p38 MAPK- and Akt-mediated p300 phosphorylation regulates its degradation to facilitate nucleotide excision repair.

Besides the primary histone acetyltransferase (HAT)-mediated chromatin remodeling function, co-transcriptional factor, p300, is also known to play a distinct role in DNA repair. However, the exact mechanism of p300 function in DNA repair has remained unclear and difficult to discern due to the phosphorylation and degradation of p300 in response to DNA damage. Here, we have demonstrated that p300 is only degraded in the presence of specific DNA lesions, which are the substrates of nucleotide excision repair (NER) pathway. In contrast, DNA double-strand breaks fail to degrade p300. Degradation is initiated by phosphorylation of p300 at serine 1834, which is catalyzed by the cooperative action of p38 mitogen-activated protein kinases and Akt kinases. In depth, functional analysis revealed that (i) p300 and CBP act redundantly in repairing ultraviolet (UV) lesions, (ii) the phosphorylation of p300 at S1834 is critical for efficient removal of UV-induced cyclobutane pyrimidine dimers and (iii) p300 is recruited to DNA damage sites located within heterochromatin. Taken together, we conclude that phosphorylated p300 initially acetylates histones to relax heterochromatin to allow damage recognition factors access to damage DNA. Thereupon, p300 is promptly degraded to allow the sequential recruitment of downstream repair proteins for successful execution of NER.

Thymoquinone up-regulates PTEN expression and induces apoptosis in doxorubicin-resistant human breast cancer cells.

The use of innocuous naturally occurring compounds to overcome drug resistance and cancer recalcitrance is now in the forefront of cancer research. Thymoquinone (TQ) is a bioactive constituent of the volatile oil derived from seeds of Nigella sativa Linn. TQ has shown promising anti-carcinogenic and anti-tumor activities through different mechanisms. However, the effect of TQ on cell signaling and survival pathways in resistant cancer cells has not been fully delineated. Here, we report that TQ greatly inhibits doxorubicin-resistant human breast cancer MCF-7/DOX cell proliferation. TQ treatment increased cellular levels of PTEN proteins, resulting in a substantial decrease of phosphorylated Akt, a known regulator of cell survival. The PTEN expression was accompanied with elevation of PTEN mRNA. TQ arrested MCF-7/DOX cells at G2/M phase and increased cellular levels of p53 and p21 proteins. Flow cytometric analysis and agarose gel electrophoresis revealed a significant increase in Sub-G1 cell population and appearance of DNA ladders following TQ treatment, indicating cellular apoptosis. TQ-induced apoptosis was associated with disrupted mitochondrial membrane potential and activation of caspases and PARP cleavage in MCF-7/DOX cells. Moreover, TQ treatment increased Bax/Bcl2 ratio via up-regulating Bax and down-regulating Bcl2 proteins. More importantly, PTEN silencing by target specific siRNA enabled the suppression of TQ-induced apoptosis resulting in increased cell survival. Our results reveal that up-regulation of the key upstream signaling factor, PTEN, in MCF-7/DOX cells inhibited Akt phosphorylation, which ultimately causes increase in their regulatory p53 levels affecting the induction of G2/M cell cycle arrest and apoptosis. Overall results provide mechanistic insights for understanding the molecular basis and utility of the anti-tumor activity of TQ.

Reversal of SIN-1-induced eNOS dysfunction by the spin trap, DMPO, in bovine aortic endothelial cells via eNOS phosphorylation.

BACKGROUND AND PURPOSE: Nitric oxide (NO) derived from eNOS is mostly responsible for the maintenance of vascular homeostasis and its decreased bioavailability is characteristic of reactive oxygen species (ROS)-induced endothelial dysfunction (ED). Because 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a commonly used spin trap, can control intracellular nitroso-redox balance by scavenging ROS and donating NO, it was employed as a cardioprotective agent against ED but the mechanism of its protection is still not clear. This study elucidated the mechanism of protection by DMPO against SIN-1-induced oxidative injury to bovine aortic endothelial cells (BAEC). EXPERIMENTAL APPROACH: BAEC were treated with SIN-1, as a source of peroxynitrite anion (ONOO⁻), and then incubated with DMPO. Cytotoxicity following SIN-1 alone and cytoprotection by adding DMPO was assessed by MTT assay. Levels of ROS and NO generation from HEK293 cells transfected with wild-type and mutant eNOS cDNAs, tetrahydrobiopterin bioavailability, eNOS activity, eNOS and Akt kinase phosphorylation were measured. KEY RESULTS: Post-treatment of cells with DMPO attenuated SIN-1-mediated cytotoxicity and ROS generation, restoration of NO levels via increased in eNOS activity and phospho-eNOS levels. Treatment with DMPO alone significantly increased NO levels and induced phosphorylation of eNOS Ser¹¹79 via Akt kinase. Transfection studies with wild-type and mutant human eNOS confirmed the dual role of eNOS as a producer of superoxide anion (O2⁻) with SIN-1 treatment, and a producer of NO in the presence of DMPO. CONCLUSION AND IMPLICATIONS: Post-treatment with DMPO of oxidatively challenged cells reversed eNOS dysfunction and could have pharmacological implications in the treatment of cardiovascular diseases.

Thymoquinone inhibits autophagy and induces cathepsin-mediated, caspase-independent cell death in glioblastoma cells.

