Chinese Journal of Natural Medicines  2019, Vol. 17Issue (8): 608-615  
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Cite this article as: 

LI Zheng, CHEN Qian-Qian, LAM Christopher Wai Kei, GUO Jian-Ru, ZHANG Wei-Jia, WANG Cai-Yun, WONG Vincent Kam Wai, YAO Mei-Cun, ZHAN GWei. Investigation into perturbed nucleoside metabolism and cell cycle for elucidating the cytotoxicity effect of resveratrol on human lung adenocarcinoma epithelial cells[J]. Chinese Journal of Natural Medicines, 2019, 17(8): 608-615.
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Research funding

This work was supported by the Macao Science and Technology Development Fund (FDCT, No. 0052/2018/A2)

Corresponding author

YAO Mei-Cun, E-mails:Yaomeicun@gmail.com
ZHANG Wei, E-mails:Wzhang@must.edu.mo

Article history

Received on: 13 May., 2019
Available online: 20 Aug., 2019
Investigation into perturbed nucleoside metabolism and cell cycle for elucidating the cytotoxicity effect of resveratrol on human lung adenocarcinoma epithelial cells
LI Zheng1 , CHEN Qian-Qian1 , LAM Christopher Wai Kei2 , GUO Jian-Ru1 , ZHANG Wei-Jia3 , WANG Cai-Yun1 , WONG Vincent Kam Wai1 , YAO Mei-Cun3 , ZHAN GWei1     
1 State Key Laboratory of Quality Research in Chinese Medicines and Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macau 999078, China;
2 Faculty of Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Macau 999078, China;
3 School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
[Abstract]: In an effort to understand the molecular events contributing to the cytotoxicity activity of resveratrol (RSV), we investigated its effects on human lung adenocarcinoma epithelial cell line A549 at different concentrations. Cellular nucleoside metabolic profiling was determined by an established liquid chromatography-mass spectrometry method in A549 cells. RSV resulted in significant decreases and imbalances of deoxyribonucleoside triphosphates (dNTPs) pools suppressing subsequent DNA synthesis. Mean-while, RSV at high concentration caused significant cell cycle arrest at S phase, in which cells required the highest dNTPs supply than other phases for DNA replication. The inhibition of DNA synthesis thus blocked subsequent progression through S phase in A549 cells, which may partly contribute to the cytotoxicity effect of RSV. However, hydroxyurea (HU), an inhibitor of RNR activity, caused similar dNTPs perturbation but no S phase arrest, finally no cytotoxicity effect. Therefore, we believed that the dual effect of high concentration RSV, including S phase arrest and DNA synthesis inhibition, was required for its cytotoxicity effect on A549 cells. In summary, our results provided important clues to the molecular basis for the anticancer effect of RSV on epithelial cells.
[Key words]: Resveratrol    LC-MS    Deoxyribonucleotides    DNA synthesis    S phase arrest    
Introduction

Resveratrol (3, 4', 5-trihydroxy-trans-stilbene, RSV) is a phytoalexin, which was first isolated from the roots of white hellebore in 1940 [1]. Since then, RSV has been found in a wide range of plant species, a number of which are components of the human diet, including mulberries, peanuts and grapes. RSV is a kind of typical polyphenolic phytoalexin that is produced in response to exogenous stress factors [2]. This molecule has been the focus of a number of studies investigating its beneficial effects on neurological, hepatic, and cardiovascular systems, including cardiovascular protective [3], antiplatelet [4], antioxidant [5], antiinflammatory [6], and blood glucose-regulating effects [7-9]. In recent years, interest in RSV has focused on its anti-cancer activity. RSV is closely related to the growth inhibition in various cancer cells, including stomach, prostate, lung, myeloid, breast, liver, pancreas, prostate, skin, and colon [10-13]. However, the anti-cancer mechanism of RSV has not yet been univocally elucidated. There is a common view that RSV can affect cell conditions by modulating the activities of various intercellular enzymes including (but not limited to) ribonucleotide reductases (RNR), lipo- and cyclooxygenases, DNA polymerases and other proteins [14-16], of which RNR inhibition effect was broadly reported in recent years.

