Bisdemethylcurcumin and structurally related hispolon analogues of curcumin exhibit enhanced prooxidant, anti-proliferative and anti-inflammatory activities in vitro

Curcumin, a component of turmeric (Curcuma longa), exhibits anti-inflammatory and anti-proliferative activities through the generation of reactive oxygen species (ROS). Curcumin (diferuloylmethane) contains two hydroxyl, two methoxy and two phenyl groups but how these groups contribute to its activity is poorly understood. We synthesized analogues that varied in inclusion of these groups and compared their activity. We found that bisdemethylcurcumin (BDC) was more potent than curcumin as an anti-inflammatory agent as indicated by suppression of TNF-induced NF-κB activation, more potent as an anti-proliferative agent, and more potent in inducing ROS. Hispolon, which lacks one aromatic unit in relation to curcumin, also exhibited enhanced anti-inflammatory and anti-proliferative activities. When synthetic curcumin (Cur-S) was compared with bisdemethylcurcumin (BDC), hispolon, hispolon methyl ether (HME), dehydroxy hispolon (DH), hydroxy hispolon (HH), methoxy hispolon methyl ether (MHME), and methoxy hispolon (MH), we found that following order of anti-inflammatory activity: BDC = Hispolon > HME > HH > Cur-S > MHME > MH > DH; for anti-proliferative: Hispolon > BDC > MHME > Cur-S > MH > HME = HH > DH; and for prooxidant: BDC > Cur-S = MHME > HH > MH + HME > DH (254-1414 mean fluorescence intensity). Thus, dehydroxy hispolon was least potent for all three activities. Overall the results indicate that the substitution of a hydroxyl group for a methoxy group at the meta positions of the phenyl rings in curcumin significantly enhanced the anti-inflammatory activity, and the removal of phenyl ring at the 7^th position of the heptadiene back bone and addition of hydroxyl group significantly increased the anti-proliferative activity of curcumin.     

Biological activities of curcumin and its analogues (Congeners) made by man and Mother Nature

Curcumin, a yellow pigment present in the Indian spice turmeric (associated with curry powder), has been linked with suppression of inflammation; angiogenesis; tumorigenesis; diabetes; diseases of the cardiovascular, pulmonary, and neurological systems, of skin, and of liver; loss of bone and muscle; depression; chronic fatigue; and neuropathic pain. The utility of curcumin is limited by its color, lack of water solubility, and relatively low in vivo bioavailability. Because of the multiple therapeutic activities attributed to curcumin, however, there is an intense search for a “super curcumin” without these problems. Multiple approaches are being sought to overcome these limitations. These include discovery of natural curcumin analogues from turmeric; discovery of natural curcumin analogues made by Mother Nature; synthesis of “man-made” curcumin analogues; reformulation of curcumin with various oils and with inhibitors of metabolism (e.g., piperine); development of liposomal and nanoparticle formulations of curcumin; conjugation of curcumin prodrugs; and linking curcumin with polyethylene glycol. Curcumin is a homodimer of feruloylmethane containing a methoxy group and a hydroxyl group, a heptadiene with two Michael acceptors, and an α,β-diketone. Structural homologues involving modification of all these groups are being considered. This review focuses on the status of all these approaches in generating a “super curcumin.”.     

Inhibition of the promotion of hepatocarcinogenesis by 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153) by the deletion of the p50 subunit of NF-kappa B in mice

