| Title | Effects f Bio-Normalizer Alone or in Combination with Anti-Cancer Drug on Cancer Cells. A Preliminary Study |
|---|---|
| Year | 1996 |
| Author | R. Ortega; M. Simonoff; J.A. Osato; L.A. Santiago |
| Publisher | Conference on Oxidative Stress and Redox Regulation: Cellular Signaling, AIDS, Cancer and other Diseases |
CENBG
UNIVERSITÉ DE BORDEAUX I
CNRS -IN2P3
Effects of Bio-Normalizer Alone or in Combination
with Anticancer Drugs on Cancer Cells. A Preliminary Study
ORTEGA Richard, SIMONOFF Monique
Centre d’Etudes Nucléaires de Bordeaux Gradignan, France.
LE HAUT-VIGNEAU, 33175 GRADIGNAN Cedex, France
Effects of Bio-Normalizer Alone or in Combination
with Anticancer Drugs on Cancer Cells
A Preliminary Study
ORTEGA Richard, SIMONOFF Monique
Centre d’Etudes Nucléaires de Bordeaux Gradignan, France.
INTRODUCTION
Since oxygen radicals have been implicated in the genotoxic events which can initiate and promote cancer development (for review: Ames et al, 1995), numerous clinical trials have been carried out involving anticarcinogenic effects of natural or synthetic antioxidants (for review: German, 1995). Moreover, experimental and clinical studies have also indicated that some natural antioxidants enhance therapeutic treatment of established carcinomas. Mechanisms for antitumor activity of antioxidant compounds generally involve cell differentiation and stimulation of the immunological defense. Recent studies have also revealed some potentiality for direct inhibition of cell growth.
Numerous experimental results are consistent with the assumption that the cellular environment becomes more prooxidizing during differentiation (Allen, 1991; Nagy et al, 1993; Nagy et al, 1995). Generally, it is assumed that antioxidants are inhibitory lo differentiation in many types of cells (Takenaga et al., 1981; Allen, 1991; Allen & Venkatraj, 1992). However, natural retinoids and carotenoids have inhibited the growth of several tumors in vivo due to increasing the rate of differentiation of malignant cells (for review: Cornic et al., 1994; Bertram & Bortkiewicz, 1995). Consequently, all-trans retinoic acid has been used with success in clinical trials, allowing complete remission in acute promyelocytic leukemia patients (Warell et al., 1991). Differentiation is mediated by nuclear retinoid receptors which play an important role in the control of gene expression (Pfhal et al., 1994).
Interestingly, natural antioxidants such as d-limoriene, β-carotene, canthaxanthin, and α-tocopherol have caused regression of tumors, exhibiting similarities in anticancer responses with certain chemotherapeutic agents (Elegbede et al., 1986; Shklar et al., 1987; Schwartz & Shklar, 1988; Schwartz et al., 1989; Shklar et al., 1989; Schwartz & Shklar, 1992). For example, α-tocopherol inhibits tumor cell growth through a process that reduces free radical production (Schwartz el al., 1993). α -Tocopherol bound to membrane associated proteins inhibits the development of peroxidation products. The reduction of peroxidation products directly alter the induction of transcription factors such a c-fos and c–myh, genetic products that promote the proliferation of tumor cells (Amstad et; al., 1990; Stoler, 1991). Available evidence also indicates that α-tocopherol inhibits the expression of mutant p53, while increasing the expression of the wild type form (Schwartz et al., 1993). α-Tocopherol may regulate phospholipid products, ultimately modifying calcium flux and membrane kinase activity. A reduction in kinase activity may affect cyclin activity enhancing antioncogene expression such as p53.
Therefore, strong indications support that natural antioxidants can exhibit antiproliferative activity and would probably yield to new drugs in cancer therapy. Nevertheless, high doses of antioxidant compounds were generally required to obtain cytostatic activity in vitro, and cause toxic side effects in vivo. Thence, the search for natural antioxidants with low toxicity and high free radical quenching activity is or critical interest. In the present study, we have investigated the biological action and toxicity of the natural free radical scavenger Bio-Normalizer (Santiago el al., 199l; Santiago el al., 1992a).
