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1.  Anti-Angiogenic Effect of Triptolide in Rheumatoid Arthritis by Targeting Angiogenic Cascade 
PLoS ONE  2013;8(10):e77513.
Rheumatoid arthritis (RA) is characterized by a pre-vascular seriously inflammatory phase, followed by a vascular phase with high increase in vessel growth. Since angiogenesis has been considered as an essential event in perpetuating inflammatory and immune responses, as well as supporting pannus growth and development of RA, inhibition of angiogenesis has been proposed as a novel therapeutic strategy for RA. Triptolide, a diterpenoid triepoxide from Tripterygium wilfordii Hook F, has been extensively used in treatment of RA patients. It also acts as a small molecule inhibitor of tumor angiogenesis in several cancer types. However, it is unclear whether triptolide possesses an anti-angiogenic effect in RA. To address this problem, we constructed collagen-induced arthritis (CIA) model using DA rats by the injection of bovine type II collagen. Then, CIA rats were treated with triptolide (11–45 µg/kg/day) starting on the day 1 after first immunization. The arthritis scores (P<0.05) and the arthritis incidence (P<0.05) of inflamed joints were both significantly decreased in triptolide-treated CIA rats compared to vehicle CIA rats. More interestingly, doses of 11∼45 µg/kg triptolide could markedly reduce the capillaries, small, medium and large vessel density in synovial membrane tissues of inflamed joints (all P<0.05). Moreover, triptolide inhibited matrigel-induced cell adhesion of HFLS–RA and HUVEC. It also disrupted tube formation of HUVEC on matrigel and suppressed the VEGF-induced chemotactic migration of HFLS–RA and HUVEC, respectively. Furthermore, triptolide significantly reduced the expression of angiogenic activators including TNF-α, IL-17, VEGF, VEGFR, Ang-1, Ang-2 and Tie2, as well as suppressed the IL1-β-induced phosphorylated of ERK, p38 and JNK at protein levels. In conclusion, our data suggest for the first time that triptolide may possess anti-angiogenic effect in RA both in vivo and in vitro assay systems by downregulating the angiogenic activators and inhibiting the activation of mitogen-activated protein kinase downstream signal pathway.
doi:10.1371/journal.pone.0077513
PMCID: PMC3810371  PMID: 24204851
2.  Triptolide inhibits human immunodeficiency virus type 1 replication by promoting proteasomal degradation of Tat protein 
Retrovirology  2014;11(1):88.
Background
Plants remain an important source of new drugs, new leads and new chemical entities. Triptolide is a diterpenoid epoxide isolated from Tripterygium wilfordii Hook F that possesses a broad range of bioactivities, including anti-inflammatory, immunosuppressive and anti-tumor properties. The antiviral activity of triptolide against human immunodeficiency virus type 1 (HIV-1) has not been reported.
Results
In this study, nanomolar concentrations of triptolide were shown to potently inhibit HIV-1 replication in vitro. To identify the step(s) of the HIV-1 replication cycle affected by triptolide, time-of-addition studies, PCR analysis and direct transfection of viral genomic DNA were performed. The results of these experiments indicated that triptolide acts at the stage of viral gene transcription. In addition, a luciferase-based reporter assay that allows quantitative analysis of long terminal repeat (LTR)-driven transcription showed that Tat-induced LTR activation was impaired in the presence of triptolide. Moreover, Western blot analysis of exogenous gene expression (driven by the human elongation factor 1 α subunit promoter) in transiently transfected cells revealed that triptolide specifically reduces the steady-state level of Tat protein, without suppressing global gene expression. Further studies showed that triptolide accelerates Tat protein degradation, which can be rescued by administration of the proteasome inhibitor MG132. Mutation analysis revealed that N-terminal domains of Tat protein and nuclear localization are required for triptolide to reduce steady-state level of Tat.
Conclusion
This study suggests for the first time that triptolide exerts its anti-HIV-1 activity by specifically prompting the degradation of the virally encoded Tat protein, which is a novel mechanism of action for an anti-HIV-1 compound. This compound may serve as a starting point for developing a novel HIV-1 therapeutic approach or as a basic research tool for interrogating events during viral replication.
doi:10.1186/s12977-014-0088-6
PMCID: PMC4205289  PMID: 25323821
HIV-1; Triptolide; Antiviral; Tat; Proteasomal degradation
3.  Triptolide, Histone Acetyltransferase Inhibitor, Suppresses Growth and Chemosensitizes Leukemic Cells Through Inhibition of Gene Expression Regulated by TNF-TNFR1-TRADD-TRAF2-NIK-TAK1-IKK Pathway 
Biochemical pharmacology  2011;82(9):1134-1144.
Triptolide, a diterpene triepoxide, from the Chinese herb Tripterygium wilfordii Hook.f, exerts its anti-inflammatory and immunosuppressive activities by inhibiting the transcription factor nuclear factor-κB (NF-κB) pathway, through a mechanism not yet fully understood. We found that triptolide, in nanomolar concentrations, suppressed both constitutive and inducible NF-κB activation, but did not directly inhibit binding of p65 to the DNA. The diterpene did block TNF-induced ubiquitination, phosphorylation, and degradation of IκBα, the inhibitor of NF-κB and inhibited acetylation of p65 through suppression of binding of p65 to CBP/p300. Triptolide also inhibited the IκBα kinase (IKK) that activates NF-κB and phosphorylation of p65 at serine 276, 536. Furthermore, the NF-κB reporter activity induced by TNF-TNFR1-TRADD-TRAF2- NIK-TAK1-IKKβ was abolished by the triepoxide. Triptolide also abrogated TNF-induced expression of cell survival proteins (XIAP, Bcl-xL, Bcl-2, survivin, cIAP-1 and cIAP-2), cell proliferative proteins (cyclin D1, c-myc and cyclooxygenase-2), and metastasis proteins (ICAM-1 and MMP-9). This led to enhancement of apoptosis induced by TNF, taxol, and thalidomide by the diterpene and to suppression of tumor invasion. Overall, our results demonstrate that triptolide can block the inflammatory pathway activated by TNF-TNFR1-TRADD-TRAF2-NIK-TAK1-IKK, sensitizes cells to apoptosis, and inhibits invasion of tumor cells.
