Rita Setroikromo

Quorum-Quenching Acylase Reduces the Virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans Infection Model

ABSTRACT The Pseudomonas aeruginosa PAO1 gene pvdQ encodes an acyl-homoserine lactone (AHL) acylase capable of degrading N-(3-oxododecanoyl)-l-homoserine lactone by cleaving the AHL amide. PvdQ has been proven to function as a quorum quencher in vitro in a number of phenotypic assays. To address the question of whether PvdQ also shows quorum-quenching properties in vivo, an infection model based on the nematode Caenorhabditis elegans was explored. In a fast-acting paralysis assay, strain PAO1(pMEpvdQ), which overproduces PvdQ, was shown to be less virulent than the wild-type strain. More than 75% of the nematodes exposed to PAO1(pMEpvdQ) survived and continued to grow when using this strain as a food source. Interestingly, in a slow-killing assay monitoring the survival of the nematodes throughout a 4-day course, strain PAO1-ΔpvdQ was shown to be more virulent than the wild-type strain, confirming the role of PvdQ as a virulence-reducing agent. It was observed that larval stage 1 (L1) to L3-stage larvae benefit much more from protection by PvdQ than L4 worms. Finally, purified PvdQ protein was added to C. elegans worms infected with wild-type PAO1, and this resulted in reduced pathogenicity and increased the life span of the nematodes. From our observations we can conclude that PvdQ might be a strong candidate for antibacterial therapy against Pseudomonas infections.

Rapid and efficient cancer cell killing mediated by high-affinity death receptor homotrimerizing TRAIL variants.

The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.

Thiotepa improves allogeneic bone marrow engraftment without enhancing stem cell depletion in irradiated mice

Thiotepa (TT) has long been considered for inclusion in clinical bone marrow transplant (BMT) conditioning regimens in an attempt to prevent allograft rejection and leukemia relapse. These studies have been encouraged by initial murine experiments showing a clear improvement in allogeneic bone marrow engraftment with addition of TT to total body irradiation (TBI) where it was assumed that TT enhances donor-type chimerism via ablation of competing stem cells in the recipient. The aim of the present study was to re-evaluate the hematological toxicity of TT among different stem cell subsets that included primitive cells capable of long-term repopulation and to assess how the combination of TT with TBI influences the development of donor engraftment in both syngeneic (B6-Gpi-1a → B6-Gpi-1b) and H-2 compatible allogeneic (BALB.B10 → B6) BMT models. At 24 h after TT (20 mg/kg) the femoral content of different stem cell subsets was determined from the frequency of transient repopulating, and the more primitive cobblestone area-forming, cells (CAFCs) growing in stroma-supported cultures. This assay showed a large TT-induced depletion (2% survival) of early clones developing at day 7 in culture but survival recovered towards normal for later appearing clones developing from more primitive CAFC subsets. The sparing of these primitive stem cells was reflected as undetectable levels of donor marrow repopulation in recipients given TT followed by syngeneic BMT. Addition of TT to TBI did not significantly improve long-term engraftment of syngeneic marrow while this combination had a dramatic effect in allogeneic BMT by preventing allograft rejection. In this respect TT shares similar properties with cyclophosphamide and suggests that the large improvement of allogeneic stem cell engraftment is attributable to the immune suppressive properties of TT rather than to its toxicity against host primitive stem cells.

Differences in heat sensitivity between normal and acute myeloid leukemic stem cells: Feasibility of hyperthermic purging of leukemic cells from autologous stem cell grafts

