Honokiol Arrests Cell Cycle, Induces Apoptosis, and Potentiates the Cytotoxic Effect of Gemcitabine in Human Pancreatic Cancer Cells

Survival rates for patients with pancreatic cancer are extremely poor due to its asymptomatic progression to advanced and metastatic stage for which current therapies remain largely ineffective. Therefore, novel therapeutic agents and treatment approaches are desired to improve the clinical outcome. In this study, we determined the effects of honokiol, a biologically active constituent of oriental medicinal herb Magnolia officinalis/grandiflora, on two pancreatic cancer cell lines, MiaPaCa and Panc1, alone and in combination with the standard chemotherapeutic drug, gemcitabine. Honokiol exerted growth inhibitory effects on both the pancreatic cancer cell lines by causing cell cycle arrest at G1 phase and induction of apoptosis. At the molecular level, honokiol markedly decreased the expression of cyclins (D1 and E) and cyclin-dependent kinases (Cdk2 and Cdk4), and caused an increase in Cdk inhibitors, p21 and p27. Furthermore, honokiol treatment led to augmentation of Bax/Bcl-2 and Bax/Bcl-xL ratios to favor apoptosis in pancreatic cancer cells. These changes were accompanied by enhanced cytoplasmic accumulation of NF-κB with a concomitant decrease in nuclear fraction and reduced transcriptional activity of NF-κB responsive promoter. This was associated with decreased phosphorylation of inhibitor of kappa B alpha (IκB-α) causing its stabilization and thus increased cellular levels. Importantly, honokiol also potentiated the cytotoxic effects of gemcitabine, in part, by restricting the gemcitabine-induced nuclear accumulation of NF-κB in the treated pancreatic cancer cell lines. Altogether, these findings demonstrate, for the first time, the growth inhibitory effects of honokiol in pancreatic cancer and indicate its potential usefulness as a novel natural agent in prevention and therapy.


Introduction
Pancreatic cancer is one of the most lethal malignancies in the United States with mortality rate increasing every coming year [1,2]. According to the estimate of American Cancer Society, 43140 Americans were diagnosed with pancreatic cancer in 2010 and 36,800 died, marking this malignancy as the fourth leading cause of death from cancer [2]. Due to its asymptomatic progression, pancreatic cancer is diagnosed at a stage when it has already metastasized or is locally advanced [3]. Therapeutic approaches against the advanced disease have largely failed and approximately .80% of patients diagnosed with this malignancy still die within 2-8 months [4]. Gemcitabine, a standard FDA approved drug for pancreatic cancer therapy, is reported to be minimally effective that improves patient's survival by couple of weeks only [3,5]. Therefore, it is of utmost importance to develop alternative therapeutic regimens and strategies for effective management of pancreatic cancer.
Several new strategies, which target growth promoting pathways alone and in combination with gemcitabine, have been tested in pancreatic cancer to improve therapeutic outcome [6]. In addition, several recent studies have identified deregulated signaling elements, such as Ras, Akt, NF-kB, miRNAs, etc., that not only promote cancer progression but also confer chemoresistance in pancreatic cancer [7][8][9]. Induction of these survival pathways results from activating gene mutations, loss of inhibitory pathways and/or potentiation through autocrine and paracrine signaling mechanisms [3,10]. In fact, it has now been shown that targeting of some of these signaling nodes can be useful in inhibiting tumor growth and progression as well as in restoring the sensitivity of tumor cells to the cytotoxic drugs [3,9,10].
Natural products have been at the core of cancer chemotherapy for past several decades and in fact, over 60% of the current anticancer drugs have their origin from natural sources [11]. In several recent studies, novel plant-derived compounds have been identified to act as anti-tumor agents through modulation of biological pathways [12]. Honokiol, a biologically active biphenolic compound isolated from the Magnolia officinalis/grandiflora, has received significant attention due to its potent anti-neoplastic and anti-angiogenic properties [13,14]. It has yielded promising data against skin, colon, lung and breast cancers [13,[15][16][17]. The striking aspect of honokiol as an anti-neoplastic drug is its potential to inhibit nuclear factor kappa B (NF-kB), which is associated with cancer cell survival and chemoresistance [9,10,18]. The NF-kB is constitutively activated in a variety of hematologic and solid malignancies, including pancreatic cancer and controls the expression of an array of genes involved in cell proliferation and survival through direct and indirect mechanisms [18][19][20]. In the present study, we have examined, for the first time, the effects of honokiol against pancreatic cancer. Our data show that honokiol inhibits the growth of human pancreatic cancer cell lines, MiaPaCa and Panc1, by causing cell cycle arrest and induction of apoptosis. Furthermore, our study provides evidence for a role of honokiol in chemosensitizing the pancreatic cancer cells to cytotoxic effects of gemcitabine.

