PEL cells (BCBL-1, JSC-1 and BC-3) and a Burkitt lymphoma cell line, BJAB, were gifts kindly provided by Professor K. Lan (Institute Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China). P3HR-1 cells (EBV-positive and KSHV-negative) were kindly provided by Professor Y. Cao (Central South University, Changsha, China). Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by using Ficoll-Paque Premium (GE Healthcare, Uppsala, Sweden) according to the manufacturer's instructions. Human renal embryonic 293T cells and HeLa cells, which were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA), were obtained from Professor H. Li (Wuhan University, Wuhan, China).

BJAB, P3HR-1, BCBL-1, BC-3, JSC-1 cells and PBMCs were maintained in RPMI-1640 medium containing 10% fetal bovine serum (FBS; Gibco-BRL, Gaithersburg, MD, USA). HeLa and 293T cells were cultured in DMEM containing 10% FBS. The whole cell lines were cultured at 37°C with 5% CO₂ and 100% humidity.

Triptolide(Sigma, St.Louis, MO, USA),12-O-Tetradecanoyl-phorbol-13-Acetate (TPA; Sigma), cidofovir (CDV; Selleck Chemicals, Shanghai, China), cycloheximide (CHX; Sigma), and MG-132 (Calbiochem, Billerica, MA, USA) were dissolved in dimethylsulfoxide (DMSO). Sodium butyrate (NaB; Sigma) was dissolved in PBS.

Plasmid DNA was purified through columns (Axygen Scientific Inc., Union City, CA, USA) as described by the manufacturer. The plasmid pSG5-LANA1 was constructed by inserting the full LANA1 sequence into the MfeI/EcoRI of pSG5 vector (YouBio, Shanghai, China).

The viability of cells was determined by the Cell Counting kit-8 assay (CCK-8; Dojindo Laboratories, Kumamoto, Japan). In brief, cells were seeded in 96-well plates at a density of 1×10⁴/100 μl culture medium. All the cells were treated with vehicle control (DMSO; 0.006%, vol/vol) or increasing concentrations of triptolide for 24, 48 and 72 h. After different treatments, 10 μl of tetrazolium substrate was added into each well of the plate. The plates protected from light were incubated at 37°C for 1 h. The optical density (OD) was determined at an absorbance of 450 nm using an ELx800 microimmunoanalyser (BioTek Instruments, Inc., Winooski, VT, USA).

For transfection, KSHV-negative HeLa cells were transiently transfected using X-tremeGENE HP DNA Transfection reagent (Roche, Basel, Switzerland) with pSG5 (the empty vector) or pSG5-LANA1 according to the manufacturer's protocol. At 4 h post-transfection, cells were treated with vehicle control (DMSO; 0.006%, vol/vol) or triptolide for further 44 h before cell harvesting. In some experiments, cells were treated in the absence or presence of MG-132 (10 μM) or CHX (25 μg/ml).

Briefly, cells treated in different conditions were collected and washed with ice-cold PBS twice. Whole cell lysates were prepared in RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) supplemented with 0.5% cocktail protease inhibitor (Roche) and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The cell lysates harvested were sonicated for 15 sec, followed by centrifugation at 12,000 × g for 10 min. The supernatants were collected and transferred into new tubes. Protein concentrations were measured by the bicinchoninic acid method using bovine serum albumin as a standard. Based on the measurement of protein concentrations of all samples in the same experiment, the loading volumes of samples were accordingly adjusted. Therefore, equal amounts of proteins mixed with 5X loading buffer [250 nM Tris-Hcl (pH 6.8), 10% SDS, 0.5% BPB, 50% glycerol, 5% β-mercaptoethanol] were subjected to 10% SDS-PAGE gels. The gels were run at 100 V, followed by transfer to PVDF membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membranes were blocked with 5% skim milk in TBST, followed by incubation with primary antibodies overnight at 4°C. After 3×5-min washes in TBST, the membranes were incubated with appropriate horseradish-peroxidase-conjugated secondary antibodies for 1 h. The primary antibodies used were as follows: GAPDH (cat. no. 10494-1-AP, 1:5,000; Proteintech, Wuhan, China), β-actin (cat. no. A5441, 1:5,000; Sigma), LANA-1 (cat. no. 13-210-100, 1:4,000; Abionline, Cambridge, UK), vIL-6 (cat no. 13-214-050, 1:1,000; Abionline), v-Cyclin (cat no. F105P, 1:1,000; Exalpha Biological, Inc., Boston, MA, USA), STAT3 (cat no. 9139, 1:1,000; Cell Signaling Technology, Danvers, MA, USA), phospho-STAT3 (cat. no. 4113, 1:1,000; Cell Signaling Technology) caspase-3 (cat. no. 9668, 1:1,000; Cell Signaling Technology), cleaved caspase-3 (cat. no. 9664, 1:1,000; Cell Signaling Technology), RTA (cat. no. 251345, 1:500; Abbiotec LLC, San Diego, CA, USA). A rabbit polyclonal antibody against v-Flip was raised in our laboratory. The secondary antibodies were horseradish-peroxidase-conjugated secondary anti-mouse IgG (1:10,000; Wuhan Kerui Technology Co., Ltd., Wuhan, China), anti-rabbit IgG (1:10,000; Wuhan Kerui Technology), or anti-rat IgG (1:5,000; Wuhan Kerui Technology). Antibody-bound proteins were detected using the ECL system (Bio-Rad Laboratories). Image quantifications were performed using ImageJ software.