Among the different analysis performed, cell proliferation assays have been carried out, on cisplatin-sensitive and -resistant human ovarian adenocarcinoma cells, in presence of Bio-Normalizer alone or in combination with two anticancer agents (cisplatin and melphalan). As a matter of fact, recent studies suggest that antioxidants may also find a role in conjunction with cytotoxic chemotherapy.
Several natural antioxidant compounds have been used to limit the toxicity due to prooxidant effects of anticancer drugs (Sundstrom el al., 1989; Bienvenu et al., 1992; Stahelin, 1993; Vasavi el a/., 1994). Furthermore, other studies have demonstrated that antioxidants can also enhance the effectiveness of antitumor drugs by increasing the sensitivity of tumor cells to these drugs. For example β-carotene and α-tocopherol were able to potentiate the activity of anticancer drugs such as cisplatin and melphalan (Schwartz et al., 1992; Teicher el at., 1994). This ability could be of particular interest in order to overcome clinical resistance to platinum or alkylating agents.
I MATERIAL and METHODS
1.1. Reagents & Drugs. Owen’s reagent, 3-(4,5-dimethyltiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4·sulfophenyl)-2H-tetrazolium (MTS), and phenazine methosulfate (PMS) were purchased from Promega, melphalan from Sigma Chemical Co., and cis diamminedichloroplatinum(II) (cisplatin) from Laboratoires Roger Bellon.
1.2. Cell lines. Two experimental cellular models were used in this study. IGROVI human ovarian adenocarcinoma cells grow as monoleyers in RPMI 1640 medium supplemented with antibiotics and 10% fetal calf serum. This cell line exhibits an epithelial character, highly tumorigenic properties and a 24 h doubling time (Bénard et al., 1985). A cisplatin-resistant subpopulation, IGROVI-DDP, was established by stepwise escalation with increasing concentrations of cisplatin in the growth medium. Resistant cells were adapted to grow in presence of 1 μg/ml cisplatin. The selection of cisplatin-resistant cells led to a cytologically heterogeneous cell population. Two morphological types coexist; small mononuclear cells, morphologically similar to parental IGROVI cells, and enlarged polynuclear cells with heterogeneous karyotype characteristics. IGROVI-DDP cells, when exposed continuously to 1 μg/ml cisplatin, have an average cell cycle time of 66 h. Growth conditions were at 37°C in a humidified atmosphere of 5% (v/v) CO2 in air. Cells were passaged when they reached confluence by treatment with trypsin/EDT A solutions (0 .05/0.02%), with a transfer factor of ¼.
1.3. Cell proliferation assays. For these experiments, lGROVI and IGROVI-DDP cells were grown in plastic 96-well microtiter plates. Cells in exponentially growth were exposed either to various concentrations of Bio-Normalizer, cisplatin or melphalan alone, or in combination. Incubation times were of 2h for cisplatin and melphalan, and 2 or 24h for Bio-Normalizer. After drug exposure, a recovery period of 48h for IGROVI and 72h for IGROVI-DDP cells was performed, then cells were incubated during 2h with 333 μg/ml MTS and 25 μM PMS. Solutions of reagents and drugs were prepared just prior to addition to assay plates. MTS is bioreduced by cells into a formazan by dehydrogenase enzymes found in metabolically active cells. The quantity of formazan produced is directly proportional to the number of living cells in culture. After 4h of reaction, the absorbance of the formazan was measured at 490 nm with a Bio-Tek Instruments EL 307 microplate reader. 12 wells · were analyzed for each condition of drug exposure in order to obtain a significant mean value of the absorbance. The statistical significance of differences between groups was assessed by the Student’s t-test for paired samples (P < 0.05).