RNR is a fundamental complex enzyme existing in almost all free living organisms, parts of double-stranded DNA viruses and some bacteria. RNR can catalyze the reduction of ribonucleosidediphosphates into corresponding deoxyribonucleoside diphosphates (dNDPs) which is a rate-limiting step for deoxyribonucleoside triphosphates (dNTPs) synthesis [17]. Human RNR is composed of three known subunits, RRM1 (large subunit), RRM2 (small subunit) and an encoded P53-controlled ribonucleotide reductase (P53R2) that are differentially regulated during the cell cycle [18]. Interestingly, the level of the relative large part RRM1 is more constant throughout the cell cycle, while the small unit RRM2 showing a distinct change in express level which indicates that RRM2 might donate more in regulating RNR's activity. Fontecave et al. suggested that a stoichiometric amount of RSV can destroy the tyrosyl radical in protein R2 causing inhibition of RNR activity [14, 17-19]. However, the subsequent effects, such as disturbing dNTPs supply for DNA replication, were not further investigated.

Ribonucleotides (RNs) and deoxyribonucleotides (dRNs) play an important role in a broad range of key cell functions. Unbalanced cellular pools of dNTPs, dNDPs and deoxyribonucleoside monophosphates (dNMPs) are suggested mutagenic. Thus, the perturbation of cellular RNs and dRNs after RSV treatment can in reverse illustrate the effect of RSV on cell function. Moreover, the changes in cell cycle regulatory processes and perturbation of nucleotide metabolism contributing to the cytotoxicity effect of RSV on A549 cells has not been deeply investigated.

In this study, the effects of RSV on cellular RNs and dRNs pools were investigated by an established liquid chromatography-mass spectrometry (LC-MS) method [20]. Cell cycle analysis and western blot analysis were performed to investigate RSV mechanisms of anti-proliferation and perturbating cellular nucleotide metabolism. Hydroxyurea (HU), an inhibitor of RNR activity which is the only ribonucleotide reductase tyrosyl radical scavengers used clincally [14], was used as a reference. We found that investigation of cellular nucleotide metabolism is an effective approach to facilitate our understanding of the anti-tumor mechanism of RSV.

Materials and Methods Chemicals and reagents

Resveratrol (purity 99%) was purchased from ZhengZhou HuaWen Chemical Co., Ltd. (HeNan, China). Human lung adenocarcinoma epithelial cell line A549 was supplied by American Type Culture Collection (ATCC, Rockville, MD, USA). Phosphate buffer saline (PBS), RPMI medium 1640, 0.25% Trypsin-EDTA solution, penicillin-streptomycin solution, and fetal bovine serum (FBS) were obtained from GIBCO Invitrogen Co. (Carlsbad, CA, USA). A Cell Cycle Analysis Kit was purchased from Signal-way Antibody Co., Ltd. (College Park, MD, USA). Trichloroacetic acid (TCA), hexylamine (HA), diethylamine (DEA), trioctylamine, 1, 1, 2-trichlorotrifluoroethane and stable isotope labeled adenosine-13C1015N5-triphosphate (ATP13C15N) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). LC-MS grade methanol, acetonitrile, dimethyl sulfoxide (DMSO) and acetic acid were purchased from Anaqua Chemical Supply Co. (Houston, TX, USA). Ultra-pure water was obtained from the Milli-Q Gradient Water System (Millipore Corporation, Billerica, MA, USA).

Cell Culture

A549 cells were cultured in RPMI Medium 1640 supplemented with 10% FBS, 100 UI·mL−1 penicillin, and 100 μg·mL−1 streptomycin in humidified air at 37 ℃ with 5% CO2.

Cell viability assay

The cytotoxic effect of RSV and HU on A549 cells was determined by the MTT (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) assay. A549 cells were seeded in 96-well plates (LabServ, Thermo Fisher Scientific Co., Beijing, China) at density of 1.5 × 104 cells/well. After 24 h incubation, cells were treated with RSV and HU at different concentrations (12.5, 25, 50, 100, 200 µmol·L−1 RSV and 1 mmol·L−1 HU) for further 24 h. MTT solution (final concentration of 0.5 mg·mL−1 in medium) was added to each well and incubated further for 4 h. Then the supernatant medium was removed, and 100 μL of DMSO was added to each well to dissolve the purple crystals of formazan. Absorbance was measured with a microplate reader (Infinite M200 PRO, Tecan Austria GmbH 5082, Grdig, Austria) at 570 nm, vs 650 nm as the reference wavelength.

