Background
Epstein-Barr virus (EBV), a γ-herpesvirus, causes asymptomatic infection in about 95% adults in the world [
1]. EBV sets up two types of infected situation in host cells, including latent infection and lytic infection [
1]. EBV replication is found in both B cells and epithelial cells. The potential oncogenic effect of EBV makes normal cell cancerization and eventually results in multiple human malignancies such as Hodgkin’s lymphoma, Burkitt’s lymphoma, nasopharyngeal carcinoma (NPC), and gastric carcinoma.
Epstein-Barr nuclear antigen 1 (EBNA1) is the only protein expressed in all three types of latent infection. Through binding to the EBV latent origin of replication (
OriP) [
2], EBNA1 regulates EBV’s DNA synthesis and the sequential partitioning of newly synthesized viral plasmids to daughter cells [
3,
4]. EBNA1 also plays an important role as an active transcription factor within viral and cellular proteins expression. EBNA1 effectively increases the activation of the family repeats element in
OriP and promotes the expressions of latent genes of EBNAs and latent membrane proteins (LMPs) [
5,
6]. Moreover, EBNA1 can contribute to activator protein 1(AP-1) activity by inducing the transcription of the AP-1 subunits c-Jun and activating transcription factor (ATF2) [
7], but decreases nuclear factor kappa B (NF-κB) activity through inhibiting the phosphorylation of the I-kappaB kinase (IKK) α/β kinase complex [
8]. By binding to herpesvirus-associated ubiquitin-specific protease(USP7/HAUSP) [
9] or protein kinase-casein kinase 2(CK2) [
10], EBNA1 is considered to cause the destabilization of p53 [
11] or promyelocytic leukemia(PML) proteins in EBV-positive cells. In addition, EBNA1 can assist malignance cells to inhibit apoptosis induced by extrinsic DNA damage drugs through destabilizing both p53 and PML proteins [
10,
11].
Triptolide, a diterpene epoxide of
Tripterygium extracts, has been demonstrated to perform a bioactive spectrum of anti-inflammatory, immunosuppressive, anti-fertility, anti-cystogenesis, and anti-cancer activities [
12]. Studies also reported that triptolide could effectively kill cancer cells originated from different human organizations, including gastric [
13], pancreas [
14‐
16], brain [
17], colon [
18], prostate [
19], blood [
20], breast [
21,
22], and ovary [
23]. It has been reported that triptolide can stimulate the activities of caspase-8, caspase-9, and caspase-3, cleave downstream PARP and activate apoptosis [
24,
25]. Caspase-9-dependent mitochondrial apoptosis pathway, rather thancaspase-8- dependent pathway, has been demonstrated as the primary way of triptolide-induced cell death [
12,
24]. Triptolide can covalently bind to the subunit of the transcription factor TFIIH-XPB and inhibit its downstream gene transcription [
26]. Triptolide decreases the expression of O-GlcNac transferase to influence the distribution of transcription factor specificity protein 1 (SP1) from the nucleus to cytoplasm in pancreatic tumor cells [
13,
16]. Triptolide also exerts a more powerful effect against leukemia when compared with adriamycin and aclacinomycin in the clinical trial [
12]. Our previous studies have indicated that triptolide could kill EBV-positive B cell lymphoma by targeting a viral oncologic protein, the latent membrane protein 1 [
27]. In addition, our another study also indicated that triptolide reduced viral titers of another γ-herpesvirus, Kaposi’s sarcoma-associated herpesvirus (KSHV), by decreasing expression of latency-associated nuclear antigen 1 (LANA1) [
28].
In this present study, our results indicated that triptolide inhibited the proliferation of EBV-positive NPC cells, which mainly targeted in inducing EBNA1 degradation and NPC cells apoptosis in a caspase-9-dependent pathway. Importantly, EBNA1 was critical for NPC cells to resist caspase-9-dependent apoptosis induced by triptolide. Finally, we revealed that triptolide significantly inhibited the growth of xenografted tumor induced by HONE1-Akata cell in BALB/c nude mice.
