Extracellular Guanosine and Guanine Nucleotides Decrease Viability of Human Breast Cancer SKBR-3 Cells

Abstract

Though the physiological effects of adenosine and adenine nucleotides on purinergic receptors in cancer cells have been well studied, the influence of extracellular guanosine and guanine nucleotides on breast cancer cells remains unclear. Here, we show that extracellular guanosine and guanine nucleotides decrease the viability and proliferation of human breast cancer SKBR-3 cells. Treatment with guanosine or guanine nucleotides increased mitochondrial production of reactive oxygen species (ROS), and modified the cell cycle. Guanosine-induced cell death was suppressed by treatment with adenosine or the equilibrium nucleoside transporter (ENT) 1/2 inhibitor dipyridamole, but was not affected by adenosine receptor agonists or antagonists. These results suggest that guanosine inhibits adenosine uptake through ENT1/2, but does not antagonize adenosine receptors. In contrast, guanosine triphosphate (GTP)-induced cell death was suppressed not only by adenosine and dipyridamole, but also by the A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA), suggesting that GTP-induced cell death is mediated in part by an antagonistic effect on adenosine A1 receptor. Thus, both guanosine and GTP induce apoptosis of breast cancer cells, but via at least partially different mechanisms.

INTRODUCTION

Breast cancer is the most common type of cancer among women and has a high mortality rate.¹⁾ It can be divided into four main subtypes according to the type of receptor expressed. These are estrogen receptor (ER)-positive, progesterone receptor (PgR)-positive, human epidermal growth factor receptor type 2 (HER2)-positive, and triple-negative (TNBC).²⁾ When the pathology of breast cancer is advanced and invasive, surgery alone does not provide sufficient therapeutic effects, and it is common to combine surgery with chemotherapy or radiotherapy. Chemotherapy for breast cancer uses anthracycline and taxane anticancer drugs, but these drugs often have side effects such as emetic effects and hair loss.³⁾ Drugs with mechanisms different from current anticancer drugs may enable more efficient breast cancer chemotherapy with reduced side effects. Molecular targeting is now common in the HER2-positive breast cancer, because traditional treatments such as surgery, chemotherapy, and radiation therapy have problems such as invasiveness and side effects and have limited effectiveness.⁴⁾ However, there are cases that are refractory to molecular-targeted drugs such as anti-HER2 antibodies, and the acquisition of resistance is also an issue.⁵⁾ Since hormone receptor-targeted agents such as tamoxifen have been shown to be largely ineffective in ER-negative tumors,⁶⁾ there is a need for new therapeutic drugs against ER-negative tumors with few side effects. In this study, we used the ER-negative, HER-2-positive breast cancer cell line SKBR-3.

Nucleosides consists of a purine base (such as adenine or guanine) or a pyrimidine base and a pentose, while nucleotides are phosphate-containing nucleosides. Nucleotides serve as energy donors in cells and participate in the synthesis of nucleic acids. It has also been found that nucleosides and nucleotides are released outside cells in response to cell stimulation and function as signaling molecules in the purinergic system.^7,8) Extracellular nucleotides are degraded by ectonucleoside triphosphate diphosphohydrolase (CD39) and ecto-5′-nucleotidase (CD73) ectoenzymes to generate nucleosides,⁹⁾ which are taken up by nucleoside transporters (NTs) into target tissues and act intracellularly. The NTs include the Na⁺-dependent concentrated nucleoside transporters (CNT) family and the Na⁺-independent equilibrium nucleoside transporters (ENT) family.¹⁰⁾ Among them, ENT is expressed in a wide range of tissues, whereas CNT is expressed organ-specifically.^11,12) Therefore, the concentration of the nucleosides themselves has a greater influence on the uptake of nucleosides via ENTs, especially subtypes ENT1/2.^11,12)

