Ваш браузер устарел. Рекомендуем обновить его до последней версии.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


 

Besucherzahler
счетчик для сайта
Antioxidant activity of DN-1/בבית חולים הדסה DN-1 מחקר של שימוש במרכיב ה/

 
. הינו אחד ממרכיבי הביו אורגנו מינרל קומפלקס, אשר קיים בכל מוצרי ד"ר נונה DN-1
 .בבית החולים הדסה עין כרםDN-1 לפניכם מובאות תוצאות השימוש ב 

 

"Antioxidant activity of DN-1 and its effect on cell proliferation and survival following irradiation or treatment by H2O2'' 
PI: N. Kuchina, A. Vexler, R. Gorodetsky and P. Gurevich
. Radiobiology and Biotechnology Lab., Hadassah University Hospital; 

Several cosmetic products that include a bacterial extract - DN-1 as one
of the main compounds have been developed in Clinica Lenom Ltd. (Israel) under supervision of Dr. Nona Kuchina (Dr. Nona International Ltd., Israel). Clinica Lenom using special methods prepared the DN-1 from red halobacteria - Archea, isolated from Dead Sea water. The aim of this project was to test the DN-1 on the presence of antioxidants and on its antioxidant and other biological activity using normal and tumor cell cultures.

Methods and materials.

Agent. The DN-1 is a red-orange extract from red halobacteria - Archea, isolated from Dead Sea water. This bacterial organic matter includes different kinds of carotenoids, proteins, and other components. The stock solution of the DN-1 was prepared in 7.5% NaCI (pH 7.0 ± 0.5) and then diluted to the following final concentration used in all experiments: 0.1% DN-1 in 0.90% NaCI, 0.3% DN-1 in 0.92% NaCI, 1.0% DN-1 in 0.97% NaCI and 3% DN-1 in 1.12 % NaCI.

Assay for testing antioxidant composition of DN-1 .

Cells. Human skin fibroblasts (HF) isolated from skin biopsies of young normal volunteers andmurine mammary adenocarcinoma cells (EMT-6) used in this project were maintained in DMEM for HF and RPMI-1640 for EMT-6 supplemented by 10°/o fetal calf serum, antibiotics and glutamine. All culture media components were purchased from GIBCO (Grand Island, NY). The cell cultures were maintained at 37°C in a water-jacketed CO2 incubator and harvested by trypsin/versen solution with 1-2 passages per week in a split ratio of 1:10 for fast proliferating EMT-6 tumor cells and 1:4 for normal HF. The 24 hrs cell culture was used in all the experiments.

Colorimetric assay for the number of viable cells and their proliferation. Cell density was evaluated by the CellTitre 96Aqeous colorimetric assay (MTS assay) based on the conversion of MTS to colored tetrazolium salt by cell dehydrogenases. The advantage of this assay with the use of 96 microwell plates is in the ability to test rapidly numerous arms of each experiment in the same conditions. Typically 200 m l with known number of cells from exponentially growing cultures were plated in 96 microwell flat bottom plates (in triplicate). Following 24 hrs in culture (to attach and to resume growth), 20 m I of the drug sample to be tested were added to each well. The cells were exposed to the agent for fixed time (typically 15 min) and then irradiated or treated by different concentration
of H2O2. In most experiments the incubation with the drug was prolonged also after treatment. At the end of the experiment, the number of viable
cells was determined by the MTS assay as follows. 30 m l of freshly prepared mixture of MTS/PMS (CellTitre 96 AQueous Assay by Promega) were added into each well. Following 2 hrs of incubation at 37°C the plates were placed on a mechanical plate shaker of a computerized automatic microwell plate spectrophotometer (Anthos HT-ll, Salzburg, Austria), shacked for 1 min and OD of the dye was read at 490 nm. The measurements were repeated following 4 and 6 hrs of incubation. The time point of the assay with optimal OD readings was chosen to assay the relative cell number. When more than
one time point fitted these criteria the results in the different time points were normalized and averaged. For both cell types used, the OD readings
of the MTS correlated well (r>0.97-0.99) with the number of seeded cells. The effect of the DN-1 on cell proliferation as well as on cell survival following irradiation or treatment with H2O2 was calculated by comparing the density
of similarly treated cell pre-incubated with or without the DN-1 .

H2O2 treatment. Typically following 15 min pre-incubation of cells with fixed DN-1 concentrations at room temperature, different concentrations of strong oxidant – H2O2 were added into the wells. Then the incubation at 37°C was prolonged till the
end of experiment.

Irradiation. The intact and DN-1 pre-incubated cells were irradiated in a dose rate
of 1 Gy/min in microwell plates with a Phillips 250 kVp X-ray machine, installed with a
Cu filter of 0.5 mm. The machine was calibrated
for the field size of interest using both special small ionization chambers and thermo-luminescence dosimetry (TLD).

Statistical analysis. The results for each variant in the different experimental designs were represented as an average of 2 - 4 experiments; each was typically performed in triplicate. Mean values and standard errors were calculated for each point from the pooled normalized data. Statistical analysis of the significance of paired data points was performed with the use of the Mann-Whitney's unpaired test with two tailed P value, assuming equal and unequal variance as dictated by the ANOVA test and the nature of the experiment.

Results.

Antioxidant composition of DN-1 . DN-1 includes two major groups of antioxidants: water-soluble and lipid-soluble. Every group contains couple
of different antioxidants, probably.......(Fig. 1).

Effect of DN-1 on cell proliferation.