Preparation of cell pellets

A549 cells were seeded in 100 mm × 20 mm dishes (LabServ, Thermo Fisher Scientific, Beijing, China). After overnight culture, cells of the treatment group were incubated with 25 or 100 µmol·L−1 RSV and 1 mmol·L−1 HU for 24 h, respectively. Control cells were incubated in medium only. At the end of the incubation, cells of each group were washed with ice-cold PBS twice and trypsinized with 0.25% trypsin-EDTA. Cells from control groups and treatment groups were then re-suspended in 10 mL ice-cold PBS and centrifuged for 5 min at 1500 r·min−1. The cell pellets were incubated with 150 µL of 15% TCA containing 7.5 µL of 20.0 µmol·L−1 ATP13C, 15N as internal standard and put on ice for 5 min for protein precipitation. After centrifugation at 13 500 r·min−1 for 15 min at 4 oC, the acidic supernatant was separated and neutralized twice with 100 µL mixture of 1, 1, 2-trichlorotrifluoroethane and trioctylamine (a volume ratio of 55 to 45). Samples were stored at –80 ℃ until analysis within two days.

Detection of cellular RNs and dRNs pools by LC-MS/MS

The proofing unit comprised a Thermo Fisher TSQ LC-MS/MS system incorporating an Accela pump, an Accela autosampler, and a Quantum Access triple quadrupole mass spectrometer (Thermo Fisher Scientific Co., San Jose, CA, USA). The Xcalibur software version 2.0.7 (Thermo Fisher Scientific Co., San Jose, CA, USA) and the Thermo LCquan 2.5.6 (Thermo Fisher Scientific Co., San Jose, CA, USA) data analysis program were used for the data acquisition and data processing, respectively. The chromatographic separation was performed on an XTerra® MS C18 column (3.5 µm, 2.1 mm × 150 mm, Waters Co., Milford, MA, USA). There were two mobile phases using in the analytic system, one was 0.5% DEA-5 mmol·L−1 HA in water, pH adjusted to 10 with acetic acid (A), the other was 50% (V/V) acetonitrile in water (B). The eluents consisted of linear gradient of A and B: 0−15 min, 100%−85% A; 15−35 min, 85%−75% A; 35−45 min, 75%−45% A; 45−50 min, 45%−100% A; 50−60 min, 100% A. For all RN and dRN, the following optimized parameters were obtained. The sheath gas pressure reached 40 psi. The ionspray voltage was set at 3000 V for negative mode and 4000 V for positive mode at a temperature of 350 ℃ and an auxiliary gas pressure of 15 psi. Quantification was performed using multiple reactions monitoring (MRM) as previously published [20].

Cell cycle analysis

A549 cells were seeded at density of 2.0 × 105 cells/well in 6-well plates (LabServ, Thermo Fisher Scientific Co., Beijing, China) in duplicate and incubated with 25, 100 µmol·L−1 RSV and 1 mmol·L−1 HU for 24 h, respectively. Control cells were incubated in medium only. Cells were harvested and fixed in 70% (V/V) cold ethanol overnight at 4 ℃. The fixed cells were collected by centrifugation and re-suspended in PBS and incubated with 5 mg·mL−1 PI (propidium iodide, Sigma-Aldrich, St. Louis, MO, USA) and 10 mg·mL−1 RNase A (Ribonuclease A from bovine pancreas, Sigma-Aldrich, St.Louis, MO, USA) at room temperature for 30 min in the dark. Then the cells were analyzed on a flow cytometer (MuseTM cell analyzer, Merck Millipore, Darmstadt, Germany). Finally, the percentages of cells in different phases (G0/G1, S and G2/M) were calculated using Modfit software (Verity Software House, USA).