Methods
Cell lines and reagents
EBV-positive NPC cell lines (HONE1/Akata, HK1/Akata, and C666–1) were kindly provided by Professor S.W. Tsao (The University of Hong Kong, Hong Kong, China). An EBV-negative NPC cell line, CNE1, was kindly given by Professor. Ya Cao (The University of Zhongnan, Chang Sha, China). Human renal embryonic 293 T cells were obtained from Professor. Zhanqiu Yang (Wuhan University, Wuhan, China).HeLa cells were kindly given by Professor Hui Li (Wuhan University, Wuhan, China). All cell lines were cultured at 37 °C with a humidified atmosphere of 5% CO2 in growth RPMI-1640 media (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA). G418 (400 ng/ml) was additionally added into the medium of HONE1/Akata and HK1/Akata cells to maintain the stability of the recombinant EBV genomes. HeLa and 293 T cells were cultured in DMEM (Hyclone, USA) containing 10% FBS. Triptolide (Sigma, St. Louis, MO, USA), MG-132, 3-MA (Calbiochem, Billerica, MA, USA), cycloheximide (CHX) (Sigma, USA) and 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma-Aldrich) were dissolved in dimethylsulfoxide (DMSO), and were diluted to working concentration with PBS before use. Sodium butyrate (SB; Sigma-Aldrich) was dissolved in PBS directly.
CNE1/Akata cell line was made as described below. HONE1/Akata cells were induced to the lytic form by adding TPA (40 ng/ml) and SB (3 mM) into culture medium for 48 h in order to produce virions. The cell culture medium was collected. After centrifugation at 2000 rpm for 5 min, the supernatant containing virions was used to infected CNE1 cells. At 24 h post-infection, G418 was added into the medium to get a concentration of 1000 ng/ml. After 24 h, uninfected cells would die. The living cells were continuously cultured in medium containing G418 (400 ng/ml). After stably growing for 5 generations, CNA1/Akata cells were permitted for further experiments.
Cell viability assay
HONE1/Akata, HK1/Akata, C666–1, and CNE1 cells (1 × 104 cells/well) were placed in 96-well plates and treated with DMSO control (0.01%) or increasing concentrations of triptolide (25, 50, 100, or 200 nM) for 24 and 48 h. Ten microliters of the Cell Counting Kit-8 (DOJINDO, Tokyo, Japan) reagent were then added to each well and the plates were incubated at 37 °C for 1 h in dark. The optical density (OD) value was detected at an absorbance of 450 nm using an ELx800 microimmunoanalyser (BioTek Instruments, Inc., Winooski, VT, USA).
HONE1/Akata, HK1/Akata, C666–1, and CNE1 cells were placed in 35 mm culture dishes (500 cells/dish) and cultured in standard medium with DMSO control (0.01%) or triptolide (1, 2, or 5 nM) for 2 weeks. Colony formation units were stained with 0.5% (w/v) crystal violet prepared in 0.6% (v/v) glutaraldehyde solution for 1 min, and then cell colony in culture dishes were photographed.
Cell cycle analysis
HONE1/Akata and HK1/Akata cells were seeded in 6-well plates and treated with DMSO control (0.01%) or triptolide (50 nM) for 24 h. NPC cells were digested with 0.25% EDTA-trypsin (Gbico, USA), and 2 × 105 cells were washed 2 times with cold PBS solution, and then resuspended with precooled75% ethanol. After preserved at − 20 °C for 24 h, cells were centrifuged at 1000 rpm for 5 min and resuspended in 0.5 ml cold PBS. Cells were mixed with reagent A (Multisciences, ShangHai, China). Following incubation in dark at 4 °C for 30 min, the whole cells were analyzed immediately by a Beckman-Coulter system (EPICS Altra II; BeckmanCoulter, Fullerton, CA, USA).
Apoptosis analysis
The apoptosis levels ofHONE1/Akata and HK1/Akatacells were evaluated with Annexin V-FITC/7-AAD apoptosis detection kit (Multisciences, Shanghai, China) as manufacturer described. Briefly, cells were placed in 6-well plates and treated with vehicle control (0.01% DMSO) or triptolide (100 or 200 nM) for 24 h. Cells were digested and 2 × 105 cells were washed 3 times with PBS and resuspended in 500 μl of 1× binding buffer, followed by adding 5 μl of Annexin V-FITC and 10 μl of 7-AAD, and incubating in dark at room temperature for 30 min. The whole cells were analyzed immediately by a Beckman-Coulter system (EPICS Altra II; BeckmanCoulter, Fullerton, CA, USA).
Cell transfection
Plasmid pSG5-EBNA1 (P-ala) containing the full-length EBNA1 was constructed by Neuron Biotech Corporation (Shanghai, China). PGEM-EBNA1 (V-val) was a gift from Professor Yixin Zeng (Sun Yat-Sen University, Guangzhou, P.R. China) and described in previous study [
29]. The plasmids were isolated using the plasmid DNA extraction kit (Cat No. CFLKP001–50, Chunfenglv Biomedical Technology, Beijing, P. R. China) according to the manufacturer’s instructions.
