The Impacts of Prepared Plasma-Activated Medium (PAM) Combined with Doxorubicin on the Viability of MCF-7 Breast Cancer Cells: A New Cancer Treatment Strategy.

Background
For many years, the chemotherapeutic agent doxorubicin (DOX) has been used to treat various cancers; however, DOX initiates several critical adverse effects. Many studies have reported that non-thermal atmospheric pressure plasma can provide novel, but challenging, treatment strategies for cancer patients. To date, tissues and cells have been treated with plasma-activated medium (PAM) as a practical therapy. Consequently, due to the harmful adverse effects of DOX, we were motivated to elucidate the impact of PAM in the presence of DOX on MCF-7 cell proliferation.


Methods
MTT assay, N-acetyl-L-cysteine (NAC) assay, and flow cytometry analysis were utilized in this research.


Results
The results demonstrated that 0.45 µM DOX combined with 3-min PAM significantly induced apoptosis (p< 0.01) through intracellular ROS generation in MCF-7 when compared with 0.45 µM DOX alone or 3-min PAM alone. In contrast, after treatment with 0.45 µM DOX plus 4-min PAM, cell necrosis was increased. Hence, DOX combined with 4-min PAM has cytotoxic effects with different mechanisms than 4-min PAM alone, in which the number of apoptotic cells increases.


Conclusion
Although further investigations are crucial, low doses of DOX plus 3-min PAM could be a promising strategy for cancer therapy. The findings from this research may offer advantageous and innovative clinical strategies for cancer therapy using PAM.


Introduction
Breast cancer is the most widespread malignancy among females worldwide, accounting for the greatest percentage of deaths (15.5%) from all cancers in women. It was estimated the number of new cases in 2020 would exceed 2.2 million (1,2). Subsequently, it seems obvious that more effective treatments than those currently in use are sorely needed. Current cancer treatments include hormone therapy, chemotherapy, radiotherapy, immunotherapy, and surgery. Some of these treatments have serious deleterious side effects in patients (3)(4)(5).
Anthracycline drugs are one of the most important drug classes in chemotherapy. Doxorubicin (DOX), an anthracycline component derived from Streptomyces peucetius, has been used to treat various cancers including those of breast, head and neck, lung, liver, and ovaries. Numerous mechanisms have been proposed to describe DOX antitumor activity; these include disruption of topoisomerase IIα function, protein and nucleic acid synthesis inhibition, DNA intercalation or alkylation, reactive oxygen species (ROS) production, and interaction with the cell membrane (6,7). However, DOX initiates several critical adverse effects including congestive heart failure (CHF), myelosuppression, and nausea (5,8). Additionally, multidrug resistance (MDR) of malignant cells to DOX remains the most significant barrier to effective treatment.
In 1928, plasma was introduced as a fourth phase of matter (9). This ionized gas is comprised of photons, electrons, ions, and neutrals, which include molecules, excited atoms, and radicals (10)(11)(12). Cold atmospheric plasma (CAP) is a plasma in which the temperature of the heavy particles is between 25 and 45 ˚C (13). Applications of plasma for acne, aesthetics, dental caries, and endoscopic applications are well known (10,(14)(15)(16). Recently, indirect and direct CAP treatment became a new subject in plasma medicine. According to the literature, non-thermal atmospheric plasma (NTAP) can be utilized for cancer treatment with few side effects (9,(17)(18)(19)(20). In indirect CAP therapy, the CAPstimulated solutions (PSS) are applied to affect malignant cell growth in vivo or in vitro by injecting PSS into the malignant tissue. One of the advantages of PSS treatment is that it can be stored for a long time at −80 °C (by optimizing the composition of the solution), so, it can be utilized without dependence on the CAP-producing device (21). Current studies reported that plasma can affect malignant cells indirectly with water irradiated by plasma or previously prepared medium, termed plasmaactivated water (PAW) and plasma-activated medium (PAM), respectively (22). The antitumor properties of PAM are due to its ability to eliminate cell survival elements and generate cell death factors such as singlet oxygen, nitric oxide (NO), superoxide, and the hydroxyl radical. These relatively short-lived death factors can be switched to other relatively long-lived species; for example, nitrate/nitrite (NOx) and hydrogen peroxide (H2O2) (18,23). Thus, PAM treatments may represent valuable and innovative tools for malignant therapy (12).
Due to resistance to DOX and its harmful adverse effects, the scientific community is obliged to design novel cancer treatments. Therefore, we aimed to determine the effect of DOX in the presence of PAM prepared at various time points on MCF-7 cell proliferation by MTT and N-acetyl-L-cysteine (NAC) assays, and flow cytometry. The results of this research could offer advantageous and innovative clinical strategies for cancer therapy in the future.

