Article pubs.acs.org/JAFC
Induction of p21Waf1/Cip1 by Garcinol via Downregulation of p38MAPK Signaling in p53-Independent H1299 Lung Cancer Sheng-Yung Yu,† Chiung-Ho Liao,‡ Ming-Hsien Chien,§,∥ Tsung-Yu Tsai,⊥ Jen-Kun Lin,# and Meng-Shih Weng*,† †
Department of Nutritional Science, Fu Jen Catholic University, New Taipei City 24205, Taiwan Division of Drug and New Technology Product, Food and Drug Administration, Ministry of Health and Welfare, Executive Yuan, Taipei 10058, Taiwan § Graduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan ∥ Wan Fang Hospital, Taipei Medical University, Taipei 110, Taiwan ⊥ Department of Food Science, Fu Jen Catholic University, New Taipei City 24205, Taiwan. # Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 10617, Taiwan ‡
ABSTRACT: Garcinol, a polyisoprenylated benzophenone, from Garcinia indica fruit rind has possessed anti-inflammatory, antioxidant, antiproliferation, and anticancer activities. However, the anticancer mechanisms of garcinol in lung cancer were still unclear. Therefore, we examine the effects of garcinol on antiproliferation in human lung cancer cells. Treatments with garcinol for 24 h exhibited morphological changes and inhibited the proliferation of H460 (p53-wild type) and H1299 (p53-null) cells in dose- and time-dependent manners. Furthermore, a significant G1 cell cycle arrest was observed in a dose-dependent treatment after H1299 cells were exposed in garcinol, whereas garcinol induced apoptosis rather than cell cycle arrest in H460 cells. Moreover, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin D1, and cyclin D3 were decreased, although cyclin E and cyclin-dependent kinase 6 (CDK6) were increased in garcinol-treated H1299 cells. Meanwhile, the protein levels of CDK inhibitors p21Waf1/Cip1 and p27KIP1 also exhibited upregulation after garcinol treatments. The enhanced proteinassociated level between p21Waf1/Cip1 and CDK4/2 rather than p27KIP1 and CDK4/2 was demonstrated in garcinol-treated cells. Additionally, knock-down p21Waf1/Cip1 by specific siRNA competently prevented garcinol-induced G1 arrest. Besides, garcinol also inhibited ERK and p38-MAPK activations in time-dependent mode. The pretreatment with p38-MAPK inhibitor but not ERK inhibitor raised garcinol-induced G1 population cells. Co-treatment with p38-MAPK inhibitor and garcinol synergistically elevated cyclin E, p21Waf1/Cip1, and p27Kip1 expressions. Meanwhile, overexpression dominant negative p38-MAPK also enhanced garcinol-induced p21Waf1/Cip1 expression in H1299 cells. Accordingly, our data suggested that garcinol induced G1 cell cycle arrest and apoptosis in lung cancer cells under different p53 statuses. The p53-independent G1 cell cycle arrest induced by garcinol might be through upregulation of p21Waf1/Cip1 triggered from p38-MAPK signaling inactivation. KEYWORDS: garcinol, lung cancer, p53, G1 arrest, p21Waf1/Cip1, p38-MAPK
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INTRODUCTION
garcinol has been categorized as a good cancer chemopreventive agent. Lung cancers are the most common health threat in the world, especially their high mortality rates.14 Human lung cancers are generally classified into two major categories, nonsmall cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC accounts for about 80% of the entire lung cancers, and about 50% of NSCLC are found in the mutation of p53. The mutation carries a worse prognosis and may result in relatively more resistance to chemotherapy and radiation.15,16 Aberrations in cell cycle control are a hallmark of lung cancer. Malignant lung cells develop the ability to bypass several cell cycle checkpoints, which are frequently associated with genetic mutation in key regulators of cell cycle, such as tumor suppressor gene p53.17 Numerous evidence indicated that
Phytochemicals are considered safe for human use and have been shown to modulate critical cellular signaling pathways, leading to their anticancer effects.1,2 The extract from the fruit of Garcinia indica, popularly known as Kokum or Mangosteen, has been recommended by Ayurvedic medicine for treatment of ailments, such as heat strokes, infections, and edema.3 Garcinol, a polyisoprenylated benzophenone, is a major component from G. indica. Numerous studies indicate that garcinol has pleiotropic functions, such as antioxidant activity,4 scavenging of free radicals,5 anti-inflammation,6 and anticancer activity.7 The anticancer activities of garcinol have been focused on modulating the apoptosis signaling pathways, including induction of caspase activation,8 death receptor expression, and inhibition of anti-apoptotic protein expression 9 in numerous cancers, such as breast, colon, prostate, and liver.9−12 Furthermore, garcinol has been identified with no toxic effects, even in normal breast epithelial MCF10A cells,8 and administrated orally up to 0.05% in a diet.13 In conclusion, © 2014 American Chemical Society
Received: Revised: Accepted: Published: 2085
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dependent apoptosis in H460 cells, although a dose-dependent G1 cell cycle arrest was observed in garcinol-treated H1299 cells. We developed an interest in the molecular mechanism of the p53-independent G1 cell cycle in garcinol-stimulated H1299 cells. We found that garcinol downregulated the expression of the D type of cyclins, CDK2, and CDK4, in contrast to upregulating cyclin E and CDK6 protein expression. Garcinol also induced p21Waf1/Cip1 and p27Kip1 expressions and concomitantly inhibited CDK2- and CDK4-mediated signals. In addition, ERK and p38-MAPK phosphorylations were also downregulated in a time-dependent manner by garcinol treatment. Treatment with p38-MAPK inhibitor enhanced garcinol-induced G1 arrest and cyclin E, p21 Waf1/Cip1, and p27KIP1 expression in H1299 cells. Our data suggested that the garcinol-induced p53-independent G1 cell cycle arrest might be through downregulation of p38-MAPK phosphorylation, resulting in accumulation of p21Waf1/Cip1 protein in H1299 lung cancer cells.
