P60-c-src suppresses apoptosis through inhibition of caspase 8 activation in hepatoma cells, but not in primary hepatocytes.

BACKGROUND/AIMS: Failure to induce apoptosis triggered by members of the death receptor family has been described in hepatocellular carcinoma (HCC) and sensitization of malignant cells to pro-apoptotic molecules such as TRAIL has been proposed as an alternative cancer therapy. Limiting to this approach are the resistance of many tumor cells to TRAIL and safety concerns about the toxicity of TRAIL in normal hepatocytes. METHODS: We here explored the possibility that the protooncogene c-Src, known to be overexpressed in a variety of tumors, could be specifically responsible for the loss of response to receptor-mediated apoptosis. RESULTS: Cotreatment of several hepatoma cell lines with the Src inhibitor PP2 potently sensitized these cells to TRAIL and CD95, dramatically decreasing effective doses of TRAIL to as low as 1 ng/ml. Remarkably, Src-inhibition did not synergize with TRAIL signaling in primary hepatocytes. Specific siRNAs showed that the effect was due to blockade of p60(c-Src) and occurred through increased recruitment of caspase 8. CONCLUSIONS: We provide evidence that p60(c-Src) is an important and effective suppressor of receptor-mediated apoptosis in hepatoma cells but not in primary human hepatocytes. Inhibition of Src sensitizes tumor cells to apoptosis and decreases effective doses of TRAIL to therapeutic concentrations.

IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and intestinal epithelial cell migration.

IL-22 is produced by activated T cells and signals through a receptor complex consisting of IL-22R1 and IL-10R2. The aim of this study was to analyze IL-22 receptor expression, signal transduction, and specific biological functions of this cytokine system in intestinal epithelial cells (IEC). Expression studies were performed by RT-PCR. Signal transduction was analyzed by Western blot experiments, cell proliferation by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay and Fas-induced apoptosis by flow cytometry. IEC migration was studied in wounding assays. The IEC lines Caco-2, DLD-1, SW480, HCT116, and HT-29 express both IL-22 receptor subunits IL-22R1 and IL-10R2. Stimulation with TNF-alpha, IL-1beta, and LPS significantly upregulated IL-22R1 without affecting IL-10R2 mRNA expression. IL-22 binding to its receptor complex activates STAT1/3, Akt, ERK1/2, and SAPK/JNK MAP kinases. IL-22 significantly increased cell proliferation (P = 0.002) and phosphatidylinsitol 3-kinase-dependent IEC cell migration (P < 0.00001) as well as mRNA expression of TNF-alpha, IL-8, and human beta-defensin-2. IL-22 had no effect on Fas-induced apoptosis. IL-22 mRNA expression was increased in inflamed colonic lesions of patients with Crohn's disease and correlated highly with the IL-8 expression in these lesions (r = 0.840). Moreover, IL-22 expression was increased in murine dextran sulfate sodium-induced colitis. IEC express functional receptors for IL-22, which increases the expression of proinflammatory cytokines and promotes the innate immune response by increased defensin expression. Moreover, our data indicate intestinal barrier functions for this cytokine-promoting IEC migration, which suggests an important function in intestinal inflammation and wound healing. IL-22 is increased in active Crohn's disease and promotes proinflammatory gene expression and IEC migration.

Inhibition of c-Jun-N-terminal-kinase sensitizes tumor cells to CD95-induced apoptosis and induces G2/M cell cycle arrest.

Loss of susceptibility to apoptosis signals is a crucial step in carcinogenesis. Therefore, sensitization of tumor cells to apoptosis is a promising therapeutic strategy. c-Jun-N-terminal-kinases (JNK) have been implicated in stress-induced apoptosis, but may also contribute to survival signaling. Here we show that CD95-induced apoptosis is augmented by the JNK inhibitor SP600125 and small interfering RNA directed against JNK1/2. SP600125 potently inhibited methyl methane sulfonate-induced phosphorylation of c-Jun, but had minimal effect on apoptosis alone. In contrast, it strongly enhanced CD95-mediated apoptosis in six of eight tumor cell lines and led to a G2/M phase arrest in all cell lines. SP600125 enhanced cleavage of caspase 3 and caspase 8, the most upstream caspase in the CD95 pathway. JNK inhibition up-regulates p53 and its target genes p21Cip1/Waf1 and CD95. However, although HCT116 p53-/- cells and p21+/+ cells were less sensitive to CD95 stimulation than their p53+/+ and p21-/- counterparts, p53 and p21 were not involved in the JNK-mediated effect. JunD, which was described to be protective in tumor necrosis factor-induced apoptosis, was not regulated by JNK inhibition on the protein level. When transcription was blocked by actinomycin D, JNK inhibition still enhanced apoptosis to a comparable extent. We conclude that JNK inhibition has antitumor activity by inducing growth arrest and enhancing CD95-mediated apoptosis by a transcription-independent mechanism.

