Search

To investigate the effects of curcumin on proliferation and apoptosis in testicular cancer cells in vitro and to investigate its molecular mechanisms of action. NTera-2 human malignant testicular germ cell line and F9 mouse teratocarcinoma stem cell line were used. The anti-proliferative effect was examined using MTT and colony formation assays. Hoechst 33258 staining, TUNEL and Annexin V-FITC/PI staining assays were used to analyze cell apoptosis. Protein expression was examined with Western blot analysis and immunocytochemical staining. Curcumin (5, 10 and 15 μmol/L) inhibited the viability of NTera-2 cells in dose- and time-dependent manners. Curcumin significantly inhibited the colony formation in both NTera-2 and F9 cells. Curcumin dose-dependently induced apoptosis of NTera-2 cells by reducing FasL expression and Bcl-2-to-Bax ratio, and activating caspase-9, -8 and -3. Furthermore, curcumin dose-dependently reduced the expression of AP transcription factor AP-2γ in NTera-2 cells, whereas the pretreatment with the proteasome inhibitor MG132 blocked both the curcumin-induced reduction of AP-2γ and antiproliferative effect. Curcumin inhibited ErbB2 expression, and decreased the phosphorylation of Akt and ERK in NTera-2 cells. Curcumin induces apoptosis and inhibits proliferation in NTera-2 cells via the inhibition of AP-2γ-mediated downstream cell survival signaling pathways.

Aspirin has been reported to trigger apoptosis in various cancer cell lines. However, the detailed mechanisms involved remain elusive. The present study aimed to investigate whether aspirin plays a role in apoptosis of MDA-MB-453 cells. The effect of aspirin on the proliferation of human MDA-MB-453 cells breast cancer cells was evaluated using MTT assay, flow cytometry and western blotting. The present study reports that aspirin induces the apoptosis of MDA‑MB‑453 breast cancer cells which was attributed to the increased expression and activation of caspase‑3. Moreover, AP‑2α, a transcription factor highly expressed in MDA‑MB‑453 cells, was identified as a negative regulator of caspase‑3 transcription and AP‑2α was attenuated following aspirin treatment. Therefore, aspirin may increase the expression of caspase‑3 by inducing the degradation of AP‑2α, which increases activated caspase‑3 expression, thereby triggering apoptosis in MDA‑MB‑453 cells. Thus, aspirin may be used in breast cancer therapy.

Formaldehyde (FA) is a human leukemogen. Since there is a latency period between initial FA exposure and the development of leukemia, the subsequent impact of FA on hematopoietic stem or progenitor cells (HSCs/HPCs) in post-exposure stage is crucial for a deep understanding of FA-induced hematotoxicity. BALB/c mice were exposed to 3mg/m3 FA for 2weeks, mimicking occupational exposure, and were monitored for another 7days post-exposure. Meanwhile, we included benzene (BZ) as a positive control, separately and together with FA because co-exposure occurs frequently. After 7-day recovery, colonies of progenitors for CFU-GM and BFU-E, and nucleated bone marrow cells in FA-exposed mice were comparable to controls, although they were significantly reduced during exposure. Levels of reactive oxygen species (ROS) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in CFU-GM and BFU-E from FA-exposed mice were higher than controls, although the increase in 8-OHdG was not significant. Granulocyte-macrophage colony stimulating factor (GM-CSF) level in the FA group was lower than controls, but the expression level for the receptor was not upregulated. It suggests that HSCs/HPCs in FA-exposed mice respond to a small amount of GM-CSF and proliferate rapidly, which may cause a possible risk of expansion of abnormal stem/progenitor cell clones. FA co-exposure with BZ was more potent for promoting CFU-GM formation and inducing ROS in BFU-E and 8-OHdG in CFU-GM during the post-exposure period. The compensation of myeloid progenitors with elevated ROS and 8-OHdG may lead to a risk of transforming normal HSCs/HPCs to leukemic stem/progenitor cells. Thus, co-exposure may pose a greater leukemia risk.

