Trichothecin Induces Cell Death in NF-κB Constitutively Activated Human Cancer Cells via Inhibition of IKKβ Phosphorylation

Constitutive activation of the transcription factor nuclear factor-κB (NF-κB) is involved in tumorigenesis and chemo-resistance. As the key regulator of NF-κB, IKKβ is a major therapeutic target for various cancers. Trichothecin (TCN) is a metabolite isolated from an endophytic fungus of the herbal plant Maytenus hookeri Loes. In this study, we evaluated the anti-tumor activity of TCN and found that TCN markedly inhibits the growth of cancer cells with constitutively activated NF-κB. TCN induces G0/G1 cell cycle arrest and apoptosis in cancer cells, activating pro-apoptotic proteins, including caspase-3, -8 and PARP-1, and decreasing the expression of anti-apoptotic proteins Bcl-2, Bcl-xL, and survivin. Reporter activity assay and target genes expression analysis illustrated that TCN works as a potent inhibitor of the NF-κB signaling pathway. TCN inhibits the phosphorylation and degradation of IκBα and blocks the nuclear translocation of p65, and thus inhibits the expression of NF-κB target genes XIAP, cyclin D1, and Bcl-xL. Though TCN does not directly interfere with IKKβ kinase, it suppresses the phosphorylation of IKKβ. Overexpression of constitutively activated IKKβ aborted TCN induced cancer cell apoptosis, whereas knockdown of endogenous IKKβ with siRNA sensitized cancer cells toward apoptosis induced by TCN. Moreover, TCN showed a markedly weaker effect on normal cells. These findings suggest that TCN may be a potential therapeutic candidate for cancer treatment, targeting NF-κB signaling.


Introduction
NF-kB transcription factors consist of five homologous subunits: RelA (p65), RelB, cRel (Rel), NF-kB1 (p50 and its precursor p105) and NF-kB2 (p52 and its precursor p100), which function as various homodimers and heterodimers [1,2]. In the canonical NF-kB pathway, cells can be stimulated by different stimuli, including reactive oxygen species, tumor necrosis factor alpha, interleukin 1beta, bacterial lipopolysaccharides, etc. Upon activation, the inhibitory subunit IkBa is phosphorylated by the IkB kinase (IKK) complex, which is then ubiquitinated and degraded through the proteasome pathway, promoting translocation of the p65/p50 complex into the nucleus and activating the expression of downstream genes [3,4].
NF-kB signaling plays an important role in regulating inflammation, tumorigenesis and cancer development [5][6][7]. In a wide variety of cancers-including hematogenous malignancies (such as leukemia, lymphoma, and multiple myeloma), and solid tumors (such as lung, breast and pancreas)-NF-kB is persistently activated [8,9]. Activation of NF-kB up-regulates the expression of antiapoptotic genes encoding Bcl-xL, XIAP, cIAP1 and cIAP2, as well as proliferative genes such as cyclin D1 and IL-6 [10][11][12][13]. NF-kB activity is also closely connected to tumor metastasis and cancer chemo-resistance. NF-kB activation induces the transcription of genes involved in angiogenesis, a critical process in tumor formation and metastasis [14]. Moreover, NF-kB inhibitors enhance sensitivity of cancers to chemotherapeutic agents, such as paclitaxol, TNF-a and TRIAL [15][16][17]. Given the connection between NF-kB and cancer, the development of NF-kB inhibitor holds great potential in suppressing certain types of cancer proliferation as well as improving existing cancer therapies [18,19].
Maytenus hookeri Loes. has been used as a folk remedy for a long time in southwest China because of its anticancer and antiinflammatory activities. Previously, maytansine was identified for its anticancer effect by interfering microtubules [20,21]. The derivative of maytansine, DM1, has been used in trastuzumab emtansine (T-DM1), a novel drug developed for treatment of HER2-positive breast cancer [22]. However, the chemical constituents responsible for the anticancer activities of this plant deserve further exploration.
Trichothecin (TCN) is isolated from the endophytic fungus of Maytenus hookeri Loes. Previous reports of ours and others demonstrated that TCN is involved with some anti-tumor activities, but the anti-tumor profiling or underlying mechanisms of these actions are lacking [23][24][25]. In the present study, we found that TCN inhibits the growth of human cancer cells by inhibiting NF-kB signaling. Our data showed that TCN suppresses the activation of IKKb by suppressing its phosphorylation, inhibits the expression of NF-kB target genes, and induces cell cycle arrest and cancer cell apoptosis. As a novel inhibitor of the NF-kB pathway, TCN may prove to be a potentially promising drug candidate in developing novel cancer therapeutics.

