Antiproliferative and Proapoptotic Activities of Triptolide
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2666 Vol. 8, 2666–2674, August 2002 Clinical Cancer Research Antiproliferative and Proapoptotic Activities of Triptolide (PG490), a Natural Product Entering Clinical Trials, on Primary Cultures of Human Prostatic Epithelial Cells1 Taija M. Kiviharju, Philip S. Lecane,2 INTRODUCTION Robert G. Sellers, and Donna M. Peehl3 Extracts of the herb Tripterygium wilfordii hook F have Department of Urology, Stanford University School of Medicine, been used for more than two centuries in traditional Chinese Stanford, California 94305 medicine to treat a variety of autoimmune and inflammatory diseases including rheumatoid arthritis (1). Triptolide (PG490), a diterpene triepoxide, is a purified compound from Triptery- ABSTRACT gium that has been identified as one of the major components Interest in exploiting traditional medicines for preven- responsible for the immunosuppressive and anti-inflammatory tion or treatment of cancer is increasing. Extracts from the effects of this herb (2). Recent explorations of the mechanisms herb Tripterygium wilfordii hook F have been used in China of action of triptolide revealed many properties relevant not only for centuries to treat immune-related disorders. Recently it to anti-inflammatory activity but also to anticancer activity. was reported that triptolide (PG490), a purified compound Antiproliferative and proapoptotic activity of triptolide has been from Tripterygium, possessed antitumor properties and in- shown with many different types of cancer cells in vitro and in duced apoptosis by p53-independent mechanisms in a vari- vivo (3, 4). Triptolide also potentiates the activities of other ety of malignant cell lines. This property of triptolide at- agents and therefore may be useful not only as a single com- tracted our attention because we have found that primary pound but also in combination with other cytotoxic drugs for cultures of human prostatic epithelial cells derived from cancer treatment. For example, triptolide sensitized tumor ne- normal tissues and adenocarcinomas are in general ex- crosis factor ␣-resistant tumor cells to tumor necrosis factor tremely resistant to apoptosis. Furthermore, the function of ␣-induced apoptosis (5) and showed cooperative proapoptotic wild-type p53 is impaired in these cells such that drugs that effects with doxorubicin. In the latter studies, triptolide was require p53 activity to induce cell death are ineffective. found to block doxorubicin-mediated induction of p21WAF1/CIP1 Therefore, the properties of triptolide and the recent ap- and accumulation of cells in G2-M (6). The investigators proval of its water-soluble form (PG490-88) for entry into proposed that triptolide, by blocking p21WAF1/CIP1-mediated Phase I clinical trials suggested that this drug was a prom- growth arrest, created conflicting cell cycle checkpoints that ising candidate to test for antitumor activity against prostate enhanced apoptosis in tumor cells. PG490-88, a succinate salt cells. Experiments presented here demonstrated that trip- water-soluble derivative of PG490 that is converted to triptolide tolide had dose-dependent effects on both normal and in the serum (7), has recently been approved for Phase I clinical cancer-derived primary cultures of human prostatic epithe- trials. lial cells. Low concentrations of triptolide inhibited cell pro- The exact targets and function (or molecular mechanism of liferation and induced a senescence-like phenotype. Higher action) of triptolide are still unknown. An attempt to discern the concentrations of triptolide induced apoptosis that was un- molecular signaling pathways triggered by triptolide was made expectedly associated with nuclear accumulation of p53. by cDNA microarray analysis of triptolide-treated normal and Paradoxically, levels of the p53 target genes, p21WAF1/CIP1 transformed bronchial epithelial cells. Triptolide reduced ex- and hdm-2, were reduced, as was bcl-2. Our preclinical pression of cell cycle regulators and survival genes such as studies suggest that triptolide might be an effective preven- cyclins D1, B1, and A1; cdc-25; bcl-x; and c-jun (8). Anti- tive as well as therapeutic agent against prostate cancer and inflammatory, antiproliferative, and proapoptotic properties of that triptolide may activate a functional p53 pathway in triptolide have also been associated with inhibition of nuclear prostate cells. factor-B (5, 8, 9). We became interested in triptolide because it was reported to direct cancer cells to undergo apoptosis independent of p53 activity (4). The tumor suppressor protein p53 responds to Received 1/28/02; revised 5/8/02; accepted 5/15/02. different forms of cellular stress such as DNA damage caused The costs of publication of this article were defrayed in part by the by ionizing radiation or chemotherapeutic drugs by targeting the payment of page charges. This article must therefore be hereby marked checkpoint genes that inhibit cell cycle progression (10) and/or advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. trigger apoptosis (review in Refs. 11 and 12). Previous studies 1 Supported in part by Department of Army Grant DAMD 17-99-1- conducted by us showed that activation of p53 in response to 9004. ␥-irradiation or other stresses such as hypoxia or DNA-damag- 2 Present address: Pharmacyclics, 995 East Arques Avenue, Sunnyvale, ing drugs is attenuated in primary cultures of normal and ma- CA 94086. 