1.4. Chemical analysis. Retinoids, β-carotene and α.-tocopherol, in 10 mg/l Bio-Normalizer solutions in K2HPO4 (5 mM, pH 8), were simultaneously quantified by liquid chromatography (HPLC). The HPLC method was a modification of that described previously, (Simonoff et al/., 1992). Absorbances of α-tocopherol, retinoids, and β -carotene were measured respectively at 292, 325 and 436 nm. Selenium content of Bio-Normalizer was analyzed by particle induced X-ray emission (PIXE), using yttrium as internal standard. The methodology for PIXE analysis in food and biological samples has been described in (Simonoff et al., 1988). Fatty acids in Bio-Normalizer lipid extracts were measured by gas chromatography. Methods of extraction and quantification of fatty acids were based on those described by (Lepage & Roy, 1986).
1.5, Cytoloeicnl observations. IGROVI and IGROVI-DDP cells were incubated during two weeks in presence of 4 mg/ml of Bio-Normalizer in complete culture medium. Cell morphology was examined during this high dose incubation by phase contrast light microscopy. Video images of the characteristic cell types of both cell lines were taken to be compared with controls. Growth characteristics (doubling times) were also controlled.
1.6. Nuce1ear Micro-Probe Analysis. NMPA was accomplished using the CENDG nuclear microprobe. The features of this device, and its capabilities for biological samples analysis, have been previously reported (Llabador et al., 1990; Moretto el al., 1993). The methodology for specimen preparation of IGROVI cells has been described in (Ortega et al., 1994). Platinum and trace metal were quantified in IGROVI cells after 5h incubation with 10μg/ml cisplatin alone, or in combination with 1000 μg/ml Bio-Normalizer. Large square areas (500×500 μm2) were irradiated to assess the average metal composition of each cell line. Samples were irradiated with a 1.0 nA beam of 2.5 MeV protons focused to a 5μm spot. Irradiation times of nearby 5 h were necessary for trace metal determination. High speed scans (I ms per step) were performed to avoid thermal damage. Simultaneous measurements of particle induced X-rays and backscaltered protons were achieved. PIXE spectra were treated with the GUPIX software (Maxwell et al., 1989). Backscattered particles were collected with a 20 mm2 silicon detector, at a scattering angle of 135°. The backscattering data were processed using an in house computer software, specifically designed for the treatment of spectra from biological samples (Moretto & Razafindrabe, 1995).
II RESULTS
II. I. Cell proliferation assays
II.Ia Bio-Normalizer alone. IGROV I and IGROVI-DDP cells were incubated with various concentrations of Bio-Normalizer: 0, 0.1, 1.0, 10, 100 and 1000 μg/ml. At these concentrations, no modification of IGROVI cell proliferation could be noticed after 2h exposure (Fig. I), or after 24h exposure (Fig. 2). In the case of IGROVI-DDP cells, after 2h exposure, proliferation was virtually not modified except a slight enhancement at 1000 μg/ml (Fig.3). After 24h incubation, IGROVI-DDP cell proliferation was slightly enhanced even at low concentrations of Bio-Normalizer (Fig. 4).
Figure 1. Effects of different concentrations of Bio-Normalizer on IGROVI cell growth (expressed as % of control), after a short treatment (2h).
Figure 2. Effects of different concentrations of Bio-Normalizer on IGROVI cell growth (expressed as% of control), after a long treatment (24h).
Figure 3. Effects of different concentrations of Bio-Normalizer on IGROVI-DDP cell growth (expressed as % of control), after a short treatment (2/h).
Figure 4. Effects of different concentrations of Bio-Normalizer on IGROVI-DDP cell growth (expressed as % of control), after a long treatment (24h).
II. 1. b Determination of cisplatin IC50. Cisplatin concentrations inhibiting 50% of cell proliferation (IC50) were determined both on IGROVI and IGROV I –DDP, after 2h of drug exposure (Fig. 5 and 6): for IGROVI, IC50 = 1μg/ml cisplatin; for IGROVI-DDP. IC50 = 85μg/ml cisplatin.
