Glutamate is an essential excitatory neurotransmitter regulating brain functions. Excitatory amino acid transporter (EAAT)-2 is one of the major glutamate transporters expressed predominantly in astroglial cells and is responsible for 90% of total glutamate uptake. Glutamate transporters tightly regulate glutamate concentration in the synaptic cleft. Dysfunction of EAAT2 and accumulation of excessive extracellular glutamate has been implicated in the development of several neurodegenerative diseases including Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. Analysis of the 2.5-kb human EAAT2 promoter showed that NF-κB is an important regulator of EAAT2 expression in astrocytes. Screening of approximately 1,040 FDA-approved compounds and nutritionals led to the discovery that many β-lactam antibiotics are transcriptional activators of EAAT2 resulting in increased EAAT2 protein levels. Treatment of animals with ceftriaxone (CEF), a β-lactam antibiotic, led to an increase of EAAT2 expression and glutamate transport activity in the brain. CEF has neuroprotective effects in both in vitro and in vivo models based on its ability to inhibit neuronal cell death by preventing glutamate excitotoxicity. CEF increases EAAT2 transcription in primary human fetal astrocytes (PHFA) through the NF-κB signaling pathway. The NF-κB binding site at −272 position was critical in CEF-mediated EAAT2 protein induction. These studies emphasize the importance of transcriptional regulation in controlling glutamate levels in the brain. They also emphasize the potential utility of the EAAT2 promoter for developing both low and high throughput screening assays to identify novel small molecule regulators of glutamate transport with potential to ameliorate pathological changes occurring during and causing neurodegeneration.

No abstract

Our recent findings show that astrocyte elevated gene-1 (AEG-1) is overexpressed in >90% of human hepatocellular carcinoma (HCC) samples, and AEG-1 plays a central role in regulating development and progression of HCC. In the present study, we elucidate a molecular mechanism of AEG-1-induced chemoresistance, an important characteristic of aggressive cancers. AEG-1 increases the expression of multidrug resistance gene 1 (MDR1) protein, resulting in increased efflux and decreased accumulation of doxorubicin, promoting doxorubicin resistance. Suppression of MDR1 by small interfering RNA or chemical reagents, or inhibition of AEG-1 or a combination of both genes, significantly increases in vitro sensitivity to doxorubicin. In nude mice xenograft studies, a lentivirus expressing AEG-1 short hairpin RNA, in combination with doxorubicin, profoundly inhibited growth of aggressive human HCC cells compared with either agent alone. We document that although AEG-1 does not affect MDR1 gene transcription, it facilitates association of MDR1 mRNA to polysomes, resulting in increased translation, and AEG-1 also inhibits ubiquitination and subsequent proteasome-mediated degradation of MDR1 protein. This study is the first documentation of a unique aspect of AEG-1 function (i.e., translational and posttranslational regulation of proteins). Inhibition of AEG-1 might provide a means of more effectively using chemotherapy to treat HCC, which displays inherent chemoresistance with aggressive pathology. Cancer Res; 70(8); 3249-58. ©2010 AACR.

Experimental evidence documents that the MDA-7/IL-24 protein (an IL-10 family cytokine) binds to IL-20 and IL-22 receptor complexes resulting in the activation of JAK/STAT signaling pathways. Recent published reports utilizing human blood derived primary lymphocytes have provided additional confirmatory evidence relating to the cytokine properties of this molecule. A notable attribute of mda-7/IL-24 is its cancer cell-specific apoptosis inducing capacity, which currently remains incompletely understood. Treatment with distinctive tyrosine kinase inhibitors (Genistein and AG18) or a JAK-selective inhibitor (AG490) did not prevent Ad.mda-7 induced apoptosis in diverse cell lines. In addition, there is no apparent correlation between patterns of expression of IL-20R1, IL-20R2, and IL-22R mRNA and susceptibility to Ad.mda-7 in different cell lines. Furthermore, Ad.mda-7 is able to induce killing in STAT/JAK deficient cells. In contrast, treatment with the p38(MAPK) selective inhibitor SB203580, partially inhibited apoptosis induced by Ad.mda-7 in different cell lines. These results demonstrate for the first time that signaling events leading to susceptibility to Ad.mda-7 induced apoptosis, might be tyrosine kinase independent and can thus be distinguished from its cytokine function related properties mediated by the IL-20/IL-22 receptor complexes that require JAK/STAT kinase activity.

