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Fe2+ as a Physiological and Selective Inhibitor of Vitamin C-Induced Cancer Cell Death  

Yoshimi Murayama, Ryoko Kashiyagura, Eiji Ohmomi, Yuki Ishida, Teruki Shinada, and Takumi Satoh  

Department of Anti-Aging Food Research, School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakura, Hachioji 192-0982, Japan  

Correspondence: satotkm@stf.teu.ac.jp (T.Satoh)  

Murayama Y et al. Reactive Oxygen Species 10(29):xxx–xxx, 2020; ©2020 Cell Med Press 

http://dx.doi.org/10.20455/ros.2020.833 

(Received: May 8, 2020; Revised: July 14, 2020; Accepted: July 16, 2020)  

ABSTRACT | High concentrations of ascorbic acid (AA) exert pro-oxidative actions and induce cancer cell death. Recent research on AA toxicity centers on the generation of H2O2, but it remains largely unknown why AA is toxic to cancer cells. In the present study we found that low concentrations (<10 μM) of Fe2+ inhibited the toxic effects of AA as well as those of isoascorbic acid (IAA), but not, as far as examined here, on any other types of cell death from H2O2, sodium nitroprusside (a NO donor), xanthine+xanthine oxidase (a superoxide inducer), A23187 (a Ca2+ ionophore), thapsigargin (an inducer of ER stress), staurosporine (a protein kinase inhibitor), cisplatin (an inducer of DNA damage), 5-fluorouracil (a DNA synthesis inhibitor), or actinomycin D (an RNA synthesis inhibitor) in COS7 cells. Fe2+ at concentrations of 1–10 μM inhibited the cell death caused by up to 5 mM AA. However, other divalent metal cations (Mn2+, Cr2+, Cu2+, Zn2+, Cd2+, and Ni2+) were not inhibitory, suggesting that just Fe2+, among divalent cations, had such an action on cancer cells at concentrations up to 100 μM. The Fe2+-inducedinhibition was commonly observed in COS7 (kidney cancer), Hela (uterine cancer), T98G (glioma), and PC-14 (lung cancer) cells, suggesting the inhibition to be a ubiquitous event among cancer cells. These results suggest that Fe2+ is a physiological and selective inhibitor of the AA-induced cancer cell death. The presence of high concentrations (around 30 μM) of Fe2+ in vivo might explain unstable effectiveness of AA and IAA infusions against various types of cancers. Conversely, a decrease in Fe2+ concentrations in vivo might potently enhance the therapeutic effects of AA infusion against various types of cancers.  

KEYWORDS | Ascorbic acid; Cancer; Chemotherapeutic drugs; Fe2+; Fe3+; Hydrogen peroxide  

ABBREVIATIONS | AA, ascorbic acid; AFR, ascorbic free radical; CAT, catalase; DFX, deferoxamine; DMEM, Dulbecco’s Modified Eagle medium;DMSO, dimethyl sulfoxide; ETC, electron transport chain; FRTA, free radical theory of aging; FCS, fetal calf serum; HIF-1, hypoxia-inducible factor-1; IAA, isoascorbic acid; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NO, nitric oxide; ROS, reactive oxygen species; SNP, sodium nitroprusside; SOD, superoxide dismutase; SD, standard deviation; XA, xanthine; XO, xanthine oxidase  

