Pristimerin

Pristimerin is a potent and reversible monoacylglycerol lipase (MGL) inhibitor with an IC50 of 93 nM.

In Vitro:Pristimerin inhibits the activity of purified MGL with an IC50 of 93±8 nM and that of non-purified MGL (cell lysates of MGL-transfected HeLa cells) with an IC50 of 398±68 nM. Pristimerin inhibits MGL through a mechanism that is rapid, reversible and non-competitive. The binding of pristimerin to MGL might be strengthened by formation of a polar interaction with a regulatory cysteine, possibly Cys208[1]. Pristimerin inhibits HFLS-RA and HUVEC cell viability in a dose- and time-dependent manner. Pristimerin decreases VEGF-induced autophosphorylation of VEGFR2 and attenuates the activation of the VEGF-induced VEGFR2-mediated signaling pathway [2].

In Vivo:Pristimerin inhibits inflammation and tumor angiogenesis. Pristimerin significantly reduces vessel density in synovial membrane tissues of inflamed joints and reduces the expression of pro-angiogenic factors in sera, including TNF-α, Ang-1, and MMP-9[2].

References:
[1]. King AR, et al. Discovery of potent and reversible monoacylglycerol lipase inhibitors. Chem Biol. 2009 Oct 30;16(10):1045-52. [2]. Deng Q, et al. Pristimerin inhibits angiogenesis in adjuvant-induced arthritic rats by suppressing VEGFR2 signaling pathways. Int Immunopharmacol. 2015 Dec;29(2):302-13.

Pristimerin induces apoptosis in imatinib-resistant chronic myelogenous leukemia cells harboring T315I mutation by blocking NF-kappaB signaling and depleting Bcr-Abl.[Pubmed:20482842]

Mol Cancer. 2010 May 19;9:112.

BACKGROUND: Chronic myelogenous leukemia (CML) is characterized by the chimeric tyrosine kinase Bcr-Abl. Bcr-Abl-T315I is the notorious point mutation that causes resistance to imatinib and the second generation tyrosine kinase inhibitors, leading to poor prognosis. CML blasts have constitutive p65 (RelA NF-kappaB) transcriptional activity, and NF-kappaB may be a potential target for molecular therapies in CML that may also be effective against CML cells with Bcr-Abl-T315I. RESULTS: In this report, we discovered that Pristimerin, a quinonemethide triterpenoid isolated from Celastraceae and Hippocrateaceae, inhibited growth and induced apoptosis in CML cells, including the cells harboring Bcr-Abl-T315I mutation. Additionally, Pristimerin inhibited the growth of imatinib-resistant Bcr-Abl-T315I xenografts in nude mice. Pristimerin blocked the TNFalpha-induced IkappaBalpha phosphorylation, translocation of p65, and expression of NF-kappaB-regulated genes. Pristimerin inhibited two steps in NF-kappaB signaling: TAK1TauIKK and IKKTauIkappaBalpha. Pristimerin potently inhibited two pairs of CML cell lines (KBM5 versus KBM5-T315I, 32D-Bcr-Abl versus 32D-Bcr-Abl-T315I) and primary cells from a CML patient with acquired resistance to imatinib. The mRNA and protein levels of Bcr-Abl in imatinib-sensitive (KBM5) or imatinib-resistant (KBM5-T315I) CML cells were reduced after Pristimerin treatment. Further, inactivation of Bcr-Abl by imatinib pretreatment did not abrogate the TNFalpha-induced NF-kappaB activation while silencing p65 by siRNA did not affect the levels of Bcr-Abl, both results together indicating that NF-kappaB inactivation and Bcr-Abl inhibition may be parallel independent pathways. CONCLUSION: To our knowledge, this is the first report to show that Pristimerin is effective in vitro and in vivo against CML cells, including those with the T315I mutation. The mechanisms may involve inhibition of NF-kappaB and Bcr-Abl. We concluded that Pristimerin could be a lead compound for further drug development to overcome imatinib resistance in CML patients.

