Triptolide, a diterpenoid triepoxide, induces antitumor proliferation via activation of c-Jun NH2-terminal kinase 1 by decreasing phosphatidylinositol 3-kinase activity in human tumor cells

BBRC Biochemical and Biophysical Research Communications 336 (2005) 1081–1086 www.elsevier.com/locate/ybbrc

Triptolide, a diterpenoid triepoxide, induces antitumor proliferation via activation of c-Jun NH2-terminal kinase 1 by decreasing phosphatidylinositol 3-kinase activity in human tumor cells q Yoshiki Miyata, Takashi Sato *, Akira Ito Department of Biochemistry and Molecular Biology, Tokyo University of Pharmacy and Life Science, School of Pharmacy, Hachioji, Tokyo 192-0392, Japan Received 23 August 2005 Available online 9 September 2005

Abstract Triptolide, a diterpenoid triepoxide extracted from the Chinese herb Tripterygium wilfordii Hook f., exerts antitumorigenic actions against several tumor cells, but the intracellular target signal molecule(s) for this antitumorigenesis activity of triptolide remains to be identified. In the present study, we demonstrated that triptolide, in a dose-dependent manner, inhibited the proliferation of human fibrosarcoma HT-1080, human squamous carcinoma SAS, and human uterine cervical carcinoma SKG-II cells. In addition, triptolide was found to decrease phosphatidylinositol 3-kinase (PI3K) activity. A PI3K inhibitor, LY-294002, mimicked the triptolide-induced antiproliferative activity in HT-1080, SAS, and SKG-II cells. There was no change in the activity of Akt or protein kinase C (PKC), both of which are downstream effectors in the PI3K pathway. Furthermore, the phosphorylation of Ras, Raf, and mitogen-activated protein/ extracellular signal-regulated kinase 1/2 was not modified in HT-1080 cells treated with triptolide. However, the phosphorylation of c-Jun NH2-terminal kinase 1 (JNK1) was found to increase in both triptolide- and LY-294002-treated cells. Furthermore, the triptolide-induced inhibition of HT-1080 cell proliferation was not observed by JNK1 siRNA-treatment. These results provide novel evidence that PI3K is a crucial target molecule in the antitumorigenic action of triptolide. They further suggest a possible triptolide-induced inhibitory signal for tumor cell proliferation that is initiated by the decrease in PI3K activity, which in turn leads to the augmentation of JNK1 phosphorylation via the Akt and/or PKC-independent pathway(s). Moreover, it is likely that the activation of JNK1 is required for the triptolide-induced inhibition of tumor proliferation.
2005 Elsevier Inc. All rights reserved. Keywords: Triptolide; Chinese herb; Antitumor proliferation; Phosphatidylinositol 3-kinase; c-Jun NH2-terminal kinase 1; Protein kinase C; Mitogenactivated protein/extracellular signal-regulated kinase; siRNA

Extracts of the Chinese herbal remedy Tripterygium wilfordii Hook f. (TWHF) have been shown to be effective in the treatment of autoimmune diseases such as rheumatoid arthritis, nephritis, and lupus erythematosus [1–3]. Triptolide (PG490), a diterpene triepoxide, has been identified as a crucial component of TWHF and possesses immunosuppressive, anti-inflammatory, and antifertility actions in vivo q

Abbreviations: PI3K, phosphatidylinositol 3-kinase; MEK, mitogenactivated protein/extracellular signal-regulated kinase; JNK, c-Jun NH2terminal kinase; PKC, protein kinase C. * Corresponding author. Fax: +81 426 76 5734. E-mail address: [email protected] (T. Sato). 0006-291X/$ - see front matter
2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.08.247

and in vitro [4–9]. In addition, it has been reported that triptolide causes the inhibition of cell proliferation in various tumor cell species in vivo and in vitro [10–15]. Therefore, triptolide may be clinically effective for tumor chemotherapy. Triptolide-induced antitumor proliferation has been reported to result from a decrease in the expression of cell-cycle promoting factors such as cyclins (A, B1, and D1) and c-myc in breast tumor MDA-435 cells [14]. It has been also reported that triptolide induces cell arrest in the S phase in human fibrosarcoma HT-1080 cells [13] and in human prostatic adenocarcinoma cells [12]. Furthermore, the in vitro and in vivo antitumor proliferation seen

