curcumin and 3-5-bis(2-fluorobenzylidene)piperidin-4-one

Curcumin has numerous properties and is used in many preclinical conditions, including cancer. It has low bioavailability, while its derivative EF24 shows enhanced solubility. However, its effects have never been explored in adrenocortical tumor cell models. The efficacy of EF24 alone or combined with mitotane (reference drug for adrenocortical cancer) was evaluated in two adrenocortical tumor cell lines, SW13 and H295R.. EF24 reduced cell viability with an IC50 (half maximal inhibitory concentration) of 6.5 ± 2.4 μM and 4.9 ± 2.8 μM for SW13 and H295R cells, respectively. Combination index (EF24 associated with mitotane) suggested an additivity effect in both cell lines. Cell cycle analysis revealed an increase in subG0/G1 phase, while motility assay showed a decrease in migratory cell capacity, and similarly, clonogenic assay indicated that EF24 could reduce colony numbers. Furthermore, Wnt/β-catenin, NF-κB, MAPK, and PI3k/Akt pathways were modulated by Western blot analysis when treating cells with EF24 alone or combined with mitotane. In addition, intracellular reactive oxygen species levels increased in both cell lines.. This work analyzed EF24 in adrenocortical tumor cell lines for the first time. These results suggest that EF24 could potentially impact on adrenocortical tumors, laying the foundation for further research in animal models.

Topics: Adrenal Cortex Neoplasms; Animals; Antineoplastic Agents; Benzylidene Compounds; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Curcumin; Drug Evaluation, Preclinical; Drug Synergism; Humans; Mice; Mitotane; Molecular Structure; Piperidones; Reactive Oxygen Species

The nuclear factor-kappa B (NF-κB) signaling pathway has been targeted for the therapy of various cancers, including lung cancer. EF24 was considered as a potent inhibitor of NF-κB signaling pathway. In this study, a series of asymmetric EF24 analogues were synthesized and evaluated for their anti-cancer activity against three lung cancer cell lines (A549, LLC, H1650). Most of the compounds exhibited good anti-tumor activity. Among them, compound 81 showed greater cytotoxicity than EF24. Compound 81 also possessed a potent anti-migration and anti-proliferative ability against A549 cells in a concentration-dependent manner. Moreover, compound 81 induced lung cancer cells death by inhibiting NF-κB signaling pathway, and activated the JNK-mitochondrial apoptotic pathway by increasing reactive oxygen species (ROS) generation resulting in apoptosis. In summary, compound 81 is a valuable candidate for anti-lung cancer therapy.

Topics: Antineoplastic Agents; Apoptosis; Benzylidene Compounds; Cell Line, Tumor; Drug Design; Humans; Lung; Lung Neoplasms; Mitochondria; NF-kappa B; Piperidones; Reactive Oxygen Species; Signal Transduction

Colorectal cancer is the most commonly diagnosed malignancy with high mortality rates worldwide. Improved therapeutic strategies with minimal adverse side effects are urgently needed. In this study, the anti-tumor effects of EF24, a novel analog of the natural compound curcumin, were evaluated in colorectal cancer cells.. The anti-tumor activity of EF24 on human colon cancer lines (HCT-116, SW-620, and HT-29) was determined by measures of cell cycle arrest, apoptosis, and mitochondrial function. The contribution of ROS in the EF24-induced anti-tumor activity was evaluated by measures of H. The findings indicated that EF24 treatment dose-dependently inhibited cell viability and caused cell cycle arrest at G2/M phase in all the tested colon cancer cell lines. Furthermore, we demonstrated that EF24 treatment induced apoptosis effectively via enhancing intracellular accumulation of ROS in both HCT-116 and SW-620 cells, but with moderate effects in HT-29 cells. We found that EF24 treatment decreased the mitochondrial membrane potential in the colon cancer cells, leading to the release of mitochondrial cytochrome c. Also, EF24 induced activation of caspases 9 and 3, causing decreased Bcl-2 protein expression and Bcl-2/Bax ratio. Pretreatment with NAC, a ROS scavenger, abrogated the EF24-induced cell death, apoptosis, cell cycle arrest, and mitochondrial dysfunction, suggesting an upstream ROS generation which was responsible for the anticancer effects of EF24.. Our findings support an anticancer mechanism by which EF24 enhanced ROS accumulation in colon cancer cells, thereby resulting in mitochondrial membrane collapse and activated intrinsic apoptotic signaling. Thus, EF24 could be a potential candidate for therapeutic application of colon cancer.