PEL cells treated with vehicle control (DMSO; 0.006%, vol/vol) or triptolide for 24 and 48 h were harvested and the total RNA was extracted using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The concentration of total RNA was determined by NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and all isolated RNA samples (A260/A280 ratio >1.8) were stored at −80°C. Total RNA (1 μg) was reversely transcribed using a PrimeScript RT reagent kit with gDNA Eraser (cat. no. RR047A; Takara Bio, Tokyo, Japan). Genomic DNA elimination reactions were performed in a volume of 10 μl containing 2 μl of 5X gDNA Eraser buffer, 1 μl of gDNA Eraser, and 7 μl of RNase-free dH₂O and the total RNA (1 μg), followed by storage at room temperature for 30 min. The reverse-transcription reaction mixture (20 μl) included 4 μl of 5X PrimeScript buffer 2, 1 μl of PrimeScript RT Enzyme Mix I, 1 μl of RT Primer Mixture containing oligo dT primer and random 6 mers, 4 μl of RNase-free dH₂O, and 10 μl of genomic DNA elimination reaction solution as mentioned above. The condition for reverse-transcription reaction was 15 min at 37°C and 5 sec at 85°C. Expression of viral LANA1 mRNAs was quantified by quantitative PCR using a SYBR-Green PCR kit (cat no. RR420A; Takara Bio). Quantitative PCR reaction mixtures (20 μl) included 1X SYBR Premix Ex Taq II (Tli RNaseH Plus), 0.4 μM each primer and 1 μl cDNA reaction mixture (equivalent to 50 ng of input RNA). Reaction was performed by a CFX96 Real-Time PCR detection system (Bio-Rad Laboratories) and included an initial 30 sec denaturation step at 95°C, followed by 40 cycles of 10 sec at 95°C, 10 sec at 60°C and 15 sec at 72°C. Melting curve analysis was performed between 65 and 95°C (with 0.5°C increments) to verify amplicon specificity. The average cycle threshold (CT) was determined by three independent samples. Template-negative (quantitative PCR reaction mixtures without cDNA) and RT-negative (RNA after genomic DNA elimination) conditions were used as controls. The relative amounts of LANA1 transcript were normalized to the housekeeping gene GAPDH and quantitative PCR data for the relative quantification were calculated with the ^ΔΔCt method. The level of LANA1 transcript in cells treated with vehicle control (DMSO; 0.006%, vol/vol) was set as 1. The primers were as follows: LANA1 forward, 5′-CGAGAGGAAGTTGTAGGAAACG-3′ and LANA1 reverse, 5′-CTTCCAGGTATAGGCAAGGTG-3′ (30); GAPDH forward, 5′-ACATCGCTCAGACACCATG-3′ and GAPDH reverse, 5′-TGTAGTTGAGGTCAATGAAGGG-3′ (30). All experiments were repeated from three independent cultures.

PEL cells were seeded in 6-well plates and treated with vehicle control (DMSO; 0.006%, vol/vol) or triptolide (50 or 100 nM) for 24 h, respectively. For apoptosis analysis, PEL cells were evaluated with Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Multisciences, Shanghai, China). Briefly, 2×10⁵ cells were washed 3 times with PBS and resuspended in 500 μl of 1X binding buffer, followed by adding 5 μl of Annexin V-FIFC and 10 μl of PI. The whole cells were analyzed immediately by a Beckman-Coulter system (EPICS Altra II; Beckman-Coulter, Fullerton, CA, USA).