Nucleosides and nucleotides play important roles in intercellular communication via membrane receptors. For example, adenosine acts on the P1 receptor, and ADP and ATP act on the P2 receptor to mediate various physiological activities.¹³⁾ Activation of P2Y2 receptor by ATP or uridine triphosphate (UTP) increases the proliferation of MDA-MB-231 breast cancer cells with high metastatic potential, and this effect is blocked by P2Y2 receptor-knockdown.¹⁴⁾ We previously reported that ATP and ADP have a radioprotective effect in normal cells.¹⁵⁾ However, although there have been many studies on the effects of adenine-based nucleotides on cancer cells, the effects of extracellular guanine-based nucleoside and nucleotides on breast cancer cells are unknown. Since guanosine has been reported to induce apoptosis of glioblastoma cells via activation of adenosine receptors, it is possible that guanosine also acts on adenosine receptors in cancer cells.¹⁶⁾

In this study, therefore, we investigated the effects of guanine-based nucleoside (guanosine) and nucleotides (guanosine triphosphate (GTP), guanosine diphosphate (GDP), GMP) on ER-negative, HER-2-positive breast cancer SKBR-3 cells, and the involvement of purinergic signaling pathways in these effects. Our results showed that both guanosine and guanine nucleotides induce apoptosis of ER-negative, HER-2-positive breast cancer cells and increased mitochondrial generation of reactive oxygen species (ROS). Interestingly, the mechanisms of guanosine-induced cell death and GTP-induced cell death appear to differ, at least in part.

MATERIALS AND METHODS

Reagents

Guanosine, GMP, GDP, GTP, ATP, inosine triphosphate (ITP), UTP, adenosine, AMP, ADP, and dipyridamole (an inhibitor of equilibrative nucleoside transporter 1/2 (ENT1/2)) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). 2-Chloro-N6-cyclopentyladenosine (CCPA) (a selective, high-affinity agonist of adenosine A1 receptor), CGS21680 (an agonist of adenosine A2A receptor), BAY60-6583 (a selective agonist of adenosine A2B receptor), PSB36 (a selective antagonist of adenosine A1 receptor), SCH442416 (a selective antagonist of adenosine A2A receptor), PSB603 (a selective antagonist of adenosine A2B receptor) and MRS3777 (a selective antagonist of adenosine A3 receptor) were purchased from Tocris Bioscience (Ellisville, MO, U.S.A.). 2-Cl-IB-MECA (MECA) (a highly selective agonist of A3 receptor) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). N-Acetyl-L-cysteine (NAC) was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).

Cell Culture

Human breast adenocarcinoma SKBR-3 cells (ATCC) were grown in RPMI 1640 medium (Wako Pure Chemical Corporation), supplemented with 10% fetal bovine serum (FBS) (Gibco, Amarillo, TX, U.S.A), 100 U/mL penicillin (Thermo Fisher Scientific, Tokyo, Japan), and 100 µg/mL streptomycin. All cultures were performed in an incubator under an atmosphere of 5% CO₂ in air, at 37 °C.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay

We examined cell viability by MTT assay as described previously.¹⁷⁾ Cells were seeded in 96-well plates at 1.0 × 10³ cells per well and cultured for 24 h. Then, inhibitor or vehicle (dimethyl sulfoxide (DMSO)) was added to the medium 30 min before addition of nucleoside or nucleotide. After 72 h, the cells were incubated with MTT solution (5 mg/mL phosphate buffered saline (PBS)) for 3 h, and the blue formazan crystals in living cells were dissolved in stop solution. The absorbance (570 nm) was measured on a WALLAC 1420 ARVO MX multilabel counter (PerkinElmer, Inc., Waltham, MA, U.S.A.). We have examined 3–4 samples in a group in an experiment and performed at least 3 independent experiments.

Colony Assay

Cell-proliferation ability was analyzed by colony assay. SKBR-3 cells were seeded in 40 mm dishes at 8.0 × 10⁴ cells/mL and cultured for 24 h. Then, nucleosides or nucleotides was added to the medium. After incubation for a further 24 h, cells were harvested by trypsinization and counted. They were re-seeded in 6-well plates at 2.0 × 10² cells/well, cultured for 10 d, and then stained with 0.5% crystal violet. Colonies containing more than 50 cells were identified as survivors and counted.

