Abstract
Zyflamend, a polyherbal preparation, was designed based on constituents which exhibit antiproliferative, antiinflammatory, antioxidant, antiangiogenic, and apoptotic activities through a mechanism that is not well defined. Because the nuclear factor NF-B has been shown to regulate proliferation, invasion, and metastasis of tumor cells, we postulated that zyflamend modulates the activity of nuclear factor (NF)-B. To test this hypothesis, we examined the effect of this preparation on NF-B and NF-B-regulated gene products. We found that zyflamend inhibited receptor activator of NF-B ligand (RANKL)-induced osteoclastogenesis, suppressed tumor necrosis factor (TNF)-induced invasion and potentiated the cell death induced by TNF and chemotherapeutic agents, all of which are known to require NF-B activation. Zyflamend suppressed NF-B activation induced by both TNF and cigarette smoke condensate. The expression of NF-B-regulated gene products involved in antiapoptosis (inhibitor-of-apoptosis protein 1/2, Bcl-2, Bcl-xL, FADD like interleukin-1 converting enzyme (FLICE)/caspase-8 inhibitory protein, TNF receptor-associated factor 1, and survivin), and angiogenesis (vascular endothelial growth factor, cyclooxygenase-2, intercellular adhesion molecule, and matrix metalloproteinase) were also downregulated by zyflamend. This correlated with potentiation of cell death induced by TNF and chemotherapeutic agents. Overall our results indicate that zyflamend suppresses osteoclastogenesis, inhibits invasion, and potentiates apoptosis through downregulation of NF-B activation and NF-B-regulated gene products.

Introduction
Zyflamend is a polyherbal formulation comprised of 10 standardized herbal extracts (rosemary, turmeric, ginger, holy basil, green tea, hu zhang, Chinese goldthread, barberry, oregano, and baikal skullcap). Each of these herbs contain unique chemical constituents that have been reported to possess anti-inflammatory
and anticancer activities (1-14). The exact mechanism how zyflamend mediates its anti-inflammatory effects is poorly understood although one published report suggests that it suppresses cyclooxygenase (COX)-1 and COX-2 activity in human prostate cancer cells (15).
Nuclear factor (NF)-B is a family of Rel-domain containing proteins present in the cytoplasm of all cells, where they are kept in an inactive state by a family of anchorin-domain-containing proteins which includes IB, IB, IB, IB, bcl-3, p105 and p100. Under resting conditions, NF-B consists of a heterotrimer of p50, p65, and IB in the cytoplasm; only when activated and translocated to the nucleus is the sequence of events leading to transcription initiated. Most carcinogens, inflammatory agents, and tumor promoters, including cigarette smoke condensate, phorbol ester, okadaic acid, H2O2, and tumor necrosis factor (TNF), have been shown to activate NF-B. The activation of NF-B involves the phosphorylation, ubiquitination, and degradation of IB and phosphorylation of p65, which in turn leads to the translocation of NF-B to the nucleus where it binds to specific response elements in the DNA. The phosphorylation of IB is catalyzed by IB kinase (IKK), which is essential for NF-B activation by most agents. NF-B has been shown to regulate the expression of a number of genes whose products are involved in tumorigenesis (16). These include antiapoptoic genes (e.g. ciap, suvivin, traf, cflip, bfl-1, bcl-2 and bcl-xl), angiogenesis (cox-2, mmp-9, vegf), genes encoding adhesion molecules, chemokines, and inflammatory cytokines; and cell cycle regulatory genes (e.g., cyclin d1, c-myc).
Because zyflamend contains constituents which can suppress tumor cell proliferation, invasion, angiogenesis, and metastasis, we postulated that this herbal preparation must mediate its effects through modulation of NF-B activation pathway. To test this postulate, we investigated the effect of zyflamend on NF-B activation induced by TNF and cigarette smoke condensate. We found that zyflamend inhibited TNF-induced NF-B activation in myeloid leukemia KBM-5 cells and cigarette smoke condensate-induced NF-B activation in lung adenocarcinoma H1299 cells. Inhibition of zyflamend led to suppression of TNF-induced cell invasion and abrogated RANKL-induced osteoclastogenesis. Zyflamend also suppressed TNF-induced expression of various antiapoptotic, proliferative, and metastasis gene products.

