curcumin and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Ketamine is a widely used anesthetic in pediatric clinical practice. Previous studies have demonstrated that ketamine induces neurotoxicity and has a modulatory effect on the cells of the immune system. Here, we evaluated the potential protective effect and underlying mechanisms of natural phenolic compound curcumin against ketamine-induced toxicity in rat thymocytes. Rat thymocytes were exposed to 100 µM ketamine alone or combined with increasing concentrations of curcumin (0.3, 1, and 3 μM) for 24 hours. Cell viability was analyzed with CCK-8 assay kit. Apoptosis was analyzed using flow cytometry and propidium iodide as well as Z-VAD-FMK and Z-LEHD-FMK inhibitors. Reactive oxygen species (ROS) production and mitochondrial membrane potential [MMP] were measured by flow cytometry. Colorimetric assay with DEVD-pNA substrate was used for assessing caspase-3 activity. Involvement of phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was tested with Wortmannin inhibitor. Ketamine induced toxicity in cells, increased the number of hypodiploid cells, caspase-3 activity and ROS production, and inhibited the MMP. Co-incubation of higher concentrations of curcumin (1 and 3 μM) with ketamine markedly decreased cytotoxicity, apoptosis rate, caspase-3 activity, and ROS production in rat thymocytes, and increased the MMP. Application of Z-VAD-FMK (a pan caspase inhibitor) or Z-LEHD-FMK (caspase-9 inhibitor) with ketamine effectively attenuated the ketamine-induced apoptosis in rat thymocytes. Administration of Wortmannin (a PI3K inhibitor) with curcumin and ketamine significantly decreased the protective effect of curcumin on rat thymocytes. Our results indicate that ketamine-induced toxicity in rat thymocytes mainly occurs through the mitochondria-mediated apoptotic pathway and that the PI3K/Akt signaling pathway is involved in the anti-apoptotic effect of curcumin.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase Inhibitors; Curcumin; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Ketamine; Membrane Potential, Mitochondrial; Oligopeptides; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinase; Rats; Rats, Wistar; Reactive Oxygen Species; Signal Transduction; Thymocytes

Curcumin, the biologically active compound from the rhizome of Curcuma longa, could inhibit cell growth and induce apoptosis in gastric carcinoma. However, the underlying mechanism of curcumin on gastric carcinoma cells still needs further investigation. In this study, morphological observation indicated that curcumin inhibited the proliferation of AGS cells in a dose-dependent manner. According to the flow cytometric analysis, curcumin treatment resulted in G2/M arrest in AGS cells, accompanied with an increased expression of cyclin B1 and a decreased expression of cyclin D1. In addition, DNA ladders were observed by gel electrophoresis. Meanwhile, the activities of caspase-3, -8, and -9 were also enhanced in curcumin-treated AGS cells. Nevertheless, the increased activities could be inhibited by benzyloxycarbonyl-Val-Ala-Asp (OME)-fluoromethylketone (z-VAD-fmk), which suggested that the apoptosis was caspase-dependent. Furthermore, downregulation of rat sarcoma (Ras) and upregulation of extracellular-signal-regulated kinase (ERK) were also observed in AGS cells treated with curcumin by Western blot. U0126, an ERK inhibitor, blocked curcumin-induced apoptosis. The results suggested that curcumin inhibited the growth of the AGS cells and induced apoptosis through the activation of Ras/ERK signaling pathway and downstream caspase cascade, and curcumin might be a potential target for the treatment of gastric carcinoma.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 3; Caspases; Cell Cycle; Cell Cycle Checkpoints; Curcuma; Curcumin; Extracellular Signal-Regulated MAP Kinases; Humans; Molecular Structure; Oligopeptides; Signal Transduction; Stomach Neoplasms