targeting the intracellular signaling pathway regulating cell cycle progression is an important strategy in lung cancer treatment.18 The comprehensive reports demonstrate that the mutation in p53 has potency to establish the novel strategies improving the lung cancer prognosis. The cell cycle is a critical regulation mechanism of cell growth, proliferation, and survival. The dysregulation of the cell cycle leads to uncontrolled cell proliferation and has been demonstrated in many different human cell tumorigeneses.19 The mammalian cell cycle comprises five sequential stages, quiescent phase G0, G1, S, G2, and mitotic (M) phase. G1 and G2 represent the gap intervals, where cells prepare themselves for the successful completion of S and M phases subsequently. The cells in the S phase receive a signal and faithfully replicate the genetic material, while the M phase portions out the cellular components into two daughter cells.20 The regulatory molecules of the successive cell cycle progression consist of cyclin and cyclin-dependent kinase (CDK) complexes. Different cyclin−CDK complexes are activated to ensure that cells successfully complete the previous phase and entering the new stage. 21 Furthermore, to ensure genome integrity and chromosomal stability, cell cycle checkpoints are important mechanisms to monitor cells transmitting precise copies of their genome into the next generation. CDK inhibitors (CDKIs) play an important role in checkpoint control of cell cycle progression. CDKIs are divided into two major families, INK4 and WAF/KIP families. The WAF/KIP family, including p21Waf1/Cip1, p27Kip1, and p57Kip2, intends to inhibit CDK2 and CDK4/cyclin complex activities via protein−complex association. Moreover, the members of INK4, containing p16INK4A, p15INK4B, p18INK4C, and p19INK4D, bind to either CDK4 or CDK6 and inhibit the action of the D type of cyclins.22 Activation of p53-p21Waf1/Cip1 signaling is an important mechanism to prevent cells from G1 entering the S phase.23,24 It has been shown that garcinol induced G1 cell cycle arrest and apoptosis in pancreatic cancer cells.25 However, the molecular mechanism of cell cycle progression in p53mutated lung cancer cells modulated via p21Waf1/Cip1 activation with garcinol treatment is still unknown. The mitogen-activated protein kinase (MAPK) family, a member of serine/threonine protein kinases, is involved in extracellular signal-regulated kinase (ERK), c-Jun-N-terminal kinase (JNK), and p38-MAPK signaling pathways. They mediate a variety of cellular functions through different extracellular stimulations.26 The ERK pathway has been linked to cell proliferation and growth and is primarily induced by mitogens, while p38-MAPK and JNK are generally activated by cellular stress and inflammatory cytokines.27 p38-MAPK is also involved in senescence, differentiation, and DNA-damage response.28 A usual fate of p38-MAPK activation through extracellular stimulation is cell cycle arrest or apoptosis.28−30 The proliferative role of p38-MAPK has also been reported in several cancers, especially in NSCLC.31−33 Furthermore, p38MAPK activation has shown the requirement during some mitogen-stimulated cell proliferation.34,35 Recently, our group reported that lipopolysaccharide (LPS)-induced COX-2 expression was inhibited by garcinol through p38 inactivation in macrophages.6 Therefore, it is important to verify the relationship between garcinol and p38-MAPK in tumorigenesis for cancer prevention or treatment. In this study, we examined antiproliferative effects of garcinol on p53-wild type H460 and p53-null H1299 lung cancer cells. Our data showed that garcinol significantly induced a dose-
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MATERIALS AND METHODS
Reagents. Garcinol (purity = 95%) was purchased from Biomol/ Enzo Life Sciences International, Inc. (Plymouth Meeting, PA). Anticyclin D1, D3, E, CDK2, CDK4, CDK6, anti-ERK, and anti-p38 antibodies were obtained from Cell Signaling (Beverly, MA). Anti-βactin antibody was received from Sigma-Aldrich (St. Louis, MO). PD98059 and SB203580 were obtained from Calbiochem (La Jolla, CA). Protein A/G plus agarose was acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Cell Culture and Cytotoxicity Assay. NSCLC p53-wild type H460 and p53-null H1299 cell lines were obtained from the American Type Culture Collection (Manassas, VA). Both of the cell lines were cultured in RPMI-1640 (Hyclone Laboratories, Logan, UT) supplemented with 5% fetal bovine serum and maintained at 37 °C in a humidified atmosphere at 95% air and 5% CO2. Cells (1 × 104/ well) were cultured in 96-well plates and incubated for 24 h. After the cells were treated with various dosages of garcinol for 24, 48, and 72 h, the incubation media were changed every 2 days during the experiments. At the end of incubation, cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell Synchronization, Drug Treatment, and Flow Cytometric Analysis. Lung cancer cells H460 and H1299 were plating for 24 h. Cells were then synchronized for 24 h. After synchronization, the media were removed and changed to complementary media containing various dosages of garcinol for 24 h. After the treatment, cells were harvested and stained with propidium iodide (50 μg/mL) (Sigma Chemical, MO) and DNA contents were measured using a FACScan laser flow cytometer analysis system (Beckman Coulter, CA). Western Blot Analysis. Control and garcinol-treated cells were rinsed twice with ice-cold phosphate-buffered saline (PBS) and then lysed in an appropriate RIPA extraction buffer [20 mM Tris−HCl at pH 7.5, 150 mM NaCl, 1 mM disodium salt of ethylenediaminetetraacetic acid (Na2EDTA), 1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1% (v/v) NP-40, 