Introduction

Triptolide is a natural, biologically active compound as a diterpenoid triepoxide originally purified from the Chinese herb Tripterygium wilfordii Hook F (TWHF). This natural product used in traditional Chinese medicine for centuries, has a myriad of therapeutic uses against inflammation and autoimmune disease (Chen, 2001; Qiu et al., 2003, Xiang and Zhang, 2005). Antiproliferative activity of triptolide has been shown with many different types of cancer cells in vitro and in vivo (Shamon et al., 1997; Tengchaisri et al., 1998; Chang et al., 2001; Kiviharju et al., 2002, Fidler et al., 2003; Yang et al., 2003). Therefore, triptolide might be clinically effective for tumor chemotherapy. Anti-inflammatory and anti-proliferative properties of triptolide have been associated with inhibition of NFκB (Lee et al., 1999; Qiu et al., 1999; Liu et al., 2000). Although these studies shed some lights on triptolide actions, there are still many gaps left and hard to explain why triptolide has such a broad biological function. The exact targets and molecular mechanism of action of triptolide are still unknown.

PLD catalyzes the hydrolysis of phosphatidylcholine to generate phosphatidic acid and choline. Two human PLD isozymes, phosphatidylcholine-specific PLD1 and PLD2, have been described (Hammond et al., 1995; Colley et al., 1997). The catalytic and regulatory features of PLD1 are remarkably different from those of PLD2 although both isozymes have about 50% of amino acid sequence homology and similar structural domain (Frohman et al., 1999; Exton 2002). Abnormalities in PLD expression and activity have been observed in many human cancers (Foster and Xu, 2003). We previously reported that overexpression of PLD isozymes causes anchorage-independent growth and induces tumorigenesis (Min et al., 2001). We and other research group have reported that PLD activity has been implicated in cell motility and tumor invasion and correlated with increased protease secretion (Williger et al., 1999; Park et al., 2009). These findings suggest that PLD is a critical molecule in tumorigenesis. Despite gathering evidence regarding the regulation of PLD activity in cell function, little is known about the functional role and regulatory mechanisms of PLD expression.

Recently, we have reported that PMA as a tumor promoter enhances PLD1 expression via NFκB-dependent signaling pathway and increases cell proliferation and invasion (Kang et al., 2008). Thus, we set out to identify compounds inhibiting PLD expression using stable cells expressing PLD promoter. We found that triptolide inhibited the expression and activity of PLD in MDA-MB-231 human breast cancer cell which is highly invasive and has high level of PLD activity (Chen et al., 2003, 2005). In this study, we demonstrate that downregulation of PLD as a new downstream target of triptolide suppresses cell proliferation.

Results

Triptolide inhibits PLD expression in MDA-MB-231 human breast cancer cells

Since PLD has emerged as a critical regulator of cell proliferation and survival (Foster and Xu, 2003), we tried to screen small molecules inhibiting the expression of PLD using cell assay system for measurement of PLD promoter activity. We found that triptolide significantly inhibited PLD promoter activity in stable cells expressing PLD promoter. We examined the effects of different concentrations of triptolide on PLD promoter activity in MDA-MB-231 breast cancer cells. MDA-MB-231 cells were transiently transfected with PLD1- or PLD2-luciferase reporter constructs and treated with various dose of triptolide. As shown in Figure 1A, triptolide suppressed both PLD1 and PLD2 promoter activity in a dose-dependent manner, with 50% decrease of expression observed at approximately 50 nM. It decreased the promoter activity of both PLD isozymes with similar inhibitory potency in MDA-MB-231 cells. We further investigated whether triptolide-induced PLD1 suppression may be regulated in a transcriptional level. Treatment with triptolide for 24 h decreased the expression level of PLD1 and PLD2 mRNA in a dose dependent manner with approximately 50% inhibition at 50 nM as analyzed by RT-PCR (Figure 1B). Triptolide also downregulated the expression level of both PLD1 and PLD2 in a time dependent manner (Figure 1B). In addition, the inhibition of PLD mRNA level by triptolide resulted in the corresponding suppression of PLD1 protein in a doe and time-dependent manner as analyzed by immunoblot (Figure 2A and 2B). MDA-MB-231 breast cancer cells expressed predominantly PLD1 protein. Dose and time kinetics of triptolide-induced suppression of PLD1 protein show a similar pattern with those of transcriptional expression. Taken together, these results suggest that expression of PLD isozymes was down-regulated by triptolide at both transcriptional and post-transcriptional levels.