Topics: Antineoplastic Agents; Apoptosis; Benzylidene Compounds; Caspases; Cell Line, Tumor; Cell Survival; Colorectal Neoplasms; Curcumin; Cytochromes c; Humans; Membrane Potential, Mitochondrial; Mitochondria; Piperidones; Reactive Oxygen Species

Diphenyl difluoroketone (EF24), a curcumin analog, exhibits potent anti-tumor activities by arresting cell cycle and inducing apoptosis. However, the efficacy and modes of action of EF24 on melanoma metastasis remain elusive. In this study, we found that at non-cytotoxic concentrations, EF24 suppressed cell motility and epithelial-to-mesenchymal Transition (EMT) of melanoma cell lines, Lu1205 and A375. EF24 also suppressed HMGA2 expression at mRNA and protein levels. miR-33b directly bound to HMGA2 3' untranslated region (3'-UTR) to suppress its expression as measured by dual-luciferase assay. EF24 increased expression of E-cadherin and decreased STAT3 phosphorylation and expression of the mesenchymal markers, vimentin and N-cadherin. miR-33b inhibition or HMGA2 overexpression reverted EF24-mediated suppression of EMT phenotypes. In addition, EF24 modulated the HMGA2-dependent actin stress fiber formation, focal adhesion assembly and FAK, Src and RhoA activation by targeting miR-33b. Thus, the results suggest that EF24 suppresses melanoma metastasis via upregulating miR-33b and concomitantly reducing HMGA2 expression. The observed activities of EF24 support its further evaluation as an anti-metastatic agent in melanoma therapy.

Topics: Antineoplastic Agents; Benzylidene Compounds; Blotting, Western; Cell Line, Tumor; Cell Movement; Curcumin; Epithelial-Mesenchymal Transition; Fluorescent Antibody Technique; HMGA2 Protein; Humans; Melanoma; MicroRNAs; Piperidones; Reverse Transcriptase Polymerase Chain Reaction; Up-Regulation

The aim of the present study was to investigate and compare the effects of diferuloylmethane (curcumin) and diphenyldifluoroketone (EF-24) on cell growth and apoptosis induction in human osteogenic sarcoma cells. This was examined by MTT assay, nuclear DAPI staining, caspase-activation assay, flow cytometry analysis and immunoblotting in Saos2 human osteogenic sarcoma cells. Curcumin and EF-24 inhibited the growth of Saos2 cells in a dose-dependent manner. The apparent potency of EF-24 was more than 3-fold higher that of curcumin. Treatment with curcumin or EF-24 resulted in nuclear condensation and fragmentation in the cells. The caspase-3/-7 activities were detected in living cells treated with curcumin or EF-24. Flow cytometry showed that the rate of apoptosis was increased by curcumin and EF-24 compared to the control. Curcumin and EF-24 promoted the proteolytic cleavages of procaspase-3/-7/-8/-9 with increases in the amount of cleaved caspase-3/-7/-8/-9. The curcumin- or EF-24-induced apoptosis in the Saos2 cells was mediated by the expression of Fas and activation of caspase-8, caspase-3 and poly(ADP-ribose) polymerase. Immunoblotting revealed the Bid and Bcl-2 proteins to be downregulated, and truncated-Bid, Bax and p53 proteins to be upregulated by curcumin and EF-24. Curcumin and EF-24 increased the Bax/Bcl-2 ratio significantly. These results suggest that the curcumin and EF-24 inhibit cell proliferation and induce apoptotic cell death in Saos2 human osteogenic sarcoma cells via both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway, and may have potential properties for anti-osteosarcoma drug discovery.

Topics: Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Benzylidene Compounds; BH3 Interacting Domain Death Agonist Protein; Bone Neoplasms; Caspase 3; Caspase 7; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Proliferation; Curcumin; Enzyme Activation; Fas-Associated Death Domain Protein; Humans; Osteosarcoma; Piperidones; Poly(ADP-ribose) Polymerases; Tumor Suppressor Protein p53