Results
Growth inhibitory effect of honokiol on human pancreatic cancer cells Two human pancreatic cancer cell lines viz. MiaPaCa and Panc1 were employed as a model system to investigate the effect of honokiol on pancreatic cancer cell growth. Cells treated with honokiol (10-60 mM) showed alterations in morphology as compared to vehicle (DMSO)-treated cells. With increasing concentration of honokiol, cells became round, shrunken and detached from the substratum ( Figure 1A), consistent with morphological changes associated with apoptosis. Subsequently, we quantified the cytotoxic effects of honokiol by measuring percent viability using WST-1 assay. Our data demonstrated that honokiol induced a dose-and time-dependent decrease in growth of both the pancreatic cancer cell lines with IC 50 values of ,43.25, 31.08 and 18.54 mM (against MiaPaCa), and ,47.44, 34.17 and 21.86 mM (against Panc1) after 24, 48 and 72 h treatments, respectively ( Figure 1B). Together, these findings indicate that honokiol has growth inhibitory effects on pancreatic cancer cells.
Honokiol causes G 1 phase cell cycle arrest and induces apoptosis in pancreatic cancer cells Suppression of cancer cell growth can be caused either by arrest of cell cycle progression or due to induction of apoptosis or both [12]. Our data on cell cycle distribution demonstrated that treatment with honokiol resulted in enrichment of pancreatic cancer cells in G 1 phase with a concomitant decrease in number of cells in S-phase (proliferative fraction) ( Figure 2). We observed a ,1.28, 2.16 and 2.46 folds (in MiaPaCa) and ,1.08, 1.53 and 1.93 folds (in Panc1) decrease in number of cells in S-phase at 20, 40 and 60 mM doses of honokiol, respectively ( Figure 2). In apoptosis assays, our data demonstrated a considerable increase in apoptotic index (PE Annexin V positive/7AAD negative cells) in a dose-dependent manner after 24 h of honokiol treatment (Figure 3). At 20, 40 and 60 mM concentrations of honokiol, we observed ,1.25, 2.04 and 3.96 folds increase in apoptotic indices of MiaPaCa and ,1.34, 1.98 and 3.32 folds increase in apoptotic indices of Panc1 cells, respectively. Altogether, our findings demonstrate that honokiol has both cytostatic and cytotoxic properties against pancreatic cancer cells.

Honokiol alters the expression of cell-cycle and survivalassociated proteins
To investigate the mechanistic basis of growth inhibitory effects of honokiol, we next examined its effect on the expression of key proteins involved in cell proliferation and survival. Our data revealed a dose-dependent decrease in the expression of cyclins (D1 and E) and cyclin-dependent kinases (Cdk2 and Cdk4); while an induced expression of cyclin-dependent kinase inhibitors (p21 and p27) was observed after honokiol treatment in both MiaPaCa and Panc1 pancreatic cancer cells ( Figure 4). Among the survival proteins, we observed a dose-dependent reduction in the levels of the anti-apoptotic protein Bcl-2 and Bcl-xL, whereas a concomitant increase in the level of pro-apoptotic protein Bax was observed ( Figure 5A) leading to an increase in the ratio of Bax/ Bcl-2 ( Figure 5B, upper panel) and Bax/Bcl-xL ( Figure 5B, lower panel). These findings demonstrate that honokiol alters the expression of proteins involved in the regulation of cell cycle and apoptosis to confer its growth inhibitory effect.
Honokiol attenuates the constitutive activation of NF-kB in human pancreatic cancer cells NF-kB is constitutively active in many cancer types, including pancreatic cancer [18][19][20] and it has been shown that the activation of this signaling node facilitates cell cycle progression [21] and apoptotic resistance [22]. Therefore, we investigated whether the treatment of pancreatic cancer cells with honokiol has an impact on NF-kB activation in pancreatic cancer cells. We first examined the effect of honokiol on the transcriptional activity of NF-kB-responsive promoter in a luciferase reporter assay. Our data indicated a dose-dependent reduction in transcriptional activity of NF-kB (,1.40, 2.08 and 4.0 folds in MiaPaCa, and ,1.29, 1.96 and 5.26 folds in Panc1 cells) at 20, 40 and 60 mM of honokiol treatment, respectively ( Figure 6A). To further support this observation, we next studied the cellular localization (cytoplasmic vs. nuclear) p65 subunit of NF-kB. Our immunoblot data demonstrated that honokiol treatment caused a marked and dose-dependent decrease in NF-kB levels in the nuclear fraction of both MiaPaCa and Panc1 pancreatic cancer cells with a simultaneous increase in the cytoplasmic fraction ( Figure 6B). Cellular distribution of NF-kB is controlled by relative expression of its biological inhibitor IkB, which keeps NF-kB sequestered in cytoplasm in an inactive complex [23]. Therefore, we analyzed the cytoplasmic extracts of honokiol-treated pancreatic cancer cells for determination of IkB-a level. Our data demonstrated a dosedependent increase in the level of the IkB-a upon honokioltreatment ( Figure 6B). This was associated with a concomitant decrease in IkB-a phosphorylation indicating increased stabilization of IkB-a after exposure to honokiol. Altogether, our data indicate that honokiol suppresses constitutive activation of NF-kB in pancreatic cancer cells.

