Cesium reversibly suppresses HeLa cell proliferation by ... - FEBS Press

Cesium reversibly suppresses HeLa cell proliferation by inhibiting cellular metabolism Daisuke Kobayashi1, Kei Kakinouchi1, Tomoki Nagae1, Toshihiko Nagai2, Kiyohito Shimura2 and Akihiro Hazama1 1 Department of Cellular and Integrative Physiology, School of Medicine, Fukushima Medical University, Japan 2 Department of Natural Sciences, School of Medicine, Fukushima Medical University, Japan

Correspondence D. Kobayashi, Department of Cellular and Integrative Physiology, School of Medicine, Fukushima Medical University, 1-Hikarigaoka, Fukushima 960-1295, Japan Fax: +81-24-547-1132 Tel: +81-24-547-1129 E-mail: [email protected] (Received 4 November 2016, revised 23 January 2017, accepted 24 January 2017, available online 21 February 2017) doi:10.1002/1873-3468.12579

The aim of the present study was to investigate the influence of Cs+ on cultured human cells. We find that HeLa cell growth is suppressed by the addition of 10 mM CsCl into the culture media. In the Cs+-treated cells, the intracellular Cs+ and K+ concentrations are increased and decreased, respectively. This leads to a decrease in activity of the glycolytic enzyme pyruvate kinase, which uses K+ as a cofactor. Cs+-treated cells show an intracellular pH shift towards alkalization. Based on these results, CsCl presumably suppresses HeLa cell proliferation by inducing an intracellular cation imbalance that affects cell metabolism. Our findings may have implications for the use of Cs+ in cancer therapy. Keywords: cell cycle; cesium; proliferation

Edited by Judit Ovadi

Cesium (Cs) is an alkali metal. Cs+ has no known beneficial function in humans; however, it induces toxicity at high concentrations [1]. Although physiological Cs+ uptake and responses in humans have not been studied well, several clinical studies reported Cs+ administration to humans [2], particularly in cancer therapy. Upon intravenous administration, Cs+ accumulates in the human brain, liver, and kidneys. The half-life of Cs+ in a human male adult ranges from 50 to 150 days [3–5]. In terms of its biological effects, very interesting attempts to use Cs as a cancer therapy have been reported. For instance, oral intake of CsCl has been used an alternative therapy for treating cancer. In these tests, 2-g doses of CsCl were administered three times per day after each meal with additional vitamins. Consequently, relief of cancer-associated pain and a rapid shrinkage of the tumor masses were reported [6,7]. Neulieb

orally treated himself with CsCl for 36 consecutive days and reported the effects [1]. No discomfort or debility of any kind was observed during the period, but symptoms of nausea and diarrhea were observed. Furthermore, in some cases, Cs intake caused polymorphic ventricular tachycardia, including torsades des pointes, and long QT syndrome [8,9]. A few cases of confirmed tumor regression have been reported; however, these cases were not theoretically or experimentally validated. Currently, there is insufficient information about the acute and chronic toxicity of Cs+ [10]. We must clarify the biological effects of Cs on cells to use Cs in clinical applications. Here, we investigated the influence of Cs+ on cultured human cells, and evaluated cell proliferation, viability, and Cs incorporation in the cells. We also examined the possible mechanism for the cellular effects of Cs.

Abbreviations 4-AP, 4-aminopyridine; BCECF-AM, 30 -O-Acetyl-20 ,70 -bis(carboxyethyl)-4 or 5-carboxyfluorescein, diacetoxymethylester; CE, capillary electrophoresis; DMEM, Dulbecco’s modified Eagle’s medium; LDH, lactate dehydrogenase; TEA, tetraethylammonium.

