Triptolide inhibits matrix metalloproteinase-9 expression and invasion of breast cancer cells through the inhibition of NF-κB and AP-1 signaling pathways

Triptolide is a diterpenoid epoxide that is endogenously produced by the thunder god vine, Tripterygium wilfordii Hook F. Triptolide has demonstrated a variety of biological activities, including anticancer activities, in previous studies. Invasion and metastasis are the leading causes of mortality for patients with breast cancer, and the increased expression of matrix metalloproteinase-9 (MMP-9) has been shown to be associated with breast cancer invasion. Therefore, the aim of the present study was to investigate the effect of triptolide on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced cell invasion and MMP-9 expression in breast cancer cells. The expression of signal molecules was examined by western blotting, zymography and quantitative polymerase chain reaction; an electrophoretic mobility gel shift assay was also used, and cell invasiveness was measured by an in vitro Matrigel invasion assay. The MCF-7 human breast cancer cell line was treated with triptolide at the highest concentrations at which no marked cytotoxicity was evident. The results demonstrated that triptolide decreased the expression of MMP-9 through inhibition of the TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK) and the downregulation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activity. In addition, a Transwell assay revealed that triptolide reduced the ability of MCF-7 cells to invade Matrigel. These data demonstrate that the anti-invasive effect of triptolide is associated with the inhibition of ERK signaling and NF-κB and AP-1 activation, and suggest that triptolide may be a promising drug for breast cancer.     

Clinical and laboratory studies of 17 patients with acute myeloid leukemia harboring t(7;11)(p15;p15) translocation

The cellular and molecular genetic aberrations of hematopoietic and lymphoid tissues are increasingly important in leukemia classification and are prognostically significant. Although some recurrent molecular cytogenetic abnormalities in AML have been extensively studied, others including t(7;11)(p15;p15) have not been well characterized. In this paper, seventeen AML patients with t(7;11)(p15;p15) were retrospectively reviewed for cell morphology, immuno-phenotype, cytogenetics as well as clinical features and prognosis. Among them, thirteen were female; nine were AML-M2. Six patients who were newly diagnosed were alive, one was lost for followed up and ten died. The median survival was 8 months. Taking together, AML with t(7;11)(p15;p15) is a rare and distinct disease. Most patients with this translocation are female at younger age and have special clinical and hematological characteristics such as M2-subtype of AML, easy to relapse and poor prognosis.     

RNA polymerase – An important molecular target of triptolide in cancer cells

Triptolide, a diterpenoid triepoxide, is the key biological component of Tripterygium wilfordii Hook. f. which was used in traditional Chinese medicine for centuries to treat inflammation and autoimmune diseases. Triptolide has shown potent activity in not only anti-inflammation and immune modulation, but also antiproliferative and proapoptotic activity in many different types of cancer cells. However, for a long time, the precise molecular target(s) of triptolide have remained elusive. Recently, several groups discovered that triptolide inhibited the activity of RNA polymerase. This review will focus on these breakthrough findings about the molecular target of triptolide and its implications for targeted-cancer therapeutics.     

Increased expression of phospho-acetyl-CoA carboxylase protein is an independent prognostic factor for human gastric cancer without lymph node metastasis

Triptolide is a traditional Chinese medicinal herb-derived antineoplastic agent. However, its antitumor activity against gynecologic carcinomas has not yet been well described. It is the purpose of this article to investigate the effect and mechanism of triptolide in human ovarian cancer using both A2780 (p53 wild) and OVCAR-3 (p53 mutated) cells. Our results showed that triptolide exerted a potent inhibitory effect on the growth and proliferation of both cell lines in a dose- and time-dependent manner and that the effect was independent of the expression of p53. In contrast, triptolide had only a marginal cytotoxicity in noncancerous ovary cells, lung fibroblast cells, and macrophage cells, indicating differential inhibitory effects of the drug on cell growth between ovarian cancer cells and normal tissue cells. Exposure of the ovarian cancer cells to triptolide induced apoptosis, as evaluated by annexin V/propidium iodide-labeled flow cytometry. Triptolide-induced apoptosis was accompanied by cytochrome c release and caspase-3 activation and was associated with downregulation of Bcl-2 and upregulation of Bax. Cell cycle analysis demonstrated that treatment with triptolide induced cell cycle S phase arrest in A2780 cells and G2/M phase arrest in OVCAR-3 cells. Further detection by Western blotting revealed that the cell cycle arrest by triptolide in both cell lines occurred in concert with increased expression of p21^CIP1/WAF1. This study shows that triptolide selectively kills ovarian cancer cells with different p53 status predominantly through regulating the coordinate and dynamic cellular processes of proliferation and apoptosis, thereby making it a promising chemotherapeutic agent against a broad spectrum of ovarian carcinomas.     