Materials
The MCF-7 human breast adenocarcinoma cell line was purchased from the Iranian Biological Resource Center (IBRC C10082), Iran.
3-(4,5 dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was acquired from Sigma Aldrich Co., USA. Doxorubicin hydrochloride (a bright red crystalline powder with the empirical formula C27H29NO11 under the trade name Adriamycin) was purchased from Hetero Labs Limited Co, India. The fetal bovine serum (FBS) and trypsin-EDTA were obtained from Gibco, USA. Dimethylsulfoxide (DMSO) and N-acetyl-Lcysteine (NAC) were obtained from Merck, Germany. Modified Eagle Medium without sodium pyruvate (DMEM/F12), streptomycin, and penicillin were obtained from Bio-idea, Iran. The Annexin FITC kit was obtained from IQ product, Netherlands.

Preparation of plasma-activated medium (PAM)
In this study, the plasma jet comprised a pyrex nozzle tube as an insulating shield (OD= 4 mm and ID= 2 mm), and the working gas was fed from the tube tip as previously described (24). The device produced a plasma jet with argon gas (99.99%) flow (flow rate was maintained constant at 2 standard L/min (slm)) by utilizing a voltage of 10 kV at room atmosphere. A 6 mm copper electrode covered the tube and was linked to the power supply. In brief, PAM was produced by exposing plasma to 3 mL of DMEM without FBS and antibiotics in 6-well plates with 35 mm well diameters. The distance between the media surface and the plasma source was 6 mm. The PAM irradiation times were 1, 2, 3, and 4 min. After that Pen Strep and FBS were added, and the PAM was added to cells immediately.

Samples preparation
Log-phase MCF-7 cells were treated with trypsin-EDTA and seeded in 96-well plates. The cells were washed with PBS buffer, 1×10 5 cells/well were seeded into 96-well plates, and 180 µl of growth medium were added to each well. The cells were randomly divided into one control and four experimental groups. The experimental groups were (1)

Cell viability measurements
After 48 h of incubation 0.5 mg/ml MTT (20 μl) was added to the control and experimental cells and the cells were incubated for 3 h in a CO2 incubator at 37 °C. Later, the insoluble formazan formed was dissolved in 100 µl of DMSO and mixed (25,26). The optical density (OD) of each well was measured at 570 nm against a reagent blank on an ELISA reader (ELx808, BioTek Instruments, Inc., USA). Each trial was repeated 4 times.

Morphology alterations research
After 48 h of incubation, the medium was removed from the wells and the cells were washed once with 2 ml of cold PBS buffer (0.01 M, pH 7.4). The cells were photographed on an inverted microscope (INV100; BEL Engineering, Italy), and morphological alterations were determined as previously described (27).

Measurement of reactive oxygen species (ROS)
The effect of DOX plus PAM on ROS generation in the absence or presence of NAC was investigated. NAC powder was dissolved in DMEM, then the cells were incubated with 0-, 3-, 4-, or 5-mM NAC for 3 h in a CO2 incubator at 37 °C. The cells were then incubated with DOX and/or PAM. After 48 h, 20 μl of 0.5 mg/ml of MTT were added to each well and cell viability was determined by MTT assay.

