E STIMATION THE EFFECTS OF PLASMA ACTIVATED MEDIUM ON SOME COMPONENTS THAT RELATED WITH GROWING BREAST CANCER CELL LINES

This study was aimed to assess the influence of plasma activated medium (PAM) on some parameters of breast cancer cell lines (MCF7 and AMJ13) in comparison to normal adipose-derived stem cells (ASCs). First, nitric oxide (NO) production within the used media (RPMI-1640 and MEM) was examined beyond different exposure time (10,15,20


INTRODUCTION
Plasma is the fourth matter of state following solid, liquid, and gas.It's found in two forms; thermal and cold plasma.The nonthermal plasma is a promising arising technology in the medical field as it's an ionized gas near room temperature, with multiple active components, such as the electrons, ions, free radicals, reactive molecules, photons, and ultraviolet radiation (27,8,12).Cold plasma can be used directly and indirectly to stimulate physiological mediators and produce reactive species around these cells.Plasma activated solution can be generated by exposing the liquid medium to plasma gas and retaining some reactive species created by the interaction with ions and electrons.Besides, the liquids that can be used in plasma activated medium (PAM) are: water, biological culture media, and other liquid forms that are used for cancer research, making it a more flexible tool for cancer treatment (15,13,17).Iraqi Cancer Registry survey indicated that breast cancer is the first cancer among women which affects about 32% of the Iraqi population and it was also mentioned that about one-third of the cancers in women is breast cancer.However, the survival percentage is better than other fatal cancers, as the breast tissues are not a vital organ for human survival (4,10).Metabolic reprogramming is considered a hallmark in cancer, as it increases from tumor aggressively.Many cancer cells prefer the anaerobic glycolysis rather than mitochondrial oxidative phosphorylation even with the presence of oxygen (11), as the high rate of glycolysis is considered advantageous to highly proliferative cancer cells for many causes: high ATP enough for rapid tumor growth, production of intermediate like glucose 6-phosphate and pyruvic acid can be used for the anabolism of fatty acids and nucleic acids, and the conversion of pyruvate to lactate which acidifies the microenvironment and makes it inappropriate for the growth of normal cells and promote the invasion of cancer one (28).The conversion of pyruvate to lactate is catalyzed by the lactate dehydrogenase enzyme which is found in all human cells.In addition, its well-recognized marker of tissue damage and can be indicated in serum due to pathological conditions such as cancers or cell necrosis (11,26).This study aimed to target the metabolic pathway of breast cancer through plasma-activated medium treatment.

Plasma jet device
Plasma jet instrument was designed and supplemented from University of Baghdad/College of sciences/Department of physics.In brief tungsten electrode was inserted in quartz tube (16cm) in length with outer and inner diameters of 0.5 and 0.3cm.The distance between the tip of the electron and the opening nozzle is 1 cm and supplied with alternating current (AC) power supply with a voltage of .Argon was the working gas and the feeding was controlled by flow meter which regulated the input of gas to 3 L/min flow rate.(5).

Cell lines cultivation
Table 1 shown the used cell lines and there culturing conditions Preparation of plasma activated medium Three ml of each type of media (RPMI-1640 and MEM) was placed in tissue culture plate of 3 cm in diameter (SPL life science, Korea) treated with plasma jet for different exposure times (5,10,15, and 20 minutes) at 0.5cm distance, left to get cold for 5 minutes at room temperature then 2 ml of PAM were added to the six well plates containing cell lines.(25).

Determination of pH
The hydrogen number was determined after applying both MEM and RPMI-1640 media to plasma jet irradiation at different time intervals (10,15,20, and 25 minutes) by using pH strips (Citotest scintfic.co., Ltd).The strip was emerged in the treated medium for one second and then removed.After 10-15 seconds the strip color were compared with the chart card with different color each one corresponds to specific pH value (21).

Nitric oxide concentration measurement
The concentration of nitric oxide in µmol/L within the cell culture media represented in (RPMI and MEM) were evaluated by NO colorimetric assay kit, following the Elabscince kit assay protocol.Briefly, the absorbance of plasma activated medium supernatant was calculated for the examined groups which were treated at different interval times (10, 15,20,and 25 min) at 550 nm by the ELISA plate reader and the concentration calculated according to the given equation: ( 9)

Statistical analysis
All data in this paper was analysis by graph pad prism software version 9, student t test and two way ANOVAs were used for the analysis .The data expressed as mean ±standard deviation (SD) of at least duplicate (5).