3 To whom requests for reprints should be addressed, at Department of lignant prostatic epithelial cells, despite the presence of the Urology, Stanford Medical Center, Stanford, CA 94305-5118. Phone: wild-type p53 gene (13). Because many chemotherapeutic drugs (650) 725-5531; Fax: (650) 723-0765; E-mail: [email protected]. use p53-mediated pathways to induce growth arrest or apopto- Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2002 American Association for Cancer Research. Clinical Cancer Research 2667 sis, we have suggested that dysfunction of p53 may partially 60-mm dish containing 5 ml of Complete MCDB 105 with explain the resistance of prostate cancer to drug treatment. The experimental factors. Cells were incubated for 10 days without prevalence of mutated p53 in advanced prostate cancer (14) may feeding and then fixed with 10% formalin and stained with also impact response to chemotherapy. We therefore hypothe- crystal violet (17). Growth was quantitated with an Artek image size that drugs using p53-independent pathways would be most analyzer (Dynatech, Chantilly, VA), which measures the total efficacious against prostate cancer. area of the dish covered by cells. This relative value has been In our study, we found that triptolide exerted two qualita- shown to be directly proportional to cell number (22). tively different types of effects on primary cultures of prostatic High Density Growth Assay. Cells were inoculated at 5 epithelial cells. Low concentrations of triptolide inhibited 10 cells/dish into collagen-coated, 60-mm dishes containing growth and induced senescence, an irreversible growth-arrested Complete MCDB 105. One day later (day 0), various concen- state that is associated with tumor suppression. Higher concen- trations (1–100 ng/ml) of triptolide were added. Cells treated trations of triptolide triggered apoptosis. Interestingly, unlike with diluent (0.001% DMSO) were included as controls. After 3 our results with any previously tested compound, triptolide- days, fresh media containing diluent or triptolide were replaced. induced apoptosis of primary cultures was accompanied by Cells in replicate dishes were counted by hemocytometer after nuclear accumulation of p53. This finding was unexpected, trypsinization on days 0, 3, and 6. given the original reports of p53-independent activity of triptol- Cell Viability. Loss of cell viability was assessed by the ide, but in fact more recent studies indicate that triptolide- trypan blue exclusion method. Cells treated with or without induced apoptosis may be at least partly mediated through triptolide were harvested by trypsinization. After incubation in activation of p53 in cells with wild-type p53 (6, 15). Down- 0.04% trypan blue (Sigma-Aldrich) for 4 min, cells were regulation of p53 target genes hdm-2 and p21WAF1/CIP1, as well counted under a hemocytometer. The number of cells that re- as bcl-2, was also observed in prostatic epithelial cells. Our tained the dye (nonviable) and the total cell number were noted. 5 preclinical findings support further investigation of chemopre- Apoptosis. Cells were inoculated at 10 cells/dish into ventive and chemotherapeutic activity of triptolide against pros- collagen-coated, 60-mm dishes containing Complete MCDB tate cancer. 105. One day later, the medium was replaced, and cells were treated with various concentrations (1–100 ng/ml) of triptolide. Cells treated with diluent (0.001% DMSO) were included as MATERIALS AND METHODS controls. After 24, 48, and 72 h, Hoechst 33342 and propidium Cell Culture and Reagents. Tissue samples were dis- iodide (Sigma-Aldrich) were added to medium at 10 and 20 sected from radical prostatectomy specimens. None of the pa- g/ml, respectively. After incubation for 15 min at 37°C, 400 tients had received prior chemical, hormonal, or radiation ther- cells from each dish were counted (from 10 randomly selected apy. Histological assessment was performed as described fields) under fluorescence to determine the proportion of viable previously (16). Epithelial cells were cultured and characterized and apoptotic