Special Issue on "Natural Compounds as Inflammation Inhibitors"
- Research Paper
Anti-inflammatory, pro-apoptotic, and anti-proliferative effects of a methanolic neem (Azadirachta indica) leaf extract are mediated via modulation of the nuclear factor-κB pathway
Genes & Nutrition volume 6, pages 149–160 (2011)
Azadirachta indica (neem tree) is used in traditional Indian medicine for its pharmacological properties including cancer prevention and treatment. Here, we studied a neem extract’s anti-inflammatory potential via the nuclear factor-κB (NF-κB) signaling pathway, linked to cancer, inflammation, and apoptosis. Cultured human leukemia cells were treated with a methanolic neem leaf extract with or without tumor necrosis factor (TNF)-α stimulation. Inhibition of NF-κB activity was demonstrated by luciferase assay and electrophoretic mobility shift assay (EMSA). Inhibition of viability by neem extracts was assessed by luminescent assays. Western blot analysis allowed assessing the inhibitory effect of the neem extract on TNF-α-induced degradation of inhibitor of κB (IκB) and nuclear translocation of the NF-κB p50/p65 heterodimer. Inhibition of IκB kinase (IKK) activity was shown as well as the effect of neem extract on the induction of apoptotic cell death mechanisms by nuclear fragmentation analysis and flow cytometry analysis. In conclusion, our data provide evidence for a strong effect of the neem extract on pro-inflammatory cell signaling and apoptotic cell death mechanisms, contributing to a better understanding of the mechanisms triggered by Azadirachta indica.
The evergreen neem tree (Azadirachta indica) exhibits several activities useful for agricultural and medicinal applications known in India for thousands of years. The plant or its preparations are extensively reported to exert insecticidal, pesticidal, and agrochemical properties. Additionally, its constituents are applied in alternative (Ayurveda, Unani, Homeopathy) and modern medicine, e.g., for the treatment of diverse infectious, metabolic, or cancer diseases [11, 28] (for chemical structures, see Scheme 1).
In this context, azadirachtin 1, a tetranortriterpenoid extracted from neem seeds over 40 years ago , was shown to act as a potent phagorepellent and growth inhibitor of insects . Neem tree constituents exhibit various multi-targeted biological activities, such as antibacterial , hypoglycemic , anti-ulcer , antimalarial , hepatoprotective , spermicidal , anti-inflammatory [9, 44, 66], chemopreventive [15, 62], and chemotherapeutic [23, 24, 41] properties. Additionally, the nutritional potential of neem oil was evaluated by Rukimini .
Related to cancer, Arivazahagan and coworkers suggest that neem leaf extract may exert its chemopreventive effects by down-regulation of lipid peroxidation, simultaneously increasing the level of glutathione (GSH) and GSH-dependent enzymes . The strong chemopreventive activity of the extract could additionally be confirmed in vivo in rodents [22, 58]. Neem extract was able to induce apoptosis and to inhibit proliferation in prostate cancer . This anti-proliferative effect might partially be due to nimbolide, a triterpene extracted from neem flower that was highly effective in several cancer cell lines, e.g., U937, HL-60, THP1, B16, and BoWe [23, 48]. Moreover, the leaf extract stimulates the immune function by the activation of killer cells . Antioxidative activities were described for gallic acid 3, catechin 5, and epicatechin 6 extracted from the bark . The antioxidative effect is mainly relied on the total content of phenolic compounds (e.g., flavonoids such as quercetin 4) . Molecular mechanisms largely remain to be elucidated.
The NF-κB transcription factor plays an important role in cancer and related diseases . This transcription factor is localized in the cytosol and is blocked by inhibitor of κB (IκB). Upon activation by pro-inflammatory cytokines or viruses, phosphorylation of IκB by IκB kinase complex (IKK) is observed. After ubiquitinylation of IκB, the latter is degraded resulting in the translocation of NF-κB to the nucleus. Here, the activated factor regulates the transcription of multiple genes responsible for regulation of cell cycle, apoptosis, or cellular invasion [17, 27]. Moreover, inhibition of NF-κB-regulated genes potentiates apoptosis [3, 5]. This study gives first mechanistic insights into the inhibitory effect of a methanolic neem leaf extract (NLE) on the NF-κB signaling pathway and subsequent induction of apoptosis in human leukemia cancer cell lines.