Honokiol chemosensitizes the pancreatic cancer cells for gemcitabine toxicity
Gemcitabine is the only FDA-approved chemotherapeutic drug against pancreatic cancer; however, it remains minimally effective due to chemoresistance [3,5,10]. Since activation of NF-kB is considered as one of the mechanisms potentiating chemoresistance, we examined if honokiol would act as a chemosensitizer in pancreatic cancer cells. Pancreatic cancer cells (MiaPaCa and Panc1) were treated with gemcitabine alone or in combination with to sub-IC 50 concentrations of honokiol and effect on growth inhibition was examined using cell viability assay. Our data demonstrated that gemcitabine inhibited the growth of pancreatic cancer cells in a dose-dependent manner and combined treatment with honokiol led to a significant reduction in the IC 50 of gemcitabine ( Figure 7A). At 10 and 20 mM doses of honokiol, respectively, a ,1.53 and 2.41 fold (in MiaPaCa) and ,1.40 and 2.08 fold (in Panc1) decrease in IC 50 of gemcitabine was observed signifying the chemosensitizing effect of honokiol ( Figure 7A). To identify a role of NF-kB, we examined its cellular localization in gemcitabine (alone or in combination with honokiol)-treated pancreatic cancer cells. Our data displayed an enhanced accumulation of NF-kB in nuclear compartment and a concomitant decrease in cytoplasmic fraction with increasing doses of gemcitabine in both MiaPaCa and Panc1 cells ( Figure 7B). Notably, we observed that honokiol (even at 20 mM dose) was effective in inhibiting the gemcitabine-induced activation of NF-kB in both MiaPaCa and Panc1 cells ( Figure 7C). These findings clearly suggest that honokiol potentiates the anti-tumor efficacy of gemcitabine by acting as a chemo-sensitizer in pancreatic cancer cells.

