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NF-κB p65 repression by the sesquiterpene lactone, Helenalin, contributes to the induction of autophagy cell death
© Lim et al.; licensee BioMed Central Ltd. 2012
Received: 20 January 2012
Accepted: 11 July 2012
Published: 11 July 2012
Numerous studies have demonstrated that autophagy plays a vital role in maintaining cellular homeostasis. Interestingly, several anticancer agents were found to exert their anticancer effects by triggering autophagy. Emerging data suggest that autophagy represents a novel mechanism that can be exploited for therapeutic benefit. Pharmacologically active natural compounds such as those from marine, terrestrial plants and animals represent a promising resource for novel anticancer drugs. There are several prominent examples from the past proving the success of natural products and derivatives exhibiting anticancer activity. Helenalin, a sesquiterpene lactone has been demonstrated to have potent anti-inflammatory and antitumor activity. Albeit previous studies demonstrating helenalin’s multi modal action on cellular proliferative and apoptosis, the mechanisms underlying its action are largely unexplained.
To deduce the mechanistic action of helenalin, cancer cells were treated with the drug at various concentrations and time intervals. Using western blot, FACS analysis, overexpression and knockdown studies, cellular signaling pathways were interrogated focusing on apoptosis and autophagy markers.
We show here that helenalin induces sub-G1 arrest, apoptosis, caspase cleavage and increases the levels of the autophagic markers. Suppression of caspase cleavage by the pan caspase inhibitor, Z-VAD-fmk, suppressed induction of LC3-B and Atg12 and reduced autophagic cell death, indicating caspase activity was essential for autophagic cell death induced by helenalin. Additionally, helenalin suppressed NF-κB p65 expression in a dose and time dependent manner. Exogenous overexpression of p65 was accompanied by reduced levels of cell death whereas siRNA mediated suppression led to augmented levels of caspase cleavage, autophagic cell death markers and increased cell death.
Taken together, these results show that helenalin mediated autophagic cell death entails inhibition of NF-κB p65, thus providing a promising approach for the treatment of cancers with aberrant activation of the NF-κB pathway.
The efforts of many researchers during the past dozen years to identify novel compounds with anticancer activity have pointed to plants and herbs used in herbal medicine. The rationale behind this approach is that herbal medicine looks back on a 5000-years tradition. Hence, it can be expected that many medicinal herbs and plants have been selected for pharmacological activity . Many studies have shown that herbal medicine is indeed a valuable resource for novel compounds with activity against tumors in vitro and in vivo[2–4]. Hence, the chances to find novel compounds with activity against tumor cells in natural product libraries are higher than in synthetic libraries.
In this regard, helenalin, a naturally occurring sesquiterpene lactone has generally been considered as a distinctly promising and potent antitumor compound. Helenalin has been shown to be a potent inhibitor of hTERT (human Telomerase Reverse Transcriptase) and telomerase in hematopoietic cancer cell , induces apoptosis in activated CD4+ T cells through the mitochondrial apoptosis pathway  and have been shown to selectively alkylate the p65 subunit of NF-κB .
In this report, we provide a mechanism by which NF-κB p65 plays a significant role in modulating autophagy induced cell death by the sesquiterpene lactone, helenalin. NF-κB p65 expression is down regulated upon helenalin treatment in a time and dose dependent manner. Down regulation of NF-κB p65 in turn induces caspase cleavage and autophagic genes Atg12 and LC3-B resulting in sub-G1 arrest and cell death. Exogenous expression of NF-κB p65 attenuates caspase cleavage and subsequently autophagy, demonstrating a mechanistic pathway of helenalin induced autophagic cell death. siRNA mediated transcriptional knockdown of NF-κB p65, Atg12 or LC3-B or inhibition of caspase cleavage using Z-VAD-fmk diminishes autophage cell death. In addition, helenalin induced apoptosis by activating the intrinsic apoptosis pathway. Taken together, we surmise that helenalin mediated apoptotic and autophagic cell death may provide a promising treatment strategy for cancers with aberrant activation of the NF-kB pathway.
Cell Culture and drug treatment
A2780 (human ovarian cancer cell line), RKO (colon carcinoma cancer cell line) and MCF-7 (breast adenocarcinoma cancer cell line) were obtained from ATCC (Manassas, VA). Cells were cultured in Dulbecco’s modified Eagle’s medium, supplemented with 10 % fetal bovine serum, 1 % penicillin-streptomycin (all from GIBCO® Invitrogen, Carlsbad, CA) in a humidified 5 % CO2 atm at 37 °C. Cells were treated with helenalin (Ambrosa-2,11(13)-dien-12-oic acid, 6-α,8-β-dihydroxy-4-oxo-, 12,8-lactone) purchased from EMD biosciences (Gibbstown, NJ). Dimethyl sulfoxide was used throughout the experiments as the vehicle control. At least three biological experiments were performed to verify observations.
