Rhus verniciflua Extract Modulates Survival of MCF-7 Breast Cancer Cells through the Modulation of AMPK-Pathway

Abstract

Rhus verniciflua STOKES (RVS) is used as an anti-cancer agent in traditional herbal medicine. However, the underlying molecular mechanism of its action is poorly understood. Here, we elucidated the mechanism of the anti-cancer mechanism of RVS in MCF-7 human breast cancer cells. We found that RVS increased the phosphorylation of AMP-activated protein kinase (AMPK) and downstream acetyl-CoA carboxylase (ACC) and suppressed cell viability in an AMPK-dependent fashion. RVS also induced an increase in reactive oxygen species (ROS) levels. RVS-induced AMPK phosphorylation was not observed in the presence of N-acetyl-cysteine (NAC), which indicated that ROS is associated with RVS-induced AMPK phosphorylation. In addition, fluorescent staining (Annexin V/propidium iodide) revealed that RVS increased the expression of Annexin V, which indicates that RVS leads to cancer-induced apoptosis. Moreover, RVS increased the phosphorylation of p53 and the expression of Bax. The inhibition of AMPK blocked RVS-induced p53 phosphorylation and Bax expression, which suggests that AMPK is involved in RVS-induced cancer apoptosis. Taken together, these results demonstrate that RVS has anti-tumor effects on MCF-7 cells through an AMPK-signaling pathway.

Rhus verniciflua STOKES (RVS) (Anacardiaceae) is a native plant to East Asian countries, including Korea, China, and Japan, and is used as a traditional herbal medicine.^1,2) Traditionally, RVS was considered to have the function of alleviating blood stasis and purging hardness.³⁾ Therefore, it could be applied for cancer treatment. RVS was used for treating various stomach diseases, including tumors, in East Asia, including Korea, during the 15th century.^4,5) Recently, several experimental studies have demonstrated that flavonoids from RVS have effective anti-proliferative and apoptotic activities on various tumor cell lines, including human lymphoma, breast cancer, osteosarcoma, and transformed hepatoma cells.^2,4–6) However, the molecular basis for the induction of apoptosis by RVS remains unclear. In this study, we explored the potential role of AMP-activated protein kinase (AMPK) activation in RVS-induced MCF-7 human breast cancer cell apoptosis.

AMPK is a critical enzyme that plays an essential role in cellular energy homeostasis and controls processes related to tumor development, including cell cycle progression,⁷⁾ cell proliferation,^8,9) protein synthesis,^10,11) and survival.^8,9) Therefore, as an anti-cancer target, AMPK has received intensive attention in recent years. Several metabolic stresses, including hypoxia, exercise, and starvation, lead to the activation of AMPK.^12,13) AMPK also induces apoptosis in several cell types.^14–17) These results suggest that AMPK signaling could be a potential therapeutic target for cancer.

In this study, we found for the first time that RVS-induced AMPK activation triggers apoptosis of MCF-7 cells. Reactive oxygen species (ROS) production is involved in AMPK activation by RVS. We provide evidence that supports AMPK-mediated apoptosis in RVS-treated breast cancer cells.

MATERIALS AND METHODS

Reagents

Anti-phospho-AMPKα2 (Thr¹⁷²), anti-AMPKα2, anti-phospho-p53, anti-Bax, antibodies were purchased from Cell Signaling Technology (New England Biolabs, Beverly, MA, U.S.A.). Anti-phospho-acetyl-CoA carboxylase (ACC) (Ser⁷⁹) and anti-ACC antibodies were purchased from Millipore (Billerica, MA, U.S.A.). Anti-β-actin antibody and the antioxidant compound N-acetyl-cysteine (NAC) were purchased from Sigma Chemical Company (St. Louis, MO, U.S.A.). 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) and Compound C, an AMPK inhibitor were obtained from Calbiochem (San Diego, CA, U.S.A.). RVS extract was obtained from the Department of Preventive Medicine, College of Oriental Medicine, Kyunghee University, and Republic of Korea.

