Generation of magnetic biohybrid microrobots based on MSC.sTRAIL for targeted stem cell delivery and treatment of cancer.

Background: Combining the power of magnetic guidance and the biological activities of stem cells transformed into biohybrid microrobots holds great promise for the treatment of several diseases including cancer. Results: We found that human MSCs can be readily loaded with magnetic particles and that the resulting biohybrid microrobots could be guided by a rotating magnetic field. Rotating magnetic fields have the potential to be applied in the human setting and steer therapeutic stem cells to the desired sites of action in the body. We could demonstrate that the required loading of magnetic particles into stem cells is compatible with their biological activities. We examined this issue with a particular focus on the expression and functionality of therapeutic genes inside of human MSC-based biohybrid microrobots. The loading with magnetic particles did not cause a loss of viability or apoptosis in the human MSCs nor did it impact on the therapeutic gene expression from the cells. Furthermore, the therapeutic effect of the gene products was not affected, and the cells also did not lose their migration potential. Conclusion: These results demonstrate that the fabrication of guidable MSC-based biohybrid microrobots is compatible with their biological and therapeutic functions. Thus, MSC-based biohybrid microrobots represent a novel way of delivering gene therapies to tumours as well as in the context of other diseases.

Fas-threshold signalling in MSCs promotes pancreatic cancer progression and metastasis.

Mesenchymal stem cells (MSCs) belong to the tumour microenvironment and have been implicated in tumour progression. We found that the number of MSCs significantly increased in tumour-burdened mice driven by Fas-threshold signalling. Consequently, MSCs lacking Fas lost their ability to induce metastasis development in a pancreatic cancer model. Mixing of MSCs with pancreatic cancer cells led to sustained production of the pro-metastatic cytokines CCL2 and IL6 by the stem cells. The levels of these cytokines were dependent on the number of MSCs, linking Fas-mediated MSC-proliferation to their capacity to promote tumour progression. Furthermore, we discovered that CCL2 and IL6 were induced by pancreatic cancer cell-derived IL1. Importantly, analysis of patient transcriptomic data revealed that high FasL expression correlates with high levels of MSC markers as well as increased IL6 and CCL2 levels in pancreatic tumours. Moreover, both FasL and CCL2 are linked to elevated levels of markers specific for monocytes known to possess further pro-metastatic activities. These results confirm our experimental findings of a FasL-MSC-IL1-CCL2/IL6 axis in pancreatic cancer and highlights the role of MSCs in tumour progression.

MSC.sTRAIL Has Better Efficacy than MSC.FL-TRAIL and in Combination with AKTi Blocks Pro-Metastatic Cytokine Production in Prostate Cancer Cells.

Cell therapy is a promising new treatment option for cancer. In particular, mesenchymal stem cells (MSCs) have shown potential in delivering therapeutic genes in various tumour models and are now on the verge of being tested in the clinic. A number of therapeutic genes have been examined in this context, including the death ligand TRAIL. For cell therapy, it can be used in its natural form as a full-length and membrane-bound protein (FL-TRAIL) or as an engineered version commonly referred to as soluble TRAIL (sTRAIL). As to which is more therapeutically efficacious, contradicting results have been reported. We discovered that MSCs producing sTRAIL have significantly higher apoptosis-inducing activity than cells expressing FL-TRAIL and found that FL-TRAIL, in contrast to sTRAIL, is not secreted. We also demonstrated that TRAIL does induce the expression of pro-metastatic cytokines in prostate cancer cells, but that this effect could be overcome through combination with an AKT inhibitor. Thus, a combination consisting of small-molecule drugs specifically targeting tumour cells in combination with MSC.sTRAIL, not only provides a way of sensitising cancer cells to TRAIL, but also reduces the issue of side-effect-causing cytokine production. This therapeutic strategy therefore represents a novel targeted treatment option for advanced prostate cancer and other difficult to treat tumours.

The future of mesenchymal stem cell-based therapeutic approaches for cancer - From cells to ghosts.

