Prachi Vilekar

CLEFMA—An anti-proliferative curcuminoid from structure–activity relationship studies on 3,5-bis(benzylidene)-4-piperidones

3,5-Bis(benzylidene)-4-piperidones are being advanced as synthetic analogs of curcumin for anti-cancer and anti-inflammatory properties. We performed structure-activity relationship studies, by testing several synthesized 3,5-bis(benzylidene)-4-piperidones for anti-proliferative activity in lung adenocarcinoma H441 cells. Compared to the lead compound 1, or 3,5-bis(2-fluorobenzylidene)-4-piperidone, five compounds were found to be more potent (IC(50) < 30 microM), and 16 compounds possessed reduced cell-killing efficacy (IC(50) > 50 microM). Based on the observations, we synthesized 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] (29 or CLEFMA) as a novel analog of 1. CLEFMA was evaluated for anti-proliferative activity in H441 cells, and was found to be several folds more potent than compound 1. We did not find apoptotic cell population in flow cytometry, and the absence of apoptosis was confirmed by the lack of caspase cleavage. The electron microscopy of H441cells indicated that CLEFMA and compound 1 induce autophagic cell death that was inhibited by specific autophagy inhibitor 3-methyladenine. The results suggest that the potent and novel curcuminoid, CLEFMA, offers an alternative mode of cell death in apoptosis-resistant cancers.

EF24 suppresses maturation and inflammatory response in dendritic cells

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.

In vivo trafficking and immunostimulatory potential of an intranasally-administered primary dendritic cell-based vaccine

BackgroundCoccidioidomycosis or Valley fever is caused by a highly virulent fungal pathogen: Coccidioides posadasii or immitis. Vaccine development against Coccidioides is of contemporary interest because a large number of relapses and clinical failures are reported with antifungal agents. An efficient Th1 response engenders protection. Thus, we have focused on developing a dendritic cell (DC)-based vaccine for coccidioidomycosis. In this study, we investigated the immunostimulatory characteristics of an intranasal primary DC-vaccine in BALB/c mouse strain that is most susceptible to coccidioidomycosis. The DCs were transfected nonvirally with Coccidioides- Ag2/PRA-cDNA. Expression of DC-markers, Ag2/PRA and cytokines were studied by flow cytometry, dot-immunoblotting and cytometric bead array methods, respectively. The T cell activation was studied by assessing the upregulation of activation markers in a DC-T cell co-culture assay. For trafficking, the DCs were co-transfected with a plasmid DNA encoding HSV1 thymidine kinase (TK) and administered intranasally into syngeneic mice. The trafficking and homing of TK-expressing DCs were monitored with positron emission tomography (PET) using 18F-FIAU probe. Based on the PET-probe accumulation in vaccinated mice, selected tissues were studied for antigen-specific response and T cell phenotypes using ELISPOT and flow cytometry, respectively.ResultsWe found that the primary DCs transfected with Coccidioides-Ag2/PRA-cDNA were of immature immunophenotype, expressed Ag2/PRA and activated naïve T cells. In PET images and subsequent biodistribution, intranasally-administered DCs were found to migrate in blood, lung and thymus; lymphocytes showed generation of T effector memory cell population (TEM) and IFN-γ release.ConclusionsIn conclusion, our results demonstrate that the intranasally-administered primary DC vaccine is capable of inducing Ag2/PRA-specific T cell response. Unique approaches utilized in our study represent an attractive and novel means of producing and evaluating an autologous DC-based vaccine.

Synthesis of radiolabeled cytarabine conjugates.

