Cordycepin Blocks Lung Injury-Associated Inflammation and Promotes BRCA1-Deficient Breast Cancer Cell Killing by Effectively Inhibiting PARP
Cordycepin has been shown to interfere with a myriad of molecular processes from RNA elongation to kinase activity, and prevents numerous inflammatory processes in animal models. Here we show in a mouse model of LPS-induced acute lung injury that cordycepin prevents airway neutrophilia via a robust blockade of expression of several inflammatory genes, including the adhesion molecule ICAM-1 and VCAM-1, the cytokine/chemokine MCP-1, MIP-1α, MIP-2 and KC, and the chemokine receptor CXCR2. Such a blockade appears to be related to a severe reduction in TNF-α expression. Interestingly, in an in vitro system of A549 epithelial cell inflammation, cordycepin effectively blocked LPS-induced, but not TNF-α-induced, VCAM-1 expression. Such effects correlated with a marked reduction in p65-NF-κB activation as assessed by its phosphorylation at serine-536 but without an apparent effect on its nuclear translocation. The effects of cordycepin on the expression of VCAM-1 and ICAM-1, and of NF-κB activation and nuclear translocation upon TNF-α stimulation resembled the effects achieved upon poly(ADP-ribose) polymerase (PARP) inhibition, suggesting that cordycepin may function as a PARP inhibitor. Indeed, cordycepin blocked H2O2-induced PARP activation in A549 cells. In a cell-free system, cordycepin inhibited PARP-1 activity at nanomolar concentrations. Similar to PARP inhibitors, cordycepin significantly induced killing of breast cancer susceptibility gene (BRCA1)-deficient MCF-7 cells, supporting its therapeutic use for the treatment of BRCA-deficient breast cancers. With added antiinflammatory characteristics, therapies that include cordycepin may prevent potential inflammation triggered by traditional chemotherapeutic drugs. Cordycepin, to the best of our knowledge, represents the first natural product possessing PARP inhibitory traits.
Airway inflammation is a complex process that can be mediated by a variety of stimuli from bacterial infection to allergen exposure. Interference with the expression of adhesion and chemotactic molecules, such as intracellular adhesion molecule (ICAM)-1 and vascular adhesion molecule (VCAM)-1, in response to inflammatory factors, such as lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α), prevents inflammatory cell adhesion and transendothelial migration of leukocytes. A number of drugs have been suggested to block inflammatory processes and they may exhibit therapeutic potential by impairing the establishment or progression of inflammatory diseases. The inflammatory response during acute lung injury (ALI) that is triggered, for instance, by LPS, is characterized by the massive recruitment of neutrophils (1) and the production of numerous cytokines and chemokines, including monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, interleukin (IL)-8, and MIP-2, as well as the expression of adhesion molecules and other inflammatory factors (2). The expression of these genes is believed to be associated with the production of TNF-α, as interference with the function or expression of TNF-α compromises the expression of the aforementioned genes (3,4) during LPS-induced ALI.
Our laboratory has investigated the role of poly(ADP-ribose) polymerase-1 (PARP-1) in tissue injury and its implications in several pathological conditions, including asthma, ALI, and atherosclerosis (5, 6, 7, 8, 9, 10, 11, 12). PARP-1 is emerging as a viable therapeutic target for the treatment of inflammatory disease (13, 14, 15). In addition to its effects on cell and tissue homeostasis through the direct utilization of nicotinamide adenine dinucleotide (NAD)+, PARP-1 is increasingly believed to contribute to inflammation by regulating the expression of a variety of inflammatory genes, including adhesion molecules ICAM-1 and VCAM-1, MCP-1, MIP-1α, and a number of other factors (reviewed in 16–18). This function is linked to the ability of PARP-1 to regulate signal transduction events that result in the activation of the nuclear factor (NF)-κB, extracellular signal-related kinase (ERK), and activator protein 1 (AP1) (19, 20, 21, 22). NF-κB is a pleiotropic transcription factor that plays a critical role in regulating the expression of numerous inflammatory genes, including ICAM-1, VCAM-1, TNF-α, MCP-1 and MIP-1α (23). We and others have reported that PARP-1 expression is required for the efficient translocation of NF-κB to the nucleus in response to LPS (10,24,25). However, we recently reported that this requirement does not apply when the stimulus is TNF-α (26). Interestingly, whereas NF-κB nuclear translocation in TNF-α-treated smooth muscle cells is sufficient for the expression of the adhesion molecule VCAM-1, ICAM-1 expression showed a critical requirement for PARP-1.