Sample Preparation

The bark of RVS (Anacardiaceae) was purchased from Omniherb (Gyeongsangbukdo, Korea) and extracted in 2010. The dried Rhus verniciflua was ground in a mill and passed through a 50 mesh sieve. One hundred grams of RVS was extracted twice by repeat sonication (30 min with 80% ethanol) at room temperature and then filtered. The filtrates were combined and concentrated using a vacuum evaporator at 40°C and freeze-dried. The extract yield of RVS was 13.7% w/w.¹⁸⁾ The RVS was extracted in 2010 and RVS stock solution was stored at −83°C until used.¹⁸⁾

Cell Culture

MCF-7 cells was grown in RPMI-1640 (GIBCO™, Auckland, NZ, U.S.A.) containing 0.584 g/L of L-glutamate and 4.5 g/L of glucose, 100 g/mL of gentamicin, 2.5 g/L of sodium carbonate, and 10% heat-inactivated fetal bovine serum (FBS).

Immunoblot Analysis

Cells were grown on 6, 12-well plates and serum-starved for a minimum of 6 h prior to treatment with the indicated agents. Following treatment of the cells, the media was aspirated and the cells were washed twice in ice-cold phosphate buffered saline (PBS) and lysed in 60 µL of lysis buffer. Then, the samples were briefly sonicated, heated for 5 min at 95°C and centrifuged for 5 min. The supernatants were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10%) gels and transferred to polyvinylidenedifluoride membranes. The blots were incubated overnight at 4°C on a shaker with primary antibodies and then washed 6 times in Tris-buffered saline/0.1% Tween 20 for 1 h prior to being incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature. The blots were then visualized via ECL (Amersham Biosciences, Buckinghamshire, U.K.). In some cases, the blots were stripped and reprobed using other antibodies.

Assay of Intracellular ROS by Confocal Microscopy

H₂DCFH-DA (Molecular Probe/Invitrogen, Carlsbad, CA, U.S.A.) is a ROS-sensitive probe that can be used for the detection of ROS production in living cells. It diffuses passively into cells where its acetate groups are cleaved by intracellular esterase, which release the corresponding dichlorodihydrofluorescein derivative H₂DCF. H₂DCF oxidation yields a fluorescent adduct dichlorofluorescein (DCF), which is trapped inside the cell. Cells were treated with RVS for 24 h. Cells were incubated at 37°C with 20 µM H₂DCFH-DA for 30 min and then the fluorescence was measured using a confocal microscope (Zeiss LSM 510, Carl Zeiss). Using an argon laser, DCF fluorescence was excited at 488 nm and emission was detected using a 530-nm long-pass filter.

3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium Bromide (MTT) Assay

The MTT assay was used as a crude measure of cell viability. MTT is converted by metabolically active cells into a colored water-insoluble formazan salt. MCF-7 cells were seeded at a density of 1×10⁵/mL in 96-well plates and grown for 24 h. The growth medium was replaced with serum-free medium 24 h prior to treatment. Subsequently, MTT reagents (10 µL/well (7.5 mg/mL in PBS)) were added, and the culture was incubated for 30 min at 37°C. The reaction was stopped by the addition of acidified triton buffer (0.1 M HCl and 10% (v/v) Triton X-100 (50 µL/well)), and the tetrazolium crystals were dissolved by mixing on a plate shaker at room temperature for 20 min. The samples were then analyzed using a plate reader (Bio-Rad 450, Richmond, CA, U.S.A.) at a 595-nm test wavelength and a 650-nm reference wavelength. At a minimum, the results were obtained from experiments repeated in triplicate.

