Bioactive Yoghurt Containing Curcumin and Chlorogenic Acid Reduces Inflammation in Postmenopausal Women.

Menopause is marked by a gradual and permanent decrease of estrogen from the ovaries, leading to metabolic and physiological changes in the body. Combined with increased body mass index, postmenopausal women have elevated systemic inflammation and metabolic disturbances leading to increased risk of developing chronic diseases. A bioactive coconut yoghurt containing curcumin and chlorogenic acid was developed with the potential to target inflammatory processes. In this randomized crossover study, healthy postmenopausal women with a BMI of 25-40 were recruited to consume 125 g of either the bioactive or placebo yoghurt. Blood samples were collected at baseline, 30 min, and 1, 2, 3 and 4 h postprandially. Plasma inflammatory markers (TNFα and IL6) and metabolic markers (triglycerides, insulin and glucose) were measured. Participants had significantly lower plasma TNFα Cmax after consumption of the bioactive yoghurt compared to placebo (mean difference = 0.3 pg/mL; p = 0.04). Additionally, plasma TNFα was significantly lower postprandially compared to baseline after consumption of the bioactive yogurt but not the placebo. No differences were observed in the metabolic markers measured. Conclusions: The bioactive yoghurt fortified with curcumin and chlorogenic acid has the potential to reduce inflammatory mediators; however, a larger and longer-term study is required to confirm these findings.

Chlorogenic Acid Potentiates the Anti-Inflammatory Activity of Curcumin in LPS-Stimulated THP-1 Cells.

The anti-inflammatory effects of curcumin are well documented. However, the bioavailability of curcumin is a major barrier to its biological efficacy. Low-dose combination of complimentary bioactives appears to be an attractive strategy for limiting barriers to efficacy of bioactive compounds. In this study, the anti-inflammatory potential of curcumin in combination with chlorogenic acid (CGA), was investigated using human THP-1 macrophages stimulated with lipopolysaccharide (LPS). Curcumin alone suppressed TNF-α production in a dose-dependent manner with a decrease in cell viability at higher doses. Although treatment with CGA alone had no effect on TNF-α production, it however enhanced cell viability and co-administration with curcumin at a 1:1 ratio caused a synergistic reduction in TNF-α production with no impact on cell viability. Furthermore, an qRT-PCR analysis of NF-κB pathway components and inflammatory biomarkers indicated that CGA alone was not effective in reducing the mRNA expression of any of the tested inflammatory marker genes, except TLR-4. However, co-administration of CGA with curcumin, potentiated the anti-inflammatory effects of curcumin. Curcumin and CGA together reduced the mRNA expression of pro-inflammatory cytokines [TNF-α (~88%) and IL-6 (~99%)], and COX-2 (~92%), possibly by suppression of NF-κB (~78%), IκB-ß-kinase (~60%) and TLR-4 receptor (~72%) at the mRNA level. Overall, co-administration with CGA improved the inflammation-lowering effects of curcumin in THP-1 cells.

JIP3 localises to exocytic vesicles and focal adhesions in the growth cones of differentiated PC12 cells.

The JNK-interacting protein 3 (JIP3) is a molecular scaffold, expressed predominantly in neurons, that serves to coordinate the activation of the c-Jun N-terminal kinase (JNK) by binding to JNK and the upstream kinases involved in its activation. The JNK pathway is involved in the regulation of many cellular processes including the control of cell survival, cell death and differentiation. JIP3 also associates with microtubule motor proteins such as kinesin and dynein and is likely an adapter protein involved in the tethering of vesicular cargoes to the motors involved in axonal transport in neurons. We have used immunofluorescence microscopy and biochemical fractionation to investigate the subcellular distribution of JIP3 in relation to JNK and to vesicular and organelle markers in rat pheochromocytoma cells (PC12) differentiating in response to nerve growth factor. In differentiated PC12 cells, JIP3 was seen to accumulate in growth cones at the tips of developing neurites where it co-localised with both JNK and the JNK substrate paxillin. Cellular fractionation of PC12 cells showed that JIP3 was associated with a subpopulation of vesicles in the microsomal fraction, distinct from synaptic vesicles, likely to be an anterograde-directed exocytic vesicle pool. In differentiated PC12 cells, JIP3 did not appear to associate with retrograde endosomal vesicles thought to be involved in signalling axonal injury. Together, these observations indicate that JIP3 may be involved in transporting vesicular cargoes to the growth cones of PC12 cells, possibly targeting JNK to its substrate paxillin, and thus facilitating neurite outgrowth.