Materials and methods
Materials and cell culture
Tumor necrosis factor-α (TNF-α) purchased from Sigma was dissolved in 10 μg/ml in PBS (1×) supplemented with 0.5% (w/v) BSA according to manufacturer’s instructions. Quercetin, gallic acid, catechin, and epicatechin were purchased from Sigma. Salannin and nimbin were purchased from Trifolio GmbH. K562 (human chronic myeloid leukemia in blast crisis) and Jurkat (human T-cell leukemia) and U937 (human histiocytic lymphoma) cells were obtained from (DSMZ) Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH and were maintained in RPMI medium (Bio-Whittaker) with 10% (v/v) fetal calf serum (FCS) (Bio-Whittaker) and 1% (v/v) antibiotic–antimycotic solution (Sigma) in an incubator (37°C, 5% CO2).
Healthy blood samples were kindly donated as buffy coats by the Red Cross (Luxembourg, Luxembourg). By applying diluted (1/3) blood onto a Ficoll layer followed by centrifugation (400g, 20 min), mononuclear cells were isolated and collected. The isolated peripheral blood mononuclear cells (PBMCs) were kept in culture at 37°C and 5% CO2 for 24 h before they were subjected to treatments.
Preparation of the neem leaf extract
Dried leaves of the neem tree were grinded in a mortar. The resulting green solid powder was dissolved in methanol, and the suspension was mixed in a shaker at 25°C for 24 h. Then, remaining powder particles were removed by filtration, and the solvent of the green methanolic solution was evaporated to dryness yielding a green crude extract (yield: 13.2% m/m of the starting material). This crude extract was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 80 mg/ml and stored at −20°.
An Agilent 1200 HPLC system with a DAD UV–VIS detector was used. The separation was performed on a reversed-phase column (ZORBAX C18 21.2 × 150 mm, 5 μm) eluting with methanol–water (0:100–100:0) at a flow rate of 1 ml/min and UV detection (280 nm). The active compound quercetin in methanolic leaf extract was identified by comparison with pure standard.
Measurement of NF-κB-inhibitory activity
The inhibitory activity against NF-κB was determined using a Dual-Glo™ Luciferase assay system from Promega. Electroporation of K562 cells was realized as described previously . Equal amounts (each 5 μg per pulse (2.5 × 106 cells)) of an NF-κB plasmid (Stratagene, p5xNF-κB containing five repeats of a consensus NF-κB site) and of a Renilla plasmid (phRG-TK, Promega) were added to each pulse. After transient transfection, cells were transferred to culture medium. After 24 h of incubation, transfected K562 cells were solubilized at a concentration of 1 * 106 cells per ml in RPMI 1640 medium containing 0.1% FCS and 1% antibiotic–antimycotic solution and incubated with different concentrations of the neem leaf extract solution. After 2 h of treatment, TNF-α was added (at 20 ng/ml). After 6 additional hours of incubation, expression of NF-κB was assessed according to the provider’s protocol by luminescence measurement using a Berthold Orion Luminometer (integration time 10 s).
The effect of neem leaf extract on cell viability was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay Kit from Promega. The reduction in the number of viable cells in percent was quantified in comparison with a vehicle-treated control according to the manufacturer’s instructions.
Electrophoretic mobility shift assay (EMSA)
K562 cells were preincubated with indicated concentrations of neem leaf extract solution. After 2 h of incubation, 20 ng/ml of TNF-α was added for additional 6 hours of incubation (37°C, 5% CO2). Then, nuclear proteins were extracted according to the method of Müller and coworkers  and stored at −80°C.
The nucleotide 5′-CGCTTGATGACTCAGCCGGAA-3′ (consensus NF-κB site) and the complementary sequence were used as probe. Ten micrograms of each nuclear protein extract was incubated for 20 min at 4°C with the 32P-labeled probe in a mixture of 15 μl containing 35 mM of spermidine, 4 μM of BSA, and 2.5 mM of poly-dIdC. In case of a super-shift experiment, the nuclear extract was first incubated with the antibody in the mixture for 15 min at 4°C, before the labeled probe was added, followed by an incubation period of 30 min at 4°C. Each reaction mixture was then loaded with 5 μl of loading buffer in a well of the gel, which was pre-run for at least 30 min. The electrophoresis was realized at 16 mA per gel for at least 2–3 h. The gel was then dried, and the imaging of the signals was performed using a Kodak BioMax XAR film.