Mesenchymal stem cells (MSCs) are multipotent stromal cells which can differentiate into a variety of cell types including osteoblasts, adipocytes and chondrocytes. They are normally resident in adipose tissue, bone marrow and the umbilical cord, but can also be found in other tissues and are known to be recruited to sites of wound healing as well as growing tumours. The therapeutic potential of MSCs has been explored in a number of phase I/II and III clinical trials, of which several were targeted against graft-versus-host disease and to support engraftment of haematopoietic stem cells (HSCs), but currently only very few in the oncology field. There are now three clinical trials either ongoing or recruiting patients that use MSCs to treat tumour disease. In these, MSCs target gastrointestinal, lung and ovarian cancer, respectively. The first study uses MSCs loaded with a HSV-TK expression construct under the control of the CCL5 promoter, and has recently reported successful completion of Phase I/II. While no adverse side effects were seen during this study, no outcomes with respect to therapeutic benefits have been published. The other clinical trials targeting lung and ovarian cancer will be using MSCs expressing cytokines as therapeutic payload. Despite these encouraging early steps towards their clinical use, many questions are still unanswered regarding the biology of MSCs in normal and pathophysiological settings. In this review, in addition to summarising the current state of MSC-based therapeutic approaches for cancer, we will describe the remaining questions, obstacles and risks, as well as novel developments such as MSC-derived nanoghosts.

Caspase-10: a molecular switch from cell-autonomous apoptosis to communal cell death in response to chemotherapeutic drug treatment.

The mechanisms of how chemotherapeutic drugs lead to cell cycle checkpoint regulation and DNA damage repair are well understood, but how such signals are transmitted to the cellular apoptosis machinery is less clear. We identified a novel apoptosis-inducing complex, we termed FADDosome, which is driven by ATR-dependent caspase-10 upregulation. During FADDosome-induced apoptosis, cFLIPL is ubiquitinated by TRAF2, leading to its degradation and subsequent FADD-dependent caspase-8 activation. Cancer cells lacking caspase-10, TRAF2 or ATR switch from this cell-autonomous suicide to a more effective, autocrine/paracrine mode of apoptosis initiated by a different complex, the FLIPosome. It leads to processing of cFLIPL to cFLIPp43, TNF-α production and consequently, contrary to the FADDosome, p53-independent apoptosis. Thus, targeting the molecular levers that switch between these mechanisms can increase efficacy of treatment and overcome resistance in cancer cells.

TRAIL-receptor preferences in pancreatic cancer cells revisited: Both TRAIL-R1 and TRAIL-R2 have a licence to kill.

BACKGROUND: TRAIL is a potent and specific inducer of apoptosis in tumour cells and therefore is a possible new cancer treatment. It triggers apoptosis by binding to its cognate, death-inducing receptors, TRAIL-R1 and TRAIL-R2. In order to increase its activity, receptor-specific ligands and agonistic antibodies have been developed and some cancer types, including pancreatic cancer, have been reported to respond preferentially to TRAIL-R1 triggering. The aim of the present study was to examine an array of TRAIL-receptor specific variants on a number of pancreatic cancer cells and test the generality of the concept of TRAIL-R1 preference in these cells. METHODS: TRAIL-R1 and TRAIL-R2 specific sTRAIL variants were designed and tested on a number of pancreatic cancer cells for their TRAIL-receptor preference. These sTRAIL variants were produced in HEK293 cells and were secreted into the medium. After having measured and normalised the different sTRAIL variant concentrations, they were applied to pancreatic and control cancer cells. Twenty-four hours later apoptosis was measured by DNA hypodiploidy assays. Furthermore, the specificities of the sTRAIL variants were validated in HCT116 cells that were silenced either for TRAIL-R1 or TRAIL-R2. RESULTS: Our results show that some pancreatic cancer cells use TRAIL-R1 to induce cell death, whereas other pancreatic carcinoma cells such as AsPC-1 and BxPC-3 cells trigger apoptosis via TRAIL-R2. This observation extended to cells that were naturally TRAIL-resistant and had to be sensitised by silencing of XIAP (Panc1 cells). The measurement of TRAIL-receptor expression by FACS revealed no correlation between receptor preferences and the relative levels of TRAIL-R1 and TRAIL-R2 on the cellular surface. CONCLUSIONS: These results demonstrate that TRAIL-receptor preferences in pancreatic cancer cells are variable and that predictions according to cancer type are difficult and that determining factors to inform the optimal TRAIL-based treatments still have to be identified.

DR4 specific TRAIL variants are more efficacious than wild-type TRAIL in pancreatic cancer.

