Targeting the vulnerability of cancer cell mitochondria to selectively induce apoptosis: Evaluating the efficacy of Pancratistatin as a non-toxic anti-cancer agent PANDEY S, GRIFFIN C, SIEDLAKOWSKI P, KARNIK A AND MCNULTY J *
Department of Chemistry & Biochemistry, University of Windsor, Windsor, Ontario, Canada.
*Department of Chemistry, McMaster University, Hamilton, Ontario, Canada
Despite the aggressive research efforts to find selective anti-cancer chemotherapeutics, cancer remains unconquered. The major difficulty with the treatment of cancer is the non-specificity with which chemotherapy kills cells. Many current treatments, including radiotherapy, are damaging to both cancerous and non-cancerous cells. Non-specific damage causes harsh side effects and DNA mutations that increase the possibility of cells becoming cancerous. Pancratistatin is a natural compound that was isolated from the spider lily in 1992, by Petit et al., and has been shown to have anti-cancer properties. We have recently demonstrated that while Pancratistatin induces apoptosis (programmed cell suicide) in cancer cells it does not affect non-cancerous cells. We have also demonstrated the non-genotoxic nature of Pancratistatin; its ability to kill cancerous cells without targeting their DNA. We have investigated the specificity and biochemical mechanism of action of Pancratistatin; our results indicate that Pancratistatin specifically and effectively induces apoptosis in human prostate cancer, breast cancer, neuroblastoma, melanoma and leukemia cell lines. Interestingly, we have demonstrated that Pancratistatin targets the mitochondria of cancer cells. Mitochondria from non-cancerous cells are not affected by this treatment, indicating that vulnerability to this compound is limited to cancer cell mitochondria. Our preliminary in vivo results with human colon and prostate cancer xenotransplants in immuno-compromised mice have indicated that Pancratistatin inhibits tumor growth and is well-tolerated at the effective dose. These results reveal a new opportunity for the development of chemotherapy that targets mitochondria of cancer cells and advance our knowledge of a novel mechanism of action for Pancratistatin.
The role of corticosteroids, male and female sex hormones in a complex with apolipoprotein A-I in the regulation of gene expression PANIN LE State Research Institute of Biochemistry SD RAMS, Timakov str. 2, Novosibirsk, Russia
Apolipoprotein A-I (apoA-I) is a universal transport species for corticosteroids, female and male sex hormones. This protein performs the addressable delivery of hormones into cell nuclei. The initial unit of this mechanism is represented by receptor mediated endocytosis. In cell nucleus, a hormone – apoA-I complex interacts with gene promoters comprising (GCC)n sequences and initiates their expression. However, the biological activity is inherent only in the steroid hormones that have a reduced Δ4,3-ketogroup in the A-ring. The molecular-biological methods, IR spectroscopy and small-angle X-ray scattering were used to reveal the mechanism of gene expression in a cell culture and eukaryotic DNA. The mechanism is related with competitive rupture of hydrogen bonds in complementary GC-pairs with participation of the OH-group in the third position of hormone A-ring. Further rupture of hydrogen bonds is caused by hydrophobic interaction between nitrous base rings and hydrophobic regions of apoA-I. This creates conditions for deposition of RNA-polymerase onto DNA matrix and enhancement of gene expression. In a complex with apoA-I, such effect is observed with tetrahydroderivatives of steroid hormones, dehydroepiandrosterone, its sulfated form (dehydroepiandrosterone sulfate), pregnenolone, androsterone, and other hormones.
In the organism, the A-ring Δ4,3-ketogroup of steroid hormones is reduced in resident macrophages that have a high 5- and 5-reductase activity. The macrophages entrap steroid hormones in HDL. The entrapment occurs via receptor mediated endocytosis. In secondary lysosomes of the cells, lipoproteins are disintegrated, which is accompanied by apoA-I release and reduction of Δ4,3-ketogroup of steroid hormones. Both compounds form the biologically active complex, which is secreted to interstitial space due to exocytosis. Further the complex is transferred to the somatic cell nuclei, where it participates in the enhancement of gene expression. The mechanism revealed in our work is nonspecific. However, it becomes specific when occurring in the cells of target organs of the corresponding hormones, for example, in lymphocytes, reproductive organs, hormone-dependent neoplasms, etc.
The trypanicide Effects of Amiodarone and the Azoles: From the Skin to the Heartbeat of a Continent. PANIZ MONDOLFI AE 1,2, PÉREZ ÁLVAREZ AM 1, MÁRQUEZ E 3, CONCEPCIÓN JL 3 1 Laboratorio de Bioquímica, Instituto de Biomedicina, Caracas, Venezuela.
