Oxygen Treatment of Cancer-2
Development of Methods and Technologies for Utilization of Oxygen Stable Isotoe O18 as a Cytostatic in a Novel Approach to Cancer Treatment Stage 2: Experimental in Vivo and Vitro Study of O18 Antitumor and Cytotoxic Activity
Tech Area / Field
3 Approved without Funding
NIIIT (Pulse Techniques), Russia, Moscow
- Moscow Oncology Research Institute, Russia, Moscow
- National Institute of Bioscience & Human-Technology, Japan, Tsukuba\nRoswell Park Cancer Institute / Grace Cancer Drug Center, USA, NY, Buffalo
Project summaryThe Project is based on the concept of cytostatic activity of oxygen heavy stable isotope O18 and its utilization as a potential cytostatic for treatment ofchancer put forward by Yu.A.Borschevsky.
Heavy molecular oxygen O218 and heavy water H2O18 are proposed as basic O18 preparations. The heavy oxygen has O18 concentration ranging from 10 to 100%, i.e. 50-500 as great as the background isotope level (0.1995%) in the atmospheric oxygen and natural water habitual for living organisms and resulting from evolution of interaction of geospheres (atmosphere, hydrosphere, stratisphere and biosphere) for 4 billion years starting from formation of prebiological structures (Borschevsky, 1975, 1980, 1981, 1984).
As known, chemical and biochemical properties of molecular oxygen as a basic metabolically active atom and electron acceptor make the basis for bioenergy processes in aerobic organisms and humans. Since relative difference between O16 and O18 isotope masses is 12.5% (weight), substitution of O18 for O16 in inhaled air must lead to a significant deceleration of tissular respiration that is a source of energy and organic substances needed to sustain all forms of life. All biochemical mechanisms of cellular respiration must decelerate and inhibit cell proliferation due to the contribution of O18 to this process. This must lead to activation of regulatory mechanisms of the body and therefore to mobilization of the immune system.
Proliferation of tumor cells with increased growth potential must be inhibited first of all.
It is supposed that the O18 antiproliferative effect can be increased by joint action on living organisms of molecular oxygen O218 and heavy water H2O18 that are the main oxygen-containing environmental components equally needed for normal functioning of living organisms and their environment.
The Council for Coordination of research conducted by institutes and enterprises of the Ministry for Atomic Energy of the Russian Federation together with the ISTC proposed the Project for financing by the ISTC. A Technical Schedule was submitted for consideration which provided for experimental study of O18 antiproliferative effect on stationary human cancer cell cultures, on human tumor bioptic specimens, experimental animal study of O18 antiproliferative effect using transplantable malignant tumors, study of O18 lexicological characteristics and pharmaceutical aspects of O18 effects on the live body, as well as clinical study of tolerance of O18 preparations by humans and O18 effects on tumors of various origin and sites. The complex experimental program was scheduled for 36 months.
As followed from analysis of scientific papers there were practically no publications on O218 and H2O18 kinetic effects in biological and biochemical processes. There was just one report by American investigators E.Boreck and D.Rittenberg (1960) who showed that the rate of growth of bacteria E.Coli B and K12 in H2O18 (92%) was 1.5-fold lower than in normal water H2O16.
In view of the absence of experimental evidence of inhibiting activity of compounds enriched with O18 the ISTC Expert Council recommended to give financial support to Project Stage I of a 12-month duration. The purpose of this stage was to demonstrate experimentally antiproliferative activity of O18 in vitro on human cancer cell cultures different in origin, growth kinetics and drug sensitivity, included inn the new screening set for potentially antitumor compound; to develop methodological aspects of in vitro study of O18 direct cytotoxicity as an active biophilic element of the environment; to carry out oxygen isotopic, methodological, scientific and technical research needed to solve the Project principal and related problems.
The main methodological problem of the Project Stage II was to create experimental in vitro conditions for substitution of O18 for O16 in all oxygen-containing components of cell metabolism which provided detection and reliable evaluation off-actual antiproliferative effect of O18.
Stage I of the research used heavy-oxygen water H2O18 as the main O18-containing preparation because in vitro tests for antiproliferative activity are based on the use of aqueous culture media.
In 1995-96 the first cytotoxic activity study of oxygen heavy water H2O18 (94.4 atom. % O18, A Matheson, USA Co.) was performed under this Project.
The following stationary cell cultures of human cancer different by origin, growth rate and drug sensitivity were chosen for the Project Stage I in vitro experimental evaluation of O18 cytotoxic activity:
- human ovarian carcinoma cells (line CaOv) with a broad spectrum of sensitivity to antitumor drugs belonging to different classes;
- human pigment melanoma cells (line MS) demonstrating low sensitivity to many antitumor drugs, but able to slow down their proliferation and to differentiate into normal phenotype under the action of some compounds.
Both cultures belong to the new screening system for substances with antitumor potential.