Depletion of intracellular glutathione (GSH) is the prime hallmark of the progression of apoptosis. Previously, we reported that curcumin induces reactive oxygen species (ROS)-mediated depletion of GSH, which leads to caspase-dependent and independent apoptosis in mouse fibroblast cells (F. Thayyullathil et al., Free Radic. Biol. Med.45, 1403-1412, 2008). In this study, we investigated the antileukemic potential of curcumin in vitro, and we further examined the molecular mechanisms of curcumin-induced apoptosis in human leukemic cells. Curcumin suppresses the growth of human leukemic cells via ROS-independent GSH depletion, which leads to caspase activation, inhibition of sphingomyelin synthase (SMS) activity, and induction of ceramide (Cer) generation. Pretreatment of leukemic cells with carbobenzoxy-Val-Ala-Asp fluoromethylketone, a universal inhibitor of caspases, abrogates the SMS inhibition and Cer generation, and in turn prevents curcumin-induced cell death. Curcumin treatment of leukemic cells also downregulates the expression of the inhibitor of apoptosis proteins (IAPs), phospho-Akt, c-Myc, and cyclin D1. Extracellular supplementation with GSH attenuates curcumin-induced depletion of GSH, caspase-dependent inhibition of SMS, Cer generation, and downregulation of IAPs, whereas, L-D-buthionine sulfoximine, a widely used inhibitor of GSH synthesis, potentiates GSH depletion, Cer generation, and apoptosis induced by curcumin. Taken together, our findings provide evidence suggesting for the first time that GSH regulates caspase-dependent inhibition of SMS activity, Cer generation, and apoptosis induced by curcumin in human leukemic cells.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Caspases; Cell Line; Ceramides; Curcumin; Glutathione; Humans; Leukemia

Previous studies have reported that the Curcuma wenyujin Y.H. Chen et C. Ling extract, which has a high furanodiene content, showed anti-cancer effects in breast cancer cells in vitro. The present study was designed to evaluate the in vitro and in vivo anti-cancer activity of furanodiene.. The in vitro effects of furanodiene were examined on two human breast cancer cell lines, MCF-7 and MDA-MB-231 cells. Assays of proliferation, LDH release, mitochondrial membrane potential (ΔΨm), cell cycle distribution, apoptosis and relevant signaling pathways were performed. The in vivo effect was determined with MCF7 tumor xenograft model in nude mice.. Furanodiene significantly inhibited the proliferation and increased the LDH release in both cell lines in a dose-dependent manner. ΔΨm depolarization, chromatin condensation, and DNA fragmentation were also observed after furanodiene treatment. Furanodiene dose-dependently induced cell cycle arrest at the G0/G1 phase. The protein expressions of p-cyclin D1, total cyclin D1, p-CDK2, total CDK2, p-Rb, total Rb, Bcl-xL, and Akt were significantly inhibited by furanodiene, whereas the protein expressions of Bad and Bax, and the proteolytic cleavage of caspase-9, caspase-7, and poly-ADP-ribose polymerase (PARP) were dramatically increased. Furthermore, the z-VAD-fmk markedly reversed the furanodiene-induced cell cytotoxicity, the proteolytic cleavage of caspase-9, and DNA fragmentation but did not affect the proteolytic cleavage of PARP, whereas the Akt inhibitor VIII increased the furanodiene-induced cytotoxicity and PARP cleavage. In addition, furanodiene dose-dependently suppressed the tumor growth in vivo, achieving 32% and 54% inhibition rates after intraperitoneal injection of 15 mg/kg and 30 mg/kg, respectively.. Taken together, we concluded that furanodiene suppresses breast cancer cell growth both in vitro and in vivo and could be a new lead compound for breast cancer chemotherapy.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; Breast Neoplasms; Caspase 7; Caspase 9; Cell Line, Tumor; Cell Proliferation; Curcuma; DNA Fragmentation; Female; Furans; G1 Phase Cell Cycle Checkpoints; Heterocyclic Compounds, 2-Ring; Humans; L-Lactate Dehydrogenase; MCF-7 Cells; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-akt; Transplantation, Heterologous

