Proposta di ricerca:
1.
State of the art
Anthracyclines were the first extremely powerful and highly versatile anticancer agents discovered
by Farmitalia Research Laboratories in the 1950’s. These “old” molecules today are still the most
effective wide spectrum antineoplastic antibiotics employed in chemotherapy. They also represent
the most prominent example when Italian pharmaceutical research has made a significant
improvement in worldwide therapeutic practice. Although active against a wide variety of solid
tumours and haematological malignancies, clinical use of anthracyclines is hindered by tumour
resistance and toxicity (e.g. cardiotoxicity) to healthy tissue. Recent progress in the conjugation of
anthracyclines with different types of carriers holds the promise to decrease the side-effects and to
improve selectivity.(1) Success of the design of next generation anticancer drug however highly
depends on our current knowledge of the mechanisms responsible for the cellular uptake and the
cytotoxic and apoptotic effects.
A recent high-throughput proteomic work analyzed the changes occurs in the entire proteome level
of HepG2 upon doxorubicin treatment.(2) The action mechanism proposed to be targeted to the
nucleus, mitochondria and cell membrane reorganization. This is in agreement with our preliminary
quantitative proteomic data obtained on the HL-60 model system by iTRAQ assay and MuDPIT
analysis.(3) The expression level of 21 proteins was shown to change significantly upon
daunomycin treatment at IC25 value. In particular, nucleolin and nucleophosmin abundant p53
binding proteins - known to be over-expressed in malignant tumors and relapsed/refractory acute
leukemia - were found to be significantly down-regulated. We found that protein DJ-1, a
multifunctional protein that has been implicated in tumor pathogenesis and up-regulated in acute
leukemia was also down-regulated. While the analyses of the entire cell proteome give very
valuable elements for the better understanding of drug mechanism of action in general, it gives only
limited information on the mechanisms of drug-conjugate delivery. Therefore, here we put forward
a proteomic study which specifically targets three compartments: the cell-surface, extracellular and
nuclear sub-proteomes. This shall provide us a higher resolution picture on the changes in the
expression levels of those proteins which are directly involved in the drug-uptake and apoptotic
action with the final aim to aid the design of new efficient drug carrier bioconjugates.
2.
Motivation for co-operation, expertise and resource
The two research groups, the Mass Spectrometry and Proteomics Laboratory at the Institute of
Protein Biochemistry (IBP) of CNR in Naples (Italy) and the Research Group of Peptide Chemistry,
Hungarian Academy of Sciences (HAS) at Eötvös Loránd University (RGPC) in Budapest
(Hungary) are working in complementary research fields and have a long standing collaboration.
Both groups interested in i) protein/peptide structural analysis, ii) quantitative analysis of protein
expression level, iii) characterization of changes in protein posttranslational modifications and iv.)
rational design of polypeptide/protein carriers. We have participated with success in the
“Programma triennale 2010/2012” financed by CNR/MTA agreement with joint project entitled
“Quantitative proteomics for the identification of differentially expressed proteins in cells treated
with daunomycin bioconjugates”. This gave us the opportunity to exchange researchers and to train
Ph.D. students through active participation in the project. In addition, this project led to interesting
scientific results in the field of proposed research (see final report). The researchers at IBP-CNR
involved in the proposal have a strong background in the application of mass spectrometry in
proteomics and biochemistry and has a well-equipped laboratory for nano-scale proteomic studies.
The group recently published studies on the characterization of transport mechanisms through
proteomic analysis of cell surface and extracellular proteins.(4-5) In addition, recently a patent
application has been made for the isolation of extracellular vesicles and their use in clinical
biomarker discovery.(6-7) The group has also experience in the characterization of phosphorylation
status of proteins. Recently they developed a selective phosphopeptide enrichment method
streamline applicable to shotgun proteomics.(8) The Research Group of Peptide Chemistry, HAS at
Budapest, on the other hand, has an outstanding international reputation in polypeptide synthesis, in
the preparation and characterization of peptide bioconjugates (9-11) and in peptide and protein
chemistry (12) in general. The RGPC has been updated by cell culture and flow cytometry
laboratories mandatory for the present proposal. In addition, the RGPC has been collaborating with
Dr. Pocsfalvi since a long time. This collaborative research has resulted in 11 joint publications and
16 communications at international conferences. In particular Dr. Schlosser visited several times the
mass spectrometry laboratory at ISA-CNR in Italy and her research activity was financed by
different agencies. During her visits in Italy Dr. Schlosser had the opportunity to learn and practice
principal and sophisticated proteomic methods and to apply it to a number of collaborative scientific
projects.