1% (v/v) sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM βglycerophosphate, 1 mM Na3VO4, and 1 μg/mL leupeptin] on ice for 30 min. The extracts were then centrifuged at 12000g for 30 min. Proteins were loaded at 40 μg/lane and separated by sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE). Thereafter, protein was transferred to Immobilon polyvinylidene difluoride membrane (Millipore, MA). Membrane was blocked by blocking buffer (3% bovine serum albumin and 0.5% Tween 20) for 40 min and incubated with different primary antibodies at room temperature for 2 h afterward. After 2 h of incubation, membrane was washed and soaked in horseradish-peroxidase-labeled secondary antibodies at 25 °C for 1 h. The blot was revealed by chemiluminescence (ECL kits, Amersham Pharmacia Biotech, Arlington Heights, IL). Band intensities were quantitated by UVP BioSpectrum Imaging System ChemiDoc-It2 2086
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Figure 1. Growth inhibition effect of garcinol in H460 and H1299 lung cancer cells. (A) Morphological changes in garcinol-treated lung cancer cells. H460 and H1299 were treated with various concentrations of garcinol for 24 h. Morphological changes were observed by a microscope at 10 × 10 magnification. (B). Effects of garcinol on cell viability in H460 and H1299 lung cancer cells. H460 and H1299 were maintained in a 96-well plate at a density of 1 × 103/well for 24 h. Cells were then treated with various doses of garcinol for 24, 48, and 72 h. The cell viability was measured using the MTT assay. Data were the mean ± SD of triplicate samples. (∗) p < 0.05 compared to control cells. 810 (UVP, LLC, Upland, CA). β-Actin was used as the internal control. Immunoprecipitation. H1299 lung cancer cells were cultured in 100 mm dishes for 24 h, following synchronization. Thereafter, cells were switched to complementary media containing 10 μM garcinol for 24 h. Cells were then harvested and extracted by lysis buffer. The cell lysate was clarified by centrifugation at 12000g at 4 °C for 30 min. A total of 250 μg of protein was incubated with anti-CDK2 or antiCDK4 antibody and protein A/G plus agarose at 4 °C for 18 h. The immunoprecipitate was washed twice with immunoprecipitation buffer [10 mM Tris−HCl at pH 7.4, 1 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.2 mM sodium vanadate, 0.2 mM phenylmethanesulfonyl fluoride (PMSF), 1% (v/v) Triton X-100, and 0.5% (v/v) NP-40]. CDK2 and/or CDK4 immunoprecipitates were resuspended in 25 μL of RIPA lysis buffer, mixed with protein loading buffer, and separated by SDS−PAGE. After transfer, membranes were blotted with antip21Waf1/Cip1 and anti-p27Kip1 primary antibodies. Blots were then stripped and reprobed with CDK2 and CDK4 as internal controls. The densities of the bands were then expressed as the relative densities compared to controls, which were taken as 1-fold. Reverse Transcription Polymerase Chain Reaction (RT-PCR). After garcinol treatment, cells were washed in ice-cold PBS and total RNA was isolated by RNA mini kit (Qiagen, Taiwan). cDNAs were prepared from the total RNA (5.0 μg) with high-capacity cDNA reverse transcription kit (Invitrogen, Taiwan) at 42 °C for 60 min. PCR was performed in a final volume of 25 μL containing deoxynucleotide triphosphates (dNTPs) (each at 200 μM), 1× reaction buffer, 2 μL of RT-cDNA products, and 50 units/mL pro Taq DNA polymerase (Promega, WI). PCR primers for p21Waf1/Cip1, p27Kip1, and GADPH were synthesized according to the following oligonucleotide sequences: p21 Waf1/Cip1 , forward primer, 5′-
GGCGCCATGTCAGAACCGGCTG-3′; reverse primer, 5′-ACCCAGCGGACAAGTGGGGAGG-3′; p27Kip1, forward primer, 5′ACGAAGAGTTAACCCGGGACTTGG-3′; reverse primer, 5′GGGCGTCTGCTCCACAGAACCG-3′; and GAPDH, forward primer, 5′-TGAAGGTCGGAGTCAACGGGTGAGTT-3′; reverse primer, 5′-CATGTAGACCCCTTGAAGAGG-3′. After an initial denaturation for p21Waf1/Cip1 PCR (851 kb) at 95 °C for 5 min, 26 cycles of amplification (95 °C for 50 s, 60 °C for 45 s, and 72 °C for 60 s) were performed and followed by an extension at 72 °C for 10 min. Similarly, for p27Kip1 PCR (452 kb), 29 cycles of amplification (95 °C for 50 s, 57 °C for 45 s, and 72 °C for 45 s) were performed and followed by an extension at 72 °C for 10 min. In GAPDH amplification, 29 cycles of reaction (94 °C for 50 s, 60 °C for 45 s, and 72 °C for 120 s) were performed and followed by an extension at 72 °C for 10 min. The PCR products were separated by electrophoresis on 1.8% agarose gel and visualized by SYBR Safe (Life Technologies, Taiwan) staining. Small Interfering RNA and Dominant Negative p38-MAPK (DN p38-MAPK) Transfection. H1299 cells were cultured to 70% confluence and then transfected with the siRNA duplexes at a final concentration of 10 nM using GenMute siRNA transfection reagent (SignaGen Laboratories, Ijamsville, MD) according to the instructions of the manufacturer. Cellular levels of the p21Waf1/Cip1 proteins specific for the siRNA transfection were checked by immunoblotting, and all experiments were performed at 6 h after transfection. DN p38-MAPK plasmid was kindly obtained from Dr. Kuo-Tai Hua. H1299 cells were transfected with DN p38-MAPK using Lipofectamine 2000 transfection reagent according to the protocol of the manufacturer (Invitrogen, Carlsbad, CA). Statistical Analysis. The results were expressed as the mean ± standard deviation (SD) calculated from the specified numbers of 2087
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determination. One-way analysis of variation (ANOVA) was used to compare individual data with the control value. A probability of p < 0.05 was taken as denoting a significant difference from the control data.