doi:10.1016/j.bcp.2011.07.062
PMCID: PMC3191321  PMID: 21820422
Triptolide; TNF; NF-κB; CBP/p300
4.  Triptolide induces anti-inflammatory cellular responses 
Tripterygium wilfordii Hook F. has been used for centuries in traditional Chinese medicine to treat rheumatoid arthritis, an autoimmune disease associated with increased production of the pro-inflammatory cytokine, tumor necrosis factor (TNF)-α. Triptolide is a compound originally purified from T. wilfordii Hook F. and has potent anti-inflammatory and immunosuppressant activities. In this study, we investigated the effect of triptolide on the global gene expression patterns of macrophages treated with lipopolysaccharide (LPS). We found that LPS stimulation resulted in >5-fold increase in expression of 117 genes, and triptolide caused a >50% inhibition in 47 of the LPS-inducible 117 genes. A large portion of the genes that were strongly induced by LPS and significantly inhibited by triptolide were pro-inflammatory cytokine and chemokine genes, including TNF-α, IL-1β, and IL-6. Interestingly, LPS also induced the expression of micro-RNA-155 (miR-155) precursor, BIC, which was inhibited by triptolide. Confirming the cDNA array results, we demonstrated that triptolide blocked the induction of these pro-inflammatory cytokines as well as miR-155 in a dose-dependent manner. Profound inhibition of pro-inflammatory cytokine expression was observed at concentrations as low as 10–50 nM. However, triptolide neither inhibited the phosphorylation or degradation of IκBα after LPS stimulation, nor affected the DNA-binding activity of NF-κB. Surprisingly, we found that triptolide not only inhibited NF-κB-regulated reporter transcription, but also dramatically blocked the activity of other transcription factors. Our study offers a plausible explanation of the therapeutic mechanism of T. wilfordii Hook F.
PMCID: PMC2776323  PMID: 19956437
Inflammation; cytokines; transcription; Chinese medicine; rheumatoid arthritis; Tripterygium wilfordii
5.  Triptolide induces apoptosis of breast cancer cells via a mechanism associated with the Wnt/β-catenin signaling pathway 
Triptolide is a diterpene triepoxide compound extracted from the medicinal plant, Tripterygium wilfordii Hook F. The aim of the present study was to determine whether triptolide inhibits the proliferation of breast cancer cells and to further investigate the associated molecular mechanisms. The effects of triptolide on the cell viability of three breast cancer cell lines, specifically, highly metastatic MDA-MB-231, human epidermal growth factor receptor 2-positive BT-474 and estrogen receptor-positive MCF7 cells, were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and apoptosis assays. Western blot analysis was performed to investigate the expression levels of β-catenin in the control and triptolide-treated cells. The results demonstrated that triptolide treatment caused cell death in the three types of malignant cell lines. Treatment with 25 nM triptolide for 48 h exhibited marked inhibitory effects on the cell viability of the three types of cells, with greater effects observed in BT-474 cells compared with the other two cell types. When compared with the cells not treated with triptolide, 50 nM triptolide treatment resulted in apoptosis of MDA-MB-231, BT-474 and MCF7 cells with apoptotic rates of ~80%. Western blot analysis indicated that triptolide treatment of MDA-MB-231, BT-474 and MCF7 cells decreased the expression levels of β-catenin to 5–10% of the levels observed in the cells treated with dimethyl sulfoxide only. Therefore, the results of the present study indicate that triptolide induces the apoptosis of breast cancer cells via a mechanism associated with the Wnt/β-catenin signaling pathway.
doi:10.3892/etm.2014.1729
PMCID: PMC4079444  PMID: 25009609
triptolide; breast cancer; Wnt; β-catenin
6.  LPS preconditioning redirects TLR signaling following stroke: TRIF-IRF3 plays a seminal role in mediating tolerance to ischemic injury 
Background
Toll-like receptor 4 (TLR4) is activated in response to cerebral ischemia leading to substantial brain damage. In contrast, mild activation of TLR4 by preconditioning with low dose exposure to lipopolysaccharide (LPS) prior to cerebral ischemia dramatically improves outcome by reprogramming the signaling response to injury. This suggests that TLR4 signaling can be altered to induce an endogenously neuroprotective phenotype. However, the TLR4 signaling events involved in this neuroprotective response are poorly understood. Here we define several molecular mediators of the primary signaling cascades induced by LPS preconditioning that give rise to the reprogrammed response to cerebral ischemia and confer the neuroprotective phenotype.
Methods
C57BL6 mice were preconditioned with low dose LPS prior to transient middle cerebral artery occlusion (MCAO). Cortical tissue and blood were collected following MCAO. Microarray and qtPCR were performed to analyze gene expression associated with TLR4 signaling. EMSA and DNA binding ELISA were used to evaluate NFκB and IRF3 activity. Protein expression was determined using Western blot or ELISA. MyD88-/- and TRIF-/- mice were utilized to evaluate signaling in LPS preconditioning-induced neuroprotection.