Figure 5. Effects of different concentrations of cisplatin in IGROVI cell growth (expressed as % of control), after 2h exposure.
Figure 6. Effects of different concentrations of cisplatin on IGROVI cell growth (expressed as % of control). after 2h exposure.
11.1.e. Bio-Normalizer and melphalan. IGROVI cells were incubated during 2h with 100 μg/ml melphalan, and simultaneously with various concentrations of Bio-Normalizer (0, 0. 1, l .0, 10, 100, 1000 μg/ml), or pre-incubated with these concentrations of Bio-Normalizer during 24h. During simultaneous exposure with Bio-Normalizer and melphalan, the resultant cytostatic activity of melphalan was significantly modified (Student’s t-test P< 0.05) (fig.12). Indeed, a moderate enhancement of cytoxicity was noticed with a cell proliferation rate ranging from 33 ± 7% to 40 ± 5 %, when cells were simultaneously exposed to 100 μg/ml melphalan and various concentrations of Bio-Normalizer. In comparison the corresponding value was 46 ± 3% when cells were exposed only to 100μg/ml melphaln. However, melphalan cytotoxicity was not modified after 24h pre-incubation with various concentrations of Bio-Normalizer. In this case cell proliferation varied between 44 ± 3 and 54 ± 3 % for melphalan and Bio-Normalizer combinations (46 ± 3 % for melphalan alone) (Fig. 13). This observation was confirmed by long treatment with high doses of Bio-Normalizer (Fig. 14). Effectively, A 24h pre-incubation of IGROV I cells with 4000 μg/ml of Bio-Normalizer in the culture medium did
not change cell proliferation rates after 2 h exposure to 100μg/ml melphalan .
Figure 1 2.Effects of different concentrations of Bio-Normalizer on IGROVI cell growth (expressed as % of control), after 2h with simultaneous exposure to 100μg/ml melphalan.
Figure13. Effects of different concentration of Bio-Normalizer on IGROVI cell growth (expressed as % of control), after 24h pre-treatment and 2h exposure to 100μg/ml melphalan.
Figure 14. Effect of Bio-Normalizer (4000 μg/ml) on IGROVI cell growth (expressed as % of control), after 24h pre-treatment with /without 2h exposure to 100 μg/ml melphalan.0
II.2. Chemical analysis
II.2.a Retinoids, β-carotene and α-tocopherol. The quantities of retinol, β-carotene and α-tocopherol extracted from solutions of 10 mg/l Bio-Normalizer in 5 mM K2HP04 were under the limit of detection. Respective limits of detection for retinol, β-carotene and α-tocopherol were approximately 500, 100 and 3600 μg/g of Bio-Normalizer. Retinoic acid has been detected within lipid extracts at 752 ± 13 μg/g of Bio-Normalizer. Characteristic chromatograms corresponding to HPLC analysis of Bio-Normalizer extracts from 10 mg/ml solutions in K2HP04, and to control solutions with standards of retinoic acid and retinyl acetate are presented in Fig. 15 & 16.
Figure 15. Chromatogram of Bio-Normalizer solution lipid extract with retinyl acetate tocopherol acetate internal standards, Peak at 1.78 min = retinoic acid 2.35 min = retinyl acetate, 6.42 min = tocopherol acetate.
Figure 16. Control chromatogram with retinoic acid and retinyl acetate internal standards.
Peak at1.79 min = retinoic acid, 2.23 min = retinyl acetate.
II.2.b Selenium. Selenium content of Bio-Normalizer was inferior to 1 μg/g, limit of detection obtained after pre-concentration and PIXE analysis.