We recently demonstrated that molecular therapy using aminoglycosides can overcome the underlying genetic defect in two zebrafish models of ocular coloboma and showed abnormal cell death to be a key feature associated with the optic fissure closure defects. In further studies to identify molecular therapies for this common congenital malformation, we now examine the effects of anti-apoptotic compounds in zebrafish models of ocular coloboma in vivo.. Two ocular coloboma zebrafish lines (pax2.1/noi(tu29a) and lamb1/gup(m189)) were exposed to diferuloylmethane (curcumin) or benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD-fmk; a pan-caspase inhibitor) for up to 8 days post-fertilization. The effects of these compounds were assessed by morphology, histology, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and western blot analysis.. The size of the coloboma in gup zebrafish mutants treated with diferuloylmethane was greatly reduced. In treated mutants a reduction in TUNEL staining and a 67% decrease in activated caspase-3 protein were observed. The release of cytochrome c from the mitochondria into the cytosol was reduced fourfold by in vivo diferuloylmethane treatment, suggesting that the drug was acting to inhibit the intrinsic apoptotic pathway. Inhibition of caspases directly with zVAD-fmk also resulted in a similar reduction in coloboma phenotype. Treatment with either diferuloylmethane or zVAD-fmk resulted in a statistically significant 1.4 fold increase in length of survival of these mutant zebrafish (p<0.001), which normally succumb to the lethal genetic mutation. In contrast, the coloboma phenotype in noi zebrafish mutants did not respond to either diferuloylmethane or zVAD-fmk exposure, even though inhibition of apoptotic cell death was observed by a reduction in TUNEL staining.. The differential sensitivity to anti-apoptotic agents in lamb1-deficient and pax2.1-deficient zebrafish models, suggests that apoptotic cell death is not a final common pathway in all ocular coloboma genotypes. When considering anti-cell death therapies for ocular colobomatous defects attention should be paid to the genotype under investigation.

Topics: Amino Acid Chloromethyl Ketones; Animals; Blotting, Western; Caspase 3; Cell Death; Coloboma; Curcumin; Cytochromes c; Disease Models, Animal; Dose-Response Relationship, Drug; Embryo, Nonmammalian; Eye; Genetic Variation; In Situ Nick-End Labeling; Longevity; Mitochondria; Mutation; Phenotype; Zebrafish; Zebrafish Proteins

Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase-3 activation, which is required for apoptosis as confirmed using the pan caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role of caspase-8 in curcumin-induced caspase-3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase-3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of proapoptotic proteins such as Bax, Bak, Bid, and Bim. Crosslinking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid, and Bim causes Bax-channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation, and apoptosis. Importantly, p21-deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement of p21 in Apaf-1 dependent caspase activation and apoptosis. Together, our findings demonstrate that Apaf-1, Bax, and p21 as novel potential targets for curcumin or curcumin-based anticancer agents.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptotic Protease-Activating Factor 1; bcl-2-Associated X Protein; Caspase 3; Caspase Inhibitors; Curcumin; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; HCT116 Cells; Humans; Jurkat Cells; Lentivirus; Mitochondria; Mitochondrial Membranes; RNA, Messenger; RNA, Small Interfering

  δ-Elemene, an antitumor component, is a chemical compound isolated from Curcuma wenyujin, a Chinese traditional herb. We examined whether δ-elemene could inhibit cell growth and cell cycle progression and induce apoptosis in human leukemia HL-60 cells. The results demonstrated that δ-elemene induces significant apoptosis of HL-60 cells, as shown by MTT assay, annexin V (AnV) binding of externalized phosphatidylserine (PS), and the mitochondrial probe JC-1 using flow cytometry. HL-60 cells treated with δ-elemene showed high percentages in the early apoptotic and late apoptoctic/necrotic stages, as well as caspase-3 activation of HL-60 cells. By monitoring the changes in cell cycle profiles, we confirmed that δ-elemene could interfere with the cell cycle in the G2/M phase and induce apoptosis in HL-60 cells in a time-dependent manner. Caspase-3 plays a direct role in proteolytic cleavage of the cellular proteins responsible for progression to apoptosis. Therefore we examined apoptosis in HL-60 cells after exposure to δ-elemene and measured caspase-3 activities with or without Z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk, a broad-spectrum caspase inhibitor) pretreatment using flow cytometric analysis. The results showed that δ-elemene could induce caspase-3 activation as detected by the decrease in δ-elemene-induced caspase-3 activities after treatment with z-VAD-fmk. In the present study, δ-elemene activated typical caspase-dependent apoptosis in HL-60 cells, as demonstrated by an inhibitory effect of z-VAD-fmk on this cell death. During δ-elemene-induced apoptosis, cytochrome c and apoptosis-inducing factor were released into the cytosol and BAX was translocated from the cytosol to mitochondria. However, these were not prevented by z-VAD-fmk. In conclusion, our study demonstrated that δ-elemene could induce G2/M cell cycle transition and trigger apoptosis through a caspase-3-dependent pathway.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Cell Division; Cell Line, Tumor; Curcuma; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; G2 Phase; HL-60 Cells; Humans; Membrane Potential, Mitochondrial; Mitochondria; Sesquiterpenes; Time Factors