Current treatment modalities for pancreatic carcinoma afford only modest survival benefits. TRAIL, as a potent and specific inducer of apoptosis in cancer cells, would be a promising new treatment option. However, since not all pancreatic cancer cells respond to TRAIL, further improvements and optimizations are still needed. One strategy to improve the effectiveness of TRAIL-based therapies is to specifically target one of the 2 cell death inducing TRAIL-receptors, TRAIL-R1 or TRAIL-R2 to overcome resistance. To this end, we designed constructs expressing soluble TRAIL (sTRAIL) variants that were rendered specific for either TRAIL-R1 or TRAIL-R2 by amino acid changes in the TRAIL ectodomain. When we expressed these constructs, including wild-type sTRAIL (sTRAIL(wt)), TRAIL-R1 (sTRAIL(DR4)) and TRAIL-R2 (sTRAIL(DR5)) specific variants, in 293 producer cells we found all to be readily expressed and secreted into the supernatant. These supernatants were subsequently transferred onto target cancer cells and apoptosis measured. We found that the TRAIL-R1 specific variant had higher apoptosis-inducing activity in human pancreatic carcinoma Colo357 cells as well as PancTu1 cells that were additionally sensitized by targeting of XIAP. Finally, we tested TRAIL-R1 specific recombinant TRAIL protein (rTRAIL(DR4)) on Colo357 xenografts in nude mice and found them to be more efficacious than rTRAIL(wt). Our results demonstrate the benefits of synthetic biological approaches and show that TRAIL-R1 specific variants can potentially enhance the therapeutic efficacy of TRAIL-based therapies in pancreatic cancer, suggesting that they can possibly become part of individualized and tumor specific combination treatments in the future.

Impact of short- and medium-chain fatty acids on mitochondrial function in severe inflammation.

BACKGROUND: Sepsis is a severe inflammatory disorder with a high mortality in intensive care units mostly due to multiorgan failure. Mitochondrial dysfunction is regarded as a key factor involved in the pathogenesis of septic disorders, leading to a decline in energy supply. The aim of the present study was to evaluate whether application of short-chain fatty acids (SCFAs) and medium-chain fatty acids (MCFAs) could improve mitochondrial function and thus might serve as a potential energy source under inflammatory conditions. MATERIALS AND METHODS: As an experimental approach, starved human endothelial cells and monocytes were incubated with hexanoic acid, heptanoic acid, octanoic acid, or glucose and subsequently subjected to high-resolution respirometry to assess mitochondrial function under baseline conditions. In a second set of experiments, cells were pretreated with tumor necrosis factor-α to mimic inflammation and sepsis. RESULTS: We demonstrated that addition of SCFAs and MCFAs increases mitochondrial respiratory capacity at baseline and inflammatory conditions in both cell types. None of the fatty acids induced changes in mitochondrial DNA content or the generation of proinflammatory cytokines, indicating a beneficial safety profile. CONCLUSION: We deduce that SCFAs and MCFAs are suitable and safe sources of energy under inflammatory conditions with the capability to partly restore mitochondrial respiration.

IFN-γ combined with targeting of XIAP leads to increased apoptosis-sensitisation of TRAIL resistant pancreatic carcinoma cells.

The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a specific and potent inducer of apoptosis in cancer cells, but the resistance of many tumour cells to TRAIL still represents a major hurdle for the clinical treatment of tumours with TRAIL. As apoptosis is regulated by the balance of activities of several anti-apoptotic factors and pro-apoptotic factors, we analysed the relative contribution of the two sides and found that down-regulation of Bcl-x(L) and in particular XIAP, but not c-Flip, sensitised the TRAIL resistant pancreatic cancer cell line Panc-1. A combination of both XIAP and Bcl-x(L) knock-downs showed no substantial added benefit indicating that both act in the same pathway. Notably, the degree of sensitisation by silencing of anti-apoptotic genes was further elevated by concomitantly increasing the pro-apoptotic potential in Panc-1 cells through over-expression of TRAIL-R1 or IFN-γ-mediated increases in caspase-8 levels. Similar sensitisation effects were obtained for another TRAIL-resistant pancreatic tumour cell line, AsPC-1. Our findings demonstrate that modulation of the balance between anti- and pro-apoptotic pathways from both sides by inhibition of apoptosis-antagonists and stimulation of pro-apoptotic factors provides the best way to enhance the anti-tumourigenic effect of TRAIL.

Both human and mouse mesenchymal stem cells promote breast cancer metastasis.