Results
Gene expression analyses revealed that LPS preconditioning resulted in a marked upregulation of anti-inflammatory/type I IFN-associated genes following ischemia while pro-inflammatory genes induced following ischemia were present but not differentially modulated by LPS. Interestingly, although expression of pro-inflammatory genes was observed, there was decreased activity of NFκB p65 and increased presence of NFκB inhibitors, including Ship1, Tollip, and p105, in LPS-preconditioned mice following stroke. In contrast, IRF3 activity was enhanced in LPS-preconditioned mice following stroke. TRIF and MyD88 deficient mice revealed that neuroprotection induced by LPS depends on TLR4 signaling via TRIF, which activates IRF3, but does not depend on MyD88 signaling.
Conclusion
Our results characterize several critical mediators of the TLR4 signaling events associated with neuroprotection. LPS preconditioning redirects TLR4 signaling in response to stroke through suppression of NFκB activity, enhanced IRF3 activity, and increased anti-inflammatory/type I IFN gene expression. Interestingly, this protective phenotype does not require the suppression of pro-inflammatory mediators. Furthermore, our results highlight a critical role for TRIF-IRF3 signaling as the governing mechanism in the neuroprotective response to stroke.
doi:10.1186/1742-2094-8-140
PMCID: PMC3217906  PMID: 21999375
Toll-like receptors; stroke; NFκB; inflammation; preconditioning; neuroprotection
7.  Triptolide Induces Growth Inhibition and Apoptosis of Human Laryngocarcinoma Cells by Enhancing p53 Activities and Suppressing E6-Mediated p53 Degradation 
PLoS ONE  2013;8(11):e80784.
Triptolide, an active compound extracted from Chinese herb Leigongteng (Tripterygium wilfordii Hook F.), shows a broad-spectrum of anticancer activity through its cytotoxicity. However, the efficacy of triptolide on laryngocarcinoma rarely been evaluated, and the mechanism by which triptolide-induced cellular apoptosis is still not well understood. In this study, we found that triptolide significantly inhibited the laryngocarcinoma HEp-2 cells proliferation, migration and survivability. Triptolide induces HEp-2 cell cycle arrest at the G1 phase and apoptosis through intrinsic and extrinsic pathways since both caspase-8 and -9 are activated. Moreover, triptolide enhances p53 expression by increasing its stability via down-regulation of E6 and E6AP. Increased p53 transactivates down-stream target genes to initiate apoptosis. In addition, we found that short time treatment with triptolide induced DNA damage, which was consistent with the increase in p53. Furthermore, the cytotoxicity of triptolide is decreased by p53 knockdown or use of caspases inhibitor. In conclusion, our results demonstrated that triptolide inhibits cell proliferation and induces apoptosis in laryngocarcinoma cells by enhancing p53 expression and activating p53 functions through induction of DNA damage and suppression of E6 mediated p53 degradation. These studies indicate that triptolide is a potential anti-laryngocarcinoma drug.
doi:10.1371/journal.pone.0080784
PMCID: PMC3828261  PMID: 24244715
8.  Triptolide downregulates Rac1 and the JAK/STAT3 pathway and inhibits colitis-related colon cancer progression 
Experimental & Molecular Medicine  2009;41(10):717-727.
Triptolide, a diterpenoid triepoxide from the traditional Chinese medicinal herb Tripterygium wilfordii Hook. f., is a potential treatment for autoimmune diseases as well a possible anti-tumor agent. It inhibits proliferation of coloretal cancer cells in vitro and in vivo. In this study, its ability to block progress of colitis to colon cancer, and its molecular mechanism of action are investigated. A mouse model for colitis-induced colorectal cancer was used to test the effect of triptolide on cancer progression. Treatment of mice with triptolide decreased the incidence of colon cancer formation, and increased survival rate. Moreover, triptolide decreased the incidence of tumors in nude mice inoculated with cultured colon cancer cells dose-dependently. In vitro, triptolide inhibited the proliferation, migration and colony formation of colon cancer cells. Secretion of IL6 and levels of JAK1, IL6R and phosphorylated STAT3 were all reduced by triptolide treatment. Triptolide prohibited Rac1 activity and blocked cyclin D1 and CDK4 expression, leading to G1 arrest. Triptolide interrupted the IL6R-JAK/STAT pathway that is crucial for cell proliferation, survival, and inflammation. This suggests that triptolide might be a candidate for prevention of colitis induced colon cancer because it reduces inflammation and prevents tumor formation and development.
doi:10.3858/emm.2009.41.10.078
PMCID: PMC2772974  PMID: 19561401
colonic neoplasms; interleukin-6; rac1 GTP-binding protein; STAT3 transcription factor; triptolide
9.  Studies on Calcium Dependence Reveal Multiple Modes of Action for Triptolide 
Chemistry & biology  2005;12(12):1259-1268.
Summary
Triptolide, a diterpene triepoxide isolated from the traditional Chinese medicinal vine Trypterygium wilfordii hook f., has been shown to induce rapid apoptosis in a myriad of cancer cell lines and inhibit NFκB transactivation. To understand further the general cellular mechanisms for this therapeutically relevant natural product, binding and biological activities were assessed. Studies showed that triptolide binding was saturable, reversible, and primarily localized to cell membranes. Depletion of calcium enhanced overall binding while differentially modulating biological function. Furthermore, triptolide's structural moieties demonstrated variability in the regulation of cell death versus inhibition of NFκB transactivation. These results implicate triptolide in the manipulation of at least two distinct cellular pathways with differing requirements for calcium and effective triptolide concentration in order to elicit each particular biological function.
doi:10.1016/j.chembiol.2005.09.009
PMCID: PMC2486259  PMID: 16356843
10.  Triptolide Inhibits the Proliferation of Prostate Cancer Cells and Down-Regulates SUMO-Specific Protease 1 Expression 
PLoS ONE  2012;7(5):e37693.
