Xiuwu Zhang

Enhancement of Hypoxia-Induced Tumor Cell Death In vitro and Radiation Therapy In vivo by Use of Small Interfering RNA Targeted to Hypoxia-Inducible Factor-1α

Hypoxia-inducible factor-1α (HIF-1α) is an important transcriptional factor that is activated when mammalian cells experience hypoxia, a tumor microenvironmental condition that plays pivotal roles in tumor progression and treatment. In this study, we examined the idea of down-regulating HIF-1α in tumor cells for therapeutic gain. We show that the expression levels of HIF-1α can be significantly attenuated by use of the recently established small interfering RNA technology in combination with adenovirus-mediated gene transfer. Down-regulation of the HIF-1α protein enhanced hypoxia-mediated tumor cell apoptosis in vitro. Subcutaneous tumor growth was also prevented from cells with attenuated HIF-1α expression. In addition, intratumoral injection of adenovirus encoding the HIF-1α-targeted small interfering RNA had a small but significant effect on tumor growth when combined with ionizing radiation. Therefore, our results provide proof of HIF-1α as an effective target for anticancer therapy. They also suggest that an adenovirus-based small interfering RNA gene transfer approach may be a potentially effective adjuvant strategy for cancer treatment.

GW112, A Novel Antiapoptotic Protein That Promotes Tumor Growth

GW112 is a novel gene that has little homology to other known genes. It is overexpressed in a number of human tumor types, especially in those of the digestive system. We show here that GW112 is associated with GRIM-19, a protein known to be involved in regulating cellular apoptosis. Functionally, GW112 could significantly attenuate the ability of GRIM19 to mediate retinoic acid-IFN-β-mediated cellular apoptosis and apoptosis-related gene expression. In addition, GW112 demonstrated strong antiapoptotic effects in tumor cells treated with other stress exposures such as hydrogen peroxide. Finally, forced overexpression of GW112 in murine prostate tumor cells led to more rapid tumor formation in a syngeneic host. Taken together, our data suggest that GW112 is an important regulator of cell death that plays important roles in tumor cell survival and tumor growth.

Abnormal hippocampal BDNF and miR-16 expression is associated with depression-like behaviors induced by stress during early life.

Some environmental stressors lead to the onset of depression via inhibiting hippocampal BDNF expression, but other environmental stressors-induced depression exhibits no change in BDNF expression. The underlying mechanisms behind the divergence remain unknown. In this study, depression-like behaviors were induced in rats by maternal deprivation (MD) and chronic unpredictable stress (CUPS). Depression-like behaviors were tested by open field test, forced swimming test, and sucrose consumption test. BDNF and miR-16 expressions in the hippocampus were examined by real-time PCR. MD and CUPS rats crawled less distance, exhibited decreased vertical activity, and produced more fecal pellets than control rats in the open field test. However, MD rats crawled less distance and produced significantly less fecal pellets than CUPS rats. In the forced swimming and sucrose consumption tests, CUPS and MD rats exhibited longer floating time and consumed less sucrose than control rats, but MD rats exhibited shorter floating time and consumed less sucrose than CUPS rats. MD but not CUPS rats showed lower BDNF mRNA and higher miR-16 expression than control rats. In MD rats, BDNF mRNA expression negatively correlated with the expression of miR-16. BDNF expression positively correlated with the total distance rats crawled and vertical activity in the open field test while miR-16 expression negatively correlated the two behaviors. BDNF positively correlated with sucrose preference rate while miR-16 negatively correlated with sucrose preference rate of the sucrose consumption test. Our study suggests that MD and CUPS induced different depression-like behaviors in rats. Depression induced by MD but not CUPS was significantly associated with upregulation of miR-16 and possibly subsequent downregulation of BDNF in hippocampus.

Reversal of cocaine-induced behavioral sensitization and associated phosphorylation of the NR2B and GluR1 subunits of the NMDA and AMPA receptors.

Cocaine abusers remain vulnerable to drug craving and relapse for many years after abstinence is achieved. We have recently shown that ondansetron (a 5-HT3 receptor antagonist) given 3.5 h after each daily cocaine injection reverses previously established behavioral sensitization. The purpose of the present investigation was two-fold. First, as cocaine cannot be used as therapy, we examined whether pergolide (a D1/D2 receptor agonist with reduced abuse potential) and ondansetron could reverse behavioral sensitization. Second, we investigated whether these behavioral changes were associated with parallel alterations in expression levels and/or phosphorylation changes in the NR2B and GluR1 subunits of the respective NMDA and AMPA receptors. Rats were injected for 5 consecutive days with cocaine or saline followed by 9 days of withdrawal. Starting on withdrawal day 10, animals were given vehicle, pergolide/saline, or pergolide/ondansetron for 5 consecutive days. Following a second 9-day period of withdrawal, all animals were challenged with cocaine for assessment of behavioral sensitization and tissues were collected on the following day for Western blot. Sensitization was associated with increased NR2B expression in the accumbens (NAc) shell and decreased Tyr1472 phosphorylation in the NAc core, as well as increased Ser845 phosphorylation of the GluR1 subunit in prefrontal cortex, NAc core, and shell. Pergolide/ondansetron treatment, but not pergolide alone, consistently reversed both the behavioral sensitization and the associated changes in the NMDA and AMPA receptor subunits. To the extent that sensitization plays a role in chronic cocaine abuse, a combination of these clinically available drugs may be useful in treatment of the disorder.

