Molecular and Cellular Pharmacology
Role of triptolide in cell proliferation, cell cycle arrest, apoptosis and histone methylation in multiple myeloma U266 cells

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Abstract

Multiple myeloma is an incurable hematological malignancy. Different studies demonstrated the occurrence of genetic and epigenetic alterations in multiple myeloma. Histone lysine methylation has emerged as a central epigenetic change in the organization of eukaryotic chromatin with far-reaching implications for the regulation of cell proliferation, cell-type differentiation, gene expression, genome stability, overall development, and genesis of cancer. Triptolide is the principal active ingredient in extracts from the Chinese herb Tripterygium wilfordii Hook.F (TwHF), and numerous studies have elucidated its antitumor property. Our experiments discovered that triptolide inhibited the proliferation of multiple myeloma cell line U266 in a time- and dose-dependent manner, induced G2/M cell cycle arrest and caspase-dependent apoptosis. Triptolide could decrease the expression of histone H3K4, H3K27 and H3K36 trimethylation in parallel with histone methyltransferases SMYD3, EZH2 and NSD1 respectively, which possibly was the anti-myeloma mechanism of triptolide.

Introduction

Multiple myeloma is a clonal B-cell neoplasm that accounts for 10% of all malignant hematologic neoplasms and that affects terminally differentiated B cells (e.g. plasma cells).There is increasing evidence that epigenetic changes play an important role in the pathogenesis of multiple myeloma. The cyclin D1 promoter was hypomethylated and hyperacetylated in multiple myeloma cell lines and patient samples in line with the upregulation of cyclin D1 which is involved in B-cell malignancies (Liu et al., 2004). Multiple myeloma SET domain (MMSET) protein which is a histone methyltransferase with characteristics of a transcriptional corepressor was overexpressed and closely associated with chromatin in myeloma cell lines carrying t(4;14) and MMSET-mediated repression was associated with increased H4K20 methylation and loss of histone acetylation (Marango et al., 2008).

Triptolide, a diterpenoid isolated from a Chinese herb, Tripterygium wilfordii Hook.F (TwHF), has been shown to have antitumor properties by suppressing cell growth and inducing apoptosis (Yang et al., 2003). Recent studies suggested that triptolide overcame dexamethasone resistance by upregulating interleukin-6-independent expression of glucocorticoid receptor (Yang et al., 2009) and enhanced PS-341-induced apoptosis via PI3k/Akt/NF-κB pathways in human multiple myeloma cells (Yang et al., 2008). Since triptolide induced apoptosis of multiple myeloma cells (Yinjun et al., 2005), and epigenetic changes seem to play an important role in the pathogenesis of multiple myeloma, we explored whether triptolide antagonized multiple myeloma through the alterations of epigenetic patterns.

Histone lysine methylation is involved in the regulation of a wide range of processes including gene activity, chromatin structure and epigenetic memory. Lysine can be in mono-, di- or trimethylated forms. In general, lysine methylation at H3K9, H3K27 and H4K20 is associated with gene silencing, whereas methylation at H3K4, H3K36 and H3K79 is associated with gene activation (Vakoc et al., 2005). Methylation of histone H3 on lysine 4 (H3K4me3) is associated with transcriptionally active regions of chromatin. H3K4 dimethylation appears to be broadly associated with active and potentially active genes, while H3K4 trimethylation is primarily a mark associated with the start site of transcription (Ng et al., 2003). The SET domain of SET and MYND domain-containing protein 3 (SMYD3) showed histone H3K4-specific methyltransferase activity, and plays an important role in transcriptional regulation as a member of an RNA polymerase complex (Hamamoto et al., 2004). Trimethylated H3K27 (H3K27me3) chromatin mark is commonly associated with silencing of differentiation genes in organisms ranging from plants to humans. Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 (PRC2), which is a highly conserved histone methyltransferase that targets histone H3K27. H3K36 trimethylation (H3K36me3) is enriched mostly in the transcribed region of active genes (Barski, et al., 2007), suppresses erroneous transcription initiation and is required for transcriptional activation. The nuclear receptor-binding SET domain-containing protein (NSD1) functions as a histone methyltransferase to methylate H3K36 and H4K20 in vitro.

To assess the significance of epigenetic alterations in multiple myeloma and the effects of triptolide, we studied the proliferation, cell cycle and apoptosis, as well as histone methylation status in association with mRNA and protein expression levels of histone methyltransferases in multiple myeloma cell line U266, trying to investigate whether triptolide exerts its anti-carcinogenetic effect by the regulation of histone methylation in multiple myeloma cells.

Section snippets

Cell line and reagents

The multiple myeloma cell line U266 were obtained from ATCC (Rockville, USA), cell lines were cultured in RPMI1640 (GibcoBRL, USA), 10% fetal bovine serum (FBS; GibcoBRL), containing 10% fetal bovine serum (Sijiqing Co. Ltd, Hangzhou, China) in 5% CO2 incubator at 37 °C. Triptolide with 98% purity was purchased from Sigma (St. Louis, MO, USA), initially dissolved in dimethylsulfoxide (DMSO), stored at − 20 °C, and then thawed before use. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Growth inhibition by triptolide in vitro

The inhibitory effect of triptolide on the U266 cell line was initially determined by the MTT viability assay with the triptolide concentrations of 80, 100, 160, 180 and 200 nmol/l for 24, 48 and 72 h. As shown in the dose–response experiments presented in Fig. 1, triptolide brought about a concentration-dependent inhibition of cell proliferation with an IC50 of 78.54 ± 0.83 nmol/l for 48 h with statistical significance (P < 0.05). With reaction time prolonging, the inhibition rate of cell

Discussion

Triptolide is one of the major active ingredients of TWHF, which is a diterpene triepoxide and has been found to have potent anti-inflammatory and immunosuppressive and antitumour properties. Plenty of studies explored the effects of triptolide on hematological malignancies, e.g., triptolide cooperated with chemotherapy to induce apoptosis in acute myeloid leukemia cells (Pigneux et al., 2008), sensitized acute myeloid leukemia cells to TRAIL-induced apoptosis via decrease of XIAP and

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by a grant from the National Natural Sciences Foundation of China (no. 30700882). The authors would like to thank the Department of Central Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China, for offering relevant experimental facilities and technical support.

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