ASSESSMENT OF THE PLATINUM-BASED DRUG EFFECTS IN COLORECTAL CARCINOMA WITH EMPHASIZE ON TUMOR CELL FUNCTIONAL GENOMICS AND IMMUNOMODULATION

Project title:
ASSESSMENT OF THE PLATINUM-BASED DRUG EFFECTS IN COLORECTAL CARCINOMA WITH EMPHASIZE ON TUMOR CELL FUNCTIONAL GENOMICS AND IMMUNOMODULATION

Duration of project: October 3rd 2011-December 31st, 2016
Granted by the Romanian Executive Agency for Higher Education, Research, Development and Innovation Funding
Project type: Exploratory Research Ideas
Project Code PN-II-ID-PCE-2011-3-1057
Contract no. 250/2011
Total funding: 1.500.000 RON
Host institution: Prof. Dr. Ion Chiricuta Oncology Institute Cluj Napoca, 34-36 Republicii St, 400015 Cluj Napoca, Romania
Project director:
Eva FISCHER-FODOR, Ph.D., senior researcher II, senior medical chemist

Research team:
VIRAG, Piroska, Ph.D., senior researcher
BALACESCU, Ovidiu, Ph.D., senior researcher
CAINAP, Calin Ioan, M.D., Ph.D., senior specialist in oncology
LAZAR, Gabriel, M.D., PhD, senior specialist in surgery
CENARIU, Mihai Cosmin, Ph.D., research scientist
TATOMIR, Corina Bianca, M.Sc. in Chemistry, researcher
FETICA, Bogdan, M.D., Ph.D. Student, senior pathologist
BALACESCU, Loredana, Ph.D. in Physics, postdoctoral researcher
BURCA Anca Meda, economist, specialist in grant management
LUNG Rodica, Ph.D. in Mathematics, senior researcher
KOCSOR, Tibor, M.Sc. in Chemistry, Ph.D. Student




Project abstract
The investigation of platinum-based drug effect on tumor stem cells responsible for tumor generation and propagation is a novel and expanding field, and in the colorectal cancer casuistry they are many features uncovered. Studies concerning the action of platinum compounds against immune mechanisms focusing activated lymphocytes involved could bring novelties in the field of cancer cell biochemistry and function. Our aim is to perform a multidisciplinary and translational study that impact patient care in colorectal carcinoma. The project objective is to evaluate the platinum-containing drugs effect on stem cells in colorectal carcinoma, this population being responsible for the resistance against chemotherapy treatment. It is feasible to assess the platinum-treated stem cells, with accent on apoptosis, multidrug resistance, metabolism and cell signaling. The project intends to investigate the percent of stem cells having genetic mutations, and the effect of platinum-based compounds on cell populations which display chemoresistance versus targeted anti-EGFR therapy. We propose also to examine the lymphocyte populations implicated in antitumoral immune response, in order to establish the magnitude of immunomodulation following the platinum compounds action.

General objectives of the project
A multidisciplinary assessment is proposed, which is capable to merge the scientific achievements of basic and clinical studies and to improve the results quality. Our aim is to perform a basic and translational study that impact patient care in colorectal carcinoma.
1. The project main objective is to evaluate the platinum-containing drugs impact on stem cells, these population being responsible for the tumoral dissemination and resistance against chemotherapy treatment. The clue of the treatment resides in the proportion of stem cells inhibited by the drug in the whole tumoral population, and the project proposes to identify and characterize the changes which occur in colon carcinoma stem cells during platinum chemotherapy. It is feasible to compare using functional genomic methods the platinum-treated stem and non-stem in the tumoral mass with accent on cells apoptosis, multidrug resistance, metabolism and cell signaling.

2. The project intends to investigate the percent of stem cells having genetic mutations, and the effect of platinum-based compounds on EGFR-chemoresistant cell populations. Our aim will be to measure the proportion of stem cells in the K-ras mutant population and to assess the platinum effect against the CD133 positive/K-ras mutant cells.