Recently, traditional Chinese medicine and medicinal herbs have attracted more attentions worldwide for its anti-tumor efficacy. Celastrol and Triptolide, two active components extracted from the Chinese herb Tripterygium wilfordii Hook F (known as Lei Gong Teng or Thunder of God Vine), have shown anti-tumor effects. Celastrol was identified as a natural 26 s proteasome inhibitor which promotes cell apoptosis and inhibits tumor growth. The effect and mechanism of Triptolide on prostate cancer (PCa) is not well studied. Here we demonstrated that Triptolide, more potent than Celastrol, inhibited cell growth and induced cell death in LNCaP and PC-3 cell lines. Triptolide also significantly inhibited the xenografted PC-3 tumor growth in nude mice. Moreover, Triptolide induced PCa cell apoptosis through caspases activation and PARP cleavage. Unbalance between SUMOylation and deSUMOylation was reported to play an important role in PCa progression. SUMO-specific protease 1 (SENP1) was thought to be a potential marker and therapeutical target of PCa. Importantly, we observed that Triptolide down-regulated SENP1 expression in both mRNA and protein levels in dose-dependent and time-dependent manners, resulting in an enhanced cellular SUMOylation in PCa cells. Meanwhile, Triptolide decreased AR and c-Jun expression at similar manners, and suppressed AR and c-Jun transcription activity. Furthermore, knockdown or ectopic SENP1, c-Jun and AR expression in PCa cells inhibited the Triptolide anti-PCa effects. Taken together, our data suggest that Triptolide is a natural compound with potential therapeutic value for PCa. Its anti-tumor activity may be attributed to mechanisms involving down-regulation of SENP1 that restores SUMOylation and deSUMOyaltion balance and negative regulation of AR and c-Jun expression that inhibits the AR and c-Jun mediated transcription in PCa.
doi:10.1371/journal.pone.0037693
PMCID: PMC3364364  PMID: 22666381
11.  Inhibition of poly(I:C)–induced matrix metalloproteinase expression in human corneal fibroblasts by triptolide 
Molecular Vision  2011;17:526-532.
Purpose
Triptolide is a major component of the herb Tripterygium wilfordii Hook f, extracts of which are used in traditional Chinese medicine, and it has been found to possess immunosuppressive and anti-inflammatory properties. Viral infection of the cornea can lead to corneal ulceration and perforation as a result of collagen degradation in the corneal stroma. We have now examined the effect of triptolide on the expression of matrix metalloproteinases (MMPs) induced by polyinosinic-polycytidylic acid [poly(I:C)], a synthetic analog of viral double-stranded RNA, in cultured human corneal fibroblasts.
Methods
Human corneal fibroblasts were cultured in the absence or presence of poly(I:C) or triptolide. Secretion of MMPs as well as the phosphorylation of mitogen-activated protein kinases (MAPKs) and the NF-κB–inhibitory protein, IκB-α, were examined by immunoblot analysis. The abundance of MMP mRNAs was determined by reverse transcription and real-time polymerase chain reaction analysis.
Results
Poly(I:C) induced the secretion of MMP-1 and MMP-3 from corneal fibroblasts in a concentration-dependent manner as well as increased the intracellular abundance of MMP-1 and MMP-3 mRNAs. Triptolide inhibited these effects of poly(I:C) on MMP expression in a concentration-dependent manner. The poly(I:C)-induced secretion of MMP-1 and MMP-3 was also attenuated by synthetic inhibitors of MAPK and NF-κB signaling pathways. Triptolide inhibited the poly(I:C)-induced phosphorylation of IκB-α but did not affect that of the MAPKs, Extracellular Signal-Regulated Kinase (ERK), p38MAPK, and c-Jun N-Terminal Kinase (JNK).
Conclusions
Triptolide inhibited the poly(I:C)-induced production of MMP-1 and MMP-3 by human corneal fibroblasts. Triptolide therefore warrants further investigation as a potential treatment for corneal ulceration associated with viral infection.
PMCID: PMC3044697  PMID: 21364906
12.  Antagonist Effect of Triptolide on AKT Activation by Truncated Retinoid X Receptor-alpha 
PLoS ONE  2012;7(4):e35722.
Background
Retinoid X receptor-alpha (RXRα) is a key member of the nuclear receptor superfamily. We recently demonstrated that proteolytic cleavage of RXRα resulted in production of a truncated product, tRXRα, which promotes cancer cell survival by activating phosphatidylinositol-3-OH kinase (PI3K)/AKT pathway. However, how the tRXRα-mediated signaling pathway in cancer cells is regulated remains elusive.
Methodology/Principal Findings
We screened a natural product library for tRXRα targeting leads and identified that triptolide, an active component isolated from traditional Chinese herb Trypterygium wilfordii Hook F, could modulate tRXRα-mediated cancer cell survival pathway in vitro and in animals. Our results reveal that triptolide strongly induces cancer cell apoptosis dependent on intracellular tRXRα expression levels, demonstrating that tRXRα serves as an important intracellular target of triptolide. We show that triptolide selectively induces tRXRα degradation and inhibits tRXRα-dependent AKT activity without affecting the full-length RXRα. Interestingly, such effects of triptolide are due to its activation of p38. Although triptolide also activates Erk1/2 and MAPK pathways, the effects of triptolide on tRXRα degradation and AKT activity are only reversed by p38 siRNA and p38 inhibitor. In addition, the p38 inhibitor potently inhibits tRXRα interaction with p85α leading to AKT inactivation. Our results demonstrate an interesting novel signaling interplay between p38 and AKT through tRXRα mediation. We finally show that targeting tRXRα by triptolide strongly activates TNFα death signaling and enhances the anticancer activity of other chemotherapies
Conclusions/Significance
Our results identify triptolide as a new xenobiotic regulator of the tRXRα-dependent survival pathway and provide new insight into the mechanism by which triptolide acts to induce apoptosis of cancer cells. Triptolide represents one of the most promising therapeutic leads of natural products of traditional Chinese medicine with unfortunate side-effects. Our findings will offer new strategies to develop improved triptolide analogs for cancer therapy.
doi:10.1371/journal.pone.0035722
PMCID: PMC3335786  PMID: 22545132
13.  Increased accumulation of hypoxia-inducible factor-1α with reduced transcriptional activity mediates the antitumor effect of triptolide 
Molecular Cancer  2010;9:268.
