Emergence and evolution of the renin-angiotensin-aldosterone system

The renin-angiotensin-aldosterone system (RAAS) is not the sole, but perhaps the most important volume regulator in vertebrates. To gain insights into the function and evolution of its components, we conducted a phylogenetic analysis of its main related genes. We found that important parts of the system began to appear with primitive chordates and tunicates and that all major components were present at the divergence of bony fish, with the exception of the Mas receptor. The Mas receptor first appears after the bony-fish/tetrapod divergence. This phase of evolutionary innovation happened about 400 million years ago. We found solid evidence that angiotensinogen made its appearance in cartilage fish. The presence of several RAAS genes in organisms that lack all the components shows that these genes have had other ancestral functions outside of their current role. Our analysis underscores the utility of sequence comparisons in the study of evolution. Such analyses may provide new hypotheses as to how and why in today's population an increased activity of the RAAS frequently leads to faulty salt and volume regulation, hypertension, and cardiovascular diseases, opening up new and clinically important research areas for evolutionary medicine

Angiotensin II induced inflammation in the kidney and in the heart of double transgenic rats

BACKGROUND: We are investigating a double transgenic rat (dTGR) model, in which rats transgenic for the human angiotensinogen and renin genes are crossed. These rats develop moderately severe hypertension but die of end-organ cardiac and renal damage by week 7. The heart shows necrosis and fibrosis, whereas the kidneys resemble the hemolytic-uremic syndrome vasculopathy. Surface adhesion molecules (ICAM-1 and VCAM-1) are expressed early on the endothelium, while the corresponding ligands are found on circulating leukocytes. Leukocyte infiltration in the vascular wall accompanies PAI-1, MCP-1, iNOS and Tissue Factor expression. Furthermore we show evidence that Ang II causes the upregulation of NF-{kappa}B in our model. METHODS: We started PDTC-treatment on four weeks old dTGR (200 mg/kg sc) and age-matched SD rats. Blood-pressure- and albuminuria- measurements were monitored during the treatment period (four weeks). The seven weeks old animals were killed, hearts and kidneys were isolated and used for immunohistochemical-and electromobility shift assay analysis. RESULTS: Chronic treatment with the antioxidant PDTC decreased blood pressure (162 plus minus 8 vs. 190 plus minus 7 mm Hg, p = 0.02). Cardiac hypertrophy index was significantly reduced (4.90 plus minus 0.1 vs. 5.77 plus minus 0.1 mg/g, p < 0.001) compared to dTGR. PDTC reduced 24 h albuminuria by 85 % (2.7 plus minus 0.5 vs. 18.0 plus minus 3.4 mg/d, p < 0.001) and prevented death significantly. Vascular injury was ameliorated in small renal and cardiac vessels. PDTC inhibited NF-{kappa}B binding activity in heart and kidney. Immunohistochemical analysis shows increased expression of the p65 NF-{kappa}B subunit in the endothelium, smooth muscles cells of damaged small vessels, infiltrated cells, glomeruli, tubuli and collecting ducts of dTGR. PDTC markedly reduced the immunoreactivity of p65. CONCLUSION: Our data show that inhibition of NF-{kappa}B by PDTC markedly reduces inflammation, iNOS expression in the dTGR most likely leading to decreased cytotoxicity, and cell proliferation. Thus, NF-{kappa}B activation plays an important role in ANG II-induced end-organ damage

Delivery of sTRAIL variants by MSCs in combination with cytotoxic drug treatment leads to p53-independent enhanced antitumor effects

