Functional P53 Is Required for Triptolide-Induced Apoptosis and AP-1 and Nuclear Factor-Κb Activation in Gastric Cancer Cells

Home , Tripterygium wilfordii

Functional p53 is required for triptolide-induced apoptosis and AP-1 and nuclear factor-kB activation in gastric cancer cells

Xiao-Hua Jiang1,2,5, Benjamin Chun-Yu Wong*,2,5, Marie Chia-Mi Lin3, Geng-Hui Zhu2, Hsiang-Fu Kung3, Shi-Hu Jiang1, Dan Yang4 and Shiu-Kum Lam2

1Department of Gastroenterology, Rui-jin Hospital, Shanghai, Peoples Republic of China; 2Department of Medicine, University of Hong Kong, Hong Kong; 3Institute of Molecular Biology, University of Hong Kong, Hong Kong; 4Department of Chemistry, University of Hong Kong, Hong Kong

Triptolide, a major component in the extract of Chinese Introduction herbal plant Tripterygium wilfordii Hook f (TWHf), has potential anti-neoplastic eect. In the present study we Tripterygium wilfordii Hook f (TWHf) has been used investigated the potential therapeutic eects and mechan- in traditional Chinese medicine for centuries. Its crude isms of triptolide against human gastric cancer cells. extracts continue to be used to treat a variety of Four gastric cancer cell lines with dierent p53 status, autoimmune diseases, such as rheumatoid arthritis, AGS and MKN-45 (wild type p53); MKN-28 and SGC- nephritis, and systemic lupus erythematosus (Qin et al., 7901 (mutant p53) were observed as to cell growth 1981; Tao et al., 1989; Jiang et al., 1994). It has been inhibition and induction of apoptosis in response to suggested that the major therapeutic eects of TWHf triptolide treatment. We showed that triptolide inhibited are from ingredients such as triptolide, tripdiolide, cell growth, induced apoptosis and suppressed NK-kB triptonide, and triptophenolide (Zhang et al., 1990). and AP-1 transactivation in AGS cells with wild-type High concentration of triptolide suppressed B and T p53. Triptolide induced apoptosis by stimulating the lymphocyte proliferation in mice and showed immuno- expressions of p53, p21waf1/cip1, bax protein, and increased suppressive eect in skin allograft transplantation (Pu the activity of caspases. In addition, it caused cell cycle et al., 1990; Yang et al., 1992). Interestingly, triptolide arrest in the G0/G1 phase. To examine the role of p53 in also has antineoplastic activity. For example, it has these functions, we showed that suppression of p53 level signi®cant anti-leukemic activity in vivo against L-1210 with antisense oligonucleotide abrogated triptolide-in- and P-388 mouse leukemia and in vitro against cells duced apoptosis and over-expression of dominant derived from human nasopharnygeal carcinomas (Kup- negative p53 abolished the inhibitory eect on NF-kB chan et al., 1972). Furthermore, triptolide signi®cantly activation. Furthermore, we demonstrated that triptolide suppresses the colony formation of breast cancer cell had dierential eects on gastric cancer cells with lines, stomach cancer cell lines and promyelocytic dierent p53 status. We showed that triptolide also leukemia cell lines (Wei et al., 1991). The exact inhibited cell growth and induced apoptosis in MKN-45 mechanism responsible for the anti-neoplastic and with wild-type p53, whereas it had no signi®cant growth- anti-in¯ammatory eect of triptolide is not clearly inhibition and apoptosis induction eects on the MKN-28 understood. One possible explanation is the induction and SGC-7901 cells with mutant p53. Our data suggest of apoptosis. It has been shown that triptolide induces that triptolide exhibits anti-tumor and anti-in¯ammatory apoptosis in T-cell hybridomas and peripheral T cells eects by inhibiting cell proliferation, inducing apoptosis (Yang et al., 1998) and it can sensitize tumor necrosis and inhibiting NF-kB and AP-1 transcriptional activity. factor-a (TNG-a)-resistant tumor cell lines to TNF-a- However, a functional p53 is required for these induced apoptosis (Lee et al., 1999). Another candidate proapoptotic, anti-in¯ammatory and anti-tumor eects. which is involved in the anti-tumor and anti-in¯amma- Oncogene (2001) 20, 8009 ± 8018. tory eect by triptolide is NK-kB. A lot of studies show that triptolide executes its eect by inhibiting the Keywords: apoptosis; NF-kB; AP-1; p53 status; gastric transcriptional activity and/or binding activity of NF- cancer. kB (Lee et al., 1999; Qiu et al., 1999; Zhao et al., 2001). Apoptosis is regulated by gene products, which are conserved from nematodes to mammals (Penninger et al., 1996). Among all apoptosis-related genes, p53 is of particular importance (Lowe et al., 1993). P53 is well *Correspondence: BC-Y Wong, Department of Medicine, University known for suppression of cellular proliferation through of Hong Kong, Queen Mary Hospital, Hong Kong; two mechanisms, each operating in a distinct manner. E-mail: [email protected] In normal ®broblasts, p53 induces G arrest in 5Contributed equally to this work 1 Received 21 February 2001; revised 22 August 2001; accepted 18 response to DNA-damaging agents, presumably allow- September 2001 ing the cells to perform critical repair functions before Functional p53, AP-1 and NF-kB X-H Jiang et al 8010 progressing through the cell cycle (Linke et al., 1997). Induction of apoptosis by triptolide in AGS On the other hand, in abnormally proliferating cells or irradiated thymocytes, induction of p53 leads to AO staining showed that AGS cells treated with triptolide apoptosis (Midgley et al., 1995). Furthermore, wild- (up to 40 nM) underwent morphologic changes of type p53 protein level was increased during apoptosis apoptosis 24 h after treatment dose-dependently (Figure induced by DNA-damaging agents (Zhan et al., 1996). 