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RESULTS Cell Viability Inhibition by Garcinol on Human H460 and H1299 Lung Cancer Cells. The cell viability inhibition by garcinol on p53-wild type H460 and p53-null H1299 cells has been examined. Cells were treated with serial dosages of garcinol (0, 2.5, 5, 7.5, and 10 μM) at the time points of 24, 48, and 72 h, respectively. The cell viability was measured by the MTT assay. H460 and H1299 cells were treated with 10 μM garcinol for 24 h, exhibiting morphological changes (Figure 1A). As shown in Figure 1A, the cell shrunk and detachment was observed in garcinol-treated H460 and H1299 cells. In addition, the cell debris was noticed only in garcinol-treated H460 cells. Furthermore, the growth inhibition effect of garcinol was observed in dose- and time-dependent manners in these cell lines (Figure 1B). These results showed that the cell viability was significantly decreased in garcinol-treated H460 cells compared to H1299 cells. Effects of Garcinol on Cell Cycle Distribution in Human H460 and H1299 Lung Cancer Cells. To further evaluate the hypothesis of the cytotoxic effect of garcinol mediated via alternations in cell cycle development, p53-wild type H460 and p53-null H1299 cells were used. Both H460 and H1299 cells were synchronized for 24 h and immediately treated with garcinol for another 24 h. At the end of treatment, cells were harvested and flow cytometry analysis was performed. As shown in Figure 2, the percentage of cells in the G1 phase increased from 63.7% in the control to 76.0% in garcinol-treated H1299 cells. Up to 13% of the G1 phase was boosted after garcinol treatment in H1299 cells. A sub-G1 fraction increased from 3.3% in the control to 11.2% in a dosedependent mode in garcinol-treated H460 cells. However, garcinol significantly induced G1 cell cycle arrest in H1299 cells. The results implicated that garcinol possessed different biological activities via distinct p53 statuses. Expression of Cell Cycle Regulatory Proteins in Garcinol-Treated H1299 Cells. In several studies, it has been demonstrated that garcinol induced apoptosis in many types of cancer cells.8,9,11,12,25 However, further studies on the G1 arrest molecular mechanism in garcinol-induced p53-null cancer cells are still deficient. Therefore, we focused on the verification of the molecular mechanism of garcinol in H1299 cells. The cyclins and CDKs are two important cell cycle regulatory protein families. It has been well-characterized that D- and E-type cyclins are associated with CDK2 and CDK4/6, which control G1 cell cycle progression.19,22 To investigate biological pathways of G1 arrest induced by garcinol via the regulatory protein expression, H1299 cells were synchronized and treated with garcinol for 24 h. After 24 h of treatment, the cells were harvested and changes in the expression of cyclin D1, D3, E, CDK2, CDK4, and CDK6 were inspected by western blots. Decreases in the protein levels of cyclin D1, D3, CDK2, and CDK4 were detected in a dose-dependent manner with garcinol treatment, as shown in Figure 3A, while increases in the expression of cyclin E and CDK6 were observed (Figure 3A). Garcinol Induced G1 Cell Cycle Arrest through the Upregulation of CDK Inhibitors. Previous results showed that CDK activity was inhibited by CDK inhibitors, p21Waf1/Cip1
Figure 2. Effects of garcinol on cell cycle distribution in H460 and H1299 lung cancer cells. (A) H460 and (B) H1299 cells were treated with garcinol for 24 h, and then cells were harvested for cell cycle distribution analyses by flow cytometry. Three samples were analyzed in each group, and the results are presented as the mean ± SD. (∗) Significant difference was observed from the control group (p < 0.05).