Flow cytometry
Annexin V binding was determined via the Annexin FITC kit. Cells were seeded into 6-well plates at 10 6 cells/well for 48 h, then treated with PAM and/or DOX. The cells were collected by centrifugation for 5 min at 1000 × g and washed twice with PBS buffer (0.01 M, pH 7.4), suspended in 100 µL of Annexin V binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl, and 2.5 mM CaCl2), and double-stained with 5 µL of propidium iodide and 5 µL of FITClabeled Annexin V. All samples were incubated for 30 min in the dark at room temperature and analyzed by flow cytometry on a BD FACSCalibur™ (BD Biosciences Inc., USA).

Statistical analysis
Significant differences were evaluated by t-test using GraphPad Prism Software (Version 8.4.3, GraphPad Software Inc., San Diego, USA). GraphPad Prism also was utilized to plot graphs. The flow cytometry data were analyzed using FlowJo software (Version 7.6.1). All data were reported as means±standard deviations.

Growth rates of MCF-7 cells
We assessed whether DOX and/or PAM affected cell viability. As described in materials and methods, MCF-7 cells were first examined after incubation with increasing DOX concentrations for 48 h. Cell viability decreased in a dose-dependent manner (Fig.  1a). The IC50 (50% inhibition concentration) of DOX was determined to be 0.49 µM. The MTT assay also demonstrated that PAM prepared with increasing irradiation periods reduced cell viability in a time-dependent manner (Fig. 1b). The addition of 1-or 2-min PAM did not affect cell viability. In the followup experiments, cells were incubated in 0.45 µM DOX (less than the IC50) plus 3-or 4-min PAM for 48 h to attain the optimum combination condition that impacted the majority of tumor cells. Cell viability was significantly less in the DOX plus 3-min PAM than in the DOX or 3-min PAM, and in the DOX plus 4-min PAM than in the DOX or 4min PAM (Fig. 1c, p< 0.01 for both). The addition of 1-or 2-min PAM to DOX did not significantly affect cell viability compared to DOX alone (Fig. 1c).

ROS measurements
To explore whether oxidative stress contributed to cytotoxicity, cells were treated with DOX, 3-or 4-min PAM, or DOX plus PAM in the presence of 0-, 3-, 4-, or 5-mM NAC. Pretreatment with NAC inhibited DOXinduced cytotoxicity at all NAC concentrations in a dose-dependent manner (p< 0.05 (for 3-mM NAC) and p< 0.01 (for 4-and 5-mM NAC)) (Fig. 3a). The results were slightly different with 3-and 4-min PAM; 3-mM NAC had almost no effect; however, 4-and 5-mM NAC inhibited PAM-induced cytotoxicity in a dose-dependent manner (p< 0.05 for 4-mM NAC and p< 0.01 for 5-mM NAC) (Fig. 3b). In the follow-up experiments, cells were treated with DOX plus 3-min PAM and DOX plus 4-min PAM in the presence of 5-mM NAC. It was observed that 5-mM NAC significantly inhibited DOX plus 3-min PAM and DOX plus 4-min PAM cytotoxicity (Fig.  3c, p< 0.01 for both).

Effects of DOX plus PAM on cell apoptosis and necrosis
The cells were incubated with 0.45 µM DOX, 3-and 4-min PAM, or DOX plus PAM, and analyzed by flow cytometry (Fig. 4). The early apoptotic cell percentages were dramatically greater in the 0. 45 (Fig. 4g). Surprisingly, the percentage of necrotic cells was dramatically greater in the DOX plus 4-min PAM-treated cells than in controls (44.8 vs 0.28%, p< 0.01), but necrosis was similar to controls for all other incubated cells (Figs. 4g).

