Induced Apoptosis in Cancer Cells
Study of targeting apoptosis in tumor cells induced by human alpha-fetoprotein: application for anticancer drug design
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
- MED-DRG/Drug Discovery/Medicine
8 Project completed
Senior Project Manager
Weaver L M
Institute of Immunological Engineering, Russia, Moscow reg., Lyubuchany
- Institute of Medical Ecology, Russia, Moscow
- University of Turku / Finnish-Russian Joint Biotechnology Laboratory, Finland, Turku\nAstraZeneca, UK, Macclesfield\nUniversidad de Navarra, Spain, Pamplona\nCNRS / Institute de Recherches sur le Cancer, France, Paris\nAboaTech Ltd, Finland, Turku
The design of the new types of anticancer drugs selectively affecting tumor survival in human without significant unspecific toxicity is considered to be one of the main problems in oncology. Cancer cells have certain mechanisms of resistance to apoptosis and high therapeutic doses of anticancer drugs are required for their elimination, which simultaneously are extremely toxic for normal healthy cells. Thus, the search for a drug capable to induce targeted triggering of apoptosis selectively in cancer cells in the absence of side effects is still on.
Recently, it had been established that oncoembryonal marker-fetoprotein (AFP) is capable to induce apoptosis selectively in cancer cells, but not normal cells. This allowed to propose possible use of AFP as a component of the targeting anticancer drugs. Samples of human AFP originated from various sources, such as embryonic, cancer or recombinant, - had been used in serial research of their anticancer activity in vitro, and also for the determination of the possible unspecific toxic effects in vivo in animal systems. The tumoricidal effect of AFP was shown to be due to the rapid induction of tumor cell apoptosis, and was observed for the various types of tumor cell lines in vitro. Our data also demonstrated the ability of AFP to modulate apoptotic signals, generated by other factors, such as tumor necrosis factor (TNF), retinoic acid, doxorubicin, steroids etc., by enhancing of the total anticancer effect. Basing on these studies, the new types of targeting anti-cancer drugs on the base of pure human AFP or this one in combination with other cytotoxic drugs (TNF, antibiotics, retinol or steroids), which characterised by high degree of tumor-selective cytotoxicity, could be designed.
Given the tumor-selectivity of AFP cytotoxicity in culture together with its ability of positive modulation of apoptotic signals mediated by other anticancer drugs and the absence of unwanted side effects for normal cells may have potential as a cancer therapeutic. In Moscow Cancer Research Center the I stage of the clinical trials of pure human AFP had been carried out to determine possible toxic effects for human. According to the results of these studies any unspecific toxicity in therapeutic doses had not been determined.
The objective of the project.
The project is devoted to the design of a new type of anticancer drug based on human AFP, selectively affecting tumor survival in humans without significant unspecific toxicity. Another objective of this project could be formulated as follows: to determine mechanisms of selectivity of AFP-mediated cytotoxicity and to decode the intracellular pathways of AFP-mediated apoptotic signal leading to the triggering of molecular mechanism of programmed cells death.
Basing our study on the experimental data obtained in the course of execution of the ISTC project #401, demonstrating that many cancer cell lines are sensitive to the cytotoxic effects of AFP, whereas most normal (non-transformed) cells are resistant, we decided to use AFP as a component for anticancer drug, that could provide targeting killing of cancer cells with minimal side effects. Also, a novel unknown property of AFP to trigger intracellular mechanism of programmed cell death (apoptosis) selectively in tumor, but not normal cells had been established. Given the tumor-selectivity of AFP-cytotoxicity in culture, and the absence of unwanted side toxic effects in mice allowed to propose perspectives of future application of this factor in tumor therapy.
The most particular feature of AFP differing it from other cytotoxic anticancer drugs is tumor-selectivity of its cytotoxic activity, realised due to the restricted ability of various types of cells to express specific AFP-receptors (AFPR), which are found on tumor cells and are not revealed on normal cells. Thus, the main difference between tumor-specific apoptotic factor AFP and other traditional anticancer drugs, which are able to induce apoptosis both in tumor and normal untransformed cells, is tumor-selectivity of AFP-mediated apoptotic activity. The explanation of the biological activity of AFP to trigger apoptosis in tumor cells has not been received yet. The molecular mechanisms of AFP-induced apoptosis in tumor cells have to be elucidated. The main objective of the project is to find the experimental solution of this task.