Annexin V-Propidium Iodide (PI) Assay

We examined cell death as described previously.¹⁵⁾ SKBR-3 cells were seeded in 40 mm dishes at 8.0 × 10⁴ cells/mL and cultured for 24 h. Then, nucleosides or nucleotides were added and incubation was continued for 24 h. Cells were stained with annexin V-fluorescein isothiocyanate (FITC)/PI (MEBCYTO^® Apoptosis Kit (Annexin V-FITC Kit), Medical & Biological Laboratories Co., Ltd., Tokyo, Japan) and analyzed on a BD FACS Calibur Flow Cytometer (Becton, Dickinson and Co., Franklin Lakes, NJ, U.S.A.). The data were analyzed with FlowJo software (FlowJo, LCC, Ashland, OR, U.S.A.).

Flow-Cytometric Analysis of Cell Cycle

We examined the cell cycle by analyzing the DNA content of cells on a BD FACS Calibur Flow Cytometer as described previously.¹⁵⁾ The data were analyzed with FlowJo software.

Flow-Cytometric Analysis of Mitochondrial ROS Production

Mitochondrial ROS production was detected by the use of Mitochondrial Superoxide Indicator (mitoSOX™ Red) (Thermo Fisher Scientific). SKBR-3 cells were seeded in 40 mm dishes at 8.0 × 10⁴ cells/mL and cultured for 24 h. Then, nucleoside or nucleotide was added to the medium. After incubation for a further 24 h, cells were harvested by trypsinization, washed with ice-cold Hank’s balanced salt solution (HBSS), and then incubated with 1 µM mitoSOX™ Red for 10 min at 37 °C in dark. The cells were washed with ice-cold HBSS and analyzed on a BD FACS Calibur Flow Cytometer. The data were analyzed with FlowJo software.

Real-Time RT-PCR

Real-time RT-PCR was performed as described previously.¹⁸⁾ RT²-qPCR^® primer assays for human SLC29A1 (ENT1) and SLC29A2 (ENT2) were purchased from Qiagen (Venlo, The Netherlands). The values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression.

Western Blotting

Western blotting was performed as described previously.^15,17,18) SKBR-3 cells were lysed in cold lysis buffer containing 1% Triton X-100 and protease inhibitor cocktail (Sigma-Aldrich) at 4 °C for 30 min. Lysates were centrifuged at 10000 × g for 15 min. To the supernatant was added 2× sample buffer (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10 mM dithiothreitol, and the mixture was boiled for 10 min at 95 °C. Protein (20 µg/lane) was analyzed by means of 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and bands were transferred onto a polyvinylidene difluoride (PVDF) membrane. The blots were blocked overnight in 1% bovine serum albumin at 4 °C. For detection, the blots were incubated with primary antibody (mouse anti-ENT1 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, U.S.A.) (1 : 1000), mouse anti-ENT2 monoclonal antibody (Santa Cruz Biotechnology) (1 : 1000) and anti-β-actin monoclonal antibody (FUJIFILM Wako Pure Chemical Corporation) (1 : 20000)) overnight at 4 °C, and further incubated with goat horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) antibody (Cell Signaling Technology, Danvers, MA, U.S.A.) (1 : 20000) for 1.5 h at room temperature. Specific proteins were visualized by using ImunoStar^® LD (FUJIFILM Wako Pure Chemical Corporation) according to the manufacturer’s instructions.

Statistics

Data are presented as the mean ± standard error of the mean (S.E.M.). The statistical significance (p-value) of differences between control and other groups was determined using Student’s t test (two-group test) or Dunnett’s test (multiple-group test) in the Prism statistical software package (GraphPad Software, San Diego, CA, U.S.A.). A value of p < 0.05 was considered statistically significant.

RESULTS

Extracellular Guanosine or Guanine Nucleotides Suppresses Survival of Breast Cancer SKBR-3 Cells

Nucleotide triphosphates (GTP, ATP, UTP or ITP) were added to SKBR-3 cells and the cell viability was evaluated after 72 h (Fig. 1A). GTP showed the greatest suppressing effect on cell viability (Fig. 1A). ATP also decreased cell viability, whereas ITP, which contains a purine base like adenine and guanine, and UTP, which contains a pyrimidine base, had no effect. Guanosine or guanine nucleotides decreased the viability of SKBR-3 cells in a dose-dependent manner at 72 h after addition (Fig. 1C). Among them, guanosine had the most potent effect. GMP, GDP and GTP all showed similar effects. Guanosine and GTP also induced a decrease of colony formation at 10 d after addition in SKBR-3 cells (Fig. 1B), indicating that they inhibited cell proliferation.