EBNA1 siRNA was designed according to the previous study, and the siRNA target DNA sequence of EBNA1 was 5’-GGACTACCGACGAAGGAAC-3′ [
30]. The sequence was synthesized by GenePharma (Suzhou, China). For transfection, cells were placed in 6-well plates with 4 × 10
5cells per well. The cells were transiently transfected by using X-tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland) with pSG5 (the empty vector) or pSG5-EBNA1 as indicated. At 4 h posttransfection, cells were washed with PBS and treated with DMSO (0.01%) or triptolide. After 24or 48 h incubation, cells were prepared for Western blot assay or real-time PCR.
RNA isolation, reverse transcription, and real-time PCR
TRIzol Reagent (Life, USA) was used to extract the total RNA according to the manufacturer’s instructions. The concentration of RNA was determined by Nanodrop 2000 (Thermo, USA). RNAs (1 μg) were reverse transcribedinto cDNA using Reverse Transcription kit (Takara, Tokyo, Japan) as the manufacturers’ instructions. Expression levels of EBNA1 mRNAs were quantified by the CFX96 Real-Time PCR Detection System using an SYBR Premix Ex Taq kit (Takara, Tokyo, Japan). The primers were as follows: EBNA1 forward, 5’-TCATCATCATCCGGGTCTCC-3′; EBNA1 reverse, 5’-CCTACAGGGTGGAAAAATGGC-3’;GAPDH forward, 5’-GGTGGCTTCTGACTTCAACA-3′; GAPDH reverse, 5’-GTTGCTGTAGCCAAATTCGTTGT-3′. The EBNA1 levels were normalized to the housekeeping gene GAPDH. All experiments were repeated independently 3 times.
Western blot assay
Cells treated in different conditions were harvested in RIPA lysis buffer (BeyotimeInstitute of Biotechnology, Shanghai, China) supplemented with 0.5% cocktail protease inhibitor (Roche) and 0.5 mMphenylmethylsulfonyl fluoride (PMSF). Followed by storing on ice for 10 min, the cell lysates were collected and sonicated for 15 s. After centrifugation at 12,000 x g for 15 min, the supernatants were collected and transferred into new tubes, and the protein concentration was measured by BCA protein assay (BioRad, USA). Equal amounts of proteins were then mixed with 5× loading buffer (250 nMTris-Hcl (pH 6.8), 0.5% BPB, 50% glycerol, 10% SDS, 5% β-mercaptoethanol) and boiled for 5 min. Then the mixtures were subjected to 10% SDS-PAGE gels in running buffer and subjected to immunoblot analyses.
The primary antibodies used were as follows: GAPDH (Cat No. 10494–1-AP, 1:5000; Proteintech, Wuhan, China); β-actin (Cat No. CFLKT001, 1:10,000, Beijing Chunfenglv Biomedical Technology Co., Ltd., Beijing, China); caspase-3 (Cat No. 9668, 1:1000; Cell Signaling Technology, USA); cleaved caspase-3 (Cat No. 9664, 1:1000; Cell Signaling Technology, USA); caspase-9 polyclonal antibody (Cat No. A2636, 1:1000; ABclonal, Boston, UK); cleaved PARP-1(Cat No. sc-56,196, 1:500; Santa Cruz Biotechnology, USA); P53 (DO-1) (Cat No. sc-126, 1:500; Santa Cruz Biotechnology, USA); Lamin-A (Cat No. 4777; 1:2000, Cell Signaling Technology, USA); Beta-Tublin (Cat No. 4777; 1:3000, Cell Signaling Technology, USA). Western blot gray values were determined by ImageJ software (National Institutes of Health, USA).
Hoechst 33,258 staining
Cell nuclear fragmentation was examined by Hoechst 33,258 (Beyotime Biotechnology, China). HONE1/Akata cells (4 × 105) were placed in 6-well plate with a glass slide in the well, then treated with DMSO control (0.01%) or triptolide (100 nM) for 24 h. Cells were fixed for 30 min at room temperature by using 4% paraformaldehyde, then washed 3 times with PBS. Followed by permeabilization with 0.1% TritonX-100 in PBS at room temperature for 15 min, cells were then washed 3 times with PBS. Then, cells were blocked with 5% bovine serum albumin (BSA) in PBS for 30 min and washed 3 times with PBS. Cells were stained with Hoechst 33,258 in dark for 5 min, and washed 3 times with PBS. The slides were covered with glycerinum and observed using a fluorescence microscope.
