Coronarin D, a labdane diterpene, inhibits both constitutive and inducible nuclear factor-κB pathway activation, leading to potentiation of apoptosis, inhibition of invasion, and suppression of osteoclastogenesis

Many plants belonging to the Zingiberaceae family are extensively used in traditional systems of medicine in India, China, and Southeast Asian countries. One such plant is the white ginger lily, which originated in the Himalayan region of Nepal and India (Hedychium coronarium and Hedychium flavescens) and is commonly called “dolan champa” in Hindi, “takhellei” in Manipuri, “sonataka” in Marathi, and “suruli sugandhi” in Kannada. The rhizome of this plant is used by certain tribal groups of Bihar (India) as a febrifuge and is considered as an antirheumatic, tonic, and excitant in Moluccas. The base of the stem is chewed and the juice is applied to reduce swelling (1). The native Hawaiians refer to this plant as “awapuhi” (native wild shampoo ginger) and use the juice of mature seeds as a hair and skin treatment. In Cuba, where it is the national flower it is known as the “flor de mariposa” (literally, “butterfly flower”). Extracts from H. coronarium have exhibited anti-inflammatory activity against rat paw edema (2) and diuretic activity in rats (3).

The active principle(s) responsible for these activities is not known. Several labdane-type diterpenes (coronarin A, B, C, D, E, and F) have been isolated from the rhizome of H. coronarium (4, 5), but only little is known about these coronarins. The studies showed that coronarin A inhibited the proliferation of human umbilical vein endothelial cells (6). Coronarin D is known to inhibit the release of β-hexosaminidase from RBL-2H3 cells (7) and acetic acid-induced vascular permeability in mice (8). Whereas coronarin D and E were reported to inhibit nitric oxide production in lipopolysaccharide (LPS)-induced mouse peritoneal macrophages (9), coronarin A has been shown to be cytotoxic in tumor cells (10). However, the pathway by which these diterpenes exert their anti-inflammatory and cytotoxic effects is unclear. One of the major pathways linked with inflammation and growth modulation is the nuclear factor-κB (NF-κB) pathway.

NF-κB is an inducible transcription factor for genes involved in cell survival, adhesion, inflammation, differentiation, and growth. It regulates genes that are critical in inflammation and in the early and late stages of aggressive cancers, including cyclooxygenase-2, 5-lipoxygenase, cyclin D1, apoptosis suppressor proteins such as Bcl-2 and Bcl-xL, and genes required for metastasis and angiogenesis such as matrix metalloproteinase (MMP) and vascular endothelial growth factor (VEGF; ref. 10).

Because NF-κB activation is a mediator of both inflammation and growth modulation, we hypothesized that the anti-inflammatory and cytotoxic effects of coronarin D are due to its ability to inhibit NF-κB and NF-κB-regulated gene expression. In our study, coronarin D inhibited the NF-κB activation induced by different agents, inhibited NF-κB-regulated gene expression, and abrogated receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. It also potentiated chemotherapeutic agent-induced apoptosis.

Dried, powdered rhizomes of H. flavescens (525 g), which belongs to the Zingiberaceae family, were collected from Munnar in Kerala, India, and extracted with 3 L hexane using a Soxhlet extraction apparatus for 24 h. Removal of solvent under reduced pressure using a rotary evaporator yielded 19.5 g of a dark brown residue. This crude extract was subjected to column chromatography using 400 g silica gel (100-200 mesh) and eluted with petroleum ether-ethyl acetate mixture of increasing polarity. Successive fractions were combined based on their behavior on TLC, and the solvent was evaporated to give 17 fraction pools. Fraction pools 10 and 11 (total 3.7 g), obtained by elution of the column with petroleum ether-ethyl acetate in an 80:20 ratio, were found to contain the major product, which was further purified by once again separating it using column chromatography. This yielded the major compound, coronarin D, as colorless needles (2.05 g; crystallized from a petroleum ether-dichloromethane mixture), mp 102-103°C. [a]_D^{(ref 1)} +10⁰. IR (KBr, u_max/cm): 3455, 3087, 2925, 2856, 1726, 1669, 1642, 1445, 1345, 1208, 939, 889. ¹H NMR (300 MHz): d 6.69 (br s, 1H), 5.94 (br s, 1H), 4.81 (s, 1H), 4.36 (br s, 1H), 2.99 (m, 1H), 2.71 (m, 1H), 2.40-2.36 (m, 2H), 2.20 (m, 1H), 2.05-1.08 (m, 12H), 0.89 (s, 3H), 0.82 (s, 3H), 0.71 (s, 3H). ¹³C NMR (75 MHz): d 170.65, 148.01, 143.45, 124.48, 107.52, 96.46, 56.02, 55.21, 41.90, 39.94, 39.12, 37.68, 33.57, 33.45, 25.58, 23.99, 21.62, 19.21, 14.23. UVl_max methanol nm: 239. LRMS: 318, C₂₀H₃₀O₃, require 318. ¹H NMR, IR, and UV were identical to that of coronarin D, as reported earlier.