PARP-1 also is emerging as a very viable target in therapies aimed at either blocking or reducing cancer burden, including that of breast cancer (27). It is noteworthy that breast cancer is the most common form of malignancy among women and a leading cause of cancer-related deaths worldwide. Deficiency in the breast cancer susceptibility gene BRCA1 contributes to familial breast tumorigenesis that has long been known to have a very poor prognosis (28). BRCA-deficient cancers can be selectively targeted by PARP inhibitors, as inhibition of the enzyme promotes cell death and sensitization to DNA-damaging agents (27,29).
Cordycepin, a natural compound and adenosine analogue derived from Cordyceps militaris, has been shown to harbor a great potential for therapeutic use against a number of human diseases including inflammatory disease and cancer (30). The purified drug has been shown to possess numerous biological activities such as induction of cell death, blockade of cell growth, inhibition of expression of a variety of inflammatory genes and reduction of cell migration. In a variety of animal models, cordycepin has been shown to block inflammation and reduce tumor formation (30). Accumulating evidence suggests that cordycepin may interfere with a number of molecular processes, resulting in either the inhibition or modulation of a number of genes pertinent to inflammation or carcinogenesis, including inhibition of substrate phosphorylation by key kinases (30) or direct interference with protein synthesis (31). Although a number of studies have addressed the mechanism by which cordycepin interferes with such molecular processes to achieve its therapeutic potential, its mode of action appears multifaceted and remains poorly understood.
In the present study, we investigated whether cordycepin blocks ALI-associated inflammatory responses and expression of related genes by testing whether such effects were related to PARP inhibition. We also examined whether the potential PARP inhibition trait promotes the killing of BRCA1-deficient breast cancer cells.
Materials and Methods
Mice (C57BL/6) were bred in a specific pathogen-free facility at the Louisiana State University Health Sciences Center (New Orleans, LA, USA) and allowed unlimited access to sterilized chow and water. Maintenance and experimental protocols were approved by the institution’s Animal Care and Use Committee. Five-wk-old mice (n ≥ 5) received a single injection intraperitoneally (i.p.) of 2 mg/kg cordycepin (Sigma-Aldrich, Saint Louis, MO, USA) suspended in ethanol and diluted 1/10 in saline, or vehicle 1 h before intratracheal administration of 50 μg/mouse LPS (Axxora, San Diego, CA, USA) as described recently (9). The dose of 2 mg/kg of cordycepin was selected based on a study by Rottenberg et al. reporting that this dose efficiently protects mice against trypanosomiasis without causing any detectable toxicity including wasting and diarrhea (32). Mice were euthanized 6 or 24 h after LPS treatment for tissue collection or bronchoalveolar lavage (BAL), respectively, essentially as described (9).
Cell Culture, Immunoblot Analysis and Immunofluorescence
The lung epithelial (A549) and the breast cancer (MCF-7) cell lines and PARP-1−/− lung smooth muscle cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin. The isolation of PARP-1−/− lung smooth muscle cells was conducted using an enzymatic digestion protocol and is described in details in the Supplementary Methods. Prior to treatment, cells at 50%–80% confluence were starved overnight by incubation in DMEM with 0.5% FBS. Cells were treated with 1 μg/mL LPS (Axxora) or 10 ng/mL TNF-α (Roche Diagnostics Corp, Indianapolis, IN, USA) for the indicated times in the absence or presence of increasing concentrations of cordycepin (Sigma-Aldrich). Cells were collected, washed and lysed using RIPA-lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein contents were assessed using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA). Immunoblot analyses were conducted essentially as described (33). Briefly, samples containing 20 μg of protein were fractionated by SDS-PAGE on 4%–20% gradient gels, and the separated proteins were transferred to nitrocellulose membranes. Membranes were stained with Ponceau S to confirm equal loading and transfer of proteins among lanes. After blocking with 5% nonfat milk in PBS + 0.05% Tween 20, the membranes were probed with antibodies to VCAM-1, ICAM-1, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), p65 or p50 NF-κB, the phosphorylated form of p65 NF-κB at serine 536, actin (all purchased from Santa Cruz Biotechnology), or to poly(ADP-ribose) (Axxora). Immune complexes were detected with appropriate secondary antibodies and chemiluminescence reagents (Pierce, Rockford, IL, USA). In some experiments, cells were treated with 500 μmol/L H2O2 (Sigma-Aldrich) with or without cordycepin or the PARP inhibitor NU1025 (Santa Cruz Biotechnology) or TIQ-A (Sigma-Aldrich) before processing for immunofluorescence or immunoblot analysis. Immunofluorescence was conducted essentially as described (25). Briefly, cells were fixed for 20 min in PBS containing 4% formaldehyde, washed with PBS. After washing, cells were permeabilized for 5 min in PBS containing 0.05% Triton X-100. Cells were then incubated overnight at 4°C with 20 μg/mL of antibodies to p65 or p50 NF-κ B or poly(ADP-ribose). After a series of washes, antibody-antigen complexes were detected with Alexa-Fluor 594 or 488-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) using a Leica DMRA2 fluorescence microscope (Leica, Buffalo Grove, IL, USA).