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with triptolide may be associated with its induction of apoptosis [12,15–17]. It is anticipated that drugs that target intracellular molecules such as protein kinases associated with tumorigenesis and/or apoptosis will be effective for cancer therapy [18]. Concerning the mechanism of triptolide-induced apoptosis and anti-inflammatory activity, it has been reported that the level of phosphorylated p38 increases and c-Jun NH2terminal kinase (JNK) phosphorylation decreases in triptolide-treated mouse bone-marrow derived dendritic cells and in the mouse macrophage cell line Raw 264.7, respectively [19,20]. Moreover, 2 distinct signal pathways, namely, phosphatidylinositol 3-kinase (PI3K)–Akt and Ras–Raf– mitogen-activated protein/extracellular signal-regulated kinase (MEK) 1/2 have been reported to play important roles as survival signals in the regulation of tumor proliferation [21,22]. However, it remains unclear whether or not triptolide-induced antitumor proliferation is caused by the modulation of the PI3K–Akt and/or the Ras–Raf–MEK1/2 pathway(s). In the present study, we demonstrated that the inhibition of tumor cell proliferation by triptolide is initiated by the decrease in PI3K activity, which in turn results in the augmentation of the activation of JNK1 via the Akt and/or protein kinase C (PKC)-independent pathway(s). Furthermore, the activation of JNK1 is necessary for triptolide-induced antitumor proliferation. Materials and methods Cell culture and treatment. Human fibrosarcoma HT-1080 cells (Health Science Research Resources Bank, Osaka, Japan) were cultured in EagleÕs minimum essential medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS) and nonessential amino acids (Invitrogen). Human squamous carcinoma SAS cells (Health Science Research Resources Bank) and human uterine cervical carcinoma SKG-II cells [23] were cultured in DulbeccoÕs modified EagleÕs medium supplemented with 10% fetal bovine serum. After reaching confluence, the cells were treated with triptolide (Alexis Biochemicals, San Diego, CA) and a PI3K inhibitor, LY-294002 (BioMol Research Laboratory, Plymouth Meeting, PA), in the serum-free medium with 0.2% lactalbumin hydrolysate (Sigma Chemical, St. Louis, MO) for up to 24 h. Cell proliferation. The cell proliferation was analyzed by Alamer blue assay [24]. Briefly, the tumor cells (1 · 104 cells/well) were treated with triptolide or LY-294002 for up to 48 h in 96-well multiplates. For the last hour of the treatment, Alamar blue (Asahi Techno Glass, Tokyo, Japan) was added to the cells, after which the fluorescence of the incorporated reagent was measured at 540 nm (excitation) and 595 nm (emission). Preparation of cytosol fraction. The cells were washed once with Ca2+and Mg2+-free phosphate-buffered saline and then homogenized in 10 mM Hepes–KOH (pH 7.8), 10 mM KCl, 0.1 mM EDTA, 0.1% Nonidet P-40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 5 lM pepstatin, 10 lM leupeptin, and 1 mM sodium orthovanadate. After centrifugation at 5000g at 4 C, the resultant supernatant was collected as the cytosol fraction and used for Western blotting for phosphorylated proteins and the measurement of PI3K activity as previously reported [25]. Western blotting. Aliquots (30 lg) of cytosol proteins were subjected to Western blotting using specific rabbit antibodies against phosphorylated Akt (Ser473), Raf (Ser259), MEK1/2 (Ser217/221), and JNK (Thr183/ Tyr185), and against unphosphorylated Raf, MEK1/2, and JNK (New England Biolaboratories, Beverly, MA) under nonreducing conditions. The Ras activity was measured via Ras Activation Assay kits (Upstate