Pristimerin, a natural anti-tumor triterpenoid, inhibits LPS-induced TNF-alpha and IL-8 production through down-regulation of ROS-related classical NF-kappaB pathway in THP-1 cells.[Pubmed:24957686]

Int Immunopharmacol. 2014 Aug;21(2):501-8.

Pristimerin, a naturally occurring quinonemethide triterpenoid compound, is known to exert a variety of pharmacological activities. In the present study, we investigated the molecular actions of Pristimerin against LPS-induced inflammatory responses in human monocytic THP-1 cells. The results showed that Pristimerin inhibited the production of TNF-alpha and IL-8 in a dose-dependent manner. To explore the possible mechanisms underlying these inhibitions by Pristimerin, we examined the intracellular ROS level and the NF-kappaB protein signaling pathway. Pristimerin clearly scavenged LPS-induced intracellular ROS production. In addition, Pristimerin prevented LPS-induced NF-kappaB activation through the inhibition of phosphorylation of IKKalpha/beta, phosphorylation and degradation of IkappaBalpha, as well as phosphorylation and nuclear translocation of NF-kappaB p65. These findings suggest that Pristimerin down-regulates the expression of pro-inflammatory mediators through blocking of NF-kappaB activation by inhibiting interconnected ROS/IKK/NF-kappaB signaling pathways.

Pristimerin, a quinonemethide triterpenoid, induces apoptosis in pancreatic cancer cells through the inhibition of pro-survival Akt/NF-kappaB/mTOR signaling proteins and anti-apoptotic Bcl-2.[Pubmed:24603988]

Int J Oncol. 2014 May;44(5):1707-15.

Lack of effective therapeutics for pancreatic cancer at the present time underscores the dire need for safe and effective agents for the treatment of this malignancy. In the present study, we have evaluated the anticancer activity and the mechanism of action of Pristimerin (PM), a quinonemethide triterpenoid, against MiaPaCa-2 and Panc-1 pancreatic ductal adenocarcinoma (PDA) cell lines. Treatment with PM inhibited the proliferation and induced apoptosis in both cell lines as characterized by the increased Annexin V-binding and cleavage of PARP-1 and procaspases -3, -8 and -9. PM also induced mitochondrial depolarization and the release of cytochrome c from the mitochondria. The induction of apoptosis by PM was associated with the inhibition of the pro-survival Akt, NF-kappaB and mTOR signaling proteins and their downstream intermediaries such as Foxo-3alpha and cyclin D1 (Akt); Cox-2 and VEGF (NF-kappaB); p-S6K1 and p-4E-BP1 (mTOR) as well as PKCepsilon. Treatment with PM also inhibited the expression of anti-apoptotic Bcl-2 and survivin but not Bcl-xL. The downregulation of Bcl-2 by PM was not due to proteasomal or lysosomal proteolytic degradation of Bcl-2, since treatment with PM in the presence of proteasomal inhibitors MG132 or lactacystin (LAC) or calpain inhibitor MG101 failed to block the downregulation of Bcl-2 by PM. On the other hand, RT-PCR analysis showed the inhibition of Bcl-2 mRNA by PM in a dose-related manner, indicating that inhibition of Bcl-2 by PM is mediated through the suppression of Bcl-2 gene expression. Thus, the mechanistic understanding of the antitumor activity of Pristimerin could facilitate in vivo efficacy studies of Pristimerin for pancreatic cancer.

Pristimerin, a naturally occurring triterpenoid, protects against autoimmune arthritis by modulating the cellular and soluble immune mediators of inflammation and tissue damage.[Pubmed:25308129]