2 Hospital “José Gregorio Hernández”, Instituto Venezolano de los Seguros Sociales, Caracas, Venezuela.
3 Laboratorio de Enzimología de Parásitos, Facultad de Ciencias, Universidad de Los Andes, Mérida, Venezuela.
Background: Cutaneous leishmaniasis and Chagas’ disease are among the most prevalent endemic zoonosis in the Latin-American continent. The occurrence of mixed infections of Trypanosoma cruzi and Leishmania speciesis becoming a common feature in Central and South America due to overlapping endemic areas. Unfortunately, the possibilities for treating flagellated kinetoplastid infections are still very limited and most of the available drugs exhibit severe side effects. Although the development of new drugs against Leishmaniasis has markedly improved in the last years, the tendency is still to employ antimonial compounds. On the other hand, Chagas’ disease treatment is only available for the acute phase with still no effective therapeutic options for chronic stage disease.
Nowadays several classes of compounds targeting validated biochemical pathways of the parasites are available, some proved to be useful in animal models, others widely used in other human pathological conditions and others still under clinical trials.
The following work extensively documents the diagnosis, successful treatment (using non-conventional approved drugs), and follow up of two complicated clinical cases of these diseases. The objective of this work is to present to the scientific community these two magic bullets as an alternative therapeutic option to treat these neglected diseases.
Methods:Two case reports are described. The first patient is a case of concurrent borderline disseminated cutaneous leishmaniasis and Chagas’ disease, and the second a case of chronic Chagas’ cardiomyopathy. Patients were diagnosed and followed up using conventional diagnostic methods as well as novel highly specific and sensitive recombinant proteins (PGR31-HIS, PGR30-HIS, anti-rTc24) for T. cruzi. Both patients were initiated on amiodarone and itraconazole, which, despite their classic therapeutic use, also target specific metabolic aspects of the parasite.
Results and conclusions:Complete cure was achieved by using these drugs. In both cases patients were demonstrated to be clinically and serologically cured. As far all authors are concerned these are the first case reports which reveal amiodarone’s anti T. cruzi andanti-Leishmania effect in human subjects.
Receptor Protein Tyrosine Phosphatase Beta/Zeta as a Possible New Target to Regulate Endothelial and Tumor Cell Migration PAPADIMITRIOU E, MIKELIS C, KOUTSIOUMPA M, THEODOROPOULOU C
Laboratory of Molecular Pharmacology, Department of Pharmacy, University of Patras, 26504 Patras, Greece
Background: Receptor protein tyrosine phosphatase β/ζ is a member of the receptor protein tyrosine phosphatases, characterized by the presence of an N-terminal carbonic anydrase-like domain, a fibronectin type III domain and a serine, glycine-rich domain for chondroitin sulfate attachment in the extracellular region. It exists as four isoforms: long and short receptor forms and long and short secreted proteins containing the extracellular domains of the long and short receptor forms respectively. The cytoplasmic domain of RPTPβ/ζ contains two tandemly repeated phosphatase domains, of which only the membrane proximal is catalytically active, as well as a C-terminal PDZ-binding motif, through which it interacts with other PDZ domain-containing proteins. It is expressed in the nervous system and is involved in neuronal cell migration, differentiation, circuit formation and regulation of neuronal plasticity. RPTPβ/ζ is known to bind several extracellular matrix proteins and cell adhesion molecules and it is a receptor of the heparin-binding growth factors pleiotrophin and midkine. We have studied expression of RPTPβ/ζ in primary endothelial cells and several tumor cell lines, as well as its involvement in endothelial and tumor cell migration.
Methods: A combination of methods was used, including immunoprecipitation and western blot analysis, migration assays, siRNA, immunofluorescence and confocal microscopy.
Results: RPTPβ/ζ is expressed in endothelial, as well as diverse tumor cells and in all cases interacts with pleiotrophin and mediates its effect on cell migration. Interestingly, the effect of RPTPβ/ζ on cell migration can be modulated by its interaction with other cell surface molecules, such as integrins. Moreover, RPTPβ/ζ seems to mediate cell migration induced by diverse factors, such as hydrogen peroxide, nitric oxide, aprotinin and vascular endothelial growth factor, an effect that may or may not be dependent on pleiotrophin.
Conclusions: RPTPβ/ζ seems to be an interesting target in order to limit endothelial and tumor cell migration.