4′,6-Diamidino-2-phenylindole (DAPI) Staining

Circular TC-treated cover slips were placed at the bottom of each well of a 24-well plate. In total, 1×10⁵ of MDA-MB-231-TXSA cells were seeded in each well and incubated for 24 h. The growth medium was removed and replaced with fresh Dulbecco’s modified Eagle’s medium (DMEM) or DMEM supplemented with 300 µg/mL of RVS. After 24 h, the medium was removed and the cells were incubated for 10 min with 1 µg/mL DAPI stain (Roche Diagnostics, Indianapolis, IN, U.S.A.). The morphology of the cell nuclei was observed using a fluorescence microscope (Olympus BH Series) at an excitation wavelength of 350 nm.

Apoptosis Analysis

MCF-7 cells were seeded in 60-mm plates and treated with RVS. After 48 h of treatment, the cells were harvested and stained using Annexin-V Alexa Fluor® 488/PI staining, as described by the Tali™ Apoptosis Kit (Invitrogen, Carlsbad, CA, U.S.A.). Apoptosis was evaluated using the Tali™ Image-based Cytometer.

Silencing of AMPKα2

MCF-7 cells were seeded in 96-well plates and allowed to grow to a confluence of 70% over 24 h. Transient transfections were performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, U.S.A.), according to the manufacturer’s protocol. Briefly, 100 nM of AMPKα small interfering RNA (siRNA) (NM_001013367; Dharmacon, Lafayette, CO, U.S.A.) and 5 µL of Lipofectamine 2000 were diluted with 95 µL of serum medium (Opti-MEM; Invitrogen) and then mixed. The mixture was incubated for 10 min at room temperature and added drop-wise to each culture well, which contained 800 µL of Opti-MEM (final siRNA concentration, 40 nM). Four hours after the transfection, the medium was changed with fresh complete medium. The transfected cells were cultivated for 48 h prior to conducting the experiments.

RESULTS

RVS Activates AMPKα2 and the Downstream Target of AMPK, ACC, in MCF-7 Cells

To determine whether RVS is involved in the regulation of AMPKα2, a key molecular target of anti-tumor control, the level of AMPKα2 phosphorylation in RVS-treated MCF-7 cells was examined. Using phosphorylation-specific antibodies, RVS stimulated AMPKα2 activation in a dose-dependent manner (Fig. 1A). AICAR, a known AMPK activator, was employed as a positive control. In addition, the administration of RVS induced a time-dependent increase in AMPKα2 phosphorylation (Fig. 1B). The phosphorylation level of Thr¹⁷², which is in the active site of the AMPKα2 subunit and is essential for enzyme activity, reached a maximum level 30 min after treatment. Consistent with the increase in AMPKα2 activity, the phosphorylation of ACC-Ser⁷⁹, the best-characterized phosphorylation site by AMPKα2, increased after RVS administration. These results demonstrate that AMPKα2 may mediate an RVS-associated signaling pathway in MCF-7 cell.

Fig. 1. RVS Activates AMPK and the Downstream Target of AMPK, ACC, in MCF-7 Cells

(A) Dose-dependent phosphorylation of AMPKα2 and ACC after RVS treatment. Cells were stimulated with different concentrations of RVS for 3 h. Cell lysates were analyzed by Western blot using anti-phospho-AMPKα2, anti-phospho-ACC, anti-AMPKα2, and anti-ACC antibodies. (B) Time-dependent phosphorylation of AMPKα2 and ACC after RVS treatment. Cells were stimulated with 200 µg/mL of RVS for the indicated times. Cell lysates were analyzed by Western blot using anti-phospho-AMPKα2, anti-phospho-ACC, anti-AMPKα2, and anti-ACC antibodies. Data in the bar graphs represent the mean ±S.E.M. values of the ratios of densities (p-AMPK/AMPK) for at least three dependent Western blot experiments. *p<0.05  vs. basal values.

