Plasma activated medium induces apoptosis in chemotherapy-resistant ovarian cancer cells: high selectivity and synergy with carboplatin

: Recently, cold atmospheric pressure plasmas (CAP) have emerged as a promising oncotherapeutic modality, through physical and chemical effects. Here, for the first time, A2780 CP and SKOV-3 cells, relevant to ovarian cancer and GCs as normal ovarian cells were evaluated through CAP directly, indirectly, and concomitant modality of plasma activated medium (PAM) with common drugs to overcome chemotherapy resistance in ovarian cancer. Our results confirm the high potential and the stronger selectivity of PAM in comparison to CAP for the selected cell lines and selectivity mechanism was related to the pH and concentration of H 2 O 2 , NO 2- , and NO 3-reactive species in the plasma stimulated medium. Compared to the combination of common carboplatin (CAR) and paclitaxel (PTX) chemotherapy treatments, the PAM-based treatment is very promising for ovarian cancer treatment. Our data verify that PAM alone and in combination with carboplatin sensitizes cancer cells to carboplatin, inhibits the SOD1 gene, and selectively induces apoptosis accompanied with high expression of p53, Bax, and activation of Caspase-3.


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Collectively, our observations revealed PAM treatment is an innovative and emerging technology for the future combination or multimodal ovarian cancer oncotherapy.

Introduction:
Ovarian cancer (OC) is one of the most widespread gynecological malignancies 1 . Carboplatin and paclitaxel are common chemotherapy drugs used for the treatment of ovarian cancer. Resistance to platins and taxanes persists for most patients, rendering them less responsive to further therapy 2 .
Any new treatment must have little or no adverse side effects and more effective with high selectivity.
Cold Atmospheric Plasma (CAP) where the temperature of heavy particles except electron is close to room temperature, is generated by feeding a noble gas through a pair of electrodes powered with alternating current (AC) or radiofrequency (RF) signals, typically in the kV range. The gaseous and liquid phase of CAP contains a mixture of highly reactive oxygen species (ROS) such as superoxide (O2 − •), hydrogen peroxide (H2O2), hydroxyl radical (•OH), singlet oxygen ( 1 O2), ozone (O3), also reactive nitrogen species (RNS), such as nitric oxide (NO), nitrogen dioxide (•NO2), nitrite (NO2 -), nitrate (NO3 -), peroxynitrite (ONOO − ), dichloride radicals (Cl2 ⋅ − ) and hypochloride anions (OCl − ). Among these long-lived reactive species, hydrogen peroxide, nitrite, nitrate, and peroxynitrite play an important role in the mechanisms of plasma therapy 3-12 . PAM is a new oncotherapeutics agent and produced using plasma irradiation to the culture medium 13 . Today, CAP and PAM have created new opportunities for cancer treatment. The selective killing of cancer cells without damaging normal cells is the most important feature of these methods. Numerous studies showed that both CAP and PAM have a selective effect toward oral cancer 14 , hepatic cancer 15 , skin cancer 16 , glioblastoma 17 , breast cancer 18 20 , neuroblastoma 21 , prostate cancer 22 , head and neck cancer 23 , lung cancer 24 , osteosarcoma 25 , leukemia 26 , and colorectal cancer 27 cells.
Here, we examine the therapeutic efficacy of cold atmospheric plasma in two direct and indirect modes on the important challenge of ovarian cancer oncotherapy. Further, we focus on the selective effect of CAP and PAM alone and in conjunction with chemotherapeutic agents on cisplatin-resistant A2780 CP, SKOV-3 cancer cells, and GCs normal cells. To recognize the mechanism underlying components of CAP and PAM, we measured H2O2, NO2 -, and NO3-as the most important chemical agents. Besides, the biological mechanisms of PAM mediated apoptosis on ovarian cancer cells (A2780 CP, SKOV3) was demonstrated via quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Our data demonstrate PAM is a new and plausible alternative method for chemotherapy-resistance ovarian cancer treatment.