1b). Analysis of DNA from AGS cells demonstrated that The increased expression of wild-type p53 can induce triptolide (510 nM) caused the generation of nucleoso- apoptosis in myeloid leukemia and colon cancers mal sized ladders of DNA fragments (Figure 1c). (Yonish-Rouach et al., 1991; Shaw et al., 1992). Unfortunately, the p53 gene is inactivated in approxi- Effect of triptolide on the cell cycle phase distribution mately 50% of tumors and p53 alteration has also been held responsible for the failure of most cancers to To further investigate triptolide-mediated dierential respond to radiotherapy and chemotherapy (Lee et al., growth inhibition, we studied the eect of triptolide 1995; MuÈ ller et al., 1996). For example, Vasey et al. treatment on the cell cycle kinetics in AGS cells. Flow (1996) reported that introduction of the dominant cytometry of three independent analysis showed that negative p53 enhanced resistance to irradiation, AGS cells (Figure 1d) were arrested in the G0/G1 phase cisplatin, doxorubicin and cytarabin treatment in after 24 h of 40 nM triptolide treatment (to 68.6+6.5% ovarian cancer cells. from 58.5+2.3% in control), and cells had a typical Nuclear factor-kB (NF-kB) and Activator protein-1 subdiploid peak (to 33.2% from 3.2%) on the DNA (AP-1) are two important transcriptional factors that histogram. may regulate a variety of in¯ammatory, apoptotic and immune responses. Triptolide has previously been Triptolide treatment resulted in enhanced expression of shown to sensitize several solid tumor cell lines to p53, p21waf1/cip1, and bax TNF-a-induced apoptosis through the inhibition of NF-kB (Lee et al., 1999). Furthermore, triptolide alone Time-course experiments were carried out to determine was able to inhibit transcriptional activation of NF-kB the protein levels of p53 after treatment. As shown in in Jurket cell and human bronchial epithelial cells (Qiu Figure 2, there was a signi®cant elevation of the p53 et al., 1999; Zhao et al., 2001). Taken as a whole, these protein level in triptolide (20 nM) treated AGS cells, results suggest that the anti-tumor and anti-in¯amma- starting as early as 2 h, reaching a maximal level which tory eects of triptolide are most likely associated with is about 16-fold of control after 8 h treatment. We also the modulation of NF-kB activation. Whether tripto- examined the p21waf1/cip1, bax, bc1-2 and c-myc protein lide has any modulation eect on AP-1 activity, expressions. In AGS cells, the protein levels of p21waf1/ however, has not been tested until now. cip1 and bax increased after 4 and 12 h of treatment, In the present study, we investigate the potential respectively. Triptolide did not alter the expression of therapeutic eects and underlying mechanisms of bc1-2 and c-myc within the time frame of this triptolide in human gastric cancer. We demonstrate that experiment (Figure 2). triptolide executes its anti-in¯ammatory and anti-tumor eect by inducing apoptosis and modulating AP-1 and Apoptosis triggered by triptolide is associated with NF-kB activity. We show that triptolide induces activation of caspase-3 (CPP-32) and cleavage of poly apoptosis in gastric cancer cells through a p53-dependent (ADP-ribose) polymerase (PARP) pathway and caspase-3 is required for triptolide-induced apoptosis. In addition, triptolide inhibited NF-kBand Caspases, especially caspase 3 (CPP-32), have been AP-1 activation in cells with wild-type p53, and forced shown to participate in apoptosis. To see if apoptosis alteration of p53 status suppresses triptolide-induced induced by triptolide was regulated by caspase 3, we apoptosis and modulation of NF-kB. Thus, our results examined several aspects of the caspase 3 activation. In suggest that functional p53 is essential for triptolide- the ®rst experiment, dierent concentrations of tripto- induced apoptosis and modulation of AP-1 and NF-kB lide were added to con¯uent AGS cells for 0, 6, 12, 24 activation in gastric cancer cells. and 48 h. After treatment, caspase protease activity was assessed in cell lysates by measuring hydrolysis of colorimetric caspase substrate DEVD ± rNA. Figure 3a illustrated that after the addition of triptolide (20 nM), Results caspase activity increased to maximal level within 24 h. In the next experiment, the expression of caspase 3 The effect of triptolide on cell growth in AGS during apoptosis was studied by Western blot analysis. We examined the antiproliferative eect of triptolide The 32 kD procaspase 3, when activated, was cleaved on human gastric cancer cell line AGS by MTT assay. into subunits of 17 kD as shown in Figure 3b. Figure 1a showed that treatment with triptolide at Furthermore, an 85 kD fragment cleaved from the concentrations ranging from 1 to 40 nM for 48 h speci®c caspase-3 substrate, 116 kD PARP, was inhibited cell proliferation dose-dependently in AGS detected in extracts from AGS cells treated with cells. After 48 h of treatment with triptolide, an triptolide. These results indicated that triptolide estimated 34% of AGS cells remained viable. activated caspase-3 in gastric cancer cells during the

Oncogene Functional p53, AP-1 and NF-kB X-H Jiang et al 8011

Figure 1 Eect of triptolide on cell growth and apoptosis in AGS cells. (a) Dose-response of triptolide on cell growth inhibition by MTT assay. *P40.05. (b) Dose-response of triptolide on apoptosis. AGS cells were treated with various concentrations of triptolide for 24 h. The percentage of apoptosis was quanti®ed by AO staining. *P40.05. (c) DNA ladder pattern formation. AGS cells were treated with dierent concentrations of triptolide for 24 h and the formation of oligonucleosomal fragments was visualized by 1.8% agarose gel electrophoresis. M, DNA markers; Lanes 1 ± 4, AGS cells treated with 40, 20, 10, 0 nM of triptolide. (d) FACS analysis. AGS cells were treated with 40 nM triptolide for 24 h and their DNA content was determined by FACS. (a) AGS control; (b) AGS treated with triptolide

time of apoptosis. However, no signi®cant alteration of 100 mM zVAD-fmk and 100 mM DEVD-fmk (data not caspase-3 activity or PARP cleavage was detected in shown). At this concentration, both zVAD-fmk and triptolide (5 ± 40 nM)-treated MKN-28 cells (data not DEVD-fmk signi®cantly suppressed triptolide-induced shown). apoptosis (Figure 3c).