Fig. 1. Effects of Extracellular Guanosine and Guanine Nucleotides on the Viability of SKBR-3 Cell

(A) SKBR-3 cells were treated with various nucleotides (100 µM). (B) SKBR-3 cells were treated with guanosine (GUO) and guanine nucleotides (50 µM), and incubated for 10 d. (C) SKBR-3 cells were treated with various concentrations of GUO and guanine nucleotides (10–100 µM), then incubated for 72 h. (A, C) Cell viability was measured by MTT assay. (B) Survival fraction was analyzed by colony assay. Statistical analysis was performed by one-way ANOVA followed by Dunnett’s test. Error bars indicate mean ± S.E.M. [(A) n = 11–12, (B) n = 9, (C) n = 10–13, three or more independent experiments]. Significant differences between control and test groups are indicated by **** (p < 0.0001), ** (p < 0.01) or * (p < 0.05).

Extracellular Guanosine/Guanine Nucleotides Affect the Apoptosis, Cell Cycle and Mitochondrial Production of Reactive Oxygen Species in SKBR-3 Cells

To detect dead cells, we analyzed the total percentage of annexin V⁺ cells, including both annexin V⁺/PI⁻ cells (early phase of apoptosis) and annexin V⁺/PI⁺ cells (necrosis or late phase of apoptosis), after treatment with guanosine, GMP, GDP, or GTP. The percentages of dead cells (annexin V⁺ cells) were significantly increased at 24 h after addition of GMP, GDP, and GTP (Fig. 2A).

Fig. 2. Effects of Extracellular Guanosine or Guanine Nucleotides on Cell Cycle and Generation of Mitochondrial ROS in SKBR-3 Cells

(A–C) SKBR-3 cells were treated with adenosine (ADO) (100 µM), GUO (50 µM) or guanine nucleotides (50 µM) and incubated for 24 h. (A) The cells were stained with annexin V-FITC and PI. The percentage of annexin V⁺ cells (A), the percentage of cells in each phase of the cell cycle (B), and mitochondrial ROS generation (C) were measured by flow cytometry. (D) SKBR-3 cells were treated with NAC (1 mM) for 30 min prior to addition of GUO, GMP, GDP or GTP (50 µM). After incubation for 72 h, cell viability was measured by MTT assay. Statistical analysis was performed by one-way ANOVA (A–C) or two-way ANOVA (D) followed by Dunnett’s test. Error bars indicate mean ± S.E.M. [(A, B) n = 3, (C) n = 4, (D) n = 12, three or more independent experiments]. Significant differences between control and test groups are indicated by **** (p < 0.0001), ** (p < 0.01) or * (p < 0.05).

Next, we examined the effects of adenosine, guanosine and guanine nucleotides on the cell cycle in SKBR-3 cells. GMP, GDP, and GTP significantly decreased the percentage of cells in the G₁/G₀ phase at 24 h after treatment (Fig. 2B).

We also examined whether guanosine or guanine nucleotide treatment affects mitochondrial ROS production. The proportion of cells highly producing Mitochondrial ROS was significantly increased 24 h after addition of guanosine, GMP, GDP, and GTP (Fig. 2C).

To investigate an involvement of ROS in the decrease of cell viability, we examined the effect of an antioxidant NAC on the decrease of cell viability induced by guanosine, GMP, GDP, or GTP. The decrease of cell viability induced by guanine nucleotides (GMP, GDP and GTP) was suppressed by co-treatment with NAC, though that induced by guanosine was not suppressed (Fig. 2D).

Extracellular Adenosine Antagonizes the Effects of Extracellular Guanosine

The decrease in cell viability induced by guanosine was attenuated by co-treatment with adenosine, AMP, ADP or ATP (Fig. 3A). Co-treatment with adenosine caused a significant increase of the IC₅₀ of guanosine (Fig. 3B), suggesting that adenosine antagonizes the effect of guanosine. Since this result might imply that extracellular guanosine competes with extracellular adenosine for interaction with adenosine receptors on the cell membrane, we co-treated SKBR-3 cells with guanosine and adenosine receptor agonists or antagonists, and monitored the cell viability. The decrease in cell viability induced by guanosine was not suppressed by co-treatment with either agonists or antagonists of adenosine A1, A2A, A2B, or A3 receptors. However, dipyridamole, an inhibitor of ENT1/2, which mediates transmembrane transport of nucleosides, significantly decreased the effect of guanosine (Fig. 3C).