Peripheral blood mononuclear cell (PBMC) isolation
Patient samples were obtained from Clinical Laboratory of Renmin Hospital of Wuhan University. Patients have been informed of the contents of the assay. All the selected patients were detected to carry EBV in plasma and were confirmed as EBV-positive patients. PBMC was isolated from whole blood by Ficoll-Hypaque density gradient centrifugation according to manufacturer’s instructions (Sigma, USA) and cultured in RPMI 1640 containing 10% FBS (Hyclone, USA), 1% glutamine (Sigma, USA), and 1% penicillin/streptomycin (Sigma, USA).
Immunohistochemistry
Immunohistochemistry was performed using antibodies against EBNA1 or cleaved caspase-3 under the manufacturer’s instructions. The tissue sections were fixed with 10% neutral buffered formalin for 12 h. After dehydration, the tissue sections were paraffin-embedded. The paraffin section was deparaffinized using xylene for 20 min and then permeated in gradient concentrations of alcohol. For antigen retrieval, the tissue section was boiled in citric acid (pH 6.0) for 20 min. Endogenous peroxidase activity was quenched with 3% H2O2 for 10 min. Then the slide was blocked in goat serum for 1 h at room temperature, followed by incubating with primary antibodies (EBNA1, Cat No. 8329, 1:1000, Abcam, USA; cleaved caspase-3, Cat No. 9664, 1:1000; Cell Signaling Technology, USA) overnight at 4 °C. After incubated for 15 min at room temperature with secondary antibody (MaxVision™ Kits, MXB, China) conjugated with horseradish peroxidase-labeled polymer, tissue section was incubated for 1 min with diaminobenzidine. The section was counterstained with hematoxylin lightly.
Immunofluorescence assay
The pre-process of immunofluorescence assay was performed as described in the part of immunohistochemistry. After incubation with primary antibody (EBNA1, Cat No. 8329,1:1000,Abcam, USA) for 30 min at room temperature, the slides were incubated for 50 min at 37 °C with the Alexa Fluor 488-conjugatedsecondary antibody (goat anti-mouse, cat. no. A32723, 1:100, Invitrogen, USA). The morphology of cell nuclei was observed by DAPI staining. Images were photographed and synthesized by MicroPublisher (Q-IMAGING, Canada).
Animal studies
HONE1/Akata cells (1 × 107) were inoculated subcutaneously into both flanks of 4-week-old male BALB/c nude mice (purchased from the ABSL-3 animal lab at Wuhan University). Seven days later, when tumor became to be palpable, mice (five per group) were treated with a single intraperitoneal injection of triptolide at 0.4 mg/kg or DMSO daily. After 21 days of treatment, mice were sacrificed by cervical dislocation. Mice weights and tumor sizes were measured, and tumor volume was calculated as 0.5 × length × width× width. Tissue samples were collected and fixed in 10% neutral buffered formalin, embedded, and sectioned at a 5 μm of thickness.
Nuclear and cytoplasmic protein extraction
HONE1/Akata cells (1 × 106) were placed in 6 cm plates and cultured for 48 h. Then the cells were subjected to extraction of nuclear and cytoplasmic proteins by NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo, USA). The detailed process of extraction was performed according to the introduction of manufacturer. After protein concentration was determined, the nuclear and cytoplasmic proteins were mixed with 5× loading buffer and boiled for 5 min, and subjected to Western blot assay.
Immunohistochemistry analysis of EBNA1 and Ki67 expression in NPC biopsies
All the NPC biopsies and correlative clinical data were obtained from the department of pathology, Renmin Hospital of Wuhan University. Expression of EBNA1 and Ki67 was analyzed by two pathologists, Dr. Heng Zhou and Dr. Wen Liu (department of pathology, Renmin Hospital of Wuhan University).
Statistical analysis
Data were shown as the mean ± standard deviation (mean ± SD) and analyzed by Student’s t-test using GraphPad Prism for Windows version 5.0 (GraphPad Software, La Jolla, USA), and p-values < 0.05 were considered as statistically significant.
