HSP90 inhibitor enhances anti-proliferative and apoptotic effects of celecoxib on HT-29 colorectal cancer cells via increasing BAX/BCL-2 ratio.

Due to the high prevalence and mortality rate of colorectal cancer (CRC), new treatment approaches like combination therapy seem to be necessary. The relationship between chronic inflammation and colorectal cancer development and progression has been shown to be important. Celecoxib, a selective COX-2 inhibitor, is the only non-steroidal anti-inflammatory drug (NSAID) that has been approved for cancer therapy and prevention. Because of cardiovascular side effects of COX-2 inhibitors, combination therapy may improve the therapeutic profile. 17-Demethoxy-17-allylamino geldanamycin (17-AAG), a heat shock protein 90 (HSP90) inhibitor, shows anti-inflammatory effects via down-regulation of the key mediators of inflammation such as Nuclear Factor κB (NF-kB), JAK/Signal Transducer and Activator of Transcription (JAK/STAT). Thus, we studied the effect(s) of combination of 17-AAG and celecoxib on HT-29 cells viability and apoptosis induction. Based on MTT results, we showed an increase in the inhibitory effect of celecoxib when combined with 17-AAG, especially at low a concentration of celecoxib.  Flow cytometry analysis demonstrated that apoptosis induction is probably the mechanism of additive inhibitory effects of 17-AAG and celecoxib combination. To explore the possible mechanism of apoptosis induction by 17-AAG and celecoxib combination, levels of BAX and BCL-2 proteins were determined by western blotting. The BAX/BCL-2 ratio in the combination group was increased compared to 17-AAG or celecoxib alone, mainly via decreasing BCL-2 levels. In conclusion, 17-AAG, increased inhibitory effects of celecoxib on HT-29 cells, probably by induction of apoptosis.

HSP90 Inhibition Suppresses PGE2 Production via Modulating COX-2 and 15-PGDH Expression in HT-29 Colorectal Cancer Cells.

The existence of multiple-interactive roles between several signaling pathways in tumorigenesis shows the significance of pharmacological factors like heat shock protein 90 (HSP90) inhibitors which control several signaling pathways simultaneously. HSP90 as a molecular chaperone supports the active conformational structure and function of several oncogenic signal proteins, termed "client" proteins, some of them act as a link between cancer and inflammation. Prostaglandin E2 (PGE2) is one of the major mediators of inflammation in colorectal cancer development and progress. However, the relationship between chaperone activity of HSP90 and PGE2 levels remains unclear. We evaluated the inhibitory effects of 17-demethoxy-17-allylamino geldanamycin (1 7-AAG), an HSP90 inhibitor, on PGE2 levels in HT-29 colorectal cancer cells. For the first time, we showed inhibitory effects of 17-AAG, on PGE2 levels in HT-29 colorectal cancer cells. 17-AAG inhibited PMA-induced cyclooxygenase-2 (COX-2) mRNA expression and protein level. We showed 15-hydroxyprostaglandin dehydrogenase (15-PGDH) expression induced by 17-AAG treatment at both mRNA and protein levels. In conclusion, we found that inhibitory effects of 17-AAG on PGE2 levels in HT-29 colorectal cancer cells were mediated through modulating COX-2 and 15-PGDH expression.

Expression levels of heat shock protein 60 and glucose-regulated protein 78 in response to trimethylamine-N-oxide treatment in murine macrophage J774A.1 cell line.

Trimethylamine N-oxide (TMAO), a common metabolite in animals and humans, can induce changes in the expression or conformation of heat shock proteins. It has also been introduced as a risk factor for atherosclerosis and a biomarker for kidney problems. On the other hand, increased levels of heat shock proteins 60 and 70 KDa are associated with increased atherosclerosis risk. This study was therefore designed to evaluate the possible effect(s) of TMAO on the expression of HSP60 and GRP78 at the mRNA and protein levels. Murine macrophage J774A.1 cells were treated with micromolar concentrations of TMAO and 4-phenylbutyric acid (4-PBA), a chemical chaperon, for different time intervals. Tunicamycin was also used as a control for induction of endoplasmic reticulum stress. Tunicamycin greatly increased both mRNA and protein levels of GRP78. Similarly but to a lesser extent compared to tunicamycin, TMAO also increased mRNA and protein levels of GRP78 in a dose and time-dependent manner. In contrast, 4-PBA failed to induce any changes. Similar to GRP78, HSP60 was also increased only at mRNA level in TMAO treated cells. 4-PBA also increased HSP60 mRNA levels, whereas, tunicamycin did not show any effect on either protein or mRNA levels of HSP60. Since both heat shock proteins are stress inducible and the elevation of GRP78 is a hallmark for endoplasmic reticulum stress induction, it can be concluded that TMAO may induce endoplasmic reticulum stress or may act through elevation of these heat shock proteins.