Discussion
Pancreatic cancer remains a devastating malignancy due to lack of effective therapy for treatment [3]. The present study demonstrated that honokiol (a natural biphenolic compound) is effective in suppressing the growth of human pancreatic cancer cells (MiaPaCa and Panc1) due to its cytostatic and cytotoxic properties. Furthermore, our studies provided evidence for a role of honokiol in chemosensitizing the pancreatic cancer cells to gemcitabine toxicity. Honokiol inhibited NF-kB activity and caused altered expression of many cell cycle and survivalassociated proteins to confer its growth suppressive and chemosensitizing effects in pancreatic cancer cells.
Deregulated growth in cancer cells is often attributed to loss of control in proliferative and apoptotic pathways [24]. In fact, molecular studies have revealed that the expression of cell cycle regulators and proteins associated with cell survival is frequently altered in multiple human cancers [25][26][27]. Cell cycle is regulated by concerted actions of cyclins, cyclin-dependent kinases (Cdks) and Cdks inhibitors [26,28]. We observed that the treatment of pancreatic cancer cells with honokiol resulted in G 1 -phase arrest of cell cycle progression, along with reduction in cyclin D1, cyclin E, Cdk2 and Cdk4 and increase in p21 and p27 at the protein level. Cyclin D1 and its catalytic partner Cdk4 dominate in G 1 phase, whereas, cyclin E and Cdk2 complex regulates the cell-cycle progression from G 1 to S [26,28]. Therefore, our findings indicate that the honokiol-induced arrest of pancreatic cancer cells in G 1 cell cycle phase might be mediated through the downregulation of cyclins and Cdks along with the upregulation of p21 and p27 proteins, which form heterotrimeric complexes with G 1 -S Cdks and cyclins to inhibit their activity [29]. These results are in accordance with earlier studies on the effect of honokiol in human lymphoid leukemia, squamous lung cancer and breast cancer cells [16,17,30].
Following G 1 -phase cell cycle arrest, cells may either undergo repair or enter the apoptotic pathway to maintain cellular integrity and elimination of erred/mutated pre-malignant and neoplastic cells [31]. Thus, the induction of apoptosis is one of the protective mechanisms against cancer initiation and progression and cancer cells have often acquired resistance to apoptosis [24]. In the present study, we observed significant induction of apoptosis in honokiol-treated pancreatic cancer cells indicating that honokiol is able to potentiate the apoptotic machinery. Cell survival is maintained by a fine balance of the ratios of pro-apoptotic (e.g., Bad and Bax) and anti-apoptotic proteins (e.g., Bcl-2 and Bcl-xL), which control the process of apoptosis through release of caspases [32,33]. Therefore, altered expression of the Bcl-2 family proteins observed upon honokiol treatment of pancreatic cancer cells in a manner that favors the increase in the ratios of Bax/Bcl-2 and Bax/Bcl-xL could underlie the observed apoptotic effect of honokiol. Deregulation of apoptosis-related proteins has also been reported in chondrosarcoma cells after honokiol treatment further supporting its role in apoptosis induction [34].
We also observed inhibition of NF-kB activity upon honokiol treatment, which was associated with inhibition of IkB-a phosphorylation and concomitant increase in its expression. Transcription factor NF-kB is constitutively activated in multiple malignancies and has been reported to be pathologically implicated in pancreatic cancer [18][19][20]. Recent studies have shown that the growth-suppressive effect of honokiol in prostate and colon cancers is mediated through the inhibition of NF-kB [35]. The NF-kB transcription factor is composed of heterodimers consisting of Rel (p65, c-Rel and RelB), p52, and p50 proteins and is localized in the cytoplasm in its inactive form in complex with IkB (inhibitor of NFkB consisting of a and b subunits that masks its nuclear localization signal [36]. Activation of NF-kB is caused, when IkB-a gets phosphorylated by the IKK (inhibitor kinase) complex leading to its ubiquitination and degradation. This results in the release of NFkB from the cytoplasm and transport into the nucleus followed by activation of NF-kB-responsive promoter. NF-kB is known to induce the expression of cyclin D1, Bcl-2 and Bcl-xL (altered upon honokiol treatment in current study) along with an array of proteins involved in cell proliferation and survival  [37,38]. Therefore, it can be suggested that growth suppression of pancreatic cancer cells by honokiol is mediated through the inhibition of NF-kB.
Clinical outcome in pancreatic cancer has remained poor due to advanced and metastatic state of the disease at the time of diagnosis and ineffectiveness of allowable drug-therapy due to chemoresistance [3,5,10]. Currently, gemcitabine is the national standard chemotherapeutic drug for pancreatic cancer treatment. Gemcitabine interferes with DNA synthesis leading to cell cycle arrest and apoptosis in ultimate course [39,40]. One of the mechanisms that limits gemcitabine efficacy is induced activation of NF-kB in response to its treatment [41], which may cause apoptotic delay or suppression. In this study, we have shown chemosensitizing effect of honokiol on pancreatic cancer cells to gemcitabine toxicity. Additionally, we showed that gemcitabine treatment induced nuclear accumulation of NF-kB, which could be effectively inhibited by co-treatment with honokiol. These parallel findings indicate that the inhibition of NF-kB activity may also mediate the chemosensitization of pancreatic cancer cells by honokiol. Indeed, it has been reported earlier that anti-cancer effects of chemotherapeutic agents can be potentiated by inhibition of NF-kB activity [22,41].
In conclusion, we have shown, for the first time, the growth inhibitory and chemosensitizing potential of honokiol in pancreatic cancer. Honokiol causes G 1 phase cell cycle arrest and induction of apoptosis by altering the expression of cell cycle and survival associated proteins. Inhibition of NF-kB may be one of the significant mechanisms in honokiol-induced growth suppressive and chemosensitizing effects in pancreatic cancer cells. We therefore believe that honokiol could be a novel promising natural agent for the treatment of pancreatic cancer and may also serve as a chemosensitizer to improve the therapeutic efficacy of gemcitabine, which is already in clinical use as a therapeutic drug.