Mesenchymal stem cells (MSCs) are able to infiltrate tumor tissues and thereby effectively deliver gene therapeutic payloads. Here, we engineered murine MSCs (mMSCs) to express a secreted form of the TNF-related apoptosis-inducing ligand (TRAIL), which is a potent inducer of apoptosis in tumor cells, and tested these MSCs, termed MSC.sTRAIL, in combination with conventional chemotherapeutic drug treatment in colon cancer models. When we pretreated human colorectal cancer HCT116 cells with low doses of 5-fluorouracil (5-FU) and added MSC.sTRAIL, we found significantly increased apoptosis as compared with single-agent treatment. Moreover, HCT116 xenografts, which were cotreated with 5-FU and systemically delivered MSC.sTRAIL, went into remission. Noteworthy, this effect was protein 53 (p53) independent and was mediated by TRAIL-receptor 2 (TRAIL-R2) upregulation, demonstrating the applicability of this approach in p53-defective tumors. Consequently, when we generated MSCs that secreted TRAIL-R2-specific variants of soluble TRAIL (sTRAIL), we found that such engineered MSCs, labeled MSC.sTRAIL DR5, had enhanced antitumor activity in combination with 5-FU when compared with MSC.sTRAIL. In contrast, TRAIL-resistant pancreatic carcinoma PancTu1 cells responded better to MSC.sTRAIL DR4 when the antiapoptotic protein XIAP (X-linked inhibitor of apoptosis protein) was silenced concomitantly. Taken together, our results demonstrate that TRAIL-receptor selective variants can potentially enhance the therapeutic efficacy of MSC-delivered TRAIL as part of individualized and tumor-specific combination treatments. © 2013 Macmillan Publishers Limited All rights reserved

Superior antitumoral activity of dimerized targeted single-chain TRAIL fusion proteins under retention of tumor selectivity

Although targeting of the death receptors (DRs) DR4 and DR5 still appears a suitable antitumoral strategy, the limited clinical responses to recombinant soluble TNF-related apoptosis inducing ligand (TRAIL) necessitate novel reagents with improved apoptotic activity/tumor selectivity. Apoptosis induction by a single-chain TRAIL (scTRAIL) molecule could be enhanced >10-fold by generation of epidermal growth factor receptor (EGFR)-specific scFv-scTRAIL fusion proteins. By forcing dimerization of scFv-scTRAIL based on scFv linker modification, we obtained a targeted scTRAIL composed predominantly of dimers (Db-scTRAIL), exceeding the activity of nontargeted scTRAIL ∼100-fold on Huh-7 hepatocellular and Colo205 colon carcinoma cells. Increased activity of Db-scTRAIL was also demonstrated on target-negative cells, suggesting that, in addition to targeting, oligomerization equivalent to an at least dimeric assembly of standard TRAIL per se enhances apoptosis signaling. In the presence of apoptosis sensitizers, such as the proteasomal inhibitor bortezomib, Db-scTRAIL was effective at picomolar concentrations in vitro (EC50 ∼2 × 10−12 M). Importantly, in vivo, Db-scTRAIL was well tolerated and displayed superior antitumoral activity in mouse xenograft (Colo205) tumor models. Our results show that both targeting and controlled dimerization of scTRAIL fusion proteins provides a strategy to enforce apoptosis induction, together with retained tumor selectivity and good in vivo tolerance

Novel SMAC-mimetics synergistically stimulate melanoma cell death in combination with TRAIL and Bortezomib

BACKGROUND: XIAP (X-linked inhibitor of apoptosis protein) is an anti-apoptotic protein exerting its activity by binding and suppressing caspases. As XIAP is overexpressed in several tumours, in which it apparently contributes to chemoresistance, and because its activity in vivo is antagonised by second mitochondria-derived activator of caspase (SMAC)/direct inhibitor of apoptosis-binding protein with low pI, small molecules mimicking SMAC (so called SMAC-mimetics) can potentially overcome tumour resistance by promoting apoptosis. METHODS: Three homodimeric compounds were synthesised tethering a monomeric SMAC-mimetic with different linkers and their affinity binding for the baculoviral inhibitor repeats domains of XIAP measured by fluorescent polarisation assay. The apoptotic activity of these molecules, alone or in combination with tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and/or Bortezomib, was tested in melanoma cell lines by MTT viability assays and western blot analysis of activated caspases. RESULTS: We show that in melanoma cell lines, which are typically resistant to chemotherapeutic agents, XIAP knock-down sensitises cells to TRAIL treatment in vitro, also favouring the accumulation of cleaved caspase-8. We also describe a new series of 4-substituted azabicyclo[5.3.0] alkane monomeric and dimeric SMAC-mimetics that target various members of the IAP family and powerfully synergise at submicromolar concentrations with TRAIL in inducing cell death. Finally, we show that the simultaneous administration of newly developed SMAC-mimetics with Bortezomib potently triggers apoptosis in a melanoma cell line resistant to the combined effect of SMAC-mimetics and TRAIL. CONCLUSION: Hence, the newly developed SMAC-mimetics effectively synergise with TRAIL and Bortezomib in inducing cell death. These findings warrant further preclinical studies in vivo to verify the anticancer effectiveness of the combination of these agents

