Introduction

Cancer is an important health problem affecting many people around the world. For this reason, great interest and economic effort is spent to discover new strategies and methods for the prevention and treatment of cancers worldwide [1]. Although chemotherapy is the main strategy in cancer treatment, drug resistance to chemotherapeutic drugs is one of the reasons that hinder success. While many cancers initially show sensitivity to chemotherapy, drug resistance appears over time in the effectiveness of various mechanisms. Clinically, drug resistance may be present before treatment (internal) or develop as a result of treatment (acquired) [2]. Deaths of among women worldwide result from ovarian cancer which is one of the ten most prevalent types of cancer. Epithelial tumor cancer account for 75% of all ovarian cancers and is the most common ovarian cancer [3].

Iron and calcium are the essential elements in the fulfillment of vital functions of cells. Calcium is a secondary messenger associated with basic physiological role, such as cell viability, migration, cycle control, apoptosis, and gene expression [4]. Most cellular events are controlled by intracellular calcium ion ([Ca²⁺]_i). The intracellular Ca²⁺ concentration is kept at very low levels (~10⁻⁷ mol/L), but the release of a very small amount of Ca²⁺ from the intracellular organelles (~10⁻⁵ mol/L) or the flow of Ca²⁺ into the cell from outside (~10⁻⁵ mol/L) the cell can generate important signals by activating the signal transduction cascades in the cell [5]. In general, opening of permeable ion channels to Ca²⁺ increases [Ca²⁺]_i, while active Ca²⁺ carriers are responsible for returning [Ca²⁺]_i to rest. Intracellular Ca²⁺ homeostasis is regulated through diverse calcium channels, pumps and exchange proteins. In non-excitable cells, ER is the main organelles that store Ca²⁺ in the cell, and the channels that give Ca²⁺ from these organelles into the cell; inositol 1,4,5-triphosphate receptors (IP₃Rs) and ryanodine receptors (RYRs) [6]. RYRs are mainly found in cells that can be stimulated, while IP₃Rs are found in cells that cannot be stimulated. So far, 3 types of IP₃ receptors as IP₃R1, IP₃R2 and IP₃R3 have been defined [4]. Stimuli that belong to different stress elements (e.g., immobilization stress, oxidative stress, and hypoxia) as well as apoptosis may modulate IP₃Rs. Expression of IP₃Rs may be increased or decreased relying on the length and strength of the stress stimuli [7].

RYRs have three isoforms in mammals as RYR1, RYR2, and RYR3. RYR1 is mainly found in skeletal muscle, while RYR2 is found in cardiac muscle. RYR3 is mostly found in the thalamus, hippocampus, corpus striatum and smooth muscle. In addition, RYR3 is found in mammalian skeletal muscle cells during development [5]. Another Ca²⁺ pump in the endo/sarcoplasmic reticulum (ER/SR) membrane is sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA). This pump helps to regulate the concentration of cytologic Ca²⁺ by pumping the calcium directly out of the cell or organelles [8, 9]. In addition, plasma membrane Ca²⁺ ATPase (PMCA) pump in the eukaryotic cell membranes conduce to the regulation of cytoplasmic Ca²⁺ concentration by removing the excess Ca²⁺ in the cell. Na⁺/Ca²⁺ exchange (NCX) proteins remove Ca²⁺ from the cell to maintain cellular homeostasis. Ion channels were shown to act as the main regulators of specific pathways associated with proliferation, migration, or survival in the progression of cancer and also play a role in different types of cancer such as colon, prostate and breast cancer [4].