Western Blot analysis

A549 cells were seeded at density of 2.0 × 105 cells/well in 6-well plates and incubated with 25 or 100 µmol·L−1 RSV and 1 mmol·L−1 HU for 24 h, respectively. Cells of control group were incubated in medium only. All groups of cells were harvested and lysed in RIPA buffer (Cell Signaling Technologies Inc. Beverly, MA, USA). Protein concentration was determined using Bradford reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA). Cell lysates were mixed with 5Х SDS-loading buffer (4 : 1, V/V) and heated at 100 ℃ with locked capping for 5 min. Samples (40 μg of protein) were resolved on 10% SDS-PAGE and then electroblotted onto polyvinylidene fluoride transfer membrane (Bio TraceTM PVDF, 0.45 μmol·L−1, Pall Life Sciences Co., Mexico) and were incubated with different antibodies including RRM1 (Cell Signaling Technology Inc., MA, USA), RRM2 and p53R2 (Abcam Ltd., Cambridge, UK) and β-tubulin (Santa Cruz Biotechnology, CA, USA) overnight at 4 ℃, respectively. The membranes were further incubated with HRP-conjugated antibodies for one hour. The protein bands were visualized using the enhanced chemiluminescence reagents (Invitrogen Co., Paisley, Scotland, UK), and were analyzed with the Image J 1.46r software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

The data was expressed as mean ± SD values from three independent replicate experiments. P value less than 0.05 (*P < 0.05, **P < 0.01, compared with control group) was considered to be statistically significant by using Student's t- test analysis.

Results and Discussion Cytotoxicity of RSV and HU on A549 cells

To examine the cellular toxicity effects of RSV and HU, MTT assay was performed with different RSV concentrations in A549 cells. As shown in Fig. 1, 12.5−25 μmol·L−1 RSV and 1 mmol·L−1 HU did not affect the cell viability after 24 h treatment. The toxicity of RSV was obvious when RSV concentration was more than 50 μmol·L−1. The A549 cell viability decreased in a dose-dependent manner after treatment with RSV at concentrations of 50, 100, 200 μmol·L−1. RSV at concentrations of 100 μmol·L−1 significantly inhibited cell viability relative to control cells (**P < 0.01) and the cytotoxicity effect of RSV was stronger than HU on A549 cells. In follow-up studies, 25 and 100 μmol·L−1 RSV were selected as the low and high concentrations of RSV, respectively.

Fig. 1 The effect of RSV and HU on A549 cell viability at different concentrations after 24 h treatment. The data was represented as mean ± SD (n = 6), **P < 0.01 vs control group
Perturbation of cellular RNs and dRNs pools after RSV and HU treatment

As shown in Table 1, low concentration RSV caused more extensive alterations in RNs pools than high concentration RSV. Significant decreases in ATP and GTP pools, increases in AMP, GDP, GMP and UDP pools were found in A549 cells with 25 μmol·L−1 RSV treatment. However, only significant increase in ATP pool occurred in A549 cells treated with 100 μmol·L−1 RSV or 1 mmol·L−1 HU. In contrast, high concentration RSV resulted in more extensive alterations of dRNs pools than low concentration RSV, which was similar with the effect of 1 mmol·L−1 HU (Table 2). Significant decreases in cellular dATP, dGTP, dTTP, dADP, dCDP, dGDP and dGMP absolute amounts were found in A549 cells after 100 μmol·L−1 RSV treatment. Of the four deoxyribonucleoside triphosphates (dNTPs), cellular dGTP pool presented the greatest decrease while cellular dCTP pool showed no significant change, consequently, imbalance of cellular dNTPs occurred after 100 μmol·L−1 RSV treatment.