II.2.c.2. Fatty acid. The total mass of fatty acids tn Bio-Normalizer is relatively low (inferior to 0.1 %). The main forms encountered in Bio-Normalizer lipid extracts were C 13:0, C14: l, C16:0, C17:0, C18:0,Cl8:1,CI8:2 and C18:3α. (Fig.17). Their quantity within Bio-Normalizer ranged from approximately 10 μg/g (dry mass), for C 18:0 and C 18: 3α, to 150 μg/g for C 14: 1 and C18:2. This composition is classical of plant extracts.
Figure I 7. A typical chromatogram of fatty acids distribution within Bio-Normalizer lipid
extracts. The main components detected are C13:0, C 14:1, C I6:0, C 17:0, C18:0, C18:1, C18:2 and C18:3α.
II.3. Cytological observations. After two weeks incubation with 4 mg/ml Bio-Normalizer, no modification of cellular morphology could be noticed on IGROV I, or on IGROVI-DDP cells (Fig. 18). However, a slight increase in the growth rate of cisplatin resistant cells was remarked, whereas IGROVI growth remained unchanged. Effectively, when IGROVI-DDP cells were exposed continuously to 1μg/ml cisplatin and 4mg/ml Bio-Normalizer, passages occurred after 7 days instead of 9 days. A decreased efficacy in cell separation with trypsin/EDTA solutions was also noticed during continuous incubation with Bio-Normalizer.
Figure 18. Phase contrast light microscopy video image of IGROVI cells (a, b), and IGROVI-DDP (c, d), cultured with Bio-Normalizer free medium (a, c). or with 4mg/ Bio-Normalizer in complete medium (b, d); bar, 50 μm.
II.4 Nuclear Micro-Probe Analysis. Essential trace metal (Mn, Fe, Cu, Zn) and platinum were quantified in IGROVI cells after 5h incubation with 10μg/ml cisplatin with, or without, 1,000μg/ml Bio-Normalizer (Table I). Only one analysis could be carried out on cells exposed to Bio-Normalizer, therefore these results must be considered as preliminary and confirmed by further analysis. According to these first experiment, platinum uptake in IGROVI cells appeared to be reduced by the action of Bio-Normalizer with an approximate decreased of75% of incorporation.
Table I. Platinum and trace metal concentration (μg/g dry mass) in IGROVI cells with/without 5h exposure to 10 μg/ml cisplatin, and. to 10 μg/ml cisplatin + 1 mg/ml Bio-Normalizer.
DISCUSSION
Bio-Normalizer at concentrations ranging from 0.1 to 1000 μg/ml have demonstrated no inhibition of cell proliferation on IGROV I or IGROVI-DDP cells, after a short or a long treatment (2 or 24 hours exposure). This observation suggests that Bio-Normalizer alone does not present any toxicity at pharmacological or supra-pharmacological concentrations on IGROVI or IGROVI -DDP cells. This lack of toxicity was confirmed by long term incubations (2 weeks), of IGROVI cells with high doses of Bio-Normalizer (4 mg/ml). In these conditions no changes were apparent on IGROVl cell rnorphology and growth. These results corroborate the fact that Bio-Normalizer is a natural non-toxic nutritional health food (Santiago et al.,1992b).
However, several other studies have demonstrated that some natural antioxidants can exhibit antiproliferative activity (Elegbede et al ., 1986; Shklar et al. , 1987; Schwartz & Shklar, 1988; Schwartz et al., 1989; Shklar et al., 1989; : Schwartz & Shklar, 1992). It was hypothesized that antioxidants inhibit tumor cell growth through a process that reduces free radical production. Therefore in vitro inhibition of growth is a function of the oxygen state of the tumor cell target. At normal oxygen pressure (like in these experiments 5% C02, 95% air), inhibition of tumor cell survival would required very high levels of antioxidants (Schwartz et al., 1993). Thus it would be particularly important to determine Bio-Normalizer antiproliferative activity on cancer cells in an oxygen-restricted environment. As a matter of fact, the level of reactive molecules should then be controlled by Bio-Normalizer free radical scavenger components, resulting in a decreased expression of transcription factors and membrane kinase activity, and ultimately a decreased cell proliferation. This outcome would represent a fundamental breakthrough as regions of low oxygen (hypoxia) are very common features of solid tumors (Vaupel & Hackel, 1995).
