Pramanicin induces apoptosis in Jurkat leukemia cells; a role for JNK, p38 and caspase activation

Pramanicin is a novel anti-fungal drug with a wide range of potential application against human diseases. It has been previously shown that pramanicin induces cell death and increases calcium levels in vascular endothelial cells. In the present study, we showed that pramanicin induced apoptosis in Jurkat T leukemia cells in a dose- and time-dependent manner. Our data reveal that pramanicin induced the release of cytochrome c and caspase-9 and caspase-3 activation, as evidenced by detection of active caspase fragments and fluorometric caspase assays. Pramanicin also activated c-jun N-terminal kinase (JNK), p38 and extracellular signal-regulated kinases (ERK 1/2) with different time and dose kinetics. Treatment of cells with specific MAP kinase and caspase inhibitors further confirmed the mechanistic involvement of these signalling cascades in pramanicin-induced apoptosis. JNK and p38 pathways acted as pro-apoptotic signalling pathways in pramanicin-induced apoptosis, in which they regulated release of cytochrome c and caspase activation. In contrast the ERK 1/2 pathway exerted a protective effect through inhibition of cytochrome c leakage from mitochondria and caspase activation, which were only observed when lower concentrations of pramanicin were used as apoptosis-inducing agent and which were masked by the intense apoptosis induction by higher concentrations of pramanicin. These results suggest pramanicin as a potential apoptosis-inducing small molecule, which acts through a well-defined JNK- and p38-dependent apoptosis signalling pathway in Jurkat T leukemia cells

Characterization of intracellular signaling cascades in 4-hydroxynonenal-induced apoptosis:

In this thesis we have studied the signaling pathways involved in HNE-induced apoptotis and the effect of resveratrol in signal transduction mechanisms leading to apoptosis in 3T3 fibroblasts when exposed to 4-hydroxynonenal (HNE). The results demonstrate the ability of HNE to induce apoptosis and ROS formation in a dose-dependent manner. In order to get insight into the mechanisms of apoptotic response by HNE, we followed MAP kinase and caspase activation pathways; HNE induced early activation of JNK and p38 proteins but downregulated the basal activity of ERK 1/2. We were also able to demonstrate HNE-induced release of cytochrome c from mitochondria, caspase-9 and caspase-3 activation. Resveratrol effectively prevented HNE-induced JNK and caspase activation hence apoptosis, as well as the formation of ROS. Activation of AP-1 along with increased c-Jun and phospho-c-Jun levels could be inhibited by pretreatment of cells with resveratrol. Additionally, overexpression of dominant negative c-Jun and JNK1 in 3T3 fibroblasts prevented HNE-induced apoptosis, which indicates a role for JNK-c-Jun/AP-1 pathway. Moreover, HNE induced decreased Bcl-2 and increased Bax, Bak and Bim protein levels, which could be prevented by resveratrol. In light of the JNK-dependent induction of c-Jun/AP-1 activation and the protective role of resveratrol, these data indicate a critical potential role for JNK in the cellular response against toxic products of lipid peroxidation. Resveratrol also prevents modulation of Bcl-2 proteins and formation of ROS by HNE. In this respect, resveratrol acting through MAP kinase and Bcl-2 protein pathways in addition to act as antioxidant-quenching reactive oxygen intermediates is a potential small molecule against apoptosis-related human pathologies

Pramanicin analog induces apoptosis in human colon cancer cells: critical roles for Bcl-2, Bim, and p38 MAPK signaling

Pramanicin (PMC) is an antifungal agent that was previously demonstrated to exhibit antiangiogenic and anticancer properties in a few in vitro studies. We initially screened a number of PMC analogs for their cytotoxic effects on HCT116 human colon cancer cells. PMC-A, the analog with the most potent antiproliferative effect was chosen to further interrogate the underlying mechanism of action. PMC-A led to apoptosis through activation of caspase-9 and -3. The apoptotic nature of cell death was confirmed by abrogation of cell death with pretreatment with specific caspase inhibitors. Stress-related MAPKs JNK and p38 were both activated concomittantly with the intrinsic apoptotic pathway. Moreover, pharmacological inhibition of p38 proved to attenuate the cell death induction while pretreatment with JNK inhibitor did not exhibit a protective effect. Resistance of Bax -/- cells and the protective nature of caspase-9 inhibition indicate that mitochondria play a central role in PMC-A induced apoptosis. Early post-exposure elevation of cellular Bim and Bax was followed by a marginal Bcl-2 depletion and Bid cleavage. Further analysis revealed that Bcl-2 downregulation occurs at the mRNA level and is critical to mediate PMC-A induced apoptosis, as ectopic Bcl-2 expression substantially spared the cells from death. Conversely, forced expression of Bim proved to significantly increase cell death. In addition, analyses of p53-/- cells demonstrated that Bcl-2/Bim/Bax modulation and MAPK activations take place independently of p53 expression. Taken together, p53-independent transcriptional Bcl-2 downregulation and p38 signaling appear to be the key modulatory events in PMC-A induced apoptosis

