Triptolide Transcriptionally Represses HER2 in Ovarian Cancer Cells by Targeting NF-κB

Triptolide (TPL) inhibits the proliferation of a variety of cancer cells and has been proposed as an effective anticancer agent. In this study, we demonstrate that TPL downregulates HER2 protein expression in oral, ovarian, and breast cancer cells. It suppresses HER2 protein expression in a dose- and time-dependent manner. Transrepression of HER2 promoter activity by TPL is also observed. The interacting site of TPL on the HER2 promoter region is located between −207 and −103 bps, which includes a putative binding site for the transcription factor NF-κB. Previous reports demonstrated that TPL suppresses NF-κB expression. We demonstrate that overexpression of NF-κB rescues TPL-mediated suppression of HER2 promoter activity and protein expression in NIH3T3 cells and ovarian cancer cells, respectively. In addition, TPL downregulates the activated (phosphorylated) forms of HER2, phosphoinositide-3 kinase (PI3K), and serine/threonine-specific protein kinase (Akt). TPL also inhibits tumor growth in a mouse model. Furthermore, TPL suppresses HER2 and Ki-67 expression in xenografted tumors based on an immunohistochemistry (IHC) assay. These findings suggest that TPL transrepresses HER2 and suppresses the downstream PI3K/Akt-signaling pathway. Our study reveals that TPL can inhibit tumor growth and thereby may serve as a potential chemotherapeutic agent.


Introduction
Triptolide (TPL) is a diterpenoid triepoxide derived from the herb Tripterygium wilfordii [1]. TPL has been demonstrated to possess a unique bioactive spectrum of antiinflammatory, immunosuppressive, antifertility, anticystogenesis, and anticancer activities [2]. Recently, it was found that TPL reduces proliferation and induces apoptosis in cancer cells [2][3][4]. Furthermore, it inhibits tumor growth and metastases in mouse xenografts of melanoma, breast cancer, gastric carcinoma, bladder cancer [3], and pancreatic cancer [4]. Previously, we found that TPL has a strong antitumor effect against oral cancer cells through caspase-mediated apoptosis [5]. In addition, TPL circumvents drug resistance and enhances the antitumor effect of 5-fluorouracil [6]. The understanding of the molecular mechanisms by which TPL inhibits cancer cell growth may shed light on cancer therapy.
The HER2 gene, also known as neu (in mouse) or erbB2, encodes a 185-kDa transmembrane receptor tyrosine kinase and is a member of the epidermal growth factor receptor family [7]. Overexpression of HER2 is found in ∼30% of human breast cancers and in many other cancer types [8].

Cell Transfection.
A liposome-mediated transfection method was used to transfect cells. Briefly, plasmids were diluted in Opti-MEM I (Gibco BRL) medium and then mixed with Lipofectamine 2000 (Gibco BRL) at room temperature for 20 min. The lipoplex complex was added to the cells for 6 h, and the cells were refreshed with complete medium.

Cytotoxicity and Viability Analysis by MTT Assay.
To evaluate growth inhibition, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) metabolic assay was performed [14]. Briefly, cells were seeded into 96-well microtiter plates and incubated overnight in 180 μL media containing 10% FBS. For the drug sensitivity assay, cells were treated with TPL at indicated concentrations for 72 h. After treatment, the viable cells were stained with 20 μL of MTT dye (5 mg/mL in PBS; Sigma-Aldrich) for 4 h, and then 170 μL medium was removed. The MTT formazan formed by metabolically viable cells was dissolved in 200 μL DMSO, and the absorbance was read at 545 nm with a reference at 690 nm (Emax, Molecular Device Inc.). All experiments were performed in triplicate and reproduced at least three times.

Quantitative Real-Time Reverse-Transcription PCR (RT-qPCR).
Total RNA was isolated using the Trizol method (Invitrogen, Carlsbad, CA), with homogenization of the cancer cells in Trizol lysis buffer followed by chloroform extraction (Life Technologies). For cDNA synthesis, 5 μg total RNA was reverse transcribed at 50 • C for 60 min using 200 units Superscript III Reverse Transcriptase (Invitrogen). Primer pairs for HER2 (sense 5 -CCC AAC CAG GCG CAG AT-3 ; antisense 5 -AGG GAT CCA GAT GCC CTT GTA-3 ), were used for gene amplification. The SYBR Green Master Mix Kit (Bio-Rad, Hercules, CA) was used for all qPCRs. Briefly, PCR was performed as follows: 94 • C for 2 min, followed by 40 cycles of denaturation at 94 • C for 15 s, annealing at 61.4 • C for 30 s, extension at 72 • C for 45 s, and a final extension at 72 • C for 10 min. Data were collected in triplicate and normalized to GAPDH expression.