Cisplatin-resistant cells. IGROVI-DDP, exhibited an increased growth rate when incubated with Bio-Normalizer. This phenomenon appeared in short treatment assays at high doses (1000 μg/ml), and was more clearly noticed after long treatments, even at low doses of Bio-Normalizer (0. l μg/ml). These observations may explain the increased growth rate of IGROVI-DDP cells noticed during continuous exposure with 4 mg/ml Bio-Normalizer. As IGROVI-DDP cells are cultivated in presence of 1μg/ml cisplatin, in order to maintain the cisplatin-resistant phenotype, it can be hypothesize that Bio-Normalizer interacts with cisplatin, and decreased its cytostatic effect. Cell proliferation assays with combinations of cisplatin and Bio-Normalizer supported this hypothesis. Simultaneous exposure of IGROVI cells to 1μg/ml cisplatin during 2h (IC50), and 0.1 to 1000μg/ml Bio-Normalizer resulted only in 25% inhibition of cell proliferation. During long treatments, this effect was enhanced and the maximum inhibition was only 12% with 10 μg/ml cisplatin and 10μg/ml Bio-Normalizer, instead of 50% expected when cisplatin was administered alone. The same was true for the cisplatin-resistant cell line IGROV1-DDP. For this cell line, IC50 corresponded to 2h exposure with 85 μg/ml cisplatin. Simultaneous exposure of IGROVI-DDP cells with 85 μg/ml cisplatin and Bio-Normalizer (0.1 to 1000 μg/ml) during 2 hours resulted in a nearly total inhibition of cisplatin antiproliferative effects. Similarly, during long treatments, the inhibitory effect of Bio-Normalizer on cisplatin cytotoxicity was also remarked. Therefore, it can be concluded that Bio-Normalizer interferes in vitro with cisplatin pharmacology, even at low doses, and prevents its cytotoxic effects.
Additionally, preliminary nuclear probe microanalysis results suggested that Bio-Normalizer interacts with cisplatin in the extracellular environment. Effectively, reduced intracellular platinum concentrations were measured in cultured cells after simultaneous exposure with cisplatin and Bio-Normalizer. Protection against organoplatinum toxicity, and more generally against heavy metals toxicity could represent an unexpected application of Bio-Normalizer. It would result of particular interest to continue these analysis in order to confirm the first observation. Effectively, this property would support the efficacy of Bio-Normalizer as a natural health food, enlarging its domain of action to the protection against heavy metals. Moreover, in the case of a strong interaction between cisplatin and Bio-Normalizer involving stable metal-binding, platinum would represent an exogenous tracer that would facilitate the identification of Bio-Normalizer components.
Chemical analysis of Bio-Normalizer revealed: that among the natural antioxidants analyzed such as retinoids, β-carotene, α-tocopherol, or selenoproteins, only retinoic acid was detected. The concentration measured (750 μg/g) is theoretically able to induce retinoic acid associated effects such as activation of cell differentiation. However, IGROVI cell proliferation is not affected by retinoic acid, even at high concentration (Caliaro et at., 1994). Therefore another cellular model should be used to test pro-differentiative effects of Bio-Normalizer.
After 2h of simultaneous treatment with Bio-Norma1izer and melphalan, a slight, but significant, enhancement in melphalan cytostatic effect: was noticed. This phenomenon was remarked even at low concentrations of Bio-Normalizer (0.1μg/ml). The maximum effect was measured at 1000 μg/ml Bio-Normalizer and corresponded 28% enhancement of growth inhibition. However, IGROV I cell proliferation, after melphalan exposure, was unchanged when pre-incubation with various concentrations or Bio-Normalizer were performed. Therefore, further studies seem necessary to confirm the potentiation of melphalan antiproliferative activity by Bio-Normalizer.