An algorithm was developed for experimental primary in vitro evaluation of direct cytotoxic effect of O18 in the form of H2O18 involving determination of stationary cell growth parameters in order to find minimal time of cell exposure to O18 needed to produce its cytotoxic effect, establishing time-effect and dose-effect relationships and evaluation of cell proneness to antitumor drugs with different mechanisms off action under standard conditions and in combination with H2O18.
A study of tumor cell growth kinetics was performed:
- to obtain information on culture reproductive potential;
- to determine optimal time for study of various drugs on cell-proliferation;
- to detect growth log phase for study of drug effects within thins phase.
The determination of cell growth cycle parameters was needed to establish optimal time of action of the agents studied on cell pision. For instance, cells are known to be more sensitive to many damaging effects during the period of the highest proliferative activity (log-phase). Cell doubling time is needed to calculate correctly the time of action of agents under study (not less than one period of cell doubling), i.e. time needed to produce metabolic effects.
The curves of cell culture growth kinetics obtained in our experiments were typical for cell cultures growing in vitro. However, the experimental cells had different phase duration: CaOv were growing slower than MS which might be important for their sensitivity to effects of some phase-specific agents.
We studied sensitivity of both cell cultures to antitumor drugs belonging to different classes: Vinca alkaloids (vincristine, vinblastine), an anthracycline antibiotic (adriablastin), an antimetabolite (methotrexate).
The effective time interval determined allows possible adaptation changes in the rate of proliferation and H2O18 direct cytotoxic effects to be differentiated.
Our experiments were the first to demonstrate that heavy water H2O18 (O18 concentration 80 atom. %) produced a direct cytotoxic effect, i.e. caused death of 50-70% of tumor cells as shown on the example of two stationary cultures of human malignant cells (CaOv and MS). We studied the effect magnitude with respect to time of exposure (24, 48, 72 hours) at O18 maximum concentration and with respect to O18 concentration (80, 60, 40, 16 atom. %) in the incubation medium at the optimal time of exposure. H2O18 cytotoxic effect reached maximum at O18 concentration in the culture medium 80 atom.%. The effect showed a direct correlation with H2O18 concentration in the culture medium and with time of cell exposure to H2O18.
Our study demonstrated that sensitivity of the two cell lines to O18 was different. At H2O18 maximum concentration (80 atom. %) after 72 h of incubation inhibition of CaOv growth was 65-70%, and that of MS not more than 54%. While the maximum effect on MS was observed already after a 24-h exposure with a large number of completely destroyed cells as discovered microscopically; as concerns CaOv the maximum effect was detected after a 48-h incubation.
Testing of O18 antiproliferative activity was performed using two different methodologies: MTT which detected the fraction of live cells only and the radiometric test which evaluated total (cytostatic and cytotoxic) effect. The testing allowed a reasonable conclusion that O18 produced both cytostatic effect and caused irreversible damage (death) of cells studied.
The difference in the cell sensitivity might be due to specificity of their metabolism which manifested itself in different cell growth rates and sensitivities to the cytostatics. Although it is also possible that the two techniques used in the experiments to evaluate O18 antiproliferative effect might reflect two different processes proceeding in the cells. The inhibition of 3H-TdR uptake in DNA was evidence of cell proliferation slowing down which might result both from the cytostatic and cytotoxic effects of the agent studied. While, as mentioned above, the MTT test detected the live cell fraction only. By these reasons the effect evaluated radiometrically was somewhat higher than that assessed by the MTT test.
Besides a study was performed to evaluate H2O18 effect on cytotoxicity of antitumor drugs cis-DDP and adriablastine at an IC50 concentration. H2O18 concentration in test samples was 16, 40 and 80 st. %. Duration of cell incubation with the agents tested was 72 hours.
The tests demonstrated that H2O18 enhanced 1.5-fold the cytotoxic effects of cis-DDP and adriablastine, the effect being directly related to O18 concentration in cell growth culture. The effect reached maximum (adriablastine 84.9%; cis-DDP 75.8%) at an H2O18 concentration 80 atom. %.
Behaviour of the “concentration-cytotoxic effect” curve in experimental study of effects of heavy oxygen water H2O18 in combination with the antitumor drugs on CaOv cells suggested a variety of targets to study the action of O18 and the cytostatics.
Basing on results of the H2O18 cytotoxic activity study first obtained under this Project we applied (application No.96123930) to the Russian Institute of State Patent Expertise (VNIIGPE) for an RF invention patent for "Tumor cell growth inhibitor" with a conception date of December 25th, 1996; authors: N.LMedvedovskaya (NIIIT), A.B.Syrkin, G.K.Gerasimova, N.K.Vlasenkova, O.S.Zhukova, T.P.Ivanova (EIDTT. CRC RAMS).
The VNIIGPE made a positive decision of February 26, 1997 to issue a RF Patent for the invention as specified in the application.
The Project findings were also presented at the 7th International Congress on Anticancer Treatment (Paris, February 1997).
The factual original findings are presented in full in the Final Project Technical Report.
