Article pubs.acs.org/JAFC
Plant Isoquinoline Alkaloid Berberine Exhibits Chromatin Remodeling by Modulation of Histone Deacetylase To Induce Growth Arrest and Apoptosis in the A549 Cell Line Arunachalam Kalaiarasi,† Chidambaram Anusha,†,‡ Renu Sankar,†,‡,§ Subbiah Rajasekaran,∥ Jayaraj John Marshal,⊥ Karthikeyan Muthusamy,⊥ and Vilwanathan Ravikumar*,† †
Department of Biochemistry, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024, India Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Avenue, Wooster, Ohio 44691, United States ∥ Department of Biotechnology, Bharathidasan Institute of Technology, Anna University, Tiruchirappalli, Tamil Nadu 620 024, India ⊥ Department of Bioinformatics, Alagappa University, Karaikudi, Tamil Nadu 630 003, India §
S Supporting Information *
ABSTRACT: Histone deacetylases (HDACs) are a group of epigenetic enzymes that control gene expression through their repressive influence on histone deacetylation transcription. HDACs are probable therapeutic targets for cancer treatment, spurring the progress of different types of HDAC inhibitors. Further, natural-source-based derived bioactive compounds possess HDAC inhibitor property. In this way, we hypothesized that plant isoquinoline alkaloid berberine (BBR) could be a HDAC inhibitor in the human lung cancer A549 cell line. BBR represses total HDAC and also class I, II, and IV HDAC activity through hyperacetylation of histones. Furthermore, BBR triggers positive regulation of the sub-G0/G1 cell cycle progression phase in A549 cells. Moreover, BBR-induced A549 cell growth arrest and morphological changes were confirmed using different fluorescence-dye-based microscope techniques. Additionally, BBR downregulates oncogenes (TNF-α, COX-2, MMP-2, and MMP-9) and upregulates tumor suppressor genes (p21 and p53) mRNA and protein expressions. Besides, BBR actively regulates Bcl-2/Bax family proteins and also triggered the caspase cascade apoptotic pathway in A549 cells. Our finding suggests that BBR mediates epigenetic reprogramming by HDAC inhibition, which may be the key mechanism for its antineoplastic activity. KEYWORDS: epigenetics, berberine, histone deacetylase, histone deacetylase inhibitor, cell cycle, apoptosis, A549 cells
■
INTRODUCTION Epigenetics is described as genetic modifications in gene expression without changing the primary genetic material sequence. The epigenetic pathway consists of DNA methylation and histone deacetylation that repress the transcription process.1 Histone modification is a predominant determinant in the epigenetic silencing of genes and regulatory role of cellular processes, such as gene transcription and proliferation.2 Histone acetylation emerges as a landmark and a determinant for chromatin function that allows interconversion between permissive and repressive chromatin. Its equilibrium is maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs). These enzymes exert fundamental roles in developmental processes, and their deregulation is a clear sign of the onset and growth of cancer.3 Particularly, HDACs have been found to be overexpressed, thereby modifying the chromatin structure through deacetylating the lysine residues of histone and non-histone proteins, leading to aberrant expression of oncogenic transcription factors in cancer cells. HDACs are classified into class I HDACs (1−3 and 8), class II HDACs (4−7, 9, and 10), class III HDACs (sirtuins 1−7), and class IV HDACs (11). All HDACs are zinc-dependent, except class III, which is not zinc-dependent and also needs NAD+ for its activation. Because HDAC inhibition triggers the apoptotic pathway in cancer cells, the discovery of HDAC inhibitors © XXXX American Chemical Society
provides an attractive avenue for drug development in cancer therapy. HDAC inhibitors prevent the subtraction of acetyl groups from lysine tails of histones by blocking the active sites of zinc-dependent HDAC enzymes. Also, they have the capacity to arrest the cell cycle and activate the tumor suppressor and apoptotic pathway in a variety of cancer cells.4 HDAC inhibitors belong to four different chemical classes, namely, hydroxamic acids, cyclic peptides, benzamides, and aliphatic fatty acids. While the benzamides display class I HDAC specificity in terms of inhibition, the other classes of inhibitors have a broader spectrum. The general pharmacophores of a HDAC inhibitor follow the schematic of cap− linker−chelator. Inhibitor specificity is derived from the cap, while the inhibitory functionality is based on chelation of zinc away from the active site of deacetylase. Recent drug development for HDAC inhibition relies on the chelation of zinc from the active site of the HDACs, which disturbs the substrate coordination, thereby targeting core active center of the HDAC enzymes. Thus far, only few HDAC inhibitors are approved by the U.S. Food and Drug Administration (FDA) for Received: October 7, 2016 Revised: November 11, 2016 Accepted: November 12, 2016
A
DOI: 10.1021/acs.jafc.6b04453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