Iron is an essential trace element and is the most plenty transition metal in the human body. Because of iron’s ability to receive and donate electrons, it is a crucial component of storage and transport molecules and enzymes involved in energy production and intermediate metabolism when converting between iron (Fe³⁺) and iron (Fe²⁺) oxidation states. Since the ribonucleotide reductase (RR) enzyme, which is responsible for the synthesis of deoxyribonucleotides, is iron-dependent, it is vitalin the process of iron cell division [10]. It has also been shown that iron availability regulates other proteins involved in DNA damage detection by modulating the cell cycle, such as murine double minute 2 (Mdm2), growth arrest and DNA damage (GADD45) and p21/WAF1. In addition, iron is the functional component of proteins containing heme and iron-sulfur cluster synthesized in mitochondria. Therefore, iron is an important element for survival, growth, and differentiation of cell [10]. Furthermore, changes are expected in iron metabolism in the tumor cells, which generally have rapid growth rates. Iron homeostasis is very well regulated in the metabolism [2]. The excess or deficiency of iron causes formation of reactive oxygen species (ROS) in the organism and the organism is damaged [2]. The formation of ROS and especially reactive hydroxyl radicals through Fenton reaction causes mutations and severe cellular damage. As a result of these reactions, studies on iron metabolism in cancer cells have shown that tumor cells require higher iron concentrations, and iron uptake protein genes have been shown to be highly expressed [2]. However, the studies are limited on general iron metabolism in drug-resistant tumor cells. In this research, we examined the effect of iron on the gene expression of major channels (IP3R1/2/3, RYR1, and RYR2), pumps (SERCA1/2/3, PMCA1/2/3, and PMCA4), and exchangers (NCX1/2, and NCX3) involved in the calcium regulation process in ovarian cancer cells and cisplatin resistant ovarian cancer cells. Iron was noted to indicate differential effects on the studied genes with a concentration-independent activity. To cope with drug resistance, defining the modelling Ca²⁺ channels/transporters/exchangers can supply a promising chemotherapy for cancer resistance.

In this study, epithelial ovarian cancer (EOC) cells and the same cell line resistant to cisplatin, fifteen calcium-associated genes (IP₃R1/2/3, RYR1/2, SERCA1/2/3, NCX1/2/3, and PMCA1/2/3/4) expressions were examined using the quantitative reverse transcription-polymerase chain reaction (RT-PCR) method.

Materials and methods

Results

Iron treatment of cells

MDAH and MDAH/DDP cells were incubated with iron for 24 h at increasing concentrations of iron. Iron was statistically determined to have no effect on the viability of MDAH and MDAH/DDP cells with the MTT test (Figure 1).

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Figure 1. 

The viability of MDAH and MDAH/DDP cells after treatment with iron for 24 h was determined by performing the MTT test as described in the Materials and Methods section. Values were results from three independent experiments. Student’s “t-test” was done

Expression of Ca²⁺ related genes

Differences in mRNA expression of fifteen genes (IP₃R1/2/3, RYR1/2, SERCA1/2/3, NCX1/2/3, and PMCA1/2/3/4) associated with Ca²⁺ were analyzed by quantitative RT-PCR in increasing iron concentrations in MDAH and MDAH/DDP cells. 2-ΔΔCt values that were calculated from the measurement of Ca²⁺-related mRNA expressions from MDAH and MDAH/DDP cells were compared. A two-fold gene expression difference (P < 0.05) was taken as the cut-off level. At increasing iron concentrations in MDAH and MDAH/DDP cells, mRNA expression levels of IP₃R1/2/3, RYR1 and RYR2 Ca²⁺ channels in the ER and releasing Ca²⁺ out of the cell were compared (Figure 2).

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Figure 2. 

Gene expression of the IP3R1/2/3, RYR1 and RYR2 Ca²⁺ channels in the ER in MDAH and MDAH/DDP cells treated with increasing iron concentrations. The levels of change in mRNA gene expression rates for each sample were determined with respect to actin by RT-PCR method. A: IP3R1; B: IP3R2; C: IP3R3; D: RYR1; E: RYR2

While no change in IP₃R1 gene expression was observed in increasing concentrations of iron in MDAH/ DDP cells, a significant rise in IP₃R1 gene expression was observed in the treatment of MDAH cells with 5 µM, 10 µM and 80 µM iron. While an increase in IP₃R2 gene expression was determined in the treatment of MDAH/DDP cells with 10 μM and 80 μM iron, a significant reduction in iron expression and IP₃R2 gene expression was determined in MDAH cells. In addition, IP₃R2 mRNA level was seen to be significantly higher in MDAH cells than other IP₃R1 mRNA and IP₃R3 mRNA expression levels. There is no change in IP₃R3 gene expression determined in increasing concentrations of iron in MDAH/DDP cells, but an increase in IP₃R3 gene expression was determined in the treatment of MDAH cells with 5, 10 and 20 µM iron (Figure 2).