Cell Lines
Human cancer cell lines, HepG2, A549, PANC-1 and HL-60, human embryonic kidney cell line HEK 293T, human bronchial epithelial cell line BEAS-2B and human kidney proximal tubule epithelial cell line HK-2 were purchased from ATCC. Human colonic epithelial cell line (CCD-841-CoN) was kindly gifted by Dr Lin, Li of the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) [26]. Cells were cultured in 5% CO 2 at 37uC in RPMI-1640, MEM or DMEM media containing 10% (v/v) fetal bovine serum (HyClone, Logan, UT).

Compounds
TCN and other tested compounds were extracted from Trichothecium roseum LZ93, an endophytic fungus isolated from Maytenus hookeri Loes., as previously described [24] (structures designated in Figure 1A and the Figure S1A).

Luciferase Reporter Assay
HEK 293T cells were transiently transfected with pNF-kB-Luc and pRL-TK (Promega, Madison, WI) plasmids using Lipofectamine 2000 for 4 h in a 96-well plate. Cells were then preincubated with different concentrations of compounds for 1 h, and subsequently activated with 25 ng/mL TNF-a for 18 h. Luciferase activities were measured using the Dual-Luciferase Reporter Assay kit.

Western Blotting
Total cell lysates were prepared by direct lysis in 26Laemmli buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% b-mercaptoethanol, and 0.004% bromophenol blue). Samples were then fractionated in 12% acrylamide gel, transferred to a PVDF membrane (Bio-Rad), and incubated with specific primary antibodies followed by the corresponding peroxidase-conjugated secondary antibodies. Proteins of interest were visualized by chemiluminescent detection on an ImageQuant LAS mini4000 (GE Healthcare).

Immunofluorescence Staining
For p65 translocation experiment, HepG2 cells were grown on chamber slides for 24 h and pre-incubated with TCN at 37uC for 1 h, followed by TNF-a stimulation for 20 min. For IKKb phosphorylation detection, cells were pre-incubated with TCN at 37uC for 1 h followed by TNF-a stimulation for 10 min. Cells were then fixed in phosphate buffered saline with 4% paraformaldehyde for 20 min, and subsequently permeabilized in phosphate buffered saline with 0.1% Triton X-100. To observe the localization of p65 subunit, cells were incubated with anti-p65 antibody and corresponding FITC conjugated secondary antibody before staining the nuclei with DAPI. For the IKKb phosphorylation detection, cells were incubated with anti-phospho-IKKb antibody and corresponding Alexa Fluor 546 secondary antibody. Images were later observed using a fluorescence microscope (Eclipse Ti, Nikon).

Cell Apoptosis Assay
Cell apoptosis was analyzed using the Annexin V-FITC/PI Apoptosis kit (BD Biosciences, Franklin Lakes, NJ) according to the manufacturer's protocols. Cells were seeded in 6-well plates at a density of 1.2610 6 cells/well. After 24h of compound treatment at the indicated concentrations, cells were collected and then washed twice with cold PBS, and then resuspended in a binding buffer containing Annexin V-FITC and propidium iodine (PI). After incubation for 15 min at room temperature in the dark, the fluorescent intensity was measured using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ).

Overexpression of IKKb CA
The construct driving the expression of a constitutively active IKKb (S177E, S181E) (IKKb CA) was obtained from Addgene (Catalog No.11105) [27]. HepG2 cells were transfected with the plasmids using Lipofectamine 2000. Some 12 h after transfection, cells were treated with TCN for additional 24 h.

IKKb Kinase Activity Assay
The IKKb kinase assay was performed with Z'-LYTE TM Kinase Assay kit following the manufacturer's protocols. In brief, recombinant human IKKb proteins were incubated with the kinase reaction mixture with increasing amounts of TCN or staurosporine, a kinase inhibitor, for 1h. The reaction was then stopped with the stop reagent and the fluorescence was detected with excitation at 400 nm and emissions at 445 nm and 520 nm using the EnVision Multimode Plate Reader.