CONTENTS  

  1. Introduction
  2. Materials and Methods

2.1. Chemicals 

2.2. Compound Application 

2.3. Cell Death Inducers 

2.4. Cell Cultures 

2.5. Statistical Analysis 

  1. Results

3.1. Fe2+ Inhibited Cell Death Induced by AA as Measured by both MTT and LDH Assays 

3.2. Effects of Fe2+ and CAT on AA/IAA-Induced Cell Death 

3.3. Effects of Fe2+ on Oxidative Stress-Induced Cell Death 

3.4. Effects of Fe2+ on Cell Death Induced by Putative Apoptosis Inducers 

3.5. Effects of Fe2+ on the Cell Death Induced by Typical Anticancer Drugs 

3.6. Iron Chelator (DFX) Eliminated the Protection Afforded by Fe2+ 

3.7. Fe2+, but not Other Divalent Cations, Prevented the AA-Induced Cell Death 

3.8. Fe2+ as an Inhibitor of the AA-Induced Death of Various Cancer Cells 

  1. Discussion
  2. Conclusions

1. INTRODUCTION  

The accumulation of reactive oxygen species (ROS) is one of the major mechanisms of aging, neurodegeneration, and inflammation [1–4]. Thus, antioxidants are considered to delay the pathological process of chronic diseases [1–4]. Ascorbic acid (AA) has been classified as a broad-spectrum antioxidant because it reacts with a wide array of ROS during various physiological and pathophysiological processes [5, 6]. AA can maintain a balanced intracellular redox state and consequently protect cells from oxidative stress-induced damage [5, 6]. AA is involved in the first line of antioxidant defense, protecting lipid membranes and proteins from oxidative damages [5, 6]. One of the most important issues about the biological effects of AA is why antioxidant AA is toxic to cancer cells [5, 6]. Clinical and pharmacokinetics studies within the past over 20 years indicate that oral AA produces low tightly controlled AA concentrations of around 50–100 μM in the blood and tissue [7, 8]. It has been demonstrated that only intravenous administration can achieve pharmacologic concentrations of AA in the plasma [9–11]. By intravenous application of AA, millimolar serum concentrations can be reached, which are preferentially cytotoxic to cancer cells [11–13]. High concentrations of AA induce selective death of cancer cells via the formation of ascorbic acid free radical (AFR) and H2O2in cell culture media [9, 12–15]. High concentrations of AA also increase the levels of AFR in the extracellular space [12, 13]. In this case, the presence of high concentrations of AA in extracellular fluid can induce cell death of cancer cells [9, 12–15]. The electron lost from AA would reduce a protein-centered metal, selectively driving H2O2 formation in the extracellular space [12, 13]. The cell death induced by AA can be divided into 2 parts, namely, the AA-induced cell death via H2O2production by AA [12, 13] and the cell death by H2O2 [9, 14, 15] as described below. (1) The production of H2O2: AA induces selective death of cancer cells via the formation of H2O2 and AFRs, whose formation is connected with the reduction of Fe3+ to Fe2+ in cell culture media. The electron lost from AA reduces a protein-centered metal, selectively driving H2O2 formation in the extracellular fluid [8, 9]; and (2)The cell death by H2O2: Fe2+ enhances the cell death by H2O2 of cancer cells through activation of the Fenton reaction (production of hydroxyl radicals), and Fe2+ was reported to also act as an inhibitor of cancer growth, because a selective chelator of Fe2+ can accelerate the death of cancer cells [9, 14, 15].  

Fe2+ is the main effector of the H2O2 toxicity by enhancing the Fenton reaction, which leads to the accumulation of highly toxic hydroxyl radicals [16, 17]. Many previous papers suggest that Fe2+ in the extracellular space increases the rate of H2O2 production [12, 13] and H2O2 toxic effects [18–20]. According to this hypothesis, increasing intracellular Fe2+ concentrations can enhance the AA-induced cytotoxicity. These standpoints of view indicate that transition between Fe3+ and Fe2+ plays an important role in AA-induced cytotoxicity [18–20]. Approaches that increase catalytic Fe2+ could potentially enhance the cytotoxicity of pharmacological AA in vivo [18–20]. Increasing the level of intracellular iron by pre-incubating cells with Fe2+ salts increases AA toxicity [18–20]. Recently, Fe2+-mediated cell death, “ferroptosis,” has been proposed; and the involvement of Fe2+ has attracted much attention in cancer research [21, 22]. In this context, Fe2+ has been proposed to be a new accelerator of cell death by acting via this ferroptosis mechanism [23, 24]. In most studies, Fe2+ is supposed to be an accelerator rather than as an inhibitor in both processes (H2O2 production and H2O2 toxicity) [25, 26].  

However, the effects of Fe2+ have been highly controversial in terms of cancer growth [25, 26]. The most basic question is whether Fe2+ kills or protects cancer cells. Here, we examined the involvement of Fe2+ in the toxic effects of AA on various types of cultured cancer cells. According to previous reports, there are opposite results regarding the role of Fe2+ in AA-induced cancer cell death [25, 26]. Some studies reported positive effects on cell death [18–20]. In contrast, others reported that physiological concentrations of Fe2+ did not accelerate AA-induced cell death but diminished the death through inhibition of H2O2 production [25, 26]. Here we examined the involvement of Fe2+ in AA-induced cancer cell death to determine whether Fe2+ is an accelerator or inhibitor of AA-mediated cancer cytotoxicity. 

2. MATERIALS AND METHODS  

2.1. Chemicals  

AA (sodium salt), hydrogen peroxide (H2O2), xanthine (XA), xanthine oxidase (XO), cisplatin, actinomycin D, 5-fluorouracil, A23187, thapsigargin, staurosporine, FeCl2, FeCl3, ZnCl2, MnCl2, CrCl2, CuCl2, CdCl2, NiCl2, deferoxamine (DFX), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Wako Junyaku (Tokyo, Japan). Stock solutions of AA (sodium salt, 100 mM or 1000 mM) were prepared in Ca2+ and Mg2+-free phosphate-buffered saline (Invitrogen, Carlsbad, CA). Stock solutions of A23187, thapsigargin, staurosporine, cisplatin, actinomycin D, and 5-fluorouracil were prepared in dimethyl sulfoxide (DMSO). These were used in the culture medium with more than a hundred-fold dilution in order not for DMSO (solvent) to induce its toxicity. Sodium nitroprusside (SNP), purchased from Wako Junyaku, was used as nitric oxide (NO) donor in the present study.  