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FEBS Letters 591 (2017) 718–727 ª 2017 Federation of European Biochemical Societies

D. Kobayashi et al.

Cesium suppresses HeLa cell proliferation

Materials and methods

Intracellular pH

Cell culture

HeLa cells were incubated in serum-free DMEM and subsequently loaded with 1 lM 30 -O-Acetyl-20 ,70 -bis(carboxyethyl)-4 or 5-carboxyfluorescein, diacetoxymethylester (BCECF-AM; Dojindo) for 30 min at 37 °C. BCECF-AM was dissolved in DMSO, according to the manufacturer’s instructions. HeLa cells were transferred to a high K buffer (30 mM NaCl, 120 mM KCl, 1 mM CaCl2, 0.5 mM MgSO4, 1 mM NaH2PO4, 5 mM glucose, 10 mM HEPES, and 10 mM PIPES) adjusted to various pH values between 6.0 and 8.0 [12]. The cells were treated with 10 lM nigericin (Sigma-Aldrich Japan K.K., Tokyo, Japan) for 30 min at 37 °C to adjust the intracellular pH to the pH of the extracellular buffer. The stained cells were immediately analyzed by flow cytometry. A pH calibration curve was generated by plotting the geometric mean of the green fluorescence value versus the pH values. The concentrations of chemicals that did not damage the cell membrane were: 10 mM of Li, Na, K, Rb, Cs, and tetraethylammonium (TEA); 3 mM 4-aminopyridine (4-AP); and 0.3 mM quinidine.

HeLa and HeLa.S-Fucci cells were routinely cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Wako Pure Chemical Industries, Ltd., Osaka, Japan) supplemented with 10% FBS at 37 °C in a 5% CO2 humidified incubator. A subline of the HeLa cell line expressing Fucci, a cell cycle marker, HeLa.S-Fucci (RCB2812), was provided by the RIKEN BRC through the National Bio-Resource Project of the MEXT, Japan [11]. For all experiments, HeLa and HeLa.S-Fucci cells were plated in DMEM, incubated overnight, and incubated in DMEM supplemented with specific concentrations of Cs or inhibitors.

Cell counting and viability assay Cells cultured in 96-well culture plates were assessed using the Cell Counting Kit (Dojindo Laboratories, Kumamoto, Japan), and lactate dehydrogenase (LDH) activity was measured using a Cytotoxicity Detection Kit (LDH) (Roche Diagnostics K.K., Tokyo, Japan, https://pim-eservices. roche.com/LifeScience/Document/dc220baa-d3ed-e311-98a100215a9b0ba8), according to the manufacturers’ instructions. Cell viability was also evaluated using the trypan blue dye exclusion assay.

Cell cycle analysis The two cell cycle Fucci probes, mKO2-hCdt1(30/120) and mAG-hGem(1/110), are often called mKO2-Cdt1 and mAG-Geminin, respectively. The red fluorescence observed upon expression of mKO2-Cdt1 indicates late G1 phase and the green fluorescence observed upon expression of mAG-Geminin indicates S/G2/M phases. HeLa.S-Fucci cells, which express the two Fucci probes, were used for the cell cycle analysis. Cells were cultivated for 2 days in DMEM at 37 °C in 5% CO2 with 10 mM CsCl or 0.1 mM quinidine. The cell cycle distribution was analyzed by measuring the two Fucci probes using a BD FACSCaliburTM (Nippon Becton Dickinson, Tokyo, Japan) equipped with a 15-mW air-cooled 488-nm argonion laser. At least 10 000 events were collected, and debris was excluded by gating based on a plot of the forward versus side scatter. We used the gating to plot mAG-Geminin versus mKO2-Cdt1 and identify the cell cycle phase. The data were analyzed with CellQuest Pro software (Nippon Becton Dickinson). The cell division cycle was measured by time-lapse microscopic observations. The microscopic photographs were captured every 10 min using the BZ-9000 fluorescent microscope system (Keyence, Osaka, Japan). One cell cycle period was defined as the period between the visualization of red fluorescence in a mother cell and daughter cell.

Intracellular Cs concentration Intracellular Cs concentrations were measured with a capillary electrophoresis (CE) analysis using a P/ACE MDQ system (Beckman Coulter K.K., Tokyo, Japan). The capillary column was prepared and CE conditions followed previously reported methods [13,14]. Fused silica capillaries (50 lm inner diameter, 375 lm outer diameter, length 30 cm) were obtained from GL Sciences (GL Sciences Inc., Tokyo, Japan). The culture media were aspirated from ~ 80–90% confluent cultures, and the cultured cells were washed with a 0.33 M mannitol solution three times. The cells were then suspended in an appropriate volume of the 0.33 M mannitol solution. The cell suspension was stored at 20 °C until analysis. The frozen cell suspension was heated at 98 °C for 5 min and then centrifuged at 15 000 9 g for 5 min. A background electrolyte consisting of 5 mM imidazole and 2 mM 18-crown-6 was used to determine the cation concentrations in the supernatant, and the pH was adjusted to 4.5 with H2SO4. The absorbance was measured at 214 nm, and the background was subtracted. The cell volumes were calculated by a diameter of cell. The diameters of suspended, detached cells (n = 400) were measured from the microscopic images. The average cell volume was 1.26 0.67 pL. Intracellular concentrations of K, Na, and Cs were calculated by the suspension of cell number and the average cell volume.