TCN Inhibits Cell Growth and Induces Apoptosis in NF-kB Constitutively Activated Cancer Cells
We investigated the anticancer activity of TCN by MTT cell viability assay in four cancer cell lines harboring constitutively activated NF-kB [29][30][31][32]. In all tested cell lines, TCN exhibited obvious growth inhibition ( Figure 1B). The IC 50 values in HL-60, HepG2, A549 and PANC-1 cells were 0.18, 0.82, 0.39, and 0.28 mM, respectively. Annexin V-FITC/PI staining was further analyzed for cell apoptosis with flow cytometry. After treatment with 5 mM TCN for 24 h, cell apoptosis in HL-60, HepG2, A549 and PANC-1 cells remarkably elevated to 61.13%, 44.03%, 34.93%, and 24.47%, respectively. Meanwhile, apoptosis in human normal cell lines BEAS-2B, HK-2 and CCD-841-CoN were not affected by TCN treatment, even at the highest concentration 5 mM, suggesting TCN possesses selectivity against cancer cells ( Figure 1C). Western blot analysis also demonstrated that TCN significantly induced the activation of caspase-8 and caspase-3, as well as the cleavage of PARP-1 in the four cancer cell lines. The protein level of Bcl-2, a key regulator of the intrinsic apoptotic pathway, was also down-regulated by TCN treatment. Moreover, the level of survivin, an anti-apoptotic protein, dramatically decreased in TCN treated cells, especially in HL-60 and HepG2 cells ( Figure 1D).

TCN Inhibits NF-kB Signaling and Induces Cell Cycle Arrest at G0/G1
The effect of TCN on NF-kB signaling was tested in HEK 293T cells transiently transfected with an NF-kB reporter (pNF-kB-Luc).The reporter gene expression was clearly activated by TNFa, which was efficiently inhibited by TCN (Figure 2A). To check whether TCN inhibits the intrinsic NF-kB in cancer cells, we studied the expression of NF-kB target genes in the four NF-kB activated cancer cell lines treated with TCN. The protein levels of p65, XIAP, cyclin D1, and Bcl-xL were clearly down-regulated in TCN treated cells ( Figure 2B). Since Cyclin D1 is required for G1/ S cell cycle progression, we investigated the effect of TCN on the cell cycle arrest. HepG2 cells were treated with 2.5 mM TCN. Obvious G0/G1 cell cycle arrest was detected as early as 8h. However, with prolong of the treatment, increase of cells of Sub-G1 phase were observed, indicating cell apoptosis induced by TCN ( Figure 2C).
We further investigated the effects of trichothecolone, a derivative of TCN, on cell growth and NF-kB activity. Trichothecolone is another metabolite isolated from endophytic fungus of Maytenus hookeri Loes., which shares the same parent nucleus with TCN, with the notable exception of a different substituent at 4-OH [24]. Like TCN, trichothecolone also inhibited cell growth and NF-kB reporter activity ( Figure S1), but both activities are much weaker, suggesting that the substituent at 4-OH of this class of compounds is critical for their biological activities.

TCN Inhibits the Phosphorylation and Degradation of IkBa and Blocks p65 Nuclear Translocation
We checked the effect of TCN on the nuclear translocation of p65, one hallmark of the activation of NF-kB signaling. TNF-a treatment induced the translocation of NF-kB from the cytoplasm to the nucleus, and TCN significantly blocked the translocation process in HepG2 cells ( Figure 3A). Overexpression of p65 markedly reversed the inhibitory effect of TCN on the transcription of the NF-kB reporter ( Figure 3B). We then tested the effect of TCN on IkBa phosphorylation and degradation. TCN inhibited TNF-a induced IkBa phosphorylation in a dose-dependent manner and markedly blocked the degradation of IkBa. More poignantly, we found that TCN inhibited p65 phosphorylation at Ser536 (Figure 3C), which contributes to the degradation of IkBa and the activation of NF-kB signaling [33,34].

TCN Inhibits the Phosphorylation of IKKb Induced by TNF-a
As IkBa and p65 are both substrates of IKKb kinase, which is activated by undergoing phosphorylation (Ser 177 and Ser 181) and subsequently phosphorylates IkBa [35,36], we checked the phosphorylation of IKKb in TNF-a treated HepG2 cells by immunofluorescence and western blot analysis. We found that the level of phosphorylated IKKb dramatically decreased following TCN treatment, with the total IKKb protein level unaltered ( Figure 4A and B). To determine whether TCN interferes with the IKKb kinase activity directly, a kinase activity assay was carried out using purified human IKKb recombinant protein using staurosporine, a potent kinase inhibitor, as the positive control. The result showed that TCN did not affect the kinase activity of recombinant IKKb ( Figure 4C). These data demonstrated that TCN inhibits NF-kB signaling by suppressing the activation of IKKb via blocking its phosphorylation.