Polychlorinated biphenyls (PCBs) are persistent and ubiquitous environmental chemicals that bioaccumulate and have hepatic tumor promoting activity in rodents. The present study examined the effect of deleting the p50 subunit of NF-κB on the hepatic tumor promoting activity of 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB-153) in mice. Both wild-type and p50−/− male mice were injected i.p. with diethylnitrosamine (DEN, 90 mg/kg) and then subsequently injected biweekly with 20 i.p. injections of PCB-153 (300 μmol/kg/injection). p50 deletion decreased the tumor incidence in both PCB- and vehicle-treated mice, whereas PCB-153 slightly (P = 0.09) increased the tumor incidence in wild-type and p50−/− mice. PCB-153 increased the total tumor volume in both wild-type and p50−/− mice, but the total tumor volume was not affected by p50 deletion in either PCB- or vehicle-treated mice. The volume of tumors that were positive for glutamine synthetase (GS), which is indicative of mutations in the beta-catenin gene, was increased in both wild-type and p50−/− mice administered PCB-153 compared to vehicle controls, and inhibited in p50−/− mice compared to wild-type mice (in both PCB- and vehicle-treated mice). The volume of tumors that were negative for GS was increased in p50−/− mice compared to wild-type mice but was not affected by PCB-153. PCB-153 increased cell proliferation in normal hepatocytes in wild-type but not p50−/− mice; this increase was inhibited in p50−/− mice. In hepatic tumors, the rate of cell proliferation was much higher than in normal hepatocytes, but was not affected by PCB treatment or p50 deletion. The rate of apoptosis, as measured by the TUNEL assay, was not affected by PCB-153 or p50 deletion in normal hepatocytes. In hepatic tumors, the rate of apoptosis was lower than in normal hepatocytes; PCB-153 slightly (P = 0.10) increased apoptosis in p50−/− but not wild-type mice; p50 deletion had no effect. Taken together, these data indicate that the absence of the NF-κB p50 subunit inhibits the promoting activity of PCB-153 and alters the proliferative and apoptotic changes in mouse liver in the response to PCBs.     

Iron tetrakis (N-methyl-4'-pyridyl) porphyrinato inhibits proliferative activity of thymocytes by blocking activation of p38 mitogen-activated protein kinase,nuclear factor-kappaB,and interleukin-2 secretion

Iron tetrakis (N-methyl-4'-pyridyl) porphyrinato (FeTMPyP) has been demonstrated to be a potent scavenger of reactive oxygens and to have antiinflammatory activities. However, the effects of FeTMPyP on the function of T cells have not been illustrated. The objective of this study was to determine whether treatment of thymocytes with FeTMPyP inhibited the proliferative activity in response to various mitogens and, if so, to further characterize the mechanism of FeTMPyP immune modulation. The results indicate that treatment of thymocytes with FeTMPyP resulted in dose-dependent inhibition of thymocyte proliferation stimulated by concanawalin (Con) A-, Interleukin (IL)-1beta; or lipopdy socchande-exposed macrophage supernatant. FeTMPyP treatment also inhibited Con A- or IL-1beta-induced DNA-binding activity of NF-kappaB and IL-2 secretion by thymocytes. Both the p38 MAP kinase inhibitor SB203580 and the extracellular signal-regulated protein kinases inhibitor PD98059 blocked proliferative activity in Con A-stimulated thymocytes, while SB203580 but not PD98059 blocked nuclear factor (NF)-kappaB activation. FeTMPyP inhibited the activation and phosphorylation of p38 mitogen-activated protein kinase (MAPK) in response to Con A. These data suggest that FeTMPyP downregulates the proliferative activity by inhibition of p38 MAPK activation, NF-kappaB activation, and IL-2 secretion during mitogenic stimulation of thymocytes. Therefore, further studies concerning the effects of FeTMPyP on the human diseases associated with both inflammatory disorders and immunologic overactivation are warranted.     

Suppression of NF-kappaB activation by curcumin leads to inhibition of expression of cyclo-oxygenase-2 and matrix metalloproteinase-9 in human articular chondrocytes: Implications for the treatment of osteoarthritis

Pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) play a key role in the pathogenesis of osteoarthritis (OA). Anti-inflammatory agents capable of suppressing the production and catabolic actions of these cytokines may have therapeutic potential in the treatment of OA and a range of other osteoarticular disorders. The purpose of this study was to examine the effects of curcumin (diferuloylmethane), a pharmacologically safe phytochemical agent with potent anti-inflammatory properties on IL-1beta and TNF-alpha signalling pathways in human articular chondrocytes maintained in vitro. The effects of curcumin were studied in cultures of human articular chondrocytes treated with IL-1beta and TNF-alpha for up to 72h. Expression of collagen type II, integrin beta1, cyclo-oxygenase-2 (COX-2) and matrix metalloproteinase-9 (MMP-9) was monitored by western blotting. The effects of curcumin on the expression, phosphorylation and nuclear translocation of protein components of the NF-kappaB system were studied by western blotting and immunofluorescence, respectively. Treatment of chondrocytes with curcumin suppressed IL-1beta-induced NF-kappaB activation via inhibition of IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation and p65 nuclear translocation. Curcumin inhibited the IL-1beta-induced stimulation of up-stream protein kinase B Akt. These events correlated with down-regulation of NF-kappaB targets including COX-2 and MMP-9. Similar results were obtained in chondrocytes stimulated with TNF-alpha. Curcumin also reversed the IL-1beta-induced down-regulation of collagen type II and beta1-integrin receptor expression. These results indicate that curcumin has nutritional potential as a naturally occurring anti-inflammatory agent for treating OA through suppression of NF-kappaB mediated IL-1beta/TNF-alpha catabolic signalling pathways in chondrocytes.     

Curcumin (diferuloylmethane) inhibits constitutive NF-kappaB activation, induces G1/S arrest, suppresses proliferation, and induces apoptosis in mantle cell lymphoma

Human mantle cell lymphoma (MCL), an aggressive B cell non-Hodgkin's lymphoma, is characterized by the overexpression of cyclin D1 which plays an essential role in the survival and proliferation of MCL. Because of MCL's resistance to current chemotherapy, novel approaches are needed. Since MCL cells are known to overexpress NF-kappaB regulated gene products (including cyclin D1), we used curcumin, a pharmacologically safe agent, to target NF-kappaB in a variety of MCL cell lines. All four MCL cell lines examined had overexpression of cyclin D1, constitutive active NF-kappaB and IkappaB kinase and phosphorylated forms of IkappaBalpha and p65. This correlated with expression of TNF, IkappaBalpha, Bcl-2, Bcl-xl, COX-2 and IL-6, all regulated by NF-kappaB. On treatment of cells with curcumin, however, downregulated constitutive active NF-kappaB and inhibited the consitutively active IkappaBalpha kinase (IKK), and phosphorylation of IkappaBalpha and p65. Curcumin also inhibited constitutive activation of Akt, needed for IKK activation. Consequently, the expression of all NF-kappaB-regulated gene products, were downregulated by the polyphenol leading to the suppression of proliferation, cell cycle arrest at the G1/S phase of the cell cycle and induction of apoptosis as indicated by caspase activation, PARP cleavage, and annexin V staining. That NF-kappaB activation is directly linked to the proliferation of cells, is also indicated by the observation that peptide derived from the IKK/NEMO-binding domain and p65 suppressed the constitutive active NF-kappaB complex and inhibited the proliferation of MCL cells. Constitutive NF-kappaB activation was found to be due to TNF, as anti-TNF antibodies inhibited both NF-kappaB activation and proliferation of cells. Overall, our results indicate that curcumin inhibits the constitutive NF-kappaB and IKK leading to suppression of expression of NF-kappaB-regulated gene products that results in the suppression of proliferation, cell cycle arrest, and induction of apoptosis in MCL.     

Anti-inflammatory effect of essential oil and its constituents from fingered citron (Citrus medica L. var. sarcodactylis) through blocking JNK,ERK and NF-κB signaling pathways in LPS-activated RAW 264.7 cells

We investigated the composition of essential oil from fingered citron (Citrus medica L. var. sarcodactylis) (FCEO) peels by GC–MS and its anti-inflammatory effects on lipopolysaccharide (LPS) – stimulated mouse macrophage (RAW 264.7) cells. Fifteen compounds, representing 98.97% of the essential oil, were tentatively identified; the main constituents were limonene (52.44%) and γ-terpinene (28.41%). FCEO significantly inhibited nitric oxide (NO) and prostaglandin E2 (PGE2) by suppressing the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, respectively. Additionally, FCEO suppressed the production of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6. FCEO attenuated LPS-induced nuclear factor-κB (NF-κB) activation via inhibition of inhibitor κB-α phosphorylation. Furthermore, FCEO blocked activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) but not that of p38 mitogen-activated protein kinase. These results indicate that FCEO inhibits LPS-stimulated inflammation by blocking the NF-κB, JNK, and ERK pathways in macrophages, and demonstrate that FCEO possesses anti-inflammatory properties.     