3. One of our objectives is to scan the lymphocyte populations implicated in antitumoral immune response, emphasizing the CD8 positive T lymphocytes and the natural killer cells, in order to establish the magnitude of the changes in human body immune response potential following the platinum compounds action. Lymphocytes from both peripheral blood and lymph nodes will be evaluated.
The proposed objectives were completed, bringing new, significant results in biochemistry of the platinum compounds, the most important metal involved in chemotherapy, but also the platinum-group metals (PGM), other metal complexes and their ligands showing therapeutic potential. We revealed multiple aspects of platinum-based chemotherapy, which join the outcomes of basic biochemical, genomic, proteomic and immune methods in order to improve the therapeutically benefits.

After accomplishing all phases of the project, the final results of the project are:
Scientific publications which acknowledges the project, indexed on Web of Science Thomson Reuters ISI:

1. Multifunctional applications of a dysprosium-based metal–organic chain with single-ion magnet behavior
Belén Fernández, Itziar Oyarzabal, Eva Fischer-Fodor, Sergiu Macavei, Ignacio Sánchez, José M. Seco, Santiago Gómez-Ruiz and Antonio Rodríguez-Diéguez
CrystEngComm, 2016, 18, 8718-8721, Royal Society of Chemistry, IF 3,849, influency score 2,348.

2. Cytotoxic activity of palladium(II) complexes of (1e,6e)-1,7-bis(4-dimethylamino) phenyl)hepta-1,6-diene-3,5-dione against human colon carcinoma
Natalia Miklasova, Roman Miklas, Piroska Virag, Corina Bianca Tatomir, Cristina Szalontai, Diana Cenariu, Ferdinand Devinsky, Eva Fischer-Fodor
Studia UBB Chemia, 2016, 61, 3(I): 117-123. IF 0,148; influency score 0,063.

3. Synthesis of structural analogues of hexadecylphosphocholine and their antineoplastic, antimicrobial and amoebicidal activity.
Lukáš Timko, Miloš Lukáč, Eva Fischer-Fodor, Gabriela Chereches, Mária Garajová, Martin Mrva, František Ondriska, Marián Bukovský, Janka Karlovská, Janka Kubincová, Ferdinand Devínsky.
European Journal of Medicinal Chemistry, 2015, 93:263-73.

4. Imunomodulatory potential of palladium(II) complexes with (1E,6E)-1,7-bis (3,4-dimethoxyphenyl) hepta-1,6-diene-3,5-dione.
Eva Fischer-Fodor, Roman Mikláš, Ludovic Tibor Krausz, Piroska Virag, Daniela Moldovan, Maria Perde Schrepler, Ioana Berindan-Neagoe, Ferdinand Devínsky, Natalia Miklášová.
Studia Universitatis Babes-Bolyai-Studia Chemia, 2015, LX(2): 93-100.

5. In vitro antitumour activity of tomato-extracted carotenoids on human colorectal carcinoma
Diana Cenariu, Eva Fischer-Fodor, Piroska Virag, Corina Tatomir, Mihai Cenariu, Emoke Pall, Adela Pintea, Andrei Mocan, Gianina Crisan.
Notulae Botanicae Horti Agrobotanici, 2015, 43(2): 293-301.

6. Modulation of the UV-B-induced oxidative stress and apoptosis in HaCaT cell line with calluna vulgaris extract
Virag Piroska, Brie Ioana, Burz Claudia, Tatomir Corina Bianca, Hodor Tudor, Orasan Remus, Decean Hana
Notulae Botanicae Horti Agrobotanici, 2015, 43(2): 313-319.


7. Platinum derivatives: generic brands vs. original, in vitro tests
Sînziana Cetean, Călin Căinap, Olga Soriţău, Corina Tatomir, Piroska Virag, Adriana Hangan, Luminiţa Oprean, Radu Oprean
Revista Română de Medicină de Laborator / Romanian Journal of Laboratory Medicine, 2015, 23(4): 439-448.

8. Gallium phosphinoarylbisthiolato complexes counteract drug resistance of cancer cells
Eva Fischer-Fodor Eva, Ana Maria Vălean, Piroska Virag, Petru Ilea, Corina Tatomir, Florica Imre-Lucaci, Maria Perde Schrepler, Tibor Krausz, Lucian Barbu Tudoran, Calin George Precup, Iulia Lupan, Evamarie Hey-Hawkins, Luminita Silaghi-Dumitrescu
Metallomics (Royal Society of Chemistry), 2014, 6(4): 833-44.