This research project is co-financed by E.U.-European Social Fund (75%) and the Greek Ministry of Development-GSRT (25%).
Neurosteroids in the treatment of neurodegeneration PAPADOPOULOS V, LECANU L
The Research Institute of the McGill University Health Centre and Department of Medicine, McGill University, 1650 Cedar Avenue, Montreal, Quebec, Canada.
Background: The brain has the ability to convert cholesterol to steroids, otherwise known as ‘neurosteroids’. Various studies using either pharmacological tools modulating endogenous neurosteroid levels and behavior or correlation of neurosteroid levels with neuropathology suggested that neurosteroids could be targeted to treat disturbances in the nervous system. Alzheimer’s disease (AD) is a yet incurable degenerative neurological illness characterized by memory loss. We reported that 22R-hydroxycholesterol (22ROHC), a steroid intermediate formed during the synthesis of pregnenolone from cholesterol, is present at lower levels in the hippocampus and frontal cortex of AD patients than in corresponding tissue from age-matched controls. 22ROHC was then found to protect against β-amyloid (Aβ42)-induced neurotoxicity. Aβ42 as well as the formation of Aβ oligomers and amyloid deposits has been linked to AD pathology.
Methods and Results: Because 22ROHC is a rapidly metabolized intermediate in pregnenolone biosynthesis, the stable naturally occurring heterospirostenol, (22R,25R)-20α-spirost-5-en-3β-yl hexanoate (caprospinol), was identified by in sislioc screening of commercial libraries as the lead substitute. Caprospinol was found to protect against Aβ42-induced neurotoxicity in vitro. This steroid binds to Aβ42, inhibits the formation of neurotoxic amyloid oligomers, prevents Aβ42 from reaching neuronal mitochondria, and protects mitochondrial function against direct insults. To investigate the in vivo efficacy of this compound we used a rat model of AD, which recapitulates the histopathological and cognitive phenotype of AD. Caprospinol treatment of diseased rats restored spatial memory, as assessed using Morris water maze tests. This recovery of cognitive function was accompanied by a reduction in hippocampal amyloid deposits and neurodegeneration. In parallel studies caprospinol administration resulted in an important accumulation of the compound at the forebrain demonstrating its ability to cross the blood-brain barrier. Caprospinol does not bind to any known steroid hormone receptors and is devoid of acute and 3-month toxicity in rodents. Conclusions: These results position caprospinol as a promising drug candidate for AD treatment.
This work was funded by Samaritan Therapeutics (Quebec, Canada)
Capecitabine-associated coronary vasospasm: a case report PAPADOPOULOS CA, WILSON H.
Emergency Assessment Unit Medicine, Norfolk and Norwich University Hospital, Colney Lane, Norwich, England
Capecitabine (Xeloda) is an oral 5-Fluorouracil pro-drug used in the treatment of two of the commonest cancers: breast and colorectal. Here, we report a 43 year old woman with metastatic cancer of the sigmoid colon who developed cardiac chest pain 5 days after starting capecitabine therapy. Capecitabine-induced cardiac symptoms have previously been reported, but infrequently. In the main they have documented pain and ECG changes associated with exercise. This case report is of a patient with minimal cardiac risk factors, who suffered ischaemic cardiac pain with widespread ECG changes at rest which resolved with a nitrate infusion. We propose coronary vasospasm as the probable mechanism for the cardiac ischaemia and dramatic ECG changes. Capecitabine is now in widespread use and so physicians will encounter an increasing number of patients using this therapy. In light of this, we think it important that doctors in Emergency and Acute Medicine are aware of its treatable cardiac side-effects.
Isolation, purification, partial characterization, biochemical properties and stability of two novel antimicrobial peptides produced by Pediococcus strains
PAPAGIANNI M
Department of Hygiene and Technology of Food of Animal Origin, School of Veterinary Medicine, Aristotle University of Thessaloniki
Background: Fermentation broths (in MRS medium) of Pediococcus acidilactici NRRL B5627 and P. pentosaceus SM-1, were found to exhibit strong antimicrobial activity against a wide range of food spoilage and food born pathogen bacteria, including Clostridium and Listeria spp. The antimicrobial factor in each case was identified to be a peptide.
Methods: The broths were subjected to repeated tricine-SDS-polyacrylamide gel electrophoresis until the antimicrobial factor was identified by overlaying the gel with nutrient agar in which the indicator microorganism Micrococcus luteus was embedded. Following gel excising and destaining, the proteins of interest were eluted directly into an aqueous solution of formic acid/water/2-propanol (1:3:2/v). Molecular mass determination by electrospray ionization mass spectrometric analysis (ESI-MS) revealed a 3.660 Da peptide in the case of P. acidilactici, while a 5.370 Da peptide in the case of P. pentosaceus.