for 30 min at 37 °C to arrest the reaction. The absorbance was obtained by an enzyme-linked immunosorbent assay (ELISA) plate reader at 400 nm. HDAC activity was expressed as relative optical density (OD) values per microgram of protein. TSA was used as a positive control. Assessment of Reactive Oxygen Species (ROS), Mitochondrial Membrane Potential (Δψm), Membrane Integrity, and Nuclear Fate. DCFH-DA (10 μM), Rhodamine 123 (10 μg/mL), AO and EB (1 mg/mL for both in PBS), and Hoechst 33258 (10 μg/ mL) were used to detect the ROS generation and Δψm alteration, assess the membrane integrity, and check the nuclei fate. For that, approximately 5 × 105 cells/well were seeded in a 6-well plate and incubated overnight. The attached cells were treated with a lethal dose concentration of BBR (24 h) or TSA (36 h) and stained with a respective dye for 30 min. The stained cells were washed using PBS, and cell images were captured using an inverted fluorescence microscope with a 20× objective (EVOS FLoid Cell Imaging Station, Thermo Fisher Scientific, Waltham, MA, U.S.A.). Reverse Transcription Polymerase Chain Reaction (RT-PCR). Total RNA was isolated from untreated or BBR (24 h) or TSA (36 h) treated A549 cells using TRIzol reagent (Invitrogen, Grand Island, NY, U.S.A.). RNA integrity was checked by agarose gel electrophoresis. A total of 2 μg of total RNA-based cDNA synthesis was achieved using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, U.S.A.). A total of 1 μL of cDNA pool was used for the RT-PCR reaction. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. A total of 10 μL of PCR product was analyzed by 1% agarose gel electrophoresis. The sequence of the primers is given in Supplementary Table 1 of the Supporting Information. Western Blotting. Dignam buffer was used to isolate the whole cell lysate from A549 control cells or treated with BBR (24 h) or TSA (36 h). The isolated protein concentration was attained using the Bradford method, and 50 μg of protein sample was loaded and separated by sodium dodecyl sulfate−polyacramide gel electrophoresis. The protein was transferred to a nitrocellulose membrane, blocked, and incubated overnight at 4 °C with specific primary antibodies (HDAC1−HDAC4 and HDAC6, H3, H4, p21, p53, Bax, Bcl2, Bcl-xl, caspases 3, 8, and 9, cytochrome c, matrix metalloproteinases 2 and 9, and β-actin). The membrane was washed and incubated with secondary antibodies [goat anti-rabbit immunoglobulin G (IgG)−alkaline phosphatase (ALP) antibody and goat anti-mouse IgG−horseradish peroxidase (HRP) antibody] for 1 h at 4 °C and developed using 3,3′-diaminebenzidine (Sigma, Mumbai, India). Cell Cycle Analysis. The attached 5 × 105 cells were treated with BBR (24 h) or TSA (36 h). After the treatment, cells were harvested and fixed with cold 75% ethanol overnight at 4 °C. Cells were then resuspended in PBS with 0.5% Triton X-100 (Sigma, Mumbai, India) and 0.1 mg/mL RNase (Sigma, Mumbai, India). After 1 h of incubation, propidium iodide (40 μL/mL, Sigma, Mumbai, India) was added and kept in a dark room for 45 min. Then, 10 000 event cells were analyzed by flow cytometry (FloJo or ModFit or FCS software), and the percentage of cells in each cell cycle phase was analyzed using CellQuest Pro software (FlowJo or ModFit, Ashland, OR, U.S.A.). Statistical Analysis. One-way analysis of variance (ANOVA), twoway ANOVA, and statistical significance were investigated by Graph Pad Prism 6.0 software. Post-hoc testing was achieved for comparisons using Tukey’s multiple comparisons. p < 0.05 was considered as statistically significant.
cancer treatment, which include romidepsin, vorinostat, belinostat, and panobinostat.5,6 Much effort has been made to the discovery of novel HDAC inhibitors from natural products after the FDA approval of romidepsin, which was derived from a nature source and used for T-cell lymphoma treatment. Therefore, there are intense interests in discovering specific small-molecule inhibitors from a natural inexhaustible source of novel chemotypes and pharmacopores against HDACs. Berberine (BBR) is an isoquinoline alkaloid found in Berberis aristata with variable pharmacological effects and a broad panel of target genes in various cancer cells. The BBR anticancer mechanism against a different variety of cancers was wellestablished. BBR inhibits cancer cell proliferation via positive regulation of reactive oxygen species and the apoptotic pathway.7,8 BBR was also reported to suppress cancer metastasis by stopping transferase activity.9 Thus far, at present, there is no credible sign of the BBR underlying molecular mechanism of targeting tumor. Hence, we assume that BBR has a characteristic of tumor targeting through specific inhibition of HDAC, which induce multiple effects on the cellular mechanism of cancer cells. On the basis of our hypothesis, we performed the in silco interaction of class I, II, and IV HDACs with BBR and found that BBR interacts with all of the classes of HDAC enzymes, with a particularly strong interaction with class I and II HDACs. In the present study, we intended to understand the mechanism of BBR regulating various classes of HDACs and, thereby, inducing antineoplastic activity in the A549 cell line.