In the scope of the project it is also planned to determine the localisation of the active site on the AFP molecule that is responsible for its interaction with receptors introduced in triggering of apoptotic signaling and to suggest the structures of new peptide analogs possessing similar activity as intact molecule.
It is proposed to investigate the synergistic action of AFP with various anticancer drugs, hormons and cytokines, such as doxorubicin, retinoic acid, estradiol, that are known to be natural ligands for AFP, as well as in combination with other new tumor-suppressors and apoptotic factors, which could be discovered during the project execution.
It is proposed to carry out the theoretical calculations of the secondary and tertiary structures of AFP molecule to propose the theoretical model of its general conformation. The available data on the general conformation of the AFP molecule would be utilized for the design of the biologically active peptide analogs of the molecule. It is planned to investigate in vitro and in vivo the biological activity of multidrug compositions of AFP with various apoptotic factors and apoptotically active peptides.
If positive results would be received (the synergistic action of different drugs with the essential decrease of therapeutic dose), it is planned to recommend medical trials of these multidrug compositions or/and to propose the preclinical and clinical trials of these compounds. The AFP-based anticancer drugs are proposed to have high tumor-directed selectivity and pro-apoptotic activity of AFP with minimized unwanted side effects in the course of the medical treatment.
It is also proposed to investigate the structural basis of the interaction of these AFP-based tumor-selective apoptotic factors with various functional cell receptors, that are responsible for apoptotic signaling: caspases 1-10, membrane apoptotic receptors Fas, TNFR, and their adapter proteins (FADD, TRADD, etc.) as well as specific cell-surface and cytosolic AFP-receptors.
Major research tasks:
· To study molecular mechanisms of AFP-induced apoptosis in tumor cells using various techniques, such as: cell-free system for apoptosis assessment, DNA-fragmentation assay, fluorescent cell sorting (FASC), assessment of changes in mitochondrial potential; functional tests for caspase activation and others.
· To study the genetic and receptor-dependent mechanisms of AFP-mediated apoptosis in tumor cells.
· To study the effects of AFP on the expression of various cytokines and growth factors in cancer cells;
· To determine the localization of the active sites on the AFP molecule, responsible for the apoptosis signaling.
· To study the possible role of membrane-bound and cytosolic AFP receptors in the effect of tumor-restricted targeting selectivity and also in triggering and regulation of AFP-mediated apoptosis in tumor cells using various cellular and cell-free systems for apoptosis assessment.
· To produce the isolation and physico-chemical and immunochemical characterization of specific AFP-receptors on tumor cells;
· To define possible localization of AFPR-binding site on the surface of AFP molecule.
· To predict structures of biologically active peptides, modeling active sites of AFP molecule, responsible for binding with specific membrane and cytosolic AFPRs and apoptosis signaling;
· To synthesize artificial peptides modeling active sites of AFP responsible for apoptosis signaling and for its binding with membrane and cytosolic AFPRs.
· To develop a laboratory technique of HepG2 cell line cultivation for high-yield AFP secretion obtaining.
· To design chemically-modified forms of AFP molecule displaying more high apoptotic activity, than native AFP in order to decrease its effective apoptotic dose.
· To study apoptotic effects and to determine therapeutic potential for tumor targeting therapy of various AFP samples: embryonic AFP, cancer-derived AFP, AFP-derived peptides, chemically modified forms of AFP.
· To study structural properties of various samples of human AFP: embryonic, cancer-derived, AFP-derived artificial peptides and other AFP derivatives using circular dichroism, scanning microcalorimetry, fluorescence and absorption spectroscopy.
· To study unspecific toxic effects of various AFP samples in normal cells in vitro and in vivo.
· To begin preclinical studies of anticancer AFP-based tumor-specific drugs, derived from the culture medium of HepG2 cells and also other AFP-related compounds demonstrated the most significant apoptotic effects on tumor cells in culture.