Effects of Triptolide on Cell Proliferation and CXCR4 Expression in Burkitt＇s Lymphoma Raji Cells In Vitro

Objective： To investigate the inhibitory effects of triptolide on cell proliferation and CXCR4 expression in Burkitt＇s lymphoma cell line Raji cells. Methods： The effects of triptolide on the growth of Raji cells were studied by 3-（4, 5-Dimethyl-2-thiazolyl）-2, 5-diphenyl-2H-tetrazolium（MTT） assay. The effects of triptolide on CXCR4 expression on Raji cells were studied by flow cytometric analysis. Chemotaxis assays were performed to observe the effects of triptolide on migration of Raji cells towards recombinant human SDF-1α （rhSDF-1α） in vitro. Results： Triptolide inhibited the proliferation of Raji cells in a dose- and time-dependent way with a 24-h IC50 value of 43.06 nmol/L and a 36-h IC50 value of 25.08 nmol/L. Triptolide could downregulate the CXCR4 expression on Raji cells in a dose-dependent manner. Furthermore, chemotaxis assays showed that triptolide could block the migration of Raji cells to rhSDF-1α in vitro, and the inhibition was dose-dependent. Conclusion： Triptolide could inhibit the proliferation and migration of Raji cells in vitro. The underlying anti-tumor mechanism of triptolide might be related to the anti-proliferative effect and the blockage of SDF-1/CXCR4 axis.     

Preclinical antileukemic activity,toxicology, toxicokinetics and formulation development of triptolide derivative MRx102

Purpose

Triptolide induces cancer cell apoptosis by inhibiting RNA synthesis and signaling pathways like NF-κB. We compared triptolide prodrug MRx102 to triptolide to determine whether it displayed comparable efficacy and improved toxicology and toxicokinetic profiles.

Methods

MV4-11 AML cells and cells from AML patients were analyzed for MRx102- and triptolide-induced cytotoxicity/apoptosis. MRx102 and triptolide were compared in toxicology/toxicokinetics studies in rat and dog using a new emulsion formulation.

Results

MRx102 induced cytotoxicity in MV4-11 cells (IC₅₀ = 15.2 nM, 7.29 nM for triptolide) and apoptosis in cells from AML patients (EC₅₀ = 40.6 nM and 2.13 nM for triptolide). MRx102 and triptolide induced apoptosis in CD34+CD38? AML stem/progenitor cells with a similar difference in activity (EC₅₀, MRx102 = 40.8 nM, triptolide = 2.14 nM). In a rat toxicology comparison using a new intravenous emulsion formulation, the MRx102 MTD was 4.5 mg/kg for males and 3 mg/kg for females; the triptolide MTD was 0.63 mg/kg for males and 0.317 mg/kg for females. The MRx102 NOAEL was 1.5–3.0 mg/kg, and the triptolide NOAEL was 0.05–0.15 mg/kg. Mean plasma concentrations for both MRx102 and triptolide decreased rapidly from a high C _max following i.v. injection. Plasma triptolide levels stabilized at a consistent level through 2 h after MRx102 injection. Triptolide T _1/2,e values for MRx102-injected rats (~0.85 to ~3.7 h) were markedly greater than triptolide-injected rats (~0.15 to ~0.39 h), indicating more extended triptolide exposure with MRx102. MRx102 dog toxicology and toxicokinetics results are presented.

Conclusions

MRx102 was 20- to 60-fold safer than triptolide comparing rat NOAELs. This may be due to the improved toxicokinetic profile of MRx102 compared to triptolide using the emulsion formulation, with no high C _max and more consistent early exposure to triptolide.     