■
MATERIALS AND METHODS
Reagents and Antibodies. BBR and trichostatin A (TSA) were obtained from Sigma-Aldrich (St. Louis, MO, U.S.A.), and fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), 10× phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO, cell culture grade), penicillin/streptomycin, 3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide (MTT), acridine orange (AO), ethidium bromide (EB), 2′,7′-dichlorofluorescein diacetate (DCFHDA), Rhodamine 123, and Hoechst 33258 were purchased from Hi Media Laboratories, Mumbai, India. Antibodies were bought from Santa Cruz Biotechnology (Dallas, TX, U.S.A.) and Cell Signaling Technology, Inc. (Danvers, MA, U.S.A.). Cell Cultures. The human A549 lung cancer cell line was obtained from the National Centre for Cell Science, Pune, India. The A549 cells were cultured in 10% FBS and 1% penicillin/streptomycin supplemented with DMEM in a 5% CO2 humidified atmosphere at 37 °C. Cytotoxicity Assay. Dose- and time-dependent BBR-induced cytotoxicity against A549 cells was confirmed using the MTT assay. For that, approximately 1 × 104 cells were added to each well of a 96well culture plate at 37 °C with 5% CO2 overnight. Then, cells were treated with various concentrations of BBR (20−200 μmol/mL) and incubated for 24, 36, and 48 h. After incubation, 20 μL of 5 mg/mL concentration of MTT was added to all of the wells and incubated for another 4 h. A total of 200 μL of DMSO was added, and the absorbance was measured at 570 nm using a microplate reader (BioRad, Hercules, CA, U.S.A.). HDAC Activity. The assay was achieved using a colorimetric HDAC activity assay kit (BioVision Research Products, Mountain View, CA, U.S.A.), conferring to the instructions of the manufacturer. Lethal dose concentrations of BBR-treated A549 cell lysate, control cell lysate, and TSA-treated cell lysate were used for quantifying the total HDAC. The entire samples in triplicate manner were added in the 96-well plates, and a final volume was made up to 85 μL using deionized water. Further, 10 μL of 10× HDAC assay buffer and 5 μL of the colorimetric substrate were added and incubated at 37 °C for 1 h. Consequently, 10 μL of lysine developer was added and incubated
■
RESULTS Effect of the Cytotoxicity Assay. We first determined the dose- and time-dependent cytotoxicity effects of BBR using the MTT assay. As presented in Figure 1, BBR suppressed the A549 cell growth in both dose- and time-dependent manners. The cell viability was reduced considerably at 24, 36, and 48 h treatments. The lower (20 μmol/mL) and higher (200 μmol/ mL) concentrations of BBR treatment showed 70 and 20% A549 cell viability at 24 h, respectively. The lethal dose B
DOI: 10.1021/acs.jafc.6b04453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
BBR Increases the Acetylation State of Histones H3 and H4. The genetic special effects of HDAC inhibitors are thought to be associated in part with modifications of the acetylation state of histone. Hyperacetylation of histones H3 and H4 was observed following BBR and TSA treatments. Interestingly, hyperacetylation protein expression was higher in BBR- than TSA-treated A549 cells (Figure 2F). BBR Induces Cell Cycle Progression in A549 Cells. To discover the inhibition of A549 cell growth arrest observation linked with cell cycle arrest, we treated the A549 cells with TSA or BBR. TSA and BBR induced cell cycle arrest significantly at sub-G0/G1, S, and G2/M phases in A549 cells (Supplementary Figure 2 of the Supporting Information). The result revealed that the proportions of cells at the sub-G0/G1 phase were increased in BBR treatment. Furthermore, the proportions of G2/M phase cells were significantly increased by BBR- and TSA-treated cells. Remarkably, a higher increase in the S phase and a nominal increase in the S phase of cells were found upon BBR and TSA treatments (Supplementary Figure 3 of the Supporting Information). BBR Induces ROS Generation, Δψ m Alteration, Membrane Loss, and Nuclear Fragmentation. The BBR apoptosis-mediated antineoplastic activity against A549 cells was primarily confirmed using different dye-based fluorescence microscopes. As displayed in Figure 3, BBR treatment generates the ROS production in the A549 cells. A substantial increase in the dichloroflurescence signal was noticed in the BBR- and TSA-treated cells compared to that in the control cells. The Rhodamine 123 staining experiments showed disruption of Δψm in the BBR- and TSA-treated cells. The decreased fluorescence intensity after the treatments reflects the collapse of Δψm in A549 cells compared to the control cells (Figure 3). The AO/EB fluorescent staining can be used to recognize the cell membrane alterations connected with the apoptotic process. The BBR treatment induced apoptotic characteristic morphological changes in the A549 cells. BBR treatment showed clear early- and late-stage apoptotic cells, including compressed and fragmented nuclei with yellow-green and orange colors. The necrotic cell nuclei were noticed in red color (Figure 3). Hoechst 33258 is a nuclear-specific stain, which is detected as blue nuclei under a fluorescence microscope. The BBR-treated cells exposed the clear nuclear fragmentation compared to the control cells (Figure 3). BBR Inhibits Tumor Necrosis Factor α (TNF-α)Mediated Cyclooxygenase-2 (COX-2) Expression. Inflammatory