While an increase in RYR1 gene expression was observed at 10 µM iron concentration of MDAH/DDP cells in iron treatment of MDAH/DDP and MDAH cells, a significant increase in 20 µM iron treatment of MDAH cells and later decrease in both cell groups were detected. On the other hand, no change was observed in RYR2 gene expression in iron treatment of MDAH/DDP cells, while a decrease in RYR2 mRNA expression in MDAH cells at 5 µM, 20 µM and 40 µM was observed. In addition, the examination of the gene expression levels of RYRs in MDAH and MDAH/DDP cells, showed that the level of RYR2 mRNA expression was quite lower in MDAH/DDP cells compared to expression in MDAH cells.

Thapsigargin sensitive SERCAs are responsible for the conservation and regeneration of agonist sensitive internal stores. No change in SERCA1 mRNA gene expression was observed in the treatment of MDAH/DDP cells with increasing concentrations of iron, while an increase in MDAH cells was determined at 10 µM (Figure 3). As a result of the treatment of MDAH/DDP and MDAH cells with increasing iron concentrations, a decrease in SERCA2 gene expression was determined in the treatment of MDAH/DDP cells with 5, 40 and 80 μM iron. However, when SERCA2 mRNA gene expression was examined in non-iron treated MDAH and MDAH/DDP cells, it was found lower in MDAH/DDP cells. In addition, a significant rise in SERCA3 gene expression was determined in MDAH cells in increasing iron concentrations, while no change was detected in MDAH/DDP cells.

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Figure 3. 

Gene expression of SERCA1/2 and SERCA3 Ca²⁺ pumps in the ER at increasing iron concentrations in MDAH and MDAH/DDP cells. The levels of change in mRNA expression rates for each sample were determined with respect to actin by RT-PCR method. A: SERCA1; B: SERCA2; C: SERCA3

NCX proteins send Ca²⁺ into cytosol or organelle using the electrochemical gradient of Na⁺. When NCX1 mRNA expressions in increasing iron concentrations of MDAH/DDP and MDAH cells were examined, no significant change was observed in both cell groups (Figure 4). While no change in NCX2 gene expression was observed in increasing concentrations of iron of MDAH/DDP cells, an increase in NCX2 mRNA expression in MDAH cells was determined in the treatment of MDAH cells with 10 μM, 40 μM and 80 μM iron. In the treatment of MDAH/DDP cells with 5μM and 20 μM iron, a decrease in NCX3 gene expression was detected, while an increase in NCX3 mRNA gene expression was determined in the treatment of MDAH cells with 5 μM iron.

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Figure 4. 

Gene expressions of NCX1/2 and NCX3 Ca²⁺ exchange carriers found in plasma and nuclear membrane at increasing iron concentrations in MDAH and MDAH/DDP cells. The levels of change in mRNA expression rates for each sample were determined with respect to β-actin by RT-PCR method. A: NCX1; B: NCX2; C: NCX3

PMCA is the Ca²⁺ channel protein responsible for keeping the intracellular critical Ca²⁺ concentration low. A significant increase in PMCA1 gene expression was detected in the treatment of MDAH/DDP cells with 10 μM and 20 μM iron (Figure 5). However, an increase in PMCA1 gene expression was detected in the treatment of MDAH cells with 5 μM and 80 μM iron. An increase in PMCA3 expression was determined in the treatment of MDAH/DDP cells with 5 μM, 40 μM and 80 μM iron, while no significant change was detected in the expression of PMCA3 in the treatment of MDAH cells with iron. No change in PMCA4 gene expression levels was observed in increasing iron concentrations of MDAH/DDP and MDAH cells. No change in PMCA2 mRNA gene expression was observed when MDAH and MDAH/DDP cells were treated with iron.

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Figure 5. 