HER2
Promoter Activity Assay. The HER2 promoter luciferase assay was described previously [13]. Briefly, cells were cotransfected with pNeulit (containing luciferase gene driven by neu promoter) or HER2 promoter deletion   constructs and pCMV-β-gal (as a reference control for transfection efficiency). Cell lysates were prepared before the luciferase activity assay by following the manufacturer's (Promega) instructions. The measured luciferase activity levels were normalized against the β-galactosidase (β-gal) standard. These experiments were repeated at least four times.

Xenograft Tumor Model.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Defense Medical Center (NDMC). The protocol was approved by the Committee on the Ethics of Animal Experiments of NDMC. Eight-week-old BALB/cAnN.Cg-Foxn1 nu / CrlNarl (National Laboratory Animal Center, Taiwan) mice were maintained in microisolators under specific pathogenfree conditions. These mice were fed sterile food and chlorinated sterile water. Eight mice were divided into two groups; each group of mice was subcutaneously injected with 5 × 10 6 SKOV-3 cells. Four mice were further treated with TPL (0.15 mg/kg BW/day/i.p.), and another four mice were injected daily with a vehicle PBS control. TPL was first injected on day 0 and continuously administered until day 18 in SKOV-3-bearing and control mice, respectively. The size of the transplanted tumors and mice body weight were measured by gauged calipers every 3 days, and the tumor volume was calculated using the formula: volume (V ) = 1/2 × (length×width 2 ). At the end of treatment, the mice were sacrificed, and the tumors were removed, weighed, and photographed.

Immunohistochemical (IHC) Stain and Quantification.
Immunohistochemical staining was done using the avidinbiotin method as described previously [15]. For quantitative evaluation of HER2 expression, at least three representative high-power fields (1000x) of each tumor were immunostained with HER2 monoclonal antibody (Dako, Osaka, Japan) and examined under an Olympus BX-50 microscope (Olympus Optical, Tokyo, Japan) with a photo-fixed halogen light source. A digital camera (Olympus C-2020Z) transmitted high-quality images into a computer. Manual mode was used to ensure that the image intensity was kept at a constant optimal level. Three ordered categories of HER2 expression were then scored 1-3, followed by the commercial HercepTest scoring system [15]. The percentage of Ki-67positive (Dako, Copenhagen, Denmark) cells was determined in each case by counting cells in three contiguous high-power fields using a cell counter until 1000 cells were assessed.

Statistical Analysis.
The results are expressed as the mean ± SEM. A t test was used to determine whether any significant relationships existed among quantitative results. P values <0.05 were considered significant. * P < 0.05; * * P < 0.01.

Downregulation of HER2 by TPL.
As TPL reduces PI3K activity [16], we investigate if TPL downregulates the expression of HER2, which is upstream of PI3K activity, in cancer  [13], SKOV-3 cells were used to explore the mechanism by which TPL suppresses HER2. We demonstrated that TPL inhibits SKOV-3 cell proliferation in vitro at 72 h in a dose-dependent manner (Figure 1(d)). The quantification of these results is shown in Figure 1(e). Moreover, western blot analysis revealed that TPL decreases HER2 expression in SKOV-3 cells in a dose-and time-dependent manner (Figures  2(a) and 2(b)). As mentioned above, our results reveal that TPL is an effective antitumor agent not only in ovarian cancer cells but also in oral and breast cancer cells.

Downregulation of PI3K/Akt Signaling by TPL.
The HER2-stimulated intracellular signaling pathways include the Ras/ERK and PI3K/Akt cascades. Because TPL suppresses HER2 protein expression, we examines if TPL interferes with HER2 and affects these two pathways in ovarian cancer cells. As demonstrated in Figure 2(c), TPL significantly suppresses both total and phosphorylated (activated) HER2 expression in SKOV-3 cells (P < 0.05) after 24 h. However, total ERK and phospho-ERK (activated form of ERK) are not altered by TPL treatment (Figure 2(d)). We then investigated if TPL affects the PI3K/Akt signaling cascade. After 24 h of TPL treatment, phospho-PI3K and phospho-Akt are both dramatically downregulated (Figures 2(c) and 2(d)). More than 50% inhibition by TPL is observed at a concentration of 50 nM (Figure 2(d)). At the same time, PI3K and Akt total protein levels remain unchanged (Figures 2(c) and 2(d)). In summary, these data demonstrate that TPL exerts inhibitory effects on HER2 and the PI3K/Akt signaling pathway.

