Optimized Attenuated Salmonella Typhimurium Suppressed Tumor Growth and Improved Survival in Mice

The gram-negative facultative anaerobic bacteria Salmonella enterica serovar Typhimurium (hereafter S. Typhimurium) has always been considered as one candidate of anti-tumor agents or vectors for delivering drug molecules. In this study, we compared several widely studied S. Typhimurium strains in their anti-tumor properties aiming to screen out the best one for further optimization and use in cancer therapy. In terms of the motility, virulence and anti-tumor efficacy, the three strains 14028, SL1344, and UK-1 were similar and obviously better than LT-2, and UK-1 showed the best phenotypes among them. Therefore, the strain UK-1 (D) was selected for the following studies. Its auxotrophic mutant strain (D1) harboring ∆aroA and ∆purM mutations was further optimized through the modification of lipid A structure, generating a new strain named D2 with stronger immunostimulatory activity. Finally, the ∆asd derivative of D2 was utilized as one live vector to deliver anti-tumor molecules including the angiogenesis inhibitor endostatin and apoptosis inducer TRAIL and the therapeutic and toxic-side effects were evaluated in mouse models of colon carcinoma and melanoma. After intraperitoneal infection, engineered Salmonella bacteria equipped with endostatin and/or TRAIL significantly suppressed the tumor growth and prolonged survival of tumor-bearing mice compared to PBS or bacteria carrying the empty plasmid. Consistently, immunohistochemical studies confirmed the colonization of Salmonella bacteria and the expression of anti-tumor molecules inside tumor tissue, which were accompanied by the increase of cell apoptosis and suppression of tumor angiogenesis. These results demonstrated that the beneficial anti-tumor efficacy of attenuated S. Typhimurium bacteria could be improved through delivery of drug molecules with powerful anti-tumor activities.

HO-3867 Induces ROS-Dependent Stress Response and Apoptotic Cell Death in Leishmania donovani

Lack of vaccine and increasing chemotherapeutic toxicities currently necessitate the development of effective and safe drugs against various forms of leishmaniases. We characterized the cellular stress induced by a novel curcumin analogue, HO-3867, encapsulated within the phosphatidylcholine-stearylamine (PC-SA) liposome for the first time against Leishmania. The liposomal formulation of HO-3867 (i.e., PC-SA/HO-3867) initiated oxidative stress-induced apoptosis in L. donovani, revealed by altered cell morphology, phosphatidylserine externalization, mitochondrial depolarization, intracellular lipid accumulation, and cell cycle arrest in promastigotes. Liposomal HO-3867 was observed to be a strong apoptosis inducer in L. donovani and L. major in a dose-dependent manner, yet completely safe for normal murine macrophages. Moreover, PC-SA/HO-3867 treatment induced L. donovani metacaspase and PARP1 activation along with downregulation of the Sir2 gene. PC-SA/HO-3867 arrested intracellular L. donovani amastigote burden in vitro, with reactive oxygen species (ROS) and nitric oxide (NO)-mediated parasite killing. These data suggest that liposomal HO-3867 represents a highly promising and non-toxic nanoparticle-based therapeutic platform against leishmaniasis inspiring further preclinical developments.

A Proteomic Analysis of the Effects of Yessotoxin and Di-Desulfoyessotoxin on Yeast and Mammalian Cells

<p>Yessotoxin (YTX) is a disulfated polycyclic polyether, produced by dinoflagellate algae. It is known to accumulate in edible shellfish, raising concerns about its potential risk to human health. YTX was initially classified as a diarrhetic shellfish poisoning toxin, due to commonly being extracted alongside toxins of this variety. However, YTX does not induce any of the effects characteristic of this group. A separate category for YTXs was established by the European Commission in 2002 and a limit of 1 mg/kg of shellfish meat was established. YTX has been shown to be an apoptosis inducer in a variety of cell lines in vitro. It has also been shown to be lethal to mice when administered by intra-peritoneal injection. However, when administered orally only limited toxicity is observed. The di-desulfated derivative (dsYTX) has also been shown to be lethal to mice following intra-peritoneal injection. However it causes damage mainly to the liver, whereas YTX appears to target the heart. The mechanism of action of YTX is still unknown. The goals of this project were to use proteomic techniques, to examine the effects of YTX and dsYTX on Saccharomyces cerevisiae and human promyelocytic leukemic blood leukocyte (HL60) cells. Young et al. (2009) showed that the major proteins affected by YTX in HepG2 cells were heterogeneous ribonucleoproteins (hnRNPs), lamins, cathepsins and heat shock proteins. HnRNPs had not previously been identified as possible targets of YTX. Alterations of hnRNP levels were also seen in HL60 cells treated with microtubule stabilising agents, peloruside A or paclitaxel (Wilmes et al., 2011, 2012). No differences in cell morphology or significant changes in protein abundance were observed when S. cerevisiae cells were exposed to YTX. A small number of significant changes in abundance were detected when these cells were exposed to dsYTX. The small number of protein changes seen is possibly due to poor toxin entrance into the cell through the yeast cell wall, lack of protein targets structurally homologous to those found in mammalian cells, or fast removal of the toxin through export pumps. Twenty-four hour incubation of HL60 cells with YTX resulted in increased cell death but no change in cell morphology. Treatment with dsYTX caused cells to aggregate into clusters and a 24% decrease in the number of live cells. Increases were found in the abundance of β-actin, hnRNP A and BiP proteins in response to dsYTX treatment. Decreases in these proteins were seen in HepG2 cells treated with YTX for 24 hours. As seen in S. cerevisiae cells, dsYTX had a greater effect in HL60 cells compared with YTX. Overall, the results provide some support for the previously identified effect on hnRNPs in mammalian cells exposed to YTX.</p>