RVS Suppresses Cell Viability through AMPK

To assess the effect of RVS on cell viability, we examined the cytotoxic effects of serial concentrations (0–300 µg/mL) of RVS in 2-d treatments using an MTT assay. Compared with the untreated cells, the viability of cells significantly decreased after 2 d of treatment with 200 µg/mL RVS (Fig. 2A). The microscopic examination of the drug-treated MCF-7 cell cultures revealed dose-dependent cell death that is characteristic of apoptosis. The treated cells ceased proliferation, rounded-up, and detached from the plate (Fig. 2B). The nuclear examination of the drug-treated cells stained with DAPI, a fluorescent DNA-binding dye, indicated condensed highly stained nuclei characteristic of apoptotic cells (Fig. 2C). To better understand the mechanism by which AMPKα2 alters cell viability, the effect of AMPKα2 knock-down on RVS-induced viability suppression was also determined. The suppression of cell viability was recovered by AMPKα2 knock-down (Fig. 2D). To examine the role of AMPKα2 on RVS induced viability suppression, RVS mediated AMPKα2 phosphorylation was examined in AMPKα2 knock-down condition. RVS-induced AMPKα2 phosphorylation was decreased in AMPKα2 knock-down condition (Fig. 2E). Taken together, these results indicate that AMPKα2 is involved in RVS-induced cellular apoptosis in MCF-7 cells.

Fig. 2. RVS Suppresses Cell Viability through AMPKα2

(A) Dose-dependent suppression of viability of MCF-7 cells after RVS treatment. Cells were treated with RVS at the indicated doses for 48 h. Values are expressed as the percentage of absorbance (MTT) relative to the control. (B) Dose-dependent morphological changes in RVS-treated MCF-7 cells. MFC-7 cells were exposed to the indicated dose for 2 d and examined by microscopy (20×). (C) The cells were treated with the indicated doses for 48 h. Fluorescent micrographs of cells stained with DAPI are shown on the left. Phase contrast photographs are shown on the right (magnification 20×). (D) Effect of AMPKα2 knock-down on the RVS-induced decrease in viability. Cells were transfected with 50 nM AMPKα siRNA and maintained for 48 h. Either non-transfected cells or AMPKα2 siRNA-transfected cells were then treated with 200 µg/mL of RVS for 48 h. Values are expressed as the percentage of absorbance (MTT) relative to control. (E) Effect of AMPKα2 knock-down on the RVS-induced AMPKα2 phosphorylation. Cells were transfected with 50 nM AMPKα2 siRNA and maintained for 48 h. Either non-transfected cells or AMPKα2 siRNA-transfected cells were then treated with 200 µg/mL of RVS for 48 h. Cell lysates were analyzed by Western blot using anti-phospho-AMPKα2, anti-AMPKα2, and anti-β-action antibodies. *p<0.05  vs. basal values.

ROS Production Is Involved in RVS-Induced AMPKα2 Activation

ROS are produced by the mitochondria during the execution phase of apoptosis. To clarify whether the ROS produced by RVS accumulated intracellularly, MCF-7 cells pretreated with 5 µmol/L DCFH-DA for 1 h at 37°C were stimulated with 200 µg/mL RVS for 30 min, and the DCF produced was scanned by confocal microscopy. The intracellular fluorescent intensities were rapidly increased (Fig. 3A). However, the increase in fluorescence observed in the MCF-7 cells exposed to RVS was significantly reduced by pretreatment with NAC (5 mM), a ROS scavenger (Fig. 3A). Next, we attempted to determine whether the phosphorylation of AMPKα2 is related to ROS induction. RVS-induced AMPKα2 phosphorylation was not observed in the presence of NAC (5 mM), which suggests that ROS are associated with RVS-induced AMPKα2 phosphorylation (Fig. 3B). To determine whether H₂O₂ is associated with AMPKα2 phosphorylation, MCF-7 cells were exposed to 100 µM H₂O₂. The Western blot analysis revealed that H₂O₂ induced an increase in the phosphorylation of AMPKα2 (Fig. 3C). To better understand the mechanism by which ROS alters cell viability, the effect of ROS on RVS-induced cell viability suppression was also determined. The suppression of cell viability was recovered by pretreatment with NAC (5 mM) (Fig. 3D). These results indicate that RVS induces an increase in phosphorylation of AMPKα2 through ROS.