2.2. Compound Application  

We focused on the toxic effects on non-attached cells because many studies showed that cancer cells have low ability for cellular attachment to the extracellular matrix[27, 28]. AA has been reported to prevent cellular detachment from an original tissue and attachment onto distant organs during cancer metastasis [27, 28]. The compounds were added just after the cells had been added to 24-well plates [27, 28]. This system might be a good model for the attachment of cancer cells onto endothelial cells of distal organs during the onset of cancer metastasis [27, 28]. AA induces cell death of non-attached cancer cells at concentrations of 100–500 μM, although a concentration of over 1000 μM is required for attached cells, suggesting that AA may interfere with the cellular attachment [29, 30], leading to potent sensitivities of cancer cells when their attachment process has not been completed [27, 28]. These background data led us to consider the possibility that coordination between AA and H2O2/NO may clear cancer cells from the microcirculation. Physiological concentrations (<100 μM) of AA do not affect the cellular survival of either attached or non-attached cells [27]. Earlier we found that physiological concentrations of AA potentiate the toxic effects by H2O2 or NO, although AA alone did not affect cellular survival at these concentrations [28]. The production of H2O2 and/or NO by endothelial cells can be activated by the attachment of malignant cancer cells to them, and these oxidants can kill the cancer cells in coordination with physiological concentrations of AA [28]. This may be an intrinsic defense mechanism against cancer metastasis to distal organs by AA and H2O2/NO at the first attachment of cancer cells onto the vascular endothelium [28]. Thus, this coordination may be a potent mechanism for the clearance of malignant cancer cells in vivo. These results led us to consider that physiological concentrations (<100 μM) of AA may be involved in the removal of cancer cells. Thus, it is highly important to examine the mechanism of this clearance of cancer cells. This research is intended to clarify the relationship between Fe2+ and AA [27, 28].  

2.3. Cell Death Inducers  

The present study used the following cell death inducers. (1)AA and IAA:The toxic effects may be closely connected with AA’s reductive power. IAA and AA share a reductive group (endiol), which is supposed to reduce Fe3+ to Fe2+ [27, 28]. The toxic effects are due to “endiol” chemical structures [27, 28]. AA has been reported to prevent cellular detachment from an original tissue and attachment onto distant organs during cancer metastasis [29, 30]. The supply of AA and Fe2+ may be involved in the cellular attachment process [31, 32]. AA enhances the supply of Fe2+ to hyper-activate the enzyme proline hydroxylase, which leads to the loss of protein conformation and results in the inhibition of the cellular attachment process [31, 32]; (2) XA+XO: Energy metabolism and ROS production are closely linked with each other. Superoxide anion (O2˙ˉ) is generated by the electron transport chain (ETC) during aerobic metabolism [1, 2]. The free radical theory of aging (FRTA) states that organisms age due to the buildup of free radicals over time because these atoms or molecules with an unpaired electron in their outermost shell are unstable and damage cells, thus contributing to aging [1, 2]. Oxidative damage is the most common form of damage initiated by O2˙ˉ, which is a byproduct of intracellular processes such as the ETC [3, 4]. The enzyme superoxide dismutase (SOD) dismutases O2˙ˉ to form hydrogen peroxide (H2O2), a still toxic ROS [3, 4]. The production of intracellular ROS mostly occurs in the mitochondria through the ETC, linking the FRTA with the mitochondria [3, 4]. The XA+XO system provides a model of O2˙ˉ cell toxic effectsin vitro, generating O2˙ˉ in the extracellular space, which provides us a suitable experimental model of FRTA at the cellular level [33, 34]; (3) H2O2 and NO: H2O2 [35, 36] and NO [37, 38] are themselves ROS, and they kill non-attached COS7 cells at concentrations within the range of 20–50 μM; and SNP, an NO donor, kills these non-attached COS7 cells at concentrations within the range of 200–1000 μM [27, 28]; (4) A23187: A23187 is generally assumed to directly facilitate the transport of Ca2+ across the plasma membrane and induces severe Ca2+ overload and cell death[39, 40]; (5) Thapsigargin: Thapsigargin is a potent inhibitor of the sarcoplasmic reticulum (SR) Ca2+ ATPase isolated from cardiac or skeletal muscle and is used as an inducer of endoplasmic reticulum (ER) stress associated with the accumulation of unfolded proteins [41, 42]; (6) Staurosporine: Staurosporine, a broad-spectrum protein kinase inhibitor, inhibiting kinases including protein kinase C, has been used to induce cell death in a wide range of cell types; and it has been used as a putative inducer of apoptosis [43, 44]; (7) Cisplatin: Cisplatin is a well-known chemotherapeutic agent. Its mode of action has been linked to its ability to crosslink with the purine bases on the DNA, thus interfering with DNA repair mechanisms, causing DNA damage and subsequently inducing apoptosis in cancer cells [45, 46]; (8) Actinomycin D: Actinomycin D is a well-known clinically used chemotherapy agent that inhibits RNA polymerase(s) in eukaryotic cells and induces apoptosis in various cancer cells [47, 48]; and (9) 5-Flurouracil: 5-Fluorouracil, a drug for chemotherapy against various cancers, acts by inhibiting DNA synthesis and inducing apoptosis [49, 50].  