Caspase inhibitors suppressed triptolide-induced caspase 3 Triptolide inhibits AP-1 and NF-kB transcriptional activation and apoptosis activity in AGS cells Although caspase activation and proteolysis are hall- Experiments were performed with a reporter plasmid marks of apoptosis, inhibition of caspases does not containing AP-1 or multiple kB elements. First we always prevent cells from undergoing apoptosis, treated the cells for dierent time period, we found that suggesting the existence of caspase-independent path- after treatment with 10 mM triptolide for 6 h, the way. To con®rm the essential role of caspase 3 in transcriptional activity of AP-1 and NF-kB showed triptolide-induced apoptosis, we next analysed the signi®cant decrease (data not shown). Then the cells eect of a pan-inhibitor of the caspase family, were treated for the dierent concentration of zVAD-fmk, and a speci®c inhibitor for caspase 3, triptolide for 6 h. As shown in Figure 4, triptolide DEVD-fmk, on apoptosis. We ®rst determined the inhibited the activation of AP-1 and NF-kB signi®- concentration of zVAD-fmk and DEVD-fmk required cantly. However, triptolide is not a general transcrip- to inhibit caspase 3 activity. Our results showed the tional inhibitor because it does not inhibit control cleavage of caspase 3 was completely blocked by CMV or SV40 promoter activity.

Oncogene Functional p53, AP-1 and NF-kB X-H Jiang et al 8012

Figure 2 Eect of triptolide on the expression of p53, p21waf1/ cip1, bax, bc1-2 and c-myc protein. (a) AGS cells were exposed to 20 nM triptolide for dierent time intervals (0, 2, 4, 8, 12, 24 h). The protein levels were determined by Western blot. (b) The relative density of p53, p21waf1/cip1 and bax expression in AGS cells detected by video densitometry

Triptolide does not inhibit DNA binding of NF-kB and AP-1 Figure 3 Eect of triptolide on caspase-3 activity and PARP To determine whether triptolide inhibits activation of expression in AGS cells. (a) Time course of triptolide eect on NF-kB and AP-1 through the inhibition of DNA caspase-3 activity. Con¯uent cells were treated with 20 nM binding of NF-kB and AP-1, we examined the eect of triptolide for 0, 8, 12, 24 and 48 h. Protease activity at each time point was determined as described in Materials and methods. triptolide on TNF-induced binding of NF-kB and AP-1 The data are expressed as the mean+s.d. of three individual by electrophoretic mobility shift assay. TNF-a induced experiments. (b) Analysis of caspase-3 and PARP expression in binding of NF-kB and AP-1 in AGS cells, and triptolide-treated AGS cells. Cells were treated with 20 nM antibodies to p65 (Rel A) and c-Jun supershifted the triptolide for 24 h. Then caspase-3 and PARP cleavage was analysed by Western blotting. (c) Eect of caspase inhibitors on complex demonstrating that p65 and c-Jun are part of triptolide-induced apoptosis in AGS cells. Cells were pretreated the NF-kB complex and AP-1 complex induced by with 100 mM zVAD-fmk or 100 mM DEVD-fmk for 12 h and TNF-a (Figure 5). Triptolide alone did not induce followed by 20 nM triptolide for another 24 h. The percentage of binding of NF-kB or AP-1. Furthermore, triptolide did apoptosis was determined by acridine orange staining. The results not aect the intensity of the NF-kB complex and AP-1 are expressed as mean+s.e. from three dierent experiments complex induced by TNF-a. Our results suggest that triptolide inhibits transactivation but not DNA binding of NF-kB and AP-1. The translocation of NF-kB to the antisense oligonucleotide and compared apoptotic nucleus is preceded by the phosphorylation, ubiquitina- response to triptolide. The phosphorothioate antisense tion, and proteolytic degradation of IkB-a.To oligonucleotide for p53 and the control CG-matched determine whether triptolide caused IkB-a degradation, randomized-sequence phosphorothioate oligonucleo- we measured the level of total IkB-a and phosphory- tides were pre-incubated with lipofectin (5 mg/ml, Life lated IkB-a. As shown in Figure 5, triptolide had no Technologies, Gaithersburg, MD, USA) for 30 min. eect on IkB-a phosphorylation and degradation. Cells growing in log phase were seeded in 24-well plates at a density of 16105 cells per well, incubated with the oligos/lipofectin mixture for 30 min, then with medium Down-regulation of p53 with antisense oligonucleotides containing oligos (®nal concentration equals to 5 mM) abolished triptolide-induced apoptosis in AGS cells for 18 h. Triptolide was added to the culture medium Since p53 expression is upregulated after triptolide for a ®xed time period for further analysis. Figure 6a treatment in AGS cell, next we downregulated p53 by showed that 5 mM p53 antisense oligonucleotide could