Fig. 3. ROS Production Is Involved in RVS-Induced AMPK Activation

(A) Microscopic analysis of RVS-induced ROS generation. MCF-7 cells were treated with 200 µg/mL of RVS for 48 h. ROS generation was detected by confocal microscopy. (B) Effect of NAC on RVS-induced AMPKα2 phosphorylation. Cells were pre-treated with NAC (20 mM) for 30 min and then stimulated with 200 µg/mL of RVS for 3 h. Cell lysates were analyzed by Western blot using anti-phospho-AMPKα2, anti-phospho-ACC, anti-AMPKα2, and anti-ACC antibodies. (C) Effect of H₂O₂ on AMPKα2 phosphorylation. Cells were stimulated with 100 µM H₂O₂ for the indicated times. Cell lysates were analyzed by Western blot using anti-phospho-AMPKα2, anti-phospho-ACC, anti-AMPKα2, and anti-ACC antibodies. (D) Effect of NAC on RVS-induced cell viability. Cells were pretreated with NAC for 30 min and then treated with 200 µg/mL of RVS for 48 h. Values are expressed as the percentage of absorbance (MTT) relative to control. Data in the bar graphs represent the mean ±S.E.M. values of the ratios of densities (p-AMPKα2/AMPKα2) for at least three dependent Western blot experiments. *p<0.05  vs. basal values.

RVS-Induced Apoptosis

To determine whether the cytotoxicity of RVS is due to apoptosis, apoptosis analysis was performed in MCF-7 cells after RVS treatment. Apoptotic induction was also analyzed using the Tali™ Apoptosis Kit, where Annexin V and propidium iodide (PI) are used for determination of apoptotic cell death. The treatment with RVS (0 and 200 µg/mL) for 2 d resulted in approximately 24% of the cells staining positive for Annexin V (early apoptosis) (Figs. 4A, B). To further understand the molecular mechanism of RVS-induced apoptosis in MCF-7 cells, we examined the expression of proteins associated with apoptosis after RVS treatment. RVS up regulated pro-apoptotic Bax expression in a dose-dependent manner (Fig. 4C). The tumor suppressor protein, p53, regulates apoptosis through the transcriptional activation of its target genes and acts as a key facilitator of many cross-talking pathways. After treatment with RVS, the levels of p53 phosphorylation were significantly increased (Fig. 4C). Taken together, these findings suggest that RVS induces apoptosis in MCF-7 cells.

Fig. 4. RVS-Induced Apoptosis

(A, B) Effect of RVS on MCF-7 cell apoptosis using Annexin-V Alexa Fluor® 488/PI staining. Cells were seeded in 60-mm plates and treated with 200 µg/mL of RVS for 48 h. Cell apoptosis was evaluated with the Tali™ Image-based Cytometer using the Tali™ Apoptosis Kit. (C) Expression of apoptosis-related proteins p53 and Bax in MCF-7 cells after treatment with 200 µg/mL of RVS for 2 d. Equal amounts of cell lysates were resolved by SDS-PAGE and analyzed by Western blot using specific antibodies. The blots were reprobed with anti-actin antibody to confirm equal protein loading. Data in the bar graphs represent the mean ±S.E.M. values of the ratios of densities (p-p53/β-actin) and (Bax/β-actin) for at least three dependent Western blot experiments. *p<0.05  vs. basal values.

AICAR Enhances RVS-Induced MCF-7 Breast Cancer Cell Death/Apoptosis

To gain insight into the role of AMPKα2 in RVS-mediated apoptosis, we measured the phosphorylation of p53. The levels of p53 phosphorylation were attenuated by treatment with Compound C, an AMPK inhibitor (Fig. 5A). To ascertain the role of RVS on AMPK phosphorylation, we examined the phosphorylation of AMPKα2 after the co-treatment of RVS and AICAR, an AMPK activator. The phosphorylation level of AMPKα2 was higher in co-treatment condition than individual treatment condition (Fig. 5B). Taken together, these findings support our hypothesis that AMPKα2 is a critical regulator in RVS-mediated cell viability inhibition.