RT-PCR (Conventional and Real-Time)
For RT-PCR, total RNA was extracted from cells using the RNeasy Plus Micro Kit (QIAGEN, Valencia, CA, USA). One μg of total RNA was used as a template to make first-strand cDNA by random priming using reverse transcriptase III (Invitrogen). Oligonucleotide primers (Supplementary Table 1) to specifically amplify a fragment of murine VCAM-1, ICAM-1, MCP-1, MIP-1α, MIP-2, TNF-α, keratinocyte-derived chemokine (KC) or β-actin, or human VCAM-1 or β-actin were purchased from Integrated DNA Technologies (Coralville, IA, USA). The PCR conditions were 30 s at 95° C followed by 30 s at 60° C then 30 s at 72° C for 40 cycles. The comparative Ct method was used as a quantitation approach for cDNA prepared from the different experimental groups by comparing the Ct values of the samples of interest with a control (samples from non-treated mice). The Ct values of both the control and the samples of interest were normalized to the housekeeping gene β-actin. Primers for the β-actin gene were designed specifically to avoid amplifying intronless actin pseudogenes. The quality (single peak and single band) of amplicons generated to test for messages of the different inflammatory genes is displayed in Supplementary Figure S1. All samples were tested in triplicates.
siRNA Transfection, Cell Viability and LDH Release Assays
MCF-7 cells were transfected transiently with specific small interfering RNA (siRNAs) (sc29219) targeting BRCA1 (Santa Cruz Biotechnology) or the negative control siRNA NEG2 (SA Bioscience Inc., Frederick, MD, USA) using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Cell viability was assessed in 96-well plates (10,000 cells/well) using the (3-[4,5-Dimethylthiozol-2-yl]-2-5) diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). Lactate dehydrogenase (LDH) assay was conducted as described previously (5). Briefly, cells cultured in 96-well plates (10,000 cells/well) were treated as described in the figures; media was collected from the different experimental groups and tested for LDH activity using the CytoTox-Fluor Cytotoxicity Assay kit (Promega, Madison WI, USA). All samples were tested in triplicates.
Poly(ADP-ribosyl)ation in vitro
Purified recombinant PARP-1 (Axxora) was incubated in a reaction mixture (total volume 25 μL) containing reaction buffer (100 mmol/L Tris-HCl [pH 7.4]), 1 mmol/L dithiothreitol [DTT], 10 mmol/L MgCl2), 10 mg protein extracts from PARP-1−/− cells, different doses of cordycepin or PARP inhibitors, 1 μg sonicated salmon sperm DNA and 2 mmol/L NAD+ (Sigma-Aldrich) for 30 min at 37°C. The reaction was terminated by the addition of an equal volume of 2 × sodium dodecyl sulfate (SDS) sample buffer (Bio-Rad) and heating at 95°C for 5 min. Samples then were subjected to immunoblot analysis with antibodies to poly(ADP-ribose) (PAR) (Axxora).
Data are presented as means ± S.E.M. of more than three separate experiments performed. Comparisons between multiple groups were performed with analysis of variance (ANOVA) with Bonferroni’s test. Statistical significance was considered significant when P < 0.05.
All supplementary materials are available online at www.molmed.org .
Cordycepin Prevents LPS-Induced Airway Neutrophilia in Mice
Cordycepin Effectively Blocks LPS-Induced, but not TNF-α-Induced, Expression of VCAM-1 in the Human Epithelial Cell Line A549
Interestingly, the traits of cordycepin greatly resembled those observed upon PARP-1 inhibition; we recently reported that PARP-1 inhibition by gene knockout reduces ICAM-1 but not VCAM-1 in response to TNF-α despite the clear nuclear translocation of p65 NF-κB (26). Supplementary Figure 2 shows that pharmacological inhibition of PARP by NU1025 or TIQ-A also failed to reduce TNF-α-induced nuclear translocation of p65 NF-κB. This observation led us to examine whether cordycepin may reduce LPS-induced airway inflammation and expression of NF-κB-dependent genes, in part, by blocking the activity of PARP-1.