Oncogene Functional p53, AP-1 and NF-kB X-H Jiang et al 8013

Figure 5 Triptolide does not inhibit NF-kB or AP-1 DNA binding activity and IkB degraduation. (a) Nuclear extracts were prepared and analysed in an electrophoretic mobility shift assay in AGS cells with a radiolabeled IgG NF-kB or AP-1 probe. Equal amounts (10 mg) of nuclear protein was loaded in each Figure 4 Modulation of AP-1 and NF-kB transcriptional lane. In lanes 3 and 7, a rabbit polyclonal antibody to p65 (Santa activity by triptolide. Cells that transiently express AP-1 or NF- Cruz Biotechnology) was added to the nuclear extract 10 min kB luciferase reporter gene construct were treated with dierent before the addition of radiolabeled probe. In lane 4, an 1006 concentrations of triptolide for 6 h. Cells were then harvested for excess of unlabeled IgG oligonucleotide was added 5 min before analysis of luciferase activity. The ®re¯y luciferase reading was the addition of radiolabeled probe. Lane 1, control; lane 2, TNF- normalized to renilla luciferase reading. Data represent the a (20 ng/ml R&D systems); Lane 3, TNF-a +cold; Lane 4, TNF- mean+s.d. from three experiments. (a) NF-kB activity in AGS a +Ab; 5, triptolide (20 nM); and lane 6, TNF-a +triptolide. (b) (*P40.05); (b) AP-1 activity in AGS (*P40.05); (c) AGS cells Eect of triptolide on IkB-a degradation. AGS cells with or were transiently transfected with 0.6 mg/well NF-kB luciferase without 20 nM triptolide treatment were collected at dierent time reporter vector alone or together with 0.6 mg/well pCMV-p53- points. The cytoplasmic IkB-a and phospho-IkB-a protein level mt135 vector. After 6 h treatment with triptolide, cells were then were assayed by Western blot analysis as described in Materials harvested for analysis of luciferase activity and methods eectively inhibit the triptolide-induced increase in p53 pCMV ± p53mt135, a mutant p53 that is defective in protein level after 12 h, whereas the control oligonu- the conformation of its core DNA-binding domain, cleotide showed no eect on p53 expression. The p53 into AGS cells. As shown in Figure 4c, forced antisense oligonucleotide also inhibited triptolide-in- expression of PCMV ± p53mt135 increased the NF-kB duced apoptosis. At 20 nM of triptolide, the percentage activation by about twofold and abolished triptolide- of apoptotic cells decreased from 20.5+5.3% to mediated inhibition of NF-kB activation in AGS cells. 8.9+2.4% by the addition of 5 mM of p53 antisense oligonucleotide (P40.05). The same concentration of Triptolide has differential effect on cell growth inhibition, control oligonucleotide showed no eect on apoptosis apoptosis induction and NF-kB and AP-1 activation in induced by triptolide (Figure 6b). gastric cancer cells To further con®rm the importance of p53 in triptolide's Wild-type p53 is essential for inhibition of NF-kB eect on gastric cancer cells, we used other gastric activation by triptolide cancer cell lines with dierent p53 status. Our further To test if p53 status is also essential for the inhibition experiments showed that the same concentration of eect of NF-kB transcriptional activity by triptolide, triptolide inhibited cell growth and induced apoptosis we cotransfected NF-kB reporter plasmid with the in MKN-45 bearing wild p53, whereas it had no

Oncogene Functional p53, AP-1 and NF-kB X-H Jiang et al 8014

Figure 6 Eect of oligonucleotides on p53 protein expression and apoptosis. (a) AGS cells were cultured with an antisense or sense oligonucleotide (5 mM) for 18 h before incubation with 20 nM triptolide for another 12 h. Lane 1, control; Lane 2, 20 nM Figure 7 Eect of triptolide on cell growth and apoptosis in triptolide; Lane 3, 20 nM triptolide+5 mM sense oligonucleotide; gastric cancer cells. (a) Dose-response of triptolide on cell growth Lane 4, 20 nM triptolide+5 mM antisense-oligonucleotide. (b) inhibition by MTT assay. (*P40.05). (b) Dose-response of AGS cells were pretreated with 5 mM sense (S) or antisense (AS) triptolide on apoptosis. Cells were treated with various concen- oligonucleotides for 18 h followed by triptolide treatment for trations of triptolide for 24 h. The percentage of apoptosis was another 24 h. The percentage of apoptotic cells was measured by quanti®ed by AO staining. (*P40.05) acridine orange staining. (*P40.05) signi®cantly dierent from control triptolide alone group

p53 was directly responsible for this dierential growth-inhibition and apoptosis induction eects on sensitivity, we showed that p53 expression was upregu- gastric cancer MKN-28 and SGC-7901 cells with lated in AGS cells after triptolide treatment, and mutant p53 (Figure 7). On the other hand, triptolide suppression of p53 expression by using p53 antisense reduced NF-kB and AP-1 transcriptional activity in oligonucleotide signi®cantly abolished the triptolide- MKN-45, however, instead of reduction, triptolide induced apoptosis. The tumor suppressor gene p53 stimulated AP-1 and NF-kB activation in MKN-28 mediates either cell cycle arrest or apoptosis in response and SGC-7901 cells with mutant p53 (Figure 8). to DNA damage, thus acting as a molecular `guardian of the genome' (Lane, 1992). Most cancers, including gastric cancer, have either mutation within the p53 gene Discussion or defects in the ability to induce p53, and this p53 de®ciency has long been suspected to be the cause of Triptolide is a promising immunosuppressive agent poor prognosis. In this study, we also demonstrated that widely used in Chinese medicine. Recently, several lines MKN-28 and SGC-7901 gastric cancer cell lines with of evidence suggest that it also has antineoplastic eect mutant p53 did not show any signi®cant change both in on human malignant cells. Unfortunately, the molecular the cell growth and apoptosis after treatment with mechanisms responsible for its anti-neoplastic eect are triptolide. Therefore, our results support the notion that poorly understood. In the present study, we explore the p53 status in human gastric cancer cells contribute to the eects of triptolide on cell growth and induction of sensitivity to anti-cancer treatment, at least in part in the apoptosis in gastric cancer cells. Our results indicate that readiness to undergo apoptosis. triptolide inhibited cell proliferation and induced The increase in p53 protein in response to various apoptosis more readily in the wild-type p53 cell lines. stresses is an important regulator of cell cycle and These results are consistent with other studies, which apoptosis (Amundson et al., 1998). In transcription- show that the presence of wt p53 can enhance sensitivity dependent p53 activation, p53 functions as a site speci®c to DNA damaging agents (Weller et al., 1998), sensitivity transcription factor that induces p53-inducible genes such to chemotherapy in breast carcinomas (Tetu et al., 1999), as p21cip1/waf1, bax, gadd45, and mdm2 (Farmer et al., and sensitivity for cisplatin in ovarian carcinomas 1992; El-Deiry et al., 1993; Miyashita et al., 1995; Zhu et (Calvert et al., 1996). We further demonstrated that al., 1999). In this study, we showed that induction of p53