Pyruvate kinase activity assay Pyruvate kinase activity was measured using an LDH assay at 25 °C. The standard assay cuvette contained 10 mM Tris-HCl (pH 7.4), 100 mM KCl, 10 mM MgCl2, 2 mM


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ADP, 2 mM phosphoenolpyruvate, 0.2 mM NADH, LDH (five unit), and pyruvate kinase from a crude HeLa cell extract in a final volume of 1 mL. The reaction was initiated by adding ADP and monitored at 340 nm. One unit of activity was represented as an absorbance change in 6.22 per minute. KCl was substituted with LiCl, NaCl, RbCl, or CsCl to test the alkali metal dependency of pyruvate kinase activity. The K+ to Cs+ ratio was 70 : 30 (70 mM KCl and 30 mM CsCl), 90 : 10 (90 mM KCl and 10 mM CsCl), and 97 : 3 (97 mM KCl and 3 mM CsCl).

Results Cs suppresses proliferation in a dose-dependent manner HeLa cell proliferation was assessed using the Cell Counting Kit, and Cs suppressed proliferation in a dose-dependent manner (Fig. 1A). For the CsCl treatments, NaCl was added to DMEM to maintain a total CsCl and NaCl concentration of 30 mM and maintain the osmotic pressure. The data were fitted using the equation: Y = A/[1 + (CsCl/EC50)h], where Y is the relative proliferation (%), A is the maximum value, CsCl is the CsCl concentration in DMEM (mM), EC50 is the effective concentration (mM), and h is a coefficient. The effective concentration of Cs+ was 10.9 0.9 mM, which is higher than effective concentration observed in C6 glioma cells, 4.75 mM [15]. Although cell

proliferation was suppressed by ~ 54% in response to 10 mM Cs, the cell viability was 80.7 2.9% (Fig. 1B). Extracellular LDH activity was measured to determine whether Cs induced plasma membrane damage (Fig. 1C). The increase in the extracellular LDH activity depends on plasma membrane leakage; therefore, LDH activity indicates plasma membrane integrity. The LDH activity in the extracellular media was not increased following the addition of 0.3–30 mM NaCl to DMEM as a control. The extracellular LDH activity and concomitant plasma membrane damage were not affected by the addition of NaCl to DMEM in doses up to 30 mM. Compared with NaCl, the addition of 0.3–10 mM CsCl to DMEM did not increase the LDH activity. In the LDH assay, the EC50 was 44 13 mM for Cs. Although the cells cultured in DMEM with 30 mM Na+ did not exhibit increased LDH activity, the same concentration of Cs+ seemed increase the LDH activity. Cs+ concentrations < 10 mM Cs+ in DMEM do not induce plasma membrane damage and do not critically decrease cell viability. Regarding the other alkaline metal salts, including LiCl, NaCl, KCl, and RbCl, a dose of 10 mM of each salt did not suppress HeLa cell growth (Fig. 2). The addition of LiCl, NaCl, KCl, or RbCl to DMEM at concentrations of up to 30 mM did not influence HeLa cell growth (data not shown). We determine whether this growth suppression effect of Cs was reversible (Fig. 3). HeLa cells were cultured in the presence or

Fig. 1. Dose-dependent relationship between the external CsCl concentrations and cell growth. (A) Cells were cultured in the presence of 0, 0.1, 0.3, 1, 3, 10, and 30 mM CsCl (n = 3) for 1 day at 37 °C with 5% CO2. The data were fitted using the equation: Y = A/[1 + (CsCl/EC50)h] (details in the text). The means of three independent measurements were calculated, and error bars indicate the standard deviations (n = 3). (B) Cell viability was estimated using the trypan blue dye exclusion assay. Significant differences between the control and CsCltreated cells are indicated by asterisks (P < 0.05 using Student’s t-test). (C) LDH assay. The data represent the means of triplicate measurements and error bars indicate the standard deviations (n = 3).