CONCLUSION- PERSPECTIVES
In this study we have investigated whether the natural free radical scavenger, Bio-Normalizer, could inhibit in vitro human ovarian carcinoma cells proliferation, and modulate cisplatin or melphalan anticancer activity.
We have demonstrated that Bio-Normalizer exhibits no cytotoxicity on human ovarian adenocarcinoma cells, even at supra-pharmacological concentrations. This lack of toxicity may represent a very positive characteristic if the hypothesis of anticancer activity through reduction of free radical production is confirmed in hypoxic conditions. It is now important to complete the investigation of Bio-Normalizer anticancer capabilities by cell proliferation assays in oxygen restricted conditions. These additional experiments would permit to conclude definitely on the potential role of Bio-Normalizer for inhibition of tumor cell growth.
Bio-Normalizer contains retinoic acid, and maybe other unidentified differentiative compounds, in pharmacological quantity. Differentiation studies should be carried on with adequate protocols and cellular models.
Unexpected interaction of Bio-Normalizer with cisplatin pharmacology results in the inhibition of its cytotoxic action. Therefore, Bio-Normalizer is not able to reverse cisplatin cellular resistance but demonstrates heavy metal protective capabilities. It would result very interesting to identify the mechanism involved in these interference. Preliminary experiments, by nuclear probe microanalysis, on cells incubated with Bio-Normalizer and cisplatin seemed to reveal a reduced uptake of platinum. If this result is confirmed, it will mean that the interaction occurs in the extracellular medium, thus providing evidence for metal chelation properties of Bio-Normalizer. Moreover, the fact that this interaction involves platinum would allow an easier identification of the active component(s) responsible for metal binding, and maybe for the free radical protection, if these two phenomena were linked.
Finally, Bio-Normalizer do not reduce the anticancer activity of alkylating agents such as melphalan. In contrast, a moderate enhancement of melphalan growth inhibition has been observed following simultaneous incubation with Bio-Normalizer. This result could be of major interest in order to modulate alkylating agents cytotoxicity, and reverse cellular resistance. Further studies should be performed on alkylating agents sensitive and resistant cell lines in order to determine the conditions for an optimal combination.
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EFFECTS OF BIO-NORMALIZER ALONE OR IN COMBINATION WITH ANTICANCER
DRUGS ON CANCER CELLS. A PRELIMINARY STUDY.
Richard Ortega*, Monique Simonoff*, J.A. Osato**, L.A. Santiago**
* CNRS URA 451, Centre d’Etudes Nucltéaires de Bordeaux-Gradignan, 33175 GRADIGNAN, FRANCE.
** OSATO Research Foundation, 12 Minami-machi Bairin, GIFU 500 JAPAN.
Experimental and clinical studies have indicated that some natural antioxidants enhance therapeutic treatment of established carcinomas (German, 1995). Natural retinoids and carotenoids have inhibited the growth of several tumors in vivo due to increasing the rate of differentiation of malignant cells (Cornic et al., 1994). Consequently, all-trans retinoic acid has been used with success in clinical trials, allowing complete remission in acute promyelocytic leukemia patients. Natural antioxidants such as dlimonene, β-carotene, cathaxanthin and α-tocopherol have caused regression of tumors, exhibiting similarities in anticancer responses with certain chemotherapeutic agents. Nevertheless, high doses of antioxidant compounds were generally requited to obtain cytostatic activity in vitro, and cause toxic side effects in vivo. Hence, the search for natural antioxidants with low toxicity and high free radical quenching activity is of critical interest. In the present study, we have investigated the biological action and toxicity of the natural free scavenger Bio-Normalizer obtained from Sun O International Inc. Gifu, Japan (Santiago et al., 1991 et 1992, Bernas et al., 1993). We have examined whether the natural free radical scavenger, Bio-Normalizer, could inhibit in vitro human ovarian carcinoma cells proliferation, and modulate cisplatin or melphalan anticancer activity.
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