Oncogene Functional p53, AP-1 and NF-kB X-H Jiang et al 8015 A recent study showed that triptolide sensitized non- small cell lung cancer cell line, ®brosarcoma cell line and breast cancer cell line to TNF-a-induced apoptosis through inhibition of NF-kB (Lee et al., 1999). In our B Activity κ study, we demonstrated that triptolide alone could inhibit NF-kB transcriptional activity dose-dependently after 6 h treatment in AGS cells. Since most cells are Relative NF- still alive at that time, this eect may not necessarily associate with triptolide-induced apoptosis. In most cells, NF-kB is inactive due to its cytoplasmic sequestration through interaction with inhibitor proteins IkBs. Upon activation of the complex, IkB sequentially undergoes phosphorylation, ubiquitina- tion, and degradation. However, in our system, we showed that the modulation of NF-kB transactivation by triptolide was not related to IkB phosphorylation and degradation. We also demonstrated that triptolide inhibited NF-kB transcriptional activity but not binding activity. Recent studies suggest that transactivation of NF-kB is enhanced through the phosphorylation of p65 at Ser-276 and Ser-529 (Zhong et al., 1998; Wong and Baldwin, 1998). Lee et al. (1999) showed that triptolide inhibited transactivation of both Figure 8 Modulation of AP-1 and NF-kB transcriptional the TA1 and TA2 regions of p65. These results suggest activity by triptolide in gastric cancer cells. Cells that transiently that triptolide might inhibit activation of NF-kBbya express AP-1 or NF-kB luciferase reporter gene construct were novel mechanism other than cytoplasmic sequestration treated with dierent concentrations of triptolide for 6 h. Cells were then harvested for analysis of luciferase activity. The ®re¯y of IkB and inhibition of NF-kB binding activity. luciferase reading was normalized to renilla luciferase reading. NF-kB signaling cross-talks closely with that of p53 Data represent the mean+s.d. from three experiments. (a) NF-kB (Webster et al., 1999). To determine whether p53 status activity; (b) AP-1 activity plays any role on the eect of triptolide on NF-kB activation, we co-transfected NF-kB reporter with a dominant negative p53 into AGS cells. Our results showed that forced suppression of normal p53 function by triptolide in AGS cells was also associated with by dominant-negative p53 markedly enhanced NF-kB waf1/cip1 upregulation of p21 , bax and G0/G1 arrest. activation in AGS cells. We then treated the cells with However, in a previous study, Chang et al. (2001) showed triptolide after alteration of p53 status. We demon- that triptolide induced p53 protein expression but strated deletion of p53 function by dominant negative inhibited basal p21 expression and doxorubicin-mediated p53 in AGS cells resulted in abolishment of inhibition induction of p21. This discrepancy might be due to the of NF-kB activation by triptolide. These results indicate dierential role of p21waf1/cip1 in modulation of apoptosis that wild-type p53 status is required to suppress the and cell survival is cell type and environment dependent NF-kB transcriptional activity. This observation is (El-Deiry et al., 1994; Kobayashi et al., 1995; Zhu et al., supported by some other studies, such as Nishizaki et 2000). Our results suggest that upregulation of p21waf1/cip1 al. (2000). They showed that overexpression of wild- is associated with the modulation of apoptosis in gastric type p53, but not mutant, inhibited NF-kB activity, and cancer cells. However, the exact role of p21waf1/cip1 in therefore induced apoptosis in human colon cancer cells triptolide-induced apoptosis needs further investigation. (Nishizaki et al., 2000). Consistent with our results, we We have shown that triptolide-induced apoptosis also showed that triptolide had dierential eect on was controlled through caspase activation, especially MKN-28 and SGC-7901 gastric cancer cells with caspase 3. Caspase 3 or CPP32 is one of the important mutant p53, instead of suppression, it increased the candidates for cell death-inducing proteases that cleave transcriptional activity of NF-kB. PARP and other vital proteins (Patel et al., 1996). In The activator protein-1 (AP-1) is a ubiquitous factor this study, triptolide induced caspase 3 activation and that regulates the transcription of genes involved in cell PARP cleavage in parallel with the induction of growth and proliferation in response to external stimuli apoptosis. Furthermore, caspase inhibitors could eec- such as growth factors, phorbol esters and ionizing tively block the cleavage of caspase 3. However, both radiation (Angel et al., 1991). AP-1 has been associated zAVD-fmk and DEVD-fmk could only partially inhibit with in¯ammation and neoplastic transformation. triptolide-induced apoptosis in AGS cells. These results Inhibitors of AP-1 activation block the neoplastic indicated that the activation of caspase 3 was involved transformation response (Li et al., 1997). We report for in triptolide-induced apoptosis. But it is likely that the ®rst time that triptolide inhibited AP-1 transcrip- triptolide could also induce apoptosis partly via a tional activity in gastric cancer cell AGS and MKN-45 caspase-independent pathway. containing wild-type p53, whereas increased AP-1