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Fig. 2. Effects of alkali metals on cell proliferation. HeLa cells were cultured in DMEM with 10% FBS at 37 °C with 5% CO2 (control). HeLa cells were cultured in this media supplemented with 10 mM chloride salts of alkali metals (Li, Na, K, Rb, and Cs) to assess the effects of alkali metals on cell proliferation. The means of three independent measurements were calculated, and error bars indicate the standard deviations. The data are presented as the means SD (n = 3).

absence of 10 mM CsCl for 3 days. The CsCl-treated cells grew more slowly than the controls. The apparent doubling time (T) was fitted using the equation: N = N0 9 2(t/T), where t is the incubation time (h), N is the cell number, and N0 is the initial cell number. The apparent doubling time of the 10 mM CsCl-treated cells was 74 25 h, which was ~ 2.2-fold slower than the control cells (33.2 1.2 h). After 3 days, the media were removed and the cells were washed with fresh media without CsCl. The removal of Cs allowed cell growth to recover, and the apparent doubling time was the same as the control cells (31.5 1.3 h; Fig. 3). Thus, Cs reversibly regulates cell proliferation. Effect of Cs on the cell cycle Potassium channel antagonists inhibit the proliferation of many types of cells, and the activation of K channels might be required for proliferation [16,17]. The addition of Cs to the culture media decreased cell proliferation; however, the proliferation arrest did not

seem to be severe. We used the Fucci-probe system (Fig. 4) and propidium iodide staining (Fig. S2, see also Data S1) to analyze the effect of Cs on the cell cycle. The probes mAG-Geminin (green fluorescent) and mKO2-Cdt1 (red fluorescent) represent S/G2/M phase and late G1 phase, respectively. As previously reported [16,17], the cell cycle arrested in G1 phase when quinidine was added to the culture media (Fig. 4C and Fig. S2B). In the Fucci-probe system analysis, Cs did not strongly arrest the cells in G1 phase compared with quinidine. Propidium iodide staining analysis also showed the same phenomena. Although the Fucci-probe system analysis revealed that the cell phase ratio was not strongly affected by Cs (Fig. 4B), the S phase ratio of CsCl-treated cells in the propidium iodide staining analysis seemed to be higher than control (Fig. S2B). To evaluate the difference between the Fucci-probe system analysis and the propidium iodide staining analysis, we attempted to measure the cell division cycle using microscopic observations (Table 1). One cell cycle period was


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Fig. 3. Extracellular Cs+ reversibly inhibited cell proliferation. HeLa cells were cultured in the presence (black circles) and absence (white circles) of 10 mM CsCl in the media for 3 days. Extracellular Cs was then washed out at the time indicated by the arrow. The data represent the means of triplicate measurements and error bars indicate the standard deviations (n = 3). The doubling time (T1/2) was fitted using the equation: T1/2 = t 9 log2/(logN logN0), where t is the incubation time (h), N is the cell number, and N0 is the initial cell number.

defined as the period between the visualization of red fluorescence (G0/G1 phase) in the nucleus of the mother cell and the nucleus of the daughter cell. The red fluorescence was observed in G0/G1 phase. S/G2 phase was defined as the period between the initial observation of yellow fluorescence in the nucleus, which was produced by the merger of red (G1 phase) and green (S phase) fluorescence, and whole cell green fluorescence. Whole cell green fluorescence represented M phase. The cell phases of the control and 3 mM CsCl-treated cells were the same, and the 10 mM CsCltreated cells extended the amount of time that cells spent in all phases of the cell cycle. The total cell division cycle of Cs-treated cells was ~ 1.6-fold longer than the control cells. Cs entered the cell and interrupted intracellular metabolism The intracellular cation concentrations in HeLa cells were analyzed to clarify whether Cs+ can be transported into the cells. Cytosolic extracts were prepared from cells cultured with or without 10 mM Cs+. Na+ and K+ were detected in the cytosolic extracts from both the control and Cs+-treated cells, but Cs+ was 722