Materials and Methods
Materials. Zyflamend, obtained from New Chapter. (St. Louis, MO), was dissolved in dimethyl sulfoxide (DMSO) as a 10 mg/ml stock solution and stored at -20oC. Bacteria-derived human tumor necrosis factor (TNF) -, purified to homogeneity with a specific activity of 5 x 107 U/mg, was kindly provided by Genentech, Inc. (South San Francisco, CA). Penicillin, streptomycin, RPMI 1640 medium, Iscove’s modified dulbecco medium (IMDM), D-MEM/F12 medium and fetal bovine serum (FBS) were obtained from Invitrogen (Grand Island, NY). The following polyclonal antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA): anti-matrix metalloproteinase (MMP)-9; anti-intercellular adhesion molecule (ICAM); anti-inhibitor-of-apoptosis protein (IAP) 1/2; anti-Bcl-2; anti-Bfl-1/A1; and anti-TNF receptor-associated factor (TRAF1). Anti-COX-2 and XIAP were obtained from BD Biosciences (San Diego, CA). Cigarette smoke condensate (CSC), prepared as described (17), was kindly supplied by Dr. G. Gairola (University of Kentucky, Lexington, KY). An anti-vascular endothelial growth factor (VEGF) was purchased from NeoMarkers (Fremont, CA). Survivin antibody was obtained from R&B Systems (Minneapolis, MN). An FADD-like interleukin-1-converting enzyme (FLICE)/caspase-8-inhibitory protein (cFLIP) antibodies were kindly provided by Imgenex (San Diego, CA).
Cell lines. The cell lines used in our studies included human non-small cell lung carcinoma (H1299), human myelogenous leukemia (KBM-5), and human multiple myeloma (U266), mouse macrophages (RAW 264.7) cell lines were obtained from the American Type Culture Collection (Manassas, VA). H1299, and U266 cells were cultured in RPMI 1640 medium with 10% FBS. KBM-5 cells were cultured in IMDM with 15% FBS. RAW 264.7 cells were cultured in D-MEM/F12 medium supplemented with 10% FBS. All the media were supplemented with 100 U/ml penicillin and 100 g/ml streptomycin.
Osteoclast differentiation assay. To determine the effect of zyflamend on receptor activator of NF-B ligand (RANKL)-induced osteoclastogenesis (18), we cultured RAW 264.7 cells, which can differentiate into osteoclasts by RANKL in vitro. RAW 264.7 cells were cultured in 24-well dishes at a density of 1 x 104 cells per well and allowed to adhere overnight. The medium was then replaced, and the cells were coincubated with different concentrations of zyflamend and 5 nM RANKL. At days 5, the cells were stained for tartrate-resistant acid phosphatase (TRAP) expression, as previously described using an acid phosphatase kit (Sigma-Aldrich), and the TRAP-positive multinucleated osteoclasts (>3 nuclei) per well were counted.
Invasion Assay. The membrane invasion culture system was used to assess cell invasion because invasion through the extracellular matrix is a crucial step in tumor metastasis. The BD BioCoat Tumor Invasion system is a chamber that has a light-tight polyethelyene terephthalate membrane with 8 m-diameter pores and is coated with a reconstituted basement membrane gel (BD Biosciences, San Diego, CA). A total of H1299 (2.5 x 104 cells) were suspended in serum-free medium and seeded into the upper wells. After incubation overnight, cells were coincubated with different concentrations of zyflamend and 1 nM TNF for a further 24 h in the presence of 1% FBS. The cells that invaded through the Matrigel (i.e., those that migrated to the lower chamber during incubation) were stained with 4 g/ml Calcein AM (Molecular Probes, Eugene, OR) in PBS for 30 min at 37 oC and scanned for fluorescence with a Victor3 multi-plate reader (Perkin Elmer Life and Analytical Sciences, Boston, MA); fluorescent cells were counted.
LIVE/DEAD assay. To measure cell death, we used the LIVE/DEAD assay (Molecular Probes, Eugene, OR), which determines intracellular esterase activity and plasma membrane integrity. This assay employs calcein, a polyanionic dye, which is retained within the live cells and provides green fluorescence (19). It also employs the ethidium monomer dye (red fluorescence), which can enter the cells only through damaged membranes and bind to nucleic acids but is excluded by the intact plasma membrane of live cells. Briefly, 1 x 106 cells were incubated with 0.5 mg/ml zyflamend for 24 h and then treated with 1 nM TNF or various chemotherapeutic agents for 16 h at 37°C. Cells were stained with the LIVE/DEAD reagent (5 M ethidium homodimer, 5 M calcein-AM) and then incubated at 37 oC for 30 min. Cells were analyzed under a fluorescence microscope (Labophot 2; Nikon, Tokyo, Japan).
Electrophoretic mobility shift assays. To determine NF-B activation, we carried out electrophoretic mobility shift assays (EMSA) essentially as previously described (20). Briefly, nuclear extracts (1 x 106 cells/ml) were incubated with 32P-end-labeled 45-mer double-stranded NF-B oligonucleotide (15 g of protein with 16 fmol of DNA) from the human immunodeficiency virus long terminal repeat, 5’-TTGTTACAA GGGACTTTC CGCTG GGGACTTTC CAGGGAGGCGTGG- 3’ (boldface indicates NF-B-binding sites), for 30 min at 37 oC, and the DNA-protein complex formed was separated from free oligonucleotide on 6.6 % native polyacrylamide gels. A double-stranded mutated oligonucleotide, 5’-TTGTTACAA CTCACTTTC CGCTG CTCACTTTC CAGGGAGGCGTGG-3’, was used to examine the specificity of binding of NF-B to the DNA. The dried gels were visualized, and the radioactive bands were quantitated using a Storm 820 phosphorimager with the ImageQuant software program (Amersham, Piscataway, NJ).