N4-Modified, novel Ara-C conjugate capable of radiolabeling with gamma ray-emitting ((99m)Tc) as well as positron emitting ((18)F) radionuclides, that is, N4-hydrazine derivative was synthesized. The radiolabeling of N4-(hydrazinonicotinyl)-1-beta-arabinofuranosyl cytosine (HAra-C) with (99m)Tc was performed with over 95% labeling yield. To label HAra-C with (18)F, 4-fluoro((18)F)-benzaldehyde was synthesized from 4-formyl-N,N,N-trimethylanilinium triflate in 30% radiochemical yield; it quantitatively formed hydrazone derivative with HAra-C within 45min. The radiolabeled conjugates were analyzed by radio-UV-RP-HPLC. The cold precursors were characterized by (1)H, (13)C NMR. Additionally, HAra-C was evaluated for cytotoxicity in lung adenocarcinoma (H441) cells and found to be comparable in cell killing efficiency to that of Ara-C. Uptake of (99m)Tc-HAra-C in cultures of H441 cells and sensitive pancreatic cancer cells (MIAPaCa-2) was inhibited by nucleoside transporter inhibitor nitrobenzylthioinosine. The results suggest that (99m)Tc-labeled HAra-C is a substrate for the membrane nucleoside transporters, and that it may be used in molecular imaging of nucleoside transporter expression for the verification of potential anticancer efficacy of nucleoside drugs, such as Ara-C and gemcitabine.

Antibacterial activity of synthetic curcumin derivatives: 3,5-bis(benzylidene)-4-piperidone (EF24) and EF24-dimer linked via diethylenetriaminepentacetic acid (EF2DTPA).

Curcumin is well known for its antimicrobial and anti-inflammatory properties. However, since systemic absorption and bioavailability of curcumin from gastrointestinal tract is considerably poor, synthetic curcuminoids are being developed as better alternatives. Two curcumin derivatives: 3,5-bis(benzylidene)-4-piperidone (EF24) and EF24-dimer linked via diethylenetriaminepentacetic acid (EF2DTPA), were included in this study. We investigated the antibacterial activity of EF24 and EF2DTPA against Gram-negative (Escherichia coli) and Gram-positive (Enterococcus faecalis, Staphylococcus aureus) bacteria. We also studied the effects of EF24 and EF2DTPA on uptake and localization of pHrodo-labeled E. coli in the acidic compartments (phagolysosomes) of dendritic cells (DCs) under in vitro conditions. Our results demonstrate that treatment with EF24 and EF2DTPA directly suppresses the bacterial growth. However, these compounds do not affect the bacterial uptake or localization in the DCs.

Hemorrhage-induced interleukin-1 receptor pathway in lung is suppressed by 3,5-bis(2-fluorobenzylidene)-4-piperidone in a rat model of hypovolemic shock.

Severe blood loss in victims of trauma creates an exaggerated inflammatory background that contributes to the development of intravascular coagulopathy and multiple organ dysfunction syndrome. We hypothesized that treatment with diphenyldifluoroketone EF24, an inhibitor of nuclear factor kappa-B, would have salutary effects in hemorrhagic shock. The objective of this study was to investigate the effect of EF24 on the expression of the interleukin-1 receptor (IL-1R) superfamily in a rat model of hypovolemic shock. Hypovolemia was induced by gradually withdrawing approximately 50% of circulating blood, and EF24 was administered intraperitoneally (0.2 mg/kg) in 50 μL of saline. After 6 h of shock, lung tissue was probed immunohistochemically and by immunoblotting to study the expression of Toll-like receptor 4 (TLR4), IL-1R, suppression of tumorigenicity 2 (ST2), and single immunoglobulin IL-1R-related (SIGIRR). The tissue-associated pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α) and IL-6, were measured by enzyme-linked immunosorbent assay. We observed a reduction in immunoreactive TLR4 and IL-1R1 in lung tissue of rats treated with EF24. Simultaneously, the pulmonary expression of ST2 and SIGIRR (the putative down-regulators of the pro-inflammatory IL-1R pathway) was increased in EF24-treated hemorrhaged rats. The concentration of hemorrhage-induced TNF-α and IL-6 in lung tissue homogenates was also reduced by EF24 treatment. These results confirm our previous in vitro observations in lipopolysaccharide-stimulated dendritic cells that EF24 beneficially modulates the IL-1R pathway and suggest that it could be investigated as an adjunct therapeutic in managing inflammation associated with hemorrhagic shock.

