Hyaluronic Acid Improves Hydrogen Peroxide Modulatory Effects on Calcium Channel and Sodium-Potassium Pump in 4T1 Breast Cancer Cell Line

Maintaining homeostasis of ion concentrations is critical in cancer cells. Under hypoxia, the levels of channels and pumps in cancer cells are more active than normal cells suggesting ion channels as a suitable therapeutic target. One of the contemporary ways for cancer therapy is oxidative stress. However, the effective concentration of oxidative stress on tumor cells has been reported to be toxic for normal cells as well. In this study, we benefited from the modifying effects of hyaluronic acid (HA) on H2O2, as a free radical source, to make a gradual release of oxidative stress on cancer cells while preventing/decreasing damage to normal cells under normoxia and hypoxic conditions. To do so, we initially investigated the optimal concentration of HA antioxidant capacity by the DPPH test. In the next step, we found optimum H2O2 dose by treating the 4T1 breast cancer cell line with increasing concentrations (0, 10, 20, 50,100, 200, 500, and 1000 μM) of H2O2 alone or H2O2 + HA (83%) for 24 hrs. The calcium channel and the sodium-potassium pumps were then evaluated by measuring the levels of calcium, sodium, and potassium ions using an atomic absorption flame spectrophotometer. The results revealed that treatment with H2O2 or H2O2+ HA led to an intracellular increase of calcium, sodium, and potassium in the normoxic and hypoxic circumstances in a dose-dependent manner. It is noteworthy that H2O2 + HA treatment had more favorable and controllable effects compared with H2O2 alone. Moreover, HA optimizes the antitumor effect of oxidative stress exerted by H2O2 making H2O2 + HA suitable for clinical use in cancer treatment along with chemotherapy.


Introduction
Nowadays, cancer therapy includes surgery, radiotherapy, chemotherapy, hormone therapy, and cytokine therapies. However, none of the methods present a complete recovery procedure and even can damage healthy cells/tissues of the body. This raises the need to understand more about the cancer biology and its cross-talk with the immune system whose regulation or activation can be manipulated to develop a new anticancer immunotherapy approach such as immune checkpoint blockade [1]. Metastatic cancers, such as breast cancer, are much more resistant to treatment, and despite the current improvement achieved in immunotherapeutic approaches, their treatment still remains a challenge. 4T1 cell line originates from mice triple-negative breast cancer with a high metastatic capacity to the lung [2].
It is known that a combination of internal and external factors are involved in the initiation, development, and progression of breast tumors [3,4]. The maintenance of ion homeostasis is crucial for normal cell life. Several pieces of literature have reported that the role of elements such as calcium (Ca 2+ ), sodium (Na + ), and potassium (K + ) in the proliferation, signaling pathways, and metastatic protection of cancer cells is undeniable. On this basis, the homeostasis of these elements is regarded as an important issue in tumor biology. For example, intracellular Ca 2+ plays a prominent role in cell function involved by enzyme regulation, gene activation, proliferation, apoptosis, cell cycle, and production/performance of transmitters/sensors [5,6]. Ca 2+ signaling in cancer is associated with various types of processes in the tumor microenvironment, such as tumorigenesis, proliferation, migration, angiogenesis, and apoptosis escape [7,8]. It has become evident that Ca 2+ is required for activities such as cell motility, cell migration, and remodeling of cytoskeleton and extracellular matrix [9]. In addition, the Na + -K + ATPase pump (NKA) as an integrated protein in the cell membrane can maintain the equilibrium Na + -K + between the two sides of the cell membrane. Homeostasis of such an equilibrium is important in metabolic activities of the cell as well as other cellular processes such as proliferation, motility, and apoptosis [10].