Melanoma differentiation-associated gene 7 (mda-7)/interleukin-24 (IL-24) is a unique member of the IL-10 gene family, which displays a broad range of antitumor properties, including induction of cancer-specific apoptosis. Adenoviral-mediated delivery by Ad.mda-7 invokes an endoplasmic reticulum (ER) stress response that is associated with ceramide production and autophagy in some cancer cells. Here, we report that Ad.mda-7-induced ER stress and ceramide production trigger autophagy in human prostate cancer cells, but not in normal prostate epithelial cells, through a canonical signaling pathway that involves Beclin-1, atg5, and hVps34. Autophagy occurs in cancer cells at early times after Ad.mda-7 infection, but a switch to apoptosis occurs by 48 hours after infection. Inhibiting autophagy with 3-methyladenosine increases Ad.mda-7-induced apoptosis, suggesting that autophagy may be initiated first as a cytoprotective mechanism. Inhibiting apoptosis by overexpression of antiapoptotic proteins Bcl-2 or Bcl-xL increased autophagy after Ad.mda-7 infection. During the apoptotic phase, the MDA-7/IL-24 protein physically interacted with Beclin-1 in a manner that could inhibit Beclin-1 function culminating in apoptosis. Conversely, Ad.mda-7 infection elicited calpain-mediated cleavage of the autophagic protein ATG5 in a manner that could facilitate switch to apoptosis. Our findings reveal novel aspects of the interplay between autophagy and apoptosis in prostate cancer cells that underlie the cytotoxic action of mda-7/IL-24, possibly providing new insights in the development of combinatorial therapies for prostate cancer. Cancer Res; 70(9); 3667-76. ©2010 AACR.

No abstract

Astrocyte-elevated gene-1 (AEG-1) expression increases in multiple cancers and plays a crucial role in oncogenic transformation and angiogenesis, which are essential components in tumor cell development, growth, and progression to metastasis. Moreover, AEG-1 directly contributes to resistance to chemotherapeutic drugs, another important hallmark of aggressive cancers. In the present study, we document that AEG-1 mediates protective autophagy, an important regulator of cancer survival under metabolic stress and resistance to apoptosis, which may underlie its significant cancer-promoting properties. AEG-1 induces noncanonical autophagy involving an increase in expression of ATG5. AEG-1 decreases the ATP/AMP ratio, resulting in diminished cellular metabolism and activation of AMP kinase, which induces AMPK/mammalian target of rapamycin-dependent autophagy. Inhibition of AMPK by siAMPK or compound C decreases expression of ATG5, ultimately attenuating AEG-1-induced autophagy. AEG-1 protects normal cells from serum starvation-induced death through protective autophagy, and inhibition of AEG-1-induced autophagy results in serum starvation-induced cell death. We also show that AEG-1-mediated chemoresistance is because of protective autophagy and inhibition of AEG-1 results in a decrease in protective autophagy and chemosensitization of cancer cells. In summary, the present study reveals a previously unknown aspect of AEG-1 function by identifying it as a potential regulator of protective autophagy, an important feature of AEG-1 that may contribute to its tumor-promoting properties.

MicroRNAs (miRNA), small noncoding RNAs, affect a broad range of biological processes, including tumorigenesis, by targeting gene products that directly regulate cell growth. Human polynucleotide phosphorylase (hPNPase old-35 ), a type I IFN-inducible 3′-5′ exoribonuclease, degrades specific mRNAs and small noncoding RNAs. The present study examined the effect of this enzyme on miRNA expression in human melanoma cells. miRNA microarray analysis of human melanoma cells infected with empty adenovirus or with an adenovirus expressing hPNPase old-35 identified miRNAs differentially and specifically regulated by hPNPase old-35 . One of these, miR-221, a regulator of the cyclin-dependent kinase inhibitor p27 kip1 , displayed robust down-regulation with ensuing up-regulation of p27 kip1 by expression of hPNPase old-35 , which also occurred in multiple human melanoma cells upon IFN-β treatment. Using both in vivo immunoprecipitation followed by Northern blotting and RNA degradation assays, we confirm that mature miR-221 is the target of hPNPase old-35 . Inhibition of hPNPase old-35 by shRNA or stable overexpression of miR-221 protected melanoma cells from IFN-β-mediated growth inhibition, accentuating the importance of hPNPase old-35 induction and miR-221 down-regulation in mediating IFN-β action. Moreover, we now uncover a mechanism of miRNA regulation involving selective enzymatic degradation. Targeted overexpression of hPNPase old-35 might provide an effective therapeutic strategy for miR-221-overexpressing and IFN-resistant tumors, such as melanoma.M icroRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs that regulate gene expression at the posttranscriptional level and play important roles in a multiplicity of biological functions, including cell differentiation, tumorigenesis, apoptosis, and metabolism (1). miRNA genes are initially transcribed principally by either RNA polymerase II or RNA polymerase III as long primary transcripts, which are further processed by the nuclear RNase Drosha and cytoplasmic RNase Dicer to produce precursor miRNAs and mature miRNAs, respectively (2). miRNAs recognize and bind to partially complementary sites in the 3′ UTRs of target mRNAs, resulting in either translational repression or target degradation (3). The steadystate levels of miRNAs, crucial for its profound impact on a wide array of biological processes (4, 5), are presumably determined by the opposing activities of miRNA biogenesis and degradation. Although the framework of miRNA biogenesis is established, factors involved in miRNA dysregulation remain unknown. Recent work from Ramachandran and Chen (6) documented that an exoribonuclease encoded by small rna degrading nuclease (sdn) gene degrades mature miRNAs in Arabidopsis. Although in human cells the posttranscriptional control of miRNA is poorly defined, it can be hypothesized that enzymes involved in miRNA metabolism evolved from enzymes that process structural and/or catalytic RNAs, a view supported by the fact that a number of known molecules involved...