Antifibrotic effects of Fraxetin on carbon tetrachloride-induced liver fibrosis by targeting NF-κB/IκBα, MAPKs and Bcl-2/Bax pathways

BackgroundLiver fibrosis is a chronic lesion which ultimately results in cirrhosis and possible death. Although the high incidence and lethality, few therapies are effective for liver fibrosis. Fraxetin (7,8-dihydroxy-6-methoxy coumarin), a natural product extracted from cortex fraxini, has exhibited a significant hepatoprotective and anti-fibrotic properties. However, the underlying mechanism of the anti-hepatic fibrotic property remains unknown.Methods48 Male Sprague Dawley rats were divided into four groups at random which were named as normal group, model group, fraxetin 25 mg/kg and 50 mg/kg group. The experimental model of liver fibrosis was founded by carbon tetrachloride (CCl₄) rats which were simultaneously treated with fraxetin (25 mg/kg or 50 mg/kg). Normal groups received equal volumes of saline and peanut oil.ResultsResults showed that fraxetin ameliorated CCl₄ induced liver damage and fibrosis. Furthermore, histopathology examinations revealed that fraxetin improved the morphology and alleviated collagen deposition in fibrotic liver. Fraxetin inhibited inflammation and hepatocytes apoptosis by modulating the NF-κB/IκBα, MAPKs and Bcl-2/Bax signaling pathways.ConclusionOur findings indicate that fraxetin is effective in preventing liver fibrosis through inhibiting inflammation and hepatocytes apoptosis which is associated with regulating NF-κB/IκBα, MAPKs and Bcl-2/Bax signaling pathways in rats.     

Anti-inflammatory effects of higenamine (Hig) on LPS-activated mouse microglia (BV2) through NF-κB and Nrf2/HO-1 signaling pathways

Microglia are the most widely equipped protective cells in the brain and play a pivotal role in the development of neurological diseases. Inflammatory response and oxidative stress are critical risk factors in the activation of microglia which may cause various neurological diseases. Higenamine (Hig), a plant-based alkaloid and isolated from Aconite tuber, exhibits various properties and is mainly applied to treat heart failure. In addition, Hig expresses potential protective effects for neurodegenerative diseases. However, the effects and mechanisms of Hig on lipopolysaccharide (LPS) activated mouse microglia has not been fully explored. Therefore, we evaluated the anti-inflammatory effects of Hig on LPS-activated BV2 microglia and revealed the underlying mechanisms. Our data showed that Hig significantly inhibited the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), reactive oxygen species (ROS) as well as NO (mediated by iNOS) and PGE₂ (mediated by COX2) in LPS-activated BV2 cells. Then we found that Hig suppressed NF-κB signaling pathway by inhibiting nuclear translocation of NF-κB/p65 subunit as well as degradation and phosphorylation of IκBα in cytoplasm, and the effect of Hig was intimately related to NF-κB inhibitor BAY-11-7082. Furthermore, we found that the anti-inflammatory effect of Hig were accompanied by the promotion of heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor-2 (Nrf2) expression, which was partly reversed by protoporphyrin (SnPP) and Nrf2 siRNA, respectively. Taken together, our results demonstrated that Hig expressed significant anti -inflammatory and -oxidative effects by inhibiting NF-κB and activating Nrf2/HO-1 signaling pathways.     