There is a piece of considerable evidence demonstrating that the amount of NKA and Ca 2+ channel is increased to exchange the ions at a higher level than that of the normal cells in cancer cells [11]. Consequently, it can be concluded that the NKA pump and Ca 2+ channels are good targets for cancer therapy. Given the ability of oxidative stress in disrupting these pumps/channels, oxidative stress can be applied to induce tumor cell death. While ROS regulation at a moderate level is crucial to maintain homeostasis of normal cells to function, higher levels of ROS cause damage to them [12]. One of the most convenient sources of ROS is H 2 O 2 . Hydrogen peroxide plays vital roles in pathways which are involved in the regulation of cell proliferation, differentiation, migration, and apoptosis [13,14]. It follows the importance of precise maintenance of ROS level which affects the flexibility of ion channels/pumps such as Ca 2+ and Na + /K + [12]. ROS can increase Na + level in several ways including (a) changing in the voltage of the Na + channel leading to Na + accumulation in the cells [15] and (b) activating the Na + /H + pump promoting Na + into the cell [16]. In a simple word, channel disruption causes failure in cell energy production [17] or inhibition of Ca 2+ extrusion. Additionally, numerous studies have reported dysregulated potassium channel expression in human cancer cases. However, knowledge about the redox modulation of K + channel activity is limited. Oxidative stress potentially influences the expression of KV1.5 potassium channel expression in both physiological and pathological conditions [18]. Preclinical studies conducted on the effects of oxidative stress and free radicals on cancer cells have shown that oxidative stress at the administered concentrations might be a possible explanation for the substantial toxicity associated with the clinical management and cancer treatment. Therefore, new approaches are necessitated to target the NKA pump and Ca 2+ channel using potent agents against cancer with no or least harm for the normal cells. It requires the utilization of reagents mediating the gradual release of free radicals. This would help us to optimize the therapeutic effects of oxidative stress. HA is one candidate to achieve this aim.
To exert and evaluate the effect of oxidative stress on Ca 2+ , K + , and Na + concentrations in 4T1 tumor cells, we utilized H 2 O 2 along with HA with the aim of suppressing Ca 2+ channels and NKA pumping and inducing apoptosis tumor cells. HA along with H 2 O 2 was used to protect normal cells from oxidative stress damages (see Figure 1).

Antioxidant
Capacity of HA by DPPH Assay. The 2,2diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay was performed according to the Blois method with some modifications [19]. Briefly, a 1.0 mM of DPPH radical solution in ethanol was prepared, and then, 1 ml of the solution was mixed with 3 ml of (0, 0.25, 0.5. 0.75, 1%) HA solutions. The absorbance was measured at 517 nm after 30 min. The proportion of DPPH radical scavenging was initially calculated as reported. All experiments were performed three times, and the average values were determined. Determination of intracellular Ca 2+ , Na + , and K +4 T1 cells was cultured in a six-well in the concentration of 1 × 10 6 cells/well, incubated for 24 hrs under the normoxic or hypoxic conditions, and treated with H 2 O 2 or H 2 O 2 + HA for 24 hrs. At the end of the incubation time, the supernatant was discarded and the cells were trypsinized and centrifuged at 1500 rpm for 5 min. 1 × 10 6 cells were collected and washed twice with Ca 2+ , Na + , and K + -free wash solution as previously described by Montrose in 1991 [20]. To prepare the solution, 130 mM tetramethylammonium (TMA) chloride (Merck, Germany, no. 822156), 1 mM MgSO4 (Merck, Germany, no.105886), 2 mM MgCl2 (Merck, Germany, no.874733), 0.1 mM EGTA (Merck, Germany, no. 99590-86-0), and 10 mM HEPES (Merck, Germany, no. 7365-45-9) were dissolved in ultrapure water, and pH was adjusted at 7.4 using TMA hydroxide (Merck, Germany, no. 75-59-2). Afterward, Ca 2+ , Na + , and K + were determined using an atomic absorption flame spectrophotometer (GBC AA 932). The equipment was calibrated with standard solutions recommended by the manufacturer. All the samples were analyzed in triplicate.

