Functional P53 Is Required for Triptolide-Induced Apoptosis and AP-1 and Nuclear Factor-Κb Activation in Gastric Cancer Cells
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Oncogene (2001) 20, 8009 ± 8018 ã 2001 Nature Publishing Group All rights reserved 0950 ± 9232/01 $15.00 www.nature.com/onc 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.