Table 1 RNs pools in A549 cells of different experimental groups (pmol/106 cells, mean ± SD, n = 3)
Table 2 dRNs pools in A549 cells of different experimental groups (pmol/106 cells, mean ± SD, n = 3)

In addition, the absolute amount of each RNs and dRNs in A549 cells was used to obtain a data matrix consisting of 24 objects (groups) and 24 variables (RNs and dRNs pools). Supervised orthogonal partial least squares discriminant analysis (OPLS-DA) model was constructed to understand and visualize the complex effect of RSV at different concentrations on RNs and dRNs pools using SIMCA-P version 14.0 (Umetrics Inc., Ume , Sweden). As shown in Fig. 2A, control groups and treatment groups showed an appreciable separation based on the total alteration in the data. Moreover, 25 μmol·L−1 RSV group could be separated from 100 μmol·L−1 RSV group, whereas 100 μmol·L−1 RSV group and 1 mmol·L1 HU group could not get clear separation.

Fig. 2 (A) Imbalance of dNTPs pools in A549 cells induced by RSV and HU. (B) Scores plot of OPLS-DA model for different groups. Scores plot describes the similarities between the Y-variables (groups) based on the X-variables (RNs and dRNs pools). The data was represented as mean ± SD (n = 3), *P < 0.05, **P < 0.01 vs control group

These results indicated that the perturbation of cellular RNs and dRNs pools induced by RSV was dependent on RSV concentrations. The perturbation range and degree of cellular RNs and dRNs pools was both different between 25 μmol·L−1 RSV group and 100 μmol·L−1 RSV group. RSV disturbed mainly RNs metabolism at low concentration, while perturbing mainly dRNs metabolism at high concentration. The latter was similar to the effect induced by 1 mmol·L−1 HU on A549 cells: 100 μmol·L−1 RSV and 1 mmol·L−1 HU caused similar perturbation of cellular RNs and dRNs pools in A549 cells, suggesting that they might possess similar mechanism of perturbating nucleotide metabolism.

Cellular dNTPs are essential precursors for DNA synthesis, which is required for replication, recombination and repair, therefore the concentration of dNTPs is strictly controlled. Perturbations in the absolute and relative concentrations of the four dNTPs may lead to inhibition of DNA replication and activation of the S-phase checkpoint. The activated S-phase checkpoint arrests cell cycle progression and activates DNA repair and at worst, leads to apoptosis [21-22]. In this study, RSV and HU resulted in significant alteration of dNTPs compositions (Fig. 2B). Treatment with RSV and HU resulted in increases in percentages of dCTP and dTTP, correspondingly, decreases in dATP and dGTP proportions in A549 cells. Moreover, the effect of dNTPs imbalance presented a positive correlativity with RSV concentrations. As sufficient levels and relative balance of dNTPs were required for DNA synthesis and DNA replication fidelity, the decreases and imbalance of dNTPs pools induced by RSV at high concentration could inhibit DNA synthesis and consequently, contribute to growth retardation and cell-cycle arrest of A549 cells in this study.

Cell cycle alteration induced by RSV and HU

Cell proliferation comprises two distinct processes: cell cycle and cell growth. The four phases composing the mammalian cell cycle, including S (DNA synthesis), M (mitosis), G1 and G2, are mainly regulated by cyclin-dependent kinases (CDKs), and E3 ubiquitin ligases [23]. Like many cytotoxic agents, RSV affects cell proliferation by disturbing the normal progress of the cell cycle. RSV could interfere with the molecular machinery of the cell cycle which involves various key regulators, such as p21, p27, p53, cyclin D1, cyclin-dependent kinase (CDK) 4 and CDK 6 [24-26]. Furthermore, cell cycle blockage induced by RSV varies according to the cell type, treatment concentration and duration [27]. For example, treatment with 100 μmol·L−1 RSV led to G1 phase cell cycle arrest in human T24 bladder cancer cells [25], whereas induced accumulation of cells at the G0−G1 phase in pancreatic cancer cells (PANC-1, AsPC-1, BxPC-3 cells) [26]. Additionally, RSV has also been reported to cause S phase arrest in many other cell types [28-30].

In this study, RSV and HU treatment resulted in similar decrease in percentage of cells at G2/M phase (Fig. 3). RSV at low concentration induced cell cycle arrest at G0/G1 phase. Indeed, RSV could block the G1/S transition of the cell cycle by regulating the protein expression of cyclin D1, D2, and the activities of various kinases as previously reported [31-32]. However, similar to HU, RSV at high concentration induced accumulation of cells at S phase as documented in previous reports [33-35]. RSV showed the function of reducing cell growth and inhibiting cell proliferation through directly acting on cell cycle arrest at S phase in A549 cells.