Fig. 3. Effects of Adenosine or Adenine Nucleotides on Guanosine-Induced Cell Death

(A) SKBR-3 cells were treated with ADO (100 µM), AMP (100 µM), ADP (100 µM) or ATP (100 µM) for 30 min prior to addition of GUO (50 µM). (B) SKBR-3 cells were treated with ADO (35 µM) prior to addition of GUO (0.1–1000 µM). (C) SKBR-3 cells were treated with ADO (100 µM), dipyridamole (DIPY) (10 µM), 2-Chloro-N6-cyclopentyladenosine (CCPA) (30 µM), CGS21680 (0.1 µM), BAY60-6583 (1 µM), 2-Cl-IB-MECA (MECA) (1 µM), PSB36 (5 µM), SCH442416 (10 µM), PSB603 (10 µM) or MRS3777 (10 µM) for 30 min prior to addition of GUO (50 µM). After incubation for 72 h, cell viability was measured by MTT assay. Statistical analysis was performed by two-way ANOVA followed by Dunnett’s test (A, C). Error bars indicate mean ± S.E.M. [(A) n = 11–16, (B, C) n = 12–16, three or more independent experiments]. Significant differences between GUO and test groups are indicated with **** (p < 0.0001), *** (p < 0.001), ** (p < 0.01) or * (p < 0.05).

Dipyridamole Inhibits the Effects of Extracellular Guanosine

We further investigated the effect of dipyridamole on guanosine-induced cell death and mitochondrial ROS production. Like co-treatment with adenosine (Fig. 3B), co-treatment with dipyridamole caused a significant increase in the IC₅₀ of guanosine (Fig. 4A), suggesting that both dipyridamole and adenosine suppress the effect of guanosine. Mitochondrial ROS production induced by guanosine was also suppressed by dipyridamole, though not by adenosine (Fig. 4A). ENT is expressed in a wide range of tissues, and ENT1/2 have also been reported to be functionally expressed in various breast cancer cell lines.¹⁹⁾ We confirmed that both ENT1 and ENT2 mRNAs are expressed in SKBR-3 cells (Fig. 4C). We also detected the protein expression of ENT1 and ENT2 in SKBR-3 cells (Fig. 4D). Though the expression of ENT2 mRNA was lower than that of ENT1 mRNA, we detected the protein expression of ENT2 as well as ENT1, suggesting that both ENT1 and ENT2 would be functionally expressed in SKBR-3 cells.

Fig. 4. Effects of Dipyridamole on Guanosine-Induced Cell Death and Mitochondrial ROS Production

SKBR-3 cells were treated with DIPY (5 µM) (A), ADO (100 µM), Dipyridamole (DIPY) (10 µM) (B), prior to addition of GUO (0.1–1000 µM) (A) or GUO (50 µM) (B), then incubated for 72 h (A) or 24 h (B). (A) Cell viability was measured by MTT assay. (B) Mitochondrial ROS production was analyzed by flow cytometry. (C) Expression of ENT1 and ENT2 mRNAs was analyzed by real time RT-PCR. (D) Protein expression of ENT1 and ENT2 was detected by Western blotting. Typical data from 3 samples was shown. Statistical analysis was performed by two-way ANOVA followed by Dunnett’s test (B). Error bars indicate mean ± S.E.M. [(A) n = 12, (B) n = 12, (C) n = 4 three or more independent experiments]. Significant differences between GUO and test groups are indicated with **** (p < 0.0001), *** (p < 0.001) or ** (p < 0.01).