Several studies have shown that curcumin can induce apoptosis and inhibit growth in human tumor cell lines. However, the mechanism is not completely understood yet. The present studies were designed to investigate the effects of curcumin on human A549 lung adenocarcinoma cells lines to better understand its effect on apoptosis and apoptosis-related genes in vitro. Apoptosis induction, mitochondria membrane potential, mitochondria structure, and apoptotic associated gene expression were examined by flow cytometric assay, confocal microscopy, Western blotting and electron microscopy. After treatment with curcumin, percentage of apoptotic cells increased dose- and time-dependently, and morphology observation revealed typical apoptotic features. Our data further indicated that the expression of Bax proteins in A549 cells was increased in a dose-dependent manner, whereas the expression of Bcl-2 was significantly decreased, thus the ratio of Bax/Bcl-2 was increased. The apoptotic process was accompanied by the change of mitochondrial function and structure which led to release of the cytochrome c, and activation of caspase-9 and caspase-3. Furthermore, curcumin also induced a dose-dependent cleavage of PARP. Caspases activation during the course of curcumin-induced apoptosis was additionally confirmed by using a broad-spectrum caspases inhibitor, Z-VAD-fmk. As expected, the inhibitor was able to decrease curcumin-induced apoptosis on A549 cells. These results suggested that mitochondria played an important role in the curcumin-induced apoptosis, and mitochondria membrane potential loss initiated apoptosis via the activation of caspases.

Topics: Adenocarcinoma; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspase 9; Cell Line, Tumor; Cell Shape; Curcumin; Cysteine Proteinase Inhibitors; Cytochromes c; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Microscopy, Confocal; Microscopy, Electron, Transmission; Mitochondria; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Time Factors

Intracerebral hemorrhage (ICH) induces neurovascular injury via poorly defined mechanisms. The aim of this study was to determine whether gliovascular communication may restrict hemorrhagic vascular injury. Hemin, a hemoglobin by-product, concentration- and time-dependently increased apoptotic cell death in mouse bEnd.3 cells and in primary human brain microvascular endothelial cells, at least in part, via a caspase-3 dependent pathway. Cell death was preceded by a NFκB-mediated increase in inflammatory gene expression, including upregulation of inducible nitric oxide synthase (iNOS) expression and activity. Functionally, inhibition of iNOS or the addition of a peroxynitrite decomposition catalyst reduced cell death. Interestingly, co-treatment with astrocyte-conditioned media (ACM) reversed hemin-induced NFκB activation, nitrotyrosine formation, and apoptotic cell death, at least in part, via the release of the endogenous antioxidant, reduced glutathione (GSH). Prior treatment of astrocytes with the GSH-depleting agent, DL-buthionine (S,R)-sulfoximine or direct addition of diethyl maleate, a thiol-depleting agent, to ACM reversed the observed protection. In contrast, neither exogenous GSH nor the GSH precursor, N-acetylcysteine, was protective in bEnd.3 cells. Together, these data support an important role for astrocyte-derived GSH in the maintenance of oxidative balance in the vasculature and suggest therapeutic targeting of the GSH system may reduce neurological injury following ICH.

Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Animals; Antioxidants; Apoptosis; Ascorbic Acid; Astrocytes; Brain; Caspase 3; Cells, Cultured; Curcumin; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glutathione; Hemin; Humans; L-Lactate Dehydrogenase; Mice; Microvessels; Neuroprotective Agents; Nitric Oxide Synthase Type II; Peroxynitrous Acid; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Time Factors

Curcumin (1), a natural polyphenolic compound, has shown strong antioxidant and anticancer activities. Several molecular mechanisms have been attributed to its inhibitory effects on a wide range of tumor cells. In this study, the response of the chronic myeloid leukemia cell line K562 cells to 1 is investigated. Curcumin inhibited the viability of K562 cells in a dose- and time-dependent manner. Furthermore, curcumin-induced cell death was associated with the formation of the apoptosome complex, the collapse of the mitochondrial membrane potential, and caspase-3 activation. Curcumin treatment also induced Bid cleavage and downregulated the expression of Bcl-2 protein. Surprisingly, even with these molecular features of apoptosis, we showed that 1 stimulated autophagy, which was evidenced by microtubule-associated protein light chain 3 (LC3) immunoreactivty. Curcumin also increased the protein levels of beclin 1 and membrane form LC3 (LC3-II). Autophagy inhibitor bafilomycin A1 and the pan-caspase inhibitor Z-VAD-fmk suppressed curcumin-induced K562 cell death. Overall, these results suggest that curcumin induces autophagic and apoptotic death of K562 cells. These findings suggest that both apoptotic and autophagic mechanisms contribute to the curcumin-induced K562 cell death.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; Autophagy; Curcumin; Humans; K562 Cells; Microscopy, Fluorescence; Molecular Structure

Evidence that curcumin may have anticancer activities has renewed interest in its potential to prevent and treat disease. In this study, we show that curcumin-mediated rapid generation of reactive oxygen species (ROS) leads to apoptosis by modulating different apoptotic pathways in mouse fibroblast L929 cells. We show for the first time that curcumin-induced rapid ROS generation causes the release of apoptosis inducing factor (AIF) from the mitochondria to the cytosol and nucleus, hence, leading to caspase 3-independent apoptosis. However, our studies also show that curcumin induces the release of cytochrome c from mitochondria, causing activation of caspase 3, and concomitant PARP cleavage, which is the hallmark of caspase-dependent apoptosis. Furthermore, curcumin-induced ROS generation leads to the induction of the proapoptotic protein p53 and its effector protein p21 and down-regulation of cell cycle regulatory proteins such as Rb and cyclin D1 and D3. Both glutathione (GSH) and N-acetylcysteine (NAC) pretreatment resulted in the complete inhibition of curcumin-induced ROS generation, AIF release from mitochondria, and caspase activation. Additionally, pretreatment of L929 cells with these antioxidants completely blocked the induction of p53-dependent p21 accumulation. In conclusion, our data show that in addition to caspase 3 activation, curcumin-induced rapid ROS generation leads to AIF release, and the activation of the caspase-independent apoptotic pathway.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antioxidants; Apoptosis; Caspase 3; Caspase Inhibitors; Cell Cycle Proteins; Cell Line; Cell Survival; Curcumin; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Dose-Response Relationship, Drug; Mice; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; Time Factors; Tumor Suppressor Protein p53

Apoptosis induced by toxic bile acids is thought to contribute to liver injury during cholestasis. The transcription factor AP-1 is involved in the induction of apoptosis depending on stimulus and cell type. It is not known whether the major human toxic bile acid, glycochenodeoxycholic acid (GCDCA), modulates AP-1 in hepatocytes. Our data show that GCDCA (75 microM, 4 h) significantly upregulates cFos and JunB as demonstrated by microarray analysis and real-time PCR in HepG2-Ntcp hepatoma cells. GCDCA (75 microM, 4 h) also induced AP-1 activation as determined by EMSA that was most distinct after 30 min. In parallel, AP-1 transcriptional activity increased by 40% after exposure to GCDCA. Curcumin, an AP-1 inhibitor, dose-dependently reduced (1-25 microM) or completely abolished (50 microM) the apoptotic effect of GCDCA. Thus, GCDCA-induced upregulation of AP-1-dependent genes appears important for the cytotoxicity of this bile acid.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Bile Acids and Salts; Cell Line; Cell Line, Tumor; Cholestasis; Curcumin; Detergents; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genes, Reporter; Glycochenodeoxycholic Acid; Hepatocytes; Humans; Immunoblotting; Liver; Luciferases; MAP Kinase Signaling System; Oligonucleotide Array Sequence Analysis; p38 Mitogen-Activated Protein Kinases; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Taurocholic Acid; Time Factors; Transcription Factor AP-1; Transcription, Genetic; Up-Regulation