Fig. 5. AICAR Enhances RVS-Induced MCF-7 Breast Cancer Cell Death/Apoptosis

(A) Phosphorylation of p53 and the expression of Bax in the presence of Compound C, an AMPK inhibitor. Cells were pre-treated with 5 µM Compound C for 10 min and then stimulated with 200 µg/mL of RVS for 3 h. Cell lysates were analyzed by Western blot with anti-phospho-p53 and Bax antibodies. An anti-β-actin antibody was used as the protein loading control. (B) Increase of the RVS-mediated AMPKα2 phosphorylation after pretreatment with AICAR. Cells were pretreated with 1 mM AICAR for 30 min and treated with 200 µg/mL of RVS for 3 h. Cell lysates were analyzed by Western blot with anti-phospho-AMPKα2 and AMPKα2 antibodies. All data are represented as the means of triplicate experiments. Data in the bar graphs represent the mean ±S.E.M. values of the ratios of densities (p-AMPKα2/AMPEα2) for at least three dependent Western blot experiments. *p<0.05  vs. basal values.

DISCUSSION

In this study, we demonstrated that RVS activates AMPKα2 and contributes to the suppression of MCF-7 cell viability. The association of AMPKα2 with the suppression of MCF-7 cell viability has raised several questions regarding the mechanism by which RVS can suppress tumor growth. The results of our study suggest that RVS-induced AMPKα2 activation plays a critical role in the suppression of tumor cell growth.

Our objective was to determine whether the viability of MCF-7 breast tumor cells was directly regulated by RVS and, if so, to identify the signaling molecules involved. The principal finding of this study is that AMPKα2 is involved in RVS-mediated suppression of MCF-7 cell viability. Our results also demonstrated that p53 and Bax are associated with RVS-mediated suppression of MCF-7cells. p53, a transcription factor that mediates cell responses to many types of harmful stressors,¹⁹⁾ is one of the most widely studied proteins. The activation of p53 induces cell cycle regulation, DNA repair, senescence, and cell death in cells that have been exposed to a variety of insults, including DNA damage, hypoxia, and oxidative stress.²⁰⁾ Metabolic alterations are a determinate of the response of cancer cells to variable nutrient conditions as well as their regulation of cell proliferation,²¹⁾ which indicates that p53 plays an important role in metabolic shifting in cancer cells. Bax forms heterodimers with multiple anti-apoptotic proteins and induces apoptosis through the release of cytochrome c.^22–24) Moreover, a previous study reported that AMPK activation stimulated Bax translocation, which suggests that AMPKα2 is involved in Bax redistribution to mitochondria.²⁵⁾

In the previous study, RVS suppressed inflammation by down-regulation of c-Jun N-terminal kinase (JNK) pathway in lipopolysaccharide (LPS) induced RAW264.7 macrophages.²⁶⁾ However, ethanol-extracted RVS activated JNK.²⁷⁾ This discrepancy may be caused by the experimental different conditions, such as cell type, extract method and the concentration of RVS. JNK is well known to be involved in oxidative stress-associated pro-apoptotic processes.²⁸⁾ It was also reported that activation of stress-activated protein kinases, such as JNK, induced apoptosis in certain cells.^29,30) In other reports also showed that the AMPKα2 leaded to apoptosis via JNK activation.^31–33) Taken together, these facts indicated that JNK may be involved in AMPK-mediated apoptosis.

These findings implicate the AMPKα2-mediated p53/Bax pathway in RVS-mediated suppression of tumor viability. In conclusion, we have demonstrated that RVS treatment activated AMPKα2 in MCF-7 cells.

These results showed that the AMPK pathway may exert a profound influence on RVS-mediated inhibition of tumor viability. Future studies will focus on elucidating the mechanism between AMPKα2 and p53/Bax in RVS-mediated signaling.