Western blot analysis
After treatment of Jurkat cells with NLE (300 μg/ml) and TNF-α (20 ng/ml), both IκBα degradation and subsequent translocation of p50 or p65 to the nucleus were analyzed in cytosolic and nuclear protein extracts, respectively, by Western blot. In a second set of experiments, K562 cells were treated with different concentrations of NLE. Caspase activation and up-regulation of anti-apoptotic proteins were evaluated through the analysis of total protein extracts. For caspase activation, a positive control was realized by using heteronemin or cisplatin [55, 60]. Cytosolic and nuclear extracts or total protein extraction of the cell lines was performed, and protein concentration of these extracts was determined by Bradford assay and analyzed by Western blot as previously described [13, 18]. Antibodies were from Santa Cruz Biotechnology (p50, p65, IκB, lamin B, β-actin) Cell Signaling Technologies (Bid, Caspases -3, -7, -8, -9), BD Pharmingen (Bcl-XL, XIAP) and Calbiochem (α-tubulin).
IKK kinase activity
The K-LISA IKKβ Inhibitor Screening kit from Calbiochem was used for the measurement of the kinase activity. IKK-2 inhibitor IV ([5-(p-fluorophenyl)-2-ureido]thiophene-3-carboxamide) (Calbiochem) was used as a positive control. The assay was performed as indicated in the manufacture’s protocol. The absorbance at 450 nm (with a reference wave length at 590 nm) was read using a SpectraCount UV-spectrometer (Canberra-Packard).
Analysis of nuclear fragmentation
The percentage of apoptotic cells was determined through the fraction of apoptotic nuclei via fluorescence microscopy (Leica-DM IRB microscope, Lecuit, Luxemburg) after staining with the DNA-specific dye Hoechst 33342 (Sigma, Bornem, Belgium), as previously described . The fraction of cells with nuclear apoptotic morphology was counted (at least 300 cells in at least three independent fields), and the images were analyzed using the Image J software (http://rsb.info.nih.gov/ij/docs/index.html).
Flow cytometric analysis
K562 cells were treated with indicated concentrations of NLE for an incubation period of 8 h; then, the cells were assayed for phosphatidylserine exposure, by using the AnnexinV–FITC Apoptosis Detection Kit I® (Becton–Dickinson Biosciences, Erembodegem, Belgium) as written in the manufacturer’s instructions. Stained samples were analyzed by FACS (FACSCalibur, Becton–Dickinson, San José, CA, USA). The data were recorded via the CellQuest software (http://www.bdbiosciences.com/features/products) for further analysis.
Results and discussion
Neem leaf extract inhibited TNF-α-activated NF-κB pathway
NF-κB assays of the pure neem standards quercetin, gallic acid catechin, epicatechin, nimbin, and salannin showed that only the product quercetin exerted high NF-κB inhibition potential at a concentration of 100 μM (Fig. 1). HPLC analysis was done to identify quercetin in methanolic neem leaf fractions. HPLC chromatograms are shown in Fig. 2a. Our results reveal that NLE contains quercetin 4 (52 min) Fig. 2b. This result confirms previous observations as quercetin has been reported to inhibit NF-κB signaling in murine intestinal epithelial cells .
Neem leaf extract (NLE) acts an inhibitor of the TNF-α-triggered induction of NF-κB in our model (Fig. 3) as the activation of the transcription factor was inhibited for more than 80 percent at a dose of 240 μg/ml of the leaf extract. An IC50 of 220 ± 20 μg/ml (K562) or 250 ± 20 μg/ml (Jurkat) was calculated using three independent experiments.
Furthermore, binding of NF-κB to DNA was suppressed after TNF-α-triggered activation of the transcription factor when NLE was applied at concentrations between 160 and 320 μg/ml to the CML cell line K562, as shown by EMSA where we observe a decrease in the p50/p65-dimer binding to a consensus NF-κB binding site (Fig. 4a). Immunodepletion experiments proved the identity of the p50/p65 and p50/p50 lanes. (Fig. 4c). Similar results were obtained for neem-treated Jurkat cell lines (Fig. 5).