It has been increasingly suggested that propofol protects against hypoxic-/ischemic-induced neuronal injury. As evidenced by hemorrhage-induced stroke, hemorrhage into the brain may also cause brain damage. Whether propofol protects against hemorrhage-induced brain damage remains unknown. Therefore, in this study, we investigated the effects of propofol on hemoglobin-induced cytotoxicity in cultured mouse cortical neurons.Neurons were prepared from the cortex of embryonic 15-day-old mice. Hemoglobin was used to induce cytotoxicity in the neurons. The neurons were then treated with propofol for 4 hours. Cytotoxicity was determined by lactate dehydrogenase release assay. Caspase-3 activation was examined by Western blot analysis. Finally, the free radical scavenger U83836E was used to examine the potential involvement of oxidative stress in propofol's effects on hemoglobin-induced cytotoxicity.We found that treatment with hemoglobin induced cytotoxicity in the neurons. Propofol enhanced hemoglobin-induced cytotoxicity. Specifically, there was a significant difference in the amount of lactate dehydrogenase release between hemoglobin plus saline (19.84% ± 5.38%) and hemoglobin plus propofol (35.79% ± 4.41%) in mouse cortical neurons (P = 0.00058, Wilcoxon Mann-Whitney U test, n = 8 in the control group or the treatment group). U83836E did not attenuate the enhancing effects of propofol on hemoglobin-induced cytotoxicity in the neurons, and propofol did not significantly affect caspase-3 activation induced by hemoglobin. These data suggested that caspase-3 activation and oxidative stress might not be the underlying mechanisms by which propofol enhanced hemoglobin-induced cytotoxicity. Moreover, these data suggested that the neuroprotective effects of propofol would be dependent on the condition of the brain injury, which will need to be confirmed in future studies.These results from our current proof-of-concept study should promote more research in vitro and in vivo to develop better anesthesia care for patients with hemorrhagic stroke.

Ngb (neuroglobin) has been identified as a novel endogenous neuroprotectant. However, little is known about the regulatory mechanisms of Ngb expression, especially under conditions of hypoxia. In the present study, we located the core proximal promoter of the mouse Ngb gene to a 554 bp segment, which harbours putative conserved NF-κB (nuclear factor κB)- and Egr1 (early growth-response factor 1) -binding sites. Overexpression and knockdown of transcription factors p65, p50, Egr1 or Sp1 (specificity protein 1) increased and decreased Ngb expression respectively. Experimental assessments with transfections of mutational Ngb gene promoter constructs, as well as EMSA (electrophoretic mobility-shift assay) and ChIP (chromatin immunoprecipitation) assays, demonstrated that NF-κB family members (p65, p50 and cRel), Egr1 and Sp1 bound in vitro and in vivo to the proximal promoter region of the Ngb gene. Moreover, a κB3 site was found as a pivotal cis-element responsible for hypoxia-induced Ngb promoter activity. NF-κB (p65) and Sp1 were also responsible for hypoxia-induced up-regulation of Ngb expression. Although there are no conserved HREs (hypoxia-response elements) in the promoter of the mouse Ngb gene, the results of the present study suggest that HIF-1α (hypoxia-inducible factor-1α) is also involved in hypoxia-induced Ngb up-regulation. In conclusion, we have identified that NF-κB, Egr1 and Sp1 played important roles in the regulation of basal Ngb expression via specific interactions with the mouse Ngb promoter. NF-κB, Sp1 and HIF-1α contributed to the up-regulation of mouse Ngb gene expression under hypoxic conditions.

Participates in actin remodeling through Rac and receptor endocytosisvia Rab5. Here, we used yeast two-hybrid systemwith Eps8 as bait to screen a human brain cDNA library.ITSN2 was identified as the novel binding factor of Eps8. Theinteraction between ITSN2 and Eps8 was demonstrated by thein vivo co-immunoprecipitation and colocalization assays andthe in vitro GST pull-down assays. Furthermore, we mappedthe interaction domains to the region between amino acids260-306 of Eps8 and the coiled-coil domain of ITSN2. In addition,protein stability assays and immunofluorescence analysisshowed ITSN2 overexpression induced the degradation ofEps8 proteins, which was markedly alleviated with the lysosomeinhibitor NH4Cl treatment. Taken together, our resultssuggested ITSN2 interacts with Eps8 and stimulates the degradationof Eps8 proteins. [BMB reports 2012; 45(3): 183-188]

To evaluate the cytotoxic effect and the mechanism of the apoptosis of oligo(styrene-co-acrylonitrile)-modified montmorillonite (PSAN-MMT), the present study is focused on the involvement of anti-proliferation, lactate dehydrogenase release, apoptosis level, caspase 3 activity, transcriptional activity of apoptosis-related gene and apoptosis-related protein and mRNA level. Non-cytotoxic montmorillonite (MMT) incubated cells were used as control. Little cytotoxicity was observed in PSAN-MMT treated cells, and lower level of apoptosis was induced by PSAN-MMT when cells were incubated with high montmorillonite content (1 g/L). The apoptosis seemed to follow a transcription-dependent route by activating proapoptotic genes such as p53 and p21. The modified MMT could be a promising candidate of biosafety controlled-release carrier for large drug molecules and for gene delivery.