· To study the apoptotic activity and to determine the potential for anti-cancer therapy of various AFP samples originated from embryonal, recombinant or cancer sources.
Those of theoretical importance:
· obtaining experimental data on the secondary and tertiary structures of various samples of human AFP, its proteolytic fragments and chemically modified forms using biophysical techniques: circular dichroism (CD), microcalorimetry (DASM), fluorescence and adsorption spectroscopy;
· determination of functionally important amino acids of AFP molecule responsible for its pro-apoptotic activity;
· localization of active sites on the AFP molecule responsible for interaction with membrane and cytosolic AFPRs;
· determination of the AFP-induced activiation of caspase cascade events in cancer cells;
· determination of the role of the specific membrane and cytosilic AFPRs in triggering apoptosis in cancer cells;
· determination of genetic mechanisms of the AFP-induced apoptosis in cancer cells.
Those of applied significance:
· elaboration of a new type of tumor-selective anticancer drug based on various forms of chemically-modified AFP-derivatives, which are characterised by the enhanced pro-apoptotic activity;
· upon receiving positive results (showing high selectivity of tumor suppressive activity for various AFP samples in vitro) the in vivo studies and the schemes of clinical application for therapy of cancer could be proposed;
· suggestion of synthetic peptide structures modelling active sites of AFP molecule, responsible for binding with membrane and cytosolic receptors;
· suggestion of the possible reccommendations of AFP engagement for therapy of various malignancies based on the determination of the most AFP-sensitive types of cancer cells.
Those of commercial significance:
· elaboration of the schemes of pre-clinical and clinical studies of the AFP-based drugs with high tumor-selective cytotoxicity and low unspecific toxicity;
· design of the novel chemically-modified AFP isotypes with enhanced effectiveness to reduce therapeutic dose with simultaneous retention of its tumor-selectivity and specificity;
· elaboration of diagnostic kits in vitro and in vivo for research and medicine on the basis of radio- or fluorescent-labeled AFP-derived peptides, intact purified AFP, or monoclonal antibodies versus AFP or AFPRs, for the determination of the specific AFPRs on the malignant cells;
· design of a novel drug based on cultural AFP derived from the cultivation medium of AFP-secreting human hepatoma cell line HepG2.
Technical Approach and Methodology
To perform the tasks set up in the project a complex of up-to-date methods would be applied, including gene engineering, site-directed mutagenesis, chromatography, PAGE-electrophoresis, immunoelectrophoresis, immunoblotting, biophysical methods for protein structure analysis. Various techniques: flow cytometry, DNA-electrophoresis, phase contrast microscopy, assays for proliferation, cytotoxicity and DNA-fragmentation, would be employed for documentation of AFP-mediated programmed cell death.
Scientific experience of project executors.
Functional and structural studies of human AFP, as well as measurement of apoptosis induced by various factors (cytokines, drugs, hormones, etc.) in tumor and normal cells had been carried out at the Institute of Immunological Engineering during the period of at least 7 years, including 2 years of ISTC Project #401 execution. The original data had been obtained at our Institute which demonstrated, that pure AFP is introduced in regulation of intracellular mechanisms of programmed cell death and growth signaling. These data demonstrated, that AFP operates as a growth factor for normal developing and embryonic cells, but functions as a suppressive factor for tumor or activated immune cells and does not affect the proliferation of normal untransformed cells, because of the absence of expression of specific membrane AFP-receptors. It was revealed, that tumor-selective AFP-cytotoxicity occurs via induction of apoptosis in tumor cells in vitro characterized by appearance of typical apoptotic cell morphology, nuclear DNA fragmentation, strong cell growth arrest and cytotoxicity. It was shown, that AFP causes apoptosis in tumor cells through activation of general effector of apoptosis caspase-3 and independently of Fas/FasL or TNFR/TNF signaling pathways. For the first time, at our Institute proteolytic fragments of human AFP had been obtained and characterized and structural and functional requirements which are necessary for AFP-mediated triggering of apoptosis had been determined. 8 articles in reviewing international journals and more that 40 abstracts had been published by our research group in the course of AFP studies.