TPA (20 ng/ml) and NaB (1.2 mM) were used to induce BCBL-1 cells entering lytic replication. At 3 h post-induction, cell medium was replaced and cultured in the absence or presence of triptolide for further 48 h. KSHV virion-associated DNA derived from the supernatants was collected according to the method previously described (31). Equal amounts of the viral lysate were used for the quantitative real-time PCR assay of the KSHV specific region, Orf K9. The primer pair for Orf K9 was: Orf K9 forward, 5′-GTCTCTGCGCCATTCAAAAC-3′ and Orf K9 reverse, 5′-CCGGACACGACAACTAAGAA-3′. Intracellular viral DNA in BCBL-1 cells was extracted with Phenol-Chloroform method according to the manufacturer's instructions. The copy numbers of KSHV genome were evaluated by a quantitative real-time PCR assay.

PEL cells were treated with or without 100 nM triptolide for 24, 48, and 72 h, respectively. Cell supernatants were collected and subjected to ELISA. IL-6 level of the cell supernatants was measured using human IL-6 ELISA kit (Multisciences) according to the manufacturer's instructions.

The animal protocol used in the present study was approved by the Medical Ethics Committee of Wuhan University. NOD/SCID mice were maintained under specific pathogen-free conditions in the Animal Experiment Center of Wuhan University, Animal Biosafety Level-II Laboratory.

BCBL-1 cells were inoculated intraperitoneally (i.p.) into the flank of 4-week-old female NOD/SCID mice (purchased from Beijing HFK Bioscience, Co., Ltd., Beijing, China). Twelve mice were injected with BCBL-1 cells at 1×10⁷ per mouse on day 0. Three days post-injection, the mice were randomly divided into vehicle and treatment groups (six mice per group), and daily i.p. injection of triptolide (0.4 mg/kg) or PBS was performed. After 21 days of treatment, all the mice were sacrificed by cervical dislocation. Tumor burden was evaluated by measuring body weight and volume of ascites. Tissue samples were collected and fixed in 10% neutral buffered formalin, embedded and sectioned at 5 μm. Hematoxylin and eosin staining for histological analysis and immunohistochemistry analysis were performed as previously described (32).

Data were presented as the mean ± standard deviation (mean ± SD) of three separate experiments. The statistical significance of the difference was determined by the Student's t-test, and P-values <0.05 were considered to indicate a statistically significant result.

To determine whether triptolide specifically targets cell viability in the KSHV-associated PEL cells, BC-3, BCBL-1 and JSC-1 cells, together with KSHV-negative BJAB cells were treated with vehicle control or a series of increasing concentrations of triptolide for 24, 48 and 72 h, respectively. Cell viability was determined by CCK-8 assays. Triptolide inhibited cell viability of BC-3 cells in a dose- and time-dependent manner, with 50% inhibitory concentration (IC₅₀) values of 108.3, 89.5 and 46.4 nM by a 24-, 48- and 72-h trip-tolide treatment, respectively (Fig. 1A). Similar experiments were conducted in the other two PEL cells. The IC₅₀ values were calculated as 143.3, 128.3 and 67.9 nM in JSC-1 cells and 153.9, 119.6 and 69.1 nM in BCBL-1 cells by 24-, 48- and 72-h triptolide treatment, respectively (Fig. 1B and C). In addition, triptolide inhibited cell proliferation to a similar degree in BJAB cells, and the IC₅₀ values were calculated as 146.6, 115.6 and 63.8 nM by 24-, 48- and 72-h triptolide treatment, respectively (Fig. 1D).

To further evaluate the antiproliferative effects of triptolide on KSHV-uninfected B cell line, P3HR-1 cells and PBMCs isolated from healthy donors were treated with vehicle control or a series of increasing concentrations of triptolide for 24, 48 and 72 h, respectively. Our previous results indicated that 100-nM triptolide treatment for 4 days decreased cell viability by <20% in P3HR-1 cells (28). As shown in Fig. 1E, a series of increasing concentrations of triptolide treatment for 24, 48 and 72 h showed slight proliferation inhibition in P3HR-1 cells. Similar results were also found in PBMCs treated by a series of increasing concentrations of triptolide (Fig. 1F). These results suggested that triptolide efficiently inhibited cell viability in PEL cells, but not significantly in PBMCs and P3HR-1 cells.