TCN Induced Cancer Cell Apoptosis is Mediated by Inhibition of IKKb Phosphorylation
TCN induced cell death was investigated in cells overexpressing constitutively active (CA) form of IKKb [27,28]. As shown in Figure 5A, overexpression of IKKb CA in HepG2 cells sufficiently activated NF-kB signaling in luciferase activity assay. TCN treatment antagonized with TNF-a in activation of NF-kB signaling, whereas IKKb CA transfection efficiently reversed the inhibitory effect of TCN. Consistently, overexpression of IKKb CA aborted the TCN induced cell apoptosis in HepG2 cells ( Figure 5B). Meanwhile, western blot analysis showed the deactivation of caspase-3, PARP-1 and upregulation of survivin by IKKb CA ( Figure 5C).
Next, we checked the effect of IKKb knockdown on the apoptotic activity of TCN. Compared with treatment with TCN alone, knockdown of IKKb with siRNA sensitized HepG2 cells to TCN-induced apoptosis, with the apoptotic ratio increasing to 43.11% (18.17% in TCN treatment alone) ( Figure 5D). As shown in Figure 5E, the expression of IKKb in HepG2 was partly decreased by the treatment of IKKb siRNA. Consistent with the increased cell apoptosis induction, down-regulation of the antiapoptotic proteins (survivin, XIAP and Bcl-2) by TCN was enhanced in IKKb knockdown cells, and the cleavage of caspase-3 was increased as well ( Figure 5E). Meanwhile, transfection with a control scrambled siRNA had no effect on the response of HepG2 cells to TCN treatment ( Figure 5D, 5E).

Discussion
Given the connection between NF-kB and cancer, the development of NF-kB inhibitor holds great potential in suppressing certain types of cancer proliferation as well as improving existing cancer therapies. Over the past few decades, a diverse variety of natural and synthetic compounds have been found capable of suppressing NF-kB signaling, but through a variety of different mechanisms. PS-341, a proteasome inhibitor, has been a first-line drug used in treating multiple myeloma for over a decade, which works by inhibiting intrinsic NF-kB activity by blocking IkBa degradation [37]. Meanwhile, Eriocalyxin B, an ent-Kauranoid isolated from Isodon eriocalyx, inhibits NF-kB activation by interfering with the binding of both p65 and p50 to the response element [38].
IKKb plays central role in the activation of both canonical and non-canonical NF-kB signaling pathway. Upon activation, NF-kB signaling leads to auto-polyubiquitination of tumor necrosis factor receptor associated factor 6 (TRAF6). The ubiquitinated TRAF6 then recruits the transforming growth factor-b-activated kinase 1 (TAK1) and the IkB kinase (IKK) complex, which consists of two catalytic subunits, IKK1 (IKKa) and IKK2 (IKKb), and a regulatory subunit, NEMO (NF-kB essential modulator, IKKc), so that TAK1 can phosphorylate and activate IKKb. The autophosphorylation also was reported to be involved in the activation of IKKb [39,40]. Due to frequently observed role that the activation of IKKb plays in cancer generation, proliferation and metastasis, IKKb has been considered a promising drug target for cancer treatments [41,42]. An FDA approved orphan drug to treat pancreatic cancer, CDDO-Me, also known as RTA 402, blocks the NF-kB pathway through direct inhibition of IKKb on Cys-179 [43].
In the current study, we described TCN, a potent NF-kB inhibitor, blocked IKKb activation by suppressing its phosphorylation and subsequently inhibited the expression of the target genes of NF-kB signaling pathway, including XIAP, cyclin D1 and Bcl-xL, which regulate cell survival and cell proliferation. As a result, TCN induced cell apoptosis and cell cycle arrest in NF-kB constitutively activated human cancer cells, without affecting normal cells with low basal NF-kB activity ( Figure S2). Considering the complex events associating with the activation of IKKb, the precise mechanisms how TCN impairs the phosphorylation of IKKb are worthy of further investigation.
Metabolites of endophytic fungi colonizing in herbal medicine have been found to possess various bioactivities [44,45]. Anticancer agents of many host plants are found in metabolites of the endophytic fungi, such as Taxol, which was isolated from endophytic fungus Taxomyces andreanae in Taxus brevifolia [46]. In the present study, TCN, along with 6b-hydroxyrosenonolactone, trichothecolone, roseocardin and roseotoxin B were isolated from endophytic fungus LZ93 of Maytenus hookeri Loes. and were tested for their anticancer activities ( Figure S1). Among the compounds we isolated, TCN proved to be the most potent. These findings indicate that properties of TCN might be one of the potential mechanisms underlying the efficacy and anti-cancer activities of Maytenus hookeri Loes.
Taken on the whole, our findings suggest that TCN, as a potent inhibitor of NF-kB signaling, has promising therapeutic value for cancer treatment and deserves further exploration.