2.4. Cell Cultures  

COS7 (a monkey kidney fibroblastoma cell line), Hela (a human uterus adenocarcinoma cell line), T98G (a human glioblastoma cell line), and PC14 (a human lung adenocarcinoma cell line) cells, all of which share the phenotype of malignant cancer cells, were cultured as described elsewhere [51, 52]. These cells were maintained in 10-cm dishes (Invitrogen) containing 10 ml of Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% (v/v) heat-inactivated (56°C, 30 min) fetal calf serum (FCS, Invitrogen). The cells were seeded into 24-well plates at a density of 4 × 104 cells/cm2. In Figures 1, 2, 7, and 8, Fe2+, catalase (CAT), or another divalent cation was added first and, just thereafter, various concentrations of AA or IAA were added. In Figures 3, 4, and 5, Fe2+ was added first, followed immediately by adding various concentrations of cell death inducers. In Figure 6, Fe2+ as well as DFX was added first and, just after, AA was added. Then, the cells were incubated for an additional 24 h. To evaluate cell survival, we performed the MTT assay. We used “non-attached cells” in all experiments in the present study. Then, the cells were incubated for an additional 24 h. Lactate dehydrogenase (LDH) assay was performed according the instructions provided by the manufacturer (Takara LDH cytotoxicity detection kit, Takara Co. Ltd., Japan). When examining the toxic effects of compounds themselves on attached cells, the compounds were added 24 h after the cells had been spread in 24-well plates. Then, the AA derivatives were added to the cultures after a 24-h incubation. To evaluate cell survival, we performed the MTT assay [51, 52].  

2.5. Statistical Analysis  

Experiments presented herein were repeated at least 3 times, with each experiment performed in quadruplicate. Data were presented as the mean ± standard deviation (SD). The statistical significance of differences was examined by performing Student’s t-test. 

3. RESULTS  

3.1. Fe2+ Inhibited Cell Death Induced by AA as Measured by both MTT and LDH Assays  

The toxic effects of AA were investigated in COS7 cells (Figures 1 and 2). By use of this system, we had supposed that Fe2+ would accelerate the cell death caused by AA. The concentrations of Fe2+ used here were supposed to be similar to or lower than those in the serum in vivo, i.e., around 20–30 μM [53–56]. Here we found that a very low concentration (10 μM) of Fe2+ completely inhibited the AA-induced cell death. This finding was also made earlier based on the results of a cellular survival assay (MTT assay) and cellular injury assay (LDH assay) [51, 52]. These results implied to us that Fe2+ may work as an inhibitor of the AA-induced cell death, but not as an accelerator. 

 

FIGURE 1. Fe2+ inhibited the death of COS7 cells induced by AA, as determined by MTT (A) and LDH (B) assays. Fe2+ at0 or 10 μM was added to the DMEM medium just after the cells had been spread into 24-well plates. Then, 500 μM AA was added, and the cells were incubated for 24 h, followed by the MTT assay for cell viability (A) and LDH assay for cellular injury (B) by using the same plates. Values, presented as a percentage of the control MTT and LDH values, are given as the mean ± SD (n = 4). *, significantly different (p < 0.01) from control vs. AA and AA vs. AA+Fe2+. 

 

FIGURE 2. Effects of Fe2+ and CAT on AA- or IAA-induced COS7 cell death. Structures of AA (A) and IAA (B): Because AA and IAA are isomers in terms of their hydroxyl group at carbon (5), AA and IAA have the common chemical structure of the “endiol” (reduced form), which can give electron(s) to other molecules. Both of them have reductive power as well as anti-cancer effects.Fe2+ inhibited the cell death of COS7 cells induced by AA (C) or IAA (D): Fe2+ at 0 (white), 1 (dark), 10 (diagonal-lined) or 100 (dotted) μMwas added to the DMEM medium just after the cells had been spread into 24-well plates. Then, various concentrations (0, 200, 300, and 500 μM) of AA (B) or IAA (C) were added; and the cells were incubated for 24 h, followed by the MTT assay. Values, presented as a percentage of the control MTT value, are given as the mean ± SD (n = 4). *, significantly different (p < 0.01) from samples without Fe2+. Catalase (CAT) inhibited the cell death induced by AA (E) or IAA (F): CAT at 0 (white), 1 (dark), 10 (diagonal-lined) or 100 (dotted) U/ml was added to the DMEM medium just after the cells had been spread into 24-well plates. Then, various concentrations (0, 200, 300, and 500 μM) of AA (E) or IAA (F) were added; and the cells were incubated for 24 h, followed by the MTT assay. Values, presented as a percentage of the control MTT value, are given as the mean ± SD (n = 4). *, significantly different (p < 0.01) from samples without CAT.  