The geldanamycin-induced degradation of ErbB-2 produces a 23-kDa carboxyl-terminal fragment, which has been isolated and subjected to amino-terminal microsequencing. The obtained sequence indicates that the amino terminus of this fragment corresponds to Gly-1126 of ErbB-2. Analysis of the residues immediately before Gly-1126 suggests that cleavage may involve caspase activity. Site-directed mutagenesis of Asp-1125 in ErbB-2 prevents geldanamycin-provoked formation of the 23-kDa fragment, consistent with the requirement of this residue for caspase-dependent cleavage in known substrates. Also, the addition of the pan-caspase inhibitor Z-VAD-FMK blocks formation of the 23-kDa ErbB-2 fragment in cells exposed to geldanamycin. Interestingly, staurosporin and curcumin are also shown to provoke the degradation of ErbB-2 with formation of the 23-kDa carboxyl-terminal fragment. The generation of this fragment by staurosporin or curcumin is likewise blocked by caspase inhibition. Caspase inhibition does not prevent accelerated degradation of the 185-kDa native ErbB-2 in geldanamycin-treated cells but does significantly prevent staurosporin-stimulated metabolic loss of ErbB-2.

Topics: Amino Acid Chloromethyl Ketones; Animals; Benzoquinones; Caspase Inhibitors; Caspases; COS Cells; Curcumin; Cytoplasm; Enzyme Inhibitors; Glycine; Humans; Immunoblotting; Lactams, Macrocyclic; Mutagenesis, Site-Directed; Mutation; Protein Binding; Protein Structure, Tertiary; Quinones; Receptor, ErbB-2; Sequence Analysis, Protein; Staurosporine; Transfection; Tumor Cells, Cultured

Curcumin, in addition to its role as a spice, has been used for centuries to treat inflammatory disorders. Although the mechanism of action remains unclear, it has been shown to inhibit the activation of NF-kappaB and AP-1, transcription factors required for induction of many proinflammatory mediators. Due to its low toxicity it is currently under consideration as a broad anti-inflammatory, anti-tumor cell agent. In this study we investigated whether curcumin inhibited the response of gammadelta T cells to protease-resistant phosphorylated derivatives found in the cell wall of many pathogens. The results showed that curcumin levels > or =30 microM profoundly inhibited isopentenyl pyrophosphate-induced release of the chemokines macrophage inflammatory protein-1alpha and -1beta and RANTES. Curcumin also blocked isopentenyl pyrophosphate-induced activation of NF-kappaB and AP-1. Commencing around 16 h, treatment with curcumin lead to the induction of cell death that could not be reversed by APC, IL-15, or IL-2. This cytotoxicity was associated with increased annexin V reactivity, nuclear expression of active caspase-3, cleavage of poly(ADP-ribose) polymerase, translocation of apoptosis-inducing factor to the nucleus, and morphological evidence of nuclear disintegration. However, curcumin led to only large scale DNA chromatolysis, as determined by a combination of TUNEL staining and pulse-field and agarose gel electrophoresis, suggesting a predominantly apoptosis-inducing factor-mediated cell death process. We conclude that gammadelta T cells activated by these ubiquitous Ags are highly sensitive to curcumin, and that this effect may contribute to the anti-inflammatory properties of this compound.

Topics: Adult; Amino Acid Chloromethyl Ketones; Annexin A5; Anti-Inflammatory Agents, Non-Steroidal; Antigens, Bacterial; Antineoplastic Agents; Apoptosis; Caspase 3; Caspases; Chemokine CCL4; Chemokine CCL5; Curcumin; Cycloheximide; Cysteine Proteinase Inhibitors; DNA Fragmentation; Electrophoresis, Agar Gel; Electrophoresis, Gel, Pulsed-Field; Enzyme Activation; Flow Cytometry; Hemiterpenes; Humans; In Situ Nick-End Labeling; Interleukin-15; Interleukin-2; Lymphocyte Activation; Macrophage Inflammatory Proteins; Molecular Weight; NF-kappa B; Organophosphorus Compounds; Phosphorylation; Protein Synthesis Inhibitors; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocyte Subsets; Transcription Factor AP-1; Tumor Necrosis Factor-alpha