Suppression of Mcl-1 via RNA interference sensitizes human hepatocellular carcinoma cells towards apoptosis induction

BACKGROUND: Hepatocelluar carcinoma (HCC) is one of the most common cancers worldwide and a major cause of cancer-related mortality. HCC is highly resistant to currently available chemotherapeutic drugs. Defects in apoptosis signaling contribute to this resistance. Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which interferes with mitochondrial activation. In a previous study we have shown that Mcl-1 is highly expressed in tissues of human HCC. In this study, we manipulated expression of the Mcl-1 protein in HCC cells by RNA interference and analyzed its impact on apoptosis sensitivity of HCC cells in vitro. METHODS: RNA interference was performed by transfecting siRNA to specifically knock down Mcl-1 expression in HCC cells. Mcl-1 expression was measured by quantitative real-time PCR and Western blot. Induction of apoptosis and caspase activity after treatment with chemotherapeutic drugs and different targeted therapies were measured by flow cytometry and fluorometric analysis, respectively. RESULTS: Here we demonstrate that Mcl-1 expressing HCC cell lines show low sensitivity towards treatment with a panel of chemotherapeutic drugs. However, treatment with the anthracycline derivative epirubicin resulted in comparatively high apoptosis rates in HCC cells. Inhibition of the kinase PI3K significantly increased apoptosis induction by chemotherapy. RNA interference efficiently downregulated Mcl-1 expression in HCC cells. Mcl-1 downregulation sensitized HCC cells to different chemotherapeutic agents. Sensitization was accompanied by profound activation of caspase-3 and -9. In addition, Mcl-1 downregulation also increased apoptosis rates after treatment with PI3K inhibitors and, to a lower extent, after treatment with mTOR, Raf I and VEGF/PDGF kinase inhibitors. TRAIL-induced apoptosis did not markedly respond to Mcl-1 knockdown. Additionally, knockdown of Mcl-1 efficiently enhanced apoptosis sensitivity towards combined treatment modalities: Mcl-1 knockdown significantly augmented apoptosis sensitivity of HCC cells towards chemotherapy combined with PI3K inhibition. CONCLUSION: Our data suggest that specific downregulation of Mcl-1 by RNA interference is a promising approach to sensitize HCC cells towards chemotherapy and molecularly targeted therapies

TRAIL sensitisation by arsenic trioxide is caspase-8 dependent and involves modulation of death receptor components and Akt

The majority of leukaemic cells are resistant to apoptosis induced by tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Here, we show that sublethal concentrations of arsenic trioxide (ATO) specifically enhanced TRAIL-induced apoptosis in leukaemic but not in other tumour cell lines. The combination of ATO and TRAIL synergistically enhanced cleavage of caspase-8, which was blocked by the caspase inhibitor IETD.fmk as well as in cells deficient for caspase-8, suggesting a requirement for the death-inducing signalling complex. Arsenic trioxide led to increased cell surface expression of DR5 (death receptor 5), inhibition of the serine/threonine kinase Akt and downregulation of the short isoform of FLIP (FLICE-inhibitory protein, FLIPS). Inhibition of the phosphatidylinositol 3 kinase (PI3K) was equally efficient in sensitising leukaemic cells to TRAIL with similar effects on DR5 and FLIPS expression, suggesting that ATO may in part act through inhibition of the PI3K/Akt signalling pathway. These results indicate that the enhancement in TRAIL-mediated apoptosis induced by ATO is due to alteration in the levels of multiple components and regulators of the death receptor-mediated pathway. These findings offer a promising and novel strategy involving a combination of TRAIL and ATO, or more specific Akt inhibitors in the treatment of various haematopoietic malignancies

Targeting Tumour-Initiating Cells with TRAIL Based Combination Therapy Ensures Complete and Lasting Eradication of Multiple Myeloma Tumours In Vivo