RESULTS AND DISCUSSION Plasma jet impact on the pH of media
Treating the media with argon plasma jet didn't influence the pH of both RPMI-1640 and MEM which was stable at 7.4.In the same context, the temperature remained below 30 ºC which was measured by scanner thermometer gun (Etekcity/ USA), The temperature was above the room temperature at about 2ºC.Together, these data suggest that the buffering capacity of the media is capable to maintain neutral pH in the range 7.2-7.4,which is suitable for the growth of the cells.As well as, addition of sodium bicarbonate to the synthetic media is increases the buffering and to counteract the CO2 effect and reduce the acidic environment resulting from metabolism (28).Prior results confirm that despite the reactive species generation, argon plasma didn't confer any change in the pH of treated RPMI 1640 (14).Furthermore, a Portugalian study indicated that medium irradiation did not result in a significant alteration in pH value which is in the range of 7.45 ± 0.02 at 20 ° C or in the temperature, which remained beneath 30°C (2).Nitric oxide measurement in culture media prior to cell line culturing: The result of nitric oxide (NO) in RPMI was increased proportionally to increasing exposure time from 10 to 25 minutes.Results were (117.9±7.85,129.4±4.35µmol/l) for 10 and 15 min.respectively, with non-significant difference was recorded.Whereas, the results of 20 and 25 min were significantly being differ as (187.5±12.5;p=0.047µmol/l) and (253.7±8.3;p=0.048 µmol/l) respectively.While, different results were shown in MEM medium as the 10,15, and 20 min data were non-significant (88.5±3.5,142±22,170±0.5 µmol/l) respectively.Unlike the 25 min which accompanied with significant differences (220±6;p=0.014µmol/l).Thus, it was suggested that the other reactive species will also be increased linearly.Regarding the concentration of NO in different media, the data of the RPMI medium shows an increase in nitric oxide concentration than MEM medium as shown in table 2 and figure 1.This may be due to the fact that the chemistry of these media is very sophisticated as there are about thirty different compositions in both media including amino acids, serum type (fetal or calf), salt, pyruvate, and another component that contribute to the cytotoxic effect.Consequently, the increase in the organic compound in the media will elevate the cytotoxicity of PAM against breast cancer cell lines.For instance, Kim and Kim, (16) mentioned that anticancer activity differs within the same type of media depending on amino acids composition.In agreement with present finding, Biscop and his group (7), reported that the indirect method is affected by media type and component.

Glucose concentration after PAM treatment
Glucose uptake by cell lines was affected by plasma activated medium treatment as shown in figure 2   Based on the results, the exposure time 20 minutes considered the best for treatment conditions.Since the glucose concentration between 20 and 25 min of incubation with plasma activated medium were non-significant and for economical prescriptive by reducing the exposure time and gas waste.Thus, the results were reinforced by treating the three cell lines for 20 min.The data of MCF7 showed significant differences between treated and non-treated cells in the first 24 hours 105± 9 mg/ml and 35.5±9 mg/ml; p=0.016.Over increasing incubation period, a stronger significant effect was demonstrated, since glucose concentration in the medium was increased in comparison to untreated cells (146.5±5.85 and 16±5.85 mg/ml; P= 0.002).In case of AMJ13 similar significant results was shown in the first day after treatment and glucose concentrations were 55± 25 mg/ml and 128±5 mg/ml in non-treated and treaded cells respectively.After 48 hours' significant alteration was generated with P value of 0.006 and the results were 17± 20.72 mg/ml and 151± 20.72 mg/ml in non-treated and treaded cells respectively, (table 4 and figure 3).In spite there is difference in the amount of glucose amount between RPMI and MEM media (2 and 1 g/ml respectively), the results show no significant difference in glucose concentration between both cell lines either after 24 or 48 hrs (figure 4).48 hrs Cancer cells express plenty of glucose channels on their surface enabling them to activate aerobic glycolysis and providing the cells with required energy.Adhikari and his group (1) found that the uptake of glucose in melanoma cell lines using cold atmospheric plasma decreased up to 80%.Together, these data suggest that low thermal plasma can be used to target these receptors as its capable of oxidizing the lipids in the outer membrane which result in lowering the barrier for glucose transportation across the membrane leading to a reduction in the uptake of glucose and increased its level in the medium (23).The result of glucose concentration in adipose derived stem cells glucose results indicated a significant variation in both incubations intervals and the results were (0±2 vs. 13 ±2; P=0.02) and (0 ± 4.5 vs. 33.5 ± 4.5; p=0.017) for 24 and 48 hours respectively.Based on the finding, normal non-cancerous cells (ASCs) consumed the entire glucose found in the medium under normal conditions.After plasma treatment glucose concentration was slightly elevated after a whole day of incubation.A moderate elevation was demonstrated after two days of treatment.Panina, and his colleagues (22) found that stem cells and multi-linage differentiation cells rely on glycolysis to maintain cells from uncontrolled recruitment as stem cells with adjacent cells serve as lactate source.Moreover, they documented that glycolysis determines the fate and functional activity of adipose cells.