3.2. Effects of Fe2+ and CAT on AA/IAA-Induced Cell Death  

The concentrations of Fe2+ are about 20–30 μM in the serum [53–56]. Thus, the biological effects induced by Fe2+ at around these concentrations may have some physiological significance [53–56]. Here, we examined the effects of Fe2+ at 1–100 μM on the toxic effects induced by AA and IAA (Figure 2). Previously, we found that AA and IAA share similar toxic effects on various cancer cells, e.g., COS7 cells at around 100–500 μM [27, 28]. Fe2+ completely inhibited the toxic effects by AA/IAA. Many investigators reported that CAT inhibits AA-induced cancer cell death. Here, CAT prevented the AA/IAA toxic effects when tested at around 1–100 units/ml, suggesting that the real effector of the cell death was H2O2.  

3.3. Effects of Fe2+ on Oxidative Stress-Induced Cell Death  

Since the overload of Fe2+ induces serious redox disturbance of cancer cells, the effects of typical inducers of oxidative stress were examined here. We employed the XA+XO (an O2˙ˉ inducer) [33, 34], H2O2 [35, 36], and sodium nitroprusside (SNP, a NO donor) [37, 38] as oxidative stress inducers (Figure 3). In the presence of XA (1 mM), the addition of XO triggered the death of COS7 cell at around 10 mU/ml, whereas XO at 3 mU/ml was only partially effective. Fe2+ (1–100 μM) did not protect against the cell death induced by XA+XO (Figure 3A). In addition, 20–50 μM H2O2 (Figure 3B) or 200–1000 μM SNP (Figure 3C) induced cell death, but the presence of Fe2+ at around 1–100 μM did not prevent the death elicited by them, suggesting that Fe2+ was ineffective at these concentrations. 

 

FIGURE 3. Fe2+did not inhibit the death of COS7 cells induced by superoxide from XA+XO (A), H2O2 (B), or NO from SNP (C). In “A”,Fe2+ at0 (white), 1 (dark), 10 (diagonal-lined), or 100 (dotted) μM was added to XA (1 mM)-containing DMEM medium just after the cells had been spread into 24-well plates. Then, various concentrations (1, 3, 10 mU/ml) of XO were added, and the cells were thereafter incubated for 24 h, followed by the MTT assay. In “B” and “C,” Fe2+ was added to the DMEM medium just after spreading the cells into 24-well plates. Then, various concentrations (0, 1, 3, 10 mU/ml) of H2O2 (B) or (200, 300, and 1000 μM) of SNP (C) were added; and the cells were incubated for 24 h, followed by the MTT assay. Values, presented as a percentage of the control MTT value, are given as the mean ± SD (n = 4).  

3.4. Effects of Fe2+ on Cell Death Induced by Putative Apoptosis Inducers  

If the site of action of the inhibition of AA-induced cancer cell death by Fe2+ is supposed to be in the upstream of the AA-induced H2O2 release, the inhibition by Fe2+ would be limited to AA-induced cell death. Perhaps other types of cell death would not be affected by Fe2+(Figure 4). To assess the possibility that Fe2+ does not inhibit cell death caused by other types of agents, we examined the effect of A23187 (a Ca2+ ionophore) [39, 40], thapsigargin (an inducer of ER stress) [41, 42], and staurosporine (a broad-spectrum inhibitor of protein kinase) [43, 44]. A23187 (Figure 4A) and thapsigargin (Figure 4B), both at 0.3–3.0 μM, induced significant death of COS7 cells, as did staurosporine at 0.03–0.3 μM. However,the presenceof1–100μMFe2+didnotaffect the cell death caused by any of these agents, suggesting that Fe2+ may be a highly selective inhibitor of the AA/IAA-induced cell death. 

 

FIGURE 4. Fe2+ did not inhibit the death of COS7 cells induced by typical apoptosis inducers. A23187 (a Ca2+ overload inducer, A), thapsigargin (an ER stress inducer, B), and staurosporine (a protein kinase inhibitor, C) were used as apoptosis inhibitors. Fe2+ at 0 (white), 1 (dark), 10 (diagonal-lined), or 100 (dotted) μM Fe2+ was added to the DMEM medium just after the cells had been spread into 24-well plates. Then, various concentrations (0, 0.3, 1.0, or 3.0 μM) of A23187 (A) or thapsigargin (B) or those (0, 0.03, 0.1, or 0.3 μM) of staurosporine (C) were added; and the cells were incubated for 24 h, followed by the MTT assay. Values, presented as a percentage of the control MTT value, are given as the mean ± SD (n = 4).  