GTP Induces Death of Cancer Cells via a Distinct Mechanism from That of Guanosine

When SKBR-3 cells were co-treated with GTP and adenosine, AMP, ADP or ATP, the GTP-induced decrease in cell viability was significantly suppressed (Fig. 5A). Since adenosine is generated from AMP, ADP or ATP by ecto-nucleotidases, the effect of GTP was cancelled by co-treatment with not only adenosine but also AMP, ADP, and ATP. We also co-treated SKBR-3 cells with GTP and adenosine receptor agonists or dipyridamole. As in the case of guanosine, the decrease in cell viability after treatment with GTP was suppressed by adenosine or dipyridamole. However, different from the case of guanosine, the decrease in cell viability was also suppressed by co-treatment with CCPA, an agonist of adenosine A1 receptor, though not by CGS21680 (an agonist of adenosine A2A receptor), BAY60-6583 (an agonist of adenosine A2B receptor) or 2-Cl-IB-MECA (an agonist of adenosine A3 receptor) (Fig. 5B). These results suggest the involvement of A1 receptor in the inhibitory action of GTP. Furthermore, co-treatment with CCPA caused an increase in the IC₅₀ of GTP (Fig. 5C), suggesting that the A1 receptor agonist blocked the activating effect of GTP on the A1 receptor.

Fig. 5. Effects of Adenosine A1 Receptor Agonist on GTP-Induced Cell Death

(A, B) SKBR-3 cells were treated with ADO (100 µM), AMP (100 µM), ADP (100 µM), ATP (100 µM) (A), ADO (100 µM), DIPY (10 µM), CCPA (30 µM), CGS21680 (0.1 µM), BAY60-6583 (1 µM) or MECA (1 µM) (B) for 30 min prior to addition of GTP (50 µM), then incubated for 72 h. (C) SKBR-3 cells were treated with CCPA (30 µM) for 30 min prior to addition of GTP (0.1–1000 µM). Cell viability was measured by MTT assay. Statistical analysis was performed by two-way ANOVA followed by Dunnett’s test (A, B). Error bars indicate mean ± S.E.M. [(A) n = 12–16, (B) n = 11–28, (C) n = 12, three or more independent experiments]. Significant differences between control or GTP and test groups are indicated with **** (p < 0.0001), ** (p < 0.01) or * (p < 0.05).

DISCUSSION

In this study, we found that extracellular guanosine and guanine nucleotides induce the death of breast cancer SKBR-3 cells, and that production of mitochondrial ROS was increased in cells treated with guanosine and guanine nucleotides. Guanine nucleotides but not guanosine reduced the cell population in the G₁/G₀ phase of the cell cycle. Similarly, guanine nucleotides but not guanosine significantly induced an increase of annexin V⁺ cells, suggesting that the decrease of the cell population in the G₁/G₀ phase might be a consequence of cell death in the G₁/G₀ phase. Guanosine and GTP also inhibited colony formation (10 d). Overall, these results suggest that guanosine and guanine nucleotides would have a potent anti-cancer effect, at least on the breast cancer SKBR-3 cell line.

The percentage of cell viability at 72 h (Fig. 1C) and colony formation at 10 d (Fig. 1B) was lower in guanosine-treated group than guanine-nucleotide treated groups. On the other hand, the percentage of dead cells at 24 h (Fig. 2A) was higher in guanine-nucleotide treated groups than guanosine-treated group, suggesting that guanine nucleotides would induce acute cell death, and also suggesting that guanosine might inhibit long term cell proliferation. These results implicates that the mechanism of decreased cell viability is different between guanosine and guanine nucleotides.

In mitochondria, superoxide anion radical, an ROS, is generated during ATP synthesis. Usually, ROS would be eliminated by endogenous antioxidants,²⁰⁾ but when mitochondrial dysfunction occurs, excess ROS production causes various cellular malfunctions,²¹⁾ including oxidative damage, cell cycle arrest, and apoptosis.²²⁾ Our results show that guanosine and guanine nucleotides increased mitochondrial ROS production. However, apoptosis and cell cycle outcomes differed between guanosine and guanine nucleotides; guanosine did not significantly induce apoptosis or cell cycle abnormality. A possible reason for this might be that the induction of cell death by guanosine is caused by the disruption of mitochondrial function, such as oxidative phosphorylation, rather than by an increase of mitochondrial ROS production. Functional mitochondria with respiratory capacity are critical for tumorigenesis and cancer cell survival. It has been shown that inhibitors of mitochondrial oxidative phosphorylation enhance the eradication of stem-like tumor cells.²³⁾ In our results, an antioxidant NAC suppressed the effect of guanine nucleotides, suggesting that ROS itself would be involved in guanine nucleotides-induced decrease of cell viability. On the other hand, NAC did not suppress the effect of guanosine, suggesting that mitochondrial disfunction rather than ROS might be involved in guanosine-induced decrease of cell viability. Therefore, we consider that guanosine might disrupt oxidative phosphorylation, a major function of mitochondria, leading to cell death and secondary generation of ROS. On the other hand, an increase of mitochondrial ROS production by guanine nucleotides would lead to the accumulation of cellular ROS, which could cause cell cycle abnormality and apoptosis, resulting in a decrease of cell viability.