Reagents
Dulbecco's modified Eagle's medium (DMEM) and Roswell park memorial institute (RPMI-1640) medium were obtained from Thermo Scientific (Logan, UT). Fetal-bovine serum (FBS) was from Atlanta Biologicals (Lawrenceville, GA). Penicillin, streptomycin and trypsin-EDTA were purchased from Invitrogen (Carlsbad, CA). Honokiol was procured from LKT Laboratories (St. Paul, MN). Gemcitabine was provided by USAMCI pharmacy. FuGENE transfection reagent, phosphatase/protease inhibitors cocktail and cell proliferation reagent WST-1 were procured from Roche Diagnostics (Mannheim, Germany). Propidium iodide/RNAse staining buffer and PE Annexin V apoptosis detection kit were purchased from BD Bioscience (San Diego, CA). Nuclear extract kit was procured from Active Motif, LLC   (Carlsbad, CA). Antibodies against Bcl-2, Bax and p-IkB-a (Ser32/36) (rabbit polyclonal), Bcl-xl and NF-kB/p65 (rabbit monoclonal), and IkB-a (mouse monoclonal) were obtained from Cell Signaling Technology (Beverly, MA). Antibodies against p21, Cdk4 (mouse monoclonal), p27, cyclin D1, cyclin E, Cdk2 (rabbit polyclonal), and horseradish peroxidase-conjugated secondary antibodies were procured from Santa Cruz Biotechnology (Santa Cruz, CA). b-Actin (mouse monoclonal) antibody was purchased from Sigma-Aldrich (St. Louis, MO). ECL plus western blotting detection kit was procured from Thermo Scientific. Figure 6. Honokiol attenuates constitutive NF-kB activation by inhibiting nuclear translocation of NF-kB/p65 in human pancreatic cancer cells. (A) MiaPaCa and Panc1cells (0.5610 6 cells/well) were seeded in 12-well plate. Next day at 60% confluence, cells were co-transfected with NF-kB luciferase reporter and TK-Renilla luciferase (control) plasmids. Twenty-four hours post-transfection, cells were treated with honokiol (20, 40, or 60 mM) for next 24 h. Protein lysates were made and luciferase (Fire-fly; test and Renilla, transfection efficiency control) activity assessed using a dual-luciferase assay system. Data is presented as normalized fold-change in luciferase activity (mean6 SD; n = 3, * p,0.05). (B) Total, nuclear and cytoplasmic extracts were prepared from cells treated with honokiol (20, 40, or 60 mM) for 6 h and expression of NF-kB/p65, p-IkB-a (S32/36) and IkBa was determined by Western blot analysis. b-actin was used as a loading control. Intensities of the immunoreactive bands were quantified by densitometry. Normalized densitometry values are indicated at the top of the bands indicating a decreased localization of NF-kB/p65 in nucleus with a concomitant increase in cytoplasm. In contrast, expression of p-IkB-a was decreased leading to increased levels of IkB-a. Altogether, these data clearly suggest that honokiol inhibits NF-kB activity through stabilization of IkB-a.

Cell culture and treatments
The human pancreatic cell lines MiaPaCa and Panc1 (ATCC, Manassas, VA) were maintained in culture as adherent monolayer in RPMI-1640 and DMEM respectively, supplemented with 10% (v/v) FBS, penicillin (100 units/mL) and streptomycin (100 mg/ mL). Cells were maintained in 5% CO 2 humidified incubator at 37uC. Growth medium was changed every 3 day and cells were split (1:3) when they reached 80% confluence. For treatments, stock solution of honokiol (10 mmol/L) was prepared in DMSO, stored at 220uC, and diluted with fresh complete medium immediately before use. Cells were treated with various concentrations of honokiol alone, gemcitabine alone or in combination (as specified in the figure legends). An equal volume of DMSO (final concentration, ,0.1%) was added to the control.

Cell growth assay
Cells were seeded in 96 well plates (1610 4 cells/well) a day prior to treatments. Cell viability in the treated cells was examined after 24-72 h by using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1, 3-benzene di-sulfonate) assay kit as per manufacturer's instructions with appropriate controls. This assay is based on the cleavage of WST-1 in metabolically active cells to form water-soluble formazan. The absorbance of the formazan was measured at a wavelength of 450 nm, with background subtraction at 630, using a Bio-Rad Benchmark microplate reader (Bio-Rad Laboratories, Hercules, CA). Growth was calculated as percent viability = [(A/B)6100], where A and B are the absorbance of treated and control cells, respectively.

