NanoBiotechnology and Cancer Research
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irk-123456789-1568252019-06-20T01:26:21Z NanoBiotechnology and Cancer Research Abstracts of RECOOP HST Research Networks’ Invited Speakers 2012 Article NanoBiotechnology and Cancer Research // Вiopolymers and Cell. — 2012. — Т. 28, № 2, доп. — С. 48-55. — англ. 0233-7657 http://dspace.nbuv.gov.ua/handle/123456789/156825 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Abstracts of RECOOP HST Research Networks’ Invited Speakers Abstracts of RECOOP HST Research Networks’ Invited Speakers |
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Abstracts of RECOOP HST Research Networks’ Invited Speakers Abstracts of RECOOP HST Research Networks’ Invited Speakers NanoBiotechnology and Cancer Research Вiopolymers and Cell |
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NanoBiotechnology and Cancer Research |
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NanoBiotechnology and Cancer Research |
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NanoBiotechnology and Cancer Research |
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Інститут молекулярної біології і генетики НАН України |
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Abstracts of RECOOP HST Research Networks’ Invited Speakers |
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NanoBiotechnology and Cancer
Research // Вiopolymers and Cell. — 2012. — Т. 28, № 2, доп. — С. 48-55. — англ. |
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Вiopolymers and Cell |
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48
NanoBiotechnology and Cancer
Research
49
Paracrine interaction drives EMT and lethal progression of prostate
cancer: from biology to therapy
Chung Leland W. K., Josson Sajni and Zhau Haiyen E.
Uro-Oncology Research, Departments of Medicine and Surgery, Samuel Oschin Comprehensive
Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA 90048
Leland.Chung@cshs.org
Prostate cancer progression toward the development of lethal bone and soft tissue
metastases are major cause of morbidity and mortality in patients. Based on the
characterization of a series of isogenic human prostate cancer cell lines recapitulating the
lethal progression of human prostate cancer metastases, we found prostate cancer cells
with bone metastatic potential, expressed bone-like proteins (osteomimicry), including
osteocalcin, osteopontin, bone sialoprotein, osteonectin, receptor activator of NF-B
(RANK), RANK ligand (RANKL), and/or osteoprotegerin (OPG).
2-Microglobulin (2-M), a light chain of MHC class 1 molecule, was identified as a
host factor from tumor microenvironment, driving Epithelial to Mesenchymal Transition
(EMT), osteomimicry, lethal bone and soft tissue metastases. A novel 2-M receptor, the
hereditary hemochromatosis gene HFE (a MHC-like protein), is responsible for the
regulation of intracellular iron through binding to transferrin and transferrin receptor, and
regulating the status of oxidative stress of cancer cells. Anti-2-M monoclonal antibody
(anti-2-M Ab) was found to target effectively EMT and cancer bone colonization in
mice.
RANKL, another target gene of 2-M, was shown to participate in osteoclastogenesis
associated with cancer bone metastases. RANKL expressed by metastatic castration
resistant prostate cancer (CRPC) cells and tissues participated directly in prostate cancer
bone metastases. These data are consistent with the clinical results where targeting
RANKL-RANK axis with bisphosphonates (Zoledronic Acid) or Denosumab (a RANKL
monoclonal antibody) reduced bone pain and skeletal related events in CRPC patients
treated with androgen deprivation therapy.
New approaches targeting osteomimicry and the converging signaling between
RANKL/RANK and HGF/SF/c-MET will be described from the perspective of
developing novel therapies for the control of bone and soft tissue metastasis and the
lethal progression of prostate cancer in patients (supported by NCI PO-1 and RO-1 grants
and PCF Challenge Award.
Keywords: prostate cancer, control of bone and soft tissue metastasis
50
NanoBioTech Studies at ICB (UA): RECOOP collaborative
potentials
Stoika R. S.
Institute of Cell Biology, NAS of Ukraine
14/16, Drahomanov Str. Lviv, Ukraine, 79005
stoika@cellbiol.lviv.ua
Aim: The following applied research approaches are used at ICB: 1) development of
novel efficient bio-targeting carriers for drug and gene (siRNA) delivery; 2) development
of novel carriers for delivery of water-insoluble drugs; 3) application of novel labeling
materials for visualization of drug delivery, action, and clearance in the organism; 4)
development of nano(micro)particles for cell isolation and separation.