NaBH₄ (100 mg, 2.64 mmol) was added in small portions to a solution of coronarin D (125 mg, 0.4 mmol) in methanol (12 mL) maintained at 15°C, and the mixture was stirred at room temperature (25°C) for 30 min. After the reaction was complete according to TLC, the methanol was completely removed under vacuum pressure and the residue was diluted with water (15 mL) and extracted with dichloromethane (3 × 25 mL). The combined organic extract was washed with brine and dried over anhydrous Na₂SO₄. Evaporation of solvent yielded the crude product, which was purified by column chromatography over silica gel. On elution with hexane/ethyl acetate (3:1), 98 mg (80%) colorless E-labda-8(17),12-diene-15-ol-16-oic acid (coronarin D acid) crystals was obtained (mp 144-146°C). IR (KBr, u_max/cm): 3438, 2942, 2901, 2854, 1682, 1641, 1418, 1264, 1199, 1122, 1046, 998, 880, 762. ¹H NMR (300 MHz): d 6.97 (t, 1H, J = 6.40 Hz), 4.83 (s, 1H), 4.39 (s, 1H), 3.74 (t, 2H, J = 6.15 Hz), 2.61 (t, 2H, J = 6.03 Hz), 2.42-1.08 (m, 14H), 0.88 (s, 3H), 0.82 (s, 3H), 0.73 (s, 3H). ¹³C NMR (75 MHz): d 172.90, 149.95, 148.22, 127.60, 1.7.71, 61.73, 56.56, 55.38, 42.03, 39.54, 39.22, 37.89, 33.57, 30.20, 24.12, 24.08, 21.73, 19.30, 14.30. C/H analysis: Calculated for C₂₀H₃₂O₃; C, 74.96%; H, 10.07%; Found: C, 74.88%; H, 10.44%.

A stock solution (50 mmol/L) of the compound was prepared in DMSO and used for further studies. Bacteria-derived human recombinant tumor necrosis factor (TNF), purified to homogeneity with a specific activity of 5 × 10⁷ units/mg, was kindly provided by Genentech. Cigarette smoke condensate, prepared as described previously (11), was kindly supplied by Dr. G. Gairola (University of Kentucky). Phorbol myristate acetate, okadaic acid, LPS, H₂O₂, and Taxol were obtained from Sigma. Penicillin, streptomycin, RPMI 1640, Iscove's modified Dulbecco's medium, DMEM, DMEM/F-12, and fetal bovine serum were obtained from Invitrogen. Anti-β-actin was obtained from Sigma-Aldrich. Anti-p65, anti-p50, anti-IκBα, anti-poly(ADP-ribose) polymerase, anti-intercellular adhesion molecule-1, MMP-9, cyclin D1, c-myc, Bcl-2, anti-cyclooxygenase-2, and anti-inhibitor of apoptosis protein 1 were obtained from Santa Cruz Biotechnology. Phosphospecific anti-IκBα (Ser³²) and anti-p65 (Ser⁵³⁶) were purchased from Cell Signaling. Anti-IκBα kinase (IKK)-α and -β antibodies were kindly provided by Imgenex.