Flow cytometry analysis
Cells were harvested after drug treatment and fixed with 70 % ethanol. Fixed cells were treated with RNase (100 μg/ml) and stained with propidium iodide (50 μg/ml). Subsequently, stained cells were analyzed for DNA content by flow cytometry using FACScalibur (Becton Dickinson, Franklin Lakes, NJ). Cell cycle fractions were quantifies using the CellQuest software (BD Biosciences, San Jose, CA). Further details can be found in . At least three biological experiments were performed to verify observations.
Cell Proliferation Assay
Inhibition of cell proliferation by helenalin was assessed using the MTT assay (Roche, Indianapolis, IN). Briefly, A2780, MCF-7 or RKO cells were plated in 96-well culture plates (5 × 104 cells/well) and treated the following day with helenalin or DMSO vehicle as described in the results section. Following helenalin treatment, cells were incubated with MTT labeling reagent for 4 h, solubilized in 10 % SDS, and the MTT metabolite formazan crystals were quantitated at 575 nm on a microplate reader (Tecan, Männedorf, Switzerland). All experiments were performed and verified using at least three biological replicates.
To determine the growth suppression effect of helenalin treatment, A2780 cells were treated with helenalin or DMSO vehicle for 24 h. After treatment, cells were replated in complete DMEM and allowed to grow for 14 days to form colonies that were then stained with crystal violet (Sigma) and quantified. All experiments were performed and verified using at least three biological replicates.
Direct fluorescence staining of apoptotic cells for flow cytometric analysis was performed with the Annexin V-FITC apoptosis detection kit (BD Pharmingen, San Jose, CA). After the indicated times, cells were harvested and stained according to the manufacturer’s protocol. Stained cells were analyzed in a flow cytometer. All experiments were performed and verified using at least three biological replicates.
Western blotting procedure was followed according to . Briefly, c ells were lysed in appropriate volume of lysis buffer (Sigma Aldrich, St. Louis, MO). 50 μg of protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membrane (Bio-Rad, Hercules, CA). The membranes were immunoblotted with primary antibodies purchased from Cell Signal Technology (Danvers, MA) or Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Blots were incubated with horseradish peroxide-conjugated goat anti-rabbit, goat anti mouse or rabbit anti-goat secondary antibodies purchased from Santa Cruz Biotechnology, Santa Cruz, CA. All experiments were performed and verified using at least three biological replicates.
A2780 cells were transfected with non-targeting control siRNA (siRNA Neg), siRNA Atg12, siRNA LC3-B or siRNA RelA p65 when cells reached 80 % confluency. After 24 h, cells were split 1:3, and treated with helenalin or DMSO the next day. Final siRNA concentration was 100nM and transfection was performed using Lipofectamine RNAimax (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Target sequences used for siRNA against Atg12, LC3-B and RelA p65 were 5′CUUAACAGAUGUGAUCUAU-3′, 5′-GUAAUUCCAGCAGUAAUUU-3′, 5′-CUCAAGAUCUGCCGAGUGA-3′ respectively. All experiments were performed and verified using at least three biological replicates.
A2780 cells were transfected with 2.0 ug of empty vector or NF-κB RelA p65 overexpressing vector (purchased from Origene Technologies, Rockville, MD; Cat # RC220780) using FuGENE 6 transfection reagent (Roche, Indianapolis, IN) following manufacturer’s instructions. All experiments were performed and verified using at least three biological replicates.
Acridine Orange staining for autophagy detection
Cell staining with Acridine orange (10 mg/ml in water, A8097, Sigma Chemical Co) was performed according to published procedures , adding at a final concentration of 1 mg/ml for a period of 15 min. Bafilomycin A1 (Sigma Chemical Co.) was dissolved in DMSO and added to the cells 45 min before addition of acridine orange. Photographs were obtained with a fluorescence microscope and percent of staining was determined by harvesting cells by trypsinization and measuring with a FACSCalibur from (Becton Dickinson) using CellQuest software. All experiments were performed and verified using at least three biological replicates.
Results and Discussion
Helenalin Inhibits Cell Proliferation and Clonogenic Survival in cancer cells
Induction of G1 Phase Arrest and cell death by Helenalin
Helenalin induces cell death via caspase cleavage and induction of autophagy
Inhibition of Atg12 and LC3-B expression reduces caspase cleavage and cell death induced by Helenalin
NF-κB p65 inhibition by Helenalin is essential for caspase cleavage and induction of autophagy
Helenalin induces autophagy cell death via suppression of NF-κB p65
Pharmacologically active natural compounds such as those from marine and terrestrial plants and animals represent a promising resource for novel anticancer drugs. There are several prominent examples from the past proving the success of natural products and derivatives exhibiting anticancer activity. This includes the Vinca alkaloids from Catharanthus roseus, the terpene paclitaxel from Taxus brevifolia, the DNA topoisomerase I inhibitor camptothecin from Camptotheca acuminata, and the semisynthetic derivatives etoposide and teniposide of the lignan podophyllotoxin from Podophyllum peltatum. Natural products alone or synthetics developed based on knowledge gained from natural products account for about 70 % of anticancer therapeutics approved between 1980s and 2002 .