Over the last years, the use of plant extracts allowed the discovery of various bioactive compounds. Indeed, Moon et al.  observed that an aqueous extract of Benincasa hispida Cogniaux reduced NF-κB promoter activity in glucose-induced vascular inflammation of human umbilical vein endothelial cells. The extract of the tropical plant Knema laurina (Myristicaceae) was used for centuries for the treatment of digestive and inflammatory diseases and inhibited NF-κB-translocation in brain tissue after inflammatory damage . Lampronti et al.  evaluated the inhibitory effects of Bangladeshi medicinal plant extracts on interactions between selected transcription factors and target DNA sequences and discovered that extracts from Terminalia arjuna and Saraca asoka appeared to be most efficient in inhibiting NF-κB binding activities. Interestingly, a purified extract called CML-1 was generated from a mixture of 13 oriental herbs widely used for the treatment of inflammatory diseases in Asia (Achyranthis Radix, Angelicae Gigantis Radix, Cinnamomi Cortex Spissus, Eucommiae Cortex, Glycyrrhizae Radix, Hoelen, Lycii Fructus, Paeoniae Radix, Rehmanniae Radix Preparata and Atractylodis Rhizoma, Zingiberis Rhizoma, Zizyphi Semen, Acori Graminei Rhizoma). This complex mixture inhibits TNF-α-induced IκB kinase activation, subsequent degradation of IκBα, and nuclear translocation of NK-κB that may explain the ability of CML-1 to suppress inflammation . Finally, an extract from stem bark of Cinnamomum cassia Blume (Lauraceae) was discovered to exert an inhibitory effect on lipopolysaccharide (LPS)-induced NF-ΚB transcriptional activity. Following activity-guided fractionation, trans-cinnamaldehyde and 2-methoxycinnamaldehyde were identified as the NF-κB inhibitors from C cassia with IC50 values of 43 μM and 31 μM, respectively .
Several groups reported anticancer, anti-inflammatory, and antioxidant activities of neem tree extracts [20, 34, 54, 57] without providing in-depth analysis of the molecular mechanisms leading to NLE-induced inhibition of NF-κB activation. Accordingly, we compared Jurkat cells treated with TNF-α with or without pretreatment by NLE. Results show in control cells that IκBα is degraded after 15 min of cytokine treatment, followed by the translocation of p50 and p65 to the nucleus (Fig. 6 left panel). However, pretreatment of Jurkat cells with 300 μg/ml of NLE resulted in a complete abrogation of IκBα degradation and a significant reduction in nuclear translocation of p50/p65 (Fig. 6 right panel) confirming previous results.
Furthermore, IKKβ assay showed that NLE significantly inhibited the kinase activity of IKKβ at a concentration from 160 to 240 μg/ml. Moreover, the inhibitory activity of NLE is comparable to the prototypical IKK inhibitor ([5-(p-fluorophenyl)-2-ureido]thiophene-3-carboxamide) provided as a positive control by the supplier (Fig. 7) again confirming previous results.
A number of extracts were described in the literature to efficiently inhibit IKK activity. No results exist so far for neem extracts, but the aqueous extract of Dichroa febrifuga, a medicinal plant well known in China and Korea, inhibits interleukin-1β and interleukin-6 production in LPS-stimulated mouse peritoneal macrophages. These effects are mediated by the inhibition of the activity of IKK/IκB/NF-κB signaling cascade . Moreover, water-soluble extracts of Panellus serotinus (Mukitake) powder showed an inhibitory effect on IKKβ, whose activation is required for NF-κB-mediated inflammatory response . Finally, medicinal value of Marasmius oreades was also described as a source for bioactive substances able to induce a direct blockage of NF-κB activation at the IKK level .
Neem leaf extract induced apoptosis in leukemia cells
Accordingly, we evaluated the percentage of induction and nature of cell death and found that NLE induced cell death in K562 cells with a LC50 of 300 ± 20 μg/ml after 8 h of incubation. At a concentration of 400 μg/ml, NLE induced 75 ± 5% of cell death to the K562 cell line (Fig. 8a). Interestingly, at concentrations that inhibit NF-kB or induce cell death in cancer cells, healthy PBMCs were not significantly affected by the treatment.