To examine the effect of trip-tolide on induction of cell cycle arrest and apoptosis, BC-3, JSC-1 and BCBL-1 cells were treated with vehicle control or indicated triptolide for 24 h. Cell cycle arrest and apoptosis were analyzed by flow cytometry. As shown in Fig. 2A, treatment of PEL cells with triptolide resulted in apoptosis in a dose-dependent manner, which was confirmed by staining with Annexin V-FITC/PI. In addition, treatment with 100 nM triptolide for 24 h could induce cell apoptosis in BJAB cells, while not in P3HR-1 cells (data not shown). For cell cycle analysis, treatment of BC-3 and JSC-1 cells with triptolide resulted in G0/G1 arrest as observed by a marked increase in the number of cells in the G1 phase (Fig. 2B). In contrast, treatment of BCBL-1 cells with triptolide resulted in cell cycle arrest in G2/M phase as observed by an increase in the percentage of cells in the G2/M phase (Fig. 2B). These results suggest that triptolide induces cell cycle arrest and apoptosis in the KSHV-associated PEL cells.

To determine whether triptolide alters expression of LANA1, three different latently infected KSHV-associated PEL cells were treated with vehicle control or triptolide for 48 h. Whole-cell extracts were prepared and subjected to western blot analysis. In addition to inhibition of LANA1 expression, two other KSHV-encoded latent proteins, v-Cyclin and v-Flip, were reduced after triptolide treatment in the three KSHV-associated PEL cells (Fig. 3A). When compared to the vehicle control, triptolide reduced the LANA1 expression level to 9.8, 16.8 and 23.4% in BC-3, BCBL-1 and JSC-1 cells, respectively. When compared to the vehicle control, triptolide reduced the v-Cyclin expression level to 3.4, 17.4 and 19.6%, the v-Flip expression level to 14.2, 22.3 and 24.6% in BC-3, BCBL-1 and JSC-1 cells, respectively. Furthermore, consistent with the results of cell apoptosis assays, triptolide resulted in increased caspase-3 cleavage in the three KSHV-associated PEL cells. In addition, expression of STAT3, which is one of transcription factors, remained unchanged with triptolide treatment, suggesting that the effect of triptolide on LANA1 was a selective event. To further determine if triptolide specifically decreases LANA1 expression, KSHV-negative HeLa cells were transiently transfected with pSG5-LANA1, followed by treatments with or without triptolide beginning at 4 h after transfection. As shown in Fig. 3B, triptolide treatment significantly decreased the expression of transfected pSG5-LANA1 in HeLa cells.

Since triptolide reduced expression of LANA1, a real-time quantitative reverse transcript polymerase chain reaction (qRT-PCR) analysis was used to determine if triptolide suppressed LANA1 at the mRNA level. BC-3, BCBL-1 and JSC-1 cells were treated with vehicle control or triptolide (100 nM) for 24 and 48 h, respectively. The total RNAs were harvested and subjected to qRT-PCR. The relative expression was detected by comparison to the housekeeping gene GAPDH. It was indicated that the mRNA levels of LANA1 in BCBL-1, BC-3 and JSC-1 cells were undetectable upon triptolide treatment (Fig. 3C).

To examine whether triptolide decreased expression of LANA1 via the proteasome pathway, HeLa cells were transiently transfected with the pSG5-LANA1 and treated with vehicle control or triptolide in the absence or presence of proteosomal inhibitor MG-132. As shown in Fig. 4A, MG-132 obviously attenuated the effect of triptolide on LANA1 expression. Similarly, MG-132 also resulted in endogenous LANA1 protein level accumulation in BCBL-1 (Fig. 4B) and BC-3 (Fig. 4C) cells after treatment with triptolide.

Previous studies had indicated that triptolide decreased the expression of LANA1 and induced proteasomal degradation of LANA1. To determine whether triptolide affect LANA1 stability through some other mechanisms, HeLa cells were transiently transfected with the pSG5-LANA1 and treated with vehicle control or triptolide in the presence of cycloheximide (CHX). As shown in Fig. 4D, triptolide reduced the half-life of LANA1 when compared to vehicle control, while not affecting GAPDH levels. Similarly, triptolide also reduced the half-life of endogenous LANA1 protein in the CHX-treated BCBL-1 (Fig. 4E) and BC-3 (Fig. 4F) cells. Notably, triptolide did not significantly affect STAT3 stability (Fig. 4D–F).