Oncogene Functional p53, AP-1 and NF-kB X-H Jiang et al 8016 activity in MKN-28 and SGC-7901 with mutant p53. propidium iodide (PI, 400 mgml71, Sigma, USA) were added However, whether the dierential modulation of AP-1 and incubated at 378C for 30 min. Sample analysis of samples transcriptional activity contributes to the anti-tumor was performed by ¯ow cytometry (Coulter Epics, XL, UK). and anti-in¯ammatory eect by triptolide needs further The cell cycle phase distribution was calculated from the investigation. resultant DNA histogram using Multicycle AV software (Phoenix Flow System, San Diego, CA, USA). The apoptotic In summary, triptolide is a novel drug with potent cells were observed as a subdiploid or `pre-G ' peak. anti-in¯ammatory and anti-proliferative eects on 1 gastric cancer cells. Triptolide induced apoptosis in gastric cancer cells through a p53-dependent pathway. DNA fragmentation analysis In addition, triptolide inhibited NF-kB and AP-1 As described previously, DNA fragmentation was analysed activation in cells with wild-type p53, forced alteration with some modi®cations (Grant et al., 1992). Brie¯y, cell of p53 status suppressed triptolide-induced apoptosis pellets were lysed in 10 mM Tris-HCL (pH 7.4) buer 71 and modulation of NF-kB. Our results suggest that containing 25 mM EDTA, 0.5% SDS and 0.1 mg ml functional p53 is required for the anti-in¯ammatory and proteinase K (Sigma) and incubated at 508C for 12 ± 18 h. anti-tumor eect by triptolide in gastric cancer cells. DNA was extracted with an equal volume of phenol/ chloroform/isoamyl alcohol (25 : 24 : 1) and precipitated with two volumes of ice-cold absolute ethanol and 1/10 volume 3M sodium acetate. Equal amounts (10 mg per well) of DNA were electrophoresed in 1.8% agarose gels Materials and methods impregnated with ethidium bromide (0.1 mgml71) for 2 h at 80 V. DNA fragments in the form of a laddering pattern Cell culture and drug treatment were visualized by ultraviolet transillumination. AGS bearing wild-type p53 was purchased from the American Type Culture Collection (ATCC, Rockville, MD, Preparation of cytoplasmic and nuclear extract USA). MKN-28, which has missense mutation in p53 (codon 251, isoleucine to leucine), and MKN-45 bearing wild-type Nuclear and cytoplasmic extracts were prepared as described p53 were purchased from RIKEN (The Institute of Physical by Dignam et al. (1983). Con¯uent cells in 10-cm dishes were and Chemical Research) Cell Bank, Japan. SGC-7901 was treated for various times with the indicated eectors. Cells kindly donated by Professor SD Xiao (Shanghai Second were then washed two times with ice-cold phosphate-buered Medical University). Cells were maintained in RPMI-1640 saline and resuspended in 400 ml of buer A (containing 71 containing 10% fetal bovine serum (FBS), 100 U ml 10 mM HEPES at pH 7.9, 1.5 mM MgCl2,10mM KCl, penicillin, 100 mgml71 streptomycin (Gibco BRL, Life 0.5 mM DTT, 0.5 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml Technologies, NY, USA). The stock solution of triptolide aprotinin, and 1 mg/ml pepstatin A). The cells were allowed to (90 mM) in ethanol was a kind gift from Dr Dan Yang swell on ice for 15 min, lysed gently with 12.5 ml of 10% (Department of Chemistry, University of Hong Kong). The Nonide P-40, and centrifuged at 2000 g for 10 min at 48C. phosphorothioate antisense oligonucleotide for p53 and the The supernatant was collected and used as the cytoplasmic control CG-matched randomized-sequence phosphorothioate extracts. The nuclei pellet was re-suspended in 40 ml of buer oligonucleotides were purchased from Biognostik (GoÈ ttingen, C (20 mM HEPES, pH 7.9, containing 1.5 mM MgCl2, Germany). 450 mM NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 1 mg/ml leupeptin, 1 mg/ml aprotinin, 1 mg/ml pepstatin A), agitated for 30 min at 48C, and the nuclear MTT assay debris was spun down at 20 000 g for 15 min. The supernatant About 5000 cells per well were grown in 96-well microtitre (nuclear extract) was collected, frozen in liquid nitrogen and plates and incubated overnight in 100 ml of culture medium. stored at 7808C until ready for analysis. Protein concentra- Cells were then treated with dierent concentrations of tion was determined using Bradford reagent. triptolide for ®xed time intervals. Ten ml MTT (Fluka, Buchs, Switzerland) labeling reagent (®nal concentration 0.5 mg/ml) Western blotting was added into each well and the cells were incubated for another 4 h at 378C. The supernatant was removed, and Twenty mg of protein were resolved and separated by 100 ml of 0.04 M hydrochloric acid in isopropanol was added electrophoresis on a 10% denaturing SDS gel. Proteins were to each well. A micro ELISA reader (BioRad, CA, USA) electroblotted onto nitrocellulose membranes. Detection was measured the absorbency at a wavelength of 595 nm. conducted by immuno-staining using speci®c primary antibodies and horseradish peroxidase-conjugated anti-IgG antibody. The protein bands were visualized by the enhanced Acridine orange (AO) staining chemiluminescence (ECL) assay (Amersham) following Single cell suspensions were ®xed in 1% formalin/PBS and manufacturer's instructions. Mouse monoclonal antibodies stained with acridine orange (AO, 10 mgml71, Sigma, St. against p53 (Ab-6), p21waf1/cip1 (Ab-1), c-myc (Ab-1) and Louis, USA). Cells were spotted on glass slides and visualized peroxidase-conjugated anti-mouse and anti-rabbit IgG were under a UV ¯uorescence microscope. A minimum of 300- purchased from CalBiochem (La Jolla, CA, USA). Mono- cells/®eld was counted for apoptotic index. clonal antibodies against bc1-2 (100) and bax (B-9) were purchased from Santa-Cruz Biotechnology (CA, USA). Rabbit polyclonal antibody against CPP-32 and mouse Flow cytometry monoclonal antibody against PARP (c-20) were purchased Cells were collected and ®xed in ice-cold 70% ethanol in from Pharmingen (San Diego, CA, USA). Rabbit polyclonal phosphate buered saline (PBS) and stored at 7208C. After antibody against IkB-a and phosphor IkB-a were purchased resuspension, 100 ml RNAase I (1 mg ml71) and 100 ml from New England Biolabs (Boston, MA, USA).