and p27Kip1. Furthermore, the activities of p21Waf1/Cip1 and p27Kip1 associated with the complexes of cyclin/CDK were repressed, resulting in cell cycle arrest.22,23 The effects of garcinol on the expression of p21Waf1/Cip1 and p27Kip1 were characterized by western blot, as shown in Figure 3B. Obviously, the protein levels of p21Waf1/Cip1 and p27Kip1 were upregulated by garcinol treatment in a dose-dependent mode (Figure 3B). The verification of the activities of garcinolinhibited CDK2 or CDK4 enhanced by p21Waf1/Cip1 and p27Kip1 was performed by H1299 cell synchronization treated with or without garcinol. The outcomes revealed that the bound 2088
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results implicated that the garcinol-induced G1 cell cycle arrest might be through upregulation of p21Waf1/Cip1 and p27Kip1 expression, resulting in downregulated CDK activities via enhancing the association. It has been confirmed that p21Cip1/Waf1 expression is regulated by p53-dependent and -independent transcriptional mechanisms.23 After the cells were treated with garcinol for 12 h, total RNA was collected and RT-PCR was perform to investigate the expression of p21Waf1/Cip1 induced by garcinol through transcriptional activation. The p21Waf1/Cip1 mRNA expression was increased in a dose-dependent manner. Conversely, the expression of p27Kip1 mRNA was declined (Figure 5A). To further verify whether garcinol-induced p21Waf1/Cip1 expression in H1299 cells correlated with G1 growth arrest, we knocked down p21Waf1/Cip1 expression by siRNA and analyzed the cell cycle distribution in garcinoltreated cells. As shown in Figure 5B, the protein level of p21Waf1/Cip1 was dramatically downregulated after specific siRNA transfection. Furthermore, garcinol-induced G1 arrest was completely prevented by transfection with p21Waf1/Cip1 siRNA into H1299 cells (Figure 5B). These results suggested that the garcinol-induced G1 arrest in H1299 cells was through upregulation of p21Waf1/Cip1 protein. Garcinol Inhibited ERK and p38-MAPK Activation. To explore the molecular mechanism of garcinol-induced G1 arrest, the phosphorylation of ERK and p38-MAPK, two important molecules in MAPK pathways involving cell cycle regulation, were analyzed in garcinol-treated H1299 cells. The outcomes exhibited that ERK phosphorylation was briefly stimulated between 0.5 and 1 h by serum significantly. Conversely, serum-stimulated ERK activation was repressed with 10 μM garcinol treatment (Figure 6). p38-MAPK was induced at 0.5 h and remained active in serum stimulation for 12 h. With garcinol treatment, the level of active p38-MAPK was diminished in a time-dependent mode (Figure 6). The results implicated that G1 arrest prompted by garcinol might be through ERK and p38-MAPK inhibition in H1299 cells. p38-MAPK Rather than ERK Involved in GarcinolInduced G1 Arrest. In the present study, the mechanism of garcinol-induced G1 arrest through p21Waf1/Cip1 and p27Kip1 activation, following the inhibition of CDK activity, via association with CDKs, was established (Figure 4). Furthermore, ERK and p38-MAPK were activated in garcinol-treated H1299 cells (Figure 6). The pharmacological inhibitors, PD98059 and SB203580, for ERK and p38-MAPK, correspondingly, were used to understand the correlation in the activated pathways between ERK and p38-MAPK and the destination of garcinol-treated cells. H1299 cells were pretreated with 10 μM PD98059 or SB203580 for 30 min before stimulated with 10 μM garcinol for 24 h. Cells were collected for flow cytometry analysis. G1 arrest was observed in H1299 cells treated with ERK and p38-MAPK inhibitors alone (Figure 7). Interestingly, the synergistic effect of the G1 arrest population was detected in combinational treatments with p38-MAPK inhibitor and garcinol unlike in the co-treatment of ERK inhibitor and garcinol. Additionally, H1299 cells were pretreated with p38-MAPK inhibitor before 10 μM garcinol stimulation to verify the cell cycle regulators participated in p38-MAPK signaling when garcinol was supplied. The results showed that the expression of CDK2 was reduced in garcinoltreated cells on western blot (Figure 8A). Moreover, the expression of CDK2 was repressed by the combinational treatment with p38-MAPK inhibitor and garcinol. The
Figure 3. Effects of garcinol on the expressions of cyclins, CDKs, and CDK inhibitors. H1299 lung cancer cells were initially synchronized by serum-free medium and then serum-supplemented medium containing various doses of garcinol (0, 2.5, 5, 7.5, and 10 μM). After the cells were harvested, western blot analyses were performed with (A) anticyclin D1, cyclin D3, cyclin E, CDK2, CDK4, and CDK6 and (B) antip21Waf1/Cip1, p27Kip1, and β-actin antibodies. The protein levels in each treatment after normalization with the levels of β-actin are shown in parentheses. Data shown are representative of at least three independent experiments.