Triptolide induces Bcl-2 cleavage and mitochondria dependent apoptosis in p53-deficient HL-60 cells

Triptolide, a bioactive component of the Chinese medicinal herb Tripterygium wilfordii Hook F., induces p53-mediated apoptosis in cancer cells. This study demonstrated that triptolide activated an alternative p53-independent apoptotic pathway in HL-60 cells. In the absence of an intact p53 and without changing Bax level, at nM range triptolide induced apoptosis with concomitant DNA fragmentation, S phase cell cycle arrest, mitochondrial cytochrome c release and the activation of caspases. Besides, both caspases 8 and 9 were activated and the simultaneous inhibition of both was required to completely block triptolide's apoptotic effect. Importantly, triptolide induced the appearance of a truncated 23kD Bcl-2 which was inhibited by the general caspase inhibitor Z-VAD-FMK. In the MCF-7 cells that possessed the wild type p53 but lacked caspases 3, triptolide induced cell death with an increase in p53 but Bcl-2 remained unaltered. On the other hand, transfected cells overexpressing the 28kD Bcl-2 became more resistant to triptolide and upon triptolide treatment accumulated in the G(1) instead of S phase. After 36h treatment, triptolide activated JNK pathways, at the same time inactivated the ERK and p38 pathways. However, SP600125, a specific JNK inhibitor, could not inhibit the triptolide-mediated cleavage of caspase 3, indicated that activation of JNK might not be related to the apoptotic effects of triptolide. Our data suggest that in the absence of an intact p53 and without altering Bax level triptolide induces apoptosis activates a positive amplification loop involving caspase-mediated Bcl-2 cleavage/activation, mitochondrial cytochrome c release and further activation of caspases.     

Activity of triptolide against human mast cells harboring the kinase domain mutant KIT

Gain-of-function mutations of the receptor tyrosine kinase KIT can cause systemic mastocytosis (SM) and gastrointestinal stromal tumors. Most of the constitutively active KIT can be inhibited by imatinib; D816V KIT cannot. In this study, we investigated the activity of triptolide, a diterpenoid isolated from the Chinese herb Tripterygium wilfordii Hook. f., in cells expressing mutant KIT, including D816V KIT. Imatinib-sensitive HMC-1.1 cells harboring the mutation V560G in the juxtamembrane domain of KIT, imatinib-resistant HMC-1.2 cells harboring both V560G and D816V mutations, and murine P815 cells, were treated with triptolide, and analyzed in terms of growth, apoptosis, and signal transduction. The in vivo antitumor activity was evaluated by using the nude mouse xenograft model. Our results demonstrated that triptolide potently inhibits the growth of both human and murine mast cells harboring not only imatinib-sensitive KIT mutation but also imatinib-resistant D816V KIT. Triptolide markedly inhibited KIT mRNA levels and strikingly reduced the levels of phosphorylated and total Stat3, Akt, and Erk1/2, downstream targets of KIT. Triptolide triggered apoptosis by inducing depolarization of mitochondrial potential and release of cytochrome c, downregulation of Mcl-1 and XIAP. Furthermore, triptolide significantly abrogated the growth of imatinib-resistant HMC-1.2 cell xenografts in nude mice and decreased KIT expression in xenografts. Our data demonstrate that triptolide inhibits imatinib-resistant mast cells harboring D816V KIT. Further investigation of triptolide for treatment of human neoplasms driven by gain-of-function KIT mutations is warranted. ( Cancer Sci 2009; 100: 1335–1343)     

The Proteasomal Inhibitor MG132 Potentiates Apoptosis of Triptolide-Treated K562 Cells by Regulating the NF-κB Signal Pathway

OBJECTIVE To explore the anticancer mechanism of triptolide in human leukemia K562 cells,and to further determine whether the proteasomal inhibitor,MG132,can potentiate apoptosis in triptolide-treated K562 cells.METHODS Apoptosis was assessed via annexin V/PI double-labeled cytometry.The expressions of the IκBα and NF-κB/p65 proteins in K562 cells was investigated using Western blo ing.RESULTS The inhibitory rates of K562 cells treated by triptolide gradually increased in a dose-and time-dependent manner,and treatment with triptolide plus MG132 potentiated the apoptotic rate.Triptolide inhibited the degradation of the IκBα protein and the nuclear localization of NF-κB/p65 proteins induced by TNF-α,and MG132 potentiated the effect of triptolide.Triptolide plus MG132 almost completely blocked the NF-κB activation induced by TNF-α.CONCLUSION The anti-proliferative activities of triptolide and MG132 were related to the NF-κB signal pathway.     