OBJECTIVES In autologous stem cell transplantation contamination of the graft with malignant cells is frequently noticed and necessitates the use of in vivo or in vitro purging modalities. The hematopoietic recovery after transplantation depends on the number of stem and progenitor cells in the transplant. Therefore, in the present study the effects of hyperthermic treatment on the human normal and acute myeloid leukemic (AML) stem cell compartment were investigated. METHODS Normal bone marrow and AML blasts were heat treated up to 120 minutes at 43 degrees C. The surviving fractions of the different stem cell subsets were determined using in vitro methylcellulose and cobblestone area-forming cell (CAFC) clonogenic assays, as well as the in vivo NOD/SCID repopulating assay. The leukemic nature of the colonies from AML cells was confirmed by RT-PCR analysis. In order to increase the therapeutic index of the hyperthermic purging modality, the heat treatment was preceded by a 3-hour incubation at 37 degrees C with the ether lipid ET-18-OCH(3) (25 microg/mL). RESULTS It could be demonstrated that normal progenitor cells are far more resistant to hyperthermia than leukemic progenitor cells (56%+/-7% vs 9.9%+/-2.6% survival after 60 minutes at 43 degrees C, respectively). Furthermore, normal hematopoietic stem cells appear to be extremely resistant to the heat treatment (94%+/-9% survival after 60 minutes at 43 degrees C). In contrast, in the leukemic stem cell compartment no significant differences in heat sensitivity between the stem cells and progenitor subsets could be observed (12.3%+/-2.9% vs 9.9%+/-2.6% survival after 60 minutes at 43 degrees C, respectively). The combined treatment resulted in a survival for normal progenitor and stem cells of 32%+/-6% and 85%+/-15% after 60 minutes at 43 degrees C, respectively. Under these conditions the number of leukemic stem cells was reduced to 1%+/-0.3%. After 120 minutes at 43 degrees C, no AML-colonies could be detected anymore. CONCLUSIONS Our data demonstrate that leukemic stem cells have an increased hyperthermic sensitivity compared to their normal counterparts and that this difference can be further increased in combination with ET-18-OCH(3). These striking differences in heat sensitivity warrant the use of hyperthermia as a clinically applicable purging modality in autologous stem cell transplantation.

Peripheral blood stem cells differ from bone marrow stem cells in cell cycle status, repopulating potential, and sensitivity toward hyperthermic purging in mice mobilized with cyclophosphamide and granulocyte colony-stimulating factor

Peripheral blood stem cells (PBSCs) are increasingly used in autologous stem cell transplantations. We investigated the mobilizing effect of a combined cyclophosphamide (CTX) and granulocyte colony-stimulating factor (G-CSF) treatment on progenitor cells (STRA) and primitive stem cells (LTRA) in normal and splenectomized CBA/H mice. This combined treatment not only resulted in mobilization but also in expansion of hematopoietic stem cell subsets. The latter phenomenon was somewhat suppressed in splenectomized animals, but in these mice an enhanced mobilization of STRA and LTRA cells into the peripheral blood was observed. Furthermore, we studied the engraftment potential of mobilized PBSCs. Mice transplanted with PBSCs engrafted significantly better compared to mice transplanted with bone marrow stem cells from control and mobilized mice. The repopulation curve was characterized by a less-deep nadir indicating that the differences occur during the initial phase after transplantation. Contamination of autologous PBSC transplants with malignant cells is noticed frequently and is the basis for urging the use of purging modalities. Here we used hyperthermia and found that the mobilized progenitor cells in peripheral blood are more resistant to hyperthermia than those in the bone marrow (i.e., a survival of 11 +/- 5% after 90 min at 43 degrees C for peripheral blood progenitors, compared to 0.5 +/- 0.4% in bone marrow of mobilized animals and 1.6 +/- 0.5% in normal animals, respectively). Hyperthermic purging does not eliminate the superior repopulating features of a PBSC graft, as is demonstrated by an increased median survival time of lethally irradiated mice transplanted with purged PBSCs. In conclusion, our data demonstrate that CTX + G-CSF-mobilized PBSCs have an enhanced engraftment potential concomitantly with a decreased cycling activity and hence a decreased hyperthermic sensitivity. These findings support the use of these mobilized PBSCs for autologous stem cell transplantation and strengthen the basis for using hyperthermia as a purging modality.