Discussion
Non-thermal plasma therapy has been investigated as a useful but challenging cancer treatment. Previous results indicated that the target of direct plasma treatment is limited to superficial lesions. More recently, PAM has been utilized as an indirect plasma therapy, which is more practical than the previous method, because tumor cells inside the peritoneal cavity are wholly and directly subjected to cytotoxic fluids (18,22). On the other hand, despite decades of good results in the application of DOX as a chemotherapeutic agent, this anticancer agent generates cumulative, dose-dependent adverse reactions in patients. Thus, this study focused on elucidating the effects of PAM and DOX combinations on MCF-7 cell proliferation. Chemically active PAM can be generated by NTAP in a culture medium (22). Thus, we first prepared PAM with various irradiation periods by plasma jet. Then, we assessed whether DOX in the presence of PAM could change cell viability. MTT identifies cell mortality at the advanced stage of apoptosis, wherein tetrazolium salt metabolization is diminished. The MTT results demonstrated that PAM prepared with different irradiation periods affected MCF-7 cell viability in a timedependent manner. The 3-and 4-min PAM reduced MCF-7 cells viability to approximately 61% and 53% of control (p< 0.01), respectively. However, in the cases of 1and 2-min PAM cell proliferation did not diminish significantly as compared with the control group (p> 0.05). This observation is consistent with earlier studies, which illustrated that plasma irradiation reduced cell viability in a time-dependent manner (4, 28-32). Adachi et al., (22) also demonstrated that PAM suppressed MCF-7 cell viability. However, it should be emphasized that their study utilized DMEM containing pyruvate and they observed that pyruvate significantly attenuated plasma-induced reductions in cell viability. Thus, in this research, we utilized a medium without added pyruvate. DOX (0.45 µM) combined with 3-or 4-min PAM killed MCF-7 cell efficiently (43% and 38% cell viability, respectively; p< 0.01) than DOX (53% cell viability) or 3-or 4-min PAM alone (61% and 54% cell viability, respectively). These data correlate with those of Ikeda et al. (33), who demonstrated that PAM plus cisplatin at low doses reduced viability of human endometrial carcinoma more effectively than cisplatin or PAM alone.
Cell health can be determined by morphological analysis. It has been widely accepted that DOX can change the cell morphology of cancer cells (34), which confirms our microscopic data. After cells treatment with 3-or 4-min PAM, cell morphology was slightly altered, and some cells became rounded. Several previous studies found the same association between PAM exposure time and cell morphology (30,33,35). Our data correlate with those of Iseki et al. (36) who proved that 3-min PAM can induce the morphological changes in the ovarian clear-cell carcinoma. Treatment with DOX plus 3-min PAM or 4-min PAM changed the cell morphology dramatically. Rasouli et al. showed that the morphology of SKOV-3 and A2780 CP cells significantly changed with the combination of PAM with carboplatin (37), which is completely consistent with our results.
As mentioned above, previous research confirmed that plasma irradiation can induce ROS production (18,23,29). Generally, NAC has been utilized as an antioxidant and can scavenge hypochlorous acid (HClO), H2O2, and hydroxyl radicals (·OH) directly but not superoxide radicals (O2 -). Our results showed that 4-and 5-mM of NAC reduced statistically significant DOX-and PAM-induced cytotoxicity (Figs. 3a and 3b). Interestingly, 5-mM NAC pretreatment significantly reduced the cytotoxic effect of DOX plus 3-or 4-min PAM (p< 0.01). Several studies showed that plasma irradiation can induce ROS production, including H2O2, NO2 − , and NO3 − within cancer cells, and in the liquid phase (extracellular culture medium), and subsequently induce cell death in cell lines, including breast cancer cells (18,30,38). Conway et al.,(39) demonstrated that NAC could not alleviate the cytotoxic effects of NTAP in relatively NTAP-resistant cells, but was protective against toxicity caused by NTAP in NTAP-sensitive cancer cells. This observation is consistent with those of Nakamura et al., (18) and Yan et al., (21) as well. According to the literature, cancer cells pretreated with NAC can only scavenge intracellular, but not extracellular, ROS (21). Increases in intracellular ROS can affect DNA structure, and protein and mitochondrial functions (40). Consequently, even though the exact components of PAM remain unclear, PAM-induced cellular ROS play a crucial role in anti-proliferative effects against cancer cells (18,35). Thus, our results suggest that ROS is one of the effectors in PAM in combination with DOX to abolish MCF-7 cell viability, i.e., MCF-7 cells undergo ROS-dependent cell death in response to PAM plus DOX. It could be hypothesized from our results and previous investigations (29) that due to MCF-7 cells' high metabolic rate, the cells were under high oxidative stress; consequently, they could not tolerate an increase in ROS; thus, cell death occurred after 48 h of incubation with DOX plus PAM.