Cell therapy has the potential to offer novel treatment modalities for a number of diseases including cancer, and stem cells and in particular mesenchymal stem cells (MSCs) have been experimentally used to deliver therapeutic transgenes. However, conflicting reports have on the one side found that human MSCs can promote metastasis, while on the other hand other studies have shown that MSCs can stall the growth of metastatic lesions. In order to clarify the role of MSCs in metastasis development, we tested whether murine MSCs would behave similarly to human cells in mice. We found that the tissue distribution of human and mouse MSCs was nearly identical after intravenous injection. In mice with MDA-MB-231 mammary carcinoma xenografts we found that a fraction of MSCs infiltrated the primary tumor mass, but that the general tissue distribution of MSCs was unaffected by the tumor-burden. About half of the tumor-burdened animals that were treated with murine and human MSCs, respectively, harbored metastatic lesions with only 17% of controls showing metastatic nodules. Hence, both human and mouse MSCs possess metastasis-promoting activity raising concerns about the safe use of MSCs, but at the same time making the use of murine transgenic model systems feasible to study the role of MSCs in metastasis development and possibly finding ways of using them safely as cell therapeutic vehicles.

Cost-effectiveness of targeted therapy with cetuximab in patients with K-ras wild-type colorectal cancer presenting with initially unresectable metastases limited to the liver in a German setting.

BACKGROUND: In patients with metastases limited to the liver (liver-limited disease [LLD]), effective therapies such as monoclonal antibodies combined with chemotherapy may facilitate metastasis resection and improve long-term survival. OBJECTIVE: This study assessed the cost-effectiveness of bevacizumab and cetuximab in the treatment of patients with colorectal cancer presenting with initially unresectable liver metastases of the Kirsten rat sarcoma viral oncogene homolog (K-ras) wild type, from the perspective of German statutory health insurance. METHODS: The health-economic modeling approach presented here made indirect comparisons between available data on bevacizumab and cetuximab treatment outcomes using evidence synthesis techniques, extrapolating from the follow-up duration of identified clinical trials to a longer time horizon of up to 10 years and inferring costs and health outcomes based on modeled patient pathways. Expert opinion and Delphi panel methods were used for some assumptions, when evidence was missing. Probabilistic sensitivity analyses and different scenario analyses were applied to test for uncertainty around input parameters and assumptions. RESULTS: For the metastatic colorectal cancer LLD population with K-ras wild-type genotype, mean overall survival estimates were 37.7 months for first-line treatment with cetuximab plus FOLFIRI (irinotecan, leucovorin, fluorouracil) and 30.4 months for bevacizumab plus FOLFOX (oxaliplatin, leucovorin, fluorouracil). Corresponding discounted survival estimates were 2.88 life-years with cetuximab plus FOLFIRI versus 2.38 life-years with bevacizumab plus FOLFOX, an average gain of 0.50 discounted life-years. The incremental cost-effectiveness ratio of cetuximab plus FOLFIRI versus bevacizumab plus FOLFOX was €15,020 (year 2010 €) per life-year gained in the base case (with a 95% CI from the probabilistic sensitivity analysis of €3806-€24,660). Results were robust in different scenario analyses as well as in the probabilistic sensitivity analysis. CONCLUSIONS: First-line treatment with cetuximab plus FOLFIRI offers a cost-effective treatment option versus bevacizumab plus FOLFOX for the metastatic colorectal cancer LLD population with K-ras wild-type genotype in Germany. K-ras testing should be performed on all presenting cases of metastatic colorectal cancer to ensure access to this treatment option.

Targeting of XIAP combined with systemic mesenchymal stem cell-mediated delivery of sTRAIL ligand inhibits metastatic growth of pancreatic carcinoma cells.

Disseminating tumors are one of the gravest medical problems. Here, we combine the tumor-specific apoptosis-inducing activity of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with the ability of mesenchymal stem cells (MSCs) to infiltrate both tumor and lymphatic tissues to target primary tumors as well as disseminated cancer cells in a human pancreatic cancer mouse model. Furthermore, we targeted X-linked inhibitor of apoptosis protein (XIAP) by RNA interference (RNAi) inside the cancer cells to make use of the apoptosis sensitization as well the antimetastatic effect that is afforded by XIAP silencing. We generated MSCs, termed MSC.sTRAIL, that express and secrete a trimeric form of soluble TRAIL (sTRAIL). MSC.sTRAIL triggered limited apoptosis in human pancreatic carcinoma cells that were resistant to soluble recombinant TRAIL, which is most likely due to the enhanced effect of the direct, cell-mediated delivery of trimeric TRAIL. MSC.sTRAIL-mediated cell death was markedly increased by concomitant knockdown of XIAP by RNAi in the cancer cells. These findings were confirmed in xenograft models, in which tumors from the parental pancreatic carcinoma cells showed only growth retardation on treatment with MSC.sTRAIL, whereas tumors with silenced XIAP that were treated with MSC.sTRAIL went into remission. Moreover, animals with XIAP-negative xenografts treated with MSC.sTRAIL were almost free of lung metastasis, whereas animals treated with control MSCs showed substantial metastatic growth in the lungs. In summary, this is the first demonstration that a combined approach using systemic MSC-mediated delivery of sTRAIL together with XIAP inhibition suppresses metastatic growth of pancreatic carcinoma.