Western blot analysis. To determine the effect of zyflamend on TNF-induced expression of COX-2, VEGF, ICAM-1, MMP-9, cIAP-1/2, survivin, Bfl-1/A1, Bcl-2, Bcl-xL, cFLIP, TRAF1, and XIAP in whole-cell extracts of treated cells (2 x 106 cells/ml), 30 g of protein was resolved on SDS–PAGE and probed by Western blot with specific antibodies as per manufacturer’s recommended protocol. The blots were washed, exposed to HRP-conjugated secondary antibodies for 1 h, and finally detected by ECL reagent (Amersham Pharmacia Biotechnology, Piscataway, NJ).

Results
The goal of this study was to investigate the effect of herbal formulation, zyflamend, on the transcription factor NF-B, on NF-B-regulated gene products, and on NF-B mediated cellular responses. The various constituents of zyflamend are shown in Figure 1A. To examine the effect of zyflamend on the NF-B activation pathway, most studies employed TNF since the pathway activated by this agent is well understood.
Zyflamend suppresses RANKL-induced osteoclastogenesis. Because RANKL, a member of the TNF superfamily, induces osteoclastogenesis through the activation of NF-B (21), we determined whether zyflamend can suppress RANKL-induced osteoclastogenesis. We found that RANKL induced osteoclast differentiation, as indicated by the expression of TRAP, and that zyflamend suppressed it (Figure 1B) in a dose-dependent manner (Figure 1C).
Zyflamend suppresses TNF-induced tumor cell invasion activity. It is known that NF-B regulates the expression of gene products (e.g., MMP-9, COX-2, and VEGF) that mediate tumor cell invasion (22). Whether zyflamend can modulate TNF-induced tumor cell invasion activity, was investigated in vitro. To determine this, tumor cells were seeded to the top chamber of the matrigel invasion chamber with TNF in the presence or absence of zyflamend, and then examined for invasion. As shown in Figure 2, TNF-induced tumor cell invasion by almost 2 fold and zyflamend suppressed this activity in a dose-dependent manner. Zyflamend alone had no effect on invasion activity.
Zyflamend potentiates the cytotoxic effects of TNF and chemotherapeutic drugs. Because NF-B activation has been shown to inhibit the apoptosis induced by various agents (23, 24) whether zyflamend will modulate the cytotoxic effects induced by TNF and chemotherapeutic agents, was investigated. The effect of zyflamend on TNF and chemotherapeutic agent-induced cell death was examined by the LIVE/DEAD assay. The LIVE/DEAD assay, which measures intracellular esterase activity and plasma membrane integrity, indicated that zyflamend enhances the apoptotic effects of TNF, taxol, and doxorubicin against tumor cells (Figure 3)
Zyflamend suppresses NF-B activation in a dose- and time-dependent manner. As NF-B plays an important role in apoptosis and in cell invasion, we examined the effect of zyflamend on activation of this transcription factor. We first investigated the effect of zyflamend on the activation of NF-B induced by TNF in human myelogenous leukemia (KBM-5) cells. DNA-binding assay (EMSA) results showed that zyflamend alone had no effect on NF-B activation. However, it inhibited TNF-mediated NF-B activation in a dose-dependent manner (Figure 4A). The suppression of NF-B activation by zyflamend was also found to be time-dependent (Figure 4B). The suppression of NF-B activation by zyflamend was not cell type dependent (data not shown).