RESULTS:
Cytotoxicity and the Selectivity index: Table 1 shows the IC50 values and selectivity indices of the CAR and PAM against the selected ovarian cells. For two chemotherapeutic agents, the selectivity indices were smaller than the three that specified for the interesting selectivity index. Also, diagram of used treatment methods demonstrated in Fig. S3.

Selectivity induced of CAP and PAM:
The experimental setup of the used CAP has been demonstrated in Figure 1  10% FBS with high selectivity is the best alternative method for ovarian cancer therapy ( Figure   2).

Synergistic effect of PAM, CAR, and PTX:
In this section, building upon the results obtained above (e.g., carboplatin inefficiency, the cytotoxic effect of paclitaxel on normal cells, the negative selectivity of the combination of carboplatin and paclitaxel, and selectivity of cold plasma in cancer treatment (Table 1,    For this purpose, we selected .075 µM (IC50) and 20 µM doses of PTX and CAR, respectively.
We evaluated the synergistic effect of PAM 10% FBS with CAR and PTX performed in three methods. The results show that, although PAM reduces the cytotoxic effect of PTX on normal cells, the combination of PTX with CAR reduces the viability of normal cells. ( Figure. 2.e,f) Our result revealed the combination of CAR and PAM as a promising method, did not reduce the viability of normal cells while cancer cells were affected and the viability of OC cells decreased to <15 % (P <0.001). Therefore, the combination of PAM and CAR can be further explored as a novel method in ovarian cancer treatment (Figure 2.g).

RONS measurement:
Due to the importance of RONS in the mechanism of PAM action, after the plasma irradiation, we measured the amount of hydrogen peroxide, nitrate, and nitrite as long-lived species than other produced species. In the first step, the amount of hydrogen peroxide was measured in RPMI-1640 culture medium supplemented with 1% FBS and 10% FBS under the plasma irradiation for 1 to 4 min and the results are shown in Table 2. The concentrations of hydrogen peroxide, nitrites, and 8 nitrates after 4 min plasma treatment in the culture medium that contains 1% FBS were 10.20 (mg/l), 13.27 (mg/l), and 21.29 (mg/l), respectively.

Morphological changes:
After 24 h of treatment, OC cells stained with H&E, and morphological differences between the treated and control cells were observed by an optical microscope ( Figure 3).

Acridine orange (AO) & propidium iodide (PI) staining:
In the present study, AO/PI is utilized to recognize live, apoptotic, and necrotic cells, which are categorized by their color (green, yellow-green, stained orange and stained red for live, early apoptotic, late apoptotic and necrotic cells, respectively. As shown in Figure 3, normal cells are not affected by the treatment, compared to cancer cells in which apoptosis and necrosis observed.
These results confirm the selectivity of the plasma and improvement of this selectivity in synergy with carboplatin.

Analysis of gene expression by real-time PCR
To clarify the expression of apoptosis related genes (Bax, Caspase 3, P53, and SOD1) for A2780 CP and SKOV-3 cells, we utilized the quantitative real-time PCR (qRT-PCR). In A2780 CP cells, While in PAM (60s) expression of p53, Bax, and caspase-3 genes were (p < 0.01), (p <0.05), and (p <0.05), respectively (Fig. 3.d). Another gene that was evaluated in this study was SOD1, which had a non-significant change in comparison to non-treatment for two cell lines in all treatments ( Figure 3.b,d).