Gene expression of PMCA1/2/3 and PMCA4 channels in the plasma membrane at increasing iron concentrations in MDAH and MDAH/DDP cells. The levels of change in mRNA gene expression rates for each sample were determined with respect to P-actin by RT-PCR method. A: PMCA1; B: PMCA2; C: PMCA3; D: PMCA4

Discussion

Cisplatin is one of the most effective anticancer agents known as cisplatinum or cis-diamminedichloroplatin (II). It has been widely used for the treatment of different types of neoplasms, including head, neck, lung, ovarian, breast, brain, kidney and testicular cancers, and leukemia. In general, cisplatin and other platinum-based compounds are considered cytotoxic drugs that inhibit DNA synthesis and mitosis, killing cancer cells and causing apoptotic cell death. It is characterized by the effect of various molecular mechanisms, production of ROS and lipid peroxidation, stimulation of p53 signaling, and cell cycle arrest, down regulation of proto-oncogenes, anti-apoptotic proteins, and oxidative stress in both internal and external pathways in apoptosis [14]. However, cisplatin chemotherapy also causes important side effects such as hepatotoxic, nephrotoxic, cardiotoxic, neurotoxic and/or hematotoxic damage. In addition, cisplatin resistance may develop, and cancer may relapse in the treatment of some patients with cisplatin [15]. Therefore, cisplatin resistance is a major barrier to successful treatment outcomes and limits its clinical application. Although cisplatin and its derivatives cause various side effects and resistance development in ovarian cancer, they are used in basic therapy. Cisplatin is used with other chemical agents or compounds to overcome barriers to the treatment of ovarian cancer with cisplatin. Therefore, it is very important to explain the resistance mechanism, and the mechanism of action of cisplatin is unknown. In this study, MDAH/DDP cells were treated with increasing iron concentrations to investigate the effect of iron on calcium channels, carriers, and exchange proteins of mRNA gene expressions in ovarian cells in which ovarian cancers developed cisplatin resistance, and 15 genes (IP₃R1/2/3, RYR1/2, SERCA1/2/3, NCX1/2/3, PMCA1/2/3/4) related to Ca²⁺ were examined by quantitative RT-PCR method and compared with MDAH cells.

The ER acts as the main Ca²⁺ storage in the cell. It is also the organelle that regulates ER cell apoptosis, survival, and ATP production. Intracellular Ca²⁺ levels are controlled by Ca²⁺ release from ER and Ca²⁺ entry from outside the cell. When calcium arrived the cytoplasm, Ca²⁺ often occurs complexes with calmodulin to regulate a diversity of kinases and cyclins, which regulate cell proliferation and apoptosis [16, 17]. Ca²⁺ is a regulater for global cellular processes in such a way that any disturbances to Ca²⁺ homeostasis via changings in expression of Ca²⁺ channels and Ca²⁺ binding proteins can disrupt the cell-cycle [18]. Ca²⁺ levels of cytosolic the activity of guanosine exchange factor (GEF), a Ras stimulator, and GTPase activating protein (GAP), a Ras inhibitor. When GEF is activated, Ras stimulates the proliferative mitogen-activated protein kinase (MAPK) pathway. This results in upregulation of cyclin D1 in the cytoplasm and phosphorylation of RB1 and then, release of the E2F transcription factor which initiates the cells transition into S-phase. This relationship between calcium and RB1 show that elevated cytosolic Ca²⁺ levels can regulate activation of the MAPK pathway, causing removal of the G1/S transition. The first stage in metastasis is the loss of cell-cell connections. Focal adhesion kinase (FAK) is a protein tyrosine kinase that increases turnover of cell-cell contacts [19]. FAK is overexpressed in a number of tumors and is a significant indicator of patient survival. Rised intracellular Ca²⁺ upregulates FAK at focal adhesions through phosphorylation by the calmodulin-dependent protein kinase II (CaMKII) [20]. Thus, abnormal signaling resulting in increased intracellular Ca²⁺ levels can lead to a raise in FAK and a higher turnover rate for cell-cell attachments [21].