protein levels of p21Waf1/Cip1 with CDK2 were increased about 3-fold in garcinol-treated cells, otherwise a 5-fold increase in bound protein levels of p21Waf1/Cip1 with CDK4 in garcinoltreated cells (Figure 4). Nevertheless, p27Kip1, bound with CDK2, was increased about 2-fold in garcinol-treated cells. The 2089
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Figure 4. Effects of garcinol on the expressions of CDK−CDKI complexes in lung carcinoma H1299 cells. H1299 cells were treated with 10 μM garcinol for 24 h. Cell lysates were then incubated with protein A/G agarose beads conjugated with (A) anti-CDK2 or (B) anti-CDK4 antibody. The immunoprecipitates were electrophoresed, and the separated proteins reacted against primary antibodies, anti-p21Waf1/Cip1, p27Kip1, CDK2, and CDK4, to monitor protein interaction by western blot. The bound levels of p21Waf1/Cip1 and p27Kip1 in each treatment normalized with the levels of immunoprecipitation loading control are shown and representative of an experiment performed in triplicate. The density of the band was then expressed as the relative density compared to that in untreated cells (control), which was taken as 1-fold.
prompted in garcinol-treated cells, and the upregulation of p21Waf1/Cip1 was associated with CDK2 and CDK4, followed by the inhibition of cyclin/CDK complex activity. Meanwhile, the upregulation of p21Waf1/Cip1 expression via garcinol stimulation was through transcriptional regulation. The studies indicated that garcinol inhibited ERK and p38-MAPK phosphorylation and merely a combination with the p38-MAPK inhibitor resulted in the enhancement of G1 cell cycle arrest. The expressions of cyclin E, p21Waf1/Cip1, and p27Kip1 were also boosted after co-treatment of the p38-MAPK inhibitor and garcinol. Tumor suppressor protein p53 has been shown to participate in numerous cellular processes.15,24,37 The pivotal roles of p53 are involved in the cell growth arrest and cell death induction in cancer treatment. However, p53 mutations are present in almost half of NSCLC and award chemoresistance and poorer survival prognosis in lung cancer patients.15,37 The application of disrupting the oncogenic effects is certainly important for the treatment and prevention of lung cancer. One of the advanced strategies for preventive or therapeutic p53 mutant tumors is the probing of active components to restore the p53-regulated pathway in the cells. Garcinol has evaluated its growth inhibition and apoptosis induction in a number of cancer models.7,9−11,25 However, the role of p53 in garcinol-treated cancer cells is still unclear. We hypothesized that p53 statuses of lung cancer cells might affect the distinct molecular pathways in garcinol-treated cells. The effects of garcinol in p53-wild type and p53-null lung cancer cells were studied to further validate our hypothesis. The results revealed that garcinol induced the morphological changes and decreased the amount of lung cancer cells. Moreover, H460 cells were more active treated with garcinol (Figure 1). To further verify the cytotoxic effects in garcinol-treated cells, the cell cycle distribution was investigated. The outcomes showed that the G1 cell cycle arrest was enhanced by garcinol in H1299 cells (Figure 2). Our results revealed that garcinol induced apoptosis and G1 growth
expression of cyclin E in the co-treatment of p38-MAPK inhibitor and garcinol was upregulated (Figure 8A). Similarly, the expression of p21Waf1/Cip1 and p27Kip1 in co-treatment with p38-MAPK inhibitor and garcinol was increased, especially in the protein level of p21Waf1/Cip1 (Figure 8B). To further evaluate the significance of the p38-MAPK signaling in garcinol-induced p21Waf1/Cip1 expression, H1299 cells transiently transfected the DN p38-MAPK. As shown in Figure 8C, ectopic expression of DN p38-MAPK enhanced garcinolinduced p21Waf1/Cip1 expression. These results revealed that the garcinol-induced p21Waf1/Cip1 expression was through inhibition of p38-MAPK activation.
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DISCUSSION Natural products represent good candidates to downregulate cancer cell growth. Major beneficial effects of these natural components as cancer-preventive agents are their comparative nontoxic effects. These non-nutritive components in a diet have been demonstrated to contribute from their pleiotropic effects, including downregulation of survival signaling and activating the death signaling pathway in cancer cells.1,2 Garcinol has been indicated to possess numerous biological activities, such as antioxidation, anti-inflammation,4−6,36 and upregulation of death signaling.7,9,11,12,25 Although there is countless literature on the anticancer properties of garcinol, the effect of garcinol is still unclear regarding the molecular mechanism of diverse p53 statuses in lung cancer. Here, we have demonstrated that p38MAPK signaling might be the essential target in p53 mutant lung cancer cell growth via garcinol treatment. Both p53-wild type H460 and p53-null H1299 lung cancer cell growth were inhibited by garcinol treatment. Garcinol significantly induced G1 cell cycle arrest in H1299 but not in H460 lung cancer cells. Furthermore, the D type of cyclin, CDK2, and CDK4 expressions were downregulated, whereas cyclin E and CDK6 expressions were upregulated in garcinol-stimulated H1299 cells. Also, the protein levels of p21Waf1/Cip1 and p27Kip1 were 2090
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Figure 5. Role of p21Waf1/Cip1 in the garcinol-induced G1 cell cycle arrest. (A) H1299 cells were treated with garcinol for 12 h, and total mRNA was extracted afterward. After the extraction of total mRNA, p21Waf1/Cip1, p27Kip1, and GAPDH RT-PCR were performed, as described in the Materials and Methods. (B) Knock-down p21Waf1/Cip1 expression prevented garcinol-induced G1 cell cycle arrest in H1299 cells. H1299 cells were transfected with p21Waf1/Cip1 siRNAs as described in the Materials and Methods. After transfection, H1299 cells were synchronized for 24 h and then treated with or without garcinol (10 μM) for an additional 24 h. Cells were harvested for Western blot and cell cycle progression analyses.