3.5. Effects of Fe2+ on the Cell Death Induced by Typical Anticancer Drugs  

Next, we investigated the effect of Fe2+ on the cell death caused by various chemotherapy drugs (Figure 5), such as cisplatin (a DNA damage inducer) [45, 46], actinomycin D (an RNA synthesis inhibitor) [47, 48], and 5-fluorouracil (a DNA synthesis inhibitor) [49, 50]. Cisplatin at 20–50 μM (Figure 5A), actinomycin D at 0.03–0.3 μM (Figure 5B), and 5-fluorouracil at 100–600 μM(Figure 5C) induced significant death of COS7 cells. However, Fe2+ examined at 1–100 μM did not affect the cisplatin-, actinomycin D-, or 5-fluorouracil-induced cell death, again suggesting that Fe2+ may be a highly selective inhibitor of the AA/IAA-induced cell death. 

 

FIGURE 5. Fe2+ did not inhibit the death of COS7 cells induced by anti-cancer drugs.Cisplatin (a DNA damage inducer, A), Actinomycin D (an RNA synthesis inhibitor, B), and 5-fluorouracil (a DNA synthesis inhibitor, C) were used as apoptosis inhibitors. Fe2+ at 0 (white), 1 (dark), 10 (diagonal-lined) or 100 (dotted) μM was added to the DMEM medium just after spreading the cells into 24-well plates. Then, various concentrations (0, 20, 30, or 50 μM) of cisplatin (A), (0, 0.03, 0.1, or 0.3 μM) of actinomycin D (B), or (0, 100, 300, or 600 μM) of 5-flurouracil (C) were added, and the cells were incubated for 24 h, followed by the MTT assay. Values, presented as a percentage of the control MTT value, are given as the mean ± SD (n = 4).  

3.6. Iron Chelator (DFX) Eliminated the Protection Afforded by Fe2+  

DFX is known as a hydrophilic and non-membrane-permeable Fe3+ chelator, and it can deplete Fe3+ in the extracellular space [57, 58]. In order to examine the involvement of Fe2+ in the extracellular space, we added DFX at concentrations of 10, 30, and 100 μM; but DFX exerted no inhibition (Figure 6A), suggesting that endogenous Fe2+ was not involved in the AA-induced toxic effects, possibly due to low concentrations of Fe2+ in the DMEM medium. In order to provide direct evidence for Fe2+ involvement, we added various concentrations (1, 3,10, and 30 μM) of Fe2+ to the cells in the absence (white columns) or presence (dark columns) of 100 μM DFX (Figure 6B). Fe2+ did not protect against the AA toxic effects in the presence of DFX, suggesting that Fe2+ was indeed involved in the inhibition of cell death. 

 

FIGURE 6. Iron chelator (DFX) reversed the protection afforded by Fe2+ in COS7 cells. (A), DFX at a 0 (white), 20 (dark), 50 (diagonal-lined) or 100 (dotted) μM concentration was added just after the introduction of the cells into 24-well plates along with various concentrations (0, 200, 300, or 500 μM) of AA. (B), A 0 (white) or 100 μM (dark) concentration of DFX was added just after the introduction of the cells into 24-well plates along with various concentrations (0, 1, 3, 10, or 30 μM) of Fe2+. Just thereafter, vehicle (control) or 500 μM AA was added. Then, the cells were incubated for 24 h. For the MTT assay, the cells were then incubated for an additional 24 h. Values, presented as a percentage of the control MTT value, are given as the mean ± SD (n = 4). *, significantly different (p< 0.01) from samples without DFX.  

3.7. Fe2+, but not Other Divalent Cations, Prevented the AA-Induced Cell Death  

Next, we compared the effects of reductive (Fe2+) and oxidative (Fe3+) forms of Fe on the inhibition of cell death (Figure 7A). Fe3+ was less active than Fe2+, suggesting the active form to be Fe2+ since Fe3+ reduced to Fe2+ by AA. In addition, we investigated whether other divalent cations (Mn2+, Cr2+, Cu2+, Zn2+, Cd2+, and Ni2+) were protective or not (Figure 7BC, and D).Noneof them wasable to inhibitAA toxic effects, suggesting that the inhibition by Fe2+ was highly selective. 