Methods: Novel polymeric nanocomposites (1) and superparamagnetic nanoparticles (2)
were synthesized at Lviv National Polytechnic University (1), Kyiv National University
(1), and Institute of Macromolecular Chemistry (IMC, Prague, Czech Republic) (2).
Those materials were functionalized for providing them bio-targeting characteristics.
Flow cytometry, light and fluorescent microscopy, Western-blot analysis of expression of
proapoptotic proteins, in vivo toxicity studies in mice, and chemotherapy treatment of
tumor-bearing mice were applied at characterization of the developed drug carriers.
Specific approaches for detection, selection and addressed targeting the apoptotic cells
have been also developed.
Results: To address the NanoBioTech research tasks within the RECOOP Association,
superparamagnetic nanoparticles were synthesized at IMC, functionalized at ICB, and
used to monitor phagocytic activity of macrophages. Besides, similar particles were bio-
functionalized by specific lectins, and used to isolate different murine lymphoma cells.
Polymeric nanoparticles were designed and decorated with specific lectins for addressed
targeting the apoptotic cells. Effective gene delivery to microorganisms and mammalian
cells was achieved by the developed polymeric nanocomposites. They showed low
toxicity and no mutagenicity.
Conclusions: ICB research team is ready to discuss common design of novel
nanocomposites, their bio-functionalization, and application for achieving different joint
research tasks within collaborative biomedical projects of the RECOOP Association.
Keywords: nanocarriers, drug delivery, gene delivery, treatment, diagnostics, apoptosis.
51
Designing, synthesis, structural and optical properties of ultrasmall
inorganic markers doped with lanthanide ions for bio-medical
applications
Podhorodecki A., Bański M., Misiewicz J., Noculak A., Sojka B.
Institute of Physics, Wrocław University of Technology,
Wybrzeże Wyspiańskiego 27, 50-370 Wrocław
artur.p.podhorodecki@pwr.wroc.pl;
Aim: Introducing to medicine and biology concept of optical markers in tremendous way
has changed the recent status of these two important disciplines. This was mainly due to
strong development in imaging techniques which recently allow us to investigate both
static as well dynamic properties of living cells, their components and their interactions
with external factors.
Method and results: Recently used molecular markers including organic dyes,
fluorescent proteins or chelates containing lanthanide ions have several significant
limitations. One of the alternatives for molecular markers are inorganic quantum dots (ie.
CdSe, CdS) which are commonly used in many academic works. However, even if they
are much better from physico-chemical point of view, from the application point of view
at this moment they are rather useless mainly because of their high risk of toxicity. One
of the solution combining advantages of both concepts is to make nontoxic inorganic
nanocrystals doped by lanthanide ions.
Conclusion: In this work, we will present optical results obtained for NaYF4, NaGdF4
and GdOF nanocrystals doped by different lanthanide ions (Eu, Tb, Nd). The aim of this
work was to design and to synthesize these markers and to understand physical processes
responsible for their emission/excitation and to optimize these processes to the physical
limits.
Keywords: nanocrystal, rare earth ions, GdOF
52
Nanoparticles for monitoring differentiated stem cells
Horák D., 1Jendelová P., Babič M., 1Vaněček V.
Institute of Macromolecular Chemistry,
Heyrovského 2, 16206 Prague 6, Czech Republic
1 Institute of Experimental Medicine,
Vídeňská 1083, 14220 Prague 4, Czech Republic
horak@imc.cas.cz
Aim: It is important in regenerative medicine to noninvasively track stem cells to
evaluate their therapeutic effect and grafting location to rule out side effects. Magnetic
nanoparticles are therefore being developed as labels visualized by MRI in in vivo cell
tracking. Before evaluating the clinical potential of nanoparticles in cell transplantation,
the effect of these particles on cultured cells should be assessed. As a model, we chose
human mesenchymal stem cells (hMSCs) as they are widely used in the regeneration of
connective tissues (bone, cartilage and fat). We studied the basic properties of hMSCs
labeled with superparamagnetic nanoparticles: labeling efficiency, cell growth,
proliferation, migration and differentiation.