Background
Hypoxia-inducible factor-1α (HIF-1α), a critical transcription factor to reduced O2 availability, has been demonstrated to be extensively involved in tumor survival, aggressive progression, drug resistance and angiogenesis. Thus it has been considered as a potential anticancer target. Triptolide is the main principle responsible for the biological activities of the Traditional Chinese Medicine tripterygium wilfordii Hook F. Triptolide possesses great chemotherapy potential for cancer with its broad-spectrum anticancer, antiangiogenesis, and drug-resistance circumvention activities. Numerous biological molecules inhibited by triptolide have been viewed as its possible targets. However, the anticancer action mechanisms of triptolide remains to be further investigated. Here we used human ovarian SKOV-3 cancer cells as a model to probe the effect of triptolide on HIF-1α.
Results
Triptolide was observed to inhibit the proliferation of SKOV-3 cells, and meanwhile, to enhance the accumulation of HIF-1α protein in SKOV-3, A549 and DU145 cells under different conditions. Triptolide did not change the kinetics or nuclear localization of HIF-1α protein or the 26 S proteasome activity in SKOV-3 cells. However, triptolide was found to increase the levels of HIF-1α mRNA. Unexpectedly, the HIF-1α protein induced by triptolide appeared to lose its transcriptional activity, as evidenced by the decreased mRNA levels of its target genes including VEGF, BNIP3 and CAIX. The results were further strengthened by the lowered secretion of VEGF protein, the reduced sprout outgrowth from the rat aorta rings and the inhibitory expression of the hypoxia responsive element-driven luciferase reporter gene. Moreover, the silencing of HIF-1α partially prevented the cytotoxicity and apoptosis triggered by triptolide.
Conclusions
The potent induction of HIF-1α protein involved in its cytotoxicity, together with the suppression of HIF-1 transcriptional activity, indicates the great therapeutic potential of triptolide as an anticancer drug. Meanwhile, our data further stress the possibility that HIF-1α functions in an unresolved nature or condition.
doi:10.1186/1476-4598-9-268
PMCID: PMC2958983  PMID: 20932347
14.  Triptolide inhibits ovarian cancer cell invasion by repression of matrix metalloproteinase 7 and 19 and upregulation of E-cadherin 
Experimental & Molecular Medicine  2012;44(11):633-641.
Triptolide, a compound extracted from the traditional Chinese medicine preparation of Tripterygium wilfordii Hook F., has been reported to have anti-inflammatory and anti-cancer activities. However, its effect on ovarian cancer invasion is unknown. We observed that MMP7 and MMP19 expression increased in ovarian cancer tissue. Triptolide treatment inhibited the migration and invasion of ovarian cancer cells SKOV3 and A2780 at the concentration of 15 nM. We also observed that triptolide suppressed MMP7 and MMP19 promoter activity in a dose-dependent manner, down-regulating the expressions of these promoters on mRNA and protein level. Moreover, triptolide enhanced E-cadherin expression in ovarian cancer cells. In vivo, triptolide inhibited tumor formation and metastasis in nude mice, and suppressed MMP7 and MMP19 expression; it also enhanced E-cadherin expression in tumor in a dose-dependent manner. Over expression of MMP7 and MMP19, or suppression of E-cadherin expression partially abolished the inhibitory effect of triptolide on invasion of ovarian cancer cells. To summarize, triptolide significantly inhibited the migration and invasion of ovarian cancer cells by suppression of MMP7 and MMP19 and up-regulation of E-cadherin expression. This study shows that triptolide is a good candidate for the treatment of ovarian cancer and reduction of metastasis.
doi:10.3858/emm.2012.44.11.072
PMCID: PMC3509180  PMID: 22902510
cadherins; matrix metalloproteinase 19; matrix metalloproteinase 7; ovarian neoplasms; triptolide
15.  Triptolide markedly attenuates albuminuria and podocyte injury in an animal model of diabetic nephropathy 
Triptolide is a major active component of Tripterygium wilfordii Hook F, which exerts marked immunosuppressive, anti-inflammatory and podocyte-protective effects. In this study, the ability of triptolide to inhibit inflammation and attenuate podocyte injury was examined in a rat model of diabetic nephropathy (DN). Type II diabetic rats with DN were treated with triptolide at a dose of 100 μg.kg−1.day−1. Following 8 weeks of triptolide treatment, the urine albumin level, kidney weight/body weight and the number of cells positive for ED-1 (a marker for rat mononuclear macrophages) in the kidney were assessed. The effects of triptolide on podocyte injury and chronic inflammation were analyzed using quantitative polymerase chain reaction (qPCR), western blotting and immunohistochemistry. Following triptolide treatment, the albuminuria in the type II diabetic rats was significantly reduced. Furthermore, the glomerular hypertrophy and foot process effacement were improved, and there was a recovery of the slit diaphragm associated with nephrin and podocin expression. The inflammation in the kidneys was also attenuated. Furthermore, triptolide significantly reduced the expression of transforming growth factor-β1 and osteopontin, and the infiltration of ED-1-positive cells into the kidney. The results demonstrated that triptolide markedly attenuated albuminuria and podocyte injury in the rat model of DN, which may have been correlated with the inhibition of inflammation and macrophage infiltration in the kidneys.
doi:10.3892/etm.2013.1226
PMCID: PMC3786875  PMID: 24137241
albuminuria; podocyte injury; triptolide
16.  Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation 
Liu, L | Li, G | Li, Q | Jin, Z | Zhang, L | Zhou, J | Hu, X | Zhou, T | Chen, J | Gao, N
Cell Death & Disease  2013;4(12):e941-.