Acknowledgment

This work was supported by Grant 2012R1A1A2041548 from the National Research Foundation of Korea, funded by the Korea Government.

REFERENCES

• 1) Kim TJ. Korea resource plants, Vol. II. Seoul University Press, Seoul, Korea (1996).
• 2) Lee JC, Lee KY, Kim J, Na CS, Jung NC, Chung GH, Jang YS. Extract from Rhus verniciflua STOKES is capable of inhibiting the growth of human lymphoma cells. Food Chem. Toxicol., 42, 1383–1388 (2004).
• 3) Bensky D, Gamble A, Kaptchuk TJ. Chinese herbal medicine: materia medica. Eastland Press, Seattle, Washington DC (1992).
• 4) Jang HS, Kook SH, Son YO, Kim JG, Jeon YM, Jang YS, Choi KC, Kim J, Han SK, Lee KY, Park BK, Cho NP, Lee JC. Flavonoids purified from Rhus verniciflua STOKES actively inhibit cell growth and induce apoptosis in human osteosarcoma cells. Biochim. Biophys. Acta, 1726, 309–316 (2005).
• 5) Son YO, Lee KY, Lee JC, Jang HS, Kim JG, Jeon YM, Jang YS. Selective antiproliferative and apoptotic effects of flavonoids purified from Rhus verniciflua STOKES on normal versus transformed hepatic cell lines. Toxicol. Lett., 155, 115–125 (2005).
• 6) Samoszuk M, Tan J, Chorn G. The chalcone butein from Rhus verniciflua STOKES inhibits clonogenic growth of human breast cancer cells co-cultured with fibroblasts. BMC Complement. Altern. Med., 5, 5 (2005).
• 7) Hardie DG. New roles for the LKB1→AMPK pathway. Curr. Opin. Cell Biol., 17, 167–173 (2005).
• 8) Imamura K, Ogura T, Kishimoto A, Kaminishi M, Esumi H. Cell cycle regulation via p53 phosphorylation by a 5′-AMP activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, in a human hepatocellular carcinoma cell line. Biochem. Biophys. Res. Commun., 287, 562–567 (2001).
• 9) Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, Birnbaum MJ, Thompson CB. AMP-activated protein kinase induces a p53-dependent metabolic checkpoint. Mol. Cell, 18, 283–293 (2005).
• 10) Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, Turk BE, Shaw RJ. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell, 30, 214–226 (2008).
• 11) Inoki K, Zhu T, Guan KL. TSC2 mediates cellular energy response to control cell growth and survival. Cell, 115, 577–590 (2003).
• 12) Hardie DG. Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology, 144, 5179–5183 (2003).
• 13) Hardie DG, Sakamoto K. AMPK: a key sensor of fuel and energy status in skeletal muscle. Physiology (Bethesda), 21, 48–60 (2006).
• 14) Garcia-Gil M, Pesi R, Perna S, Allegrini S, Giannecchini M, Camici M, Tozzi MG. 5′-Aminoimidazole-4-carboxamide riboside induces apoptosis in human neuroblastoma cells. Neuroscience, 117, 811–820 (2003).
• 15) Kefas BA, Cai Y, Kerckhofs K, Ling Z, Martens G, Heimberg H, Pipeleers D, Van de Casteele M. Metformin-induced stimulation of AMP-activated protein kinase in beta-cells impairs their glucose responsiveness and can lead to apoptosis. Biochem. Pharmacol., 68, 409–416 (2004).
• 16) Meisse D, Van de Casteele M, Beauloye C, Hainault I, Kefas BA, Rider MH, Foufelle F, Hue L. Sustained activation of AMP-activated protein kinase induces c-Jun N-terminal kinase activation and apoptosis in liver cells. FEBS Lett., 526, 38–42 (2002).