Comparison of PD-L1 Level as Malignancy Marker in
Hypoxic and Normoxic Conditions. To determine how hypoxic or normoxic condition affects 4T1 cell line, we used PD-L1 as a marker of malignancy. PD-L1 is one of those markers whose expression is increased under hypoxia conditions and enables tumors to escape from the adaptive immune system [21]. Flow cytometry analysis of 4T1 cells under the normoxic or hypoxic circumstances revealed that the PD-L1 surface expression under the hypoxic conditions significantly increased compared to 4T1 cells under the normoxic condition, as shown in Figure 2. + HA also led to increased intracellular Ca 2+ content in a dose-dependent manner though the extent of increase was about half that of the treatment with H 2 O 2 alone (see Figure 4). Notably, the intracellular Ca 2+ accumulation in H 2 O 2 + HA-exposed cells had a gentler slope in the increment rate than that of the H 2 O 2 -treated cells in either normoxic or hypoxic conditions. In addition, the intracellular Ca 2+ accumulation in hypoxic conditions is lower compared to the normoxic condition. This may be due to the adaptation of cancer cells with their specific hypoxic condition of the microenvironment, as demonstrated in Figure 4.

Evaluation of
3.4. Treatment with H 2 O 2 and H 2 O 2 + HA Affects Na + and K + Levels under Normoxia and Hypoxia. In order to investigate the NKA pump function, we examined the changes in intracellular Na + and K + levels. As shown in Figure 5, treatment of cancer cells with increasing concentration of H 2 O 2 under normoxic conditions led to increment in intracellular K + and Na + levels as well. However, the rate of increase in K + 3 Oxidative Medicine and Cellular Longevity was far greater than in Na + . Instead, cancer cells treated with H 2 O 2 + HA under normoxic conditions had lower intracellular K + and Na + accumulation levels.
The results under hypoxic conditions were similar to those of normoxia. Treatment of cancer cells with H 2 O 2 increased the intracellular Na + and K + levels in a dose-dependent manner. In contrast, treatment of the cells with H 2 O 2 + HA caused milder changes compared to the treatment with H 2 O 2 .
Overall, the comparison of intracellular Na + and K + in cancer cells under hypoxia and normoxia showed that the cells under hypoxia were more resistant to H 2 O 2 and H 2 O 2 + HA treatment than normoxia (see Figure 5).

Discussion
Hypoxia is one of the important features of solid tumors. Several mechanisms are presumed to involve in the development of hypoxic conditions within the tumor foci. They include limited perfusion and/or delivery of O 2 delivery [22]. Tumor cells adapt to persistent hypoxic and harsh conditions and consequently become more invasive and metastatic. This is evidenced by the treatment resistance of tumor cells to a number of anticancer agents. Normal cells typically die in hypoxic conditions while tumor cells adapt to hostile hypoxic microenvironments and remain viable due to hypoxia-mediated proteomic and genomic changes within tumor cells [23].
Despite significant advances toward cancer biology in the last decade, the survival rate of cancer patients has remained insignificant. The approximate failure of the treatment procedure and also the increased cancer cell resistance might be due to several reasons such as (1) hypoxic condition in a cancer microenvironment that plays an essential role in  Oxidative Medicine and Cellular Longevity tumor survival and resistance, (2) increasing the number of pumps/channels on cancer cells to bypass the apoptotic pathways, and (3) inefficiency of anticancer drugs because of the host condition and side effects. The development, survival, and progression of tumors depend on a combination of internal and external factors of which is the balance of ions [3,4]. Maintaining the ionic homeostasis and optimal electrical potential is vital for survival in cell biological settings. On this basis, a new approach of drug designation has been developed focusing on ion transport pumps/channels in cell membranes to remove cancer cells. Oxidative stress is one of the novel approaches for cancer therapy. In this regard, many points have not yet been addressed and examined about the pathophysiological effects. This study was based on the premise that ROS kills cancer cells by disrupting the ion transport pumps/channels in cancer cell membranes and eventually induce apoptosis and death in cancer cells.