Clin Immunol. 2014 Dec;155(2):220-30.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder affecting the synovial joints. The currently available drugs for RA are effective only in a proportion of patients and their prolonged use is associated with severe adverse effects. Thus, new anti-arthritic agents are being sought. We tested Pristimerin, a naturally occurring triterpenoid, for its therapeutic activity against rat adjuvant arthritis. Pristimerin effectively inhibited both arthritic inflammation and cartilage and bone damage in the joints. Pristimerin-treated rats exhibited a reduction in the pro-inflammatory cytokines (IL-6, IL-17, IL-18, and IL-23) and the IL-6/IL-17-associated transcription factors (pSTAT3 and ROR-gammat), coupled with an increase in the immunomodulatory cytokine IL-10. Also increased was IFN-gamma, which can inhibit IL-17 response. In addition, the Th17/Treg ratio was altered in favor of immune suppression and the RANKL/OPG ratio was skewed towards anti-osteoclastogenesis. This is the first report on testing Pristimerin in arthritis. We suggest further evaluation of Pristimerin in RA patients.

Pristimerin enhances recombinant adeno-associated virus vector-mediated transgene expression in human cell lines in vitro and murine hepatocytes in vivo.[Pubmed:24461592]

J Integr Med. 2014 Jan;12(1):20-34.

OBJECTIVE: In the present study, we systemically evaluated the ability of two bioactive compounds from traditional Chinese medicine, celastrol and Pristimerin, to enhance recombinant adeno-associated virus (rAAV) serotype vector-mediated transgene expression both in human cell lines in vitro, and in murine hepatocytes in vivo. METHODS: Human cell lines were infected with rAAV vectors with either mock treatment or treatment with celastrol or Pristimerin. The transgene expression, percentage of nuclear translocated viral genomes and the ubiquitination of intracellular proteins were investigated post-treatment. In addition, nonobese diabetic/severe combined immunodeficient gamma (NSG) mice were tail vain-injected with rAAV vectors and co-administered with either dimethyl sulfoxide, celastrol, Pristimerin or a positive control, bortezomib. The transgene expression in liver was detected and compared over time. RESULTS: We observed that treatment with Pristimerin, at as low as 1 mumol/L concentration, significantly enhanced rAAV2 vector-mediated transgene expression in vitro, and intraperitoneal co-administration with Pristimerin at 4 mg/(kg.d) for 3 d dramatically facilitated viral transduction in murine hepatocytes in vivo. The transduction efficiency of the tyrosine-mutant rAAV2 vectors as well as that of rAAV8 vectors carrying oversized transgene cassette was also augmented significantly by Pristimerin. The underlying molecular mechanisms by which Pristimerin mediated the observed increase in the transduction efficiency of rAAV vectors include both inhibition of proteasomal degradation of the intracellular proteins and enhanced nuclear translocation of the vector genomes. CONCLUSION: These studies suggest the potential beneficial use of Pristimerin and Pristimerin-containing herb extract in future liver-targeted gene therapy with rAAV vectors.

Antiproliferative activity of pristimerin isolated from Maytenus ilicifolia (Celastraceae) in human HL-60 cells.[Pubmed:18296021]

Toxicol In Vitro. 2008 Jun;22(4):854-63.

Pristimerin has been shown to be cytotoxic to several cancer cell lines. In the present work, the cytotoxicity of Pristimerin was evaluated in human tumor cell lines and in human peripheral blood mononuclear cells (PBMC). This work also examined the effects of Pristimerin (0.4; 0.8 and 1.7 microM) in HL-60 cells, after 6, 12 and 24h of exposure. Pristimerin reduced the number of viable cells and increased number of non-viable cells in a concentration-dependent manner by tripan blue test showing morphological changes consistent with apoptosis. Nevertheless, Pristimerin was not selective to cancer cells, since it inhibited PBMC proliferation with an IC50 of 0.88 microM. DNA synthesis inhibition assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation in HL-60 cells was 70% and 83% for the concentrations of 0.4 and 0.8 microM, respectively. Pristimerin (10 and 20 microM) was not able to inhibit topoisomerase I. In AO/EB (acridine orange/ethidium bromide) staining, all tested concentrations reduced the number of HL-60 viable cells, with the occurrence of necrosis and apoptosis in a concentration-dependent manner, results in agreement with trypan blue exclusion findings. The analysis of membrane integrity and internucleosomal DNA fragmentation by flow cytometry in the presence of Pristimerin indicated that treated cells underwent apoptosis. The present data point to the importance of Pristimerin as representative of an emerging class of potential anticancer chemicals, exhibiting an antiproliferative effect by inhibiting DNA synthesis and triggering apoptosis.