Available resources: Proteomics related scientific instruments including a two dimensional nano
HPLC system (LCPackings) and a triple quadrupole time of flight mass spectrometer equipped with
a nano-ESI source (QStar-Elite), a surface enhanced laser desorption (SELDI)-TOFMS, HPLC
systems and robotics are available at IBP-CNR.
All the necessary equipment for cellular biology including flow cytometry as well as laboratories
and equipment for synthetic chemistry are available at ELTE-MTA.
Human resources: In this proposal we put together two research teams including researchers and
students at different levels of their carriers with high level of interdisciplinarity and complementary
research interests. Grants of Ph.D. students have been obtained separately but their activity will be
complementary and compatible with their Ph.D. works.
3.
Workplan
During the three year period, research activities will be organized in the following stages:
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4.
1st YEAR: Systematic characterization of the changes in the cell NUCLEAR PROTEOME
upon treatment with different daunomycin (Dau)-peptide conjugates.
2nd YEAR: Changes in the cellular SECRETOME to target cellular trafficking of Daupeptide conjugate treated HL-60 cells.
3rd YEAR: Study the influence of different CARRIER MOLECULES on the nuclear
proteome, cellular trafficking, and apoptosis in HL-60 cells treated by Daunomycin
conjugated to different polypeptide/protein carriers.
Chemical, biomolecular and analytical methodologies:
First, the drug-peptide bioconjugates will be prepared. HL-60 human leukemia cell line will be used
as a model system because daunomycin is widely used in the treatment of leukemia. Time- and
concentration-dependency of cellular uptake will be determined by flow cytometry to assign the
appropriate treatment time and concentration, which can evoke changes in protein expression
profile. This part of the work will be performed at RGPC in Budapest.
Cellular sub-proteome fractions (membrane surface, nuclear and extracellular) will be prepared and
analyzed by proteomics using two control samples (untreated and Dau-treated cells). Protein
expression profiles of control samples will be compared with that of Dau-peptide conjugates treated
cells. In-solution proteomics will be applied using MudPIT (multidimensional protein identification
technology) with iTRAQ (isobaric tag for relative and absolute quantitation) labeling. In this
workflow, protein samples will be first enzymatically digested and then labeled. After iTRAQ
labeling samples are pooled and fractioned by two-dimensional nano HPLC using SCX separation
in the first and reverse phase C18 chromatography in the second dimension. Separated peptides then
will be on-line analyzed by nano-ESI tandem mass spectrometry (MS/MS) which facilitate
multiplex quantitative analysis of the four samples. Low molecular mass reporter ions are used to
relatively quantify the peptides and the proteins from which they originate. This part of the work
will be performed at IBP-CNR (Naples).
5.
Expected results and benefits
The main expected scientific outcome will be the lists of differentially expressed proteins and
phosphoproteins upon treatment using the different drug-bioconjugates as new vehicles to
anticancer drug targeting. The rationalization of nuclear, membrane and secreted protein expression
profiles will help to better understand apoptotic action, cellular uptake and transport mechanisms.
This information is mandatory to improve rational design of drug delivery system which is the
ultimate goal of this project. The project by collaborating with other national and international
research groups, clinics and SMEs will also serves to put together new ideas and to find new ways
to continue our collaboration in the field. We also expect to develop new methods using
nanomaterials for phosphoprotein characterization. The results will be published in international
journals and newly developed methods (if any) will be patented and applied to solve biochemical
and medical problems.