Multiple myeloma (MM) remains an incurable disease despite improvements to available treatments and efforts to identify new drug targets. Consequently new approaches are urgently required. We have investigated the potential of native tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), in combination with doxorubicin, to induce apoptotic cell death in phenotypically distinct populations of myeloma cells in vitro and in vivo. The cytotoxic potential of TRAIL alone, and in combination with DOX, was assessed in vitro in purified CD138+ and CD138− cells from the MM cell lines and samples from patients with MM. Mouse xenografts obtained by implanting CD138− MM cells were used to assess the efficacy of TRAIL, alone and in combination with DOX, in vivo. CD138− cells were shown to be more resistant to the cytotoxic activity of TRAIL than CD138+ cells and have reduced expression of TRAIL death receptors. This resistance results in preferential killing of CD 138+ cells during exposure of MM culture to TRAIL. Furthermore, prolonged exposure results in the appearance of TRAIL-resistant CD138− cells. However, when TRAIL is combined with doxorubicin, this results in complete eradication of MM cells in vivo. Most importantly, this treatment successfully eliminates CD138− cells implicated in tumour initiation and growth maintenance. These findings may explain the failure of current therapies and offer a promising new approach in the quest to cure MM and disseminated cancers

Synergistic induction of cell death in liver tumor cells by TRAIL and chemotherapeutic drugs via the BH3-only proteins Bim and Bid

Although death receptors and chemotherapeutic drugs activate distinct apoptosis signaling cascades, crosstalk between the extrinsic and intrinsic apoptosis pathway has been recognized as an important amplification mechanism. Best known in this regard is the amplification of the Fas (CD95) signal in hepatocytes via caspase 8-mediated cleavage of Bid and activation of the mitochondrial apoptosis pathway. Recent evidence, however, indicates that activation of other BH3-only proteins may also be critical for the crosstalk between death receptors and mitochondrial triggers. In this study, we show that TNF-related apoptosis-inducing ligand (TRAIL) and chemotherapeutic drugs synergistically induce apoptosis in various transformed and untransformed liver-derived cell lines, as well as in primary human hepatocytes. Both, preincubation with TRAIL as well as chemotherapeutic drugs could sensitize cells for apoptosis induction by the other respective trigger. TRAIL induced a strong and long lasting activation of Jun kinase, and activation of the BH3-only protein Bim. Consequently, synergistic induction of apoptosis by TRAIL and chemotherapeutic drugs was dependent on Jun kinase activity, and expression of Bim and Bid. These findings confirm a previously defined role of TRAIL and Bim in the regulation of hepatocyte apoptosis, and demonstrate that the TRAIL–Jun kinase–Bim axis is a major and important apoptosis amplification pathway in primary hepatocytes and liver tumor cells

TRAIL treatment provokes mutations in surviving cells

Chemotherapy and radiotherapy commonly damage DNA and trigger p53-dependent apoptosis through intrinsic apoptotic pathways. Two unfortunate consequences of this mechanism are resistance due to blockade of p53 or intrinsic apoptosis pathways, and mutagenesis of non-malignant surviving cells which can impair cellular function or provoke second malignancies. Death ligand-based drugs, such as tumor necrosis factor-related apoptosis inducing ligand (TRAIL), stimulate extrinsic apoptotic signaling, and may overcome resistance to treatments that induce intrinsic apoptosis. As death receptor ligation does not damage DNA as a primary mechanism of pro-apoptotic action, we hypothesized that surviving cells would remain genetically unscathed, suggesting that death ligand-based therapies may avoid some of the adverse effects associated with traditional cancer treatments. Surprisingly, however, treatment with sub-lethal concentrations of TRAIL or FasL was mutagenic. Mutations arose in viable cells that contained active caspases, and overexpression of the caspase-8 inhibitor crmA or silencing of caspase-8 abolished TRAIL-mediated mutagenesis. Downregulation of the apoptotic nuclease caspase-activated DNAse (CAD)/DNA fragmentation factor 40 (DFF40) prevented the DNA damage associated with TRAIL treatment. Although death ligands do not need to damage DNA in order to induce apoptosis, surviving cells nevertheless incur DNA damage after treatment with these agents