cytokines can contribute to the high level of the COX-2 expression in inflamed cells and tissues. We investigated the effect of BBR on TNF-α and its target gene COX-2 mRNA expression in A549 cells. An exposure of cells to BBR diminished the expressions of COX-2 and TNF-α mRNA genes (Supplementary Figure 3 of the Supporting Information). These results explained that BBR possesses an inhibitory effect on the COX-2 activation pathway. BBR Triggers the Activation of Apoptosis-Regulating Proteins. Because p53 and p21 play a crucial role in apoptosis, we inspected the effect of BBR on p53 and p21 mRNA and protein expressions. The alterations in p53 and p21 mRNA expression levels in response to BBR and TSA were observed (Figure 4A). The effects are concomitant with p53 and p21 protein expressions (Figure 4B). Moreover, we also examined the constitutive protein level of the Bcl-2/Bax family after the treatment with BBR. Western blot analysis revealed a substantial decrease in anti-apoptotic protein (Bcl-xl and Bcl-
Figure 1. Dose- and time-dependent cytotoxic activities of BBR in A549 cells: (A) bar chart and (B) line chart.
concentration (IC50) of BBR against A549 cells was 100 μmol/ mL at 24 h (Figure 1). HDAC Activity Assay. After the successful detection of the lethal dose concentration, we were concerned with checking the BBR role in regulation of HDACs. For that, we measured the total HDAC activity using the lethal dose concentrations of BBR (100 μmol/mL) and TSA (9 μmol/mL) treated A549 cell lysate. As exposed in Supplementary Figure 1 of the Supporting Information, colorimetric assay analysis of the total HDAC activity was significantly decreased in BBR and TSA treatment. The HDAC activity of BBR-treated cells was significantly decreased in comparison to standard HDAC inhibitor (TSA) treated and control A549 cells. Effect of BBR on Regulating Various Classes of HDACs. The colorimetric total HDAC assay result gave the sign that BBR significantly arrested the HDAC activity and could be a strong HDAC inhibitor. Furthermore, to prove its activity, we checked different classes of HDAC mRNA and protein expressions after the treatment with BBR. The result showed that BBR-treated cells reduced the mRNA expression of all classes of zinc-dependent HDACs (class I, II-a, II-b, and IV). However, the subtypes of classes I and II were strongly reduced by BBR compared to the class IV subtypes of HDACs (panels A−D of Figure 2). The results suggest that strong inhibition of class I and II HDACs and moderate inhibition of class IV HDACs by BBR might be the possible mechanism for its antineoplastic activity. The results were intensely supported by the protein expression of class I and II HDACs (Figure 2E). The BBR-downregulated HDAC mRNA and protein expressions were comparable to the TSA treatment. C
DOI: 10.1021/acs.jafc.6b04453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 2. Role of BBR on regulating various classes of HDACs in A549 cells: (A) class I (1−3 and 8), (B) class II-a (4, 5, 7, and 9), (C) class II-b (6 and 10), and (D) class IV (11) HDAC mRNA expression by RT-PCR. GAPDH was used as an internal control. (E) Western blotting analysis of HDAC1−4 and HDAC6 protein expressions. (F) Western blotting analysis of histone H3 and H4 protein expressions. β-Actin was used as an internal control (∗, p < 0.05; ∗∗, p < 0.01).
■
DISCUSSION Epigenetic is a principal control agent for oncogenes and tumor suppressor genes during cancer development. The main epigenetic modifications in humans are DNA methylation and post-translational histone modifications (acetylation and methylation). However, among the histone modifications, acetylation shows a remarkable role in chromatin remodeling induced by HATs and HDACs to regulate gene transcription. A recent report explained that HDACs can regulate a wide number of gene expressions by direct interaction with transcription factors, such as Stat3, p53, and retinoblastoma proteins.10,11 Furthermore, HDACs also control non-histone proteins, which regulate cellular homeostasis, such as differentiation, cell cycle progression, and apoptosis.12 Aberrant recruitment of HDACs has been related to the development of
2) and a considerable increase in the expression of proapoptotic protein Bax (Figure 5). Furthermore, the substantial release of cytochrome c (Figure 6B) mediated activation of caspases 8, 9, and 3 (panels A and B of Figure 6) was observed in the BBR-treated cells compared to TSA-treated and control A549 cells. BBR Induces Downregulation of MMP-2 and MMP-9. We determined the mRNA and protein expressions of MMP-2 and MMP-9 in BBR-treated cells. The influence of BBR on the expression of mRNA was well-understood with downregulation of MMP-2 and MMP-9 genes compared to control cells (Figure 7A). The BBR-induced changes in the transcript levels of MMP-2 and MMP-9 coincide well with the protein results, showing the regulatory effect of BBR (Figure 7B). D
DOI: 10.1021/acs.jafc.6b04453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 3. Assessment of ROS, Δψm, membrane integrity, and nuclear fate of A549 cells after the treatment with BBR (scale bar, 80 μm; orange arrows, intracellular ROS generation; blue arrors, Δψm alteration; green arrows, live cells, red arrows, apoptotic cells, purple arrows, necrotic cells; and cyan arrows, nuclear fragmentation).