Oncogene Functional p53, AP-1 and NF-kB X-H Jiang et al 8017 (EG&G B, Australia). The Fire¯y luciferase luminescence Caspase-3 activity assay activity were normalized to the control Renilla luciferase Caspase activity was determined using the Clontech ApoAlert activity and expressed as a relative ratio. CPP32 Assay Kit according to the manufacturer's instructions (Clontech, Palo Alto, CA, USA). These assays Electrophoretic mobility shift assay measured the cleavage of a speci®c colorimetric caspase substrate, DEVD ± rNA (Asp-glu-val-asp-r-nitroanilide). Six mg of nuclear proteins were incubated with 1 mg each of Cells were plated into 60 mm dishes at 26106 cells/dish poly(dI.dC) and poly(dG.dC) in the presence of 10 fmol of and cultured for 24 h before treatment. After treatment, cells [g-32P] ATP end-labeled double-stranded Ap1 probe (5'-CGC were collected by scraping in cold PBS, centrifuged TTG ATG AGT CAG CCG GAA-3' Promega) or Nf-kBB 2000 r.p.m., 8 min) and lysed in the cell lysis buer provided (5'-AGT TGA GGG GAC TTT CCC AGG C-3' Santa Cruz) in the kit on ice for 10 min. Extracts were then frozen and for 30 min at room temperature in a total volume of 20 ml. maintained at 7708C until the time of assay. At that time, Oligonucleotide competition experiments were performed in the extracts were thawed and reacted with an equal volume of the presence of 50-fold excess of cold AP1 and NF-kB oligos. 26reaction buer containing DTT (10 mM) and the Supershift experiments were done by preincubating nuclear colorimetric caspase substrate (DEVD ± rNA). The mixtures extract proteins with 2 ml antibody for 1 h at 48C before the were maintained in a water bath at 378C for 45 min and then addition of labeled DNA probe. DNA complexes were analysed in a spectrophotometer at 405 nm. resolved from free probe with 4% nondenaturing polyacry- lamide gels in 0.56 Tris-borate-EDTA (pH 8.3) and visualized by ¯uorography. Polyclonal antibody c-Jun (N) Transient transfection and luciferase assay X for AP1 and NF-kB p65 (c-20) for NF-kB from Santa NF-kB luciferase and AP-1 reporter plasmid were con- Cruz Biotechnology (Santa Cruz, CA, USA) was used for structed individually by inserting the IL-6 promoter region supershift experiments. (7194 to 7114 containing four repeats of NF-kB binding sites) and collagenase promoter region (773 to +67 Statistical analysis containing one AP-1 binding site) into a luciferase reporter vector pGL-3-basic (Promega Corp., Madison, WI, USA) (Li The data shown were mean values of at least three dierent et al., 1997; Angel et al., 1987). Expression plasmids pCMV ± experiments and expressed as means+s.d. Student's t-test p53mt135, which expresses a dominant-negative mutant, were was used for comparison. A P-value of less than 0.05 is purchased from Clontech (CA, USA). For transient transfec- considered statistically signi®cant. tion experiments, AGS cells were seeded in 12-well plates to 70 ± 80% con¯uence. The cells were transfected with 0.6 mg/ well NF-kB or AP-1 reporter plasmid with the presence or absence of dierent concentrations of pCMV ± p53mt135 Abbreviations (0.1 ± 0.6 mg/well) using lipofectamine 2000. pRL ± CMV TWHf, Tripterygium wilfordii Hook f; NF-kB, nuclear vector (0.01 mg/well, Promega) was cotransfected as internal factor-kB; AP-1, Activator Protein-1 control. After transfection for 6 h, cells were changed to normal medium and allowed to recover overnight, and then treated with dierent concentrations of triptolide for another 6 h. Cells were harvested for analysis of luciferase activity Acknowledgments using the dual luciferase reporter assay system (Promega). This study is supported by Gastroenterological Research Luminence was measured by a LB 9507 Luminometer Fund, University of Hong Kong.