The human cell lines KBM-5 (chronic myeloid leukemia), A293 (embryonic kidney carcinoma), H1299 (lung adenocarcinoma), U266 (multiple myeloma), 253JBV (bladder cancer), MCF-7 (breast cancer), SKOV3 (ovarian cancer), HT29 (colon cancer), and PANC-1 (pancreatic cancer) and the murine monocytic cell line RAW 264.7 were obtained from the American Type Culture Collection. Cells were cultured as follows: KBM-5 in Iscove's modified Dulbecco's medium with 15% fetal bovine serum; H1299, K562, MCF-7, SKOV3, and U266 in RPMI 1640; A293, HT29, and PANC-1 in DMEM; and RAW 264.7 in DMEM/F-12 supplemented with 10% fetal bovine serum. Culture medium was supplemented with 100 units/mL penicillin and 100 μg/mL streptomycin.

To determine NF-κB activation, we used electrophoretic mobility shift assays (EMSA) as described previously (12). In brief, nuclear extracts prepared from TNF-treated cells were incubated with ³²P-end-labeled 45-mer double-stranded NF-κB oligonucleotide (15 μg protein with 16 fmol DNA) from the HIV long terminal repeat 5′-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3′ (boldface indicates NF-κB binding sites) for 30 min at 37°C. The DNA-protein complex that formed was separated from the free oligonucleotide on 6.6% native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5′-TTGTTACAACTCACTTTCCGCTGCTCACTTTCCAGGGAGGCGTGG-3′, was used to evaluate the specificity of the NF-κB binding to the DNA. The specificity was also determined through competition with the unlabeled oligonucleotide. The dried gels were visualized and the radioactive bands quantitated with a Storm 220 PhosphorImager (Amersham Biosciences) using ImageQuant software Molecular Dynamics.

To determine the levels of protein expression in the cytoplasm and nucleus, we fractionated extracts using SDS-PAGE as described previously (13). The proteins were then electrotransferred to nitrocellulose membranes and blotted with each antibody, and the expression of proteins was detected with an enhanced chemiluminescence reagent (Amersham Biosciences). We quantitated the bands with NIH imaging software.

To determine the effects of coronarin D on TNF-induced IKK activation, we used the IKK assay as described previously (13). In brief, to determine the total amounts of IKK-α and IKK-β in each sample, we resolved 50 μg whole-cell protein by using 7.5% SDS-PAGE, electrotransferred the proteins to a nitrocellulose membrane, and blotted them with anti-IKK-α or anti-IKK-β antibodies.

We performed a NF-κB-dependent reporter gene expression assay as described previously (14). The effects of coronarin D on NF-κB-dependent reporter gene transcription as induced by TNF, TNF receptor (TNFR), TNFR-associated death domain (TRADD), TNFR-associated factor 2 (TRAF2), NF-κB-inducing kinase (NIK), IKK-β, and p65 were analyzed using a secretory alkaline phosphatase (SEAP) assay as described previously (14).

The effects of coronarin D on p65 nuclear translocation were evaluated by an immunocytochemical analysis as described previously (13).

The effect of coronarin D on the cytotoxic effects of chemotherapeutic agents was determined by the MTT uptake method as described previously (15). Briefly, 5,000 cells per well were incubated with different chemotherapeutic agents such as doxorubicin (KBM-5 and U266; 1-100 nmol/L), gemcitabine (PANC-1 and 253JBV; 1-100 nmol/L), 5-fluorouracil (H1299 and HT29; 0.1-5 μmol/L), cisplatin (OSC 19; 0.01-0.5 μg/mL), and docetaxel (SKOV3 and MCF-7; 0.5-5 nmol/L) in combination with coronarin D (10 μmol/L) for 24 h and the cell viability was measured and the IC₅₀ was determined.

To determine membrane permeability, we used the Live/Dead assay (Molecular Probes), which measures intracellular esterase activity and plasma membrane integrity. This assay was done as described previously (13).

An early indicator of apoptosis is the rapid translocation and accumulation of the membrane phospholipid phosphatidylserine from the cytoplasmic interface of the membrane to the extracellular surface. This loss of membrane asymmetry can be detected by using the binding properties of Annexin V. To identify apoptosis, we used an Annexin V antibody, which was conjugated with a fluorescent dye, FITC, as described previously. Briefly, cells were preincubated with coronarin D, treated with TNF for 16 h at 37°C, and subjected to Annexin V staining. The cells were washed in PBS, resuspended in 100 μL binding buffer containing a FITC-conjugated anti-Annexin V antibody, and then analyzed with a flow cytometer (FACSCalibur, BD Biosciences; ref. 13).