CXCR4 and CXCL12 are inversely expressed in colorectal cancer cells and modulate cancer cell migration, invasion and MMP-9 activation.

Colorectal cancer (CRC) is characterized by a distinct metastatic pattern resembling chemokine-induced leukocyte trafficking. This prompted us to investigate expression, signal transduction and specific functions of the chemokine receptor CXCR4 in CRC cells and metastases. Using RT-PCR analysis and Western blotting, we demonstrated CXCR4 and CXCL12 expression in CRC and CRC metastases. Cell differentiation increases CXCL12 mRNA levels. Moreover, CXCR4 and its ligand are inversely expressed in CRC cell lines with high CXCR4 and low or not detectable CXCL12 expression. CXCL12 activates ERK-1/2, SAPK/JNK kinases, Akt and matrix metalloproteinase-9. These CXCL12-induced signals mediate reorganization of the actin cytoskeleton resulting in increased cancer cell migration and invasion. Moreover, CXCL12 increases vascular endothelial growth factor (VEGF) expression and cell proliferation but has no effect on CRC apoptosis. Therefore, the CXCL12/CXCR4 system is an important mediator of invasion and metastasis of CXCR4 expressing CRC cells.

IL-28A and IL-29 mediate antiproliferative and antiviral signals in intestinal epithelial cells and murine CMV infection increases colonic IL-28A expression.

Human cytomegalovirus virus (CMV) is a major cause of morbidity and mortality in immunocompromised individuals. Recently, a novel group of cytokines [interleukin (IL)-28A/B and IL-29, also termed interferon (IFN)-lambdas] has been described. Here, we demonstrate that intestinal epithelial cell (IEC) lines as well as murine and human colonic tissue express the IFN-lambda receptor subunits IL-28R and IL-10R2. IL-28A and IL-29 binding to their receptor complex activates ERK-1/2 and stress-activated protein kinase/c-Jun NH2-terminal kinase MAPKs and Akt, resulting in increased IL-8 protein expression. IFN-lambdas also induce phosphorylation of signal transducer and activator of transcription 1 and significantly increase mRNA expression of suppressor of cytokine signaling 3 and the antiviral proteins myxovirus resistance A and 2',5'-oligoadenylate synthetase. These signals result in an up to 83% reduction of cells positive for human CMV immediate-early protein after human CMV infection. In mice, IL-28A mRNA expression is upregulated after infection with murine CMV in vivo. Both IL-28A and IL-29 significantly decrease cell proliferation but have no effect on Fas-induced apoptosis. In conclusion, IECs express functional receptors for IFN-lambdas, which mediate antiviral and antiproliferative signals in IECs, suggesting a potential for therapeutic use in certain viral infections and as (antiproliferative) anticancer therapy.

SOCS-1 inhibits expression of the antiviral proteins 2',5'-OAS and MxA induced by the novel interferon-lambdas IL-28A and IL-29.

Recently, we have shown that SOCS-1/3 overexpression in hepatic cells abrogates signaling of type I interferons (IFN) which may contribute to the frequently observed IFN resistance of hepatitis C virus (HCV). IFN-lambdas (IL-28A/B and IL-29), a novel group of IFNs, also efficiently inhibit HCV replication in vitro with potentially less hematopoietic side effects than IFN-alpha because of limited receptor expression in hematopoietic cells. To further evaluate the potential of IFN-lambdas in chronic viral hepatitis, we examined the influence of SOCS protein expression on IFN-lambda signaling. First, we show that hepatic cell lines express the IFN-lambda receptor complex consisting of IFN-lambdaR1 (IL-28R1) and IL-10R2. Whereas in mock-transfected HepG2 cells, IL-28A and IL-29 induced STAT1 and STAT3 phosphorylation, overexpression of SOCS-1 completely abrogated IL-28A and IL-29-induced STAT1/3 phosphorylation. Similarly, IL-28A and IL-29 induced mRNA expression of the antiviral proteins 2',5'-OAS and MxA was abolished by overexpression of SOCS-1. In conclusion, we assume that despite antiviral properties of IFN-lambdas, their efficacy as antiviral agents may have similar limitations as IFN-alpha due to inhibition by SOCS proteins.

Modulation of apoptosis as a target for liver disease.