Zyflamend blocks NF-B activation induced by cigarette smoke condensate. We next examined the effect of zyflamend on the activation of NF-B induced by cigarette smoke condensate in non-small lung adenocarcinoma H1299 cells. DNA-binding assay (EMSA) results showed that zyflamend suppressed the NF-B activation induced by cigarette smoke condensate (Figure 4C). These results suggest that zyflamend acted at a step in the NF-B activation pathway that is common to TNF and cigarette smoke condensate.
Zyflamend inhibits TNF-induced NF-B dependent gene products involved in the proliferation and metastasis of tumor cells.
We also investigated whether zyflamend can modulate NF-B dependent gene products involved in the proliferation and metastasis of tumor cells. TNF has been shown to induce COX-2, MMP-9, ICAM-1, and VEGF, all of which have NF-B binding sites in their promoters (16, 25-27). As shown in Figure 5A, TNF treatment induced the expression of COX-2, VEGF, ICAM-1, and MMP-9 gene products and zyflamend abolished the expression.
Zyflamend inhibits TNF-induced NF-B dependent anti-apoptotic gene products.
NF-B upregulates the expression of the antiapoptotic proteins IAP1, IAP2, survivin, Bfl-1/A1, Bcl-2, Bcl-XL, cFLIP, TRAF1, and XIAP (28-35). We next investigated whether zyflamend affects the expression of these gene products. We found that zyflamend inhibited the TNF-induced as well as the basal expression of all of these proteins (Figure 5B).

Discussion
The present study was designed to investigate the effect of zyflamend on the NF-B activation pathway and on the NF-B-regulated gene products which control tumor cell survival, proliferation, invasion, angiogenesis, and metastasis. We found that zyflamend inhibited RANKL-induced osteoclastogenesis, TNF-induced invasion, and potentiated cell death-induced by TNF and chemotherapeutic agents in various tumor cell lines. The expression of gene products involved in antiapoptosis (IAP1, Bfl-1/A1, Bcl-2, TRAF1, and cFLIP), and metastasis (MMP-9, COX-2, ICAM-1, and VEGF) were also downregulated by zyflamend.
Our results indicate that zyflamend suppresses NF-B activated by TNF in human myeloid leukemia KBM-5 cells and cigarette smoke condensate in lung adenocarcinoma H1299 cells. This is the first report to examine the effect of zyflamend on NF-B activated by different stimuli. These results suggest that zyflamend must act at a step common to both these agents. The genes that are involved in the proliferation and metastasis of cancer have been shown to be regulated by NF-B (16). We show in this report that zyflamend inhibits the expression of COX-2, MMP-9, and VEGF regulated by NF-B. Recent reports showed that zyflamend could suppress both COX-1 and COX-2 enzymatic activities (15). Our results show that zyflamend can also inhibit the expression of COX-2 protein.
Our results also imply that zyflamend exercises its anti-cancer properties through the inhibition of NF-B. NF-B is known to regulate the expression of IAP1, xIAP, Bfl-1/A1, TRAF1, Bcl-2, cFLIP, and survivin, and their overexpression in numerous tumors has been linked to survival, chemoresistance, and radioresistance (36). Our results indicate that zyflamend treatment downregulates most of these gene products. Previous report has shown that zyflamend induces apoptosis through a caspase-mediated pathway in human prostate cancer cells (15). Our studies also show that zyflamend potentiated the apoptotic effects of TNF, taxol, and doxorubicin. These effects are similar to that reported with a specific inhibitor of NF-B (37).
Zyflamend is a polyherbal preparation consisting of ten different plant extracts (see Fig. 1). Several of the phytochemicals present in these plant extracts have been previously shown to suppress NF-kB activation. These include ursolic acid ( ), eugenol ( ), curcumin ( ), gingerol ( ), epigallocatechin gallate ( ), resveratrol ( ), berberine ( ), baicalein ( ), and wogonin ( ). Most of these phytochemicals have also been shown to induce apoptosis (Shishodia and Aggarwal, 2006-BP). However, only curcumin has been shown to inhibit invasion ( ) and osteoclastogenesis ( ). Thus it is possible that the activities of zyflamend described here are due to combination of various phytochemicals. Whether these phytochemicals mediate their effects through additive, synergistic or antagonistic manner, is not understood. Curcumin has been shown to interact with epigallocatechin gallate (EGCG) in synergistic manner in some assays (Khafif A, Schantz SP, Chou TC, Edelstein D, Sacks PG. Related Articles, Links Abstract Quantitation of chemopreventive synergism between (-)-epigallocatechin-3-gallate and curcumin in normal, premalignant and malignant human oral epithelial cells.
Carcinogenesis. 1998 Mar;19(3):419-24. ) and antagonistic manner in another (Balasubramanian S, Eckert RL. Related Articles, Links Free Full Text Green tea polyphenol and curcumin inversely regulate human involucrin promoter activity via opposing effects on CCAAT/enhancer-binding protein function.
J Biol Chem. 2004 Jun 4;279(23):24007-14. ).
Overall our results indicate that antiproliferative, pro-apoptotic, anti-invasive, anti-osteoclastogenic, anti-angiogenic, and anti-metastatic effects assigned to zyflamend may be mediated through suppression of NF-B-regulated gene products. Whereas each of the herbs that are used in the formulation of zyflamend are known to contain unique anti-inflammatory and anticancer compounds (1-15), one common property of each component of zyflamend is the ability to suppress NF-B activation. Based on the results presented here, the anticancer potential of zyflamend requires further investigation.