Methods: hMSCs were labeled with neat or poly (L-lysine) (PLL)-coated
superparamagnetic γ-Fe2O3 nanoparticles prepared by an original procedure. Labeling
efficiency, cell proliferation and migration were determined by counting the number of
Prussian Blue-stained and unstained cells using a cell analyzer. Cell migration was
studied using SDF1 factor as a chemoatractant. The osteogenic, chondrogenic and
adipogenic differentiation potential of nanoparticle-labeled MSCs was also determined.
Results: The efficiency of cell labeling for PLL-coated and uncoated γ-Fe2O3 was 91.5 %
and 45 %, respectively. The magnetic label slightly reduced cell proliferation during the
first 10 h; however, the final PLL-γ-Fe2O3-labeled cell index was high after 24 h.
Compared to unlabeled cells, cell migration towards the chemoattractant was not
affected. No difference was found in the gene expression of LPL and PPARG adipogenic
markers, while osteogenic gene expression (ALPL and RunX2) slightly decreased.
However, histological examination revealed that osteogenic (Alizarin Red), chondrogenic
(Alcian Blue) and adipogenic (Red Oil O) differentiation were not affected.
Conclusions: Even a highly efficient cell label, PLL-γ-Fe2O3, does not significantly
affect basic stem cell properties and therefore can be considered as a suitable contrast
agent for in vivo monitoring of transplanted stem cells in different applications, such as
the transplantation of MSCs into cartilage or bone defects.
Keywords: magnetic, stem cells, labeling
Supported by KAN401220801 and P304/12/1370
53
Immobilization of recombinant human arginase I onto gold and
silver nanoparticles and their potential use
1,2Gonchar M., 3Stasyuk N., 1Gayda G.
1Institute of Cell Biology, NAS of Ukraine,
Drahomanov Str. 14/16, 79005 Lviv, Ukraine;
2University of Rzeszow, Rzeszow, Poland;
3Ivan Franko National University of Lviv,
Kyryla and Mefodia Str. 6, 79005 Lviv, Ukraine
gonchar@cellbiol.lviv.ua
Aim: Metal nanoparticles (NPs), such as gold (Au) and silver (Ag), have recognized
importance in chemistry, physics, and biology due to their unique optical, electrical, and
photothermal properties. AuNPs have photothermal properties that can be exploited for
localized heating which results in drug release, thus increasing their potential for
therapeutic applications of some malignant diseases (melanoma, hepatocarcinoma).
The aim of the present study was to immobilize the recombinant human arginase I on
AuNPs and AgNPs and to characterize the obtained enzyme-NPs: analyses of their size,
structure, enzymatic activity and stability.
Methods: The size and structure of the AgNPs and AuNPs were characterized using
TEM, AFM and XRD analyses.
Results: The synthesis of silver and gold nanoparticles was carried out by reduction of
silver nitrate by glucose and reduction of tetrachloroauric acid by sodium citrate,
respectively. Recombinant arginase I was immobilized using the carbodiimide method on
the surface of NPs functionalized with ω-mercaptohexadecanoic acid. Recombinant
human arginase I was successfully linked on both NPs with a binding efficiency of 85%
for AgNPs and 87% for AuNPs (in the range of added enzyme concentration 0.15-0.5
mg/mL). The nanocomposite-bound arginase was used to construct potentiometric and
amperometric biosensors for monitoring of arginine level during arginase-based
enzymotherapy of some cancers.
Conclusions: Thus, the synthesized gold and silver nano-carriers have a stabilizing effect
on recombinant human arginase I due to its fixation on the surface of the NPs, preventing
enzyme inactivation. So, the immobilization procedures open extensive possibilities for
the construction of very sensitive and stable biosensors for wide biochemical
applications. A high thermostability of immobilized on NPs arginase I preparations and a
wide working pH range of this enzyme is predicted to be a perspective tool for
bioanalytical purposes, namely, for arginine monitoring in pharmaceuticals, in food
products and in blood.
Keywords: silver and gold nanoparticles, covalent enzyme immobilization.
54
Multispectral quantum dot labeling detects elevated C-MET cell
signaling mediators in castration resistant and invasive human
prostate cancers
Zhau H. E., Hu P., Liu C., Wang R., 1Wang Y., and Chung L. W. K.