Apoptosis mediated via extrinsic or intrinsic pathways is essential for maintaining cellular homeostasis in the liver. The extrinsic pathway is triggered from the cell surface by engagement of death receptors as CD95, TRAIL (TNF-related apoptosis inducing ligand) and TNF (tumour necrosis factor) or TGF-beta (transforming growth factor beta) receptors. The intrinsic pathway is initiated from the mitochondria and can be influenced by Bcl-2 family members. Both pathways are intertwined and play a physiological role in the liver. Dysregulation of apoptosis pathways contributes to diseases as hepatocellular carcinoma, viral hepatitis, autoimmune hepatitis, ischaemia-reperfusion injury, iron or copper deposition disorders, toxic liver damage and acute liver failure. The apoptosis defects are often central pathogenetic events; hence molecular mechanisms of apoptosis give not only insight into disease mechanisms but also provide potential corresponding therapeutic candidates in liver disease. The focus of this review is the identification of apoptotic signalling components in the liver as therapeutic targets.

DRO1, a gene down-regulated by oncogenes, mediates growth inhibition in colon and pancreatic cancer cells.

Neoplastic progression in human tissues appears to be paralleled by a series of genetic and epigenetic alterations. In human colorectal cancers, defect Wnt/beta-catenin/T-cell factor and RAS/RAF signaling pathways have a major contributing role in tumor initiation and progression. To date, much of the research on the consequences of beta-catenin activation has been focused on genes whose expression is believed to be activated by beta-catenin-associated T-cell factor-dependent transcription. Little is known about genes whose expression may be down-regulated secondary to beta-catenin activation. Using a subtractive suppression hybridization approach, we identified a gene with markedly decreased expression in rat RK3E epithelial cells neoplastically transformed by beta-catenin. Because expression of this gene was also down-regulated in RK3E transformed by several other oncogenes, the gene was named DRO1 for "down-regulated by oncogenes 1." Compared with corresponding normal tissues, DRO1 expression was found to be very reduced in colon and pancreatic cancer cell lines as well as in most colorectal cancer specimens. The predicted DRO1 protein contains three repetitive elements with significant similarity to the carboxyl-terminal regions of the predicted proteins from DRS/SRPX/ETX1 and SRPUL genes, suggesting the existence of a new protein family. Ectopic expression of DRO1 in neoplastically transformed RK3E or colorectal and pancreatic cancer cell lines lacking endogenous DRO1 expression resulted in substantial inhibition of growth properties. DRO1 was found to suppress anchorage independent growth and to sensitize cells to anoikis and CD95-induced apoptosis. Our findings suggest that inhibition of DRO1 expression may be an important event in the development of colorectal and pancreatic cancers.

Suramin inhibits death receptor-induced apoptosis in vitro and fulminant apoptotic liver damage in mice.

Suramin is a polysulfonated derivative of urea and has been widely used both to treat infections and as a chemotherapeutic drug. Suramin has been shown to inhibit growth factor signaling pathways; however, its effect on apoptosis is unknown. Here we show that suramin inhibits apoptosis induced through death receptors in hepatoma and lymphoma cells. It also inhibits the proapoptotic effect of chemotherapeutic drugs. The antiapoptotic mechanism is specific to cell type and is caused by reduced activation, but not altered composition, of the death-inducing signaling complex (DISC), and by inhibition of the initiator caspases 8, 9 and 10. Suramin also shows similar effects in in vivo models: apoptotic liver damage induced by CD95 stimulation and endotoxic shock mediated by tumor-necrosis factor (TNF) are inhibited in mice, but necrotic liver damage is not inhibited in a rat model of liver transplantation. Thus, the antiapoptotic property of suramin in the liver may be therapeutically exploited.

Cephalostatin 1 selectively triggers the release of Smac/DIABLO and subsequent apoptosis that is characterized by an increased density of the mitochondrial matrix.

Cephalostatin 1 is a bis-steroidal marine natural product with a unique cytotoxicity profile in the in vitro screen system of the National Cancer Institute, suggesting that it may affect novel molecular target(s). Here we show that cephalostatin 1 induces a novel pathway of receptor-independent apoptosis that selectively uses Smac/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis-binding protein with a low isoelectric point) as a mitochondrial signaling molecule. At nanomolar concentrations, cephalostatin 1 triggers dose- and time-dependent DNA fragmentation in leukemia Jurkat T cells. Apoptosis was found to be dependent on caspase activity because the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone blocks cephalostatin 1-mediated DNA fragmentation. The CD95 death receptor as well as other caspase-8-requiring death receptors were not involved because Jurkat T cells lacking the CD95 receptor or caspase-8 and control cells responded equally to cephalostatin 1. Although cephalostatin 1 affects mitochondria by dissipating the mitochondrial membrane potential, neither cytochrome c nor apoptosis-inducing factor is released, as shown by Western blot analysis. Interestingly, cephalostatin 1 selectively triggers the mitochondrial release of the inhibitor of apoptosis antagonist Smac/DIABLO. Overexpression of the antiapoptotic protein Bcl-x(L) delayed both Smac/DIABLO release and onset of apoptosis, suggesting that Smac/DIABLO is required for cephalostatin 1-induced apoptosis. This new mitochondrial pathway is accompanied by marked structural changes of mitochondria as shown by transmission electron microscopy.