9. Anti-cancer Applications of Titanocene-Functionalised Nanostructured Systems: An Insight into Cell Death Mechanisms
Jesus Ceballos-Torres, Piroska Virag, Mihai Cenariu, Sanjiv Prashar, Mariano Fajardo, Eva Fischer-Fodor, Santiago Gómez-Ruiz.
Chemistry – A European Journal (John Wiley & Sons), 2014, 20(34):10811-28.

10. Synthesis and characterization of new biologically active palladium(II) complexes with (1E,6E)-1,7-bis(3,4-diethoxyphenyl)-1,6-heptadiene-3,5-dione
Authors: Natalia Miklášová, , Eva Fischer-Fodor, Roman Mikláš, Lenka Kucková, Jozef Kožíšek, Tibor Liptaj, Olga Soritau, Jindra Valentová, Ferdinand Devínsky.
Journal: Inorganic Chemistry Communications (Elsevier), 2014, 46: 229–233.

11. Oxaliplatin induces different cellular and molecular chemoresistance pattern in colorectal cancer cell lines of identical origins
Authors: Piroska Virag, Eva Fischer-Fodor, Maria Perde-Schrepler, Ioana Brie, Corina Tatomir, Loredana Balacescu, Ioana Neagoe, Ovidiu Balacescu
Journal: BMC Genomics (BioMed Central Publisher), 2013,14: 480.

Among the published papers, 5 were awarded under the frame of UEFISCDI PRECISI prizes for the scientific research activity in 2013, 2014 and 2015.

Papers in journals indexed by international databases:
12. Iron, inflammation and invasion of cancer cells
Eva Fischer-Fodor, Natalia Miklasova, Ioana Berindan-Neagoe, Bhaskar Saha.
Clujul Medical, 2015, 88(3): 272-277.

13. Vitis vinifera seeds extract for the modulation of cytosolic factors bax-α and NF-κβ involved in UVB-induced oxidative stress and apoptosis of human skin cells
Hana Decean, Eva Fischer-Fodor, Corina Tatomir, Maria Perde Schrepler, Lidia Somfelean, Claudia Burz, Tudor Hodor, Remus Orasan, Piroska Virag
Clujul Medical, 2015, 89(1): 72-81.

The PN-II-ID-PCE-2011-3-1057 project endowed logistic support for three bilateral projects projects:
Romanian-Slovakian Bilateral Cooperation , PN II, Capacities, Module III, SK-RO 0006-10471, contract no. 471/ 2011, acronym: BILATSLOVROM, 2011-2012, co-directors: Dr.Eva Fischer-Fodor (IOCN Cluj Napoca) and Prof. Dr. Ing. Ferdinand Devinsky from the Comenius University in Bratislava.
Romanian-Hungarian Bilateral Cooperation, PN II, Capacities Module III, contract no. 678, acronym: BILATHUROM II, 2013-2014, co-directors: Dr. Piroska Virag (IOCN Cluj Napoca) and Dr. Katalin Lumniczky from Joliot Curie Radiobiology and Radiohygiene Institute, Budapest.
Romanian- South African Bilateral Cooperation, PNIII-P3-116/24.06.2016, contract no. 9BM/2016, acronym: AFROMET, co-directors: CSII.Dr. Eva Fischer-Fodor, IOCN Cluj-Napoca and Prof.Dr. Gregory S. Smith, University of Cape Town, South Africa.