Discussion
As reported, EBV infection is significantly associated with increased risks and poor prognosis of NPC [
36]. The main function of EBNA1 is to regulate DNA synthesis of EBV and maintain mitotic segregation of EBV episomes to daughter cells [
2‐
4]. Our previous study has proposed EBNA1 as a new molecular target for antiviral and anticancer treating strategies [
33‐
35,
37]. In this study, we found that a traditional extract of herbal medicine, triptolide, effectively suppresses NPC cell growth and induces NPC apoptosis in vivo and in vitro. Besides, the low-toxicity triptolide decreased EBNA1 expression in EBV-positive PBMCs from patients. Triptolide significantly decreased the expression of EBNA1 through a proteasome-ubiquitin pathway. Furthermore, we found that over-expression of EBNA1attenuates the caspase-9-dependent apoptosis induced by triptolide in NPC cells. In addition, EBNA1 was expressed in 100% NPC samples from patients and the nuclear distribution of EBNA1 indicated a low growth speed of NPC.
Triptolide is the most effective bioactive compound from
Tripterygium wilfordii extracts [
38]. Studies have suggested that triptolide killed cancer cells originated from blood, ovary, breast, lung prostate, and brain with IC
50 values ranging from 2.5 to 50 nM [
12]. Here, our results showed that the IC
50 of triptolide acting on NPC cells were from 1.12to 75.56 nM. Our previous study found that the cell cycle of EBV-positive B lymphoma cells was retarded with a reduction in S phase [
27]. Here, we observed that the cell cycles of EBV-positive epithelial NPC cells treated with triptolide were ceased in S phase, which indicates that triptolide may encumber DNA synthesis of EBV-positive NPC cells. Some studies have suggested that triptolide induces caspase-8, − 9, and − 3 activation and then activates downstream PARP in different cells [
24,
25]. However, more evidence indicated that the essential apoptotic pathway is mitochondrial pathway rather than death receptor pathway [
12,
24,
39,
40]. Our findings suggested that NPC cells were induced to apoptosis through a caspase-9 pathway with triptolide treatment. P53 was reported to be necessary for triptolide to induce apoptosis in some cancer cells [
41,
42]. Our results demonstrated that triptolide actives p53-dependent apoptotic pathway in NPC cells, which was similar to other cancer cells treated with triptolide [
41,
42].
Triptolide was reported to cause transcription inhibition by targeting the largest subunit of RNA polymerase II (RPB1)-XPB and then induce apoptosis or immune/inflammatory responses [
26]. Our previous study found that the transcription level of LMP1 was inhibited by triptolide in B lymphoma cells [
27]. Interestingly, we first found that triptolide increased the transcription levels of EBNA1 in NPC cells, but decreased protein level of EBNA1. Our previous study has certificated that triptolide inhibited LANA1, a critical latency antigen protein of KSHV, in KSHV-associated primary effusion lymphoma cells through proteasome-ubiquitin pathway [
28]. Hence, we speculated whether triptolide has the similar effect on EBNA1. Although the exact mechanism of how triptolide inhibits EBNA1expression still remains unclear, our results that triptolide induced EBNA1instability and degradation through proteasome-ubiquitin pathway suggested triptolide decreased EBNA1 expression through the post-translation pathway. Previous studies found that Gly-Ala repeat sequence plays important roles in inhibiting or abrogating EBNA1 from degradation by interfering its interaction with the 26S proteasome [
43,
44]. However, these conclusions were challenged when EBNA1 was artificially fused an Ub to its N terminus, it was efficiently and completely degraded which suggested that GAr can’t prevent Ub-EBNA1 from being degraded. In addition, EBNA1 and EBNA1ΔGAr had the similar half-life, which suggested that ΔGAr does not increased the stability of EBNA1 [
45‐
47]. Interestingly, our results showed that high concentration and long-time treatment of MG-132 significantly increased EBNA1 expression, suggesting that EBNA1 degrades through proteasome-ubiquitin pathway, which is consistent with the finding of Chrysoula and his colleagues [
46]. Similarly, the studies have reported that triptolide could induce degradations of various critical proteins from host or virus by promoting proteasome-ubiquitin pathway, including α-synuclein [
48] and Tat [
49].