The diterpene triepoxide triptolide is a major active component of Tripterygium wilfordii Hook F, a popular Chinese herbal medicine with the potential to treat hematologic malignancies. In this study, we investigated the roles of triptolide in apoptosis and cell signaling events in human leukemia cell lines and primary human leukemia blasts. Triptolide selectively induced caspase-dependent cell death that was accompanied by the loss of mitochondrial membrane potential, cytochrome c release, and Bax translocation from the cytosol to the mitochondria. Furthermore, we found that triptolide dramatically induced ROCK1 cleavage/activation and MLC and MYPT phosphorylation. ROCK1 was cleaved and activated by caspase-3, rather than RhoA. Inhibiting MLC phosphorylation by ML-7 significantly attenuated triptolide-mediated apoptosis, caspase activation, and cytochrome c release. In addition, ROCK1 inhibition also abrogated MLC and MYPT phosphorylation. Our in vivo study showed that both ROCK1 activation and MLC phosphorylation were associated with the tumor growth inhibition caused by triptolide in mouse leukemia xenograft models. Collectively, these findings suggest that triptolide-mediated ROCK1 activation and MLC phosphorylation may be a novel therapeutic strategy for treating hematological malignancies.
doi:10.1038/cddis.2013.469
PMCID: PMC3877542  PMID: 24309928
triptolide; leukemia; apoptosis; ROCK1; MLC; MYPT
17.  Triptolide-induced suppression of phospholipase D expression inhibits proliferation of MDA-MB-231 breast cancer cells 
Experimental & Molecular Medicine  2009;41(9):678-685.
In spite of the importance of phospholipase D (PLD) in cell proliferation and tumorigenesis, little is known about the molecules regulating PLD expression. Thus, identification of small molecules inhibiting PLD expression would be an important advance for PLD-mediated physiology. We examined one such here, denoted "Triptolide", which was identified in a chemical screen for inhibitors of PLD expression using cell assay system based on measurement of PLD promoter activity. Triptolide significantly suppressed the expression of both PLD1 and PLD2 with sub-µM potency in MDA-MB-231 breast cancer cells as analyzed by promoter assay and RT-PCR. Moreover, triptolide abolished the protein level of PLD in a time and dose-dependent manner. Triptolide-induced PLD1 downregulation was also observed in all the cancer cells examined, suggesting a general phenomenon detected in various cancer cells. Decrease of PLD expression by triptolide suppressed both basal and PMA-induced PLD activity. In addition, triptolide inhibited activation of NFκB which increased PLD1 expression. Ultimately, downregulation of PLD by triptolide inhibited proliferation of breast cancer cells. Taken together, we demonstrate that triptolide suppresses the expression of PLD via inhibition of NFκB activation and then decreases cell proliferation.
doi:10.3858/emm.2009.41.9.074
PMCID: PMC2753661  PMID: 19478552
breast neoplasms; cell proliferation; gene expression regulation, neoplastic; NF-κB; phospholipase D; triptolide
18.  Triptolide Inhibits Proliferation and Migration of Colon Cancer Cells by Inhibition of Cell Cycle Regulators and Cytokine Receptors 
The Journal of surgical research  2009;168(2):197-205.
Background
Phytochemicals are an important source of emerging preventive and therapeutic agents for cancer. Triptolide/PG490, an extract of the Chinese herb Tripterygium wilfordii Hook F, is a potent anti-inflammatory agent that also possesses anticancer activity. While its anti-proliferative effects are well-established, the potential anti-migratory effects of triptolide have not been characterized.
Material and Methods
Effects of triptolide on the proliferation and invasion of colon cancer cells and expression of cancer-related genes and proteins were assessed.
Results
Triptolide potently inhibited HT29 and HCT116 colon cancer cell growth and reduced basal and stimulated HCT116 migration through collagen by 65–80%. Triptolide inhibited mRNA expression of the positive cell cycle regulatory genes c-myc, and A, B, C, and D-type cyclins in multiple colon cancer cell lines. Additionally, we show that triptolide treatment decreased expression of VEGF and COX-2, which promote cancer progression and invasion, and inhibited the expression of multiple cytokine receptors potentially involved in cell migration and cancer metastasis, including the thrombin receptor, CXCR4, TNF receptors and TGF-β receptors.
Conclusions
Triptolide is a potent inhibitor of colon cancer proliferation and migration in vitro. The downregulation of multiple cytokine receptors, in combination with inhibition of COX-2 and VEGF and positive cell cycle regulators, may contribute to the anti-metastatic action of this herbal extract.
doi:10.1016/j.jss.2009.07.002
PMCID: PMC2949684  PMID: 19922946
triptolide; colorectal cancer; herbal extract; growth factor receptors; cell cycle; chemokine
19.  Triptolide inhibits MDM2 and induces apoptosis in acute lymphoblastic leukemia cells through a p53-independent pathway 
Molecular cancer therapeutics  2012;12(2):184-194.