Basal levels and patterns of anticancer drug-induced activation of nuclear factor-kappaB (NF-kappaB), and its attenuation by tamoxifen,dexamethasone, and curcumin in carcinoma cells

Nuclear factor-kappaB (NF-kappaB) has been implicated in the development of drug resistance in cancer cells. We systematically examined the baseline levels of NF-kappaB activity of representative carcinoma cell lines, and the change of NF-kappaB activity in response to a challenge with four major anticancer drugs (doxorubicin, 5-fluorouracil, cisplatin, and paclitaxel). We found that the basal level of NF-kappaB activity was heterogeneous and roughly correlated with drug resistance. When challenged with various drugs, all the cell lines examined responded with a transient activation of NF-kappaB which then declined to basal level despite variation in the concentration of the agent and the timing of the treatment. In contrast to tumor necrosis factor-alpha (TNF-alpha), which activates NF-kappaB in minutes, NF-kappaB activation induced by anticancer drugs usually occurred more than 1hr after stimulation. A gradual increase of total NF-kappaB and its nuclear translocation, and cytoplasmic translocation of nuclear IkappaBalpha and its degradation were involved in this process. In particular, when cells were pretreated with common biologic modulators such as tamoxifen, dexamethasone, and curcumin, the doxorubicin-induced NF-kappaB activation was attenuated significantly. This inhibition may play a role in sensitizing cancer cells to chemotherapeutic drugs. This study has demonstrated that activation of NF-kappaB is a general cellular response to anticancer drugs, and the mechanism of activation appears to be distinct from that induced by TNF-alpha. These observations may have implications for improving the efficacy of systemic chemotherapy for cancer patients.     

Suppression of MyD88- and TRIF-dependent signaling pathways of Toll-like receptor by (-)-epigallocatechin-3-gallate, a polyphenol component of green tea

Toll-like receptors (TLRs) play an important role in recognition of microbial components and induction of innate immunity. The microbial components trigger the activation of two downstream signaling pathways of TLRs; MyD88- and/or TRIF-dependent pathways leading to activation of NF-kappaB. (-)-Epigallocatechin-3-gallate (EGCG), a flavonoid found in green tea, is known to inhibit NF-kappaB activation induced by many pro-inflammatory stimuli. EGCG was shown to inhibit the activity of IKKbeta which is the key kinase in the canonical pathway for NF-kappaB activation in MyD88-dependent pathway of TLRs. However, it is not known whether EGCG inhibits TRIF-dependent pathway through which more than 70% of lipopolysaccharide (LPS)-induced genes are regulated. Therefore, we attempted to identify the molecular target of EGCG in TRIF-dependent pathways of TLR3 and TLR4. EGCG inhibited the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C], or the overexpression of TRIF. The inhibition of IRF3 activation by EGCG was mediated through the suppression of the kinase activity of TBK1. However, EGCG did not inhibit activation of IRF3 induced by overexpression of constitutively active IRF3. These results suggest that the molecular target of EGCG is TBK1 in TRIF-dependent signaling pathways of TLR3 and TLR4. Therefore, our results suggest that green tea flavonoids can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs and subsequent inflammatory target gene expression.     

Biochemical mechanism of modulation of human P-glycoprotein (ABCB1) by curcumin I, II, and III purified from Turmeric powder

P-glycoprotein (Pgp, ABCB1) is an ATP-dependent drug efflux pump linked to development of multidrug resistance (MDR) in cancer cells. Previously [Biochem Pharmacol 2002;64:573-82], we reported that a curcumin mixture could modulate both function and expression of Pgp. This study focuses on the effect of three major curcuminoids--curcumin I, II and III purified from a curcumin mixture--on modulation of Pgp function in a multidrug resistant human cervical carcinoma cell line (KB-V1). The similar IC(50) values for cytotoxicity of curcuminoids of KB-V1, and KB-3-1 (parental drug sensitive cell line) suggest that these curcuminoids may not be substrates for Pgp. Treating the cells with non-toxic doses of curcuminoids increased their sensitivity to vinblastine only in the Pgp expressing drug resistant cell line, KB-V1, and curcumin I retained the drug in KB-V1 cells more effectively than curcumin II and III, respectively. Effects of each curcuminoid on rhodamine123, calcein-AM, and bodipy-FL-vinblastine accumulation confirmed these findings. Curcumin I, II and III increased the accumulation of fluorescent substrates in a dose-dependent manner, and at 15 microM, curcumin I was the most effective. The inhibitory effect in a concentration-dependent manner of curcuminoids on verapamil-stimulated ATPase activity and photoaffinity labeling of Pgp with the [(125)I]-iodoarylazidoprazosin offered additional support; curcumin I was the most potent modulator. Taken together, these results indicate that curcumin I is the most effective MDR modulator among curcuminoids, and may be used in combination with conventional chemotherapeutic drugs to reverse MDR in cancer cells.     