Under normal conditions, intracellular Ca²⁺ stimulates cell survival; however, increased Ca²⁺ levels under the influence of stimuli such as cisplatin and thapsigargin may trigger apoptosis. The release of Ca²⁺ from ER into the cell takes place by the channels of two different classes, IP₃Rs and RYRs [15]. Luminal ER Ca²⁺ levels are protected with SERCA (SERCA1/2/3) calcium pumps [22]. The activation of phospholipase C-coupled receptors (some G-protein coupled receptors) causes IP₃ formation and then the release of Ca²⁺ from calcium-sensitive channels (IP₃R1, IP₃R2, IP₃R3) in the ER membrane [23]. The cytosolic calcium signal induced by IP₃ is highly dependent on the expression profile of IP₃R and the sensitivity of IP₃R subtypes to IP₃ is also different [24]. In addition, IP₃R regulates cellular functions such as secretion, motion, and autophagy [23]. Gene expression of IP₃R has been shown to decrease in cancer and especially IP₃R3 isoform in glioblastoma, gastric, small, and non-small lung, and colorectal cancer [25]. In addition, IP₃R may play a role in cancer progression and metastasis, but its mechanism of action has not been revealed [25]. In our study, no change in IP₃R1 and IP₃R3 mRNA gene expression was observed in iron treatment of MDAH/DDP cells. However, an increase in the IP₃R1 mRNA level was observed in iron treatment of MDAH cells, while a decrease in the IP₃R3 mRNA level was determined. An increase in IP₃R2 gene expression was determined in iron treatment of MDAH/DDP cells while a significant reduction in iron treatment of MDAH cells was determined. In addition, the comparison of MDAH/DDP cells and MDAH cells without iron conditions showed that IP₃R2 expression was 100 times higher in MDAH cells. These findings suggest that IP₃R2 may have an important role in MDAH/ DDP and MDAH cells in Ca²⁺ homeostasis influenced by iron.

While RYRs, a class of Ca²⁺ releasing channels, are rarely associated with cancer, they were reported to regulate proliferation of melanocytes and migration of T cells and astrocytes [25, 26]. RYR1 is mainly expressed in skeletal muscle and although in small amounts, it is found in the heart muscle, smooth muscle, stomach, kidney, thymus, cerebellum, Purkinje cells, adrenal gland, testicle in the ovaries and B-lymphocytes [27]. RYR2 is mostly found in the heart muscle, while in small amounts, it is expressed in Purkinje cells, stomach, kidney, adrenal gland, ovary, thymus, and lung. RYR3 is expressed in hippocampal neurons, thalamus, Purkinje cells, corpus striatum, skeletal muscle, coronary smooth muscle cells, lung, kidney, ileum, jejunum. High conductivity RYR ion channels can form rapid transient increases of cytosolic Ca²⁺. Analysis of the primary structure of RYRs reveals various functional motifs seen in other proteins; however, the role of these motifs in the function of RYR has not been clarified yet [28]. In this study, an increase in gene expression levels of RYR1 was determined in iron treatment of MDAH and MDAH/DDP cells. In MDAH/DDP cells, there was no change in the effect of iron in the level of RYR2 mRNA, whereas in MDAH cells, the effect of iron was decreased. While differences in RYR2 mRNA gene expression levels are observed in MDAH cells, the absence of a significant change in MDAH/DDP cells indicates that RYR2 is not affected by iron concentrations. However, significant increase in RYR1 expression and subsequent reduction in increasing concentrations of iron in resistant MDAH cells showed that RYR1 can contribute to regulation of Ca²⁺ levels in resistant MDAH cells.

The SERCA pump is from the family of P-type ATPases and functions in intracellular calcium signal and homeostasis. SERCA has 3 isoforms. SERCA1/2 is expressed in some tissues, while SERCA3 is ubiquitous. Change in SERCA expression has been recorded in oral squamous cancer, choroid plexus, thyroid, lung, colon, acute promyelocytic leukemia, cervical and breast cancers [29–34]. During tumorigenesis in colon cancer, SERCA3 gene expression is gradually decreasing, while becoming almost zero in poorly differentiated tumors. Similarly, SERCA3 gene has not been expressed when compared to normal forms such as ductal carcinomas, acute promyelocytic leukemia cells and Burkitt lymphoma cells [33]. When SERCA1/2/3 mRNA gene expressions in iron effect were examined in cisplatin resistant MDAH/DDP cells, there was no change in SERCA1 and SERCA3 mRNA expressions, whereas SERCA2 mRNA gene expression was decreased. In iron treatment of MDAH cells, an increase in SERCA1/3 mRNA levels was determined, while the effect of iron was decreased in the level of SERCA2 mRNA. Changes in SERCA2 mRNA levels in cisplatin resistant cells under the influence of iron suggest that there may be a contribution to SERCA2 in the regulation of Ca²⁺ homeostasis in these cells. It also shows that changes in SERCA1/2/3 mRNA levels in MDAH cells under the influence of iron may play a role in regulating Ca²⁺ homeostasis in ovarian cancer epithelial cells.