Antiangiogenic action of redox-modulating Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP5+, via suppression of oxidative stress in a mouse model of breast tumor

MnTE-2-PyP(5+) is a potent catalytic scavenger of reactive oxygen and nitrogen species, primarily superoxide and peroxynitrite. It therefore not only attenuates primary oxidative damage, but was found to modulate redox-based signaling pathways (HIF-1alpha, NF-kappaB, SP-1, and AP-1) and thus, in turn, secondary oxidative injury also. Cancer has been widely considered an oxidative stress condition. The goal of this study was to prove if and why a catalytic SOD mimic/peroxynitrite scavenger would exert anti-cancer effects, i.e., to evaluate whether the attenuation of the oxidative stress by MnTE-2-PyP(5+) could suppress tumor growth in a 4T1 mouse breast tumor model. Tumor cells were implanted into Balb/C mouse flanks. Three groups of mice (n=25) were studied: control (PBS) and 2 and 15 mg/kg/day of MnTE-2-PyP(5+) given subcutaneously twice daily starting when the tumors averaged 200 mm(3) (until they reached approximately 5-fold the initial volume). Intratumoral hypoxia (pimonidazole, carbonic anhydrase), HIF-1alpha, VEGF, proliferating capillary index (CD105), microvessel density (CD31), protein nitration, DNA oxidation (8-OHdG), NADPH oxidase (Nox-4), apoptosis (CD31), macrophage infiltration (CD68), and tumor drug levels were assessed. With 2 mg/kg/day a trend toward tumor growth delay was observed, and a significant trend was observed with 15 mg/kg/day. The 7.5-fold increase in drug dose was accompanied by a similar (6-fold) increase in tumor drug levels. Oxidative stress was largely attenuated as observed through the decreased levels of DNA damage, protein 3-nitrotyrosine, macrophage infiltration, and NADPH oxidase. Further, hypoxia was significantly decreased as were the levels of HIF-1alpha and VEGF. Consequently, suppression of angiogenesis was observed; both the microvessel density and the endothelial cell proliferation were markedly decreased. Our study indicates for the first time that MnTE-2-PyP(5+) has anti-cancer activity in its own right. The anti-cancer activity via HIF/VEGF pathways probably arises from the impact of the drug on the oxidative stress. Therefore, the catalytic scavenging of ROS/RNS by antioxidants, which in turn suppresses cellular transcriptional activity, could be an appropriate strategy for anti-cancer therapy. Enhancement of the anti-cancer effects may be achieved by optimizing the dosing regime, utilizing more bioavailable Mn porphyrins (MnP), and combining MnP treatment with irradiation, hyperthermia, and chemotherapy. Mn porphyrins may be advantageous compared to other anti-cancer drugs, owing to their radioprotection of normal tissue and the ability to afford pain management in cancer patients via prevention of chronic morphine tolerance.

Soluble TGFβ TYPE II receptor gene therapy ameliorates acute radiation-induced pulmonary injury in rats

PURPOSE To assess whether administration of recombinant human adenoviral vector, which carries soluble TGFbeta1 Type II receptor (TbetaRII) gene, might reduce the availability of active TGFbeta1 and thereby protect the lung from radiation-induced injury. METHODS AND MATERIALS Female Fisher 344 rats were given a single 30 Gy dose of right hemithoracic irradiation 24 h after the injections of control (AdGFP) or treatment (AdexTbetaRII-Fc) vectors. Different end points were assessed to look for lung tissue damage. RESULTS There was a significant increase in the plasma level of soluble TbetaRII 24 h and 48 h after injection of treatment vector. In the radiation (RT) + AdexTbetaRII-Fc group, there was a significant reduction in respiratory rate at 4 weeks after treatment as compared to the RT-alone group. Histologic results revealed a significant reduction in lung damage and decrease in the number and activity of macrophages in the RT + AdexTbetaRII-Fc group as compared to the RT-alone group. The tissue level of active TGFbeta1 was significantly reduced in rats receiving RT + AdexTbetaRII-Fc treatment. There was also an upregulation of transmembrane TbetaRII in lung tissue in the RT-alone group as compared to the RT + gene therapy rats. CONCLUSIONS This study shows the ability of AdexTbetaRII-Fc gene therapy to induce an increase in circulating levels of soluble receptors, to reduce the tissue level of active TGFbeta1, and consequently to ameliorate acute radiation-induced lung injury.