Investigation of natural ventilation in a public place in winter season with the comparison of experimentation and CFD results

In educational buildings, efficient ventilation is vital for better indoor air quality and less contaminant level. High level of carbon dioxide concentration causes to drop in concentration and learning capacities of students in addition to headache, dizziness and rapid spreading of infections. In order to provide high indoor air quality; indoor temperature, carbon dioxide level, air humidity and air flow rate should be in the comfortable zone defined by standards. Natural ventilation is preferable solution since it provides improved indoor air quality with reduced energy consumption. The present study investigates the applicability of natural ventilation in the Middle East Technical University Library Reserve Section in Ankara, Turkey for desired indoor air quality defined by ASHRAE air quality standards. Temperature, carbon dioxide concentration and relative humidity data are collected during experimentation period. In the analyses, single zone model is considered. Computational fluid dynamics (CFD) analysis will are also performed for the comparison of results. Comfortable air flow rate is examined using CFD analysis. Energy saving for winter conditions is calculated. According to the results, solutions are provided for a better indoor air quality and application of natural ventilation. Experimental and numerical results clarify that natural ventilation can be used in winter times in order to reduce the carbon dioxide concentration inside the zone. Mathematical results show that there is an energy saving about 8,34 percent with usage of natural ventilation. The carbon dioxide concentration in the zone is decreased from 1147 ppm to 1000 ppm during 3 hours with the help of natural ventilation without usage of electricity. CFD results will be compared with these result

Aspirin inhibits TNF alpha- and IL-1-induced NF-kappa B activation and sensitizes HeLa cells to apoptosis

Rel/nuclear factor-kappa B (NF-κB) transcription factors are involved in transcription of several target genes that modulate proliferation, apoptosis and cell growth. TNFα- and IL-1-induced NF-κB activation pathways mainly involve the phosphorylation and degradation of IκBα by a signalsome complex followed by nuclear translocation of NF-κB and target gene expression. NF-κB mediates the balance between cell death and survival as most cancer cells that have rather constitutive or inducible activation of NF-κB are resistant to apoptosis even by strong apoptotic agents such as TNFα. In this study we demonstrate that proinflammatory cytokines TNFα and IL-1 induced NF-κB activation in human cervical carcinoma HeLa cells. Our studies reveal that acetylsalicylic acid (aspirin) prevents TNFα- and IL-1-induced NF-κB activation in a dose-dependent manner through inhibition of phosphorylation and degradation of IκBα and IκBβ. Moreover, aspirin sensitizes HeLa cells to TNFα-induced apoptosis. These results suggest that aspirin could be used to potentiate the effectiveness of TNFα-based therapeutic interventions in cancer treatment

Antibody array based immunosensor for detecting cardiovascular disease markers

Quantitative detection of proteins in multiplexed platforms presents technical advantages at clinical and laboratory settings compared to the monoplex ELISA method. With this purpose, we implemented a pilot study using in-house-designed sandwich-type antibody array for multiplexed detection of seven cardiovascular disease (CVD) risk markers and compared the performance of our immunosensor to conventional ELISA kits. Results indicated that our immunosensor can determine serum amyloid A (SAA), vascular cell adhesion molecule (VCAM), and myoglobin (MYO) concentrations accurately, precisely, and above all very much similar to ELISA. Hence, multiplexed detection and quantification of SAA, VCAM, and MYO with our immunosensor can be considered as a potential multiplexed alternative to the ELISA method

Antibody array-based immunosensor for detecting cardiovascular disease risk markers

Quantitative detection of proteins in multiplexed platforms presents technical advantages at clinical and laboratory settings compared to the monoplex ELISA method. With this purpose, we implemented a pilot study using in-house-designed sandwich-type antibody array for multiplexed detection of seven cardiovascular disease (CVD) risk markers and compared the performance of our immunosensor to conventional ELISA kits. Results indicated that our immunosensor can determine serum amyloid A (SAA), vascular cell adhesion molecule (VCAM), and myoglobin (MYO) concentrations accurately, precisely, and above all very much similar to ELISA. Hence, multiplexed detection and quantification of SAA, VCAM, and MYO with our immunosensor can be considered as a potential multiplexed alternative to the ELISA method