Upregulation of major histocompatibility complex class I on liver cells by hepatitis C virus core protein via p53 and TAP1 impairs natural killer cell cytotoxicity.

The mechanisms of immune evasion and the role of the early immune response in chronic infection caused by hepatitis C virus (HCV) are still unclear. Here, we present evidence for a cascade of molecular events that the virus initiates to subvert the innate immune attack. The HCV core protein induced p53-dependent gene expression of TAP1 (transporter associated with antigen processing 1) and consecutive major histocompatibility complex (MHC) class I upregulation. Moreover, in p53-deficient liver cell lines, only reconstitution with wild-type p53, but not mutated p53 lacking DNA binding capacity, showed this effect. As a consequence of increased MHC class I expression, a significantly downregulated cytotoxic activity of natural killer (NK) cells against HCV core-transfected liver cells was observed, whereas lysis by HCV-specific cytotoxic T cells was not affected. These results demonstrate a way in which HCV avoids recognition by NK cells that may contribute to the establishment of a chronic infection.

An unexpected role for FosB in activation-induced cell death of T cells.

The CD95 (APO-1/Fas) system plays a major role in induction of apoptosis in lymphoid and nonlymphoid tissues. The CD95 (APO-1/Fas) ligand (CD95L) is induced in response to a variety of signals including TCR/CD3 stimulation or application of chemotherapeutic drugs. Here we report that an AP-1 site located in the 5' untranslated region of the CD95L gene is required for TCR/CD3-mediated induction of the human CD95L promoter. Electrophoretic mobility shift assays using nuclear extracts of Jurkat T cells as well as TCR/CD3-restimulated primary human T cells demonstrated specific binding of AP-1, predominantly composed of c-Jun and FosB, to this sequence. Ectopic expression of transdominant negative Jun mutants strongly reduced CD95L promoter activity and activation-induced cell death (AICD), confirming the functional significance of FosB/c-Jun binding. Thus, our results demonstrate an important novel function for FosB dimerized with c-Jun in TCR/CD3-mediated AICD in human T cells.

Restoration of p53 expression sensitizes human papillomavirus type 16 immortalized human keratinocytes to CD95-mediated apoptosis.

To understand the function of the individual oncogenes of HPV16 in modulating the cellular response to apoptogenic signals, we used human keratinocytes immortalized with either E6, E7 or E6/E7 oncoproteins as model system. Applying CD95 antibodies or recombinant CD95 ligand, only the E7-immortalized cells underwent extensive apoptosis. In contrast, E6- and E6/E7-expressing keratinocytes were resistant. Dominance of E6 correlated with significant down-regulation of p53, c-Myc, p21 and Bcl-2. CD95 was found to be reduced in resistant HPV-positive cells, while there were no quantitative differences in expression levels of FADD, FLICE/caspase-8 or caspase-3. Notably, in contrast to primary human keratinocytes, all immortalized cells showed a general reduction of c-FLIP, an inhibitory protein which normally prevents unscheduled CD95-induced apoptosis. E6- and E6/E7-positive keratinocytes, however, can be sensitized to CD95 apoptosis by blocking proteasome-mediated proteolysis. CD95-resistant HPV-positive cells underwent apoptosis within 3-5 h upon co-incubation with MG132 and agonistic antibodies or CD95 ligand, which was preceded by a strong re-expression of p53 and c-Myc, but not of other half-life controlled proteins such as Bax or IkappaBalpha. Blockage of proteasomal activity alone did not result in apoptosis, although the same set of pro-apoptotic proteins was up-regulated. Performing similar experiments with cervical carcinoma cells expressing mutated p53 (C33a) or with p53-'null' lung carcinoma cells (H1299), no CD95 cell killing occurred even though c-Myc was strongly induced. These data indicate that the reduced bioavailability of p53 is a key-regulatory event in perturbation of CD95 signaling in HPV16 immortalized keratinocytes.