only detected in the cytosolic extracts of the Cs+-treated cells (Fig. 5A). The cell volume was first measured to estimate the intracellular ion concentrations. The diameters of suspended, detached cells were measured from the microscopic images and the cell volumes were calculated. The average cell volume was 1.26 pL. The intracellular Na+ and K+ concentrations in the control cells were 28.5 13.0 and 107.6 0.5 mM, respectively, but Cs+ was not detected in the control cells. The intracellular Na+, K+, and Cs+ concentrations in the CsCl-treated cells were 25.9 7.6, 78.0 6.4, and 22.1 0.9 mM, respectively (Fig. 5B). The intracellular Na+ concentrations were the same in the Cs-treated and control cells, but the intracellular K+ concentrations were decreased and the Cs+ concentrations were increased in the Cs-treated cells compared to the control cells. However, the sum of the intracellular K+ and Cs+ concentrations in the Cstreated cells was similar to the intracellular K+ concentrations in the control cells. The intracellular Cs concentration was higher than the extracellular Cs concentration; therefore, Cs was incorporated in the intracellular space. Pyruvate kinase, which catalyzes the final step of glycolysis in the cytosol, produces pyruvate and ATP from the substrates phosphoenolpyruvate and ADP. In this catalysis step, pyruvate kinase requires K+ as a cofactor [18]. In general, the K+ channel transports K+, and Cs+ acts as a competitive inhibitor. Hence, we verified the behavior of Cs+ as a competitor for K+ in a pyruvate kinase reaction. Pyruvate kinase activity in a crude HeLa cell extract was 44.6 0.7 lmole min1 (mg protein) 1, using K+ as a cofactor. The activity was significantly decreased in the presence of the other alkali metals, Li+, Na+, Rb+, and Cs+ (Fig. 6). For Li+, Na+, and Cs+, the measured activity was half the activity observed with K+. The tendency of alkali metals to inhibit pyruvate kinase activity agreed with a previous study [18]. Furthermore, pyruvate kinase activity was also measured by changing the K+ to Cs+ ratio. Concentrations of up to 10% Cs (90 mM K+ and 10 mM Cs+) in the reaction solution did not inhibit the pyruvate kinase activity, but a 30% Cs solution (70 mM K+ and 30 mM Cs+) decreased the activity. The decrease in pyruvate activity by Cs+ was also shown in a purified pyruvate kinase (Fig. S1, see also Data S1). The purified pyruvate kinase activity with 100 mM K+ was 7.6 0.3 lmole min1 mL1. In comparison to the activity using K+, pyruvate kinase activity with 100 mM Cs+ and 30% Cs+ (70 mM K+ and 30 mM Cs+ in the reaction solution) was 4.9 0.1 and 6.8 0.1 lmole min1 mL1, respectively. The intracellular K+ to Cs+ ratio was ~ 20%. Thus,


D. Kobayashi et al.

104

B

Control

100 Early G1 Late G1 G1/S S/G2/M

Percentage of cell (%)

Late G1 102

G1/S

100

101

mKO2

103

A


S/G2/M

Early G1 101

102 mAG

103

104

100

104

Late G1

G1/S

60

40

20

102

0

Control

CsCI

Quinidine

100

101

mKO2

103

CsCl

80

S/G2/M

Early G1 101

102 mAG

104

100

103

104

C

Late G1 102

G1/S

101

mKO2

103

Quinidine

100

Control 100

Early G1 101

102 mAG

Cs

Quinidine

S/G2/M 103

104

Fig. 4. Cell cycle phase distribution of Cs-treated cells. (A) Representative flow cytometry dot plots of HeLa.S-Fucci cells treated with 10 mM CsCl and 0.1 mM quinidine. Each cell cycle phase was determined using the indicated gates. (B) The bar graph showed the percentage of cells in each cell cycle phase. The data represent the means of triplicate measurements and error bars indicate the standard deviations (n = 3). (C) Fluorescence microscopic image of cells treated with 10 mM CsCl and 0.1 mM quinidine. The red fluorescence of mKO2-Cdt1 indicates G1 phase and the green fluorescence of mAG-Geminin indicates S/G2/M phases. The scale bar indicates 20 lm.