Malignant cell death may occur for various reasons. Thus, to differentiate between apoptosis and necrosis, flow cytometry analysis was performed. In the flow cytometry assessment, PI is a dead cell marker and annexin V identifies apoptotic cells by binding externalized phosphatidylserine residues (41,42). Apoptosis serves as a critical mechanism to defend organs and tissues from numerous kinds of cell damage and stress. Since apoptosis does not initiate inflammatory responses as is the case with necrosis, apoptosis induction in malignant cells is more beneficial than that of necrosis in cancer therapy (30). The percentages of apoptotic cells were increased by DOX alone (32.08% of cells were Annexin V-FITC + /PI − (p< 0.01) and 15.21% of cells were Annexin V-FITC + /PI + (p< 0.05)) and 4-min PAM (29.09% of cells were Annexin V-FITC + /PI − (p< 0.05) and 14.70% of cells were Annexin V-FITC + /PI + (p< 0.05)). This observation correlates with those of Yoshikawa et al., (32) who demonstrated that the percentage of late apoptotic cells increased significantly in endometrial cancer cells after treatment with PAM. Contrary to our data, Xiang et al., (43) found no observable induction of apoptosis in MCF7 cells after treatment with 5-min PAM. Yan et al., (44) proposed that the anti-tumor capacity of PAM alters with the gap between the media and the source of plasma, i.e., decreasing the gap between the media and the plasma source produces PAM with stronger anti-tumor activity without increasing the treatment time. In the Xiang et al. study, the distance between the medium surface and the source of plasma was 13 mm, however, in our study, this distance was 6 mm. Furthermore, as mentioned above, the addition of pyruvate to the growth medium attenuated PAM-induced reductions in cell viability, and in this research, we utilized a medium without sodium pyruvate. Consequently, this might be another reason for the difference observed between our results and those of Xiang et al. When cells were treated with DOX plus 3-min PAM, the percentage of apoptotic cells increased considerably (41.39% of cells were Annexin V-FITC + /PI − , early apoptotic cells (p< 0.01)) and 15.22% of cells were Annexin V-FITC + /PI + (late apoptotic cells (p< 0.05)). Surprisingly, after treatment with DOX plus 4min PAM, 44.8% of the cells were Annexin V-FITC -/PI + (p< 0.01), which recognizes primary necrotic cells. Thus, our results demonstrated that DOX plus PAM effectively generates cell death in MCF-7 cells; however, this process is irradiation time-dependent. The underlying process of cell death generated by PAM has been examined by many researchers and they assumed that the mechanism is related to increased apoptosis through intracellular ROS accumulation (21,32,45). Other mechanisms proposed by researchers include senescence induction, and reduction in cell surface protein expression; for example, FAK and integrins (46). Earlier studies confirmed that normal cells are mostly unaffected by PAM treatment (30,36), i.e., normal cells are less sensitive than cancer cells (35). Hence, based on our results, although the relative mechanisms need to be explored further, DOX combined with PAM represents an alternative strategy for cancer treatment while having minimal harmful side effects on normal adjacent cells or tissue.
Briefly, we demonstrated that 3-min PAM plus DOX, which induced apoptosis through intracellular ROS generation, had greater antiproliferative effects on MCF-7 cells than did DOX or PAM alone. We also showed that in 4-min PAM plus DOX the percentage of necrotic cells increased dramatically. Hence, DOX combined with 4-min PAM has cytotoxic effects with different mechanisms than 4-min PAM alone, in which the number of apoptotic cells increases. Although further investigations are crucial, PAM combined with DOX could be a promising cancer treatment strategy, contributing to a more positive therapeutic outlook.