Figure 4. Effect of BBR on tumor suppressor gene mRNA and protein expressions in A549 cells: (A) mRNA expression of p21 and p53 analyzed by RT-PCR and (B) protein expression of p21 and p53 analyzed by western blotting. β-Actin was used as an internal control (∗∗, p < 0.01) (∗, P < 0.05).
Figure 5. Effect of BBR on pro-apoptotic (Bax) and anti-apoptotic (Bcl-2 and Bcl-xl) protein expressions in A549 cells was analyzed by western blotting (∗, p < 0.05; ∗∗, p < 0.01).
different varieties of cancers. Hence, HDACs are among the most promising targets for cancer treatment, which inspired researchers in the development of HDAC inhibitors. Natural compounds possess a lot of bioactive ingredients, which actively regulate tumor genesis by interacting with various molecular targets. These bioactive components have the ability to
interfere with various epigenetic targets. Bioactive ingredients, such as curcumin, genistein, tea polyphenols, resveratrol, and sulforaphane, have been reported to alter the DNA methylation and histone modifications in cancer cells.13 Recently, the FDA approved a nautral bacterium-based derived romidepsin compound for the treatment of cutaneous and peripheral TE
DOI: 10.1021/acs.jafc.6b04453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 6. Effect of BBR on the regulation of apoptosis mRNA and protein expressions in A549 cells: (A) caspases 3 and 8 mRNA expressions analyzed using RT-PCR and (B) cytochrome c and caspases 3, 8, and 9 pre-cleaved and cleaved protein expressions analyzed by western blotting (∗∗, p < 0.01) (∗, P < 0.05).
Figure 7. Effect of BBR on the regulation of matrix metalloproteinase (MMP-2 and MMP-9) expression in A549 cells: (A) MMP-2 and MMP-9 mRNA expressions by RT-PCR and (B) MMP-2 and MMP-9 protein expressions by western blotting. β-Actin was used as an internal control (∗∗, p < 0.01).
cell lymphoma in patients.14 This holds considerable promise on bioactive compounds as a rich source for the discovery of the lead structure against molecular targets involved in tumorigenesis. BBR is a naturally occurring isoquinoline alkaloid derived from medicinal herbs, such as B. aristata, Berberis vulgaris
(barberry), Hydrastis canadensis, and Berberis aquifolium. The potential effectiveness of BBR against various cancer cells through different forms of molecular targets has been reported in numerous studies.15 BBR diminishes the side population cells and reversed drug resistance by inhibiting ABCG2 protein expression in breast cancer cells.16,17 BBR can also reverse the F
DOI: 10.1021/acs.jafc.6b04453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry drug resistance by targeting cancer stem cells.18 A recent study reports that BBR suppresses proliferation and migration of cancer cells by binding to vasodilator-stimulated phosphorotein in mouse xenograft models.19 BBR actively engaged in modulation and regulation of mitogen-activated protein kinase (MAPK) signaling, RhoA signaling, and HIF-1α in cancer cells.20−22 Apart from these, there have been numerous studies carried out to illustrate the potential impact of BBR in cancer. Plenty of reports are available on BBR-induced anticancer activity, but the fundamental path of BBR-induced cancer cell death is not fully understood. In the present study, we establish for the first time that BBR downregulates HDACs in the A549 cell line. Initially, we performed the molecular docking studies by using GLIDE (Schrödinger). The active site was predicted using Sitemap (Schrödinger). The ligands of BBR showed high binding affinity against the HDAC family (PDB IDs 3C5K, 3C10, 3MAX, 3SFF, 4A69, 4BKX, and 2VQM). They exactly fit into the active site region, and the ligands formed more hydrogenbond interactions (Supplementary Figure 4 and 5 of the Supporting Information). The recent study predicted the potential molecular target of BBR by a gene expression signature-based approach of using the Connectivity Map (CMap) database.23 They predicted that BBR may inhibit protein synthesis, HDAC, and AKT/mammalian targets of rapamycin pathways, which were very well-correlated with our in silico prediction, exhibiting BBR having a high binding affinity with the HDAC family, suggesting the potential role of BBR in epigenetic modulation. We found that BBR inhibits the growth of A549 cell viability, as determined by the MTT assay. Consistent with our prediction, we opted to ensure the HDAC activity and histone acetylation