Figure 1
figure 1

Triptolide downregulates transcriptional level of PLD isozymes in MDA-MB-231 breast cancer cells. (A) MDA-MB-231 cells were transiently transfected with PLD1 or PLD2 reporter constructs and treated with the indicated concentration of triptolide for 18 h, and luciferase activity was subsequently measured. Each value represents the mean ± S.D. of five independent experiments. *P < 0.05 versus non-treatment (B) MDA-MB-231 cells were treated with the indicated concentration of triptolide for 24 h or 100 nM of triptolide for the indicated times, and then PLD expression was analyzed by RT-PCR. The level of PLD1 expression was determined by densitometer analysis. The data are representative of results obtained from three experiments.

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Figure 2
figure 2

Triptolide suppresses expression of PLD protein in MDA-MB-231 cells. MDA-MB-231 cells were treated with the indicated concentration of triptolide for 24 h (A) or 100 nM of triptolide for the indicated time. The cell lysates were analyzed by Western blot using antibody to PLD. The level of PLD1 expression was determined by densitometer analysis. The data are representative of results obtained from three experiments.

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Downregulation of PLD by triptolide in various human cancer cells

To examine whether triptolide-induced expression of PLD1 is specific to the MDA-MB-231 cells or also occurs in other human cancer cells, various cancer cells including the glioblastoma (U373) and colon cancer cells (DKO, HCT-116, SW480, DLD-1) and breast cancer cells (HS578T, MCF-7) were treated with triptolide for 24 h and analyzed by Western blot (Figure 3). In most cells examined, PLD1 protein was predominantly detected but PLD2 was not detectable. Triptolide-induced suppression of PLD1 expression was noted in all of the cancer cells examined. Therefore, it is suggested that downregulation of PLD by triptolide is a general phenomenon.

Figure 3
figure 3

Triptolide-induced PLD suppression occurs in various human cancer cells. Various human cancer cells were treated with triptolide (100 nM) for 24 h. The cell lysates were analyzed by western blot using the antibody to PLD or α-tubulin. The level of PLD1 expression was determined by densitometer analysis. These blots are representative of results obtained from three experiments.

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Triptolide decreases both basal and PMA-induced PLD activation

Next, we examined whether suppression of PLD1 expression by triptolide decrease its enzymatic activity. As shown in Figure 4A, treatment with triptolide inhibited basal PLD activity in a dose-dependent manner in MDA-MB-231 cells. Moreover, triptolide inhibited PLD activity stimulated by PMA which is known as a PLD activator (Figure 4B). These data demonstrate that triptolide-induced suppression of PLD expression leads to a decrease of both basal and PMA-induced PLD activity.

Figure 4
figure 4

Triptolide inhibits both basal and PMA-induced PLD activation. MDA-MB-231 cells were labeled with [3H]myristate for 12 h, and were treated with the indicated concentration of triptolide for 30 min (A) or pretreated with triptolide (100 nM) for 30 min and treated with PMA (100 nM) for 1 h (B). Then, the PLD activity was measured as described under "Methods." Each value represents the mean ± S.D. of five independent experiments.

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Triptolide suppresses PLD expression via inhibition of NFκB transcriptional activity

Next, we tried to investigate the molecular mechanism for triptolide-induced PLD suppression. Recently, we have reported that PMA induces selectively PLD1 expression via NFκB (Kang et al., 2008). Ectopic expression of NFκB (p65) enhanced the promoter activity, protein expression, and enzymatic activity of PLD1, but did not significantly affect those of PLD2 in HCT116 colorectal cancer cells (Kang et al., 2008). Two NFκB binding sites present in the PLD1 promoter was responsible for PMA-induced PLD1 expression (Kang et al., 2008). We found that NFκB also enhanced PLD1 expression but not PLD2 in MDA-MB-231 cells (Figure 5A). Therefore, we examined whether triptolide affects transcriptional activity of NFκB in MDA-MB-231 cells. As shown in Figure 5B, triptolide significantly inhibited transactivation of NFκB in a dose-dependent manner. These data suggest that triptolide might suppress PLD1 expression via inhibition of NFκB transactivation and abolish PLD2 expression via mechanism independent of NFκB transactivation.