To test the hypothesis that central changes in sympathoregulation might contribute to sympathoexcitation after cyclic intermittent hypoxia (CIH) we exposed male Sprague-Dawley rats to CIH or to room air sham (Sham) for 8 hours/d for 3 weeks. After completion of the exposure we assessed heart rate, mean arterial pressure and renal sympathetic nerve activity in conscious animals before and after intracerebroventricular (i.c.v.) administration of endothelin-1 (ET-1, 3 pmol). CIH-exposed animals had a significantly greater sympathetic response to ET-1 than did Sham-exposed animals (CIH 137.8 ± 15.6% of baseline; Sham 112.2 ± 10.0 % of baseline; CIH vs. Sham, P = 0.0373). This enhanced sympathetic response to i.c.v. ET-1 was associated with greater expression of endothelin receptor A (ETA) protein in the subfornical organs of CIH-exposed relative to Sham-exposed rats. We conclude that 3-week CIH exposure enhances central ET-1 receptor expression and the sympathetic response to i.c.v. ET-1 suggesting central endothelin may contribute to the sympathetic and hemodynamic response to cyclic intermittent hypoxia.

TNFAIP1 is a protein which can be induced by tumor necrosis factoralpha (TNFalpha) and interleukin-6 (IL-6), it may play roles in DNA synthesis, DNA repair, cell apoptosis and human diseases. However, very little has been known about how TNFAIP1 acts in these physiological processes. In this paper, CK2beta was identified as a partner of TNFAIP1 by screening the HeLa cDNA library in yeast two-hybrid system with TNFAIP1 as a bait. Furthermore, it was demonstrated that CK2 could phosphorylate TNFAIP1 in vitro and in vivo, which facilitated the distribution of TNFAIP1 in nucleus and enhanced its interaction with PCNA. It is suggested that the phosphorylation of TNFAIP1 may be required for its functions.

Mouse zinc finger CCHC domain containing 12 gene (ZCCHC12) has been identified as a transcriptional co-activator of bone morphogenetic protein (BMP) signaling, and human ZCCHC12 was reported to be related to non-syndromic X-linked mental retardation (NS-XLMR). However, the details of how human ZCCHC12 involve in the NS-XLMR still remain unclear. In this study, we identified a novel nuclear localization signal (NLS) in the middle of human ZCCHC12 protein which is responsible for the nuclear localization. Multiple-tissue northern blot analysis indicated that ZCCHC12 is highly expressed in human brain. Furthermore, in situ hybridization showed that ZCCHC12 is specifically expressed in neuroepithelium of forebrain, midbrain, and diencephalon regions of mouse E10.5 embryos. Luciferase reporter assays demonstrated that ZCCHC12 enhanced the transcriptional activities of activator protein 1 (AP-1) and cAMP response element binding protein (CREB) as a co-activator. In conclusion, we identified a new NLS in ZCCHC12 and figured out that ZCCHC12 functions as a transcriptional co-activator of AP-1 and CREB.

Exposure to cyclic intermittent hypoxia (CIH) is associated with elevated arterial pressure and sustained sympathoexcitation, but the causes of the augmented sympathetic activity remain poorly understood. We recorded arterial pressure, heart rate, and renal sympathetic nerve (RSN) activity in conscious rats previously exposed to either CIH or Sham for 3 weeks during acute exposure to hypoxia (15% and 10% O(2)) or hypercapnia (7% CO(2)). Hemodynamic responses to both hypercapnia and hypoxia were similar between CIH-exposed and Sham-exposed rats, although the pattern of response was different for hypoxia (tachycardia with no change in arterial pressure) and hypercapnia (bradycardia and increased arterial pressure). RSN responses as a percent of the baseline were, however, significantly greater in CIH-exposed animals (CIH-exposed: 15% O(2) - 123.4+/-0.06%; 10% O(2) - 136.7+/-0.12%; 7% CO(2) - 138.3+/-0.18%; Sham-exposed: 15% O(2) - 106.6+/-0.03%; 10% O(2) - 107.6+/-0.01%; 7% CO(2) - 103.0+/-0.14% P0.01 for all conditions). These data indicate that in conscious rats exposure to CIH enhances sympathetic responses to both hypoxia and hypercapnia.