Cell-cycle analysis
The effect of honokiol treatment on cell cycle progression was determined by flow cytometry following staining with propidium iodide (PI). In brief, cells (1610 6 cells/well) were seeded in 6 well plate and synchronized by culturing them in serum free media. After 48 h, medium was replaced with complete medium containing desired concentrations of honokiol or DMSO. Floating and attached cells were collected after 24 h of treatment and fixed in 70% ethanol overnight at 4uC. The cells were then stained with propidium iodide, using PI/RNase staining buffer for 1 h at 37uC. Stained cells were analyzed by flow-cytometry on a BD-FACS  viability was measured by WST-1 assay. Data are presented as relative percent viability with respect to untreated or honokiol only-treated cells to control for the growth suppressive effects of honokiol (mean6 SD; n = 3. *, p,0.05). A significant decrease in IC 50 values of gemcitabine was observed in cells co-treated with honokiol. (B) Cells were treated with gemcitabine (5, 10 and 20 mM) for 6 h and expression of NF-kB/p65 in nuclear, cytoplasmic and total cell lysates was determined by Western blot analysis. Gemcitabine treatment of cells resulted in enhanced nuclear accumulation of NF-kB/p65 in a dose dependent manner (C) Nuclear and cytoplasmic extracts were prepared from cells treated with gemcitabine (10 mM), honokiol (20 mM) alone or in combination for 6 h. Expression of NF-kB/p65 in nuclear, cytoplasmic and total cell lysates was determined by Western blot analysis. Data shows that honokiol co-treatment restricted gemcitabine-induced NF-kB/p65 nuclear accumulation. Together, these findings suggest that honokiol acts as a chemosensitizer and increases the cytotoxic effects of gemcitabine in pancreatic cancer. doi:10.1371/journal.pone.0021573.g007 Canto TM II (Becton-Dickinson, San Jose, CA) to calculate the percentage of cell population in various phases of cell cycle using Mod Fit LT software (Verity Software House, Topsham, ME).

Apoptosis analysis
MiaPaCa and Panc1 cells were seeded (1610 6 cells/well) in 6 well plate. After overnight incubation, cells were treated with either control vehicle (DMSO) or various concentrations of honokiol for 24 h. Following treatment, cells were harvested, and stained with 7-Amino-Actinomycin (7-AAD) and PE Annexin V, using PE Annexin V Apoptosis Detection Kit I, followed by flow cytometry. Percentage of cell population in apoptosis was calculated using Mod Fit LT software.

Nuclear and cytoplasmic fractionation
The preparation of cytoplasmic and nuclear extracts was performed using the Nuclear Extract Kit. In brief, cells were washed following treatment with 1 mL ice-cold PBS/phosphatase inhibitors, lysed in 500 mL hypotonic buffer and then centrifuged at 14,000 g for 30 s at 4uC. After collecting supernatant (cytoplasmic fraction), pellets were resuspended in 50 mL complete lysis buffer, and centrifuged at 14,000 g for 10 min at 4uC, and supernatant (nuclear fraction) were stored at 280uC.

NF-kB transcriptional activity assay
To measure the NF-kB transcriptional activity, pancreatic cancer cells were seeded (0.5610 6 cells/well) in 12-well plate. After 60% confluence level, the cells were transiently transfected with 1 mg of NF-kB -luciferase promoter-reporter construct (pGL4.32 [luc2P/NF-kB -RE/Hygro]) and 0.5 mg of control reporter plasmid (pRL-TK), containing Renilla reniformis luciferase gene downstream of the TK promoter. Transfections were carried out using FuGENE as a transfection reagent according to the manufacturers' recommendations. Twenty-four hours after transfection, the cells were treated with honokiol for next 24 h, washed with ice cold PBS, and harvested in reporter lysis buffer. Luciferase activity was measured using the Dual Luciferase Assay System. All experiments were carried out in triplicate and relative luciferase activity reported as the fold induction after normalization for transfection efficiency.

Statistical analysis
All the experiments were performed at least three times, independently. A logistic regression model was fit to the data using R statistical software to calculate the IC 50 . The data obtained were expressed as 'mean 6 standard deviation'. Wherever appropriate, the data were also subjected to unpaired two tailed Student's t-test. A value of p,0.05 was considered as significant.