There are currently therapeutic approaches known as radiotherapy and photodynamic therapy that function based on the production of oxygen radicals within cancer cells [24]. Notably, pretreatment with H 2 O 2 in radiotherapy can optimize the cancer microenvironment and improve the efficiency of clinical outcomes [25]. In this approach, ROS production is the mechanism by which apoptosis is induced in tumour cells [24,[26][27][28][29]. Given the point that cancer cells are highly dependent on ROS homeostasis compared to normal cells, they are more sensitive to further variation in their redox homeostasis compared to normal cells. This is because ROS can act as a double-edged sword and affect tumour cells more than normal cells [30]. Presumably, H 2 O 2 kills tumour cells via direct induction of ROS production in cancer cells and inhibition of antioxidant machinery as a defence mechanism of cancer cells. The applied concentration of H 2 O 2 to kill tumour cells should be optimized depending on the antioxidant potential of cancer cells [31,32]. Previous studies utilized H 2 O 2 as a radiosensitizer material and even H 2 O 2 along with sodium hyaluronate have further been used as a radiosensitizer. These approaches are targeted to inactivate cell antioxidant enzymes and produce oxygen from H 2 O 2 to decrease hypoxia [33,34]. Given the important role of oxygen deprivation in tumour adaptation and development, it has been proposed that oxygenation can restore health by destroying cancer cells. Supporters of oxygen therapy claim that low levels of oxygen enable tumour cells to adapt and thrive. Accordingly, oxygenation of tumour cells interferes with their proteomic and genomic changes and destroys them. Hydrogen peroxide is one of the oxygenating agents. The initial idea for the medical use of hydrogen peroxide  5 Oxidative Medicine and Cellular Longevity dates back to decades ago when antineoplastic hydrogen peroxide was observed [35][36][37][38]. However, claims that hydrogen peroxide therapy can increase cellular levels of oxygen have been a matter of debate. Since hydrogen peroxide decomposition and the type of product mainly depend on the microenvironment [39], it may produce OH radical or water and oxygen. On the other hand, HA is one of the chemicals which facilitate the production of oxygen from hydrogen peroxide. Accordingly, it has been shown that hyaluronic acid, commonly used as a drug delivery vehicle [40], is capable of increasing the toxic effects of the oxidative stress mediated by H 2 O 2 on tumour cells [41]. This is consistent with the fact that CD44, as major receptor HA, is abundantly expressed on cancer stem cells [42] which facilitates targeting of toxic effects of H 2 O 2 on cancer cells. In this study, we utilized hydrogen peroxide along with HA to investigate how it affects 4T1 cell line in vitro. H 2 O 2 could alternatively be a source of free radicals to exert oxidative stress or could be an oxygen-producing source on cancer cells.
Hydrogen peroxide is one of the inhibitors of PI3K/AKT and the PTEN pathway [18]. Cancer cells adapted themselves to hypoxic conditions to escape from the immune response against cancer. Some of these escape mechanisms include the increase in PDL-1 receptor and elevation of NKA pump and Ca 2+ channel levels involved in cellular ion regulation [11]. In this study, the simulation of hypoxic conditions in vivo was confirmed in cancer cells by increasing the PDL-1 expression. Previous reports have shown that Ca 2+ plays a prominent role in tumor proliferation, angiogenesis, invasion, and apoptosis by remodeling of cytoskeleton and extracellular matrix [7,8,43]. Ca 2+ homeostasis is regulated directly and indirectly by different molecules [7,26,28]. ROS disrupts these molecules, and subsequently, the membrane channels lose their regulatory function. As a result, disruption of these channels and related molecules changes intracellular Ca 2+ level, increases Ca 2+ uptake by mitochondria, and triggers apoptotic pathways leading to cancer cell death. Our results also showed that treatment of cancer cells with H 2 O 2 under normoxic and hypoxic conditions increased intracellular Ca 2+ level suggesting that the Ca 2+ channels were disrupted. Interestingly, HA softened the H 2 O 2 effect and made a gradual release of free radicals under both hypoxic and normoxic conditions. ROS inhibits and disrupts the NKA pump and increases the Na + levels in the cell in several ways including (1) changes the voltage of the NKA pump [15], (2) activates the NKA pump and releases Na + into the cell [44], (3) degrades NKA proteasomal complex resulting in reduced expression of the  Oxidative Medicine and Cellular Longevity pump at the membrane surface [45], and (4) activates protein kinase C (PK C) which induces NKA phosphorylation and membrane depolarization [46,47] all of which lead to cell death. However, knowledge about the redox modulation of the K + channel activity is limited. It has been shown that an increase in calcium can alter the K + channels and increase K + in the cell. Additionally, current data on the effect of oxidative stress on Ca 2+ -activated K + channel (BK Ca ) activity in vascular smooth muscle cells suggest that O 2 and H 2 O 2 enhance the BK Ca channel activity [48]. It has also been shown that oxidative stress (O 2 and H 2 O 2 ) increases the K ATP channel activity in cardiac myocyte vasculature [49]. Moreover, enhanced ATP-dependent K + channel activity by H 2 O 2 has also been observed in rabbit airway smooth muscle [50]. Our study showed that treatment of cancer cells with H 2 O 2 under normoxia or hypoxia led to an increased intracellular Na + and K + in a dose-dependent manner indicating impaired NKA pumping. In contrast, treatment of cells with H 2 O 2 + HA made these changes gradual. This again confirmed the modifying effects of HA on the oxidative stress capacity exerted by H 2 O 2 consistent with previous reports in other settings [51]. Unique HA properties include biocompatibility, nontoxicity, and biodegradability which make it suitable to be applied in biomedical applications [52,53].