The main expected benefit is that both research groups can use the different instrumentations and to
learn different approaches from each other’s experience. Both groups can profit from joint results
especially in the fields of solid phase affinity chemistry, nano-separation, proteomics and drug
carrier bioconjugate design and synthesis. The Hungarian researchers could use HT proteomic
instrumentation not yet available in Hungary. Young Hungarian Ph.D. students will be trained for
the use of modern mass spectrometric techniques and proteomics not yet available for them. The
Italian researchers can learn peptide and bioconjugation chemistry, cell culture, sample preparation
methods and techniques to study the surface chemistry process developed in Budapest. They also
can use the advanced online library search facility during their visits at the RGPC. The scientific
results will be published in international journals and newly developed methods will be applied to
solve biochemical and medical problems.
Additionally, we aim to organize a consortium to promote EU-FP7 project proposal including the
participants of the present application.
References: 1. Miklán, Zs. et la. (2009) Cells, Pep. Sci. 92, 489-501.; 2. Hammer, E. et al. (2010)
Proteomics, 10, 990-114; 3. Schlosser, G. et a. (2012) (in preparation); 4. Palmieri, G. (2009) Mol
Cell Proteomics, 8, 2570-2581; 5. Pocsfalvi, G. (2011) et al. Journal of Proteome Research 10,
5326-5337; 6. Pocsfalvi, G., et al., (2011) Ufficio Italiano Brevetti e Marchi, UIBM, Italy; 7. Raj,
D.A.A., et al (2012) 81, 1263–1272; 8. Pocsfalvi, G. (2009 )In Methods in Enzymology, 457, 8196; 9. Szabó, I. et al (2009) Bioconjug. Chem. 20, 656-65; 10. Bánóczi, Z., et al. (2010) Bioconjug.
Chem. 2, 1948-1955; 11. Miklán, Zs. et al. J. (2011) Peptide Science, 17, 805-811; 12. TQke, O., et
al. (2011) Eur. Biophys. J., 40, 447-462.
Obiettivi:
The principal aim during the three-year collaborative research is to characterize the changes in
specific cellular sub-proteomes induced by treatment with three different daunomycin-containing
bioconjugates using a quantitative proteomic approach. The existing long-standing collaboration
between the two groups makes feasible to achieve this ambitious goal. The peptide bioconjugates
will be prepared and characterized by the RGPC group in Budapest because they have a strong
expertise in this field. Considering their different uptake properties three candidate carrier peptides
have been chosen for this work: 1. oligoarginine which enters the cell mainly by macropinocytosis,
2. IELLQAR peptide ligand which binds to E-selectin, and 3. gonadotropin-releasing hormone
(GnRH), which binds to GnRH receptor. Cellular phenotypes, uptake, cytostatic and apoptotic
effect of these conjugates will be monitored in preliminary experiments. Quantitative proteomics
will be performed at the Mass Spectrometry and Proteomics Laboratory at IBP-CNR using peptide
tagging to measure the differences in protein expressions of Dau-conjugates treated vs. Dau-treated
and untreated cells. Based on previous work on the action mechanisms of anthracyclines we
propose to perform a targeted study which will enable us to define the changes in sub-proteomes
with a higher resolution and a deeper extent than previous global analysis could do. In the first stage
we aim to apply quantitative nuclear proteomics which gives the opportunity to monitor protein
effectors that contribute to cellular phenotype. In a second step membrane surface and extracellular
fractions will be analyzed in order to gain knowledge on cellular trafficking. The later could be
particularly of value for the rational design of new carrier molecules. In addition, we also aim to
monitor protein phosphorylation status in the nucleus upon bioconjugate-drug treatment using a
phosphoproteomic approach recently developed by the group at IBP-CNR.(8) In this context we
aim to further develop our method by applying nano-sized ZrO2 particles for the batch-wise
enrichment of phosphopeptides.
Besides the scientific objectives this project aims to provide high quality training for young Ph.D.
students and researchers mainly in the fields of the synthesis and characterization of bioconjugates,
cellular biology and proteomics. In particular, the Hungarian researchers could use the state-of-the
art instrumentation of IBP-CNR not yet available in Hungary. The Ph.D. students will be trained for
the use of nano-scale two-dimensional HPLC and mass spectrometric techniques and their
application in quantitative proteomics. The Italian researchers can learn synthetic peptide chemistry,
methodologies in cellular biology, bioconjugates synthesis, and techniques to study the surface
chemistry process developed in Budapest.