Nuclear factor kappa B (NF-κB) is a key signaling molecule in the elaboration of the inflammatory response. Data indicate that curcumin, a natural ingredient of the curry spice turmeric, acts as a NF-κB inhibitor and exhibits both anti-inflammatory and anti-cancer properties. Curcumin analogs with enhanced activity on NF-κB and other inflammatory signaling pathways have been developed including the synthetic monoketone compound 3,5-Bis(2-fluorobenzylidene)-4-piperidone (EF24). 3,5-Bis(2-pyridinylmethylidene)-4-piperidone (EF31) is a structurally-related curcumin analog whose potency for NF-κB inhibition has yet to be determined. To examine the activity of EF31 compared to EF24 and curcumin, mouse RAW264.7 macrophages were treated with EF31, EF24, curcumin (1-100 μM) or vehicle (DMSO 1%) for 1h. NF-κB pathway activity was assessed following treatment with lipopolysaccharide (LPS) (1 μg/mL). EF31 (IC(50)~5 μM) exhibited significantly more potent inhibition of LPS-induced NF-κB DNA binding compared to both EF24 (IC(50)~35 μM) and curcumin (IC(50) >50 μM). In addition, EF31 exhibited greater inhibition of NF-κB nuclear translocation as well as the induction of downstream inflammatory mediators including pro-inflammatory cytokine mRNA and protein (tumor necrosis factor-α, interleukin-1β, and interleukin-6). Regarding the mechanism of these effects on NF-κB, EF31 (IC(50)~1.92 μM) exhibited significantly greater inhibition of IκB kinase β compared to EF24 (IC(50)~131 μM). Finally, EF31 demonstrated potent toxicity in NF-κB-dependent cancer cell lines while having minimal and reversible toxicity in RAW264.7 macrophages. These data indicate that EF31 is a more potent inhibitor of NF-κB activity than either EF24 or curcumin while exhibiting both anti-inflammatory and anticancer activities. Thus, EF31 represents a promising curcumin analog for further therapeutic development.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Benzylidene Compounds; Cell Line, Tumor; Cell Proliferation; Cell Survival; Curcumin; DNA-Binding Proteins; I-kappa B Kinase; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Macrophages; Mice; Mitogen-Activated Protein Kinase Kinases; NF-kappa B; Phosphorylation; Piperidones; Protein Transport; Signal Transduction; Tumor Necrosis Factor-alpha

Synthetic curcuminoid EF24 was studied for its effect on the maturation and inflammatory response in murine bone marrow derived immortalized JAWS II dendritic cells (DCs). EF24 reduced the expression of LPS-induced MHC class II, CD80 and CD86 molecules. It also abrogated the appearance of dendrites, a typical characteristic of mature DCs. These effects were accompanied by the inhibition of LPS-induced activation of transcription factor nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB). Simultaneous reduction of pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, IL-6] both at the mRNA and secreted levels was also observed. To investigate the dependency of LPS effects on MyD88 adaptor protein, we transfected JAWS II DCs with dominant negative MyD88 plasmid construct (MyD88-DN). EF24 reduced NF-κB activity and TNF-α secretion in a MyD88-dependent manner. These results suggest that EF24 modulates DCs by suppressing their maturation and reducing the secretion of inflammatory cytokines. Further, it appears that EF24 acts at or upstream of MyD88 in the LPS-TLR4/MyD88/NF-κB pathway.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Benzylidene Compounds; Bone Marrow Cells; Cell Differentiation; Cell Line, Transformed; Cells, Cultured; Curcumin; Cytokines; Dendritic Cells; Inflammation Mediators; Lipopolysaccharides; Mice; Mice, Inbred C57BL; NF-kappa B; Piperidones; Transcriptional Activation

Nitrosative stress has recently been demonstrated as a causal in a select sporadic variant of Parkinson's (PD) and Alzheimer's (AD) diseases. Specifically, elevated levels of NO disrupt the redox activity of protein-disulfide isomerase, a key endoplasmic reticulum-resident chaperone by S-nitroso modification of its redox-active cysteines. This leads to accumulation of misfolded AD- and PD-specific protein debris. We have recently demonstrated in vitro that polyphenolic phytochemicals, curcumin and masoprocol, can rescue S-nitroso-PDI formation by scavenging NOx. In this study, using dopaminergic SHSY-5Y cells, we have monitored the aggregation of green-fluorescent protein (GFP)-tagged synphilin-1 (a known constituent of PD Lewy neurites) as a function of rotenone-induced nitrosative stress. Importantly, we demonstrate a marked decrease in synphilin-1 aggregation when the cell line is previously incubated with 3,5-bis(2-flurobenzylidene) piperidin-4-one (EF-24), a curcumin analogue, prior to rotenone insult. Furthermore, our data also reveal that rotenone attenuates PDI expression in the same cell line, a phenomenon that can be mitigated through EF-24 intervention. Together, these results suggest that EF-24 can exert neuroprotective effects by ameliorating nitrosative stress-linked damage to PDI and the associated onset of PD and AD. Essentially, EF-24 can serve as a scaffold for the design and development of PD and AD specific prophylactics.