Results: N-terminal sequencing determined by Edman degradation in both cases showed that the peptides have 19 amino acid residues and the consensus sequence of -YGNGV- near the N-terminal. The last characteristic classifies the peptides to the class IIa of bacteriocins, known as pediocins. The names of pediocin SA-1 from P. acidilactici and pediocin SM-1 from P. pentosaceus were assigned to the isolated peptides. The purified pediocins were examined for their sensitivity to proteolytic enzymes and for their stability to cold, heat and pH treatments (the parameters ranging widely). The most remarkable characteristics were the stability to heat treatment -both peptides were heat stable for up to 60 min at 121oC and to cold storage- peptides remained stable for one year at -80 oC and -20 oC. Pediocin SA-1 remained active after treatment with trypsin, -chymotrypsin, pepsin and papain, but lost its activity with proteinase K, while pediocin SM-1 lost its activity in most cases. The mode of action of both pediocins was found to be bactericidal. Fermentation kinetics studies in bioreactors revealed the primary metabolite nature of the peptides.
Conclusions: Antimicrobial peptides produced by lactic acid bacteria are believed to be a potential answer to the growing problem of resistance to conventional antibiotics. The fact that the two isolated pediocins are produced by well-known food grade bacteria, makes them important candidates for use as biopreservatives. The fact however, that the producer bacteria belong to the human intestinal lactic acid bacteria, makes them attractive targets for further research with the aim of the production of novel antimicrobial drugs or innovative drug delivery systems.
A Novel, Safe, and Effective Clinical Treatment to Eliminate Resistance in Ectoparasites MEINKING T, VILLAR ME, RIVERA H, PAQUET D, EYERDAM D, VICARIA M
Institution: Global Health Associates of Miami, 7800 SW 57th Ave Suite 219, 33143 Miami FL, USA
Background: Head lice have been on the increase for over a decade, in large part due to the fact that they have become resistant to conventional treatment. Head lice (Pediculus capitis) feed every 4 to 6 hours on human blood by attaching to the scalp .The adult female louse usually lays eggs (nits) near the scalp and cements them to a hair shaft. If they are not killed or
removed, nits hatch in 7-10 days and propagate the infestation. We are studying a botanical derived product (BGC20-582 Lice Treatment Gel) that kills both lice and their eggs.
Methods: Pediculicidal activity (in vitro) was evaluated for BGC20-0582 in concentrations of 0% (vehicle), 2.5%, 7.5%, 10.0% and 12.5%. NIX®, an FDA approved 1% permethrin product, was used as an active control and water as a negative control. Five centimeters disks were cut from 100% cotton towels and placed in the bottom of 15 X 60 mm Petri dishes. Each formulation was measured at 0.7cc and spread across the bottom of the disk. Adults lice and nymphs, were distributed evenly between test samples and controls. Pediculicidal activity was observed over an exposure period of 5 minutes to 1 hour for each treatment. Ovicidal activity (in vitro) was evaluated with the same concentrations and controls. Groups of 10 hairs, each with a viable egg, were cut to 2-cm long strands and grouped together at one end with small adhesive labels. Each group was immersed in a test product for 30 minutes, and then rinsed with purified water and air dried prior to being transferred into 1 dram sterile vials and labeled accordingly. The eggs were placed in an incubator for two weeks with 80 degree F. temperature and relative humidity of 70%-80%. Ovicidal activity was determined by observing the number of eggs failing to hatch following the 14 day incubation period.
Results: Based on preclinical study results it is proposed that the required concentration and exposure time for BGC20-0582 to achieve maximal pediculicidal and ovicidal activity in vitro is treatment with a concentration of 10% for a period of 30 minutes.
Conclusion: We have just concluded a 4 arm, randomized double blinded-placebo controlled dose ranging study on approximately 230 subjects between 6 months of age and 70 years. This population is known to have permethrin and pyrethrin resistant head lice. The results are being analyzed and will be available shortly.
Erythropoiesis-stimulating agents may improve survival in low-risk MDS patients PARK S, KELAIDI C, GREENBERG P, FENAUX P, DREYFUS F.
For the GFM group (Groupe Francophone des Myélodysplasies). Hôpital Cochin, Paris, FRANCE.