pyruvate kinase activity was decreased in a manner dependent on the intracellular Cs+ content. Intracellular pH determination The decrease in pyruvate kinase activity was postulated to cause a decrease in intracellular metabolism, which induced a reduction in the acidic metabolite levels. The intracellular pH was measured in cells cultured with alkali metals and some K+ channel blockers using flow cytometry (Fig. 7). A representative histogram of the BCECF fluorescence (FL1-H) data obtained from the flow cytometry measurements is shown in Fig. 7A. The geometric mean BCECF

fluorescence depends on the intracellular pH of the cells. The intracellular pH was measured in cells cultured with alkali metals and some K+ channel blockers using a standard pH curve, which was generated at each measurement. The intracellular pH of the control cells was 6.94 0.10. The cells cultured with Cs+ (pH = 7.66 0.25) and quinidine (pH = 7.08 0.04) were alkalized, and the cells cultured with Li+ (pH = 6.52 0.18) and K+ (pH = 6.51 0.28) were acidified. The cells cultured with the alkali metals Na+ (pH = 6.87 0.20) and Rb+ (pH = 7.04 0.45) and the K+ channel blockers TEA (pH = 6.99 0.06) and 4-AP (pH = 6.99 0.07) displayed the same pH as the control cells.


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Table 1. Cell division cycle measurement base on microscopy observation. Cell phase (h) G0/G1 phase Control (n = 10) 3 mM CsCl (n = 12) 10 mM CsCl (n = 4)

S/G2 phase

M phase

Division cycle

9.0 1.6

14.2 1.5

1.1 0.3

24.3 1.6

9.9 2.2

12.9 1.5

1.0 0.5

23.9 2.9

14.4 6.6

22.5 1.6

2.3 0.7

39.2 7.9

Discussion Cesium and K are alkaline metals. The Cs concentration in the Earth’s crust is ~ 3 mgkg1 [19]. People usually ingest stable Cs through crops, which take up Cs from arable land. Concentrations of Cs in the soil and in potatoes from fields in Japan were 1– 11 mgkg1 and 0.004–0.13 mgkg1 dry weight, respectively, in 1991–1994 [20], and those in paddy soil and polished rice were 1.2–53 mgkg1 and 0.0005– 0.0065 mgkg1 dry weight, respectively, in 1996–1997 [21]. Cs ingested via food is assumed to be distributed to the tissues throughout the body in its ionic state through pathways similar to those for distribution of K+. After intraperitoneal administration of 134Cs to rats as a chloride salt, it was found to be distributed in the intestinal tract, muscle, liver, lungs, heart, kidneys, testes, stomach, and spleen [22]. Radioactive Cs was also released into the environment around the nuclear power plants in Fukushima Prefecture after the Fukushima nuclear accident on March 11, 2011. The effect of this Cs, which was consumed via food and entered the human body, has not yet been

Fig. 6. Pyruvate kinase activity of crude HeLa cell extracts. Pyruvate kinase activity was measured in the presence of each alkali metal (Li+, Na+, K+, Rb+, and Cs+), and the K+ to Cs+ ratio was 70 : 30, 90 : 10, and 97 : 3. The data are presented as the means SD (n = 3). Significant differences between the control and treated cells are indicated by asterisks (P < 0.01 using Student’s t-test).

elucidated. In addition, we still do not know the exact pathway by which Cs+ enters cells. Although K+ channel blockers such as 4-AP, quinidine, TEA, and Cs+, have been used in K+ transport studies, there is insufficient information available to describe how Cs+ blocks K+ channels or whether Cs+ competes with K+ in cases where Cs+ passes through the K+ channel pore. Furthermore, there are few studies which have focused on the characteristics of Cs+ transporters across a range of organisms from bacteria to mammals. In the Cs+-treated HeLa cells used in the present study, Cs+ was detected in the cytoplasmic

Fig. 5. Extracellular Cs+ passed through the cell membrane and was present in the intracellular fraction. (A) The intracellular fraction was used for a CE analysis. A.U.: arbitrary units. (B) The intracellular K+, Na+, and Cs+ concentrations were calculated. N.D.: not detected. The data represent the means of three independent measurements and error bars indicate the standard deviations (n = 3).