DISSEMINATIONS
The results of the project were presented at international or national conferences, as follows:

- The 7th International Conference of Oxidative Stress in Skin Biology and Medicine, 1-4 September 2016, Andros, Greece:
Eva Fischer-Fodor, Piroska Virag, Roman Miklas, Maria Perde Schrepler, Corina Tatomir, Natalia Miklasova: New knoevenagel condensates target superoxid dismutases in epidermoid carcinoma cells in vitro, Abstract Book, pages 106-107.
P. Virag, M. Perde-Schrepler, C. Tatomir, H. Decean, E. Fischer-Fodor: UVB-activated signaling pathways and their modulation with Vitis vinifera seeds extract in human skin cells, Abstract Book, pages 23-25.
- The 27th International Conference on Organometallic Chemistry, 17-22 July 2016, Melbourne, Australia, participant: E. Fischer-Fodor.
- The 1st Conference of the Romanian Association of Laboratory Medicine with international participation, 18-21st May 2016,Cluj-Napoca, Romania; participants; P. Virag, E. Fischer-Fodor.
- At the „First Conference of the Romanian Association of Laboratory Medicine”, Sighisoara, Romania, 20-23 May 2015, the poster presentation with the title: „Evaluation of K-ras mutations in colorectal cancers by pyrosequencing method”, authors: Eva Fischer-Fodor, Loredana Bălăcescu, Gabriel Lazăr, Călin Căinap, Bogdan Fetică, Piroska Virág, Ovidiu Bălăcescu; abstract published in: Romanian Journal of Laboratory Medicine/Revista Romana de Laborator, 2015, Vol 23, Supplem 1, pages S108-S109.
-The 5th International Conference on Metallomics, Beijing, China, 9-12 Sept 2015, the oral presentation with the title: “The platinum cellular uptake as biomarker for uterine cervix carcinoma therapy” , authors: Eva Fischer-Fodor, Petru Ilea, Ioana Carmen Brie, Piroska Virag, Corina Tatomir, Florica Imre-Lucaci, Gabriela Chereches, Luminita Silaghi Dumitrescu, Viorica Magdalena Nagy.
-The III International Conference on Cellular Environmental Stressors in Biology and Medicine: Focus on Redox Reactions, held in Ferrara, Italy, 25-27 June 2014.Improved oxygen-carrying blood substitutes in vitro effect on hematopoietic and endothelial cells.Authors: Eva Fischer-Fodor, Denisa Hathazi, Florina Scurtu, Augustin C. Mot, Grigore Damian, Piroska Virag, Radu Silaghi-Dumitrescu.Poster presentation.
-The 6th International Conference on Oxidative Stress in Biology and Medicine, held in Andros, Greece between 28-31 August 2014: Metal-based therapeutically agents targeting the malignant stem cells: the role of reactive oxygen species. Authors: Eva Fischer-Fodor, Piroska Virag, Gabriel Lazar, Maria Perde Schrepler, Mihai Cenariu, Luminita Silaghi Dumitrescu, oral presentation
- The 14th Congress of the European Academy of Dento-Maxillo-Facial Radiology, Cluj Napoca, June 25-28, 2014: Cytogenetic modifications induced by computed tomography-derived low-doses of irradiation in human leukocytes, Authors: Piroska Virag, Ioana Brie, Maria Perde Schrepler, Eva Fischer-Fodor, Valentin Cernea, oral presentation.
- Presentations held at the “Oncology Days” June 2014 in Cluj-Napoca, Romania:
Eva Fischer-Fodor: Titanocene nanostructures modulate PARP-1 and Bax-α expression;
Piroska Virag: Potential biomarkers for therapeutic response in colorectal cancers;
Corina Tatomir: Novel antitumor immunomodulatory metallodrugs.
- Oral presentation at the 4th International Symposium on Metallomics, Oviedo, Spain, 8-11 July 2013, published in The Abstract Book of the conference: Gallium phosphinoarylbisthiolato complexes counteract drug resistance of cancer cell. Authors: Eva Fischer-Fodor, Ana-Maria Valean, Petru Ilea, Piroska Virag, Corina Tatomir, Maria Perde Schrepler, Florica Imre-Lucaci, Lucian Barbu Tudoran, Evamarie Hey-Hawkins, Luminita Silaghi-Dumitrescu.
- Presentation at the 22nd Biennial Congress of the European Association for Cancer Research: From Basic Research to Personalised Cancer Treatment, Barcelona, Spain, July 7-10, 2012: Alterations of cellular and molecular patterns in oxaliplatin resistant colorectal adenocarcinoma cell lines, Authors: P. Virag, O. Balacescu, E. Fischer-Fodor, M. Perde Schrepler, C. Tatomir, L. Balacescu, I. Neagoe. Published in European Journal of Cancer, 2012, Vol 48, Supplem. 5, p. S60.
- Oral presentation held at OECI Cancer Molecular Pathobiology in the Clinics: Highlights, Cluj-Napoca, Romania, April 18-20, 2012: Superior cytotoxicity and DNA cross-links induction by oxaliplatin vs cisplatin at lower cellular uptake in colorectal cancer cell line. Author: Piroska Virag, Published in:Proceedings of the Cancer Molecular Pathobiology in the Clinics Conference, pag 33-34.
- Oral presentation at the International OECI Workshop, Cluj Napoca, Romania, September 10-12, 2012: Molecular profiling and innovation in cancer, Author: Piroska Virag,
- The Gordon Research Conference- Metals In Medicine, Andover, NH, USA, June 24-29, 2012: Novel antitumoral platinum and palladium complexes of curcumin merge antiproliferative effect against tumor cells with immunomodulatory capacity. Authors: Eva Fischer-Fodor, Natalia Miklasova, Roman Miklas,Piroska Virag, Corina Tatomir, Maria Perde Schrepler, Tibor L. Krausz, Calin Precup, Ferdinand Devinsky, poster presentation
- Participation at the 4th Interamerican Oncology Conference, Buenos Aires, Argentina, June 6-7, 2011, Current Status and Future of Targeted Therapies, Eva Fischer-Fodor.