Discussion:
This was the first study of the effect of CAP and PAM alone or in combination with carboplatin and paclitaxel on the A2780 CP and GCs cells. Our results reveal that, while the CAP showed better selectivity than carboplatin and paclitaxel, PAM treatment presented an even stronger selectivity towards OC cells.
Contrary to the previous results suggesting that PAM has similar anti-cancer effects as direct CAP treatment 28 , we conclude that these two methods performed differently. The anti-cancer effect of PAM is based on long-lived reactive oxygen and nitrogen species while, in CAP treatments, in addition to the long-life reactive species, short-life reactive species, and physical effects such as UV radiation, electromagnetic fields, shock waves, and a number of RONS that are still unknown 11 also affect the outcomes 29 . It appears that healthy cells are more sensitive than cancer cells to CAP treatment while cancer cells do not show much difference in their response between the CAP and PAM treatments. One possibility is that healthy cells are more sensitive to the mentioned physical factors in comparison to the cancer cells. This is an important finding that PAM has a very strong selectivity performance than CAP because PAM with greater permeability to the depth of tissue, ability to be maintained, the possibility of synergistic and uniform treatment with conventional treatments such as chemotherapy can complement some shortcomings of direct CAP treatment 30,31 .   that it is required for selective apoptosis induction in tumor cells as well as the production of peroxynitrite and primary oxygen singlet 3,33 . This study confirmed that pH of the culture medium decreased after the plasma exposure 37 . Indeed, the pH of the two culture media decreased after 4 min of the plasma exposure. Our findings on the simultaneous effect of H2O2 and NO2and also, the acidification of our culture medium which is the basic factor to produce primary and secondary 1 O2, are consistent with the selectivity mechanism of Bauer et al. 3,7 During this study, we investigated the action of p53 among tumor suppressor genes. In ovarian cancer cells, p53 is a crucial apoptotic cell death mediator 38 . Mechanisms of apoptosis induction by p53 have been identified in prior studies. One of these mechanisms is that p53 induces apoptosis by transcriptional up-regulation of proapoptotic genes such as Bax. Bax is classified as proapoptosis and a member of the Bcl-2 family proteins which are critical regulators of apoptosis.
Caspase-3 is part of the family of cysteine proteases that cleave certain vital structural proteins and proteolytically activate latent enzymes that contribute to cell apoptosis 39,40 . From these points, the results of this study demonstrate that the PAM with the mechanism presented in the previous 14 section causes the increased expression of p53, Bax, and Caspase-3 genes, inhibit proliferation and induces p53-dependent mitochondrial apoptosis in A2780 CP and SKOV-3 cells (Figure 4. The next part of this study was the study of the effect of PAM with CAR and PTX. In this regard, these drugs were investigated alone and together with PAM. We stress that A2780 CP and SKOV-3 cells are also resistant to carboplatin. Also, CAR did not have a significant cytotoxic effect on selected cells and these cells remained unaffected under the doses that are commonly used in clinical trials. On the other hand, we observed that PTX alone and in the combination with CAR showed negative selectivity so that normal GCs cells are more affected than cancerous cells.
Therefore, this treatment method re-sensitized A2780 CP and SKOV-3 to CAR and induced selective apoptosis in cancer cells.
CAP helps reduce the resistance of cancer cells to chemotherapy drugs 45 , e.g., TRAIL-resistant colorectal cancer cells 46 . Here we examined one of the most important resistance mechanics. We found that all of the combination treatments with PAM restore carboplatin sensitivity in resistant A2780 CP and SKOV-3 ovarian cancer cells. However, only the synergy of PAM and CAR restored carboplatin sensitivity, targeted cancer cells, and left normal cells unscathed. 15 Another promising finding was that the combination of CAR and PAM affects apoptosis regulation as discussed above. Consequently, ovarian cancer cells, A2780 CP, and SKOV-3, which were resistant to carboplatin, follow mitochondrial pathways of apoptosis if treated synergistically with PAM.
Collectively, our strategy to target the resistance mechanisms, in particular reducing the absorption of carboplatin into the tumor and inhibiting apoptosis, was the simultaneous combination of PAM and carboplatin. The mechanism of action of PAM and carboplatin can be attributed to several factors. The first factor is the penetration of carboplatin into the tumor due to the effect of plasma on reducing the pH of the culture medium. Lower pH causes more carboplatin to penetrate the tumor 47 . The other factor is the production of RONS ( H2O2, NO2 -) by CAP in culture media. Another possibility, yet to be confirmed, is the activation of sphingolipids and ceramide production which is a promising strategy for overcoming drug resistance 48 .