TRAIL-induced apoptosis is preferentially mediated via TRAIL receptor 1 in pancreatic carcinoma cells and profoundly enhanced by XIAP inhibitors.

PURPOSE: We previously reported that small molecule X-linked inhibitor of apoptosis (XIAP) inhibitors synergize with soluble TRAIL to trigger apoptosis in pancreatic carcinoma cells. Because cancers may preferentially signal via 1 of the 2 agonistic TRAIL receptors, we investigated these receptors as a therapeutic target in pancreatic cancer in the present study. EXPERIMENTAL DESIGN: We examined TRAIL receptor expression and cytotoxicity of specific monoclonal antibodies to TRAIL-R1 (HGS-ETR1, mapatumumab) or TRAIL-R2 (HGS-ETR2, lexatumumab) and of TRAIL receptor selective mutants alone and in combination with small molecule XIAP inhibitors in pancreatic cancer cell lines, in primary specimens, and in a xenotransplant model in vivo. RESULTS: The majority of primary pancreatic carcinoma samples and all cell lines express one or both agonistic TRAIL receptors. Nine of 13 cell lines are more sensitive to mapatumumab-induced apoptosis, whereas lexatumumab requires cross-linking for maximal activity. Similarly, TRAIL-R1 selective mutants display higher cytotoxicity than TRAIL-R2 selective mutants. Small molecule XIAP inhibitors preferentially act in concert with mapatumumab to trigger caspase activation, caspase-dependent apoptosis, and suppress clonogenic survival. Also, primary cultured pancreatic carcinoma cells are more susceptible to mapatumumab than lexatumumab, which is significantly enhanced by a XIAP inhibitor. Importantly, combined treatment with mapatumumab and a XIAP inhibitor cooperates to suppress tumor growth in vivo. CONCLUSIONS: Mapatumumab exerts antitumor activity, especially in combination with XIAP inhibitors against most pancreatic carcinoma cell lines, whereas lexatumumab requires cross-linking for optimal cytotoxicity. These findings have important implications for the design of TRAIL-based protocols for pancreatic cancer.

The TRAIL-receptor-1: TRAIL-receptor-3 and -4 ratio is a predictor for TRAIL sensitivity of cancer cells.

The tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in many cancer cells. However, a significant proportion of tumours are TRAIL-resistant erecting a major hurdle for a successful TRAIL-based treatment regimen in the future. In this context, it would be a major advantage to be able to identify the tumours that respond to TRAIL. The existence of two apoptosis-inducing receptors (TRAIL-R1 and TRAIL-R2) and two receptors that cannot transmit an apoptotic signal and have an inhibitory function (TRAIL-R3 and TRAIL-R4) make TRAIL signalling complicated. We analysed the surface expression of all four membrane-bound TRAIL receptors in cancer cell lines of various origin and primary cancer and normal cells and found a good correlation between TRAIL-sensitivity and the expression of TRAIL-R1 alone, but an even better correlation when a ratio of TRAIL-R1/TRAIL-R3+TRAIL-R4 was analysed. Experimental overexpression of TRAIL-R1 alone or in combination with TRAIL-R4 in PANC-1 cells confirmed our correlation results. Similar to the surface expression-apoptosis correlation analysis we found a high correlation between TRAIL-sensitivity and the mRNA level ratio of TRAIL-R1/TRAIL-R3+TRAIL-R4. A value of <0.85 for the ratio predicted TRAIL resistance in both protein and RNA analysis. Hence, TRAIL receptor RNA expression analysis by real-time PCR might be a feasible approach to predict possible TRAIL-responses in individual tumour samples.

A non-apoptotic role for caspase-9 in muscle differentiation.