The final phase of the project, PHASE VI (December 10th 2015- December 31st 2016) was implemented by completing the following objective:
6. Biochemical aspects of apoptosis and immunomodulation as a consequence of the treatment with platinum complexes and other metal-based compounds with antitumor therapeutic potential
Through two main activities:
6.1. Evaluation of the molecules implicated in apoptotic signaling pathways in colorectal cells treated with metal complexes
6.2. Assessment of the metal compounds effect on lymphocyte subsets and cellular adhesion molecules

The final phase of the project, PHASE VI (December 10th 2015- December 31st 2016) was implemented by completing the following objective:

6. Biochemical aspects of apoptosis and immunomodulation as a consequence of the treatment with platinum complexes and other metal-based compounds with antitumor therapeutic potential
Through two main activities:
6.1. Evaluation of the molecules implicated in apoptotic signaling pathways in colorectal cells treated with metal complexes
In order to identify new biomarkers connected to the apoptotic processes induced by metal compounds, the biologic effect of a new class of dysprosium complexes was studied; the antiproliferative effect of the Dy compounds was compared with that of standard platinum-based drugs. The single-magnet metal complex Dy(III)Na(ampy)4]n, having as ligand the 5-aminopyridine-2-carboxylic acid ligand (H-ampy), exhibited biologic activity against DLD-1, HT-29 si Caco-2 human colon tumor cells in vitro. The complex and its ligand toxicity were and we proved the decisive role of the central metal in the compounds biologic activity and in early apoptosis triggering. The treated tumor cell populations were exposed to a uniform magnetic filed, with an intensity which does not affected the cells viability, and we observed that unlike to the ligand or the platinum drugs, the magnetic field applied concomittant with in Dy(III)Na(ampy)4]n treatment induced a stronger growth inhibitory effect on tumor populations. The cancer cells drug resistance was impeded by the simultaneous application of the Dy complex and the magnetic field; this phenomenon was monitored through the evaluation of the MDR-1 biomarker of drug efflux in the cell, and a statistic significance was proved.

The results of the research were published in the manuscript entitled:
Multifunctional applications of a dysprosium-based metal–organic chain with single-ion magnet behavior, Belén Fernández, Itziar Oyarzabal, Eva Fischer-Fodor, Sergiu Macavei, Ignacio Sánchez, José M. Seco, Santiago Gómez-Ruiz and Antonio Rodríguez-Diéguez.
CrystEngComm, 2016, 18, 8718-8721.