Triptolide, a natural product derived from the Chinese plant Tripterygium wilfordii, is reported to exhibit antitumor effects in a broad range of cancers. The antitumor activity of triptolide is associated with its biological activities, as it inhibits various pro-proliferative or anti-apoptotic factors that are dominantly expressed in given types of cancer cells. Herein, we demonstrate that triptolide induced apoptosis in a subgroup of acute lymphoblastic leukemia (ALL) cells overexpressing the MDM2 oncoprotein, by inhibiting MDM2 expression. More specifically, we found that triptolide inhibited MDM2 at the transcriptional level by suppressing its mRNA synthesis. This MDM2 inhibition led in turn to increased levels of p53 protein; however, p53 functionality was not activated, due to the fact that triptolide-treated cells lacked induction of p21 and PUMA as well as in G1 cell-cycle arrest. Triptolide-mediated downregulation of MDM2 increased inhibition of XIAP, its translational target, in a manner distinct from reactions to cellular stress and DNA-damaging agent ionizing radiation (IR) that induce XIAP due to p53-activated MDM2. These results suggest that increased inhibition of XIAP due to downregulation of MDM2 may play a critical role in triptolide-induced apoptosis in MDM2-overexpressing cancers.
doi:10.1158/1535-7163.MCT-12-0425
PMCID: PMC3570632  PMID: 23243057
20.  Effects of triptolide from Radix Tripterygium wilfordii (Leigongteng) on cartilage cytokines and transcription factor NF-κB: a study on induced arthritis in rats 
Chinese Medicine  2009;4:13.
Background
Triptolide, an active compound of Radix Tripterygium wilfordii, is immunosuppressive, cartilage protective and anti-inflammatory both in human and animal studies of various inflammatory and autoimmune diseases, including rheumatoid arthritis, but its therapeutic mechanism remains unclear. The aim of this study is to investigate the effects of triptolide on cartilage cytokines in the CIA model.
Methods
Sprague Dawley rats were immunized with type II collagen and orally administered with triptolide. The arthritic scores and incidence changes of the rats were observed. The expression of TNF-α, IL-6, COX-2 and NF-κB in paw cartilage was studied with immunohistochemical staining.
Results
Triptolide, at both high and low doses, significantly lowered the arthritic scores, delayed the onset of arthritis and lowered the arthritis incidence. Triptolide treatment at both high and low doses lowered the expression of TNF-α, IL-6, COX-2 and NF-κB in paw cartilage in arthritic rats.
Conclusion
Triptolide lowers the arthritic scores, delays the onset of collagen induced arthritis and reduces the expressions of TNF-α, IL-6, NF-κB and COX-2 in paw cartilage in arthritic rats.
doi:10.1186/1749-8546-4-13
PMCID: PMC2709898  PMID: 19570240
21.  Triptolide inhibits cell proliferation and tumorigenicity of human neuroblastoma cells 
Molecular Medicine Reports  2014;11(2):791-796.
Triptolide is a diterpene triepoxide, extracted from the Chinese herb Tripterygium wilfordii Hook F, which has been shown to have antitumor activity in a number of cancers. Neuroblastoma is an aggressive extracranial pediatric solid tumor, with significant chemotherapeutic resistance. In this study, triptolide was hypothesized to be a potential therapeutic agent for neuroblastoma. The effects of triptolide on neuroblastoma cell growth and tumor development were investigated. Cell growth and proliferation were evaluated using a cell counting kit-8 assay and a 5-bromo-2-deoxyuridine staining assay. Cell cycle and apoptosis were detected by flow cytometry. Reverse transcription-quantitative polymerase chain reaction was conducted to detect the expression levels of the apoptosis-associated proteins, caspase-3 and caspase-9. The tumorigenicity of neuroblastoma cells was assessed by a soft agar clonogenic assay and an in vivo tumorigenic assay. The results demonstrated that exposure of BE(2)-C human neuroblastoma cells to triptolide resulted in a reduction in cell growth and proliferation, and the induction of cell death and apoptosis, together with cell cycle arrest in the S phase. A soft agar assay indicated that triptolide inhibited the colony-forming ability of BE(2)-C neuroblastoma cells. The xenograft experiment showed that triptolide significantly reduced tumor growth and development in vivo. The data suggested that this Chinese herb may be a potential novel chemotherapeutic agent for neuroblastoma.
doi:10.3892/mmr.2014.2814
PMCID: PMC4262511  PMID: 25354591
neuroblastoma; triptolide; cell proliferation; tumorigenicity; cell cycle arrest; apoptosis
22.  Activation of Nrf2 Protects against Triptolide-Induced Hepatotoxicity 
PLoS ONE  2014;9(7):e100685.
Triptolide, the major active component of Tripterygium wilfordii Hook f. (TWHF), has a wide range of pharmacological activities. However, the toxicities of triptolide, particularly the hepatotoxicity, limit its clinical application. The hepatotoxicity of triptolide has not been well characterized yet. The aim of this study was to investigate the role of NF-E2-related factor 2 (Nrf2) in triptolide-induced toxicity and whether activation of Nrf2 could protect against triptolide-induced hepatotoxicity. The results showed that triptolide caused oxidative stress and cell damage in HepG2 cells, and these toxic effects could be aggravated by Nrf2 knockdown or be counteracted by overexpression of Nrf2. Treatment with a typical Nrf2 agonist, sulforaphane (SFN), attenuated triptolide-induced liver dysfunction, structural damage, glutathione depletion and decrease in antioxidant enzymes in BALB/C mice. Moreover, the hepatoprotective effect of SFN on triptolide-induced liver injury was associated with the activation of Nrf2 and its downstream targets. Collectively, these results indicate that Nrf2 activation protects against triptolide-induced hepatotoxicity.
doi:10.1371/journal.pone.0100685
PMCID: PMC4079517  PMID: 24988078
23.  Heme activates TLR4-mediated inflammatory injury via MyD88/TRIF signaling pathway in intracerebral hemorrhage 
Background
Inflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced neurological deficits; however, the signaling pathways are not apparent by which the upstream cellular events trigger innate immune and inflammatory responses that contribute to neurological impairments. Toll-like receptor 4 (TLR4) plays a role in inflammatory damage caused by brain disorders.