Biotechnology, Lake Placid, NY) using aliquots (500 lg) of the cytosol proteins, according to the manufacturerÕs instructions [25]. Immunoreactive GTP-bound Ras, Raf, MEK1/2, and JNK, and phosphorylated Akt, Raf, MEK1/2, and JNK were detected through the use of enhanced chemiluminescence-Western-blotting detection reagents (Amersham Bioscience, Tokyo, Japan) after being complexed with horseradish peroxidase-conjugated goat anti-rabbit IgG (New England Biolaboratories). Relative amounts of the immunoreactive proteins were quantified via densitometric scanning, using an Image Analyzer LAS-1000 Plus (Fuji Film, Tokyo, Japan). Kinase assay. For measurement of PI3K activity, aliquots (750 lg) of the cytosol proteins were incubated with rabbit anti-(PI3K, p85) antibody (Upstate) for 18 h at 4 C, following which the immunoreactive complex bound to added protein A–Sepharose (Amersham Bioscience) was further incubated for 2 h at 4 C, as previously reported [25]. After centrifugation at 10,000g at 4 C, the resultant precipitate containing PI3K was resuspended in 75 lL of TNE buffer [10 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 5 mM EDTA], and then incubated with 10 lL of phosphatidylinositol (2 mg/mL) in TNE buffer and 10 lL of 100 mM MgCl2. Next, an enzymic reaction was started by adding 5 lL of [c-32P]ATP (5.5 kBq) (Dupont NEN, Boston, MA) and carried out for 15 min at 37 C. After the reaction was terminated by adding 20 lL of 1 M HCl, the synthesized 32P-labeled phosphatidylinositol 3-phosphate was extracted with chloroform/methanol (1:1, v:v), analyzed by TLC on a silica-gel 60 F254-coated plate (Merck, Darmstadt, Germany) in a chloroform/methanol/H2O/25% ammonia solution (60:47:11.3:2, v:v:v:v), and then detected by exposing the plate to X-ray film at 80 C. Relative amounts of the 32P-labeled phosphatidylinositol 3-phosphate were quantified via densitometric scanning, using an Image Analyzer LAS-1000 Plus. To assess PKC activity, the cytosol fraction (50 lg) was measured using a PKC Enzyme Assay System (Amersham Bioscience) and [c-32P]ATP (7.5 kBq) (Dupont NEN), according to the manufacturersÕ instructions. Construction of JNK1 knockdown HT-1080 cells. Synthesized JNK1 siRNA cocktail (1 lg) (GeneWorld, Tokyo, Japan) [26,27] was transfected to HT-1080 cells (1 · 105 cells/mL) in a 6- or 48-multiwell plate (2.0 and 0.2 · 105 cells/well, respectively) using Lipofectamine 2000 reagent (Invitrogen) for 24 h, after which the cell lysate (50 lg) was subjected to Western blotting for the confirmation of endogenous JNK1 levels. The JNK1 knockdown cells were treated with triptolide (14–56 nM) for 15 h, and the resulting cell proliferation was analyzed by Alamer blue assay as described above. Statistical analysis. A one-way analysis of variance (ANOVA) was used for statistical analysis. The Fisher test was applied when multiple comparisons were performed.

Results The decrease in PI3K activity by triptolide leads to the inhibition of HT-1080 cell proliferation We first examined the effect of triptolide on the proliferation of HT-1080, SAS, and SKG-II cells. As shown in Table 1, triptolide inhibited the proliferation of HT-1080 cells (maximum inhibition, 39% at 56 nM; p < 0.001) in a dose-dependent manner. A similar anti-proliferation effect was observed in triptolide-treated SAS and SKG-II cells (maximum inhibition, 27% and 19% at 56 nM, respectively; p < 0.001). Since intracellular-signal pathways of PI3K–Akt and Ras–Raf–MEK1/2 play important roles in the regulation of tumor cell proliferation [21,22], we investigated whether these signal pathways might be associated with the triptolide-induced inhibition of cell proliferation, using HT-1080 cells. As shown in Fig. 1, triptolide was found to inhibit the activity of

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Table 1 Triptolide inhibits the tumor cell proliferation Treatments

Proliferative activity (·103fluorescence/well) (% of untreated cells) HT-1080

SAS

SKG-II

None Triptolide (nM) 14 28 56

4.74 ± 1.08 (100)

4.99 ± 0.48 (100)

1.33 ± 0.16 (100)

3.72 ± 0.26 (79)* 3.31 ± 0.40 (70)** 2.89 ± 0.49 (61)***

3.98 ± 0.74 (80)** 4.09 ± 0.70 (82)* 3.66 ± 0.58 (73)***

1.20 ± 0.13 (90)* 1.07 ± 0.06 (80)*** 1.08 ± 0.02 (81)***

Tumor cells (1 · 104 cells/well) in 96-well multiplates were treated with triptolide, and the resulting cell proliferation was measured by Alamar blue assay, as described in the text. Data (n = 6) are shown as means ± SD. *, **, and *** indicate results that were significantly different from untreated control cells (none) at p levels of <0.05, <0.01, and <0.001, respectively.