References

Amundson SA, Myers TG and Fornace AJ. (1998). Farmer G, Bargonetti J, Zhu H, Friedman P, Prywes R and Oncogene, 17, 3287 ± 3299. Prives C. (1992). Nature, 358, 83 ± 86. AngelP,BaumannI,SteinB,DeliusH,RahmsdorgHJand Grant S, Jarvis WD, Swerdlow PS, Turner AJ, Traylor RS, Herrlich P. (1987). Mol. Cell. Biol., 7, 2256 ± 2266. Wallace HJ, Lin PS, Pettit GR and Gewirtz DA. (1992). Angel P and Karin M. (1991). Biochem. Biophys. Acta., 1072, Cancer Res., 52, 6270 ± 6278. 129 ± 157. Jiang X. (1994). Pediatr. Nephrol., 8, 343 ± 344. CalvertAH,GhokulS,Al-AzraqiA,WrightJ,LindMand Kobayashi T, Consoli U, Andree M, Shiku H and Zhang Bailey N. (1999). Semin. Oncol., 16, S90 ± S94. W. (1995). Oncogene, 11, 2311 ± 2316. Chang WT, Kang JJ, Lee KY, Wei K, Anderson E, Gormare Kupchan SM, Court WA, Dailey RG, Gilmore CJ and Bryan S, Ross JA and Rosen GD. (2001). J. Biol. Chem., 276, RF. (1972). Amer. Chem. Soc., 94, 7194 ± 7195. 2221 ± 2227. Lane DP. (1992). Nature, 358, 15 ± 16. Dignam JD, Lebovitz RM and Roeder RG. (1983). Nucleic Lee JM and Bernstein A. (1995). Cancer Metastasis Rev., 14, Acids Res., 11, 1475 ± 1489. 149 ± 161. El-Deiry WS, Harper JW, O'Connor PM, Velculescu VE, LeeKY,ChangWT,QiuDM,KaoPMandRosenGD. Canman CE, Jackman J, Pietenpol JA, Burrell M, Hill (1999). J. Biol. Chem., 274, 13451 ± 13455. DE, Wang Y, Wiman KG, Mercer WE, Kastan MB, Kohn Li JJ, Westergaad D, Ghosh P and Colburn NH. (1997). KW, Elledge SJ, Kinzler KW and Vogelstein B. (1994). Cancer Res., 57, 3569 ± 3576. Cancer Res., 54, 1169 ± 1174. Linke SP, Clarkin KC and Wahl GM. (1997). Cancer Res., El-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons 57, 1171 ± 1179. R, Trent JM, Lin D, Mercer WE, Kinzler KW and Vogelstein B. (1993). Cell., 75, 817 ± 825.

Oncogene Functional p53, AP-1 and NF-kB X-H Jiang et al 8018 Lowe SW, Ruley HE, Jacks T and Housman DE. (1993). Wang D and Baldwin AS. (1998). J. Biol. Chem., 273, Cell, 74, 957 ± 967. 29411 ± 29416. Midgley CA, Owens B, Briscoe CV, Thomas DB, Lane DP Webster GA and Perkins ND. (1999). Mol. Cell. Biol., 19, and Hall PA. (1995). J. Cell Sci., 108, 1843 ± 1848. 3485 ± 3495. Miyashita T and Reed JC. (1995). Cell, 80, 293 ± 299. Wei YS and Adachi L. (1991). Acta. Pharmacologica Sinica., MuÈ ller H and Eppenberger U. (1996). Anticancer Res., 16, 12, 406 ± 410. 3845 ± 3848. Weller M. (1998). Cell Tissue Res., 292, 435 ± 445. Nishizaki M, Roth JA and Tanaka N. (2000). Oncogene, 19, Yang SX, Gao HL, Xie SS, Zhang WR and Long ZZ. (1992). 726 ± 736. Int. J. Immunopharmacol., 14, 963 ± 969. Patel T, Gores GJ and Kaufmann SH. (1996). FASEB J., 10, YangY,LiuZ,TolosaE,YangJandLiL.(1998). 587 ± 597. Immunopharmacology, 40, 139 ± 149. Penninger JM and Kroemer G. (1996). Adv. Immunol., 68, Yonish-Rouach E, Resnitzky D, Lotem J, Sachs L, Kimchi A 51 ± 144. and Oren M. (1991). Nature, 352, 345 ± 347. Pu LX and Zhang TM. (1990). Acta. Pharmacologica Sinica., Zhao G, Vaszar LT, Qiu D, Shi L and Kao PN. (2001). Am. 11, 76 ± 79. J. Physiol. Lung Cell Mol. Physiol., 279, L958 ± L966. Qin WZ, Liu CH and Yang SM. (1981). Chin.Med.J. Zhan Q, Alamo I, Yu K, Boise LH, Cherney B, Tosato G, (Engl.), 94, 827 ± 830. O'Connor PM and Fornace AJ. (1996). Oncogene, 13, QiuD,ZhaoG,AokiY,ShiL,UyeiA,NazarianS,NgJC 2287 ± 2293. and Kao PN. (1999). J. Biol. Chem., 274, 13443 ± 13450. Zhang LX, Yu FK, Zhang QY, Fang Z and Pan DJ. (1990). Shaw P, Bovey R, Tardy S, Sahli R, Sordat B, Costa J. Acta. Pharmaceutica Sinica., 25, 573 ± 577. (1992). Proc. Natl. Acad. Sci. USA, 89, 4495 ± 4499. Zhong H, Voll RE and Ghosh S. (1998). Mol. Cell. Biol., 1, TaoX,SunY,DongY,XiaoYL,HuDW,ShiYP,ZhuQL, 661 ± 671. Huan D and Zhang NZ. (1989). Chin.Med.J.(Engl.), Zhu GH, Wong BC, Eggo MC, Yuen ST, Lai KC and Lam 102, 327 ± 332. SK. (1999). Dig. Dis. Sci., 44, 2020 ± 2026. Tetu B, Brisson J, Plante V and Bernard P. (1998). Mod. ZhuGH,WongBC,SlosbergED,EggoMC,ChingCK, Pathol., 11, 823 ± 830. YuenST,LaiKC,SohJW,WeinsteinIBandLamSK. Vasey PA, Jones NA, Jenkins S, Dive C and Brown R. (2000). Gastroenterology, 118, 507 ± 514. (1996). Mol. Pharmacol., 50, 1536 ± 1540.

Oncogene