In a next step, we performed a long-term viability assay over 3 days on three different leukemias (K562, Jurkat, and U937) and one prostate cancer (PC-3) cell lines (Fig. 9). K562 showed to be the most resistant cell line, whereas U937 was the most sensitive one (Table 1). At a concentration of 320 μg/ml of neem leaf extract, almost all PC-3 cells and K562 cells were killed, in contrast to Jurkat and U937 cells, for which a concentration of 200 μg/ml was sufficient to reach the same target. These concentrations seem to be very high in contrast to known cancer drugs such as taxol ; however, neem leaves are known to be nontoxic to humans, and the pure compound of this extract responsible for this activity will have a lower IC50 (in μM) than that of the crude extract.
In order to determine the precise modality of cell death and mechanisms, we proceeded with nuclear morphology analysis of cells, stained with Hoechst, via fluorescence microscopy. Treated cells clearly displayed apoptotic fragmentation after 8 h of treatment with 240 μg/ml. Figure 10 shows the kinetic evolution of apoptotic cell death induced by NLE and confirmed the data of the viability assay. The flow cytometry analysis clearly indicated that NLE induced a slight level of apoptotic cell death to the K562 cell line with a maximum of 18 percent at a concentration of 240 μg/ml (Fig. 11) with a high number of cells in late apoptosis. Next, we evaluated whether NLE activated a caspase-dependent apoptosis by using Western blot analysis (Fig. 12). Caspase-3, -7, -8, and -9 activation could be observed after 6 h of treatment at a concentration of 200 μg/ml; the sesterterpene heteronemin and the known cancer drug cisplatin were used as positive control [55, 60]. Additionally, the level of the anti-apoptotic proteins Bcl-xL, Bid, and XIAP was down-regulated after the identical treatment. These results proved that NLE progressively induced apoptotic cell death most likely via the mitochondrial cell death pathway.
Our results corroborate with published data of an ethanolic neem extract able to induce apoptosis in cancer cells from rodents and men [29, 57]. Interestingly, to the best knowledge of the authors, no effect of neem extract on XIAP was reported in literature; recently, the inhibition of XIAP emerged as an important cancer drug target [14, 19, 52]. Similar pro-apoptotic effects were described for extracts from various plants including Physalis minima L. , Cassia tora Linn (Leguminacea) , Serenoa repens , Uncaria rhynchophylla , Hibiscus , Smilax glabra Roxb. , Arnebia nobilis , Cimicifuga foetida , Brassica oleracea , Calendula officinalis , Pereskia bleo (Kunth) DC. (Cactaceae) , and Catha edulis (Khat) .
Altogether, we show here that NLE acted on different levels of the NF-κB pathway and that it induced apoptosis in leukemia cancer cell. This is, to the best of our knowledge, the first report of these TNF-α-induced, anti-inflammatory, and anti-proliferative treatment properties of NLE in human leukemia cancer cell lines. Since the observed effects are beneficial, the extract and its constituents including quercetin might be promising candidates for future studies.
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This work was inspired by Sister Marie-Odile (Evelyne Rehlinger) of the Carmelite Third Order. The authors thank E Henry and J Ghelfi for technical support and Mr Pierre Lutgen for interesting discussion. This work was supported in part by the “Recherche Cancer et Sang” foundation, Télévie and the “Recherches Scientifiques Luxembourg (RSL)” association. M.S., C.C., C.S., and S.R. are supported by a Télévie grant. The authors thank the “Action Lions Vaincre le Cancer” and “Een Häerz fir kriibskrank Kanner” asbl for additional support.
Marc Schumacher and Claudia Cerella contributed equally to this work.
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Schumacher, M., Cerella, C., Reuter, S. et al. Anti-inflammatory, pro-apoptotic, and anti-proliferative effects of a methanolic neem (Azadirachta indica) leaf extract are mediated via modulation of the nuclear factor-κB pathway. Genes Nutr 6, 149–160 (2011). https://doi.org/10.1007/s12263-010-0194-6
- Azadirachta indica
- Methanolic neem leaf extract
- Nuclear factor-kappa B