Methods
In this study, we investigate the role of TLR4 signaling in ICH-induced inflammation. In the ICH model, a significant upregulation of TLR4 expression in reactive microglia has been demonstrated using real-time RT-PCR. Activation of microglia was detected by immunohistochemistry, cytokines were measured by ELISA, MyD88, TRIF and NF-κB were measured by Western blot and EMSA, animal behavior was evaluated by animal behavioristics.
Results
Compared to WT mice, TLR4−/− mice had restrained ICH-induced brain damage showing in reduced cerebral edema and lower neurological deficit scores. Quantification of cytokines including IL-6, TNF-α and IL-1β and assessment of macrophage infiltration in perihematoma tissues from TLR4−/−, MyD88−/− and TRIF−/− mice showed attenuated inflammatory damage after ICH. TLR4−/− mice also exhibited reduced MyD88 and TRIF expression which was accompanied by decreased NF-κB activity. This suggests that after ICH both MyD88 and TRIF pathways might be involved in TLR4-mediated inflammatory injury possibly via NF-κB activation. Exogenous hemin administration significantly increased TLR4 expression and microglial activation in cultures and also exacerbated brain injury in WT mice but not in TLR4−/− mice. Anti-TLR4 antibody administration suppressed hemin-induced microglial activation in cultures and in the mice model of ICH.
Conclusions
Our findings suggest that heme potentiates microglial activation via TLR4, in turn inducing NF-κB activation via the MyD88/TRIF signaling pathway, and ultimately increasing cytokine expression and inflammatory injury in ICH. Targeting TLR4 signaling may be a promising therapeutic strategy for ICH.
doi:10.1186/1742-2094-9-46
PMCID: PMC3344687  PMID: 22394415
Toll-like receptor 4; MyD88; TRIF; Inflammation; Intracerebral hemorrhage; Heme
24.  Triptolide Inhibits IL-12/IL-23 Expression in APCs via CCAAT/Enhancer-Binding Protein α 
Triptolide is a biologically active component purified from Chinese herbal plant Tripterygium wilfordii Hook F. It is widely used in East Asia for treatment of systemic lupus erythematosus, rheumatoid arthritis, nephritis, Bechect’s disease, psoriasis, atopic dermatitis, and asthma. However, its immunological mechanisms are poorly understood. IL-12 and IL-23 are closely related heterodimeric cytokines that share the common subunit p40. They are produced by APCs and are key factors in the generation and effector functions of Th1 and Th17 cells, respectively. They have been strongly implicated in the pathogenesis of several autoimmune disorders. In this study, we investigated the molecular mechanism whereby triptolide inhibits the expression of the p40 gene in APCs. We demonstrate that triptolide does so at the transcriptional level in part through targeting CCAAT/enhancer-binding protein-α (C/EBPα), which directly interacts with the p40 promoter and inhibits its transcription in inflammatory macrophages. Triptolide can activate the transcription of C/EBPα, and phosphorylation of Ser21 and Thr222/226 critical for C/EBPα inhibition of p40. Further, activation of C/EBPα by triptolide is dependent on upstream kinases ERK1/2 and Akt-GSK3β. This study provides mechanistic insights into the immunomodulatory capacity of triptolide and has strong implications for its therapeutic applications in autoimmune diseases.
doi:10.4049/jimmunol.0903417
PMCID: PMC2965075  PMID: 20194724
25.  Triptolide reverses hypoxia-induced epithelial–mesenchymal transition and stem-like features in pancreatic cancer by NF-κB downregulation 
Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies characterized by an intense tumor stroma with hypoperfused regions, a significant inflammatory response and pronounced therapy resistance. New therapeutic agents are urgently needed. The plant-derived agent triptolide also known as “thunder god vine” has a long history in traditional Chinese medicine for treatment of rheumatoid arthritis and cancer and is now in a clinical phase II trial for establishing the efficacy against a placebo. The authors mimicked the situation in patient tumors by induction of hypoxia in experimental models of pancreatic cancer stem cells (CSCs) and evaluated the therapeutic effect of triptolide. Hypoxia led to induction of colony and spheroid formation, aldehyde dehydrogenase 1 (ALDH1) and NF-κB activity, migratory potential and a switch in morphology to a fibroblastoid phenotype, as well as stem cell- and epithelial–mesenchymal transition-associated protein expression. Triptolide efficiently inhibited hypoxia-induced transcriptional signaling and downregulated epithelial–mesenchymal transition (EMT) and CSC features in established highly malignant cell lines, whereas sensitive cancer cells or nonmalignant cells were less affected. In vivo triptolide inhibited tumor take and tumor growth. In primary CSCs isolated from patient tumors, triptolide downregulated markers of CSCs, proliferation and mesenchymal cells along with upregulation of markers for apoptosis and epithelial cells. This study is the first to show that triptolide reverses EMT and CSC characteristics and therefore may be superior to current chemotherapeutics for treatment of PDA.
What's new?
Current treatment for pancreatic cancer does not directly target tumor hypoxia, a major mediator of aggressive growth, early metastasis, and therapy resistance. The plant-derived agent triptolide has a long history of use in rheumatoid arthritis and cancer in traditional Chinese medicine and has been shown to have potent therapeutic properties in a variety of studies. Here, the authors show for the first time that triptolide effectively inhibits hypoxia-induced signaling, leading to downregulation of NF-κB activity, epithelial-mesenchymal transition, and stem cell-like features. Triptolide may therefore be superior to current chemotherapeutics for treatment of pancreatic cancer.
doi:10.1002/ijc.28583
PMCID: PMC4255690  PMID: 24615157
pancreatic cancer; novel antitumor agents; hypoxia; epithelial–mesenchymal transition; cancer stem cells

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