Fig. 3 Cell cycle distribution of A549 cells after RSV and HU treatment. The images of cell cycle distribution are presented as one experiment representative of 3 experiments with similar results. The quantitative data was represented as mean ± SD (n = 3), *P < 0.05, **P < 0.01 vs control group
Cellular RNR expression after RSV and HU treatment

It has been suggested that the perturbation nucleotide metabolism could be caused by the inhibitory effects of RSV on the enzyme RNR. RNR is the key enzyme of de novo DNA synthesis. The enzyme needs a tyrosl free radical for its activity and can therefore be inhibited by free radical scavengers, such as RSV and HU [36-39]. The ability of RSV to destroy the tyrosyl radical was correlated with its strong dose- dependent inhibitory effects on RNR activity [36-37]. In this study, RSV at different concentrations and HU have no effect on protein expression of RRM1, RRM2 and P53R2 in A549 cells after 24 h treatment (Fig. 4). These findings were consistent with previous reports that the inhibition effect of RSV on RNR could derive from reducing RNR activity by scavenging tyrosyl free radicals like HU does rather than decreasing RNR expression [14, 19, 36-39].

Fig. 4 Western blot analysis of the effect of RSV and HU on expression of RNR subunits M1 (RRM1) and M2 (RRM2), P53-controlled ribonucleotide reductase (P53R2). The images of cell cycle distribution are presented as one experiment representative of 3 experiments with similar results. The quantitative data was represented as mean ± SD (n = 3), *P < 0.05, **P < 0.01 vs control group
Dual effects of high concentration RSV on A549 cells

RSV at high concentration had a significant cytotoxicity effect on A549 cells while exerting no cytotoxicity at low concentration after 24 h treatment. Our results revealed that, in A549 cells, the effect of RSV on nucleotide metabolism varied dramatically depending on the concentration. RSV at 25 μmol·L−1 caused no remarkable perturbation of dRNs pools. However, when treatment concentration was increased to 100 μmol·L−1, severe decreases in many dRNs, especially dNTPs were observed, which could subsequently inhibit DNA synthesis. Simultaneously, RSV at 100 μmol·L−1 induced enrichment of A549 cells in S phase, in which the cells should be actively proliferating with highest dNTPs pools than other phases. However, the inhibitory effect of RSV at 100 μmol·L−1 on DNA synthesis limited the subsequent progression through S phase in A549 cells. Nevertheless, HU caused similar dNTPs perturbation but slighter S phase arrest as high concentration RSV does, consequently no cytotoxicity effect. Therefore, the dual effect of high concentration RSV including induction of S phase and inhibition of DNA synthesis, may be responsible for its cytotoxicity effect (Fig. 5).

Fig. 5 Putative mechanisms elucidating the cytotoxicity effect of resveratrol on A549 cells. RSV at high dose induces a dual effect including S phase arrest and DNA synthesis inhibition, which may respond for its cytotoxicity effect. The quantitative data was represented as mean ± SD (n = 3), *P < 0.05, **P < 0.01 vs control group
Conclusion

Our findings confirmed that the effects of RSV on perturbation of cellular nucleotide pools and cell cycle distribution were correlated with treated concentrations. RSV at high concentration resulted in significant decreases in dNTPs pools which could inhibit DNA synthesis, while RSV at low concentration had no remarkable effect on dRNs pools. Meanwhile, RSV at high concentration induced cell cycle arrest at S phase, in which the cells required the highest dNTPs supply than other phases for DNA replication. However, the inhibition of DNA synthesis induced by RSV at 100 μmol·L−1 limited the subsequent progression through S phase in A549 cells. Therefore, this dual effects of high concentration RSV on two important processes in cell cycle progression, S phase arrest and DNA synthesis inhibition, may be responsible for its cytotoxicity effect. The unique features of RSV provide important clues to the molecular mechanisms of its anticancer effect on epithelial cells, and suggest its potential usefulness as an adjuvant in chemotherapy of lung adenocarcinoma.

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