Figure 5
figure 5

Triptolide suppresses PLD expression via inhibition of NFκB transcriptional activity. (A) MDA-MB-231 cells were cotransfected with pGL4-PLD and NFκB (p65) and then luciferase activity was subsequently measured. Each value represents the mean ± S.D. of five independent experiments. *P < 0.05 versus vehicle for pGL4-PLD1 (B) The cells were transfected with the NFκB-dependent reporter plasmid pGL2-3X NFκB for 20 h and treated with the indicated concentration of triptolide and then their luciferase activity was measured. A Renilla luciferase control vector was co-transfected to normalize the transfection efficiency. Each value represents the mean ± S.D. of five independent experiments. *P < 0.05 versus non-treatment.

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Triptolide-induced PLD suppression inhibits the proliferation of breast cancer cells

Next, we investigated the biological significance of triptolide-induced PLD downregulation. The proliferation of MDA-MB-231 cells was examined by treatment with triptolide as measured by MTT assay. Triptolide inhibited cell viability in a dose-dependent manner (Figure 6A). Moreover, depletion of PLD1 using siRNA led to decreased proliferative activity (Figure 6B). These data suggest that inhibitory effect of triptolide on cell proliferation is mediated at least by triptolide-induced PLD repression.

Figure 6
figure 6

Triptolide-induced PLD downregulation inhibits proliferation of breast cancer cells. (A) MDA-MB-231 cells were treated with the indicated concentration of triptolide for 24 h. The cell viability was measured using MTT assay. Each value represents the mean ± S.D. of three independent experiments. *P < 0.05 versus non-treatment (B) The cells were transfected with siRNA for control or PLD1 and the cell viability was measured using MTT assay. The data represents the mean ± S.D. of five independent experiments. *P < 0.05 versus control siRNA.

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Discussion

In the present study, we demonstrate that triptolide downregulates expression and activity of PLD and then inhibits proliferation of breast cancer cells. These effects appear to be mediated through suppressing the transactivation of NFκB. Multiple protein targets both upstream and downstream of PLD have been linked to propagation of survival signals and metastasis in cancer progression.

Overexpression and elevated activity of PLD result in cellular transformation and has been implicated in multiple human cancers including colorectal (Saito et al., 2007; Kang et al., 2008), breast (Noh et al., 2000), renal (Zhao et al., 2000), gastric carcinoma (Uchida et al., 1999), and thyroid cancers (Kim et al., 2008). Stable cells overexpressing PLD1 and PLD2 demonstrate anchorage-independent growth and tumorigenesis in nude mice (Min et al., 2001; Buchanan et al., 2005). Thus, it is suggested that PLD plays an important role in cancer progression and that PLD could be a target for therapy in cancer. It thus is conceivable that a strategy aiming to block the effect of PLD would decrease expression and activity of the PLD, breaking down a tumor-promoting loop and there reducing tumor growth.

We have identified triptolide via chemical screen for inhibitors of PLD expression using cell assay system based on measurement of PLD promoter activity. Triptolide has been investigated for many of its potential therapeutic uses, including reduction of solid tumor masses, and is in clinical trials based on its potent antitumor effects in a prostate cancer model (Kiviharju et al., 2002). Because the natural source of triptolide, the medicinal vine, Tripterygium wilfordii Hook F, has a long history in Chinese traditional medicine, it is hoped that triptolide will be promising therapeutic candidate for the treatment of cancer. Although several intracellular pathways such as NFκB, Bcl-2, PI3K, and c-myc have been reported to be responsible for the mechanisms underlying the action of triptolide (Lee et al., 1999; Kiviharju et al., 2002; Miyata et al., 2005; Ko et al., 2007), our results show that downregulation of PLD by triptolide is a key mechanism for reduction of cell proliferation.

Triptolide abolished expression of both PLD1 and PLD2 at the transcriptional level. Moreover, triptolide inhibited both basal and PMA-induced PLD activity. Triptolide-induced PLD suppression inhibited proliferation of breast cancer cells. Recently, we have reported that tumor prosmoter signal such as PMA stimulates selectively PLD1 expression by increasing the binding of NFκB to PLD1 promoter (Kang et al., 2008). NFκB did not affect PLD2 expression (Kang et al., 2008).