Hemorrhage-inducedIL-1R pathway in lung is suppressed by 3,5-bis(2-fluorobenzylidine)-4-piperidone (EF24) in a rat model of hypovolemic shock

Severe blood loss in victims of trauma creates an exaggerated inflammatory background that contributes to the development of intravascular coagulopathy and multiple organ dysfunction syndrome. We hypothesized that treatment with diphenyldifluoroketone EF24, an inhibitor of nuclear factor kappa-B, would have salutary effects in hemorrhagic shock. The objective of this study was to investigate the effect of EF24 on the expression of the interleukin-1 receptor (IL-1R) superfamily in a rat model of hypovolemic shock. Hypovolemia was induced by gradually withdrawing approximately 50% of circulating blood, and EF24 was administered intraperitoneally (0.2 mg/kg) in 50 μL of saline. After 6 h of shock, lung tissue was probed immunohistochemically and by immunoblotting to study the expression of Toll-like receptor 4 (TLR4), IL-1R, suppression of tumorigenicity 2 (ST2), and single immunoglobulin IL-1R-related (SIGIRR). The tissue-associated pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α) and IL-6, were measured by enzyme-linked immunosorbent assay. We observed a reduction in immunoreactive TLR4 and IL-1R1 in lung tissue of rats treated with EF24. Simultaneously, the pulmonary expression of ST2 and SIGIRR (the putative down-regulators of the pro-inflammatory IL-1R pathway) was increased in EF24-treated hemorrhaged rats. The concentration of hemorrhage-induced TNF-α and IL-6 in lung tissue homogenates was also reduced by EF24 treatment. These results confirm our previous in vitro observations in lipopolysaccharide-stimulated dendritic cells that EF24 beneficially modulates the IL-1R pathway and suggest that it could be investigated as an adjunct therapeutic in managing inflammation associated with hemorrhagic shock.

Hemorrhage-Induced Interleukin-1 Receptor Pathway in Lung Is Suppressed by 3,5-Bis(2-Fluorobenzylidene)-4-Piperidone in a Rat Model of Hypovolemic Shock: Hemorrhage-Induced Inflammation

Severe blood loss in victims of trauma creates an exaggerated inflammatory background that contributes to the development of intravascular coagulopathy and multiple organ dysfunction syndrome. We hypothesized that treatment with diphenyldifluoroketone EF24, an inhibitor of nuclear factor kappa-B, would have salutary effects in hemorrhagic shock. The objective of this study was to investigate the effect of EF24 on the expression of the interleukin-1 receptor (IL-1R) superfamily in a rat model of hypovolemic shock. Hypovolemia was induced by gradually withdrawing approximately 50% of circulating blood, and EF24 was administered intraperitoneally (0.2 mg/kg) in 50 μL of saline. After 6 h of shock, lung tissue was probed immunohistochemically and by immunoblotting to study the expression of Toll-like receptor 4 (TLR4), IL-1R, suppression of tumorigenicity 2 (ST2), and single immunoglobulin IL-1R-related (SIGIRR). The tissue-associated pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-α) and IL-6, were measured by enzyme-linked immunosorbent assay. We observed a reduction in immunoreactive TLR4 and IL-1R1 in lung tissue of rats treated with EF24. Simultaneously, the pulmonary expression of ST2 and SIGIRR (the putative down-regulators of the pro-inflammatory IL-1R pathway) was increased in EF24-treated hemorrhaged rats. The concentration of hemorrhage-induced TNF-α and IL-6 in lung tissue homogenates was also reduced by EF24 treatment. These results confirm our previous in vitro observations in lipopolysaccharide-stimulated dendritic cells that EF24 beneficially modulates the IL-1R pathway and suggest that it could be investigated as an adjunct therapeutic in managing inflammation associated with hemorrhagic shock.