TPL Represses HER2 Gene Activity.
To elucidate the mechanisms by which TPL downregulates HER2, we assayed HER2 mRNA level over a treatment time course. Consistent with the HER2 protein level, RT-qPCR analysis revealed that HER2 mRNA is significantly downregulated by TPL after 3 hours of treatment (Figure 3(a)). Only about 20% of HER2 mRNA remains after 24 h. The transcriptional activity of the HER2 promoter is also analyzed. In the transient expression assays, using NIH3T3 (Figure 3(b)) as recipient cells, cotransfected with pNeulit and pCMV-β-gal, TPL treatment causes a significant decrease in neu promoter luciferase activity in a dose-dependent manner. Because pNeulit is a mouse neu promoter construct, we cloned the human HER2 promoter which was subsequently divided it into four fragments, F1-F4, containing bp from −1067 to −103, from −871 to −103, from −495 to −103, and from −207 to −103, respectively. These fragments were then subcloned into the luciferase reporter as described in Section 2, and the transcriptional activity of the HER2 reporter constructs was monitored following a transient transfection in NIH3T3 cells. The results shown in Figure 3(c) demonstrate that about 40% of all four promoter activities are suppressed by TPL. Thus, the F4 region may be the target site for TPL. Through bioinformatics research, we identified a putative NF-κB binding site within this region.

TPL Suppresses the Expression of HER2 and Ki-67 In
Vivo. To examine whether TPL inhibits the growth of HER2-overexpressing tumor cells in vivo, tumorigenicity test is performed in athymic nude mice growing SKOV-3 tumor. The mean tumor volumes of nude mice treated with TPL are significantly smaller than those treated with PBS ( Figure 5(a)). These data demonstrate that TPL significantly inhibits the proliferation of HER2-overexpressing tumor cells in nude mouse xenografts. The body weights of these TPL-treated mice were not significantly changed ( Figure 5(b)). Furthermore, we also tested whether TPL inhibits HER2 protein and Ki-67 expression (a proliferation marker) in nude mice. Tumor sections were immunostained for HER2 and Ki-67. We applied the semiquantitative Her-cepTest scoring system to measure the expression of HER2 [15]. The HercepTest score in the PBS control and TPLtreated groups were 3+ and 2+, respectively ( Figure 5(c)).
In an in vitro study, TPL suppressed Akt activity, which is a key mediator of antiapoptosis and cell proliferation. We also examined the Ki-67 proliferation index in these tumor sections. As shown in Figure 5 significantly lower than the PBS-treated control group (16.7% versus 26.5%). These immunohistochemistry data are consistent with the protein levels reported above ( Figures  1 and 2). All together, these data suggest that TPL significantly inhibits HER2 and Ki-67 expression in vivo.