status in A549 cells. We show that BBR arrested total HDAC activity in A549 cancer cells, which was accompanied by histone hyperacetylation, suggesting that inhibitory modulation of HDAC classes shown by this class of compounds always parallels changes in H3 and H4 acetylation.24 This phenomenon was further confirmed by western blotting, speculating the altered expression of class I HDACs upon BBR treatment. Western blotting revealed a concomitant increase in acetylated histones H3 and H4. A reduced expression of histone H4 was detected in 80% carcinomas and 39% adenomas, which was well-associated with an advanced tumor stage.25 BBR indeed inhibited HDAC activity and induced histone acetylation in A549 cells. Existing data all together provide the first evidence that HDAC inhibition by BBR accomplishes epigenetic alterations by modulating HDAC activity and hyperacetylates histones in cancer cells. It is clear that BBR regulates histone hyperacetylation gene expression, which might be the key factor of its antineoplastic action. We further extended our studies to understand the impact of epigenetic modulation by BBR on cell cycle and apoptosis regulating proteins. Histone acetylation is regulated in the S phase, and its interruption activates cell cycle arrest within the G2/M phase. The G2/M phase is related to HDACinhibitor-mediated hyperacetylation of the centromere, permitting the release of heterochromatin protein, resulting in atypical chromosomal segregation and nuclear damage.26 Therefore, it is possible that deregulated histone acetylation during the S phase associated with sub-G0/G1 cell cycle arrest may be necessary for BBR-induced cell death. The BBR treatment upregulated ROS generation, altered Δψm, and activated
apoptosis-mediated cell death. Moreover, the clear sign of DNA fragmentation was noticed in the BBR-treated cells. The results were well-correlated with our previous study, where we proved that plant-leaf-extract-mediated copper oxide nanoparticles activate ROS generation and tempted Δψm-alterationmediated apoptosis cell death in A549 cells.27 In addition, a recent study demonstrates that excessive deacetylation of critical regulatory proteins in the COX-2 activation pathway is involved in carcinogenesis.28 In line with the above results, specifically our data suggest that BBR suppresses COX-2 expression, through the arrest of HDACs in lung cancer cells. Bcl-2 is an oncogene-derived protein, which works as a negative control in the cellular suicide pathway. Bax is a Bcl-2 homologous protein, which promotes cell death by competing with Bcl-2. If a Bcl-2/Bax heterodimer is formed, it promotes a survival signal for the cells, unless Bax/Bax homodimer formation acts as an apoptosis inducer. Bcl2 and Bax are transcriptional targets for p53, which induces apoptosis. Herein, BBR induces Bax activation in A549 cells, which disturbs the Δψm, thereby allowing for the p53-pathway-mediated release of cytochrome c to cytosol. This event leads to the subsequent activation of caspase-signaling pathway results in apoptosis. Additionally, there is considerable evidence that MMP-2 and MMP-9 are associated with tumor invasion and metastasis. Increased histone acetylation by HDAC inhibitors in the promoter region is the suppressor of invasion and metastasis, including metalloproteinases in carcinogenesis.29 The downregulation of HDAC blocked cancer cell invasion and migration through MMP.30 In this study, HDAC downregulation by BBR resulted in a decrease in MMP-2 and MMP-9 mRNA and protein levels. In conclusion, the present study demonstrated that BBR possesses the ability to enforce epigenetic modifications by downregulation of HDAC enzymes accompanied by histone hyperacetylation. This deregulated histone acetylation is associated with sub-G0/G1 cell cycle arrest and preventing deacetylation of regulatory proteins in COX-2 activation. It is conceivable that BBR activates the Bcl-2/Bax family, allowing for the release of cytochrome c, eventually leading to the activation of caspase-mediated apoptosis in A549 cells. This study also demonstrated that BBR suppresses the invasion and metastasis by hyperacetylation of metalloproteinases, namely, MMP-2 and MMP-9. Altogether, we provide mechanistic evidence that BBR-induced apoptosis in A549 cells is mediated through HDAC inhibition and activation of hyperacetylation in a lung cancer cell line.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b04453. HDAC activity of BBR in A549 cells (Supplementary Figure 1), effect of BBR on cell cycle analysis in A549 cells (Supplementary Figure 2), effect of BBR on TNF-α and COX-2 mRNA expressions in A549 cells, analyzed by RT-PCR (Supplementary Figure 3), in silico analysis of BBR interaction with class I (1, 2, 3, and 8) and class II (4, 7, and 6) HDACs (Supplementary Figure 4), binding modes of BBR with class I (1, 2, 3 and 8) and class II (4, 7 and 6) HDACs (Supplementary Figure 5), and sequence of the primers used for RT-PCR (Supplementary Table 1) (PDF) G
