributes to CRC progression.

Cleavage of CD95 by matrix metalloproteinase-7 induces apoptosis resistance in tumour cells

The ability of tumour cells to resist apoptosis-inducing signals by cytotoxic T cells may decide the success or failure of tumour elimination. An important effector of apoptosis is the CD95/CD95 ligand system (APO-1/Fas) that mediates perforin-independent cytotoxic T-cell killing of tumour cells. We propose a new strategy by which tumour cells can resist CD95-induced apoptosis. We identified matrix metalloproteinase-7, MMP-7 (Martilysin), as the first physiologically relevant protease that can specifically cleave CD95. MMP-7 is of unique importance because it is produced by the tumour cells themselves at early stages of tumour development. Microsequencing of the positions in CD95 cleaved by MMP-7 revealed two sites in the N-terminal extracellular domain of CD95, important for preligand assembly of CD95. MMP-7 cleavage of CD95 results in reduced CD95 surface expression and decreased CD95-mediated apoptosis sensitivity of tumour cells. Treatment of MMP-7-positive HT-29 tumour cells with MMP-7-antisense oligonucleotides led to an increase in CD95-mediated apoptosis sensitivity. Finally, specific cytotoxic T-cell killing was reduced in the presence of MMP-7. Thus, MMP-7 expression in tumour cells may contribute to an apoptosis-resistant phenotype, which ultimately promotes immune escape. This activity may account for the well-established role of MMP-7 in early tumour development.

Expression of Hugl-1 is strongly reduced in malignant melanoma

The human gene Hugl-1 (Llgl/Lgl1) has significant homology to the Drosophila tumor suppressor gene lethal(2)giant larvae (lgl). The lgl gene codes for a cortical cytoskeleton protein, Lgl, that is involved in maintaining cell polarity and epithelial integrity. We speculate that Hugl-1 might play a role in epithelial–mesenchymal transition (EMT) and that loss of Hugl-1 expression plays a role in the development or progression of malignant melanoma. Thus, we evaluated melanoma cell lines and tissue samples of malignant melanoma for loss of Hugl-1 transcription. We found that Hugl-1 was downregulated or lost in all cell lines and in most of the tumor samples analysed, and that these losses were associated with advanced stage of the disease. Reduced Hugl-1 expression occurred as early as in primary tumors detected by both immunohistochemical and reverse transcription–polymerase chain reaction (RT–PCR) analysis. Functional assays with stable Hugl-1-transfected cell lines revealed that Hugl-1 expression increased cell adhesion and decreased cell migration. Further, downregulation of MMP2 and MMP14 (MT1-MMP) and re-expression of E-cadherin was found in the Hugl-1-expressing cell clones supporting a role of Hugl-1 in EMT. Our studies thus indicate that loss of Hugl-1 expression contributes to melanoma progression.

Sirtuin-6-dependent genetic and epigenetic alterations are associated with poor clinical outcome in hepatocellular carcinoma patients.

Sirtuin 6 (SIRT6) is a member of the sirtuin family of NAD+–dependent deacetylases. Genetic deletion of Sirt6 in mice results in a severe degenerative phenotype with impaired liver function and premature death. The role of SIRT6 in development and progression of hepatocellular carcinoma is currently unknown. We first investigated SIRT6 expression in 153 primary human liver cancers and in normal and cirrhotic livers using microarray analysis. SIRT6 was significantly down‐regulated in both cirrhotic livers and cancer. A Sirt6 knockout (KO) gene expression signature was generated from primary hepatoctyes isolated from 3‐week‐old Sirt6‐deficient animals. Sirt6‐deficient hepatocytes showed up‐regulation of established hepatocellular carcinoma (HCC) biomarkers alpha‐fetoprotein (Afp), insulin‐like growth factor 2 (Igf2), H19, and glypican‐3. Furthermore, decreased SIRT6 expression was observed in hepatoma cell lines that are known to be apoptosis‐insensitive. Re‐expression of SIRT6 in HepG2 cells increased apoptosis sensitivity to CD95‐stimulation or chemotherapy treatment. Loss of Sirt6 was characterized by oncogenic changes, such as global hypomethylation, as well as metabolic changes, such as hypoglycemia and increased fat deposition. The hepatocyte‐specific Sirt6‐KO signature had a prognostic impact and was enriched in patients with poorly differentiated tumors with high AFP levels as well as recurrent disease. Finally, we demonstrated that the Sirt6‐KO signature possessed a predictive value for tumors other than HCC (e.g., breast and lung cancer). Conclusion: Loss of SIRT6 induces epigenetic changes that may be relevant to chronic liver disease and HCC development. Down‐regulation of SIRT6 and genes dysregulated by loss of SIRT6 possess oncogenic effects in hepatocarcinogenesis. Our data demonstrate that deficiency in one epigenetic regulator predisposes a tumorigenic phenotype that ultimately has relevance for outcome of HCC and other cancer patients. (Hepatology 2013;53:1054–1064)

Immune evasion by tumours: involvement of the CD95 (APO-1/Fas) system and its clinical implications

T cells can cause the death of tumour cells by two mechanisms, one involving CD95 and the other involving perforin. T-cell activity or reduced tumour-cell responsiveness towards CD95 stimulation might result in an impaired anti-tumour immune response and tumour cell outgrowth. Recent data suggest that de novo expression of the CD95 ligand (CD95L) in tumours might result in elimination of CD95+ anti-tumour lymphocytes, and that tumours might therefore be privileged sites. However, conflicting data on the role of CD95L in transplantation experiments indicate that CD95L expression alone might not be sufficient to confer the status of immune privilege.

Role of SIRT1 and FOXO factors in eNOS transcriptional activation by resveratrol.

Many of the cardiovascular protective effects of resveratrol are attributable to an enhanced production of nitric oxide (NO) by the endothelial NO synthase (eNOS). Resveratrol has been shown to enhance eNOS gene expression as well as eNOS enzymatic activity. The aim of the present study was to analyze the molecular mechanisms of eNOS transcriptional activation by resveratrol. Treatment of human EA.hy 926 endothelial cells with resveratrol led to a concentration-dependent upregulation of eNOS expression. In luciferase reporter gene assay, resveratrol enhanced the activity of human eNOS promoter fragments (3500, 1600, 633 and 263bp in length, respectively), indicating that the proximal promoter region is required for resveratrol-induced eNOS transcriptional activation. Knockdown of the NAD(+)-dependent protein deacetylase sirtuin 1 (SIRT1) by siRNA prevented the upregulation of eNOS mRNA and protein by resveratrol. Forkhead box O (FOXO) transcription factors are established downstream targets of SIRT1. siRNA-mediated knockdown of FOXO1 and FOXO3a abolished the effect of resveratrol on eNOS expression, indicating the involvement of these factors. Resveratrol treatment enhanced the expression of FOXO1 and FOXO3a in EA.hy 926 cells. Reporter gene assay using promoter containing forkhead response elements showed increased FOXO factor activity by resveratrol. In electrophoretic mobility shift assay, the enhanced binding of nuclear proteins to the eNOS promoter regions by resveratrol could be blocked by antibodies against FOXO1 and FOXO3a. In conclusion, resveratrol enhances the expression and activity of FOXO transcription factors. The SIRT1/FOXO factor axis is involved in resveratrol-induced eNOS transcriptional activation.