Under the frame of the 6.1. phase it was tested as well a group of new compounds: polynuclear complexes of ferrocene with thiosemicarbazide ligands, having the formula C192H208Fe8N14 (1), C63H66Fe3N4 (2) and C92H104Fe4N6 (3). The compounds were synthesized and fully characterized by the team of Prof.G.S. Smith from the University of Cape Town, and published in J.Organomet.Chem, 2016, 819:166-172. The complexes 1-3 were tested in vitro in order to elucidate their biologic activity against human colon cells in vitro. The inhibitory effect against the tumor cell populations was tested by MTT method and the degree of the toxicity was quantified using the IC50 half inhibitory concentration parameter, which revealed a superior toxicity against the invasive, mutant colon cancer cells. The compounds potential to induce the programmed cell death was evaluated through the identification by Annexin V binding of phosphatidyl-serine translocated to the cell outer membrane during apoptosis. In parallel, the number of necrotic cells was evaluated. Using the flow cytometry, we studied the phases of the cell cycle in tumor cell populations treated with ferrocenyl complexes 1-3; a G0/G1 phase arrest was observed in the treated tumor cell proliferation. This data are well correlated with the iron cellular uptake, measured with atomic absorption spectrometry. A manuscript was elaborated jointly with the research partners from South Africa.

6.2. Assessment of the metal compounds effect on lymphocyte subsets and cellular adhesion molecules
The activity implied to achieve the phase VI objective was the study of two complexes of the Platinum Group Metals (PGM), steric isomers, having as central metal Pd (II) and the ligand (1e,6e)-1,7-bis(4-dimethylamino)phenyl) hepta-1,6-diene-3,5 dione, in two different conformations. Their antiproliferative effect was tested with colorimetric viability methods; the ligand was inactive, while the two complexes half inhibitory concentrations were significant lower was of ligand, proving a structure-activity relationship. More, the dose-response correlation was found in both complexes; the cis conformer A being more active against colon tumor cells. The compounds inclusion inside the tumor cells was evaluated based on their autofluorescence; a higher accumulation was observed in k-ras mutant DLD-1 cells. The second research direction was to elucidate the role of A si B, on cellular adhesion molecules. In this purpose we studied the effect of the metal complexes A and B against the peripheral blood mononuclear cells (PBMC), emphasizing on the compounds modulator effect on the neural cell adhesion molecule NCAM or CD56 biomarker, which characterizes the cytotoxic natural killer cell subset. In the antitumor immune response an important role is of adhesion and activation marker CD11a (ITGAL) and of tumor necrosis factor CD27; their expression was quantified using the flow cytometry and a statistic significant correlation was found with the presence of CD56 markers on the PBMC cell membrane. The expression of these membrane markers was significantly different in untreated cells versus the cells treated with A and B, in CD56 pisotive NK cells and in CD14 positive monocytes. In the 95% confidence interval a correlation was confirmed between the compound B effect and the inhibition of CD56, and overexpression of CD11a.

The research results were published in Studia Universitatis UBB Chemia, 2016, 61, 3 (I): 117-123, with the title: Cytotoxic activity of palladium(II) complexes of (1e,6e)-1,7-bis(4-dimethylamino) phenyl)hepta-1,6-diene-3,5-dione against human colon carcinoma, authors: N. Miklasova, R. Miklas, P. Virag, C.B. Tatomir, C. Szalontai, D. Cenariu, F. Devinsky, E. Fischer-Fodor. The journal is indexed in Thomson Reuters Web of Science Core Collection, and can be accessed freely: http://chem.ubbcluj.ro/~studiachemia/issues/ chemia2016_3/tom1/11Miklasova_etal_109_116.pdf.

Analogous structures of PGM metal complexes Pd (1) and Pt (2) with the ligand (1e,6e)-1,7-bis(4-dimethylamino)phenyl) hepta--3,5 dione were tested for their immunomudulator effects. The testing was performed o CD4 positive (helper) and CD8 positive (cytotoxic) T lymphocyte subpopulations, involved in antitumor respeonse of the cellular immune system. We targeted the membrane markers of lymphocytes subjected to prolonged exposure (24 hours) to the PGM complexes, focusing on CD25 and CD28 cellular activation markers, the tumor necrosis factors GITR (TNFRSF18) and CD154 (CD40L), the adhesion molecule L-lectin sauor CD62L, and CD127, respectively, a marker of suppressor cells within a lymphocyte population. Complexes 1 and 2 have a significant influence on lymphocyte activation through CD25 signaling, GITR and CD154 augmentation. They strongly upregulate the CD4+CD25+CD127+ subset and exhibit a modest influence on CD28 membrane marker expression. The ligand alone has a slight effect on the studied membrane markers, and complex 1 strongly interfere with the costimulatory prcesses in the cellular immune system. Using proteomic methods, a quantitatively evaluation was performed on the intracellular and extracellular production of interleukines IL-2, IL-6, IL-8 andIL-10, and the date were correlated with the adhesion and activation markers expression. We concluded that these group of metal complexes exhibited in vitro a significant immune modulator potential, besides the cytotoxic effect, through the main signalling pathways CD25-CD69-IL 2 and GITR-T regulator cells.