Inhibitory action of pristimerin on hypoxiamediated metastasis involves stem cell characteristics and EMT in PC-3 prostate cancer cells.[Pubmed:25571882]

Oncol Rep. 2015 Mar;33(3):1388-94.

The aim of the present study was to investigate whether Pristimerin affects the bone metastasis, stem cell characteristics and epithelial-mesenchymal transition (EMT) of prostate cancer (PCa) PC-3 cells subjected to hypoxia. The PC-3 cells were cultured under hypoxia or normoxia for 48 h and were then treated with increasing concentrations of Pristimerin from 0 to 0.8 micromol/l, under normoxia. Hypoxiainducible factor-1alpha (HIF-1alpha) was detected by western blotting. Proliferation was assessed with the CCK-8 assay. Transwell invasion assay was used to analyze the potency of invasion. Stem cell characteristics were detected by sphere formation, colony formation assay and western blotting, including CD44, KLF4, OCT4 and AGO2, which are stem cell characteristic-related markers. EMT was confirmed by the expression changes of EMT-related markers, including N-cadherin, fibronectin, vimentin and ZEB1, which were evaluated by western blotting. The addition of Pristimerin to the medium reduced the hypoxia-induced PC-3 cell proliferation in a dose-dependent manner. Pristimerin effectively inhibited hypoxiainduced invasion of the PCa cells in vitro. Moreover, the treatment of cells with Pristimerin induced the reversal of hypoxia-induced stem cell characteristics and EMT, which was confirmed by sphere formation, colony formation assay and the expression changes of CSC- and EMT-related markers. The reversal of hypoxiainduced stem cell characteristics and EMT in the PCa cells by low-dose Pristimerin was dosedependent. These results showed that treatment with Pristimerin may be a potential strategy for the suppression of hypoxia-induced metastasis through the reversal of hypoxia-induced stem cell characteristics and EMT in cancer cells, which justifies the potential use of Pristimerin as a practical chemopreventive approach for patients with PCa.

Antifungal properties of pristimerin and celastrol isolated from Celastrus hypoleucus.[Pubmed:15593077]

Pest Manag Sci. 2005 Jan;61(1):85-90.

Pristimerin and celastrol isolated from the roots of Celastrus hypoleucus (Oliv) Warb f argutior Loes exhibited inhibitory effects against diverse phytopathogenic fungi. Pristimerin and celastrol were found to inhibit the mycelial growth of Rhizoctonia solani Kuhn and Glomerella cingulata (Stonem) Spauld & Schrenk in vitro by 83.6 and 62.6%, respectively, at 10 microg ml(-1). Pristimerin showed good preventive effect (96.7% at 100 microg ml(-1)) and curative effect (66.5% at 100 microg ml(-1)) against wheat powdery mildew in vivo. For celastrol, the preventive and curative effects against wheat powdery mildew were 80.5 and 45.4%, respectively, at 100 microg ml(-1).

Reactive oxygen species-dependent activation of Bax and poly(ADP-ribose) polymerase-1 is required for mitochondrial cell death induced by triterpenoid pristimerin in human cervical cancer cells.[Pubmed:19574249]

Mol Pharmacol. 2009 Oct;76(4):734-44.