Previous studies supported that H 2 O 2 increases cell death induced by inhibition of PKCε, PI3K, pAKT, pJAK-2, and pSTAT3 [54]. H 2 O 2 also prevents the interaction of AP-1, NF-κB, and STAT3 transcription factors with DNA [55,56]. Our results on the effect of H 2 O 2 on the NKA pump and Ca 2+ channels can also be added to the above list as the cause of apoptosis induction by H 2 O 2 in cancer cells.
Our results demonstrated that the control cells showed higher K + and lower Na + concentrations which is supported by previous studies. Increasing evidence suggests that ion channels and pumps not only regulate membrane potential, ion homeostasis, and electric signaling in excitable cells but also play important roles in cell proliferation, migration, apoptosis, and differentiation [57]. In addition, recent analysis has revealed complex interconnections between oncogenic activity, ion channels, hypoxia signaling, and metabolic pathways that are dysregulated in cancerous cells. Normal and tumor cells frequently express distinct pump isoforms. Several changes in the expression of NKA have been observed in cancer cells, such as elevation of activity during the transformation of malignant cells. Roles in cell survival, proliferation, adhesion, and migration have also been described [58][59][60]. Alterations in K + channel subtypes have been confirmed in breast and colon cancer [61]. To maintain the homeostasis of cancer cells, these subunits increase and in some cases decrease; for example, the K2P channel KCNK9 is overexpressed in breast and lung cancer [62]. Recent work has demonstrated that oncogenic stress increases the KCNA1 expression and promotes its relocation from the cytoplasm to the plasma membrane, which is required for oncogeneinduced senescence. Ectopic expression of KCNA1 inhibits the RAS-induced transformation, which is related to decreased aggressiveness in breast cancer [63]. In this context, variations in the expression of the different subunits of the enzyme compared to the normal tissues have also been described. On this basis, cancer has also been named as a channelopathy. Collectively, based on our knowledge, the differences in the expression, function, and activity of ion channels and pumps in cancer cells depend on different factors. These include types of channels, the differences in pump and ion channel isoforms on cancer cells, the type and class of cancer, the metastasis and invasiveness degree, the stage of cancer, and the duration of hypoxia.
We previously evaluated the effect of H 2 O 2 or H 2 O 2 + HA on 4T1 cancer cells and demonstrated that treatment of 4T1 cells with concentrations higher than 100 μM of H 2 O 2 or 200 μM of H 2 O 2 + HA leads to decreased survival, increased apoptosis, cell cycle disturbance, and decreased expression of MMP2, MMP9, and VEGF genes [26].
Overall, numerous preclinical studies have been done on the effects of oxidative stress on cancer cells with considerations on the dose as oxidative stress at some concentrations may be of higher toxicity and side effects [17]. So finding a way to get this problem under control is essential. In this study, we used HA to overcome toxic and side effects with optimal results. As a result, the effects of H 2 O 2 combined with HA may provide us the opportunity to develop and modify cancer treatment using oxidative stress.

Conclusion
Our finding suggests considering the inactivation of the NKA pump and Ca 2+ channels and triggering of cancer cell apoptosis when using chemotherapeutic agents containing oxidative stress. This study also gives an initial image on the rate and ratio of H 2 O 2 plus HA usage to put control on our targeted selective and effective oxidative stress therapy.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that they have no conflict of interest.