CONCLUSION:
To our knowledge, this is the first report that investigates the effect of CAP and PAM on A2780 Ovarian Granulosa cells (GCs) were extracted from NMRI mice 21-24 aging days (Royan Institute). After the mice sacrificed, GCs cells were extracted by a combination of enzymatic and mechanical methods (Fig. S2). GCs cells were cultured in the α-MEM (Alpha Minimum Essential Medium Eagle, Gibco-Invitrogen, Grand Island, USA) culture medium supplemented with 10% of fetal bovine serum (FBS), and 1% penicillin/streptomycin at 37 °C in a humidified atmosphere with 5% CO2 and flow cytometry analysis performed to evaluated isolated cells (Fig. S7).

Plasma device and characterization:
Among the four plasma jets that were made at the start of the work, a jet with the longest plasma plume and lower temperature operated under the low inlet gas and voltage conditions was selected (Fig. S1). We used four of the plasma therapies included, direct plasma exposure contains 1% FBS (direct 1% FBS), direct plasma exposure contains 10% FBS (direct 10% FBS), PAM 1% FBS, and PAM 10% FBS. In the direct exposure method, we set cells in contact with CAP at different treatment durations ranging from 0 to 240 s. Also, to prepare PAM, we exposed a fresh culture medium to the CAPJ similarly to the CAP treatment conditions. The distance between the 96 well cell culture plates and the plasma jet was kept constant at 10 mm during the experiment. Further details of the plasma device operation and diagnostics can be found in Supplementary Information   (Figs. S5, S6 ).

Cell viability assay:
The viability of cells after treatment with drugs and plasma in conditions mentioned in the above sections was assessed using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) For the measurement of H2O2, NO2 -, and NO3in the culture medium after plasma exposure, we used a colorimetric assay. Further details of the assay can be found in Supplementary Information. Hematoxylin and eosin staining: 19 Hematoxylin-eosin staining was performed to the check morphological change of normal and cancer cells, 24 h after treatment using a light microscope (Zeist, Germany). Briefly, cells were washed 3 times with distilled water, hematoxylin (Bio-Idea, Iran) dye was added to the cells, and after five minutes the cells were again washed with distilled water, then eosin (Bio-Idea, Iran) added to cells for one minute. As a final step, we fixed the cell used alcohols 70%, 96%, and 100% and finally observed changes of them with a microscope.

Detection of apoptosis by dual acridine orange/ propidium iodide staining:
In this study, we performed AO/PI staining to observed apoptosis and necrotic cells 24 h after treatment. In brief, we removed the culture medium and cells trypsinized, then Acridine orange (Bio-Idea, Iran) (100 μg/mL) and Propodium iodide (Bio-Idea, Iran) (100 μg/mL) (1:1) added to cells. The cells were immediately moved on to slides and observed under a fluorescence microscope (Zeist, Germany) for the evaluation of the cells undergoing apoptosis or necrosis.

Selectivity index calculation:
The selectivity index corresponded to the IC50 value determined for the activity of carboplatin (CAR) and paclitaxel (PTX) on GCs cells divided by the IC50 determined for ovarian cancer cells.
Our basis for specifying the selective effect was the selectivity index specified by Bézivin et al. 49 Statistical analysis: Statistical analysis was performed using Graphpad Prism 8 (GraphPad Software, CA). The results are expressed as the mean ± standard deviation (SD) of at least three independent experiments were performed in triplicate. One-way ANOVA and Two-way ANOVA analysis of variance and Tukey's and Dunnett's post hoc multiple-comparisons tests were performed to analyze differences