Apoptotic protease activating factor-1(Apaf1) and caspase-9 dependent mitochondrial apoptosis is activated to response to intracellular stress factors and ultimately leads to DNA damage and cell death. Our results firstly reported that EBNA1 inhibited the triptolide-induced apoptosis through inhibiting the caspase-9-dependent mitochondria apoptosis. Notably, our results showed that this inhibitory effect didn’t rely on EBV genome and suggested that EBNA1 is an anti-apoptosis protein acting on mitochondria apoptosis pathway. V-val EBNA1 is the predominant subtype in NPC patients living in southeast China [
31,
32]. Hence, we compared the anti-apoptosis effect of V-val with that of P-ala subtype of EBNA1. As speculated, V-val EBNA1 had the anti-apoptosis effect similar to P-ala EBNA1. Our previous studies have reported that EBNA1 could assist malignance cells to inhibit berberine [
34] or 17-DMAG [
33]-induced cancer cells death. Here, both P-ala and V-val EBNA1 attenuated the killing effects of triptolide on EBV-positive and EBV-negative NPC cell lines, suggesting that targeting on EBNA1 is a substantial strategy of treating EBV-positive NPC. It is a further reasonable interpretation how EBNA1 resists NPC cells death. However, the exact molecular mechanism how EBNA1 inhibits procaspase-9 split into active caspase-9 is still unclear. In addition, proteomic methods revealed that EBNA1 binds to a cellular ubiquitin-specific protease -USP7/HAUSP. Because USP7can stabilize p53 and Mdm2 by interacting with these proteins, EBNA1 can competitively bind to the binding pocket in the N-terminal domain of USP7 and ultimately cause p53 destabilization in EBV-positive cells [
9,
11]. By inducing degradation of PML proteins, EBNA1 binds to USP7 and the host CK2 kinase and recruits these proteins to PML nuclear bodies and causes PML nuclear bodies disruption [
9,
10,
50]. These mechanisms contribute to elucidating why EBV-positive cancer cells can resist DNA damage and apoptosis after treatment with DNA damaging agents, which may result in the development of NPC and gastric cancer.
The effect of triptolide on EBV-positive NPC cells was further confirmed by the animal experiments, since HONE1/Akata cell-induced xenograft tumors in BALB/c nude mice were significantly inhibited by triptolide. One group found that the positive rate of EBNA1 was about 92.5% in Indian population [
51]. Here, the expression of EBNA1in NPC biopsy of Chinese population was first investigated. Our present data showed a higher sensitivity (100%) in EBV-positive tumor tissue of Chinese people, which suggested that detecting EBNA1 by immunohistochemistry assay may be a possible method to determine EBV infection in clinical application. Furthermore, our results showed that there were two statuses of EBNA1 expression. At all the biopsies, EBNA1 was expressed in cytoplasm in cancer cells. However, EBNA1 could be expressed in both cytoplasm and nucleus in some cases (23.36%). These results which were consistent with previous studies suggested EBNA1 may exert different functions in tumor cells based on its various distributions [
51,
52]. Importantly, the result that EBNA1 expressed in cells nuclear revealed a low proliferation speed of cancer cells. Limited studies have focused on the function of EBNA1 in cytoplasm. As a transcriptional factor, EBNA1 could inhibit NF-κB pathway in carcinoma cell lines by decreasing the phosphorylation of IKKα/β, and negatively modulate oncogenesis [
8]. Ki67 expression is upregulated by EBNA1 in LCL cell lines [
53]. It’s consistent with our results that EBNA1 expressed in the nucleus was associated with a low tumor proliferation. Interestingly, the expression of EBNA1 in cytoplasm can only be found in cells of squamous cell carcinoma. In contrast, the expression of EBNA1 was found in nucleus of normal tissues. However, due to the restricted numbers of clinical samples, much more clinical samples deserve to be examined to confirm the phenomenon.
Our previous studies have suggested that low-toxicity 17-DMAG reduced the expression of EBNA1 in LCLs and inhibited the growth of tumor cells [
33]. Here in this study, our results found that low-toxicity triptolide decreased EBNA1 expression in EBV-positive PBMCs. Although triptolide showed a super anti-tumor activity compared with adriamycin or aclacinomycin in clinical research, serious toxicities restricted the further clinical application of triptolide [
12]. Overall, our results have indicated that triptolide inhibits EBNA1 expression at a low concentration, suppresses growth of NPC cells but has no significant toxic effect on PBMCs. These results suggested that a safe concentration of triptolide can be used in treatment of NPC in the future.
Acknowledgments
We thank S.W. Tsao (The University of Hong Kong, Hong Kong, China), Ya Cao (The University of Zhongnan, Chang Sha, China), Zhanqiu Yang (Wuhan University, Wuhan, China), HuiLi (Wuhan University, Wuhan, China) and Yixin Zeng (Sun Yat-Sen University, Guangzhou, China) for providing cell lines or plasmid.