EBV LMP1-C terminal binding affibody molecule downregulates MEK/ERK/p90RSK pathway and inhibits the proliferation of nasopharyngeal carcinoma cells in mouse tumor xenograft models.

Nasopharyngeal carcinoma (NPC), is an Epstein-Barr virus (EBV) associated malignancy most common in Southern China and Southeast Asia. In southern China, it is one of the major causes of cancer-related death. Despite improvement in radiotherapy and chemotherapy techniques, locoregional recurrence and distant metastasis remains the major causes for failure of treatment in NPC patients. Therefore, finding new specific drug targets for treatment interventions are urgently needed. Here, we report three potential ZLMP1-C affibody molecules (ZLMP1-C15, ZLMP1-C114 and ZLMP1-C277) that showed specific binding interactions for recombinant and native EBV LMP1 as determined by epitope mapping, co-localization and co-immunoprecipitation assays. The ZLMP1-C affibody molecules exhibited high antitumor effects on EBV-positive NPC cell lines and displayed minimal cytotoxicity towards EBV-negative NPC cell line. Moreover, ZLMP1-C277 showed higher antitumor efficacy than ZLMP1-C15 and ZLMP1-C114 affibody molecules. The ability of ZLMP1-C277 decrease the phosphorylation levels of up-stream activator phospho-Raf-1(Ser338), phospho-MEK1/2(Ser217/Ser221), phospho-ERK1/2(Thr202/Thr204), thereby leading to downstream suppression of phospho-p90RSK(Ser380) and transcription factor c-Fos. Importantly, tumor growth was reduced in tumor-bearing mice treated with ZLMP1-C277 and caused no apparent toxicity. Taken together, our findings provide evidence that ZLMP1-C277 as a promising therapeutic agent in EBV-associated NPC.

Artemether confers neuroprotection on cerebral ischemic injury through stimulation of the Erk1/2-P90rsk-CREB signaling pathway.

Ischemic stroke is one of the leading causes of death and disability among adults. Despite the economic burden of the disease, available treatment options are still very limited. With the exception of anti-thrombolytics and hypothermia, current therapies fail to reduce neuronal injury, neurological deficits and mortality rates, suggesting that the development of novel and more effective therapies against ischemic stroke is urgent. In the present study, we found that artemether, which has been used in the clinic as an anti-malarial drug, was able to improve the neurological deficits, attenuate the infarction volume and the brain water content in a middle cerebral artery occlusion (MCAO) animal model. Furthermore, artemether treatment significantly suppressed cell apoptosis, stimulated cell proliferation and promoted the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), P90rsk and cAMP responsive element-binding protein (CREB). Artemether protective effect was attenuated by PD98059, an ERK1/2 inhibitor, administration. Similarly, in oxygen-glucose deprivation/reperfusion (OGD/RP) cell models, artemether pre-treatment induced the suppression of the intracellular ROS, the down-regulation of LDH activity, the reduction of caspase 3 activity and of the apoptosis cell rate and reversed the decrease of mitochondrial membrane potential. As with MCAO animal model, artemether promoted the activation of Erk1/2-P90rsk-CREB signaling pathway. This effect was blocked by the inhibition or knock-down of ERK1/2. The present study provides evidences of the neuroprotective effect of artemether unravelling its potential as a new therapeutic candidate for the prevention and treatment of stroke.

Mitogen- and Stress-Activated Protein Kinase 1 Mediates Alcohol-Upregulated Transcription of Brf1 and tRNA Genes to Cause Phenotypic Alteration.