Triptolide inhibits interferon-gamma-induced programmed death-1-ligand 1 surface expression in breast cancer cells

Triptolide, a natural compound purified from the Chinese herb Tripterygium wilfordii, has been reported to inhibit the growth and metastasis of tumors in vivo. However, the effects of triptolide on the immune responses of cancer cells remain unknown. Up-regulation of programmed death-1-ligand 1 (PD-L1) in cancer cells is an important mechanism of tumor immune evasion. In the present study, we demonstrated that triptolide was able to inhibit interferon-γ-induced PD-L1 surface expression in human breast cancer cells. Therefore, by down-regulating PD-1/PD-L1 pathway, triptolide may also serve as a modulator to promote cancer cell-reactive immune responses.     

Triptolide Inhibits Breast Cancer Cell Metastasis Through Inducing the Expression of miR-146a,a Negative Regulator of Rho GTPase

Triptolide, an extract of Tripterygium wilfordii, has been shown to have a potent anticancer activity. In the present study, it was found that triptolide could effectively induce apoptosis and inhibit proliferation and invasion in malignant MDA-MB-231 breast cancer cells. The study focused on its effect on inhibiting invasion, which has not been extensively reported to date. We predicted that triptolide may change invasion activity via microRNAs (miRNAs), which have been recognized as important regulators of gene expression. miRNAome variation in MDA-MB-231 cells with or without triptolide treatment demonstrated that miR-146a was upregulated following treatment with triptolide. Our previous studies have shown that miR-146a can inhibit migration and invasion by targeting RhoA in breast cancer. This time, we found that miR-146a can target Rac1, another key member of the Rho GTPase family. Luciferase reporter containing Rac1 3′-UTR was constructed to prove this hypothesis. In addition, following treatment with triptolide, the expression of RhoA and Rac1 was found to be decreased. These results indicated that triptolide exerts its anti-invasion activity through a miRNA-mediated mechanism, which indirectly regulates the expression of Rho GTPase. Triptolide combined with miR-146a could improve the effect of triptolide treatment on breast cancer.     

Triptolide functions as a potent angiogenesis inhibitor

Triptolide is a key anti‐inflammatory compound of the Chinese herbal medicine Tripterygium wilfordii Hook. f. (Celastraceae). It also possesses potent antitumor activity. In this study, we show that triptolide is an angiogenesis inhibitor based on various angiogenesis assays. The IC₅₀ in in vitro assays was 45 nM, which was much lower than the plasma concentrations of triptolide in the rat or human administered with T. wilfordii extracts for treating inflammation. When dosed in vivo, triptolide potently inhibited angiogenesis at 100 nM in Matrigel plug assay. Triptolide at 0.75 mg/kg/day significantly blocked tumor angiogenesis and tumor progression in murine tumorigenesis assay. The underlying mechanism of triptolide correlated with downregulation of proangiogenic Tie2 and VEGFR‐2 expression in human umbilical vein endothelial cell by semiquantitative RT‐PCR and western blot analysis. Although Tie2 inhibition appeared to be a later event as compared with VEGFR‐2, Tie2 overexpression significantly attenuated the inhibitory effect of triptolide on endothelial proliferation and network formation. By contrast, Tie2 knockdown mimicked the inhibitory effect of triptolide on endothelial network formation. Our findings suggest that antitumor action of triptolide is partly via inhibition of tumor angiogenesis by blocking 2 endothelial receptor‐mediated signaling pathways, and triptolide can be a promising antiangiogenic agent.     

Knockdown of IARS2 Inhibited Proliferation of Acute Myeloid Leukemia Cells by Regulating p53/p21/PCNA/eIF4E Pathway

IARS2 encodes mitochondrial isoleucine-tRNA synthetase, which mutation may cause multiple diseases. However, the biological function of IARS2 on acute myeloid leukemia (AML) has not yet been identified. In the present study, qRT-PCR was used to determine the expression of IARS2 in K562, THP1, and HL-60 leukemia cells. Additionally the mRNA levels of IARS2 in CD34 cells and AML cells obtained from patients were detected by qRT-PCR. IARS2-shRNA lentiviral vector was established and used to infect acute myeloid leukemia HL-60 cells. qRT-PCR and Western blot analysis were employed to assess the knockdown effect of IARS2. The proliferation rate and cell cycle phase of HL-60 cells after IARS2 knockdown were evaluated by CCK-8 assay and flow cytometry. The PathScan Antibody Array was used to determine the expression of cell cycle-related proteins in HL-60 cells after IARS2 knockdown. The expression of proliferation-related proteins in HL-60 cells after IARS2 knockdown was determined by Western blot analysis. Results showed that IARS2 expression was stable and much higher in HL-60, THP-1, and K562 leukemia cells and AML cells obtained from patients than that of human CD34 cells. Compared with cells of the shCtrl group, IARS2 was markedly knocked down in cells that were transfected with lentivirus encoding shRNA of IARS2 in HL-60 cells (p<0.05). IARS2 knockdown significantly inhibited the proliferation and induced cycle arrest at the G₁ phase in HL-60 cells. Additionally IARS2 knockdown significantly increased the expression of p53 and p21, and decreased the expression of PCNA and eIF4E in HL-60 cells. In conclusion, IARS2 knockdown can inhibit acute myeloid leukemia HL-60 cell proliferation and cause cell cycle arrest at the G₁ phase by regulating the p53/p21/PCNA/eIF4E pathways.     