Post-transcriptional regulation of VEGF-A mRNA levels by mitogen-activated protein kinases (MAPKs) during metabolic stress associated with ischaemia/reperfusion.

Angiogenesis is a well-characterised response to the metabolic stresses that occur during ischaemia/reperfusion, but the signalling pathways that regulate it are poorly understood. We tested whether activation of mitogen-activated protein kinases (MAPKs) was involved in regulating the expression of pro-angiogenic growth factors by the metabolic stresses associated with ischaemia/reperfusion in H9c2 rat cardiomyoblasts. Metabolic stress had no effect on vascular endothelial growth factor (VEGF) mRNA levels, but recovery after metabolic inhibition led to a strong induction of VEGF-A mRNA (3.8 ± 0.5-fold at 4 h), a modest rise in VEGF-C mRNA levels (1.7 ± 0.3-fold at 4 h), with no effect on VEGF-B or -D. A VEGF-A promoter reporter construct was unresponsive to metabolic inhibition/recovery and increases in VEGF-A mRNA were not blocked by the transcription inhibitor actinomycin D suggesting that increases in VEGF mRNA were due to enhanced VEGF-A mRNA stability. In addition, studies using reporter constructs demonstrated that regions within the 5' untranslated region (UTR) contributed to enhanced mRNA stability following recovery from metabolic stress. Increases in VEGF-A mRNA were abolished by inhibition of extracellular signal-regulated kinase or c-jun N-terminal kinase MAPKs, suggesting that these kinases may promote angiogenesis in response to metabolic stress during ischaemia/reperfusion by increasing VEGF-A message stability.

Disassembly of the JIP1/JNK molecular scaffold by caspase-3-mediated cleavage of JIP1 during apoptosis.

We report here the cleavage of the c-Jun N-terminal Kinase (JNK) pathway scaffold protein, JNK Interacting Protein-1 (JIP1), by caspases during both Tumour Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) and staurosporine-induced apoptosis in HeLa cells. During the initiation of apoptosis, maximal JNK activation is observed when JIP1 is intact, whereas cleavage of JIP1 correlates with JNK inactivation and progression of apoptosis. JIP1 is cleaved by caspase-3 at two sites, leading to disassembly of the JIP1/JNK complex. Inhibition of JIP1 cleavage by the caspase-3 inhibitor DEVD.fmk inhibits this disassembly, and is accompanied by sustained JNK activation. These data suggest that TRAIL and staurosporine induce JNK activation in a caspase-3-independent manner and that caspase-3-mediated JIP1 cleavage plays a role in JNK inactivation via scaffold disassembly during the execution phase of apoptosis. Caspase-mediated cleavage of JIP scaffold proteins may therefore represent an important mechanism for modulation of JNK signalling during apoptotic cell death.

p38 MAPK/MK2-mediated induction of miR-34c following DNA damage prevents Myc-dependent DNA replication.

The DNA damage response activates several pathways that stall the cell cycle and allow DNA repair. These consist of the well-characterized ATR (Ataxia telangiectasia and Rad-3 related)/CHK1 and ATM (Ataxia telangiectasia mutated)/CHK2 pathways in addition to a newly identified ATM/ATR/p38MAPK/MK2 checkpoint. Crucial to maintaining the integrity of the genome is the S-phase checkpoint that functions to prevent DNA replication until damaged DNA is repaired. Inappropriate expression of the proto-oncogene c-Myc is known to cause DNA damage. One mechanism by which c-Myc induces DNA damage is through binding directly to components of the prereplicative complex thereby promoting DNA synthesis, resulting in replication-associated DNA damage and checkpoint activation due to inappropriate origin firing. Here we show that following etoposide-induced DNA damage translation of c-Myc is repressed by miR-34c via a highly conserved target-site within the 3(') UTR. While miR-34c is induced by p53 following DNA damage, we show that in cells lacking p53 this is achieved by an alternative pathway which involves p38 MAPK signalling to MK2. The data presented here suggest that a major physiological target of miR-34c is c-Myc. Inhibition of miR-34c activity prevents S-phase arrest in response to DNA damage leading to increased DNA synthesis, DNA damage, and checkpoint activation in addition to that induced by etoposide alone, which are all reversed by subsequent c-Myc depletion. These data demonstrate that miR-34c is a critical regulator of the c-Myc expression following DNA damage acting downstream of p38 MAPK/MK2 and suggest that miR-34c serves to remove c-Myc to prevent inappropriate replication which may otherwise lead to genomic instability.

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells.

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.