There are 3 different isoforms of NCX proteins, NCX1, 2, and 3, that allow Ca²⁺ to be excreted from the plasma membrane. Pelzl et al. [35] showed that mRNA levels of NCX3 in cisplatin resistant ovarian cells increased in relation to the sensitive cell line. Moreover, as a result of pre-incubation of cells with an NCX inhibitor KB-R7943, the inhibitor showed a blunted effect on [Ca²⁺]_i, and significantly augmented the cisplatin-induced cell death of cisplatin resistant ovary carcinoma cells without effecting cisplatin-induced cell death of cisplatin sensitive ovary carcinoma cells. In another study by Pelzl et al. [36], therapy sensitive D283, and therapy resistant UW228-3 medulloblastoma cells were used and they found that NCX3 silencing enhanced the apoptosis. In our study, we also found that NCX3 gene expression levels were increased in MDAH/DDP cells at presence of iron. Our results support to previous studies and it is thought that NCX3 plays an important role in the drug resistance mechanism.

PMCA is expressed ubiquitously, and has critical role for maintaining low resting ([Ca²⁺]i) in all eukaryotic cells. Cytotoxic Ca²⁺ overload has such a central role in cell death and the PMCA takes a role for the delicate balance between cell survival, and cell death. In general, the decreased PMCA expression leads to cytotoxic Ca²⁺ overload and Ca²⁺ dependent cell death both in apoptosis and necrosis. Therefore, PMCA is suggested to have a paradoxical role in cell survival in drug resistance. Our results also showed that the upregulation of PMCA1 at 10 µM, and 20 µM iron and PMCA3 at 5 µM, 40 µM and 80 µM in MDAH/DDP cells. Upregulation of PMCA1 and PMCA3 mRNA levels in cisplatin resistant EOCs under the influence of iron may suggest that PMCAs may be a separate pathway for resistant cells to escape from cell death and/or, perhaps a new route through which they efflux drugs out. In addition, some isoforms of PMCA in colorectal and breast cancers have been shown to have significant changes in gene expression [37–42] and colorectal tumor tissues decreased PMCA4 compared to normal colon cancers.

Barabas and Faulk [43] have shown that doxorubicin-resistant human chronic myelogenous leukemia cells (K562) and promyelocytic leukemia cells (HL60) have up-regulated transferrin receptor (TfR) expression. They have also found that the number of transferrin receptors have been down-regulated by calcium channel blockers, such as verapamil, which is a well-known P-glycoprotein inhibitor and a calcium channel blocker. Doxorubicin-resistant HL60 cells did not overexpress P-glycoprotein, in comparison to drug-sensitive cells, by down regulating TfR. Davies et al. [44] have determined that TfR can be upregulated by growth hormones and this induction has been demonstrated to be triggered by calcium channels [45] and the blockade of such mechanism could re-sensitize drug-resistant cells [46]. This may explain the association of TfR with drug resistance through the function of calcium-sensitive receptors [37]. Finally, we may suggest in accordance with our results that there is a relation between calcium homeostasis and iron uptake and that this response is different in cisplatin-resistant cells than in cisplatin-sensitive cells.

In conclusion, changes were observed in expression of IP₃R1, IP₃R2, IP₃R3, RYR1, RYR2, SERCA1, and SERCA3 genes in iron treatment of MDAH cells, while changes were determined in IP₃R2, RYR1, SERCA3, PMCA1, and PMCA2 gene expression in iron treatment of MDAH/DDP cells. These results suggest that changes in the expression of calcium channels, pumps and exchange proteins in the EOC cell line and cisplatin-resistant EOC cells may have an important role in iron regulation of calcium homeostasis. Due to the differences in gene expressions involved in regulation of calcium levels under the effect of iron, drug resistance can be prevented by introducing a new perspective on the use of inhibitors and activators of these genes and thus cytostatic treatment strategies.