p < 0.001), also in a dose-dependent manner (Fig. 1C). The antitumor proliferation activity of LY-294002 was similarly observed in SAS and SKG-II cells (maximum inhibition, 21% in each at 20 lM, p < 0.001) (data not shown). Furthermore, although LY-294002 has been shown to decrease both PI3K activity and Akt phosphorylation in HT-1080 cells (data not shown) [25], triptolide was found to not modify the level of phosphorylated Akt in HT-1080 cells (Fig. 2A). We confirmed that the activity of PKC, another predictive downstream effector of the PI3K pathway [28,29], was not altered by treatment with triptolide for up to 60 min (Table 2). Moreover, as shown in Fig. 2, there was no change in the level of GTP-bound Ras (panel B), phosphorylated and unphosphorylated Raf (panel C), and MEK1/2 (panel D) in HT-1080 cells treated with triptolide. Therefore, these results suggest that the inhibition of PI3K activity by triptolide causes the antiproliferative activity in HT-1080 cells through the Akt and/or the PKC-independent pathway(s). Augmentation of JNK1 activation is required for the triptolide-induced antitumor proliferation

Fig. 1. The decrease in PI3K activity by triptolide leads to the inhibition of HT-1080 cell proliferation. (A,B) Confluent HT-1080 cells were treated with triptolide (14–56 nM) for 1 h, and the resulting harvested cytosol fractions were subjected to Western blotting for PI3K (A) and measurement of PI3K activity (B) as described in the text. Relative amounts of PI3K and phosphatidylinositol 3-phosphate (PIP3) were determined by densitometric analysis. Data for PI3K activity are shown as means ± SD, based on three independent experiments. (C) HT-1080 cells (1 · 104 cells/ well) in 96-well multiplates were treated with LY-294002 (5–20 lM) for 18 h; the resulting cell proliferation was measured by Alamar blue assay as described in the text. Data (n = 6) are shown as means ± SD. ** and *** indicate results that were significantly different from untreated control cells at p levels of <0.01 and <0.001, respectively.

PI3K (maximum inhibition, 63% at 56 nM, p < 0.001) (panel B), whereas there was no change in the level of PI3K protein (panel A). In addition, a PI3K inhibitor, LY-294002, was found to inhibit the proliferation of HT-1080 cells (maximum inhibition, 49% at 20 lM,

To clarify downstream signal effector(s) in the triptolidemodulated PI3K pathway, we investigated the activation of JNK, which is a mitogen-activated kinase associated with PI3K signal transduction [25,30], in triptolide-treated HT-1080 cells. As shown in Fig. 3, JNK1 and its phosphorylated form were constitutively detected in HT-1080 cells, whereas this occurred to a lesser extent with JNK2 (data not shown). The phosphorylation of JNK1 was found to increase in both triptolide- and LY-294002-treated HT1080 cells (2.8- and 2.5-fold, respectively). As shown in Fig. 4, when JNK1 expression was repressed by its specific siRNA (inserted panel), the proliferation of HT-1080 cells was found to decrease to 56% among untransfected cells (p < 0.001). In the JNK1 knockdown cells, the triptolideinduced anti-proliferation was not observed, whereas there was dose-dependent inhibition of cell proliferation in the untransfected HT-1080 cells. Therefore, these results strongly suggest that triptolide inhibition of PI3K leads to the activation of JNK1, which is a crucial signal for the antitumor proliferation induced by triptolide.

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Fig. 2. No effects of triptolide on Akt phosphorylation and Ras–Raf–MEK1/2 signal pathway in HT-1080 cells. The harvested cytosol fractions as described in Fig. 1 were subjected to Western blotting for phosphorylated Akt (p-Akt) (A), GTP-bound Ras (GTP-Ras) (B), phosphorylated Raf (p-Raf) and Raf (C), and phosphorylated MEK1/2 (p-MEK) and MEK1/2 (D) as described in the text. The relative amounts of each protein were determined by densitometric analysis. Data for the phosphorylated proteins (A, C, and D) and the GTP-bound Ras (B) are shown as means ± SD, based on three independent experiments. Lane 1, untreated control cells; lane 2, triptolide-treated cells (56 nM).