We also determined cytotoxicity using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method, which is used to evaluate DNA strand breaks during apoptosis using an in situ cell death detection reagent. This assay was done as described previously (13).

Extracellular matrix invasion is a crucial step in tumor metastasis. Therefore, we determined how invasion was affected by coronarin D with an invasion assay as described previously (15). A Matrigel basement membrane matrix extracted from a Engelbreth-Holm-Swarm mouse tumor (BD Biosciences) was reconstituted and used for this assay.

To determine whether coronarin D inhibits osteoclastogenesis, we performed an osteoclast differentiation assay as described previously (16). In brief, 1 × 10⁴ RAW 264.7 cells per well were cultured in 24-well dishes and allowed to adhere overnight. They were then exposed to RANKL, with or without coronarin D, for different numbers of days and examined for osteoclast formation.

We isolated coronarin D from the rhizomes of H. flavascens and confirmed the structure using ¹H NMR, IR, and UV analysis. The structure of coronarin D is shown in Fig. 1A. We investigated the effects of coronarin D on the NF-κB activation pathway as induced by various carcinogens and inflammatory stimuli. We also evaluated the effect of coronarin D on NF-κB-regulated gene expression and NF-κB-mediated cellular responses. Because TNF is one of the most potent proinflammatory cytokines and the most potent activator of NF-κB, and because the TNF-induced NF-κB activation pathway has been well characterized, we determined the effects of coronarin D on TNF-induced NF-κB activation.

Because TNF, H₂O₂, phorbol myristate acetate, okadaic acid, interleukin-1β, LPS, and cigarette smoke condensate are potent activators of NF-κB (10, 17–19), we determined the effect of coronarin D on NF-κB activation by all these agents. Although all agents activated NF-κB in human myeloid leukemia KBM-5 cells, coronarin D blocked this activation (Fig. 1B), and cells were fully viable at this concentration and exposure time. These results suggest that coronarin D acts at a step in the NF-κB activation pathway that is common to all these agents.

TNF is one of the most potent activators of NF-κB, and the mechanism of NF-κB activation has been well established; therefore, we determined the effects of coronarin D on TNF-induced NF-κB activation. In KBM-5 cells, coronarin D suppressed TNF-induced NF-κB activation in a dose-dependent manner, and this inhibition was observed at a concentration even as low as 10 μmol/L: TNF-induced NF-κB activation was completely suppressed after 8 h at 50 μmol/L coronarin D (Fig. 1C, top).

Next, we determined the minimum exposure time required for coronarin D to inhibit TNF-mediated NF-κB activation. The cells were exposed to the inhibitor for 2, 4, 8, or 12 h and then treated with TNF for 30 min. We found that coronarin D started to inhibit TNF-induced NF-κB activation after only 2 h and completely suppressed it after 8 h (Fig. 1C, bottom). However, under the same conditions, coronarin D alone (in the absence of TNF) had no effect on NF-κB activation.

Because the signal transduction pathway mediated by NF-κB may be distinct in different cell types, we also determined whether coronarin D blocked TNF-induced NF-κB activation in K562 and H1299 cells (Fig. 1D, top). We found that coronarin D inhibited TNF-induced NF-κB activation in these cells, indicating that the coronarin D-induced inhibition of NF-κB activation is not cell type specific.

Most tumor cells express constitutively active NF-κB (20), although the mechanism of constitutive activation is not well understood. In this study, we found that incubation with coronarin D suppressed the constitutive activation of NF-κB in U266 (multiple myeloma), 253JBV (bladder cancer), and PANC-1 (pancreatic cancer) cells, which are known to express constitutively active NF-κB (Fig. 1D, bottom).