The Proteasomal Inhibitor MG132 Potentiates Apoptosis of Triptolide-Treated K562 Cells by Regulating the NF-KB Signal Pathway

OBJECTIVE To explore the anticancer mechanism of triptolide in human leukemia K562 cells,and to further determine whether the proteasomal inhibitor,MG132,can potentiate apoptosis in triptolide-treated K562 cells.METHODS Apoptosis was assessed via annexin V/PI doublelabeled cytometry.The expressions of the IκBα and NF-κB/p65 proteins in K562 cells was investigated using Western blotting.RESULTS The inhibitory rates of K562 cells treated by triptolide gradually increased in a dose-and time-dependent manner,and treatment with triptolide plus MG132 potentiated the apoptotic rate.Triptolide inhibited the degradation of the IκBα protein and the nuclear localization of NF-κB/p65 proteins induced by TNF-α,and MG132 potentiated the effect of triptolide.Triptolide plus MG132 almost completely blocked the NF-κB activation induced by TNF-α.CONCLUSION The anti-proliferative activities of triptolide and MG132 were related to the NF-κB signal pathway.     

Effects of triptolide on histone acetylation and HDAC8 expression in multiple myeloma <Emphasis Type="Italic">in vitro</Emphasis>

Objective: Multiple myeloma is a kind of malignant plasma cell disease that originated from B lymphocyte and secrete great amount of monoclonal immunoglobulin. It is still one of the refractory diseases at present. Numerous studies show that there is an intensive relationship between the disequilibrium of histone acetylation and the occurance of multiple myeloma. Here we investigated the effect of triptolide(TPL) on the proliferation, apoptosis, histone H3 and H4 acetylation and expression of histone deacetylase 8 (HDAC8) in vitro, to explore its anti-myeloma mechanism.Methods: The effect of triptolide on the growth of RPMI8226 was studied by 3-(4,5-Dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium(MTT) assay. Apoptosis was detected by Hoechst 33258 staining. The protein expressions of acetyl-histone H3 and H4 were determined by Western blot, and the expression of HDAC8 was assessed by RT-PCR, Western blot and confocal microscopy.Results: Triptolide inhibited the proliferation of RPMI8226 and induced apoptosis in a time- and dose-dependent manner. The 36h IC50 value was (105.370±0.189)nmol/L. Triptolide increased the acetylation of histone H3 and H4 greatly. Furthermore, triptolide significantly down-regulated the mRNA and protein expression of HDAC8.Conclusion: Triptolide can inhibit proliferation and induce apoptosis of RPMI8226 significantly. Triptolide reduces the expression of HDAC8 in order to increase the histone H3 and H4 acetylation, which is possibly the anti-myeloma mechanism of triptolide.     

Triptolide inhibits proliferation and induces apoptosis of human melanoma A375 cells

Triptolide, a diterpenoid obtained from Tripteryglum wilfordii Hook.f, has attracted interest for its anti- tumor activities against human tumor cell lines in recent years. This report focuses on anti-proliferative and pro-apoptotic activities in human melanoma A375 cells assessed by CCK8 assay, Hoechst 33258 staining and flow cytometry. In addition, triptolide-induced arrest in the S phase was also observed. Caspase assays showed the apoptosis induced by triptolide was caspase-dependent and probably through intrinsic apoptotic pathways. Furthermore, expression of NF-κB (p65) and its downstream factors such as Bcl-2, Bcl-XL was down-regulated. Taken together, the data indicate that triptolide inhibits A375 cells proliferation and induces apoptosis by a caspase-dependent pathway and through a NF-κB-mediated mechanism.