Table 2 Effect of triptolide on PKC activity in HT-1080 cells Treatments

PKC activity (nmol phosphate/min/mg protein) Treatment periods (min) 10

None Triptolide (nM) 56

30

60

96.7 ± 4.8

104.3 ± 3.4

97.7 ± 4.2

101.4 ± 4.8

104.4 ± 5.5

100.6 ± 3.1

The harvested cytosol fractions (50 lg) shown in Fig. 1 were subjected to measurement of PKC activity, as described in the text. Data (n = 3) are shown as means ± SD.

Fig. 4. Abolishment of triptolide-induced antiproliferation in JNK1 knockdown HT-1080 cells. HT-1080 cells were transfected with a JNK1 siRNA cocktail (1 lg) for 24 h, and the resulting cell lysate (50 lg) was subjected to Western blotting for JNK1 (inseted panel). The proliferation of JNK1 knockdown HT-1080 cells in the presence or absence of triptolide (14–56 nM) was analyzed by Alamar blue assay, as described in the text. Open circles, untransfected control cells. Closed squares, JNK1 knockdown cells. Three independent experiments were reproducible and the typical resulting data are shown. Data for the cell proliferation are shown as means ± SD of five individual wells. *** indicates result that was significantly different from the untransfected control cells without triptolide treatment at p level of <0.001. Fig. 3. Triptolide augments the phosphorylation of JNK1 in HT-1080 cells. Confluent HT-1080 cells were treated with triptolide (56 nM) or LY294002 (20 lM) for 1 h, and the resulting harvested cytosol fractions were subjected to Western blotting for the phosphorylated JNK1 (p-JNK1) (upper panel) and unphosphorylated JNK1 (middle panel), as described in the text. Data for the phosphorylated JNK1 are shown as means ± SD, based on three independent experiments. * and ** indicate results that were significantly different from untreated control cells at p levels of <0.05 and <0.01, respectively.

Discussion Triptolide possesses beneficial antitumorigenic effects against various tumor cell species, both in vivo and in vitro [10–15]. Regarding the molecular mechanisms for this antitumor proliferation, Yang et al. [14] reported that tri-

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ptolide reduces the level of cell cycle-promoting factors such as cyclins and c-myc in human breast carcinoma MDA-435 cells. In addition, the antitumor proliferation induced by triptolide has been reported to be associated with the down-regulation of NF-jB activity in human multiple myeloma cells [15]. However, it remains unclear how triptolide modulates initial intracellular signal(s) to lead to the regulation of the cell cycle and the subsequent inhibition of tumor proliferation. In the present study, we demonstrated that triptolide decreased the activity of PI3K and inhibited the cell proliferation in HT-1080, SAS, and SKG-II cells. Furthermore, LY294002, a PI3K inhibitor, mimicked the triptolide-induced antiproliferation in the three cell lines. PI3K has been reported to contribute to the regulation of tumorigenesis and the apoptosis of tumor cells [21,22]. We further confirmed that both triptolide and LY-294002 induced the apoptosis of HT-1080 cells as shown by flow cytometric analysis of Annexin V positive cells and by electrophoresis of DNA fragmentation (data not shown). Therefore, these results provide novel evidence that the decrease in PI3K activity by triptolide results in the inhibition of tumor cell proliferation, which may subsequently induce apoptosis. JNK has been identified as a downstream mitogen-activated protein kinase linked to the PI3K signal pathway [28]. Levresse et al. [30] reported that the activation of JNK is negatively regulated by the PI3K–Akt pathway in PC12 cells. We recently reported that the inhibition of the PI3K pathway by LY-294002 augments the phosphorylation of JNK in phorbol ester-treated HT-1080 cells [25]. In the present study, triptolide augmented the phosphorylation of JNK1 without the alteration of its protein level in HT-1080 cells. LY-294002 was found to similarly increase the JNK1 phosphorylation. Furthermore, the inhibition of cell proliferation by triptolide was not observed in the JNK1-knockdown HT-1080 cells. Taken together with the finding that JNK1 negatively regulates tumorigenesis in vivo [31], these results strongly suggest that JNK1 is a pivotal signal effector for the triptolide-induced antitumor proliferation, which is linked to the initial inhibition of PI3K activity. A recent report of microarray analysis by Du et al. [32] showed that triptolide decreases the level of the PI3K gene in T-cell leukemia Jurkat cells, whereas our finding showed that there was no change in the level of PI3K protein in HT-1080 cells. Furthermore, Kim et al. [20] reported that triptolide inhibits the lipopolysaccharide-augmented JNK phosphorylation in macrophage Raw 264.7 cells. Liu et al. [19] also reported no effect of triptolide on the regulation of JNK phosphorylation in mouse dendritic cells. Taken together with our finding, therefore, it is likely that the regulatory mechanism of PI3K and JNK activation by triptolide may differ among different cell species. Nonetheless, we suggest that PI3K and its downstream effector, JNK1, are crucial target molecules for at least triptolide-induced antitumor proliferation.