Tumor necrosis factor-alpha induces long-term potentiation of C-fiber evoked field potentials in spinal dorsal horn in rats with nerve injury: the role of NF-kappa B, JNK and p38 MAPK

Compelling evidence has shown that in hippocampus tumor necrosis factor alpha (TNF-alpha) at pathological concentration inhibits long-term potentiation (LTP), a synaptic model of learning and memory. In the present work we investigated the role of TNF-alpha in LTP of C-fiber evoked field potentials in spinal dorsal horn, which is relevant to pathological pain. We showed that spinal application of TNF-alpha affected neither basal synaptic transmission mediated by C-fibers nor spinal LTP of C-fiber evoked field potentials induced by tetanic stimulation in intact rats. However, in rats with neuropathic pain, produced by either lumbar 5 ventral root transection (L5 VRT) or spared nerve injury (SNI), spinal application of TNF-alpha induced LTP of C-fiber evoked field potentials. Spinal application of JNK inhibitor (SP600125) or p38 MAPK inhibitor (SB203580) did not affect the spinal LTP induced by tetanic stimulation in intact rats, but completely blocked LTP induced by TNF-alpha in L5 VRT rats. NF-kappa B (NF-kappaB) inhibitor (PDTC) also blocked LTP induced by TNF-alpha. These results suggest that TNF-alpha and its downstream molecules may have no acute effect on spinal synaptic transmission in intact animals and induce LTP in rats with neuropathic pain produced by nerve injury.     

Inhibition of lipopolysaccharide-inducible nitric oxide synthase and IL-1beta through suppression of NF-kappaB activation by 3-(1'-1'-dimethyl-allyl)-6-hydroxy-7-methoxy-coumarin isolated from Ruta graveolens L

The Ruta graveolens L. plant is used in traditional medicine to treat a large number of diseases. The methanol (50%) extract of the whole plant was observed to inhibit the expression of inducible nitric oxide synthase (iNOS) and the cycloxygenase-2 (COX-2) gene in lipopolysaccharide (LPS)-induced macrophage cells (J774A.1, [Raghav, S.K., Gupta, B., Agrawal, C., Goswami, K., Das, H.R., 2006b. Anti-inflammatory effect of Ruta graveolens L. in murine macrophage cells. J. Ethnopharmacol. 104, 234-239]). The effect of whole plant extract on the expression of other pro-inflammatory genes such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-12, interferon-gamma (IFN-gamma) and the activation of nuclear factor-kB (NF-kappaB) were investigated in LPS stimulated macrophage cells. An active compound was isolated from this methanol extract by further solvent fractionation and reverse phase high performance liquid chromatography (RP-HPLC). The purified compound was identified as 3-(1'-1'-dimethyl-allyl)-6-hydroxy-7-methoxy-coumarin having IUPAC nomenclature of 6-hydroxy-7-methoxy-3-(2-methyl but-3-en-2yl)-2H-chromen-2-one by ESI-MS, MALDI, FT-IR and NMR. Effect of this purified compound was assessed on iNOS, COX-2 and various pro-inflammatory cytokine genes and was observed to inhibit both the protein and mRNA expression of iNOS and IL-1beta in LPS challenged macrophages. Electrophoretic mobility shift assay (EMSA) and Western blot analyses indicated that the plant extract and the isolated active compound blocked the LPS-induced activation of NF-kappaB through the prevention of inhibitor-kB (IkB) degradation. The purified compound also showed the anti-oxidant activity. The active compound at a dose of 40 mg/kg body weight was observed to inhibit the iNOS and IL-1beta gene expression significantly in endotoxin-induced inflammatory model of BALB/c mice. The low level of nitric oxide production was also observed in the sera of compound treated mice. The normal behavioral condition in LPS challenged BALB/c mice was noticed when these were treated with active compound.