Upregulation of Brf1 (TFIIB-related factor 1) and Pol III gene (RNA polymerase III-dependent gene, such as tRNAs and 5S rRNA) activities is associated with cell transformation and tumor development. Alcohol intake causes liver injury, such as steatosis, inflammation, fibrosis, and cirrhosis, which enhances the risk of HCC development. However, the mechanism of alcohol-promoted HCC remains to be explored. We have designed the complementary research system, which is composed of cell lines, an animal model, human samples, and experiments in vivo and in vitro, to carry out this project by using molecular biological, biochemical, and cellular biological approaches. It is a unique system to explore the mechanism of alcohol-associated HCC. Our results indicate that alcohol upregulates Brf1 and Pol III gene (tRNAs and 5S rRNA) transcription in primary mouse hepatocytes, immortalized mouse hepatocyte-AML-12 cells, and engineered human HepG2-ADH cells. Alcohol activates MSK1 to upregulate expression of Brf1 and Pol III genes, while inhibiting MSK1 reduces transcription of Brf1 and Pol III genes in alcohol-treated cells. The inhibitor of MSK1, SB-747651A, decreases the rates of cell proliferation and colony formation. Alcohol feeding promotes liver tumor development of the mouse. These results, for the first time, show the identification of the alcohol-response promoter fragment of the Pol III gene key transcription factor, Brf1. Our studies demonstrate that Brf1 expression is elevated in HCC tumor tissues of mice and humans. Alcohol increases cellular levels of Brf1, resulting in enhancement of Pol III gene transcription in hepatocytes through MSK1. Our mechanism analysis has demonstrated that alcohol-caused high-response fragment of the Brf1 promoter is at p-382/+109bp. The MSK1 inhibitor SB-747651A is an effective reagent to repress alcohol-induced cell proliferation and colony formation, which is a potential pharmaceutical agent. Developing this inhibitor as a therapeutic approach will benefit alcohol-associated HCC patients.

Modulation of hypothalamic S6K1 and S6K2 alters feeding behavior and systemic glucose metabolism.

The mTOR/S6Ks signaling is one of the intracellular pathways important for metabolic control, acting both peripherally and centrally. In the hypothalamus, mTOR/S6Ks axis mediates the action of leptin and insulin and can modulate the expression of neuropeptides. We analyzed the role of different S6Ks isoforms in the hypothalamic regulation of metabolism. We observed decreased food intake and decreased expression of agouti-related peptide (AgRP) following intracerebroventricular (icv) injections of adenoviral-mediated overexpression of three different S6Ks isoforms. Moreover, mice overexpressing p70-S6K1 in undefined periventricular hypothalamic neurons presented changes in glucose metabolism, as an increase in gluconeogenesis. To further evaluate the hypothalamic role of a less-studied S6K isoform, p54-S6K2, we used a Cre-LoxP approach to specifically overexpress it in AgRP neurons. Our findings demonstrate the potential participation of S6K2 in AgRP neurons regulating feeding behavior.

Mitogen- and stress-activated protein kinase 1-induced neuroprotection in Huntington's disease: role on chromatin remodeling at the PGC-1-alpha promoter.

Huntington's disease (HD) is a neurodegenerative disorder due to abnormal polyglutamine expansion in huntingtin protein (Exp-Htt). This expansion causes protein aggregation, leading to neuronal dysfunction and death. We have previously shown that mitogen- and stress-activated kinase (MSK-1), a nuclear protein kinase involved in chromatin remodeling through histone H3 phosphorylation, is deficient in the striatum of HD patients and model mice. Restoring MSK-1 expression in cultured striatal cells prevented neuronal dysfunction and death induced by Exp-Htt. Here we extend these observations in a rat model of HD based on striatal lentiviral expression of Exp-Htt (LV-Exp-HTT). MSK-1 overexpression attenuated Exp-Htt-induced down-regulation of DARPP-32 expression 4 and 10 weeks after infection and enhanced NeuN staining after 10 weeks. LV-MSK-1 induced constitutive hyperphosphorylation of H3 and cAMP-responsive element binding protein (CREB), indicating that MSK-1 has spontaneous catalytic activity. MSK-1 overexpression also upregulated peroxisome proliferator-activated receptor γ coactivator alpha (PGC-1α), a transcriptional co-activator involved in mitochondrial biogenesis. Chromatin immunoprecipitation indicated that transcriptional regulation of PGC-1α is directly linked to increased binding of MSK-1, along with H3 and CREB phosphorylation of the PGC-1α promoter. MSK-1 knock-out mice showed spontaneous striatal atrophy as they aged, as well as higher susceptibility to systemic administration of the mitochondrial neurotoxin 3-NP. These results indicate that MSK-1 activation is an important and key event in the signaling cascade that regulates PGC-1α expression. Strategies aimed at restoring MSK-1 expression in the striatum might offer a new therapeutic approach to HD.