AFP is known to bind noncovalently with the high affinity different ligands, such as steroid hormones, fatty acids, heavy metals and others and to deliver these biologically active substances into the malignant cell. We used this carrier AFP function to deliver cytotoxic drugs to the tumor cells. A covalent conjugate of estron (this hormone can bind to AFP) with anticancer drug doxorubicin (Dox-Est) was prepared. This complex maintained the ability to bind AFP. Complexes of AFP and Dox-Est could penetrate to tumor cells and kill them. These toxic complexes did not enter into the healthy cells because of the absence of AFP receptors on their surface. This new type of drug selectively targeted only tumor cells, without any toxicity directed to the healthy cells. It was proposed to use this new type of drug with the high selectivity of AFP and high antitumor activity of doxorubicin for the therapy of AFP positive tumors, such as hepatoma, mammary carcinoma, colon carcinoma and others.
Basing on the ability of AFP to penetrate selectively to tumor cells, but not normal cells, during the last five years at the Moscow Research Institute of Medical Ecology bicomponent anticancer drugs composed of the human AFP covalently linked with the molecule of cytotoxic compound had been designed. These complexes are taken up selectively by tumor cells and kill them without unwanted side toxic effects against normal cells. Preclinical studies demonstrated high effectiveness of these complexed AFP-based anticancer drugs with the simultaneous significant decrease of the unspecific toxicity. Additionally at the Moscow Institute of Medical Ecology monoclonal antibodies to AFP and its membrane receptors had been obtained and characterized. One of the possible applications of these monoclonal antibodies is their use for determination of AFPR+ tumors, which is necessary for cancer diagnostics in vivo and in vitro and for the determination of their sensitivity to targeting anticancer therapy with AFP-based multicomponent drugs. More than 50 articles and abstracts had been published according to the results received.
The main goal of the project
The execution of this project would make a valuable contribution to the development of fundamental and applied biology and medicine, as well as to the solution of a social problem, namely: it would give scientists from the Institute of Immunological Engineering, who fulfilled earlier the State orders of the Ministry of Defense, the possibility to redirect their scientific interests and to apply their scientific experience for carrying out of fundamental and applied investigations, dealing with the solution of international scientific problems in the fields of biology and medicine in pacific goals, and for creation of long-term perspectives of fruitful functional activity within the frames of international scientific collaboration.
The important and urgent character of the problem.
The importance of the problem is determined by the necessity to create a targeting antitumor drug selectively affecting tumor survival in humans without significant toxicity. The second very important problem to resolve is to find possibilities to overcome multiple drug-resistance of certain types of cancer, being another great problem in oncology. The investigations in this scope are very important both in fundamental and in applied sciences, to open a new approach in anticancer therapy.
Within the scope of the project it is proposed to elaborate a new compound based on human AFP and its peptide and chemically-modified derivatives which could be applied for the high-effective tumor-selective therapy of cancer. It has been proposed, that a new type of AFP-based drugs could be used to overcome multiple drug-resistance of certain types of tumors by specific triggering of apoptotic mechanisms selectively in tumor cells. Another project aim is to compare the fragments of three-dimensional structures of AFP molecule to find the sites responsible for interaction with their membrane and cytosolic receptors and for its apoptotic activity and to predict the structures of the peptides anticipated on the basis of such investigations.
Investigations on the structural basis of the interaction of these AFP-derived compounds with their functional cell receptors are also proposed. It is planned to investigate the structural properties of human AFP and its derivatives using biophysical techniques: CD, DASM, fluorescence and adsorption spectroscopy.
The next aim is to study tumor-suppressive and apoptotic activity of the original peptides and AFP-derivatives and their interaction with functional receptors: AFPRs, caspases, nuclear receptors and other apoptosis-associated proteins.
Another problem to resolve is the investigation of specific membrane and cytosolic AFPRs, by using monoclonal antibodies directed against these receptors and artificial peptides, modeling binding sites of AFP responsible for its interaction with AFPRs.