The antitumor proliferation induced by triptolide has been reported to be associated with the down-regulation of NFκB activity (YinJun et al., 2005). We found that triptolide inhibited transactivation of NFκB in MDA-MB0231 breast cancer cells. Thus, it is suggested that triptolide-mediated PLD1 repression might be due to inhibition of NFκB activity by triptolide. However, it is likely that triptolide inhibits PLD2 expression via distinct pathway from that of PLD1 since NFκB is not responsible for PLD2 expression. Until now, the lack of potent and isoform-selective inhibitors has limited progress in defining the cellular roles of PLD. Recently, isoform-selective PLD inhibitors have been developed (Scott et al., 2009) and may facilitate a better understanding of the biological roles played by the respective PLD isozymes. We have identified triptolide as dual inhibitor for PLD1 and PLD2 expression. In summary, we have identified PLD as a candidate target for the therapeutic action of the traditional Chinese medicine-derived natural product triptolide. Triptolide could be used as dual inhibitor for PLD1 and PLD2 expression. It is expected that drugs modulating function of PLD as a therapeutic target will develop as a promising strategy applied to a broad spectrum of cancer treatment.

Methods

Cells and material

Breast cancer cells (MDA-MB-231, MCF7, HS578T), colorectal cancer cells (HCT116, DLD-1, DKO, SW480) and glioma cells (U373) were purchased from ATCC (Rockville, MA). MCF7, HS578T, DLD-1, and HCT116 cells were incubated with RPMI 1640 medium containing 10% heatinactivated FBS with penicillin-streptomycin (Gibco BRL, Grand Island, NY). MDA-MB231, DKO, SW480 and U373 cells were grown in DMEM supplemented with 10% FBS. Triptolide and PMA was purchased from Sigma Chemical Company (St. Louis, MO). The promoter region of human PLD1 and PLD2 gene has been cloned (Kang et al., 2008).

Transient transfection and luciferase assay

Cells were grown to 50-60% confluence. Plasmid or siRNA was transiently transfected into MDA-MB231 cells using Lipofectamine Plus reagent (Invitrogen) according to the manufacturer's instruction. For luciferase reporter assay, the cells were seeded in 24-well plates and transiently transfected with 100 ng of promoter constructs and 10 ng of pRL-TK (internal control). The activities of firefly and renilla luciferase in the cellular extracts were measured using the Dual-Luciferase Assay kit (Promega) according to the manufacturer's instructions. For the NFκB transactivation assay, pGL2-3X NFκB, which contained three tandem repeats of the NFκB binding motif, was used.

RNA isolation and RT-PCRRNA isolation and RT-PCR

Total RNA was isolated from cells using TRIzol reagent. First-strand cDNA was synthesized using 5 µg total RNA with Moloney murine leukemia virus reverse transcriptase (MMLV-RTase) (Promega, Mannheim, Germany). The reaction was incubated at 80℃ for 5 min, 42℃ for 90 min, and MMLV-RTasse was inactivated at 95℃ for 5 min. The synthesized cDNA was amplified using PLD1, PLD2 and β-actin primers with an eppendorf thermocycler (Eppendorf Scientific, Westbury, NY). PCR products were analyzed by agarose gel electrophoresis with ethidium bromide staining.

Western blot analysis

The cells were analyzed by immunoblotting as described previously (Kang et al., 2008). Enhanced chemiluminescence (ECL) reagents were obtained from Amersham Biosciences (Piscataway, NJ). ECL was used to detect the signal. The following antibodies were used: anti-β-tubulin antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-PLD antibody that recognizes both PLD1 and PLD2 was generated as described previously (Min et al., 2001).

PLD activity assay

PLD activity was assessed by measuring the formation of [3H] phosphatidylbutanol, the product of PLD-mediated transphosphatidylation, in the presence of 1-butanol. Cells were subcultured in six-well plates at 2 × 105/well, and then incubated in the presence of 3 µCi/ml [3H] myristic acid. After overnight labeling, the cells were washed thrice with 5ml of PBS and preequilibrated in serum-free DMEM for 1 h. PLD catalyzed transphosphatidylation in the presence of 0.5% 1-butanol, and the extraction and characterization of lipids by thin layer chromatography were done as previously described (Ahn et al., 2003).

Cell viability assay

For the cell viability assay, an MTT assay was performed. Absorbance was measured using a spectrophotometer at 540 nm, and viability was expressed relative to the control.

Statistics

The results are expressed as means ± S.D. of the number of determinations indicated. Statistical significance of differences was determined by analysis of variance. A P value less than 0.05 was considered to indicate statistical significance.