Discussion
TPL, a pure compound extracted from the traditional Chinese medicinal plant T. wilfordii, has significant cytotoxic effects on different types of tumors. The mechanisms of action of TPL, for a long time, were hypothesized to specifically target some transcription factors. It elicits inhibitory effects upon nuclear factor-κB (NF-κB), heat shock transcription factor1 (HSF1), activator protein-1 (AP-1), nuclear factor of activated T-cells (NF-AT), and hypoxia-induced factor-1α (HIF-1α), while it has no impact on cytomegalovirus-luciferase or SV40-luciferase reporters [2]. In this study, we demonstrated that TPL inhibits the proliferation of HER2-overexpressing human ovarian cancer SKOV-3 cells. Further analysis indicated that TPL effectively inhibits HER2 expression in oral, breast, and ovarian cancer cell lines (Figures 1 and 2). In addition, HER2 promoter activity and mRNA level are reduced by TPL (Figures 3(a) and 3(b)), suggesting that TPL downregulates HER2 expression by diminishing HER2 gene transcription. In addition, recently, TPL is demonstrated to cause transcriptional inhibition, initially believed to target specific transcription factors but recently revealed to cause global transcription inhibition via the largest subunit of RNA polymerase II (RPB1). Two pathways are proposed that TPL interacts with XPB and CDK7 to cause transcriptional inhibition. TPL binds to human XPB, a subunit of the transcription factor TFIIH, and inhibits its DNA-dependent ATPase activity, which leads to the inhibition of RNA polymerase II-(RNAPII-)mediated transcription [2,18]. In addition, TPL also triggers CDK7 to phosphorylate Rpb1 and induces degradation of RNAPII [19]. Thus, the downregulation of HER2 and NF-κB gene expression by TPL may be also due to its effects on RNAPII.
In promoter deletion assays, because the transient transfection rate of SKOV-3 cells is low [13], NIH3T3 cells were used as recipient cells to measure luciferase activity. We found that TPL-mediated reduction of HER2 promoter activity is specifically prevented by NF-κB (Figures 4(a) and 4(b)). By comparing the results of Figures 4(a) and 4(b), we discovered that the F4 fragment contains a binding site for NF-κB-p65 but not NF-κB-p50. In addition, Figure 4 also reveals that NF-κB-p65, but not NF-κB-p50, is constitutively expressed in SKOV-3 and OVCAR-3 ovarian cancer cell lines. However, TOV-21G cells express endogenously both NF-κB-p65 and NF-κB-p50.
HER2 activation is often associated with invasion and proliferation through its downstream Ras/Raf/MAPK and PI3K/Akt signaling pathways [9,10]. It is noteworthy that our results (Figures 2(c) and 2(d)) illustrate that in the HER2-overexpressing SKOV-3 (p53-null) cancer cells, PI3K/ Akt signaling is dominantly activated as a result of increased HER2 activation and in the absence of p53, which has been reported to repress PI3K/Akt activity [20,21]. In other words, although MAPK is also activated in ovarian carcinoma cells, it may not be the major pathway for TPLmediated suppression of HER2 signaling in SKOV-3 cells ( Figure 6).
In LPS-stimulated macrophages, TPL downregulates the gene expression of COX-2, iNOS, and IL-1β [22]. Clinical trials using the extract of T. wilfordii or TPL have been reported. Wu and Guo studied the clinical effects of TPL in patients with psoriasis vulgaris and observed about 75% effectiveness [23,24]. Tao et al. conducted a randomized double-blind study to assess the efficacy and tolerability of T. wilfordii root extracts in patients with rheumatoid arthritis (RA) and found therapeutic benefits in 60% of patients with high tolerability at the tested doses [24,25]. In addition to immunosuppressive effects, TPL inhibits proliferation, angiogenesis, and invasion of a variety of cancer cells [2]. Our present work demonstrates that NF-κB protein expression is inhibited by TPL treatment, which is consistent with most of the previous studies [11,26,27]. However, there is also evidence to suggest that TPL inhibits NF-κB transcriptional activity by blocking the association of the p65 subunit with CREB-binding protein/p300 [11]. Thus, TPL may exert dual effects, through interactions with p65/CBP and by reducing p65 protein expression.
NF-κB is a complex consisting of a heterodimer of p50 and p65 subunits. IκB binds NF-κB in the cytoplasm and prevents NF-κB activation. The phosphorylation, ubiquitination, and degradation of IκB lead to the release of the p50-p65 heterodimer, which translocates into the nucleus and then associates with the downstream promoter regions of multiple target genes, including vascular endothelial growth factor (VEGF) [28]. Angiogenesis typically occurs in RA, retinopathy, and tumor growth, as they are collectively called "angiogenic diseases" [29]. Thus, drugs used to treat RA and other angiogenic diseases may possess antitumor activity through antiangiogenic effects. VEGF has been identified as the most potent inducer of angiogenesis. Huang et al. demonstrated a concordant increase in NF-κB activity with elevated VEGF mRNA in ovarian cancer cells, suggesting that the regulation of VEGF by NF-κB is mediated at the transcriptional level [30]. We found that NF-κB induces HER2 gene expression and subsequently stimulates Akt activity (data not shown). Akt has been implicated in the regulation of VEGF, which is activated by the mTOR/p70S6K pathway. mTOR is a 289-kDa phosphoinositide kinase-related serine/threonine kinase [31]. Through the formation of multimolecular complexes, rictor or raptor, the evolutionarily conserved TOR pathway controls many fundamental cellular functions, such as translation initiation, protein stability, transcription of ribosome and stress response genes, ribosomal biogenesis, and transfer RNA synthesis, and thereby plays a central role in the regulation of cell growth, proliferation, and survival [32,33]. Therefore, we assume that TPL can inhibit angiogenesis through suppression of VEGF. Indeed, He et al. demonstrated that T. wilfordii and its terpenoids (TPL) possess antiangiogenic activity [34], which is consistent with our hypothesis.
Increasing evidence indicates that a small subpopulation of tumor cells, called tumor-initiating cells (TIC) or cancer stem cells, is not only responsible for the generation of the phenotypically diverse tumor cell populations but is also capable of long-term self-renewal, thereby supporting the growth and dissemination of cancers [35]. Several recent studies report that HER2 regulates TIC sphere formation and tumorigenicity [35][36][37]. Moreover, Notch, an important receptor in normal and malignant stem cells, may bind and regulate the HER2 promoter [37]. Thus, HER2 may become a superior target for TIC chemotherapy. Another way to identify putative TIC is through incubation with Hoechst dye and analysis by flow cytometry; a small population of cells that does not accumulate dye is called a side population [38]. The side population phenotype is mediated by the ATPbinding cassette (ABC) transporter protein family. These transporters are also called multidrug-resistant (MDR) ABC