To examine the effect of triptolide on antiviral activity, BCBL-1 cells uninduced or induced by TPA and NaB for 3 h were treated with indicated concentrations of triptolide for 48 h. Whole cell lysates, supernatants, and genomic DNA were prepared at 48 h post-incubation. qRT-PCR and western blotting were used to analyze the effect of triptolide on lytic and latent replication of KSHV. As shown in Fig. 5A, triptolide decreased viral genome copies in both uninduced and induced BCBL-1 cells. When compared to the vehicle control in induced BCBL-1 cells, intracellular viral DNA replication was reduced by 5-fold after triptolide (100 nM) treatment for 48 h. Production of progeny virion extracted from the BCBL-1 cell supernatants was calculated according to the standard curve derived from the Orf K9 construct (Fig. 5B). Results indicated that the production of progeny virion was dose-dependently reduced after triptolide treatment (Fig. 5C). As expected, CDV, which was used as a positive control, efficiently reduced the lytic replication and virion production of KSHV as previously described (33). Replication and transcription activator (RTA), a lytic switch protein, is necessary for KSHV reactivation and lytic DNA replication. Western blotting was used to detect the effect of triptolide on the expression of RTA in BCBL-1 cells. As shown in Fig. 5D, triptolide reduced expression of LANA1 and RTA in both uninduced and induced BCBL-1 cells. In addition, vIL-6, encoded by KSHV Orf K2, contributed to the growth, survival and spread of PEL. Our results as shown in Fig. 5D also indicated that triptolide decreased vIL-6 expression in induced BCBL-1 cells.

LANA1, a transcriptional co-activator of STAT3, modulates STAT3 activity in PEL cells (20). STAT3 is constitutively phosphorylated and is essential to cell survival in PEL cells (34). Since STAT3 represents a potential target for treatment of PEL, the STAT3 activity affected by triptolide was determined. Results shown in Fig. 6A indicated that triptolide dephosphorylated STAT3 in BCBL-1, BC-3 and JSC-1 cells. It has been reported that KSHV encoded LANA1 upregulated IL-6 expression by inducing transcription of the IL-6 promoter (35). IL-6 and vIL-6 secreted by KSHV-positive cells are multifunctional cytokines that contribute to the pathogenesis of PEL (35). To determine effect of triptolide on secretion of IL-6, KSHV-associated PEL cells were treated with vehicle control or 100 nM triptolide for 24, 48 and 72 h. Cell supernatants were collected and subjected to ELISA. As shown in Fig. 6B, triptolide resulted in reduced secretion of IL-6 in the supernanants of BCBL-1, BC-3 and JSC-1 cells. These results suggests that downregulation of LANA1 by triptolide results in inhibition of IL-6/STAT3 pathway in PEL cells.

Since our previous results indicated efficacy of triptolide for treatment of PEL in vitro, effects of triptolide in vivo were also assessed in an immunodeficient xenograft mouse model. NOD/SCID mice were inoculated intraperitoneally with 1×10⁷ BCBL-1 cells. A dose of 0.4 mg/kg triptolide or PBS alone was administrated via intraperitoneal injection daily for 3 weeks from day 3 after cell inoculation. As the dose of triptolide from intraperitoneal injection had already been reported (23), the dosage of triptolide in vivo in the present study was expected to be safe. The body weight of the PBS alone treated mice significantly increased compared to that of triptolide treated mice (28.9±1.79 vs. 19.2±2.21 g, n=6, P<0.01; Fig. 7A). Moreover, mice treated with triptolide had a significantly lower volume of ascites (1.45±0.27 vs. 0.17±0.07 ml, n=6, P<0.01; Fig. 7B). In addition, IL-6 level in the supernatants of ascites was significantly decreased in comparison to that of triptolide treatment (Fig. 7C). Western blotting results of ascites cells indicated that triptolide significantly cut down the expression of LANA1 and induced ascites cell apoptosis via caspase-3 pathways (Fig. 7D). Representative photos of mice treated with PBS alone and triptolide treated are shown in Fig. 7E.

Organ invasion by BCBL-1 cells on day 24 was evaluated by hematoxylin and eosin staining, as well as LANA1 immu-nostaining. As shown in Fig. 7F, spleens of mice treated with triptolide were significantly smaller compared to the group treated with PBS alone. The mice inoculated with BCBL-1 cells exhibited infiltration in the spleen and the number of LANA1 positive cells in triptolide treated mice was significantly reduced (Fig. 7G). These results suggest that triptolide efficiently inhibits growth and infiltration of PEL cells in vivo.