garcinol-treated p53 wild-type A549 cells does not show apoptosis and/or G1 arrest induction.38 In our study, garcinolinduced significant apoptotic or G1 arrest cell accumulation was observed upper 5 μM garcinol treatment in two cell lines
arrest in lung cancer cells in different p53 statuses. Tumor suppressor protein p53 might be regarded as a key factor in control cell apoptosis or G1 growth arrest in garcinol-treated lung cancer cells. However, a low concentration (4 μM) of 2091
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Figure 6. Effects of garcinol on ERK and p38-MAPK expressions in H1299 lung cancer cells. H1299 cells were treated with garcinol (10 μM) for indicated time intervals, and western blot analyses were performed with anti-p-ERK, ERK, p-p38-MAPK, p38-MAPK, and β-actin antibodies. The ratio of phospho-form/non-phospho-form protein in each treatment normalized with the levels of β-actin are shown in parentheses. Data shown are representative of at least three independent experiments. (∗) Significant differences were compared to the control and determined by one-way ANOVA (p < 0.05).
Figure 7. Effect of ERK and p38-MAPK inhibitors on cell cycle distribution in garcinol-treated H1299 cells. H1299 cells were treated with 10 μM PD98059 or SB203580 for 30 min and then stimulated with or without 10 μM garcinol for 24 h. Cells were harvested and stained with propidium iodine subsequently. Cell cycle distribution was determined by FACScan analysis. Three samples were analyzed in each group. (∗) Significant differences were determined by Student’s t test using the mean ± SD from the control group (p < 0.05).
Hep3B cells.12 Although treatment with a high dosage of garcinol might induce p53-independnet apoptosis in cancer cells, other side effects might emerge in clinical application. It has been well-known that garcinol induced the apoptosis of cancer cells through intrinsic and extrinsic pathways.8,9,11,12
(Figure 2). These results suggested that the effective dosage of garcinol should be carefully concerned in anticancer actions. Furthermore, treatment with a high dosage of garcinol (50 μM) has been demonstrated to induce ROS-dependent apoptosis rather than G1 arrest in p53-negative hepatocellular cancer 2092
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Figure 8. Effects of p38-MAPK inhibitor and garcinol co-treatment on the expressions of cyclins, CDKs, and CDKIs in H1299 cells. H1299 cells were treated with 10 μM SB203580 for 30 min and then stimulated with or without 10 μM garcinol for 24 h. Cells were harvested, and western blot was implemented with (A) anti-cyclin D1, cyclin D3, cyclin E, CDK2, and CDK4 and (B) anti-p21Waf1/Cip1, p27Kip1, and β-actin antibodies. (C). H1299 cells were transiently transfected with DN p38-MAPK, and then treated with 10 μM garcinol for 24 h. The protein levels of p21Waf1/Cip1, p38MAPK, and β-actin were determined by western blotting. The protein levels in each treatment after normalization with the levels of β-actin were shown in parentheses. Data shown were representative of at least three independent experiments. The significant differences were determined by one-way ANOVA (a) compared to the control (p < 0.05) and (b) compared to the garcinol-treated group (p < 0.05).
Besides, Collins et al. demonstrated that, with garcinol-induced p53 expression, acetylation accompanied G1 phase population accumulation in breast cancer cells.39 Additionally, the proliferation of the nicotine-stimulated breast cancer cell is blocked by garcinol, which diminished nicotine receptor signal cascade to mediate cyclin D3 expression and induce p53p21Waf1/Cip1 signaling, resulting in G1 arrest.10 The reports confirm that garcinol induced G1 cell cycle arrest through p53related signaling pathways. However, the effects of garcinol in cell cycle regulation on p53-null cancer cells are still mysterious. The results indicated that the D type of cyclins, CDK2, and
CDK4 were reduced, whereas the protein level of cyclin E was increased, in garcinol-treated H1299 cells (Figure 3A). The progression of the cell cycle is tightly regulated by the complexes of cyclins and CDKs. In the transition from the G1 to S phase, cyclin D/CDK4/6 and cyclin E/CDK2 control the cycle entry.19 Furthermore, the activities of cyclin−CDK complexes are modulated through CDK inhibitors. The prominent CDK inhibitors are the WAF/KIP family, including p21Waf1/Cip1, p27Kip1, and p57 Kip2.23 We demonstrated that CDK inhibitors, p21Waf1/Cip1 and p27Kip1, were enhanced in a dose-dependent manner (Figure 3B). The association between 2093
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p21Waf1/Cip1 and CDK2/4 was raised in garcinol-treated cells. However, p27Kip1 was only complexed with CDK2 (Figure 4). The data disclosed that the garcinol-induced G1 cell cycle arrest might be through p53-independent upregulation of p21Waf1/Cip1 rather than p27Kip1, resulting in the inhibition of cyclin−CDK activities via the strengthening binding activity. It is well-established that p21Waf1/Cip1 is necessary and sufficient for p53-dependent repressing of genes regulating cell cycle progression.23 Post-translational modifications of p21Waf1/Cip1, such as phosphorylation, increasing protein stability following cell accumulation at the G1 phase, were acknowledged.29 In this study, the gene expression of p21Waf1/Cip1 was further examined after the treatment with garcinol. The RNA level of p21Waf1/Cip1 was considerably increased in