Whether coronarin D-induced inhibition of NF-κB activation is reversible was examined. To determine this, U266 cells were treated with coronarin D for 8 h, washed with PBS two times, resuspended in a fresh medium, harvested after 0, 2, 4, 8, 12, and 24 h, prepared the nuclear extracts, and analyzed for NF-κB activity by EMSA. Our results show that coronarin D-induced NF-κB inhibition is not reversible (Supplementary Fig. S1).³

To determine how coronarin D inhibits TNF-induced NF-κB activation, we exposed the cells to coronarin D for 8 h and then treated them with TNF for different periods. Nuclear extracts were prepared and analyzed for NF-κB; cytoplasmic extracts were prepared and analyzed for IκBα. We found that coronarin D completely inhibited TNF-induced NF-κB activation (Fig. 2A) and also suppressed TNF-induced IκBα degradation (Fig. 2B, middle) as determined by Western blot analysis. The results also showed that coronarin D inhibited TNF-induced IκBα phosphorylation (Fig. 2B, top).

IKK is required for the TNF-induced phosphorylation of IκBα and the phosphorylation of p65 (20). Because coronarin D inhibited the phosphorylation of IκBα, we analyzed its effects on TNF-induced IKK activation using immune complex kinase assays. We found that coronarin D suppressed TNF-induced IKK activation (Fig. 2C). However, neither TNF nor coronarin D affected the expression of IKK proteins. Whether coronarin D inhibits IKK directly was also examined. The results show that coronarin D directly inhibits the IKK activity (Supplementary Fig. S2).³

Above, we have shown that coronarin D can suppress constitutive active NF-κB. Whether this inhibition is mediated through inhibition of IKK was examined. Our results show that coronarin D inhibits constitutive active IKK in these cells (Supplementary Fig. S3).³

The phosphorylation of p65 is required for the transactivation of NF-κB. Using phosphospecific anti-p65 (Ser⁵³⁶) antibody, we determined that TNF did induce p65 phosphorylation, which was substantially inhibited by coronarin D (Fig. 2D, top).

Whether coronarin D inhibits TNF-induced p65 nuclear translocation was examined by the immunocytochemical method. We found that TNF induced the nuclear translocation of p65 and coronarin D blocked the translocation (Fig. 2D, bottom).

Because DNA binding alone is not always related to NF-κB-dependent gene transcription (21), we also analyzed the effects of coronarin D on TNF-induced reporter activity. After cells were transiently transfected with the NF-κB-regulated SEAP reporter construct, incubated with coronarin D, and stimulated with TNF, we found that TNF induced NF-κB reporter activity and that this activity was substantially suppressed by coronarin D (Fig. 3A). These results suggest that coronarin D inhibits TNF-induced gene expression. In addition, DN-IκBα plasmid suppressed TNF-induced reporter activity, indicating the specificity of the plasmid.

TNF-induced NF-κB activation is mediated through the sequential interaction of TNFR and TRADD, TRAF2, NIK, IKK, and p65 and results in the phosphorylation of IκBα (22). Therefore, to further examine the effects of coronarin D on TNF-induced NF-κB activation, we transiently transfected cells with a NF-κB-regulated SEAP reporter construct and TNFR1-, TRADD-, TRAF2-, NIK-, IKK-β-, or p65-expressing plasmids. Cells were then treated with coronarin D and monitored for NF-κB-dependent SEAP expression. We found that coronarin D suppressed the NF-κB activation induced by TNFR1, TRADD, TRAF2, NIK, IKK-β, and p65 (Fig. 3B).

NF-κB activation has been shown to regulate the expression of c-myc, cyclin D1, and cyclooxygenase-2 (23–25), gene products that are involved in the proliferation of various tumor cells. Therefore, we determined whether the expression of these gene products is modulated by coronarin D. As shown in Fig. 4A, TNF induced the expression of all these gene products, and coronarin D suppressed it.

NF-κB is key to the survival of tumor cells, as its activation induces the expression of antiapoptotic gene products. Because earlier experiments in this study showed that coronarin D inhibits TNF-induced NF-κB activation, we hypothesized that coronarin D would also inhibit the expression of TNF-induced antiapoptotic gene products, such as cellular inhibitor of apoptosis protein-1, TRAF-2, survivin, and Bcl-2, all known to be regulated by NF-κB (26–31). Using Western blot analyses, we found that coronarin D did inhibit the expression of all these proteins (Fig. 4B).