Cordycepin Induces Killing of BRCA-Deficient Breast Cancer Cells
After the promising use of PARP inhibitors as therapies for the sensitization of cancers that are deficient in BRCA, the search for even better inhibitors continues. PARP inhibitors have a bright future in the fight against not only cancer but also against various inflammatory diseases (27,29). Our laboratory has been actively pursuing the search for mechanisms by which PARP-1 regulates inflammatory processes using both cell culture systems and animal models of diseases. Indeed, we have reported that PARP inhibition prevents the manifestation of asthma-related traits in an animal model of the disease (5, 6, 7), reduces atherogenesis in high fat diet-induced atherosclerosis using the ApoE−/− mouse model (10), and promotes the regression of already established atherosclerotic plaques (36). We associate such effects with the involvement of PARP-1 in these molecular processes, leading to the expression of inflammatory genes, primarily those regulated by NF-κB (5,9,25,26) and signal transducer and activator of transcription (STAT)-6 (37). The results of the present study provide evidence that cordycepin, a natural compound, inhibits PARP-1 with a rather potent efficacy. These findings shed new light on the mechanism behind the ability of cordycepin to modulate inflammation, inhibit the expression of a number of inflammatory genes and enhance the killing of cancer cells. Clearly, the ability of cordycepin to inhibit PARP could have an important clinical impact as it could be tested in the treatment of triple-negative breast cancers and reduce the inflammation associated with a number of chemotherapeutic strategies.
Cordycepin has been shown to exhibit a number of properties that block or reduce inflammation, angiogenesis, tumorigenesis, dislipidemia, microbial growth, aging and neurotoxicity (30). A number of molecular processes appear to be affected by cordycepin. Unfortunately, a large number of these studies have utilized high doses of the drug that would be rather difficult to translate into the clinic. In this study, we attempted to focus primarily on low micromolar concentrations to avoid overwhelming intracellular molecular processes, given the nucleotide-like structure of the drug. Although our finding relevant to the protective effects of cordycepin against LPS-induced ALI is novel, it was not completely unexpected given that many studies have demonstrated protective effects of the drug in other inflammatory disease models (30). According to our results, it appears that the ability of cordycepin to inhibit TNF-α expression in vivo is a primary cause of the reduction of the other inflammatory genes, especially given that most of the tested genes could be induced by TNF-α. This suggestion is further supported by our finding that cordycepin differentially affects the expression of adhesion molecules ICAM-1 and VCAM-1. It is important to note that these two genes, though tightly regulated by NF-κB, may also be differentially affected by other factors as well.
The remarkable aspect of these findings is that the differential effect of cordycepin on the expression of ICAM-1 and VCAM-1 directed our attention to the possibility that the drug may be acting as a PARP inhibitor. Our previous studies delineated certain aspects of the mechanism by which PARP-1 regulates the expression of ICAM-1 and VCAM-1 via the regulation of NF-κB activation (26). We showed that while p65 NF-κB nuclear translocation in TNF-α-treated cells is sufficient for the expression of VCAM-1, expression of ICAM-1 showed a critical requirement for PARP-1. Similar effects were observed when cordycepin was used in our cell culture system. The ability of the drug in blocking poly(ADP-ribose) formation upon H2O2 treatment provided initial evidence for the potential action of cordycepin as a PARP inhibitor. The PARP inhibitory action of cordycepin was fully confirmed using a cell-free system with purified recombinant PARP-1. How cordycepin inhibits PARP-1 is unclear, but because of its ability to inhibit the enzyme at very low concentrations, noncompetitive inhibition is likely, since competitive inhibitors such 3-aminobenzamide can only achieve full inhibition of PARP-1 at millimolar concentrations. However, to gain a precise mode of action and determine the IC50 of the drug, additional and precise experimentation is required.
The ability of cordycepin to inhibit PARP-1 suggests a promising use in the killing of BRCA-deficient breast cancer cells. It is noteworthy that a number of PARP inhibitors have already passed through a number of clinical trials and represent a promising strategy in the treatment of not only breast but also ovarian cancers (27,29,38). Cordycepin, to the best of our knowledge, represents the first natural product possessing PARP inhibitory traits that could function both as a promising drug for the treatment of breast and ovarian cancers in humans and as an initial structure to use for future modifications that may render it even more potent in inhibiting PARP-1 and killing cancer cells. These possibilities become even more significant given the antiinflammatory characteristics of cordycepin: if coadministered with traditional chemotherapeutic drugs, it could act to counteract any associated inflammatory processes triggered by such traditional therapies.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported, in part, by grant HL072889 from the NIH and grant RSG-116608 from the American Cancer Society as well as funds from the Louisiana Cancer Research Consortium (New Orleans, LA) to H Boulares.
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