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Fig. 7. Intracellular pH measurements using flow cytometry. Cells were stained with the pH-sensitive fluorescent dye BCECF-AM in DMEM and incubated at 37 °C for 1 h. The detached cells were analyzed with a FACSCalibur flow cytometer. Raw data from the green fluorescent channel (FL1-H) are shown in the left panel (A). The intracellular pH was estimated using a calibration curve ranging from pH 6.0–8.0 (B). Significant differences between the control and treated cells are indicated by asterisks (P < 0.01 using Student’s t-test).

extracts (Fig. 5) and the concentration was higher than its extracellular concentration. Thus, HeLa cells possess at least one Cs+ uptake pathway. The addition of Cs+ to the culture medium at concentrations up to 10 mM was less toxic toward the cytoplasmic membrane of HeLa cells, as assessed by the trypan blue dye exclusion and LDH assays (Fig. 1). This result suggests that the addition of Cs+ to the medium did not disrupt the cytoplasmic membrane or cause leakage of the intracellular contents, including LDH. The proliferation of the Cs-treated cells was slow (Fig. 2), and removal of Cs+ from the medium restored their proliferation rate (Fig. 3). HeLa cell growth was recovered by excluding Cs+ from the extracellular medium. Thus, HeLa cell proliferation was not disrupted by Cs+. HeLa cell proliferation was inhibited by the addition of 10 mM Cs to the culture medium, whereas this effect was reversed by removing Cs from the culture medium (Fig. 3). Therefore, the inhibitory effect of Cs on cell proliferation was reversible. These results were in good agreement with the reversible inhibition of cell proliferation by the K+ channel blockers glibenclamide and quinidine in MCF7 human breast cancer tumor cells [17]. However, K+ channel blockers did not always inhibit cell growth. Charybdotoxin, iberiotoxin, margatoxin, and apamin did not inhibit the growth of human MCF-7 cells, but linogliride, 4-AP, and TEA suppressed their proliferation. However, these drugs did not show reversible effects. Glibenclamide and quinidine, which inhibited ATP-sensitive K+ channels, induced cell cycle arrest during the G1 phase, thereby preventing cell proliferation [16,17]. Furthermore, Weigher et al. [15] reported

that the growth of mouse C6-glioma cells was also inhibited by quinidine and Cs. The half-effective concentration required to induce cytotoxicity was 112 lM for quinidine and 4.75 mM for Cs, as estimated using the LDH assay. Therefore, Cs might act as an ATPsensitive K+ channel blocker, similarly to glibenclamide and quinidine as observed in previous studies. Although excessive drug use inhibits cell proliferation, the reversible inhibition of cell proliferation by Cs+ is assumed to be physiologically mild. We analyzed the cell division cycle by microscopic observations using Fucci indicator probes. Cells treated with Cs concentrations of up to 3 mM Cs did not display any difference in the cell division cycle; however, 10 mM Cs treatment extended the amount of time that cells spent in all phases of the cell cycle (Table 1). Although the K+ channel blocker quinidine induced cell cycle arrest during the G1 phase, Cs+ did not show the same effect (Fig. 4). Although the Fucciprobe system analysis could not clearly separate the S phase from the G1 and G2 phases by flow cytometry, the other propidium iodide staining analysis revealed the S phase. This latter analysis indicated that 10 mM CsCl treatment increased the proportion of cells in the S phase and decreased that in the G2/M phase compared with those in the control (Fig. S2). The time that Cs-treated cells spent in each phase of the cell cycle was longer than that spent by the control cells (Table 1). During 2 days of cultivation, the control cells were thought to divide approximately twice; however, the Cs-treated cells were thought to divide once and then proceed to the second cell division (Table 1). Prior to the propidium iodide staining analysis, the