Potassium channel tetramerization domain-containing 10 gene (KCTD10) belongs to the polymerase delta-interacting protein 1 (PDIP1) gene family. Mouse KCTD10 was thought to interact with proliferating cell nuclear antigen and the small subunit of polymerase delta, and to have roles in DNA repair, DNA replication and cell-cycle control. To better understand the regulatory mechanism of KCTD10 expression, we characterized the promoter of human KCTD10 containing a 639 bp fragment of the 5'-flanking region (-609/+30). A primer extension assay identified the transcription start site as an A, 63 bp upstream of the start codon. The promoter region contains neither a TATA box nor a CCAAT box, but a CpG island was found near to the transcription start site. Deletion mutagenesis showed that the region from -108 to +30 was indispensable to the promoter activity, and site-directed mutation analysis in this proximal promoter region of KCTD10 revealed two important transcription regulatory elements of specificity protein 1 (SP1) and activating protein-2 (AP-2). An in vivo chromatin immunoprecipitation assay further demonstrated that SP1 and AP-2alpha could bind to this proximal promoter region. Moreover, using a luciferase reporter assay, quantitative real-time RT-PCR and western blot analysis, both overexpression and RNA interference of SP1 and AP-2alpha indicated that the binding of SP1 to the proximal promoter region stimulated the promoter activity and endogenous KCTD10 expression, whereas binding of AP-2alpha to this region showed opposite effects.

Although large quantities of glutamate are found in the carotid body, to date this excitatory neurotransmitter has not been assigned a role in chemoreception. To examine the possibility that glutamate and its N-methyl-d-aspartate (NMDA) receptors play a role in acclimatization after exposure to cyclic intermittent hypoxia (CIH), we exposed male Sprague-Dawley rats to cyclic hypoxia or to room air sham (Sham) for 8 h/day for 3 wk. Using RT-PCR, Western blot analysis, and immunohistochemistry, we found that ionotropic NMDA receptors, including NMDAR1, NMDAR2A, NMDAR2A/2B, are strongly expressed in the carotid body and colocalize with tyrosine hydroxylase in glomus cells. CIH exposure enhanced the expression of NMDAR1 and NMDAR2A/2B but did not substantially change the level of NMDAR2A. We assessed in vivo carotid sinus nerve activity (CSNA) at baseline, in response to acute hypoxia, in response to infused NMDA, and in response to infused endothelin-1 (ET-1) with and without MK-801, an NMDA receptor blocker. Infusion of NMDA augmented CSNA in CIH rats (124.61 ± 2.64% of baseline) but not in sham-exposed rats. Administration of MK-801 did not alter baseline activity or response to acute hypoxia, in either CIH or sham animals but did reduce the effect of ET-1 infusion on CSNA (CSNA after ET-1 = 160.96 ± 8.05% of baseline; ET-1 after MK-801 = 118.56 ± 9.12%). We conclude that 3-wk CIH exposure increases expression of NMDA functional receptors in rats, suggesting glutamate and its receptors may play a role in hypoxic acclimatization to CIH.

Since its discovery in 1998 RNA interference (RNAi), a potent and highly selective gene silencing mechanism, has revolutionized the field of biological science. The ability of RNAi to specifically down-regulate the expression of any cellular protein has had a profound impact on the study of gene function in vitro. This property of RNAi also holds great promise for in vivo functional genomics and interventions against a wide spectrum of diseases, especially those with "undruggable" therapeutic targets. Despite the enormous potential of RNAi for medicine, development of in vivo applications has met with significant problems, particularly in terms of delivery. For effective gene silencing to occur, silencing RNA must reach the cytoplasm of the target cell. Consequently, various strategies using chemically modified siRNA, liposomes, nanoparticles and viral vectors are being developed to deliver silencing RNA. These approaches, however, can be expensive and in many cases they lack target cell specificity or clinical compatibility. Recently, we have shown that RNAi can be activated in vitro and in vivo by non-pathogenic bacteria engineered to manufacture and deliver silencing shRNA to target cells. This new approach, termed TransKingdom RNAi (tkRNAi), has several key advantages. First, tkRNAi may provide a viable means to accomplish therapeutic RNAi since non-pathogenic bacteria have a proven safety record in clinical applications. Second, tkRNAi eliminates the cost of siRNA manufacture since silencing shRNA are produced inside bacteria. Moreover, the intracellular mechanism of shRNA release inherent to tkRNAi may circumvent, or mitigate, the activation of host immune responses. Finally, tkRNAi may facilitate high-throughput in vivo functional genomics screening since bacteria-based RNAi libraries can be easily constructed, stored, reproduced and amplified, thereby allowing for the creation of a stable gene silencing system.