Also using Western blot analysis, we determined that coronarin D inhibited the TNF-induced expression of three metastatic gene products, all of which have been shown elsewhere to be regulated by NF-κB: intercellular adhesion molecule-1, a cell surface adhesion factor (32); MMP-9, which is involved in tumor cell invasion and metastasis (33); and VEGF, the most potent angiogenic factor (ref. 34; Fig. 4C).

Because NF-κB activation can inhibit TNF expression and, as shown above, chemotherapeutic agents can induce apoptosis through the expression of the antiapoptotic gene products intercellular adhesion molecule-1, MMP-9, and VEGF (35–37), we used several methods to measure different aspects of apoptosis to determine whether coronarin D enhances the apoptosis induced by TNF and other cytotoxic agents. Using a Live/Dead assay, which measures cell membrane permeability, we found that coronarin D enhanced TNF-induced cytotoxicity from 9% to 57% and Taxol-induced cytotoxicity from 12% to 76% (Fig. 5A). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and Annexin V staining also showed that TNF-induced apoptosis was enhanced by incubation with coronarin D (Fig. 5B and C). Using a caspase-3-activated poly(ADP-ribose) polymerase cleavage assay, we found that coronarin D had a dramatic effect on TNF-induced poly(ADP-ribose) polymerase cleavage (Fig. 5D). These results suggest that the apoptotic effects of TNF and Taxol (paclitaxel) are enhanced by coronarin D alone, at a dose at which coronarin D exhibits minimum cytotoxicity.

Whether coronarin D can also potentiate the effect of chemotherapeutic agents was also examined. We found that coronarin D potentiated the cytotoxic effects of doxorubicin, gemcitabine, 5-fluorouracil, cisplatin, and docetaxel against different tumor cell types (Table 1).

MMPs have been found to provide critical assistance to tumor cells during metastasis, and because earlier experiments in this study showed that coronarin D suppresses TNF-induced MMP-9 expression, we sought to determine the effects of coronarin D on the TNF-induced MMP-9 invasion. After seeding H1299 cells in the top chamber of a Matrigel invasion chamber in the absence of serum and incubating them with TNF with or without coronarin D for 24 h, we found that coronarin D suppressed TNF-induced cell invasion (Fig. 6A).

Given that the agents that suppress RANKL signaling may inhibit bone resorption or osteoclastogenesis (38), we examined the effect of coronarin D on osteoclastogenesis. Toward that end, we treated RAW 264.7 cells with 5 μmol/mL coronarin D in the presence of RANKL. Cells were then allowed to grow and differentiate into osteoclasts. Using osteoclast differentiation assays, we found that coronarin D substantially decreased RANKL-induced osteoclast differentiation (Fig. 6B). In fact, a 5 μmol/L concentration of coronarin D was sufficient to reduce osteoclastogenesis by more than 70%. Under these conditions, the RAW 264.7 cells remained fully viable (data not shown).

We also compared the activity of coronarin D with its acidic form (Fig. 6C) and found that coronarin D acid also suppressed TNF-induced NF-κB activation but was less active (Fig. 6D).

The present study reports for the first time that coronarin D inhibits NF-κB. Specifically, coronarin D inhibited the NF-κB activation induced by different carcinogens and proinflammatory molecules and inhibited constitutive NF-κB expression. It also inhibited NF-κB by down-regulating IKK activation, which led to the inhibition of IκBα phosphorylation and degradation, p65 phosphorylation, and translocation. Coronarin D also inhibited NF-κB-regulated gene products that are involved in antiapoptotic activity, proliferation, invasion, and angiogenesis. Coronarin D also potentiated TNF- and Taxol-induced apoptosis. The coronarin D-modulated inhibition of NF-κB activity also led to the inhibition of TNF-induced invasion and RANKL-induced osteoclastogenesis.