• 17) Saitoh M, Nagai K, Nakagawa K, Yamamura T, Yamamoto S, Nishizaki T. Adenosine induces apoptosis in the human gastric cancer cells via an intrinsic pathway relevant to activation of AMP-activated protein kinase. Biochem. Pharmacol., 67, 2005–2011 (2004).
• 18) Choi HS, Seo HS, Kim SR, Choi YK, Jang BH, Shin YC, Ko SG. Anti-inflammatory and anti-proliferative effects of Rhus verniciflua STOKES in RAW264.7 cells. Mol. Med. Rep., 9, 311–315 (2014).
• 19) Levine AJ. p53, the cellular gatekeeper for growth and division. Cell, 88, 323–331 (1997).
• 20) Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature, 408, 307–310 (2000).
• 21) Gottlieb E, Vousden KH. p53 regulation of metabolic pathways. Cold Spring Harb. Perspect. Biol., 2, a001040 (2010).
• 22) Eskes R, Desagher S, Antonsson B, Martinou JC. Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol. Cell. Biol., 20, 929–935 (2000).
• 23) Finucane DM, Bossy-Wetzel E, Waterhouse NJ, Cotter TG, Green DR. Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL. J. Biol. Chem., 274, 2225–2233 (1999).
• 24) Smaili SS, Hsu YT, Sanders KM, Russell JT, Youle RJ. Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potential. Cell Death Differ., 8, 909–920 (2001).
• 25) Capano M, Crompton M. Bax translocates to mitochondria of heart cells during simulated ischaemia: involvement of AMP-activated and p38 mitogen-activated protein kinases. Biochem. J., 395, 57–64 (2006).
• 26) Jung CH, Kim JH, Hong MH, Seog HM, Oh SH, Lee PJ, Kim GJ, Kim HM, Um JY, Ko SG. Phenolic-rich fraction from Rhus verniciflua STOKES (RVS) suppress inflammatory response via NF-kappaB and JNK pathway in lipopolysaccharide-induced RAW264.7 macrophages. J. Ethnopharmacol., 110, 490–497 (2007).
• 27) Hong MH, Kim JH, Lee SY, Go HY, Kim JH, Shin YC, Kim SH, Ko SG. Early antiallergic inflammatory effects of Rhus verniciflua STOKES on human mast cells. Phytother. Res., 24, 288–294 (2010).
• 28) Kim EK, Choi EJ. Pathological roles of MAPK signaling pathways in human diseases. Biochim. Biophys. Acta, 1802, 396–405 (2010).
• 29) Chan WH, Yu JS, Yang SD. Apoptotic signalling cascade in photosensitized human epidermal carcinoma A431 cells: involvement of singlet oxygen, c-Jun N-terminal kinase, caspase-3 and p21-activated kinase 2. Biochem. J., 351, 221–232 (2000).
• 30) Park HJ, Kim BC, Kim SJ, Choi KS. Role of MAP kinases and their cross-ta in TGF-beta1-induced apoptosis in FaO rat hepatoma cell line. Hepatology, 35, 1360–1371 (2002).
• 31) Sauer H, Engel S, Milosevic N, Sharifpanah F, Wartenberg M. Activation of AMP-kinase by AICAR induces apoptosis of DU-145 prostate cancer cells through generation of reactive oxygen species and activation of c-Jun N-terminal kinase. Int. J. Oncol., 40, 501–508 (2012).
• 32) Meisse D, Van de Casteele M, Beauloye C, Hainault I, Kefas BA, Rider MH, Foufelle F, Hue L. Sustained activation of AMP-activated protein kinase induces c-Jun N-terminal kinase activation and apoptosis in liver cells. FEBS Lett., 526, 38–42 (2002).
• 33) Kefas BA, Cai Y, Ling Z, Heimberg H, Hue L, Pipeleers D, Van de Casteele M. AMP-activated protein kinase can induce apoptosis of insulin-producing MIN6 cells through stimulation of c-Jun-N-terminal kinase. J. Mol. Endocrinol., 30, 151–161 (2003).