Glioblastoma is the most aggressive and common type of malignant brain tumor in humans, with a median survival of 15 months. There is a great need for more therapies for the treatment of glioblastoma. Naturally occurring phytochemicals have received much scientific attention because many exhibit potent tumor killing action. Thymoquinone (TQ) is the bioactive compound of the Nigella sativa seed oil. TQ has anti-oxidant, anti-inflammatory and anti-neoplastic actions with selective cytotoxicity for human cancer cells compared to normal cells. Here, we show that TQ selectively inhibits the clonogenicity of glioblastoma cells as compared to normal human astrocytes. Also, glioblastoma cell proliferation could be impaired by chloroquine, an autophagy inhibitor, suggesting that glioblastoma cells may be dependent on the autophagic pathway for survival. Exposure to TQ caused an increase in the recruitment and accumulation of the microtubule-associated protein light chain 3-II (LC3-II). TQ also caused an accumulation of the LC3-associated protein p62, confirming the inhibition of autophagy. Furthermore, the levels of Beclin-1 protein expression were unchanged, indicating that TQ interferes with a later stage of autophagy. Finally, treatment with TQ induces lysosome membrane permeabilization, as determined by a specific loss of red acridine orange staining. Lysosome membrane permeabilization resulted in a leakage of cathepsin B into the cytosol, which mediates caspase-independent cell death that can be prevented by pre-treatment with a cathepsin B inhibitor. TQ induced apoptosis, as determined by an increase in PI and Annexin V positive cells. However, apoptosis appears to be caspase-independent due to failure of the caspase inhibitor z-VAD-FMK to prevent cell death and absence of the typical apoptosis related signature DNA fragmentation. Inhibition of autophagy is an exciting and emerging strategy in cancer therapy. In this vein, our results describe a novel mechanism of action for TQ as an autophagy inhibitor selectively targeting glioblastoma cells.

Diesterified nitrone rescues nitroso-redox levels and increases myocyte contraction via increased SR Ca(2+) handling.

Nitric oxide (NO) and superoxide (O(2) (-)) are important cardiac signaling molecules that regulate myocyte contraction. For appropriate regulation, NO and O(2) (.-) must exist at defined levels. Unfortunately, the NO and O(2) (.-) levels are altered in many cardiomyopathies (heart failure, ischemia, hypertrophy, etc.) leading to contractile dysfunction and adverse remodeling. Hence, rescuing the nitroso-redox levels is a potential therapeutic strategy. Nitrone spin traps have been shown to scavenge O(2) (.-) while releasing NO as a reaction byproduct; and we synthesized a novel, cell permeable nitrone, 2-2-3,4-dihydro-2H-pyrrole 1-oxide (EMEPO). We hypothesized that EMEPO would improve contractile function in myocytes with altered nitroso-redox levels. Ventricular myocytes were isolated from wildtype (C57Bl/6) and NOS1 knockout (NOS1(-/-)) mice, a known model of NO/O(2) (.-) imbalance, and incubated with EMEPO. EMEPO significantly reduced O(2) (.-) (lucigenin-enhanced chemiluminescence) and elevated NO (DAF-FM diacetate) levels in NOS1(-/-) myocytes. Furthermore, EMEPO increased NOS1(-/-) myocyte basal contraction (Ca(2+) transients, Fluo-4AM; shortening, video-edge detection), the force-frequency response and the contractile response to ß-adrenergic stimulation. EMEPO had no effect in wildtype myocytes. EMEPO also increased ryanodine receptor activity (sarcoplasmic reticulum Ca(2+) leak/load relationship) and phospholamban Serine16 phosphorylation (Western blot). We also repeated our functional experiments in a canine post-myocardial infarction model and observed similar results to those seen in NOS1(-/-) myocytes. In conclusion, EMEPO improved contractile function in myocytes experiencing an imbalance of their nitroso-redox levels. The concurrent restoration of NO and O(2) (.-) levels may have therapeutic potential in the treatment of various cardiomyopathies.

Acute DPP-4 inhibition modulates vascular tone through GLP-1 independent pathways.

Evidence from both clinical and experimental studies indicates that Di-peptidyl peptidase-IV (DPP-4) inhibition may mediate favorable effects on the cardiovascular system. The objective of this study was to examine the acute effects of DPP-4 inhibition on vascular responses and to study the underlying mechanisms of alteration in tone. Aortic segments from C57BL/6 mice were treated with vasoconstrictors and exposed to various doses of alogliptin, a selective DPP-4 inhibitor. Vasodilator responses were evaluated using pathway specific antagonists to elucidate mechanisms of response. In parallel experiments, cultured human umbilical vein endothelial cells (HUVEC) were exposed to varying concentrations of alogliptin to evaluate the effects on candidate vasodilator pathways. Alogliptin relaxed phenylephrine and U46619 pre-constricted aortic segments in a dose dependent manner. Relaxation responses were not affected by the glucagon-like peptide-1 (GLP-1) receptor antagonist, exendin fragment 9-39 (88 ± 6 vs. 91 ± 2, p < 0.001). Vascular relaxation to alogliptin was significantly decreased by endothelial denudation, L-N(G)-monomethyl-arginine citrate (L-NMMA) and by the soluble guanylate cyclase inhibitor ODQ. DPP-4 inhibition induced relaxation was completely abolished by a combination of L-NMMA, charybdotoxin and apamin. Incubation of HUVECs with alogliptin resulted in eNOS and Akt phosphorylation (Ser(1177) and Ser(473) respectively) paralleled by a rapid increase in nitric oxide. Inhibition of Src kinase decreased eNOS and Akt phosphorylation, in contrast to a lack of any effect on insulin mediated activation of the eNOS-Akt, suggesting that alogliptin mediates vasodilation through Src kinase mediated effects on eNOS-Akt. DPP-4 inhibition by alogliptin mediates rapid vascular relaxation via GLP-1 independent, Src-Akt-eNOS mediated NO release and the activation of vascular potassium channels.