DOI: 10.1021/acs.jafc.6b04453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
■
human cholangiocarcinoma QBC939 cells. J. Pharmacol. Sci. 2012, 119, 341−348. (9) Ho, Y. T.; Yang, J. S.; Li, T. C.; Lin, J. J.; Lin, J. G.; Lai, K. C.; Ma, C. Y.; Wood, W. G.; Chung, J. G. Berberine suppresses in vitro migration and invasion of human SCC-4 tongue squamous cancer cells through the inhibitions of FAK, IKK, NF-κB, u-PA and MMP-2 and -9. Cancer Lett. 2009, 279, 155−162. (10) Lin, H. Y.; Chen, C. S.; Lin, S. P.; Weng, J. R.; Chen, C. S. Targeting histone deacetylase in cancer therapy. Med. Res. Rev. 2006, 26, 397−413. (11) Ropero, S.; Esteller, M. The role of histone deacetylases (HDACs) in human cancer. Mol. Oncol. 2007, 1, 19−25. (12) Minucci, S.; Pelicci, P. G. Histone deacetylase inhibitors and the promise of epigenetic (and more) treatments for cancer. Nat. Rev. Cancer 2006, 6, 38−51. (13) Meeran, S. M.; Ahmed, A.; Tollefsbol, T. O. Epigenetic targets of bioactive dietarycomponents for cancer prevention and therapy. Clin. Epigenet. 2010, 1, 101−116. (14) Petrich, A.; Nabhan, C. Use of class I histone deacetylase inhibitor romidepsin incombination regimens. Leuk. Lymphoma 2016, 57, 1755−1765. (15) Tang, J.; Feng, Y.; Tsao, S.; Wang, N.; Curtain, R.; Wang, Y. Berberine and Coptidis rhizoma as novel antineoplastic agents: A review of traditional use and biomedical investigations. J. Ethnopharmacol. 2009, 126, 5−17. (16) Kim, J.; Ko, E.; Han, W.; Shin, I.; Park, S.; Noh, D.-Y. Berberine diminishes the side population and ABCG2 transporter expression in MCF-7 breast cancer cells. Planta Med. 2008, 74, 1693−1700. (17) Park, S. H.; Sung, J. H.; Chung, N. Berberine diminishes side population and down regulates stem cell-associated genes in the pancreatic cancer cell lines PANC-1 and MIA sssPaCa-2. Mol. Cell. Biochem. 2014, 394, 209−215. (18) Ma, X.; Zhou, J.; Zhang, C.-X.; Li, X.-Y.; Li, N.; Ju, R.-J.; Shi, J.F.; Sun, M.-G.; Zhao, W.-Y.; Mu, L.-M.; Yan, Y.; Lu, W.-L. Modulation of drug-resistant membrane and apoptosis proteins of breast cancer stem cells by targeting berberine liposomes. Biomaterials 2013, 34, 4452−4465. (19) Su, K.; Hu, P.; Wang, X.; Kuang, C.; Xiang, Q.; Yang, F.; Xiang, J.; Zhu, S.; Wei, L.; Zhang, J. Tumor suppressor berberine binds VASP to inhibit cell migration in basal-like breast cancer. Oncotarget 2014, DOI: 10.18632/oncotarget.9968. (20) Li, H.-L; Wu, H.; Zhang, B.-B; Shi, H.-L; Wu, X.-J. MAPK pathways are involved in the inhibitory effect of berberine hydrochloride on gastric cancer MGC 803 cell proliferation and IL-8 secretion in vitro and in vivo. Mol. Med. Rep. 2016, 14, 1430−1438. (21) Tsang, C. M.; Lau, E. P.; Di, K.; Cheung, P. Y.; Hau, P. M.; Ching, Y. P.; Wong, Y. C.; Cheung, A. L.; Wan, T. S.; Tong, Y.; Tsao, S. W.; Feng, Y. Berberine inhibits Rho GTPases and cell migration at low doses but induces G2 arrest and apoptosis at high doses in human cancer cells. Int. J. Mol. Med. 2009, 24, 131−138. (22) Lin, S.; Tsai, S. C.; Lee, C. C.; Wang, B. W.; Liou, J. Y.; Shyu, K. G. Berberine inhibits HIF-1α expression via enhanced proteolysis. Mol. Pharmacol. 2004, 66, 612−619. (23) Lee, K. H.; Lo, H. L.; Tang, W. C.; Hsiao, H. H.; Yang, P. M. A gene expression signature based approach reveals the mechanisms of action of the Chinese herbal medicine berberine. Sci. Rep. 2014, 4, 6394. (24) Gurvich, N.; Tsygankova, O. M.; Meinkoth, J. L.; Klein, P. S. Histone deacetylase is a target of valproic acid-mediated cellular differentiation. Cancer Res. 2004, 64, 1079−1086. (25) Myzak, M. C.; Dashwood, W. M.; Orner, G. A.; Ho, E.; Dashwood, R. H. Sulforaphane inhibits histone deacetylase in vivo and suppresses tumorigenesis in Apcmin mice. FASEB J. 2006, 20, 506− 508. (26) Taddei, A.; Maison, C.; Roche, D.; Almouzni, G. Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases. Nat. Cell Biol. 2001, 3, 114−120.