Uro-Oncology Research Program, Department of Medicine, Samuel Oschin Comprehensive Cancer
Institute, Cedars-Sinai Medical Center; Los Angeles, CA, USA 90048;
1BC Cancer Agency, University of British Columbia, 5Molecular Pathology Lab of the Vancouver
Prostate Center at Vancouver General Hospital, Vancouver, B.C., Canada V6H 3Z6
Haiyen.Zhau@cshs.org
Aim: To test the hypothesis that activation of c-MET-mediated signaling in prostate
cancer cells supports cancer cell growth and survival and promotes progression and
metastasis. We focused on the simultaneous detection and quantification of 5 proteins in
the c-MET activation pathway, receptor activator of NF-κB ligand (RANKL), VEGF,
NRPLN-1, p-c-MET and p-p65 (NF-κB) and epithelial to mesenchymal transition (EMT)
known to be associated with human prostate cancer progression and bone metastasis.
Methods: We established a novel technology of multiplexed quantum dot labeling,
MQDL, to detect the expression and activation of multiple c-MET pathway and EMT
mediators in tissue specimens and subject to signal intensity quantification. Three
experimental systems were used: 1) human prostate cancer cell model exhibiting
activated c-Met signaling and bone metastasis; 2) xenograft tissues from an established
LTL313 castration-resistant human prostate cancer (CRPC) model; and 3) clinical
prostate cancer tissue specimens. Nuance software was used for image capturing,
unmixing and inForm software for per cell base intensity quantification of the five
prostate cancer progression associated proteins in the c-MET activation pathway,
RANKL, VEGF, NRPLN-1, p-c-MET and p-p65 (NF-κB) and EMT biomarkers
EpCAM, N-cadherin and RANKL.
Results: Cell based MQDL quantification data showed that the c-MET activation
mediators, RANKL, VEGF, NRPLN-1, p-c-MET and p-p65 (NF-κB) and mesenchymal
biomarkers, N-cadherin and RANKL were all elevated in CRPC human prostate mouse
xenograft model and in the invasive human prostate cancer with statistical significance.
Results were confirmed by real-time RT-PCR and western blots in a metastatic human
prostate cancer cell model.
Conclusions: Our results showed that activation of c-MET-mediated signaling occurs in
prostate cancer cells through increased phosphorylated c-MET in all three tested systems.
The downstream survival signaling network was mediated by NF-κB and EMT driven by
RANKL, in clinical prostate cancer specimens and the xenograft model. MQDL is a
powerful tool for assessing biomarker expression and it offers molecular insights into
cancer progression at both the cell and tissue level with high degree of sensitivity.
Keywords: multiplexed quantum dot labeling, c-MET mediators, castration-resistant
prostate cancer, epithelial to mesencymal transition, metastasis
55
The role of epigenetic regulation in cervical carcinogenesis
Kónya J.
Dept. Med. Microbiol, University Debrecen,
Nagyerdei krt 98 Debrecen 4032 Hungary
konya@med.unideb.hu
High-risk or oncogenic human papillomaviruses (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52,
56, etc) are causally linked to the development of cervical cancer. The E6 and E7
oncoproteins of high-risk HPVs are responsible for the transforming activity of the virus.
The virus genome is usually found in an episomal form in benign and premalignant
lesions, while it is frequently integrated in the host cell genome in malignant cancers. The
E6 and E7 oncogenes are always retained and expressed in the transformed cells.
During cervical carcinogenesis, genetic and epigenetic alterations disrupting the cell
cycle control are needed to acquire immortal phenotype and to progress further to overt
malignant and invasive phenotype. The stepwise accumulation of these alterations is
manifested in the well-defined clinical stages.
Epigenetic alterations include those heritable (covalent) modifications of DNA and
histone proteins that do not result in changes of the genomic DNA sequence. In
vertebrates, CpG methylation of DNA acts in context with post-translationally modified
histones and other chromatin-remodelling factors to establish the characteristic gene
expression profile of individual somatic tissues (2). In cancer, the aberrant functions of
chromatin modifier enzymes result in altered DNA methylation patterns and histone
modifications and the epigenetic alterations involved in uncontrolled cellular
proliferation and viral expression during the multistep process of cervical carcinogenesis.
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