 

FIGURE 7. Among the divalent cations tested, Fe2+as the only inhibitor of the AA-induced death of COS7 cells. The cells were introduced into 24-well plates along with various concentrations (0, 1, 3, 10, 30, or 100 μM) of Fe2+ (white columns) or Fe3+ (dark columns; A), those of Mn2+ (white columns) or Cr2+ (dark columns; B) or those of Cu2+ (white columns) or Zn2+ (dark columns; C) or those of Cd2+ (white columns) or Ni2+ (dark columns; D). Just thereafter, vehicle (control) or 500 μM AA was added. Then, the cells were incubated for 24 h. For the MTT assay, the cells were then incubated for an additional 24 h. Values, presented as a percentage of the control MTT value, are given as the mean ± SD (n = 4). *, significantly different (p < 0.01) from AA-treated samples without divalent cations.  

3.8. Fe2+ as an Inhibitor of the AA-Induced Death of Various Cancer Cells  

Next, we examined if other cancer cell lines would be affected similarly (Figure 8). The presence of Fe2+ at 10 μM totally inhibited the cell death induced by 5 mM AA. We examined the effects of Fe2+ on higher concentrations of AA corresponding to those when AA is infused [7, 8]. Fe2+ inhibited the AA-induced cell death even when such high concentrations (up to 5 mM) of AA were present. Such inhibition was observed for COS7 (Figure 8A), Hela (Figure 8B), PC14 (Figure 8C), and T98G (Figure 8D) cells, suggesting that the inhibition by Fe2+ of the AA-induced cell death was conserved among various types of cancer cells. 

 

FIGURE 8. Fe2+ as a selective inhibitor of the AA-induced death of various types of cancer cells. To COS7 (A), Hela (B), PC14 (C), or T98G (D) cell cultures, Fe2+ at0 (white) or 10 (dark) μM Fe2+ was added just after the introduction of the cells into 24-well plates along with various concentrations (0, 1000, 2000, or 5000 μM) of AA. For the MTT assay, the cells were then incubated for an additional 24 h. Values, presented as a percentage of the control MTT value, are given as the mean ± SD (n = 4). *, significantly different (p < 0.01) from samples without Fe2+. 

4. DISCUSSION  

Previously we reported that H2O2 is the main effector of the XA+XO-induced cell death [33, 34]. Thus, we supposed that Fe2+ accelerated the cell death by enhancing the Fenton reaction. However, Fe2+, at up to 100 μM, did not affect the cell death induced by XA+XO, H2O2, or NO, as was shown in Figure 3. Thus, the site of action by Fe2+ is not the cell death pathways themselves, at least at around the physiological concentrations of Fe2+; but the real site of action is the upstream H2O2 production. The sequential eventsduringtheoxidativestress-inducedcelldeath are summarized in Figure 9A. By the combination of results shown in Figures 2 and 3, sites 1–5 (indicated in Figure 9A) were not affected by Fe2+, nor were sites 6 and 7. It seems that Fe2+ inhibited at sites 8 and 9, showing that the H2O2 release by AA/IAA was sensitive to physiological concentrations of Fe2+. 

 

FIGURE 9. Schematic illustration of the proposed mechanisms. (A) Fe2+ as an inhibitor of H2O2 release by AA/IAA. XA+XO: Basically, XA+XO induces the release of O2 in the extracellular space (reaction “1” in Figure 9). Oxidative damage is the most common form of damage initiated by O2, which is a byproduct of intracellular processes such as the electron transfer chain. The enzyme superoxide dismutase (SOD) forms H2O2 (reaction “2”), which strongly activates cell death pathway through formation of hydroxyl radicals (reactions “3”, “4”, “6”, and “7”). NO directly activates cell death pathway as does H2O2 (reaction “5”). AA/IAA firstly induces H2O2 production (reactions “8” and ‘9”) followed by the reactions “6” and “7”. Because Fe2+ did not affect the cell death induced by XA+XO, H2O2, or NO, Fe2+ must not inhibit the H2O2 toxicity itself. Thus, it is highly possible that Fe2+ inhibits H2O2 production (reactions “8” and “9”). (B) Fe2+ as a selective inhibitor of AA/IAA toxic effects: AA and IAA induce cancer cell death by 2 distinctive steps, the first is H2O2 production and the second, cell death by H2O2. Fe2+, an inhibitor of H2O2 production, and catalase, by removing H2O2, can prevent the toxic effects by AA.  

As shown in Figure 9B, the production of H2O2is considered to be the main effector of AA- and IAA-induced cell death, because many investigators have reported that CAT inhibits such cell death [9–15]. Here we confirmed that CAT effectively abolished the toxic effects elicited by AA (Figure 2). In addition, the most cited papers [12, 13] proposed that Fe2+ is the main effector of H2O2 production in the extracellular space [12, 13]. Thus, we had supposed that Fe2+ would enhance cell death by increasing H2O2 production. The result was surprising that Fe2+ totally suppressed the toxic effects of AA (Figure 1).  