Topics: Benzylidene Compounds; Carrier Proteins; Cell Line, Tumor; Curcumin; Flavonoids; Free Radical Scavengers; Green Fluorescent Proteins; Humans; Lewy Bodies; Nerve Tissue Proteins; Nitric Oxide; Parkinson Disease; Phenols; Piperidones; Polyphenols; Reactive Oxygen Species; Stress, Physiological

The nuclear factor-kappaB (NF-kappaB) signaling pathway has been targeted for therapeutic applications in a variety of human diseases, includuing cancer. Many naturally occurring substances, including curcumin, have been investigated for their actions on the NF-kappaB pathway because of their significant therapeutic potential and safety profile. A synthetic monoketone compound termed 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24) was developed from curcumin and exhibited potent anticancer activity. Here, we report a mechanism by which EF24 potently suppresses the NF-kappaB signaling pathway through direct action on IkappaB kinase (IKK). We demonstrate that 1) EF24 induces death of lung, breast, ovarian, and cervical cancer cells, with a potency about 10 times higher than that of curcumin; 2) EF24 rapidly blocks the nuclear translocation of NF-kappaB, with an IC(50) value of 1.3 microM compared with curcumin, with an IC(50) value of 13 microM; 3) EF24 effectively inhibits tumor necrosis factor (TNF)-alpha-induced IkappaB phosphorylation and degradation, suggesting a role of this compound in targeting IKK; and 4) EF24 indeed directly inhibits the catalytic activity of IKK in an in vitro-reconstituted system. Our study identifies IKK as an effective target for EF24 and provides a molecular explanation for a superior activity of EF24 over curcumin. The effective inhibition of TNF-alpha-induced NF-kappaB signaling by EF24 extends the therapeutic application of EF24 to other NF-kappaB-dependent diseases, including inflammatory diseases such as rheumatoid arthritis.

Topics: Benzylidene Compounds; Cell Death; Cell Line, Tumor; Cell Nucleus; Curcumin; Drug Screening Assays, Antitumor; Humans; I-kappa B Kinase; Inhibitory Concentration 50; Models, Biological; NF-kappa B; Phosphorylation; Piperidones; Protein Processing, Post-Translational; Protein Transport; Signal Transduction; Tumor Necrosis Factor-alpha

Curcumin, the yellow pigment of the spice turmeric, has emerged as a promising anticancer agent due to its antiproliferative and antiangiogenic properties. However, the molecular mechanism of action of this compound remains a subject of debate. In addition, curcumin's low bioavailability and efficacy profile in vivo further hinders its clinical development. This study focuses on the mechanism of action of EF24, a novel curcumin analog with greater than curcumin biological activity and bioavailability, but no increased toxicity. Treatment of MDA-MB231 breast and PC3 prostate cancer cells with EF24 or curcumin led to inhibition of HIF-1alpha protein levels and, consequently, inhibition of HIF transcriptional activity. This drug-induced HIF inhibition occurred in a VHL-dependent but proteasome-independent manner. We found that, while curcumin inhibited HIF-1alpha gene transcription, EF24 exerted its activity by inhibiting HIF-1alpha posttranscriptionally. This result suggested that the two compounds are structurally similar but mechanistically distinct. Another cellular effect that further differentiated the two compounds was the ability of EF24, but not curcumin, to induce microtubule stabilization in cells. EF24 had no stabilizing effect on tubulin polymerization in an in vitro assay using purified bovine brain tubulin, suggesting that the EF24-induced cytoskeletal disruption in cells may be the result of upstream signaling events rather than EF24 direct binding to tubulin. In summary, our study identifies EF24 as a novel curcumin-related compound possessing a distinct mechanism of action, which we believe contributes to the potent anticancer activity of this agent and can be further exploited to investigate the therapeutic potential of EF24.

Topics: Antineoplastic Agents; Benzylidene Compounds; Cell Hypoxia; Curcumin; Cytoskeleton; Dose-Response Relationship, Drug; Down-Regulation; Drug Evaluation, Preclinical; Drug Resistance, Neoplasm; Epothilones; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Microtubules; Mitosis; Neoplasms; Paclitaxel; Piperidones; Proteasome Endopeptidase Complex; Tumor Cells, Cultured; Von Hippel-Lindau Tumor Suppressor Protein