Caspases, a family of cysteine proteases most often investigated for their roles in apoptosis, have also been demonstrated to have functions that are vital for the efficient execution of cell differentiation. One such role that has been described is the requirement of caspase-3 for the differentiation of skeletal myoblasts into myotubes but, as yet, the mechanism leading to caspase-3 activation in this case remains elusive. Here, we demonstrate that caspase-9, an initiator caspase in the mitochondrial death pathway, is responsible for the activation of caspase-3 in differentiating C2C12 cells. Reduction of caspase-9 levels, using an shRNA construct, prevented caspase-3 activation and inhibited myoblast fusion. Myosin-heavy-chain expression, which accompanies myoblastic differentiation, was not caspase-dependent. Overexpression of Bcl-xL, a protein that inhibits caspase-9 activation, had the same effect on muscle differentiation as knockdown of caspase-9. These data suggest that the mitochondrial pathway is required for differentiation; however, the release of cytochrome c or Smac (Diablo) could not be detected, raising the possibility of a novel mechanism of caspase-9 activation during muscle differentiation.

Mesenchymal stem cells expressing TRAIL lead to tumour growth inhibition in an experimental lung cancer model.

Lung cancer is a major public health problem in the western world, and gene therapy strategies to tackle this disease systemically are often impaired by inefficient delivery of the vector to the tumour tissue. Some of the main factors inhibiting systemic delivery are found in the blood stream in the form of red and white blood cells (WBCs) and serum components. Mesenchymal stem cells (MSCs) have been shown to home to tumour sites and could potentially act as a shield and vehicle for a tumouricidal gene therapy vector. Here, we describe the ability of an adenoviral vector expressing TRAIL (Ad.TR) to transduce MSCs and show the apoptosis-inducing activity of these TRAIL-carrying MSCs on A549 lung carcinoma cells. Intriguingly, using MSCs transduced with Ad.enhanced-green-fluorescent-protein (EGFP) we could show transfer of viral DNA to cocultured A549 cells resulting in transgenic protein production in these cells, which was not inhibited by exposure of MSCs to human serum containing high levels of adenovirus neutralizing antibodies. Furthermore, Ad.TR-transduced MSCs were shown not to induce T-cell proliferation, which may have resulted in cytotoxic T-cell-mediated apoptosis induction in the Ad.TR-transduced MSCs. Apoptosis was also induced in A549 cells by Ad.TR-transduced MSCs in the presence of physiological concentrations of WBC, erythrocytes and sera from human donors that inhibit or neutralize adenovirus alone. Moreover, we could show tumour growth reduction with TRAIL-loaded MSCs in an A549 xenograft mouse model. This is the first study that demonstrates the potential therapeutic utility of Ad.TR-transduced MSCs in cancer cells and the stability of this vector in the context of the blood environment.

Immunomodulation by n-3- versus n-6-rich lipid emulsions in murine acute lung injury--role of platelet-activating factor receptor.

OBJECTIVE: Cytokines, platelet-activating factor (PAF), and eicosanoids control local and systemic inflammation. Conventional soybean oil-based lipid emulsions used for parenteral nutrition may aggravate the leukocyte inflammatory response or adhesion to the vessel wall. Fish oil-based lipid emulsions, in contrast, may exert an anti-inflammatory effect. DESIGN: We investigated the impact of lipid emulsions on leukocyte invasion, protein leakage, and cytokines in two murine models of acute inflammation. SETTING: Research laboratory of a university hospital. SUBJECTS: Wild-type mice and PAF-receptor knockout mice. INTERVENTIONS: Mice received an infusion of normal saline, fish oil- or soybean oil-based lipid emulsions before lipopolysaccharide challenge. MEASUREMENTS AND MAIN RESULTS: Preinfusion with soybean oil resulted in increased leukocyte invasion, myeloperoxidase activity, and protein leakage and exaggerated release of tumor necrosis factor (TNF)-alpha as well as macrophage inflammatory protein (MIP)-2 into the alveolar space after intratracheal lipopolysaccharide challenge. In contrast, preinfusion with fish oil reduced leukocyte invasion, myeloperoxidase activity, protein leakage, and TNF-alpha as well as MIP-2 generation. Corresponding profiles were found in plasma following intraperitoneal lipopolysaccharide application: Soybean oil increased but fish oil decreased the TNF-alpha and MIP-2 formation. When PAF-receptor-deficient mice were challenged with lipopolysaccharide, leukocyte invasion, lung tissue myeloperoxidase, cytokine generation, and alveolar protein leakage corresponded to those observed in wild-type animals. Fish oil and soybean oil lost their diverging effects on leukocyte transmigration, myeloperoxidase activity, leakage response, and cytokine generation in these knockout mice. Similarly, the differential impact of both lipid emulsions on these lipopolysaccharide-provoked changes was suppressed after pretreating animals with a PAF-receptor antagonist. CONCLUSIONS: Fish oil- vs. soybean oil-based lipid infusions exert anti- vs. proinflammatory effects in murine models of acute inflammation. The PAF/PAF-receptor-linked signaling appears to be a prerequisite for this differential profile.