Acknowledgments

We also thank Dr. Bryant Darnay for supplying the RANKL protein. Dr. Bharat B. Aggarwal is a Ransom Horne, Jr., Professor of Cancer Research.
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Figure legends
Fig. 1. (A) Composition of Zyflamend. Percentage of each plant extract in Zyflamend is indicated in parenthesis.
Fig. 1 (B) Zyflamend suppresses RANKL-induced osteoclastogenesis: RAW 264.7 cells (1 x 104 cells/well) were incubated either alone or in the presence of 5 nM RANKL with 0.8 mg/ml zyflamend for 5 days and stained for TRAP expression. TRAP-positive cells were photographed (original magnification, 100). (C) RAW 264.7 cells (1 x 104 cells/well) were incubated either alone or in the presence of 5 nM RANKL with zyflamend at the indicated concentrations for 5 days and stained for TRAP expression. Multinucleated (three nuclei) osteoclasts were counted.

Fig. 2. Zyflamend suppresses TNF-induced tumor cell invasion activity. H1299 cells (2.5 x 104 cells/well) were seeded into the upper wells of a Matrigel invasion chamber overnight in the absence of serum, coincubated with 0.3 mg/ml zyflamend and 1 nM TNF for 24 h in the presence of 1 % serum, and then subjected to invasion assay. The value for no zyflamend and no TNF was set to 1.0.

Fig. 3. Zyflamend enhances cell death induced by TNF and chemotherapeutic agents. (A) Human multiple meloma U266 cells (1 x 106 cells/ml) were serum starved for 24 h and then incubated with 1 nM TNF, 1 nM taxol and 300 nM doxorubicin alone or in combination with (0.5 mg/ml) zyflamend as indicated for 24 h. Cell death was determined by calcein AM based LIVE/DEAD assay as described under “Materials and Methods”. Red color highlights dead cells, and green color highlights live cells.

Fig. 4 (A) Zyflamend suppresses TNF-induced NF-B activation in a dose- and time-dependent manner. KBM-5 cells (2 x 106 cells/ml) were preincubated with indicated concentrations of zyflamend for 1 h, treated with 0.1 nM TNF for 30 min. The nuclear extracts were assayed for NF-B activation by EMSA. (B) KBM-5 cells (2 x 106 cells/ml) were preincubated with 1 mg/ml of zyflamend for either 5 min (-5), 15 min (-15) and 30 min (-30) before exposing to TNF; or same time as TNF (0); or 5 min (+5), 15 min (+15) and 30 min (+30) after TNF treatment. TNF treatment time was 30 min. The nuclear extracts were assayed for NF-B activation by EMSA. (C) Zyflamend inhibits cigarette smoke-induced NF-B activation. H1299 cells were preincubated with zyflamend (1 mg/ml) for 1 h, then treated with cigarette smoke (10 g/ml) for 1 h. The nuclear extracts were assayed for NF-B activation by EMSA.

Fig. 5 . Zyflamend suppresses TNF-induced NF-B regulated gene products.
(A) Proliferative and metastatic proteins, (B) Anti-apoptotic proteins. KBM-5 cells (2 x 106 cells/ml) were incubated with 1 mg/ml zyflamend for 1 h and then treated with 1 nM TNF for the indicated times. Whole-cell extracts were prepared, subjected to four different gels, electrotransferred to the nitrocellulose membrane, sliced accordingly to the molecular weight, and then subjected to Western blot analysis using the indicated antibodies.