Elsevier

Leukemia Research

Volume 29, Issue 1, January 2005, Pages 99-105
Leukemia Research

Triptolide inhibits transcription factor NF-kappaB and induces apoptosis of multiple myeloma cells

https://doi.org/10.1016/j.leukres.2004.05.014Get rights and content

Abstract

Triptolide has been reported to be effective in the treatment of auto-immune diseases. This study investigates the cytotoxic function of triptolide on multiple myeloma (MM) cells. We found that triptolide inhibited the proliferation of both RPMI8226 and U266 cells in a dose-dependent manner (10–80 ng/mL). Triptolide induced apoptosis in MM cells through activation of the cystein protease caspase 8, 9 and 3, and subsequent cleavage of the DNA repair enzyme poly (ADP-ribose) polymerase. Apoptosis was confirmed with cell-cycle analysis and annexin V staining. Moreover, triptolide down-regulated nuclear factor (NF)-kappaB activity in MM cell lines. In addition, triptolide also induced chemosensitivity to doxorubicin and suppressed cell proliferation of fresh MM cells. Therefore, triptolide appears to be a potent inducer of apoptosis in myeloma cells, and might have some benefit in the treatment of myeloma patients.

Introduction

Multiple myeloma (MM) is characterized by accumulation of malignant plasma cells in the bone marrow associated with bone marrow failure, renal dysfunction, osteolytic lesions, hypercalcemia, and immunodeficiency. MM accounts for 1% of all cancers and more than 10% of all hematologic cancers. Although conventional chemotherapy and high-dose therapy with hematopoietic stem-cell rescue can prolong survival [1], few, if any, patients are cured. Thalidomide has shown promise [2], however, new treatments are still urgently needed.

Recently, it was reported that nuclear factor-kappaB (NF-kappaB), which, up-regulates the transcription of proteins that promote cell survival, stimulate growth, and reduce susceptibility to apoptosis, is constitutively activated in the majority of primary MM specimens [3]. NF-kappaB activation also induces drug resistance in myeloma cells and up-regulates the expression of adhesion molecules involved in the resistance of myeloma cells to drugs. In addition, it modulates the secretion by bone marrow stromal cells of cytokines that mediate the growth, survival, and migration of myeloma cells [4], [5], [6], [7], [8], [9]. Thus, strategies to inhibit NF-kappaB, and thereby block growth and survival pathways regulated by NF-kappaB, may represent a useful approach to more durable MM therapy. Proteasome inhibition PS-341, which blocked nuclear translocation of NF-kappaB, blocked NF-kappaB DNA binding, is a promising new treatment of relapsed and refractory MM [10], [11], [12].

Triptolide (Diterpenoid triepoxide), a purified component of a traditional Chinese medicine, is extracted from a shrub-like vine named Tripterygium wilfordii Hook F (TWHF). It has been reported to be effective in the treatment of autoimmune diseases, especially rheumatoid arthritis [13], [14], and it can also induce anti-neoplastic activity on several human tumor cell lines [15], [16], [17], [18], [19], [20], [21], [22], [23], [24]. We also found triptolide can induce apoptosis in K562 cells [25]. Interestingly, Triptolide has previously been shown to sensitize several tumor cell lines to TNFα-induced apoptosis through the inhibition of NF-kappaB [23], [26]. Triptolide alone was able to inhibit transcriptional activation of NF-kappaB in Jurkat cell and human bronchial epithelial cells [27], [28], [29]. Furthermore, triptolide also has been shown to down-regulate the expression of various NF-kappaB-regulated genes, including IL-6, cIAP, XIAP, bcl-2, COX-2, IL-1, IL-2, TNF, and the adhesion molecules [21], [29], [30]. It was also reported triptolide inhibit vascular endothelial growth factor expression, which is believed to play a role in tumor angiogenesis [31]. So we examined whether triptolide affects the survival of MM cells and investigated the molecular mechanism of triptolide-induced apoptosis.

Section snippets

Cell culture and drugs

The MM cell lines RPMI8226 and U266 were obtained from ATCC (Rockville, USA), cell lines were cultured in RPMI1640 (GibcoBRL, USA), 10% fetal bovine serum (FBS; GibcoBRL), 50 μg/mL streptomycin, 50 IU/mL penicillin, and 2 mM glutamine in a 5% humidified CO2 atmosphere at 37 °C. BM aspirates were obtained from three patients with high tumor burden. The mononuclear cell fraction was enriched by Ficoll (ShangHai, China) density gradient sedimentation at 600 g for 20 min and washed twice in PBS. The

Effect of triptolide on the growth of MM cell lines, MM patients’ cells

Using MTT assays, we first determined the effect of triptolide on growth of human MM cell lines (RPMI8226 and U266). Fig. 1 shows the dose-related effect of triptolide in the 10–80 ng/mL range after 48 h of exposure. The triptolide IC50 for both cell lines was about 25 ng/mL. In the three plasma-cell preparations that were cultured in the presence of different concentrations of triptolide, a dose-dependent inhibition of cell growth was observed, with a 50% inhibitory concentration on the order of

Discussion

NF-kappaB regulates adhesion molecule expression on MM cells and BMSCs and their related binding, and also regulates both constitutive and MM cell-adhesion-induced cytokine transcription and secretion in BMSCs. NF-kappaB activation also confers drug resistance in MM cells. We became interested in triptolide because it was reported that triptolide is a potent inhibitor of NF-kappaB activation. We therefore decided to test the effects of triptolide on MM cells to see if this compound would show

Acknowledgements

Contributions. (1) Conception and design: Dr. Jinjie/Dr. Lou yinjun; (2) Analysis and interpretation of data: Dr. Lou yinjun/Dr. Jinjie; (3) Drafting the article: Dr. Lou yinjun; (4) Critical revision of the article for important intellectual content: Dr. Jinjie, Dr.Wang yungui; (5) Final approval of article: Dr.Jinjie/Dr. Lou yinjun; (6) Provision of study materials or patients: Dr.Jinjie, Dr. Tong xiangming, Dr. Wang yungui; (7) Statistical expertise: Dr. Lou yinjun; (8) Obtaining funding:

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