Directional Regulation of Programmed Cell Death (Apoptosis) of Nervous Tissue Cells under Influence of Laser Radiation
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
- PHY-OPL/Optics and Lasers/Physics
3 Approved without Funding
Institute of Physiology, Belarus, Minsk
- B.I. Stepanov Institute of Physics, Belarus, Minsk
- Laser und Medizin-Technologie, Germany, Berlin\nUniversity of Bologna, Italy, Bologna\nInstitute for Physiological Chemistry II / University of Duesseldorf Hospitals and Clinics, Germany, Düsseldorf
Project summaryThe purpose of the project is to clarify functional metabolic modifications and the basic possibility of initiation and/or inhibition of apoptosis in cultured nervous tissue under laser radiation, including cases with the presence of sensitizers.
State of the art and main idea of the project
Apoptosis (programmed cell death, PCD) is a fundamental process lying in the basis of mechanisms of embryogenesis, morphogenesis and maintenance of cell homeostasis. It was discovered relatively recently, in the early 1970s. Now it is clear that two main types of cell death exist, necrosis and apoptosis, which are phenomenologically distinct from each other.
It is known that apoptosis is a complex biological, genetically programmed phenomenon, which can be initiated and controlled both by endogenous physiological signals and exogenous factors. It has been demonstrated that, similarly to proliferation and differentiation, PCD is controlled by conservative genes, such as bcl/ced-9, etc., and continuing research discovers new genes with pro- and antiapoptosis action. A doubtless role in the initiation of apoptosis is played by the active oxygen species, resulting from oxidative stress, the disbalance in the system of pro- and antioxidants.
Molecular and cellular mechanisms of apoptosis remain insufficiently studied. Nevertheless, the directional regulation of this process represents apparent interest. Thus, at uncontrolled proliferation, tumor growth, the initiation of apoptosis would allow degradation of a tumor without necrotic changes in the tissue, which essentially complicates the recovery of the organ structure. It is especially significant for the nervous tissue, particularly in brain tumors, which are located deeply or in the proximity of centers controlling the vital functions.
To regulate apoptosis, it appears very attractive to use light (laser) radiation as the application of chemical compounds can result in a number of negative effects because of possible polyvalence of their action, long-lived circulation, toxicity, etc.
At present, laser radiation is most efficiently applied in many areas of clinical medicine in the treatment of different diseases. The effect of low intensity laser radiation on the mitotic activity of cultured tissues has been found. A number of studies showed that living systems rearrange their metabolism in response to weak luminous fluxes. The spectral ranges of about 760, 630, and 580nm, corresponding to the absorption bands of molecular oxygen, have been detected, in which a sustained stimulating effect was observed practically in all cell cultures studied.
At the same time, analysis of modern scientific literature displays that research of the medico-biological effect of laser radiation, on nervous tissue in particular, is fragmentary, molecular and cellular mechanisms of biological effects of laser radiation are under-studied. The possible use of laser radiation for initiation or inhibition of apoptosis of the nervous tissue also remains obscure, though some findings allow one to admit the existence of such an effect.
The possibility of cell death by apoptosis has been established in cases of laser exposure in the presence of photosensitizers during photodynamic therapy (PDT), a method intensively developed and widely applied for treatment of many tumor diseases. The destruction of tumor tissues and cells with PDT occurs in response to the active oxygen species, with a whole set of responses, systematic and cellular, switching on. The character of cell death depends both on the intensity of photic exposure, and intracellular localization of a pigment. Depending on a combination of these factors, either apoptosis or necrosis can be induced.
Initiation of an effect similar to the photodynamic one during exposure of cells in the oxygen absorption bands in the absence of sensitizers (light-oxygen effect) was also assumed. The light-oxygen effect may appear upon exposure of biological systems to laser light with wavelengths corresponding to one of the five absorption maximums of molecular oxygen 3О2 (1270nm, 1065nm, 762nm, 630nm, 577nm). A number of 3О2 absorption bands lie in the most penetrating to biotissue, near the infrared (IR) range. The photo-destruction of Lewis carcinosarcoma cells in the absence of exogenous sensitizers was experimentally shown under laser exposure both of cell suspensions and at body level. Though the mechanism of cell death in these experiments was not analyzed, it is necessary to state the identity of stages of cell photoreactivity at PDE and light-oxygen effect. It follows that apoptosis may also occur under the light-oxygen effect. Thus, in principle, there are some mechanisms of influence on the process of programmed cell death by laser radiation with definite wavelength.
During project fulfillment research is proposed of the functional and metabolic properties of cultures of PC12 and C6 cell lines, accepted models for the nervous tissue, when exposed to laser radiation in a wide range of wavelengths lying in near IR, visible and UV- spectral ranges, including maximum absorption bands of molecular oxygen 3O2 (1.35m, 1.27m; 1.06m; 980-990nm; 780-830nm; 762nm, 660-680nm; 633nm; 532nm; 470nm; 266nm). In experiments the main markers of apoptosis will be monitored, which are the chromatin state of a cell nucleus, manifestations of apoptosis- specific degradation of DNA, and the activity of certain caspases.
Expected Results and their Application
During the course of project implementation, the following is supposed:
Development and creation of an experimental set-up for investigation of the effect of dosed laser radiation with wavelength from near IR to UV (including wavelength coinciding with the absorption maxima of molecular oxygen) on nervous tissue cultures in conditions of their sterility (task 1).