The effect of the DN-1 on cell growth was tested on both HF and EMT-6 cells. The agent was added in 4 concentrations (from 0.1% till 3% solution from stock extract) to equal number of cells. Following 3-4 days of cell growth the coif number was measured using MTS colorimetric assay. The effect of
 DN-1 on cell proliferation was evaluated by comparing the cell number with and without added DN-1 .

It was found that the DN-1 did not effect on the proliferation of fast proliferating tumor EMT-6 cells in all concentrations used (Fig. 2).
In contrast, the statistical significant increase of proliferation of slow dividing normal HF (115-140%) was observed following the DN-1 treatment (Fig. 2). The offed of the DN-1 increased with the agent concentration excluding its 3% highest concentration with hyperosmolaric NaCI (1,2%) that may have some inhibitory influence on cell growth.

Possibly the stimulating effect of the DN-1 on cell proliferation of slow dividing cells such as HF may be associated with the reach composition of this produd extracted from halobacteria that survive and proliferate in
strong hyperosmolaric conditions of the Dead Sea water.

Anti-oxidant effect of DN-1 on cells treated by H2O2.

The strong oxidant H2O2 decreased extremely cell survival of both types
 of cultured cells used. The offed was time and dose dependent. In the experiments with short-term incubation it was shown that 5 min incubation of HF with 300 m M H2O2 resulted in ~30% cell survival but more prolonged incubation - 15 min and 30 min produced almost 100% cell death (Fig. 3). In the experiments with long-term incubation the same inhibitory effect of H2O2 was observed yet at very low H2O2concentration of 10 m M (Fig. 4 and 9). EMT-6 cells were found to be less sensitive to H2O2 than HF. In these cells only 100 m M H2O2 resulted in the same inhibitory effect on cell survival in the conditions of long-term incubation (Fig. 10).

A possible antioxidant activity of DN-1 on the cell survival was evaluated on both types of cells. The cells were pre-incubated for 15 min with different concentrations of DN-1 . Then the fixed concentrations of H2O2 were added to the cells and the incubation in these conditions were prolonged till the end of experiment when the number of survived cells was measured by MTS assay.

Pre-incubation of HF with 0.1% DN-1 (Fig. 5) and 0.3°/o DN-1 (Fig. 6) did not protect the cells from inhibitory offed of H2O2. The 1% DN-1 proteds the cells in the case of the 10 m M H2O2 but it was found to be non-effective at higher concentrations of H2O2 
(Fig. 7). More concentrated 3% DN-1 had protective effect not only at 10 m M H2O2 but also in the case of 30 m M H2O2 (Fig. 8). At the highest concentration of H2O2 all used concentrations
of DN-1 were found to be non-effective (Fig. 9).

In the same long-term experiments the protective effect of DN-1 on EMT-6 cell survival was found only at the highest 3% DN-1 that neutralized the inhibitory offed of 100 luM H2O2 (Fig. 10) and moreover of 300 m M H2O2 (Fig. 11).

The mechanism of this protective effect is possibly associated with interaction of antioxidant components of DN-1 with H2O2 in the medium. Another possibility is that
the DN-1 changes the cell membrane and this resulted in higher cell survival.

Effect of DN-1 on irradiated cells.

The radio-protective offed of DN-1 was evaluated on both cell lines - HF and EMT-B. These cell lines are known to differ on their sensitivity to irradiation (Vexler et al., 1998). In these experiments HF were also less sensitive (Fig. 12) than EMT-6 cells (Fig. 19).

To test radio-protective offed the cells were pre-incubated 15 min with different concentrations of DN-1 and then irradiated with the dose of 2, 4 and 6 Gy. In the dose range used in these experiments the survival of HF decreased only on 10-15%. Therefore, it was practically impossible to detect the radio-protective offed of DN-1 on cell survival at all concentrations used (Figs. 13 - 17). But the stimulating effect of the DN-1 on HF proliferation was also observed on the irradiated survived cells (Fig. 18)
and it was similar to this offed on the intad cells (Fig. 2).

Oppositely, the irradiation of EMT-6 cells in the same dose range decreased significantly their survival till ~60% at 6 Gy (Fig. 19). The pre-incubation of EMT-6 cells with the DN-1 did not increase the survival of irradiated cells (Figs. 20 - 24). Also the DN-1 did not stimulate the proliferation of the irradiated survived cells (Fig. 25) that is similar to the absence of this effect on the intact cells (Fig. 2).

One of the possible explanations of the lack of radio-protedive effect of DN-1 may be associated with the fail of this agent to penetrate into the cells and cell nuclei that are responsible for the killing effect of ionizing radiation.

Conclusions.

1. DN-1 contains water-soluble and lipid-soluble anti-oxidant compounds.

2. DN-1 is not damaging agent to cells; only at the highest 3% DN-1 it was found some inhibitory effect on cell proliferation comparing to the offed of 1% DN-1 that may be associated with high concentration of NaCI. 

3. DN-1 increases the proliferation of normal HF intact as well as irradiated and does
not timulate the proliferation of tumor EMT-6 cells. 

4. DN-1 protects both cell lines from the strong oxidant - H2O2

5. DN-1 does not prated cells from radiation in dose range used. 

Suggestions (for company used only)

1. To study antioxidant's activities of DN-1 versus other oxidants. 

2. To study mechanism of stimulating offed of DN-1 on normal cells proliferation. 

3. To test protective offed of DN-1 on cell survival after UV irradiation. 

4. To study the penetration of DN-1 into cells. 

5. To test cell survival after irradiation with long-term preincubation with
 DN-1 .