However, we found that Fe2+ did not enhance or suppress various types of cell death other than that caused AA and IAA (Figures 2–5). In terms of Fe2+ involvement, the cell death induced by AA and IAA has a highly distinctive feature from other types of cell death, suggesting that Fe2+ does not affect the death pathway itself. Transition metals including Fe2+ may regulate cellular redox and affect AA-induced cell death. As far as we examined here, no other divalent metals affected such cell death, suggesting that Fe2+ may be a highly selective inhibitor. In contrast, other divalent cations did not have such a protective effect on cancer cells, as was shown in Figure 7. The inhibition of the AA-induced cell death by Fe2+ was observed in COS7, Hela, PC14, and T98G cells (Figure 8) as well as K252a (lymphoma cells), LN-Cap, and PC-3 (lung cancer cells) in other reports [59, 60], suggesting that this inhibition can be observed in many cancer cell types.  

Here, Fe2+is considered to be a potent negative regulator of the AA-induced cancer cell death, at least as far as physiological concentrations of Fe2+ are concerned. Thus, we have a strong doubt regarding the proposal that Fe2+ really has positive effects on the AA-induced cancer cell death. If physiological concentrations of Fe2+ can really prevent the H2O2 production by AA as suggested by other reports, the Fe2+-mediated toxic effects by AA might possibly be just a “a pharmacological event” solely when pharmacologically high concentrations of Fe2+ are applied [61]. In contrast, traditionally in the clinical field, iron-depletion treatment is also known to suppress tumor growth in vivo, suggesting that Fe2+ is a negative regulator of cancer cell death. Iron depletion has generally been thought to help ordinary chemotherapy and a standard therapeutic strategy in the treatment of cancer[62–65]. Thus, it occurs to us that Fe2+ may inhibit the actions of anticancer agents used generally in clinics [62–65].  

One possible reason why this controversy is spreading is the difference in Fe2+ concentrations between in vitro and in vivo settings [61]. Although the concentrations in the serum are reported to be around 30 μM in vivo, DMEM supplemented with 10% FCS contains < 3 μM Fe2+ [61]. Because allin vitro experimental conditions suffer from deficiency of Fe2+, most investigators might miss the contribution of Fe2+ [61]. In contrast, under in vivo conditions, the anticancer effects found with AA infusion are hardly obtainable because of the presence of high concentrations of Fe2+[59, 60]. The inhibitory concentrations of Fe2+ found presently were on the order of 1 to 10 μM, as in other reports [59, 60]. It was reported thatAAinfusioncansuppresscancervolumeunder iron removal, suggesting that Fe2+ suppresses the anticancer effects of AA [59, 60]. Further, these cited reports as well as one by another group [59, 60] stated that H2O2 production is severely inhibited by the presence of Fe2+ at around 10 μM. One possible mechanism is inhibition of hypoxia-inducible factor-1 (HIF-1) activities by these concentrations of Fe2+[66, 67]. In this case, HIF-1 might be involved in the production of H2O2 by AA and IAA [30, 31].

5. CONCLUSIONS 

In the present study, we found that low concentrations of Fe2+ fully suppressed AA-induced cell death of non-attached tumor cells, suggesting that Fe2+ may be a physiological and selective inhibitor of such cell death. We propose Table 1 summarizing the differential effects of Fe2+ on the AA-induced cancer cell death. In the presence of physiological concentrations (< 100 μM) of Fe2+, the toxic effects of AA on cancer cells are diminished through inhibition of H2O2 production. Although AA is reported to be a potent inhibitor of the HIF-1 transcriptional pathway, Fe2+ reactivates this pathway to inhibit the cell death pathway of AA. In contrast, when pharmacological concentrations (> 100 μM) of Fe2+ are added, the cell death by AA may be potentiated through the Fenton reaction (H2O2 toxic effects), which may overcome the inhibition of H2O2 production. Thus, the concentration of Fe2+ may be one of the most important determinant factors for whether AA infusion or dietary intake is active or not regarding the prevention of cancer progression. 

TABLE 1. Differential effects of Fe2+ on AA-induced cancer cell death
Concentration Effect Site of action Reported mechanism References
Physiological Fe2+ concentrations (< 100 µM)

Inhibition

 

Inhibition of

H2O2 production

Reactivation of HIF-1 pathway  [59-61]
Pharmacological Fe2+concentrations (> 100 µM) Potentiation

Potentiation of

H2O2 toxic effects

Potentiation of Fenton reaction  [18-20]

ACKNOWLEDGMENTS 

The authors thank Larry D. Frye for editorial help with the manuscript. This work was supported in part by Grant for Research and Education of Undergraduate School of Tokyo University of Technology and Grant for Research and Education of Graduate School of Tokyo University of Technology. 

CONFLICTS OF INTEREST STATEMENT

The authors declare no conflict of interest. 

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