A Proteomic Analysis of the Effects of Yessotoxin and Di-Desulfoyessotoxin on Yeast and Mammalian Cells

<p>Yessotoxin (YTX) is a disulfated polycyclic polyether, produced by dinoflagellate algae. It is known to accumulate in edible shellfish, raising concerns about its potential risk to human health. YTX was initially classified as a diarrhetic shellfish poisoning toxin, due to commonly being extracted alongside toxins of this variety. However, YTX does not induce any of the effects characteristic of this group. A separate category for YTXs was established by the European Commission in 2002 and a limit of 1 mg/kg of shellfish meat was established. YTX has been shown to be an apoptosis inducer in a variety of cell lines in vitro. It has also been shown to be lethal to mice when administered by intra-peritoneal injection. However, when administered orally only limited toxicity is observed. The di-desulfated derivative (dsYTX) has also been shown to be lethal to mice following intra-peritoneal injection. However it causes damage mainly to the liver, whereas YTX appears to target the heart. The mechanism of action of YTX is still unknown. The goals of this project were to use proteomic techniques, to examine the effects of YTX and dsYTX on Saccharomyces cerevisiae and human promyelocytic leukemic blood leukocyte (HL60) cells. Young et al. (2009) showed that the major proteins affected by YTX in HepG2 cells were heterogeneous ribonucleoproteins (hnRNPs), lamins, cathepsins and heat shock proteins. HnRNPs had not previously been identified as possible targets of YTX. Alterations of hnRNP levels were also seen in HL60 cells treated with microtubule stabilising agents, peloruside A or paclitaxel (Wilmes et al., 2011, 2012). No differences in cell morphology or significant changes in protein abundance were observed when S. cerevisiae cells were exposed to YTX. A small number of significant changes in abundance were detected when these cells were exposed to dsYTX. The small number of protein changes seen is possibly due to poor toxin entrance into the cell through the yeast cell wall, lack of protein targets structurally homologous to those found in mammalian cells, or fast removal of the toxin through export pumps. Twenty-four hour incubation of HL60 cells with YTX resulted in increased cell death but no change in cell morphology. Treatment with dsYTX caused cells to aggregate into clusters and a 24% decrease in the number of live cells. Increases were found in the abundance of β-actin, hnRNP A and BiP proteins in response to dsYTX treatment. Decreases in these proteins were seen in HepG2 cells treated with YTX for 24 hours. As seen in S. cerevisiae cells, dsYTX had a greater effect in HL60 cells compared with YTX. Overall, the results provide some support for the previously identified effect on hnRNPs in mammalian cells exposed to YTX.</p>

Structure of PDE3A–SLFN12 complex and structure-based design for a potent apoptosis inducer of tumor cells

Molecular glues are a class of small molecular drugs that mediate protein-protein interactions, that induce either the degradation or stabilization of target protein. A structurally diverse group of chemicals, including 17-β-estradiol (E2), anagrelide, nauclefine, and DNMDP, induces apoptosis by forming complexes with phosphodiesterase 3A (PDE3A) and Schlafen 12 protein (SLFN12). They do so by binding to the PDE3A enzymatic pocket that allows the compound-bound PDE3A to recruit and stabilize SLFN12, which in turn blocks protein translation, leading to apoptosis. In this work, we report the high-resolution cryo-electron microscopy structure of PDE3A-SLFN12 complexes isolated from cultured HeLa cells pre-treated with either anagrelide, or nauclefine, or DNMDP. The PDE3A-SLFN12 complexes exhibit a butterfly-like shape, forming a heterotetramer with these small molecules, which are packed in a shallow pocket in the catalytic domain of PDE3A. The resulting small molecule-modified interface binds to the short helix (E552-I558) of SLFN12 through hydrophobic interactions, thus “gluing” the two proteins together. Based on the complex structure, we designed and synthesized analogs of anagrelide, a known drug used for the treatment of thrombocytosis, to enhance their interactions with SLFN12, and achieved superior efficacy in inducing apoptosis in cultured cells as well as in tumor xenografts.