Cequent Pharmaceuticals, Inc. is a recently established biopharmaceutical company that aims to develop clinically compatible therapies based on RNAi, a potent gene-silencing mechanism discovered in 1998. The company’s proprietary technology, transkingdom RNAi (tkRNAi), uses nonpathogenic bacteria to produce and deliver shRNA into target cells to induce RNAi. Our initial focus is on the development of a tkRNAi-based therapy for familial adenatomous polyposis, an inherited form of colon cancer. Cequent’s first tkRNAi-based drug for familial adenatomous polyposis, CEQ501, is currently in advanced preclinical testing. As part of its ongoing activities, Cequent plans to develop additional tkRNAi-based products for indications within and outside the GI tract. Our overall goal is to establish tkRNAi as a platform for developing a wide range of RNAi-based therapies.

Intersectin-2Long (ITSN2L) is a multi-domain protein participating in endocytosis and exocytosis. In this study, RABEP1 was identified as a novel ITSN2L interacting protein using a yeast two-hybrid screen from a human brain cDNA library and this interaction, specifically involving the ITSN2L CC domain and RABEP1 CC3 regions, was further confirmed by in vitro GST (glutathione-S-transferase) pull-down and in vivo co-immunoprecipitation assays. Corroboratively, we observed that these two proteins co-localize in the cytoplasm of mammalian cells. Furthermore, over-expression of ITSN2L promotes RABEP1 degradation and represses RABEP1-enhanced endosome aggregation, indicating that ITSN2L acts as a negative regulator of RABEP1. Finally, we showed that ITSN2L and RABEP1 play opposite roles in regulating endocytosis. Taken together, our results indicate that ITSN2L interacts with RABEP1 and stimulates its degradation in regulation of endocytosis.

Transcription factor AP-2α is involved in many types of human cancers, but its role in hepatocellular carcinogenesis is largely unknown. In this study, we found that expression of AP-2α was low in 40% of human hepatocellular cancers compared with adjacent normal tissues by immunohistochemical analysis. Moreover, AP-2α expression was low or absent in hepatocellular cancer cell lines (HepG2, Hep3B, SMMC-7721 and MHHC 97-H). Human liver cancer cell lines SMMC-7721 and Hep3B stably overexpressing AP-2α were established by lentiviral infection and puromycin screening, and the ectopic expression of AP-2α was able to inhibit hepatocellular cancer cell growth and proliferation by cell viability, MTT assay and liquid colony formation in vitro and in vivo. Furthermore, AP-2α overexpression decreased liver cancer cell migration and invasion as assessed by wound healing and Transwell assays, increasing the sensitivity of liver cancer cells to cisplatin analyzed by MTT assays. Also AP-2α overexpression suppressed the sphere formation and renewed the ability of cancer stem cells. Finally, we found that AP-2α is epigenetically modified and modulates the levels of phosphorylated extracellular signal-regulated protein kinase (ERK), β-catenin, p53, EMT, and CD133 expression in liver cancer cell lines. These results suggested that AP-2α expression is low in human hepatocellular cancers by regulating multiple signaling to affect hepatocellular cancer cell growth and migration. Therefore, AP-2α might represent a novel potential target in human hepatocellular cancer therapy.