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cells were synchronized at the M phase by nocodazole in normal DMEM; subsequently, the cells were transferred to fresh DMEM in the presence or absence of 10 mM CsCl. Both the control and Cs-treated cells underwent cell division. However, CsCl treatment slowed down the progression through the cell cycle. The effect of Cs+ on proliferation was not to interrupt cell division but to slow down the cell cycle and thus extended the cell division time. Further studies are required to understand the relationship between Cs+ and the cell cycle. The proposed mechanisms by which Cs+ suppresses cell proliferation are described below. Cs+ was transferred to the intracellular space through the cell membrane via a K+ transporter(s); thus, Cs+ influx increased and K+ influx decreased (Fig. 5). The cytoplasmic space had lower K+ and higher Cs+ concentrations than the general cytosolic cation composition. In the cytosolic environment, an enzyme associated with glycolysis, pyruvate kinase, which requires K+ as a cofactor for catalysis, did not show high activity (Fig. 6, Fig. S1). A decrease in glycolysis might inhibit pyruvate kinase, and, subsequently, alter intracellular metabolite production, which probably decreased the levels of the metabolite. The metabolites are generally acidified compounds; therefore, the decrease in acidic metabolite production induced by the Cs treatment shifted the intracellular pH to an alkaline level (Fig. 7). Alkalization of the intracellular environment also suppressed cell proliferation. Subsequently, cell proliferation was suppressed by Cs+ intake. Further experiments, such as the detection of intracellular metabolites, are necessary to confirm this hypothesis. Moreover, in future studies, we must investigate other cell lines to determine whether these phenomena are specific to HeLa cells. From a physiological perspective, the types of ion channel or transporter involved in transporting Cs through cell membranes must be identified. For people living in Fukushima, Japan, after the Fukushima nuclear disaster, information on the effects of Cs+ on the human body is particularly important. Studying the dynamics of Cs+ at the cellular level is thus an important task for scientists living in Fukushima. We speculated that HeLa cells also possess at least one Cs+-export pathway. We will investigate this pathway (s) involved in transporting Cs across the cell membrane in future studies. Intraperitoneal administration of different doses of CsCl in mice resulted in the rapid transfer of Cs+ from the blood to the tissue compartments. Mice that were intraperitoneally injected with CsCl for 14 consecutive days prior to sarcoma implantation exhibited 726

reduced tumor-induced mortality compared with controls [23]. Intraperitoneal administration of different doses of CsCl in mice was associated with the rapid transfer of Cs+ from the blood to the tissue compartments. Mice that were intraperitoneally injected with CsCl for 14 consecutive days prior to sarcoma implantation exhibited reduced tumor-induced mortality compared with the controls [23]. A group of mice were administered 28 once-daily intraperitoneal injections of CsCl, at 5 mEqkg1day1. Twenty-eight days after the final injection, the mice tissues were shown to have accumulated Cs during this period, in the following order: skeletal muscle > ileum > brain > liver > lung > blood. The Cs content was 11.5– 144 mEqkg1 wet tissue in the skeletal muscle and 10– 20 mEqkg1 in the blood [24]. These levels were in good agreement with the distribution of Cs-134 in the adult rat after intraperiotoneal administration [22]. From these reports, it can be concluded that Cs could accumulate in some tissues to a level of ~ 10 mM. If the distribution of Cs concentration in humans is the same as that in mice and rats, oral intake of 6 g CsClday1 [1,6,7] in adult humans would lead to concentrations of over 10 mEqkg1 of CsCl in the skeletal muscle, ileum, liver, and lung. Incorporation of Cs in the tissues was expected to decrease tumor progression. In this study, Cs+ suppressed cell proliferation, and pyruvate kinase activity in the glycolysis pathway was involved in suppressing cell growth. These results are in good agreement with the Warburg effect on tumor cell metabolism. If we control Cs+ uptake by the cells, Cs+ would play a key role in suppressing tumor progression in a clinical context. In conclusion, the results of our investigation of the effects of Cs+ on cultured human cells suggested that Cs+ suppressed cell proliferation without inducing severe damage in the cells. These findings support the idea of using Cs to inhibit tumor growth without damaging the normal tissue. Our ultimate aim is to identify the Cs ion channels or transporters.

Acknowledgements This work was supported by Maekawa Houonkai Foundation and JSPS KAKENHI Grant Number 26460299.

Author contributions DK and AH conceived and designed the study, edited and revised the manuscript and approved the final version of the manuscript; DK, KK, TN, and TN performed the experiments and analyzed the data; DK,


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AH, TN, and KS interpreted the results of the experiments; DK prepared the figures and drafted the manuscript.

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Disclosures

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The authors have no conflicts of interest, financial or otherwise, to declare.

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Supporting information Additional Supporting Information may be found online in the supporting information tab for this article: Fig. S1. Pyruvate kinase activity of purified enzyme. Fig. S2. Cell cycle phase distribution of Cs-treated cells. Data S1. Materials and Methods supplement.


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