Lactate dehydrogenase enzyme activity
The present study measured the level of LDH in both media supernatant and supernatant of cell lysate to detect if there is a potential membrane damage-causing LDH release into the media.The results of LDH level in MCF7 showed no activity in the untreated cells (supernatant from cell lysate) in both incubations hours.On the other hand, the treated cell showed a highly significant elevation in LDH after 24 hours of incubation (17.75± 0.25; p=0.003), and a significant reduction followed the 48 hrs treatment (12.75± 0.25; p=0.022).Regarding the LDH activity level in culture media, result revealed non-significant results after 24 hrs of incubation (0.03±1 and 4±1).While a significant change was recorded after48 hrs of cell incubation (3.25± 0.34 versus 8.25±0.25;p=0.008) (table 5), figure (5).Lactate dehydrogenase represented high activity in the cell lysate.After whole day incubation, a nonsignificant alteration was detected (177±10.6, 210± 10.6).While, 48 hours of incubation accompanied by significant differences (133± 4.5, 233±4.5;p=0.002).Non-significant changes were shown in both incubation times in the tested media and the results were (8±2.2,16±2.2) after 24hrs and (8±2, 10±2) after 48 hrs respectively The findings suggested that LDH activity was decreased within the cells as the incubation time increased.The possible mechanism of cold plasma on LDH was summarized by Tolouie et al (24), they proposed that prolonged plasma treatment have the ability to alter the 3D structure of protein molecule (LDH) by decreasing α helix turns and increasing the β plated sheet, thus the secondary structure of the protein will be change and eventually, loss of function will result.While the enzyme that was released to extracellular media in the first 24 hours increases which indicates damage in the cell membrane as lactate dehydrogenase enzyme is a cytosolic enzyme that is released to the extracellular matrix only after cell membrane damage, like apoptosis or necrosis (10).Consistent study to these findings produced by (29), they treated cancer cells with cold atmospheric plasma (direct and indirect methods) and revealed that the activity of the enzyme decreases steadily in a dose-dependent manner irrespective of treatment route.

Conclusion
Plasma activated medium proved their capability to effects breast cancer cell line metabolic pathway by decreasing both glucose uptake and lactate dehydrogenase production in a dose dependent pattern.In addition, PAM overcomes the metabolic flexibility of cancer cells and eventually effects the energy production.Nitric oxide was sufficiently produced which is essential for generating the required physiological influences and its production is proportional to the exposure periods.In addition, it was found that PAM may negatively influence the metabolic program of adipose-derived stem cells through the stimulation of obesity, since it increased glucose concentration in the circulation which considered as substrate for fatty acid synthesis.

Figure 1 .
Figure 1.Nitric oxide concentration at different exposure time in RPMI-1640 and MEM medium Table 2. Nitric oxide concentration (mean ±SD) at different irradiation time for two types of media

Figure 2 .
Figure 2. glucose concentration at 10, 15, 20, and25 minutes of two incubation intervals in which (A) represents 24 hours incubation while (B) represent 48 hours of incubation

Table 4 .Figure 3 .
Figure 3. Concentration of glucose in culture media after 24 hrs and 48hrs incubation times

Figure 4 .
Figure 4.This figure shown the comparison between glucose level in MCF7 grown in MEM medium and AMJ13 cell line grown in RPMI-1460 medium treated for 24 and48 hrs Cancer cells express plenty of glucose channels on their surface enabling them to activate aerobic glycolysis and providing the cells with required energy.Adhikari and his group (1) found that the uptake of glucose in melanoma cell lines using cold atmospheric plasma decreased up to 80%.Together, these data suggest that low thermal plasma can be used to target these receptors as its capable of oxidizing the lipids in the outer membrane which result in lowering the barrier for glucose transportation across the membrane leading to a reduction in the uptake of glucose and increased its level in the medium(23).The result of glucose concentration in adipose derived stem cells glucose results indicated a

Table 1 . The used cell lines and their culturing conditions Cell line Cultivation and incubation conditions Origin MCF7 This cell line is isolated from Caucasian women of age 69 years old in 1970. A concentration of 1× 𝟏𝟎 𝟓 cell/ml was cultured in MEM culture medium (Biochrom, German) with the addition of fetal bovine serum 10% (FBS; Biochrom, Germany), 1%penicillin-streptomycin (TROGE, Germany). Incubated at 37°C with 5% CO2.(18) All cell lines were provided by the Experimental therapy department / Iraqi Center for Cancer and Medical Genetics Research (ICCMGR) AMJ13 This cell line was cultured in the Iraqi Center for Cancer Research and medical genetics from70 of age Iraqi female. were cultivated in RPMI-1640 (USA), at a concentration of 1×𝟏𝟎 𝟓 cell/ml, 10% FBS and 1% antibiotic (penicillin- streptomycin) were added. Cells were incubated at 37°C in 5% CO2 (4) ASCs (Adipose-derived stem cells) This cell line was cultured in the the Iraqi Center for Cancer Research and medical genetics. It was cultivated in RPMI-1640 (USA), at a concentration of 1×𝟏𝟎 𝟓 cell/ml, 10% FBS and 1% antibiotic (penicillin-streptomycin) were added. Cells were incubated at 37°C in 5% CO2 (20).
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