a dose-dependent profile (Figure 5A). The data suggested that the garcinol-induced expression of p21Waf1/Cip1 might be through transcriptional regulation. Furthermore, to further investigate the relationship between garcinol-induced p21Waf1/Cip1 expression and G1 arrest, we knocked down p21Waf1/Cip1 expression by specific siRNA before garcinol stimulation. Our results revealed that downregulation of p21Waf1/Cip1 by siRNA transfection completely prevented the garcinol-induced G1 arrest (Figure 5B). According to our results, garcinol-induced G1 growth arrest was through p21Waf1/Cip1 upregulaion. MAPK pathways have been demonstrated to play an important role in cell cycle regulation, especially ERK and p38-MAPK.26,28,30,40 ERK kinases are activated in various cell types as a survival signal through mitogenic and cytokine stimulation. Inhibition of active ERK has been investigated in blockage of cell cycle progression.26 Moreover, p38-MAPK is generally activated by inflammatory cytokines and cellular stress and then results in G1 arrest, which mediated differentiation.26−28 Emerging evidence points out the roles of p38-MAPK in cell cycle regulation, proliferation, and metastasis in different cells. The level of active p38-MAPK is higher in 18 analyzed primary NSCLC tumor tissues than normal lung tissue. Active p38-MAPK is discovered in the nucleus and cytoplasm of malignant and dysplastic cells.33 p38-MAPK has been proven to promote melanoma cell proliferation through ATF-2-meidated upregulation of cyclin D1.34 Faust et al. indicate that mitogen- and stress-induced activations of p38MAPK have different functions. The duration of p38-MAPK activation determines the cell’s destination, proliferation, or induction of cell cycle arrest.27 Mitogen stimulation induces a weak and transient p38-MAPK phosphorylation, which is required for the G1−S transition. However, stress induces the strong and sustained p38-MAPK activation, following increasing cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) phosphorylation and cell arrest.27 Furthermore, our recent report showed that chrysin induced sustained p38-MAPK activation and upregulation of p21Waf1/Cip1 expression, following downregulated C6 glioma cell growth.29 The results also revealed that p38-MAPK phosphorylation was detected at 0.5 h and persisted up to 6 h after serum stimulation. Moreover, serum-stimulated p38-MAPK activation was suppressed by garcinol (Figure 6). Inconclusively, garcinol inhibited serum-stimulated p38-MAPK activation, resulting in the disruption of p38-MAPK-mediated cell proliferation via G1 arrest. Recent reports show that p21Waf1/Cip1 is positively regulated via p38-MAPK phosphorylation and subsequent cell accumulation at the G1 phase.29,30 Nevertheless, p38-MAPK also
affects the cell fate to arrest the G1 phase in the survival cascade of several cancer cell types.27,32,35 These dual roles of p38MAPK in cell cycle regulation may depend upon the cell type and other related factors. In our studies, garcinol inhibited serum-stimulated ERK and p38-MAPK activation in a timedependent profile (Figure 6). The co-treatment of SB203580 and not PD98059 plus garcinol revealed the substantial G1 accumulation compared to garcinol treatment alone (Figure 7). The data suggested that the garcinol-induced G1 cell cycle arrest might be partially proceeded by the means of p38-MAPK inhibition. Also, co-treatment with SB203580 and garcinol enhanced cyclin E and p21Waf1/Cip1 expressions compared to garcinol-treatment alone in H1299 cells (Figure 8). Furthermore, transiently transfecting the DN p38-MAPK also enhanced garcinol-induced p21Waf1/Cip1 expression (Figure 8C). Therefore, the expression of p21Waf1/Cip1 induced by garcinol might be through downregulation of p38-MAPK activity. However, recent evidence reveals that nuclear factor-κB (NF-κB) is a transcription factor regulating cell growth, apoptosis, and inflammation6,8,41 and also involved in the p21Waf1/Cip1-mediated cell cycle arrest of breast and colon cancer cells.41,42 Knock-down NF-κB expression by siRNA enhances 4-O-methylhonokiol, resulting in increasing p21Waf1/Cip1 expression in colon cancer cells.41 Accordingly, NF-κB is a key regulator in the expression of p21Waf1/Cip1. NFκB-mediated proliferative and inflammatory gene expressions have previously been identified through the p38-MAPK signaling pathway.6,41 In addition, the downregulation of NFκB via garcinol-augmented cell death has been demonstrated in breast and pancreatic cancer cells.8,25 It was speculated that p53-independent p21Waf1/Cip1 expression induced by garcinol might be as a consequence of the downregulation of p38MAPK-mediated NF-κB activity. In summary, the G1 cell cycle evoked by garcinol was triggered by p53-independent p21Waf1/Cip1 activation by means of the negative regulation of p38-MAPK activation in H1299 lung cancer cells. Our finding revealed that garcinol might become a candidate of the chemopreventive agent for p53-independent lung cancer cells in the future.
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AUTHOR INFORMATION
Corresponding Author
*Telephone: 886-2-2905-3776. Fax: 886-2-2902-1215. E-mail:
[email protected]. Funding
This work was supported by grants from the National Science Council of Taiwan, Republic of China, NSC100-2313-B-030003. Notes
The authors declare no competing financial interest.
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REFERENCES
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