Purging of acute myeloid leukaemia cells from stem cell grafts by hyperthermia : enhancement of the therapeutic index by the tetrapeptide AcSDKP and the alkyl-lysophospholipid ET-18-OCH3

Hyperthermia has been shown to be a potential purging modality in autologous stem cell transplantation settings owing to its selective toxicity towards leukaemic cells. We describe two approaches to further increase the therapeutic index of the hyperthermic purging modality by using normal murine bone marrow cells and a murine model for acute myeloid leukaemia. First, the tetrapeptide AcSDKP was used to protect the normal haematopoietic progenitor cells against hyperthermic damage. Pretreatment for 8 h at 37°C with 1 × 10−9 mol/l AcSDKP resulted in a decrease in hyperthermic sensitivity of only normal haematopoietic progenitor cells. This combined treatment protocol revealed a therapeutic index (ratio of surviving fractions of normal vs. leukaemic cells) of > 500, which was considered to be sufficient for purging. This was confirmed in vivo by the survival of lethally irradiated recipients transplanted with purged simulated remission bone marrow (1 × 106 normal bone marrow cells and 5 × 104 leukaemic cells). A further increase of the therapeutic index cells was achieved by the alkyl‐lysophospholipid ET‐18‐OCH3. An incubation for 4 h at 37°C with 25 μg/ml in the presence of 5% fetal calf serum preferentially enhanced the cytotoxic effect towards the leukaemic stem cell. The combination of AcSDKP and ET‐18‐OCH3 with hyperthermia resulted in a therapeutic index of > 5000. This enabled a reduction of the hyperthermic treatment and will further minimize the toxicity to normal haematopoietic stem cell subsets, while a therapeutic index far above the required value is achieved. This tripartite purging treatment therefore offers a safe and fast purging protocol for the elimination of residual leukaemic cells in autografts.

Synthetic constrained peptide selectively binds and antagonizes death receptor 5

Apoptosis or programmed cell death is an inherent part of the development and homeostasis of multicellular organisms. Dysregulation of apoptosis is implicated in the pathogenesis of diseases such as cancer, neurodegenerative diseases and autoimmune disorders. Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is able to induce apoptosis by binding death receptor (DR)4 (TRAIL‐R1) and DR5 (TRAIL‐R2), which makes TRAIL an interesting and promising therapeutic target. To identify peptides that specifically interact with DR5, a disulfide‐constrained phage display peptide library was screened for binders towards this receptor. Phage‐displayed peptides were identified that bind specifically to DR5 and not to DR4, nor any of the decoy receptors. We show that the synthesized peptide, YCKVILTHRCY, in both monomeric and dimeric forms, binds specifically to DR5 in such a way that TRAIL binding to DR5 is inhibited. Surface plasmon resonance studies showed higher affinity towards DR5 for the dimeric form then the monomeric form of the peptide, with apparent Kd values of 40 nm versus 272 nm, respectively. Binding studied on cell lines by flow cytometry analyses showed concentration‐dependent binding. Upon co‐incubation with increasing concentrations of TRAIL, the peptide binding was reduced. Moreover, both the monomeric and dimeric forms of the peptide reduced TRAIL‐induced cell death in Colo205 colon carcinoma cells. The peptide, YCKVILTHRCY, or its derivates, may be a useful investigative tool for dissecting signalling via DR5 relative to DR4 or could act as a lead peptide for the development of therapeutic agents in diseases with dysregulated TRAIL‐signalling.