RNA Aptamer-targeted Inhibition of NF-κB Suppresses Non-small Cell Lung Cancer Resistance to Doxorubicin

Due to the prevalence of tumor chemoresistance, the clinical response of advanced non-small cell lung cancer (NSCLC) to chemotherapy is poor. We suppressed tumor resistance to doxorubicin (Dox) in A549 cells, a human NSCLC cell line, both in vitro and in vivo in a lung tumor xenograft model, using a novel adenoviral expression system to deliver an RNA aptamer (A-p50) that specifically inhibits nuclear factor-κB (NF-κB) activation. By achieving selective, targeted, and early inhibition of NF-κB activity, we demonstrate that NF-κB plays a critical role in Dox-induced chemoresistance by regulating genes involved in proliferation (Ki-67), response to DNA damage (GADD153), antiapoptosis (Bcl-XL), and pH regulation (CA9). This Dox-induced NF-κB activation and subsequent chemoresistance is dependent on expression of p53. We also demonstrate that NF-κB promotes angiogenesis in the presence of Dox via the hypoxia-inducible factor-1α/vascular endothelial growth factor (HIF-1α/VEGF) pathway, revealing a previously unknown mechanism of NSCLC resistance to Dox. These studies provide important insights into the mechanisms of Dox-induced chemoresistance, and they demonstrate a novel, effective, and clinically practical strategy for interfering with these processes.

A Novel Conditionally Replicative Adenovirus Vector Targeting Telomerase-Positive Tumor Cells

Purpose: To develop a novel conditionally replicative adenovirus vector that targets telomerase-positive cancer cells. Experimental Design: A telomerase gene-derived promoter was used to control the expression of the E1a gene so that the E1a gene is only expressed in telomerase-positive tumor cells. In addition, a reporter gene was also engineered into the vector so that its infection and replication can be monitored easily. Results: A novel recombinant adenovirus vector that could selectively replicate in telomerase-positive cancer cells was made successfully. This vector showed active replication in a panel of cancer cells and minimal replication in normal human fibroblast or epithelial cells. The recombinant vector could effectively lyse various cultured tumor cells even at very low multiplicity of infection. The replication efficiency in tumor cells is over 103-fold more than normal fibroblast and epithelial cells. In s.c. tumor models, the newly developed telomerase-selective adenovirus vectors exhibited significantly more virus replication and reporter gene expression. Conclusions: The telomerase-targeted adenovirus vector has significant potential as an oncolytic virus as well as a tumor-specific therapeutic gene delivery vehicle.

Aptamers selected against the unglycosylated EGFRvIII ectodomain and delivered intracellularly reduce membrane-bound EGFRvIII and induce apoptosis.

Abstract Epidermal growth factor receptor variant III (EGFRvIII) is a glycoprotein uniquely expressed in glioblastoma, but not in normal brain tissues. To develop targeted therapies for brain tumors, we selected RNA aptamers against the histidine-tagged EGFRvIII ectodomain, using an Escherichia coli system for protein expression and purification. Representative aptamer E21 has a dissociation constant (Kd) of 33×10-9 m, and exhibits high affinity and specificity for EGFRvIII in ELISA and surface plasmon resonance assays. However, selected aptamers cannot bind the same protein expressed from eukaryotic cells because glycosylation, a post-translational modification present only in eukaryotic systems, significantly alters the structure of the target protein. By transfecting EGFRvIII aptamers into cells, we find that membrane-bound, glycosylated EGFRvIII is reduced and the percentage of cells undergoing apoptosis is increased. We postulate that transfected aptamers can interact with newly synthesized EGFRvIII, disrupt proper glycosylation, and reduce the amount of mature EGFRvIII reaching the cell surface. Our work establishes the feasibility of disrupting protein post-translational modifications in situ with aptamers. This finding is useful for elucidating the function of proteins of interest with various modifications, as well as dissecting signal transduction pathways.

Oxidative Stress Mediates Radiation Lung Injury by Inducing Apoptosis

PURPOSE Apoptosis in irradiated normal lung tissue has been observed several weeks after radiation. However, the signaling pathway propagating cell death after radiation remains unknown. METHODS AND MATERIALS C57BL/6J mice were irradiated with 15 Gy to the whole thorax. Pro-apoptotic signaling was evaluated 6 weeks after radiation with or without administration of AEOL10150, a potent catalytic scavenger of reactive oxygen and nitrogen species. RESULTS Apoptosis was observed primarily in type I and type II pneumocytes and endothelium. Apoptosis correlated with increased PTEN expression, inhibition of downstream PI3K/AKT signaling, and increased p53 and Bax protein levels. Transforming growth factor-β1, Nox4, and oxidative stress were also increased 6 weeks after radiation. Therapeutic administration of AEOL10150 suppressed pro-apoptotic signaling and dramatically reduced the number of apoptotic cells. CONCLUSION Increased PTEN signaling after radiation results in apoptosis of lung parenchymal cells. We hypothesize that upregulation of PTEN is influenced by Nox4-derived oxidative stress. To our knowledge, this is the first study to highlight the role of PTEN in radiation-induced pulmonary toxicity.