Determination of laser radiation regimes (wavelength, power density, irradiation dose), which bring about apoptosis or modifications of metabolic properties of cell cultures PC12 and C6 (model cultures of neuron and glial cells) (tasks 2 and 3).
Determination of laser radiation regimes (wavelengths, power density, irradiation dose), which influence chemically induced apoptosis in PC12 and C6 cells, i.e. leading to its acceleration or inhibition (task 4).
Selection of the most suitable photosensitizers for PC12 and C6 cell cultures and determination of laser radiation regimes, which evoke apoptisis of the cells under photodynamic effect (task 5)
Determination of the possible regulation of metabolic processes by laser radiation with defined characteristics (wavelength, intensity/power density, duration, and modes of exposure), including those that modify processes of apoptosis just in nervous tissue, will facilitate the following:
elucidation of photosensitive factors and reactions, whose profound study will essentially contribute to the understanding of physical and chemical aspects of apoptosis;
creation of a basis for further research for the regulation of apoptosis of nerve cells at an organism level.The project is classified as "basic research". However, in future the obtained results can underlie applied studies in the treatment of human diseases (brain tumors, Alzheimer’s disease, diseases of prion etiology, etc).
Work on the given project will be performed jointly by teams from the Institute of Physiology and the Institute of Physics of the National Academy of Sciences of Belarus.
The workers from the Institute of Physiology have accumulated considerable experience of research into the structure and function of nervous tissue (including research into cultures of primary dissociated tissue and transplanted cells) under the action of certain factors of chemical and biological nature, in the mechanism and regulation of proteolytic reactions, processes occurring with the involvement of active oxygen species, and original factors regulating proliferation, differentiation, and apoptosis.
The team from the Institute of Physics includes specialists in laser physics, who have considerable experience in the development and research of coherent radiation sources, control of their characteristics, and the production of instruments for medical and biological applications based on laser diodes. The team also includes specialists with much experience in studies of the mechanisms of photodynamic effects of light with the use of molecules of a porphyrinic series as sensitizers. They have created chlorin е6, a new effective drug for diagnosis and photodynamic therapy of cancer, which is now used in medical practice. Experts in spectroscopy of organic compounds will be also enlisted to the project.
Meeting ISTC Goals and Objectives
While working on the project, a team of highly skilled specialists in chemical and biological weapons, and also experts in the field of optoelectronic systems applied in missile technologies, will be reorientated to the development of problems not related to the creation of military objects and technologies.
Even during the first phases of the project the basis for a broad integration of specialists in neurobiology, experimental oncology, biochemistry, physiology and pathology of cells into the international scientific community will be established.
The project will also facilitate further hygienic standardization of laser radiation effects on humans, i.e. it will contribute to applied studies in environmental protection.
Scope of Activities
The fulfillment of the project will take three years. Total project efforts are 186.8 man*months, including 137,5 man*months of total project effort for scientists whose activities were connected with weapons technology.
The purpose of the project will be reached by fulfilling five interralated tasks: production of an experimental installation and metrological support of research at all stages (task 1); fulfillment of a series of experiments on laser radiation effects on model cell cultures of nervous tissue, investigation of possible manifestations of apoptosis and a number of biochemical parameters related to cell metabolism (tasks 2,3); studies of the possible inhibition of already initiated apoptosis under laser radiation (task 4); research of apoptosis of nervous cells at photodynamic effect, i.e. at laser exposure of a cell in the presence of sensitizers (task 5).
The fulfilment of all tasks supposes the close collaboration of the teams of biologists and physicists at all experimental stages and in discussions of the findings.
Role of Foreign Collaborators
In accordance with the scope of the work under the project, the following joint activities with foreign collaborators are planned:
periodic information exchange
collaborator comments to project technical reports (annual and final)
collaborator participation in technical inspection of project activities
preparation of joint publications
collaborator assistance to project participants in attending international scientific conferences, relevant to the project
collaborator participation in seminars and workshops within the framework of the project, conducted at the Institutes of Physiology or Institute of Physics of NASB.
Technical Approach and Methodology
Laser exposure of cell cultures will be performed with the use of fiber optics in conditions of their sterility. The IR, visible and UV spectral ranges, including wavelengths of molecular oxygenium absorption, will be overlapped by radiation of laser diodes on the basis of InGaAs, AlGaAs, InGaAsP, He-Ne laser, Nd3+:KGW and Nd3+:YAG solid state lasers with laser diode pumping, forsterite and titanium sapphire lasers.
The cells will be cultured in RPMI-1640 medium, containing 10-15% embryonic calf serum in a СО2- incubator. The apoptosis specific fragmentation of DNA will be assessed with the help of a TACS apoptotic DNA laccering kit (Sigma). The metabolic processes in a cell will be studied with methods of histochemistry, colorimetry, fluorescence analysis, microphotometry, spectrophotometry, chemiluminescence, affinity chromatography, electrophoresis, gel- and ion-exchange chromatography, radioactive labeling and other biochemical methods. The specificity of binding of photosensitizers to cells and their spectral- energy characteristics will be studied with special equipment by luminescence and flash-photolysis methods.