Neuroglobin (Ngb) has been demonstrated to be neuroprotective against stroke and neurodegenerative diseases, thus upregulating Ngb might be a novel approach for neuroprotection. In this study we aimed to establish cell-based Ngb reporter systems for screening neuroprotective compounds targeting Ngb upregulation. We developed both mouse and human stable Ngb reporter systems containing a luciferase reporter gene directed by mouse and human Ngb promoter, respectively. To validate these reporter systems, we used them to screen a pool of natural plant compounds. RT-PCR was used to verify the Ngb-upregulating effects of selected compounds, and neurotoxicity assay was used to test their neuroprotection effects in primary cultured neurons. We identified polydatin, genistein, daidzein, biochanin A and formononetin that can upregulate both mouse and human Ngb promoter activity. RT-PCR confirmed that polydatin, genistein and formononetin significantly increased Ngb mRNA expression in primary neurons. Furthermore, formononetin significantly decreased oxygen-glucose deprivation (OGD)-induced neurotoxicity. Moreover, inhibition of cAMP response element-binding protein (CREB) showed that CREB is required for formononetin-induced Ngb upregulation. These results suggest that these Ngb reporter systems are suitable for neuroprotective compound screening, which will be used to screen larger compound libraries for more potent neuroprotectants. This preliminary study will facilitate the development of Ngb-targeted therapeutics for stroke and neurodegenerative diseases.

MicroRNAs (miRNAs) are critical post-transcriptional regulators. Based on a previous genome-wide association (GWA) scan, we conducted a polymorphism in microRNA target sites (poly-miRTS)-centric multistage meta-analysis for lumbar spine (LS)-, total hip (HIP)- and femoral neck (FN)-bone mineral density (BMD). In stage I, 41 102 poly-miRTSs were meta-analyzed in seven cohorts with a genome-wide significance (GWS) α = 0.05/41 102 = 1.22 × 10(-6). By applying α = 5 × 10(-5) (suggestive significance), 11 poly-miRTSs were selected, with FGFRL1 rs4647940 and PRR5 rs3213550 as top signals for FN-BMD (P = 7.67 × 10(-6) and 1.58 × 10(-5)) in gender-combined sample. In stage II in silico replication (two cohorts), FGFRL1 rs4647940 was the only signal marginally replicated for FN-BMD (P = 5.08 × 10(-3)) at α = 0.10/11 = 9.09 × 10(-3). PRR5 rs3213550 was also selected based on biological significance. In stage III de novo genotyping replication (two cohorts), FGFRL1 rs4647940 was the only signal significantly replicated for FN-BMD (P = 7.55 × 10(-6)) at α = 0.05/2 = 0.025 in gender-combined sample. Aggregating three stages, FGFRL1 rs4647940 was the single stage I-discovered and stages II- and III-replicated signal attaining GWS for FN-BMD (P = 8.87 × 10(-12)). Dual-luciferase reporter assays demonstrated that FGFRL1 3' untranslated region harboring rs4647940 appears to be hsa-miR-140-5p's target site. In a zebrafish microinjection experiment, dre-miR-140-5p is shown to exert a dramatic impact on craniofacial skeleton formation. Taken together, we provided functional evidence for a novel FGFRL1 poly-miRTS rs4647940 in a previously known 4p16.3 locus, and experimental and clinical genetics studies have shown both FGFRL1 and hsa-miR-140-5p are important for bone formation.

Over the last few years, RNA Interference (RNAi), a naturally occurring mechanism of gene regulation conserved in plant and mammalian cells, has opened numerous novel opportunities for basic research across the field of biology. While RNAi has helped accelerate discovery and understanding of gene functions, it also has great potential as a therapeutic and potentially prophylactic modality. Challenging diseases failing conventional therapeutics could become treatable by specific silencing of key pathogenic genes. More specifically, therapeutic targets previously deemed "undruggable" by small molecules, are now coming within reach of RNAi based therapy. For RNAi to be effective and elicit gene silencing response, the double-stranded RNA molecules must be delivered to the target cell. Unfortunately, delivery of these RNA duplexes has been challenging, halting rapid development of RNAi-based therapies. In this review we present current advancements in the field of siRNA delivery methods, including the pros and cons of each method.