A New Smoothened Antagonist Bearing the Purine Scaffold Shows Antitumour Activity In Vitro and In Vivo

The Smoothened (SMO) receptor is the most druggable target in the Hedgehog (HH) pathway for anticancer compounds. However, SMO antagonists such as vismodegib rapidly develop drug resistance. In this study, new SMO antagonists having the versatile purine ring as a scaffold were designed, synthesised, and biologically tested to provide an insight to their mechanism of action. Compound 4s was the most active and the best inhibitor of cell growth and selectively cytotoxic to cancer cells. 4s induced cell cycle arrest, apoptosis, a reduction in colony formation and downregulation of PTCH and GLI1 expression. BODIPY-cyclopamine displacement assays confirmed 4s is a SMO antagonist. In vivo, 4s strongly inhibited tumour relapse and metastasis of melanoma cells in mice. In vitro, 4s was more efficient than vismodegib to induce apoptosis in human cancer cells and that might be attributed to its dual ability to function as a SMO antagonist and apoptosis inducer.

Evaluation of the PBMC Proliferation, Apoptosis and Cytokines Profiling in Cattle Infected with Mycoplasma bovis Strain 07801

Bovine respiratory diseases are widespread and too costly disease impacting contributes to economically essential diseases like mastitis and pneumonia worldwide. The present study aimed to explore the influence of M. bovis field strain 07801 on the status of PBMCs in the challenged cattle group and the immunized one. For this aim, the PBMCs proliferation, apoptosis, and cytokine profile changes were determined. In this study, M. bovis strain Mb 07801 and reference strain PG45 were used; ten calves (2-3 months old) were arranged into two groups (5 calve each): 1) PBS-challenged group (with M. bovis 07801 at about 1010 CFU/ml), and 2) immunized group (by Inactivated M. bovis 07801); both groups were treated nasally and intra-tracheal. Blood samples were obtained from both groups and examined for PBMCs proliferation and apoptosis, as well as serum cytokine profile, before infection and at day zero and days 7, 14, 21, and 36 post bacterial treatment. The results revealed that M. bovis strain (07801and PG45) antigen increased the proliferative response of the stimulated PBMC compared with the unstimulated cells and ConA-alone stimulated. The PBMC apoptosis showed a non-significant increase in both challenged and immunized groups compared to the negative and positive control (treated with apoptosis inducer) groups. Besides, the levels of cytokines profile showed a significant up-regulation in IFN-γ, IL-4, IL-2, IL-18, TNF-α, IFN-α, IL-6, IL-10 IL-1β, and IL-13, in both groups, except IL-4 and IL-18 those recorded a down-regulation in the immunized group at days 7,14, 21, and 36 post-M. bovis infection. In conclusion, immunization markedly ameliorated the immune deterioration induced by M. bovis strain 07801.

Rapid in vitro quantification of TDP-43 and FUS mislocalisation for screening of gene variants implicated in frontotemporal dementia and amyotrophic lateral sclerosis

Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests these two diseases are related. Post-mortem, ~ 95% of ALS and ~ 50% of FTD patients show redistribution of the nuclear protein TDP-43 to the cytoplasm within affected neurons, while ~ 5% ALS and ~ 10% FTD show mislocalisation of FUS protein. We exploited these neuropathological features to develop an unbiased method for the in vitro quantification of cytoplasmic TDP-43 and FUS. Utilising fluorescently-tagged cDNA constructs and immunocytochemistry, the fluorescence intensity of TDP-43 or FUS was measured in the nucleus and cytoplasm of cells, using the freely available software CellProfiler. Significant increases in the amount of cytoplasmic TDP-43 and FUS were detectable in cells expressing known FTD/ALS-causative TARDBP and FUS gene mutations. Pharmacological intervention with the apoptosis inducer staurosporine and mutation in a secondary gene (CYLD) also induced measurable cytoplasmic mislocalisation of endogenous FUS and TDP-43, respectively. These findings validate this methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used for initial prioritisation of predicted FTD/ALS-causative mutations.