Previous activities:

The objective of Phase V (December 10th 2014- December 31st 2015) of the project was: 5.1. Assesment of the platinum-based drugs effect on immune system and tumor microenvironment, achieved in proportion of 100% through two major activities:
-Evaluation of platinum compounds effect on effector lymphocyte subpopulations and on stem cells isolated from metastatic lymphatic node in colorectal carcinoma patients.
Based on the hypothesis that the platinum-based drugs and other metal compounds action modify the tumor microenvironment and these modifications conduct to the tumor cells suppression or contrary, to the development of drug resistance, we examined the ex vivo modulation of different cell types, the principal components of the tumor stroma, and we evaluated as well the lymphocyte infiltration in lymph nodes, emphasizing on CD8 positive cytotoxic T cell subset. Following the written informed consent of the colorectal cancer patients from the Institute of Oncology “Prof.Dr.I.Chiricuta”, tissue samples from lymphatic nodes were harvested. The histological type of the tumors was adenocarcinoma, and all the lymphatic nodes have had tumoral infiltrations; primary cell cultures were carried out, treated in vitro with the antitumor drug oxaliplatin, and than the cell subpopulations were analyzed using the flow cytometry. The CD8 positive lymphocytes were detected using the standard fluorescence labeling, the endothelial cells were characterized using the surface marker CD105 and the anti-fibroblast antibodies enabled the quantification of fibroblast expression in tumor mass. The percent of the stem-like CD133 positive cells and EpCAM (CD326 positive) tumor cells presence was detected in the lymphatic tissue samples, which confirmed the tumor infiltration, the marker being a specific feature of epithelial tumor cells. As a result of the platinum drug therapy, a raise in the fibroblast and a decrease in the endothelial cells proportion, which can influence the proliferation indirectly, acting on tumor microenvironment.
-Validation of over-expressed or down-regulated molecules.
Two directions were approached; one of them analyzed the implications of platinum-based neoadjuvant chemotherapy treatment on lymphocyte subsets involved in antitumor immune response in colorectal cancers, the activation processes in cells obtained from lymphatic nodes resection. The testing was conducted in vitro, they were studied the effector T cells isolated from tissue samples, activated through CD25, CD69 and CD27 molecules; the expression of these surface markers was assessed through flow-cytometry, and an experimental validation was made using molecular techniques (qT PCR) Elisa testing for the soluble form of the molecules. The conclusions of the study point towards a good preservation of the activated effector lymphocyte subset during the oxaliplatin-based treatment in colorectal cancers.
The second line of the study was to elucidate the in vitro effect exerted by the metal compounds having a central metal from the platinum group, against the cells implicated in antitumor immune response, and the validation of this outcome by measuring the soluble form of the involved molecules, especially interleukins. Two novel synthesized Palladium (II) compounds were studied, having a curcuminoid ligand: (1E,6E)-1,7-bis(3,4-dimethoxypenyl)hepta-1,6-diene-3,5-dione, which showed limited toxicity against the normal peripheral blood mononuclear cells (PBMC). The ligand only and its two complexes 1 and 2 induced a moderate toxicity against PBMC’s and does not influenced the cell signaling through CD45R and CD25 molecules. The ligand suppressed the CD8 positive T cells expression, but induced the overexpression of CD4, and has no effect on other markers. Despite the fact that complex 1 slightly inhibited the CD8 expression, it has a benefic effect on other cellular subpopulations implicated in the immune response. Complex 2 has had an inhibitory effect not only on CD8 positive lymphocytes, but also against CD19 positive B cells and induced reduction in GITR positive cell population, which predicts a moderate immune suppression. Overall, the compounds exhibit applicability potential for antitumor prodrugs development.
5.2. Evaluation of the metal-based compounds in vitro effect on colorectal tumor stem-like cells; having the activities:
- Comparison between original versus generic platinum drugs, emphasizing on stem cell proliferation and multidrug resistance
There were assessed the differences between the generic and original platinum-based drugs, in order to elucidate some of the reasons why the patients respond differently to the treatment when generic or original drugs are used, an aspect which concern the scientific community and the clinicians as well. Under the frame of this activity an evaluation of the in vitro cytotoxicity was made for the original and generic versions of oxaliplatin and carboplatin on tumor cell lines DLD-1, HT-29 and A2780. The inhibitory effect the original oxaliplatin drug against colon cell lines was not significantly higher, but the effect on the stem-like CD133 positive cells isolated from the whole cell population was superior in the K-ras mutant DLD-1 cell line after 24 hours exposure. The chemoresistance towards the platinum-based drugs was higher in the HT-29 Braf-mutant cell line, the toxicity being below of that for DLD-1. We found morphologic evidences such prevailing membrane structure alteration, chromatin condensation in the tumor cells treated with original drugs. The phenomenon studied in vitro can be extrapolated to the clinics to anticipate the drugs efficacy, and to predict at some extent the hypersensibility reactions and the adverse effects of the drugs.
- Outcome of platinum group metal compounds and their ligands on membrane and cytoplasmic markers modulation and transcription factors in colorectal stem cells
Under the frame of this activity it was examined the modulation of the tumor necrosis factors CD27 and TNFRSF18, and the NF-kβ intracellular transcription factor, by complexes having natural compounds, in this case licopen, as ligands linked to the central metal. The experiment was conducted in vitro on human colorectal cell lines, the toxicity was evaluated with colorimetric methods, and the simultaneous expression of the molecules was quantified using the flow cytometry. The transcription factors were correlated with the expression of the molecules implicated in VEGF signaling. Overall, the studied compounds exhibit antiproliferative effect against the tumor cells in vitro, they influenced the NF-kβ signaling and the angiogenesis. In addition, we evaluated alchylphosphocholine ligands of metal compounds, highlighting their antimicrobial and antitumor activity.