Naturally occurring triterpenoid compounds have long been used as anti-inflammatory, antimalarial, and insecticidal agents. It has become evident that some of the natural or synthetic triterpenoids have promising clinical potential as both a therapeutic and chemopreventive agent for cancer. However, the molecular basis for the antitumor activity of triterpenoid has yet to be defined. In this study, we show that Pristimerin, a natural triterpenoid, induces mitochondrial cell death in human cervical cancer cells and that reactive oxygen species (ROS)-dependent activation of both Bax and poly(ADP-ribose) polymerase-1 (PARP-1) is critically required for the mitochondrial dysfunction. We also showed that c-Jun N-terminal kinase (JNK) is involved in ROS-dependent Bax activation. Treatment of Pristimerin induced an increase in intracellular ROS, JNK activation, conformational change, and mitochondrial redistribution of Bax, mitochondrial membrane potential loss, and cell death. The PARP-1 was also found to be activated by Pristimerin treatment. An antioxidant, N-acetyl-l-cysteine (NAC), inhibited Pristimerin-induced JNK activation, Bax relocalization, and PARP-1 activation, as well as mitochondrial cell death. Moreover, inhibition of JNK clearly suppressed conformational change and mitochondrial translocation of Bax and subsequent mitochondrial cell death but did not affect PARP-1 activation. Inhibition of PARP-1 with 1,5-dihydroxyisoquinoline (DIQ) or with small interfering RNA of PARP-1 significantly attenuated Pristimerin-induced mitochondrial membrane potential loss and cell death but did not affect JNK activation and Bax relocalization. These results indicate that the natural triterpenoid Pristimerin induces mitochondrial cell death through ROS-dependent activation of both Bax and PARP-1 in human cervical cancer cells and that JNK is involved in ROS-dependent Bax activation.

Discovery of potent and reversible monoacylglycerol lipase inhibitors.[Pubmed:19875078]

Chem Biol. 2009 Oct 30;16(10):1045-52.

Monoacylglycerol lipase (MGL) is a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Previous efforts to design MGL inhibitors have focused on chemical scaffolds that irreversibly block the activity of this enzyme. Here, we describe two naturally occurring terpenoids, Pristimerin and euphol, which inhibit MGL activity with high potency (median effective concentration, IC(50) = 93 nM and 315 nM, respectively) through a reversible mechanism. Mutational and modeling studies suggest that the two agents occupy a common hydrophobic pocket located within the putative lid domain of MGL, and each reversibly interacts with one of two adjacent cysteine residues (Cys(201) and Cys(208)) flanking such pocket. This previously unrecognized regulatory region might offer a molecular target for potent and reversible inhibitors of MGL.

Identification of a potent natural triterpenoid inhibitor of proteosome chymotrypsin-like activity and NF-kappaB with antimyeloma activity in vitro and in vivo.[Pubmed:19096011]

Blood. 2009 Apr 23;113(17):4027-37.

As multiple myeloma tumors universally dysregulate cyclin D genes we conducted high-throughput chemical library screens for compounds that induce suppression of cyclin D2 promoter transcription. The top-ranked compound was a natural triterpenoid, Pristimerin. Strikingly, the early transcriptional response of cells treated with Pristimerin closely resembles cellular responses elicited by proteosome inhibitors, with rapid induction of heat shock proteins, activating transcription factor 3 (ATF3), and CHOP. Enzymatic assays and immunoblotting confirm that Pristimerin rapidly (< 90 minutes) and specifically inhibits chymotrypsin-like proteosome activity at low concentrations (< 100 nM) and causes accumulation of cellular ubiquitinated proteins. Notably, cytotoxic triterpenoids including Pristimerin inhibit NF-kappaB activation via inhibition of IKK alpha or IKK beta, whereas proteosome inhibitors instead suppress NF-kappaB function by impairing degradation of ubiquitinated I kappaB. By inhibiting both IKK and the proteosome, Pristimerin causes overt suppression of constitutive NF-kappaB activity in myeloma cells that may mediate its suppression of cyclin D. Multiple myeloma is exquisitely sensitive to proteosome or NF-kappaB pathway inhibition. Consistent with this, Pristimerin is potently and selectively lethal to primary myeloma cells (IC(50) < 100 nM), inhibits xenografted plasmacytoma tumors in mice, and is synergistically cytotoxic with bortezomib--providing the rationale for pharmaceutical development of triterpenoid dual-function proteosome/NF-kappaB inhibitors as therapeutics for human multiple myeloma and related malignancies.