Since it is reported that guanosine acts on adenosine receptor,^16,24) we tried co-treating SKBR-3 cells with adenosine and guanosine. The guanosine-induced reduction in cell viability was suppressed by adenosine. This was also the case for guanine nucleotides. However, the guanosine-induced cell death was not suppressed by co-treatment with adenosine receptor agonists and antagonists, indicating that guanosine action on adenosine receptors is not implicated in the death of SKBR-3 cells.

Thus, we next considered the possibility that guanosine induces cell death by inhibiting transporters that transport nucleosides across the cell membrane. Cancer cells express ENT and actively supply nucleosides for nucleotide synthesis via the salvage pathway for DNA synthesis and cell proliferation.²⁵⁾ We found that dipyridamole, an inhibitor of ENT1/2, blocked the effect of guanosine. These results suggest two possible mechanisms by which guanosine induces cell death. First, since dipyridamole increases the extracellular adenosine concentration by inhibiting ENT1/2-mediated intracellular transport, extracellular adenosine might block the guanosine-induced cytotoxicity. Second, if transport of guanosine into cells through ENT1/2 results in an induction of cell death, dipyridamole might block the entry of guanosine into cells through ENT1/2. Since we found that mitochondrial ROS production by guanosine was suppressed by co-treatment with dipyridamole, but not adenosine, the latter explanation seems more likely. Though we cannot measure concentration of intracellular guanosine, it is also important to determine the change in concentration of intracellular guanosine after treatment with guanosine.

It has been reported that guanosine affects intracellular nucleotide pools in Jurkat cells.²⁶⁾ Therefore, another possibility is that guanosine reduces the amount of ATP in cells, and that supplementation with adenosine restores the ATP level, suppressing cell death. AMP, ADP, or ATP suppressed the effect of guanosine less effectively than did adenosine. This is probably because adenosine is easily taken up into cells, whereas no transporter responsible for intracellular translocation of adenine nucleotides has been identified. This would be consistent with a report that adenosine increases intracellular ATP levels.²⁷⁾

On the other hand, the effect of GTP was blocked by an A1 receptor agonist, suggesting that GTP may antagonize basal activation of A1 receptors by extracellular adenosine, which might be important for cell survival. Since dipyridamole increases extracellular adenosine concentration via ENT1/2 inhibition, the effect of GTP on A1 receptor might be cancelled by the increased level of extracellular adenosine in the presence of dipyridamole. Thus, the mechanism of GTP-induced cell death is at least partly different from that of guanosine-induced cell death.

We also observed that ATP, ADP, and AMP but not adenosine also induced decrease of cell viability. Though the mechanism is not revealed in this study, it is also interesting and important to identify the mechanism in future study. The difference of mechanism between adenine nucleotides and guanine nucleotides/nucleoside is also an important issue.

In conclusion, our findings indicate that extracellular guanosine and guanine nucleotides induce death of ER-negative and HER-2-positive breast cancer SKBR-3 cells by affecting mitochondrial function and proliferation in different ways. These findings may open up new therapeutic targets for ER-negative and HER-2-positive breast cancer. We further plan to investigate the effect of guanosine and guanine nucleotides on other types of breast cancer cells such as TNBC.

Acknowledgments

This work was supported in part by JSPS KAKENHI Grant Numbers: JP 19K08185 and 22K07780 (Grant-in-Aid for Scientific Research (C)) (to MT).

Conflict of Interest

The authors declare no conflict of interest.

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