Our results also showed that coronarin D suppressed the NF-κB activation induced by inflammatory stimuli, such as TNF, interleukin-1β, and LPS; prooxidants, such as H₂O₂; and carcinogens, such as okadaic acid, tumor promoters, and cigarette smoke condensate. These results suggest that coronarin D acts at a step that is common to all these agents. We also found that coronarin D inhibited NF-κB activation by inhibiting IKK activation, which led to the suppression of IκBα phosphorylation, p65 phosphorylation, and nuclear translocation. However, the mechanism by which coronarin D inhibits IKK activation is still unclear, as it requires an understanding of how IKK is activated, which itself has not been established. Over a dozen different kinases have been linked with IKK activation (39). One of these, TAK1, has been linked with IKK activation through TNF (40, 41). It is possible that coronarin D inhibits IKK through the inhibition of TAK1. However, we found that coronarin D directly inhibits IKK.

Coronarin D also inhibits the constitutive NF-κB activation commonly seen in a wide variety of tumors (42–48). Although the reason NF-κB is constitutively active in tumor cells is unknown, various mechanisms have been implicated. These include the constitutive expression of TNF (36, 49) or interleukin-1 (33), increased proteasome activity (50), overexpression of the epidermal growth factor receptor (51), mutation of IκBα (52), and mutation of ras (53). Although most agents activate NF-κB through IKK-dependent mechanisms, some function through IKK-independent mechanisms (54, 55). Further investigation is required to determine whether coronarin D activates NF-κB in various tumor cells through IKK-dependent or IKK-independent mechanisms.

Coronarin D also inhibits the TNF-induced expression of gene products, such as c-myc, cyclin D1, and cyclooxygenase-2, which are essential for cellular proliferation and survival (56–58). Expression of the c-myc oncoprotein prevents cell cycle arrest in response to growth-inhibitory signals, differentiation stimuli, and mitogen withdrawal. Moreover, myc activation in quiescent cells is sufficient to induce cell cycle entry in the absence of growth factors. Cyclin D1 exercises powerful control over the mechanisms that regulate the mitotic cell cycle. The inhibition of the expression of these three genes by coronarin D could account for the growth-inhibitory effects linked with certain coronarins (9). The cytotoxic effects of coronarin A reported previously against tumor cells (10) may involve suppression of NF-κB pathway. Moreover, coronarin D potentiated the cytotoxic effects of different chemotherapeutic agents in various tumor cell types. Besides tumor cells, NF-κB has been shown to be active in cancer stem cells. Thus, coronarin D is also likely to enhance the effect of chemotherapeutic agents against cancer stem cell.

Our finding that coronarin D also inhibits the TNF-induced invasion of H1299 cells suggests that coronarin D blocks not only primary tumor development but also malignant progression. The suppression of TNF-induced MMP-9 activity, which plays a key role in invasion, could account for the anti-invasive activity of coronarin D. The suppression of VEGF and intercellular adhesion molecule-1 could also contribute to the anti-invasive activity associated with coronarin D. Our results therefore imply that coronarin D should also exhibit antimetastatic activity in vivo.

RANKL has been shown to play a critical role in osteoclastogenesis, the loss of bone commonly associated with aging and cancer. The results of our study revealed that coronarin D inhibits RANKL-induced osteoclastogenesis, an effect that is most likely mediated through the suppression of NF-κB, as were many of the other effects associated with coronarin D to have.

Our results also show that coronarin D is more active than coronarin D acid. Unlike coronarin D acid, coronarin D can open to give an aldehyde group. The presence of this aldehyde group in coronarin D makes its more active than coronarin D acid.

Previously, it has been shown that coronarin D and E can inhibit nitric oxide production in LPS-induced mouse peritoneal macrophages (9). It is possible that this effect of coronarin D is mediated through suppression of NF-κB-mediated inducible nitric oxide synthase expression as described here. Previous studies have shown that coronarin A inhibits the proliferation of human umbilical vein endothelial cells (6) and acetic acid-induced vascular permeability in mice (8). It is possible that these effects are mediated through suppression of NF-κB-mediated VEGF expression as reported here.

Overall, our results suggest that coronarin D exhibits antiproliferative, proapoptotic, anti-invasive, antiangiogenic, antiosteoclastogenic, and anti-inflammatory effects through the suppression of NF-κB and NF-κB-regulated gene products. However, in vivo studies are needed to validate these findings.

No potential conflicts of interest were disclosed.

We thank Angelique L. Geehan for carefully editing the article.