The activities completed during the phases I to IV of the project (October 3rd 2011- December 10th 2014) were:
4.1. Assesment of immunomodulatory effects of chemotheray in colorectal carcinoma, objective completed through two major activities: The evaluation of peripheral blood mononuclear cell subsets proportion and activation status following the treatment with standard platinum drugs, and In vitro evaluation of immune system modulation and cytotoxicity of novel synthesized metal-based compounds with prodrug potential in colon carcinoma
4.2. Correlation of in vitro and ex vivo outcome resulted from neoadjuvant clinical treatment in colorectal cancer in order to locate potential prediction factors, accomplished by the activity: Prediction factors elucidation in colorectal cancer pacients, and the treatment follow-up.
3.2. Evaluation of the biochemical features of platinum-based drugs effect evaluation in tumoral stem cells.
3.1. In vitro assessment of platinum-drug effects on stem cells isolated from human tumoral cell lines and human tissues. Correlations between in vitro testing and clinical results.
2.2. The study of platinum and palladium compounds effect on genetically mutant tumor tissues.
2.1. Ex-vivo evaluation of platinum-based compounds effect on stem cells from colorectal tumors and colorectal cell lines.
1.1 Biochemical aspects monitoring of apoptosis triggering, DNA damages and multidrug resistance mechanisms in K-ras mutant and K-ras wild type malignant colorectal cells treated with metal complexes.





Contact person: Dr. Eva Fischer-Fodor
Senior Research Scientist II, Senior Medical Chemist
Research Department,
Institute of Oncology “Prof.Dr.Ion Chiricuta”
34-36 Republicii, 400015 Cluj-Napoca, Romania
E-mail: fischer.eva@iocn.ro
Phone/Fax: 0264-439260