In situ trapping of initiator caspases reveals intermediate surprises.

A novel method to identify initiator caspases is an in situ trapping approach using a cell-permeable biotinylated caspase inhibitor valine-alanine-aspartate-fluoromethyl ketone (bVAD) that binds covalently and irreversibly to the active cysteine site of caspases. This inhibits apoptosis and should allow precipitation of initiator caspases in their uncleaved forms. However, in our experiments TRAIL and FasL-induced apoptosis and bVAD labelling did not result in streptavidin precipitation of the procaspase-8 forms, but led to the pull-down of the intermediate and to a lesser extent fully cleaved forms (p41/43 and p18). These findings are contrary to other reports and are of relevance to apoptosis research as they challenge the general concept of the bVAD approach that procaspases are being trapped. We show that (partially) processed forms of initiator caspases rather than procaspases might be precipitated with this method.

Constitutively activated nuclear factor-kappaB, but not induced NF-kappaB, leads to TRAIL resistance by up-regulation of X-linked inhibitor of apoptosis protein in human cancer cells.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in most, but not all, cancer cells. The molecular factors regulating the sensitivity to TRAIL are still incompletely understood. The transcription factor nuclear factor-kappaB (NF-kappaB) has been implicated, but its exact role is controversial. We studied different cell lines displaying varying responses to TRAIL and found that TRAIL can activate NF-kappaB in all our cancer cell lines regardless of their TRAIL sensitivity. Inhibition of NF-kappaB via adenoviral expression of the IkappaB-alpha super-repressor only sensitized the TRAIL-resistant pancreatic cancer cell line Panc-1. Panc-1 cells harbor constitutively activated NF-kappaB, pointing to a possible role of preactivated NF-kappaB in protection from TRAIL. Furthermore, we could reduce X-linked inhibitor of apoptosis protein (XIAP) levels in Panc-1 cells by inhibition of constitutively activated NF-kappaB and sensitize Panc-1 cells to TRAIL by RNA interference against XIAP. These results implicate elevated XIAP levels caused by high basal NF-kappaB activity in TRAIL resistance and suggest that therapeutic strategies involving TRAIL can be abetted by inhibition of NF-kappaB and/or XIAP only in tumor cells with constitutively activated NF-kappaB.

Manganese superoxide dismutase induces p53-dependent senescence in colorectal cancer cells.

The mitochondrial enzyme manganese superoxide dismutase (MnSOD) is known to suppress cell growth in different tumor cell lines. However, the molecular mechanism of this growth-retarding effect is not fully understood. Here we show that overexpression of MnSOD slows down growth of HCT116 human colorectal cancer cells by induction of cellular senescence. MnSOD overexpression causes up-regulation of p53 and its transcriptional target, the cyclin-dependent kinase inhibitor p21. Adenovirus-mediated knockdown of p53 by RNA interference rescues MnSOD-overexpressing clones from growth retardation. Accordingly, the overexpression of MnSOD in HCTp53(-/-) cells does not lead to senescence, whereas in HCTp21(-/-) cells we found induction of senescence by forced expression of MnSOD. These results indicate a pivotal role of p53, but not p21, in the observed effects. Analysis of the mitochondrial membrane potential revealed reduced polarization in MnSOD-overexpressing cells. In addition, depolarization of the mitochondrial membrane by mitochondrial inhibitors such as rotenone or antimycin A led colorectal cancer cells into p53-dependent senescence. Our data indicate that uncoupling of the electrochemical gradient by increased MnSOD activity gives rise to p53 up-regulation and induction of senescence. This novel mitochondrially mediated mechanism of tumor suppression might enable strategies that allow reactivation of cellular aging in tumor cells.