Tetracycline-regulated expression of OLIG2 gene in human dental pulp stem cells lead to mouse sciatic nerve regeneration upon transplantation.

Numerous studies have indicated dental pulp stem cells (DPSCs) potency to differentiate into several types of cell lineages. Oligodendrocyte lineage transcription factor 2 (OLIG2) plays an important role in the oligodendrogenic pathway. In this study, a tetracycline (Tet)-inducible system expressing OLIG2 gene was transfected into human DPSCs to direct their differentiation toward oligodendrocyte progenitor cells (OPCs). Following induction, the expression of stage-specific markers was studied by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR), immunocytochemistry and western blotting. In the following, the cells were transplanted into the mouse model of local sciatic demyelination damage by lysolecithin. Recovery of lysolecithin-induced lesions in sciatic nerve was studied by treadmill exercise, von Frey filament test and hind paw withdrawal in response to a thermal stimulus. Improvement of behavioral symptoms was efficiently observed from the second week to the sixth week post-transplantation. Our findings showed that exogenous expression of the OLIG2 gene by a Tet-regulated system could be used as an efficient way to induce the differentiation of DPSCs into functional oligodendrocytes. Meanwhile, the DPSC-derived OPCs have relevant therapeutic potential in the animal model of sciatic nerve injury and therefore might represent a valuable tool for stem cell-based therapy in inflammatory and degenerative diseases of the peripheral and central nervous systems (CNSs).

The role of GABAergic system on the inhibitory effect of ghrelin on food intake in neonatal chicks.

Ghrelin is a gut-brain peptide that has a stimulatory effect on food intake in mammals. In contrast, this peptide decreases food intake in neonatal chicks when injected intracerebroventricularly (ICV). In mammals, neuropeptide Y (NPY) mediates the orexigenic effect of ghrelin whereas in chicks it appears that corticotrophin releasing factor (CRF) is partially involved in the inhibitory effect of ghrelin on food intake. Gamma aminobutyric acid (GABA) has a stimulatory effect on food intake in mammals and birds. In this study we investigated whether the anorectic effect of ghrelin is mediated by the GABAergic system. In Experiment 1, 3h-fasted chicks were given an ICV injection of chicken ghrelin and picrotoxin, a GABA(A) receptors antagonist. Picrotoxin decreased food intake compared to the control chicks indicating a stimulatory effect of GABA(A) receptors on food intake. However, picrotoxin did not alter the inhibitory effect of ghrelin on food intake. In Experiment 2, THIP hydrochloride, a GABA(A) receptor agonist, was used in place of picrotoxin. THIP hydrochloride appeared to partially attenuate the decrease in food intake induced by ghrelin at 30 min postinjection. In Experiment 3, the effect of ICV injection of chicken ghrelin on gene expression of glutamate decarboxylase (GAD)(1) and GAD(2), GABA synthesis enzymes in the brain stem including hypothalamus, was investigated. The ICV injection of chicken ghrelin significantly reduced GAD(2) gene expression. These findings suggest that ghrelin may decrease food intake in neonatal chicks by reducing GABA synthesis and thereby GABA release within brain feeding centers.

Oxyntomodulin reduces expression of glucagon-like peptide 1 receptor in the brainstem of chickens.

Oxyntomodulin (OXM) is a peptide released from the gut and attenuates food intake by acting on hypothalamus. However, its role at the molecular level is not well studied. In the first section of this study, we analysed the effect of OXM on food intake behaviour after injecting into the lateral ventricle of chickens. The outcome showed that food intake decreased significantly after administering 4 nmol of OXM. In the second part, the expression of glucagon-like peptide 1 receptor (GLP-1R) in the brainstem was analysed by real-time RT-PCR. The results showed that expression of GLP-1R was reduced to 27% and 16% at 30 and 90 mins after injection of OXM respectively. In saline-injected chickens, no reduction in GLP-1R was seen. It can be concluded that OXM has a down regulatory effect on the responding receptor, GLP-1R and OXM in chicks has the same reductive effect on food intake as in the mammals.