Background: Correction of anemia is generally the primary therapeutic goal in low risk MDS. EPO and its derivative darbepoietin alfa (DAR) are important treatments of anemia in low- risk MDS. The objectives of the present study were (i) to confirm prognostic factors of response and duration of response to rEPO+/- G-CSF in 403 MDS patients from the GFM (ii) to compare outcome of our cohort (in terms of progression to AML and survival) to that of untreated patients included in the International MDS risk Analysis Workshop( IMRAW) database (P.Greenberg) that was used to establish IPSS.
Methods: 403 patients with MDS according to FAB criteria from 25 French and Belgian hematological centers of the GFM who had received rEPO (ie epoetin alfa or beta, or Darbepoietin DAR) +/-G-CSF treatment at weekly doses of 60000 U for epoetin and 300µg for DAR during at least 12 weeks between 1998 and May 2006 were included in the study. Main inclusion criteria were (i)IPSS Low and Int-1 MDS (ii) Hb<10g/dl or need for more than 2 red blood cell units of transfusions during the two months preceding the date of inclusion, (iii) serum EPO level<500mUI/ml (iv) de novo MDS, excluding therapy relatedcases.
Results: 62% (43% major and 20% minor) erythroid responses were seen, and median response duration was 20 months according to IWG 2000 criteria. Significantly higher response rates were observed with <10% blasts, Low and Int-1 IPSS, RBC transfusion independence, serum EPO level<200UI/l. Significantly longer response duration was associated with major response (IWG 2000 criteria), IPSS low-INT-1, blasts<5% and absence of multilineage dysplasia. Multivariate adjusted comparisons of survival between our cohort and the untreated MDS cohort used to design IPSS, showed similar rate of progression to AML in both cohorts, but significantly better overall survival in our cohort with five year- overall survival (OS) of 64% in the French-EPO cohort and of 39% in the IMRAW cohort. This survival benefit, in the French cohort, was restricted to patients who responded to rEPO. In multivariate analysis, rEPO treatment was independently associated with better survival.
Conclusions: Major prognostic factors of response to EPO±G were confirmed. Multilineage dysplasia was not associated with lower response rates, but with shorter response duration. Epoetin or DAR treatment may have a favourable survival impact in low-risk MDS.
Glial Cell Line-derived Neurotrophic Factor Family Artemin-Transcriptional Regulation, Neurite Outgrowth and Actin Polymerization in Mature Dorsal Root Ganglia Neurons PARKS, JEONG DG
Dongduk Women’s University, Seoul, Korea.
Background: Neurotrophic factors are signaling molecules that regulate multiple aspects of the development of the nervous systems. The glial cell line-derived neurotrophic factor (GDNF) family includes artemin, persephin, and neurturin. GDNF was found to reverse the posttraumatic changes into nerve regeneration and neuropathic pain treatment. Artemin expression is observed in the adult brain and spinal cord, suggesting that artemin may play a role in the post-developmental stage. We performed a microarray analysis and real-time PCR experiment to investigate the patterns of gene expression underlying the effect of artemin on the mature DRG.
Methods: An in vitro isolated DRG model was used to study the effects of artemin on the adult rat neuronal system and investigate differentially regulated genes. For the DRG neuron culture, the ganglia were trypsinized. Total RNA was extracted and reverse-transcribed to double-stranded cDNA using an oligo-dT primer. The cDNA microarray containing a set of 5088 rat cDNA was used. The cultures were stained for vesicle endocytosis with FM 1-43 and for F-actin with phalloidin–Alexa568.
Results: 285 genes were differentially transcribed by artemin after 3 hour of treatment. A series of genes involved in the regulation of actin dynamics, including coronin, Myr 5, Wiskott-Aldrich syndrome protein interacting protein, cofilin, drebrin and dynamin were down-regulated by artemin, suggesting a previously undefined role in the regulation of actin polymerization and synaptic vesicle movement. Artemin also down-regulated the expression of genes related to cell adhesion and matrix assembly, including biglycan, plectin, nestin, neuronatin and the neuron-glia-CAM-related cell adhesion molecule, which is functionally relevant to neurite elongation in DRG neurons. Artemin resulted in increases in total neurite length and branching of the DRG neurons. Also artemin caused an increase of synaptic vesicle clustering. The inhibition of DNA methylation suppressed the artemin-dependent neurite growth.
Conclusions: 1) The mature DRG neurons showed some response to artemin, suggesting that they exhibited a developmental shift to the ligand as prenatal DRG neurons. 2) DNA methylation seems to provide a mechanism for artemin-dependent genetic regulation responsible for axonal growth.