AUTHOR INFORMATION
Corresponding Author
*Telephone: +91-431-2407071. E-mail: ravikumarbdu@gmail. com. ORCID
Vilwanathan Ravikumar: 0000-0003-4197-9684 Author Contributions ‡
Chidambaram Anusha and Renu Sankar contributed equally to this study. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors thank the Department of Science and Technology (DST), Fund for Improvement of S&T Infrastructure in Universities and Higher Educational Institutions (FIST) for their infrastructure support to their department. Arunachalam Kalaiarasi is grateful to Dr. K. Jayaraman, Department of Educational Technology, Bharathidasan University, Tiruchirappalli, India, for the constant encouragement and financial support throughout her career. We thank Dr. C. Prahalathan, Dr. A. Antony Joseph Velanganni, Department of Biochemistry, Bharathidasan University, Tiruchirappalli, India, for their help with gel documentation and fluorescent microscopy studies. and Dr. K. Natarajaseenivasan, Department of Microbiology, Bharathidasan University, Tiruchirappalli, India for his help with gel documentation studies. The authors are grateful to Dr. S. Rajakumar, Department of Marine Biotechnology, and Dr. V. Rajesh kannan, Department of Microbiology, Bharathidasan University, Tiruchirappalli, India for their advice and support. Dr. Subbiah Rajasekaran acknowledges funding from Ramalingaswami fellowship (Award number: BT/RLF/Re-entry/ 36/2013), the Department of Biotechnology, Government of India and from the Department of Science and Technology, Government of India, award number YSS/2014/000125.
■
REFERENCES
(1) Villar-Garea, A.; Esteller, M. Histone deacetylase inhibitors: Understanding a new wave of anticancer agents. Int. J. Cancer 2004, 112, 171−178. (2) Geng, L.; Cuneo, K. C.; Fu, A.; Tu, T.; Atadja, P. W.; Hallahan, D. E. Histone deacetylase (HDAC) inhibitor LBH589 increases duration of γ-H2AX Foci and confines HDAC4 to the cytoplasm in irradiated non-small cell lung cancer. Cancer Res. 2006, 66, 11298− 11304. (3) Verdone, L.; Caserta, M.; Di Mauro, E. Role of histone acetylation in the control of gene expression. Biochem. Cell Biol. 2005, 83, 344−353. (4) Frank, C. L.; Manandhar, D.; Gordân, R.; Crawford, G. E. HDAC inhibitors cause site-specific chromatin remodeling at PU. 1-bound enhancers in K562 cells. Epigenet. Chromatin 2016, 9, 15. (5) Zahnow, C. A.; Topper, M.; Stone, M.; Murray-Stewart, T.; Li, H.; Baylin, S. B.; Casero, R. A. Inhibitors of DNA Methylation, Histone Deacetylation, an Histone Demethylation: A Perfect Combination for Cancer Therapy. Adv. Cancer Res. 2016, 130, 55− 111. (6) Liao, D. Profiling technologies for the identification and characterization of small molecule histone deacetylase inhibitors. Drug Discovery Today: Technol. 2015, 18, 24−28. (7) Liu, B.; Wang, G.; Yang, J.; Pan, X.; Yang, Z.; Zang, L. Berberine inhibits human hepatoma cell invasion without cytotoxicity in healthy hepatocytes. PLoS One 2011, 6, e21416. (8) He, W.; Wang, B.; Zhuang, Y.; Shao, D.; Sun, K.; Chen, J. Berberine inhibits growth and induces G1 arrest and apoptosis in H
DOI: 10.1021/acs.jafc.6b04453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry (27) Sankar, R.; Maheswari, R.; Karthik, S.; Shivashangari, K. S.; Ravikumar, V. Anticancer activity of Ficus religiosa engineered copper oxide nanoparticles. Mater. Sci. Eng., C 2014, 44, 234−239. (28) Tong, X.; Yin, L.; Giardina, C. Butyrate suppresses Cox-2 activation in colon cancer cells through HDAC inhibition. Biochem. Biophys. Res. Commun. 2004, 317, 463−471. (29) Wang, F.; Qi, Y.; Li, X.; He, W.; Fan, Q. X.; Zong, H. HDAC inhibitor trichostatin A suppresses esophageal squamous cell carcinoma metastasis through HADC2 reduced MMP-2/9. Clin. Invest. Med. 2013, 36, 87−94. (30) Liu, L. T.; Chang, H. C.; Chiang, L. C.; Hung, W. C. Histone deacetylase inhibitor up-regulates RECK to inhibit MMP-2 activation and cancer cell invasion. Cancer Res. 2003, 63, 3069−3072.
I
DOI: 10.1021/acs.jafc.6b04453 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