The ER stress inducer DMC enhances TRAIL-induced apoptosis in glioblastoma

Glioblastoma multiforme (GBM) is the most aggressive malignant brain tumour in humans and is highly resistant to current treatment modalities. We have explored the combined treatment of the endoplasmic reticulum (ER) stress-inducing agent 2,5-dimethyl-celecoxib (DMC) and TNF-related apoptosis-inducing ligand (TRAIL WT) or the DR5-specific TRAIL D269H/E195R variant as a potential new strategy to eradicate GBM cells using TRAIL-resistant and -sensitive GBM cells. GBM cell lines were investigated for their sensitivity to TRAIL, DMC and combination of both agents. Cell viability was measured by MTS assay and apoptosis was assessed by Annexin V/PI and acridine orange staining. Caspase activation and protein expression levels were analysed with Western blotting. Death Receptor (DR) cell surface expression levels were quantified by flow cytometry. DR5 expression was increased in U87 cells by ectopic expression using a retroviral plasmid and survivin expression was silenced using specific siRNAs. We demonstrate that A172 expresses mainly DR5 on the cell surface and that these cells show increased sensitivity for the DR5-specific rhTRAIL D269H/E195R variant. In contrast, U87 cells show low DR cell surface levels and is insensitive via both DR4 and DR5. We determined that DMC treatment displays a dose-dependent reduction in cell viability against a number of GBM cells, associated with ER stress induction, as shown by the up-regulation of glucose-regulated protein 78 (GRP78) and CCAAT/-enhancer-binding protein homologous protein (CHOP) in A172 and U87 cells. The dramatic decrease in cell viability is not accompanied by a correspondent increase in Annexin V/PI or caspase activation typically seen in apoptotic or/and necrotic cells within 24h of treatment. Although DMC did not affect DR5 expression in the GBM cells, it increased TRAIL-induced caspase-8 activation in both TRAIL-sensitive and -resistant cells, indicating that DMC potentiates initiator caspase activation in these cells. In A172 cells, sub-toxic concentrations of DMC greatly potentiated TRAIL-induced apoptosis. Furthermore, DMC strongly reduced survivin expression in A172 and U87 cells and silencing of this anti-apoptotic protein partially sensitized cells to TRAIL-induced apoptosis. Our findings corroborate that DMC is a promising agent against GBM, and uncovers a potential synergistic cooperation with TRAIL in this highly malignant cancer.

Towards Metabolic Engineering of Podophyllotoxin Production: Natural Products and Cancer Drug Discovery

The pharmaceutically important anticancer drugs etoposide and teniposide are derived from podophyllotoxin, a natural product isolated from roots of Podophyllum hexandrum growing in the wild. The overexploitation of this endangered plant has led to the search for alternative sources. Metabolic engineering aimed at constructing the pathway in another host cell is very appealing, but for that approach, an in-depth knowledge of the pathway toward podophyllotoxin is necessary. In this chapter, we give an overview of the lignan pathway leading to podophyllotoxin. Subsequently, we will discuss the engineering possibilities to produce podophyllotoxin in a heterologous host. This will require detailed knowledge on the cellular localization of the enzymes of the lignan biosynthesis pathway. Due to the high number of enzymes involved and the scarce information on compartmentalization, the heterologous production of podophyllotoxin still remains a tremendous challenge. At the moment, research is focusing on the last step(s) in the conversion of deoxypodophyllotoxin to (epi)podophyllotoxin and 4′-demethyldesoxypodophyllotoxin by plant cytochromes. Keywords: etoposide, podophyllotoxin, Podophyllum hexandrum, Anthriscus sylvestris, metabolic engineering

Novel RANKL DE-loop mutants antagonize RANK-mediated osteoclastogenesis

Bone is a dynamic tissue that is maintained by continuous renewal. An imbalance in bone resorption and bone formation can lead to a range of disorders, such as osteoporosis. The receptor activator of NF‐κB (RANK)–RANK‐ligand (RANKL) pathway plays a major role in bone remodeling. Here, we investigated the effect of mutations at position I248 in the DE‐loop of murine RANKL on the interaction of RANKL with RANK, and subsequent activation of osteoclastogenesis. Two single mutants, RANKL I248Y and I248K, were found to maintain binding and have the ability to reduce wild‐type RANKL‐induced osteoclastogenesis. The generation of RANK‐antagonists is a promising strategy for the exploration of new therapeutics against osteoporosis.