Herbal medicine has been applied in the clinic for thousands of years in Asian countries such as China, Japan and Korea . However, because of its complicated chemical composition and lack of concrete evidence of its biological activity, herbal medicine is still not widely accepted by the Western medical community . Unlike modern drugs in the form of a single active compound, herbal medicine is usually prepared from aqueous extracts of a few herbs and contains hundreds or even thousands of different compounds . However, only a few compounds are responsible for the pharmacological effects . Furthermore, the bioactive components are generally present at low level. Some components are useless or even toxic. Thus, systematic characterization of active chemicals in herbal medicinal preparations and their mechanisms of action are important for providing the rationale for their efficacy and for transforming herbal medicine practices into evidence-based medicine.
Helenalin, an extracted component of Arnica Montana and Arnica chamissonis is a sesquiterpene lactone with potent anti-inflammatory and antitumor activity . The use of helenalin has been demonstrated to reduce the growth of Staphylococcus aureus and Plasmodium falciparum. In addition, previous studies have implicated helenalin to selectively inhibit the transcription factor NF-κB  and human telomerase activity , suggesting an underlying molecular mechanism for its antitumor activity.
Our ensuing findings derived from experiments performed in cancer cells treated with helenalin consistently resulted in an increase in cell death via apoptosis and autophagy. The increased sensitivity to cell death when exposed to helenalin was associated with increased levels of caspase cleavage. Indeed, when caspase cleavage was blocked using a specific inhibitor, cell death was considerably reduced. Given that several anticancer agents exert their effects by triggering autophagy, we postulated whether helenalin’s action in triggering cell death was through the activation of autophagy. Treatment with helenalin resulted in an increase in defined autophagy markers, which when transcriptionally silenced using siRNA resulted in decreased cell death. Interestingly, transcriptionally silencing Atg12 and LC3-B, both essential for induction of autophagy cell death also resulted in a decrease of caspase activity. This result suggests that caspase activation is dependent on the expression of Atg12 and LC3-B. These observations are in agreement with previous studies where a decrease in LC3-B levels was associated with reduced autophagy and cells treated with LC3-B or Beclin 1 siRNA inhibited caspase-3/8 activation . To further validate our findings, we performed Acridine Orange staining assays to measure acidic vesicular organelle (AVO) formation, a key indicator of autophagy initiation. AVO formation increased with increasing concentrations of helenalin and was suppressed with the use of bafilomycin A1, a highly potent and selective inhibitor of vacuolar H + −ATPases used in preventing the re-acidification of synaptic vesicles leading to the autophagy process [23–26].
We next examined whether helenalin’s mechanism of action was through the transcription factor NF-κB. Previous reports had revealed helenalin as a potent inhibitor of NF-κB  and that its binds with RelA disrupting its transcriptional activity . In addition, NF-κB is a key regulator of several biological processes, including proliferation, differentiation, apoptosis and autophagy [29, 30]. NF-κB has been demonstrated to play an essential role after heat shock treatment by modulating autophagy by a mechanism to increase cell survival, possibly through the elimination of irreversibly damaged proteins [31–33]. With this regard, we observe that upon helenalin treatment, the level of NF-κB p65 (RelA) was reduced. Reintroducing RelA exogenously via an over-expression construct we observed that caspase activation was reduced together with the levels of autophagy markers, resulting in decreased cell death. Conversely, transcriptionally silencing NF-κB p65 had the outcome of increasing caspase cleavage, autophagy markers and cell death. These results strongly advocate the reliance of NF-κB p65 for helenalin induced autophagy cell death. We speculate helenalin downregulates NF-κB p65 expression via ubiquitination-mediated degradation. Previous reports have shown that tumor necrosis factor-α (TNFα) polyubiquitinatates RelA at the lysine 195 residue which is critical for degradation of p65 . The precise mechanism of p65 degradation needs to be further investigated.
In summary, we have shown that helenalin induces cell death via a mechanism involving the repression of NF-κB p65 expression resulting in an increase of autophagy markers and caspase activation. This provokes the clinically relevant question as to helenalin’s use as a therapeutic intervention in patients with aberrant activation of NF-κB. Clinically, acute myeloid leukemia (AML) is an aggressive cancer with median survival rates of 2 to 3 months, and inhibition of NF-κB is considered one of the therapeutic strategies for treatment [30, 35–39]. Oncogenic addiction of activated NF-κB could be inhibited with the use of helenalin, and as such could favorably be used in a therapeutic setting to augment tumor sensitivity to conventional chemotherapeutic drugs. Further work is necessary before helenalin can be considered as a lead compound and a treatment strategy. Specificity, toxicology, pharmacokinetics and metabolism needs to be investigated and studied further before it is introduced into the market.
CBL and PYF are co-first authors.
This work was supported by the National Medical Research Council New Investigator Grant (NMRC/NIG/0036/2008), Singapore, to ZY.
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