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Akt and PKC have been reported to act as downstream effectors of PI3K signaling and to participate in the regulation of tumor development, both in vivo and in vitro [21,22,25,29]. In contrast with the finding of a negative regulator of Akt against JNK [25,30], Bra¨ndlin et al. [33] reported that PKCg positively regulates the activation of JNK in HEK293 cells. We also reported that PKCbII and/or e is involved in the phorbol-ester induced JNK phosphorylation in HT-1080 cells [25]. In the present study, there was no change in the levels of phosphorylated Akt and PKC activity in triptolide-treated HT-1080 cells. Furthermore, triptolide did not alter the constitutively activated signal of Ras–Raf–MEK1/2, which is associated with tumor cell proliferation [21,22]. Taken together with previous reports of Akt-independent PI3K signaling [34,35], therefore, these results allow us to speculate that the inhibition of PI3K by triptolide may cause the antitumor proliferation through the Akt and/or PKC-independent pathway in HT-1080 cells. Further experiments will be required to clarify possible downstream signal pathway(s) of PI3K leading to triptolide-induced antitumor proliferation. In conclusion, we suggest a novel mechanism for the observed triptolide-induced antitumor proliferation, in that triptolide initially decreases the activity of PI3K and thereby causes the activation of JNK1 through the Akt and/or PKC-independent pathway(s). Moreover, the activation of JNK1 is likely to be required for the triptolide-induced inhibition of tumorigenesis. Finally, these findings may provide new insight for developing the clinical application of triptolide in cancer therapy. Acknowledgment This work was supported by a Matching Fund Subsidy for Private School of Japan. References [1] W.Z. Qin, G.D. Zhu, S.M. Yang, K.Y. Han, J. Wang, Clinical observations on Tripterygium wilfordii in treatment of 26 cases of discoid lupus erythematosus, J. Tradit. Chin. Med. 3 (1983) 131–132. [2] X.L. Tao, Y. Sun, Y. Dong, Y.L. Xiao, D.W. Hu, Y.P. Shi, Q.L. Zhu, H. Dai, N.Z. Zhang, A prospective, controlled, double-blind, crossover study of tripterygium wilfordii hook F. in treatment of rheumatoid arthritis, Chin. Med. J. 102 (1989) 327–332. [3] X. Jiang, Clinical observations on the use of the Chinese herb Tripterygium wilfordii Hook for the treatment of nephrotic syndrome, Pediatr. Nephrol. 8 (1994) 343–344. [4] M.A. Chan, J.E. Kohlmeier, M. Branden, M. Jung, S.H. Benedict, Triptolide is more effective in preventing T cell proliferation and interferon-gamma production than is FK506, Phytother. Res. 13 (1999) 464–467. [5] D. Qiu, G. Zhao, Y. Aoki, L. Shi, A. Uyei, S. Nazarian, J.C. Ng, P.N. Kao, Immunosuppressant PG490 (triptolide) inhibits T-cell interleukin-2 expression at the level of purine-box/nuclear factor of activated T-cells and NF-jB transcriptional activation, J. Biol. Chem. 274 (1999) 13443–13450. [6] A.P. Hikim, Y.H. Lue, C. Wang, V. Reutrakul, R. Sangsuwan, R.S. Swerdloff, Posttesticular antifertility action of triptolide in the male

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