Our experimental findings demonstrated good prospects for the use of O18 in the form of H2O188 as a potential antitumor agent and the necessity of further, more profound study of cytotoxic and antitumor activities of H2O18 and other O18 compounds on a broader range of human cancer cell lines and on in vivo models (transplantable animal and human tumors). The main emphasis should be made on study of biochemical mechanisms of the O18 cytotoxic effects discovered.
It is important to emphasize that according to S.Z.Roginsky and S.E.Shnol (1961) water molecular weight plays a certain role in cellular regulatory processes. It may be therefore supposed that two isotopic types of heavy water (H2O18 and D2O16) with practically the same molecular weight are identical by effects in biological systems. Analysis of D2O isotope kinetic effects in biological systems (Lobyshev, Kalinichenko, 1978) showed that D2O produced a significant inhibiting effect, and complete substitution of concentrated D2O for H2O was incompatible with life even of simple autotrophic organisms.
It was also established that the higher the organization of a living organism, the more variety in the D2O effects is observed and the more sensitive the living organism is to increase in D2O concentration.
The D2O critical concentration in experiments in vitro is 70% versus 30% in experiments in vivo.
It may be expected that H2O18 will produce antitumor and toxic effects in vivo at a concentration 30 to 40%.
From the standpoint of the last scientific findings on the importance of mechanisms of antitumor drug action on live organisms it seems reasonable to study some aspects of mechanism of H2O18 cytotoxic effect together with further study of H2O18 cytotoxicity spectrum in vivo (murine and human transplantable tumors).
As known, mechanism of antitumor action of all known effective antitumor drugs involves apoptosis, i.e. physiological cell death. This problem arose much interest over the last years due to the discovery of a cell death gene and oncogenes regulating its action.
Normal somatic cells completing their functions or excessive differentiating cells die as a result of apoptosis activation. The cell death activation leads to DNA fragmentation and the nucleus pides into several apoptic bodies while adjacent cells experience no damage.
If mechanism of cell death involves necrosis, primary alterations arise on cell surface membranes.
It is of much interest to know mechanisms of both the H2O18 own cytotoxicity and H2O18 effect on activity of conventional anticancer agents.
Simple morphological techniques can determine the mechanisms of cell death which may further be verified at biochemical level.
H2O is the principal component of cell environment that is a place where many enzymatic reactions occur including those with oxygen. Therefore, there are reasons to suppose that the alteration of H2O isotopic composition (H2O enrichment with O18) will inhibit considerably mitochondrial functioning, in particular oxygen consumption. It is of interest to study H2O18 effect on cytochrome system and rate of oxidation of various substrates in cell mitochondria.
The above mentioned suppositions determined the main objectives and tasks of Stage II of the Project study.
1. To evaluate spectrum of H2O18 cytotoxic activity against stationary human and animal tumor cell cultures.
2. To study mechanism of death under the effect of H2O18.
3. To study H2O18 effect ongrosowth oftransplantable animal tumors of different origin and drug sensitivity as well as H2O18 effect on activity of conventional antitumor agents.
1. To study H2O18 effect an growth of human (lung carcinoma A-549, mammary carcinoma MCF-1, colonic carcinoma HT-29, apigment melanoma Bro) and animal (leukemia L-1210) tumor cell cultures.
2. To study mechanisms H2O18 own cytotoxicity and H2O18 effect on activity of conventional anticancer agents from different classes in vivo:
- tumor cell death mechanism (necrosis, apoptosis);
- kinetics of enzymatic actions with oxygen participation in H2O18-containing medium in cell-free systems.
3. To evaluate H2O18 effect on life span of mice bearing transplantable leukemia L-1210. To establish dose-effect and time-effect relationships.
4. To evaluate H2O18 effect on life span of mice bearing transplantable solid tumors. To establish dose-effect and time effect relationships.
5. To evaluate H2O18 effect on growth of human tumors transplanted to athymic mice.
6. To evaluate H2O18 effect on antitumor activity of conventional cytostatics (cis-DDP, adriablastin) in vivo.
The discovery of H2O18 cytotoxic activity against human tumor cells in vitro at Project Stage I suggests that H2O18 should reproduce antitumor effect in vivo. Basing on D2O kinetic effects in biological systems it is reasonable to suppose that H2O18 effect in vivo will manifest itself at lower concentrations as compared with in vitro systems.
Results of Project Stage B will provide starting information on H2O18 toxicity and critical in vivo concentration, on these antitumor effect magnitude and duration, character of cell death, the antitumor effect selectivity in the body and on H2O18 effect on activity of antitumor agents.
All this will be a rationale for development of a novel method for cancer treatment and consideration whether this method may be tested clinically.
The original factual findings of the biological experimental and isotopic study for evaluation of H2O18 cytotoxic and antitumor activity, cell death mechanism, will be valuable information for further study of peculiarities of biological processes in normal and neoplastic cells.
In case of positive results the fulfillment of this Project will contribute of improvement of cancer treatment efficacy that is an actual problem of clinical medicine of great socioeconomic significance.
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