The etiology and significance of genomic instability (GIN), a hallmark of human cancers, remains controversial. The paradigm that inactivation of tumor suppressors [e.g. p53 or adenomatous polyposis coli (APC) genes] leads to GIN is largely based on experiments in vitro and in animal models. It remains unclear whether GIN is a cause or a result of cancer, particularly in patients. Precancerous Barrett's esophagus (BE) provides a clinical model to investigate GIN in cancer progression. We analyzed specimens from endoscopic biopsies or esophagectomies in patients with BE (ten cases, including five cases with multilayered epithelium (ME)), BE-associated esophageal adenocarcinoma (ten cases), or with normal gastro-esophageal junction (five cases). Chromosomal enumeration probe Cep 7, 11, 12, 17 and 18 were detected by fluorescence in situ hybridization (FISH). Expression of p53 and APC were determined by immunohistochemistry. Increased p53 expression, a measurement of p53 mutations, was observed in BE with high grade dysplasia (HGD) and in BE-associated esophageal cancer (EC). The expression of wild type APC was decreased in BE with HGD and in advanced EC. Chromosomal abnormalities were found in all EC samples. Numeric changes of chromosome 7, 11 and 12 were observed in BE in 14%, 64% and 43% of cases, respectively. Aneusomy of chromosome 11 and 12 were found in ME and in BE without dysplasia, in the presence of normal expression pattern of p53 and APC. Our results suggest that GIN is an early event that occurs at precancerous stages prior to changes in tumor suppressor genes (p53 and APC) in BE-associated tumorigenesis in patients, suggesting that GIN may serve as a causative link between chronic inflammation and cancer.

As a member of the polymerase delta-interacting protein 1 (PDIP1) gene family, potassium channel tetramerisation domain-containing 10 (KCTD10) interacts with proliferating cell nuclear antigen (PCNA) and polymerase δ, participates in DNA repair, DNA replication and cell-cycle control. In order to further investigate the physiological functions of KCTD10, we generated the KCTD10 knockout mice. The heterozygous KCTD10(+/-) mice were viable and fertile, while the homozygous KCTD10(-/-) mice showed delayed growth from E9.0, and died at approximately E10.5, which displayed severe defects in angiogenesis and heart development. Further study showed that VEGF induced the expression of KCTD10 in a time- and dose-dependent manner. Quantitative real-time PCR and western blotting results revealed that several key members in Notch signaling were up-regulated either in KCTD10-deficient embryos or in KCTD10-silenced HUVECs. Meanwhile, the endogenous immunoprecipitation (IP) analysis showed that KCTD10 interacted with Cullin3 and Notch1 simultaneously, by which mediating Notch1 proteolytic degradation. Our studies suggest that KCTD10 plays crucial roles in embryonic angiogenesis and heart development in mammalians by negatively regulating the Notch signaling pathway.

To date, tissue type plasminogen activator (tPA)-based thrombolytic stroke therapy is the only FDA-approved treatment for achieving vascular reperfusion and clinical benefit, but this agent is given to only about 5% of stroke patients in the USA. This may be related, in part, to the elevated risk of symptomatic intracranial hemorrhage, and consequently limited therapeutic time window. Clinical investigations demonstrate that poststroke hyperglycemia is one of the most important risk factors that cause intracerebral hemorrhage and worsen neurological outcomes. There is a knowledge gap in understanding the underlying molecular mechanisms, and lack of effective therapeutics targeting the severe complication. This short review summarizes clinical observations and experimental investigations in preclinical stroke models of the field. The data strongly suggest that interactions of multiple pathogenic factors including hyperglycemia-mediated vascular oxidative stress and inflammation, ischemic insult, and tPA neurovascular toxicity in concert contribute to the BBB damage-intracerebral hemorrhagic transformation process. Development of combination approaches targeting the multiple pathological cascades may help to attenuate the hemorrhagic complication.

To date, tissue type plasminogen activator (tPA)-based thrombolytic stroke therapy is the only FDA-approved treatment for achieving vascular reperfusion and clinical benefit, but this agent is given to only about 5% of stroke patients in the USA. This may be related, in part, to the elevated risk of symptomatic intracranial hemorrhage, and consequently limited therapeutic time window. Clinical investigations demonstrate that poststroke hyperglycemia is one of the most important risk factors that cause intracerebral hemorrhage and worsen neurological outcomes. There is a knowledge gap in understanding the underlying molecular mechanisms, and lack of effective therapeutics targeting the severe complication. This short review summarizes clinical observations and experimental investigations in preclinical stroke models of the field. The data strongly suggest that interactions of multiple pathogenic factors including hyperglycemia-mediated vascular oxidative stress and inflammation, ischemic insult, and tPA neurovascular toxicity in concert contribute to the BBB damage-intracerebral hemorrhagic transformation process. Development of combination approaches targeting the multiple pathological cascades may help to attenuate the hemorrhagic complication.

Transcription factor activator protein 2α (AP-2α) is a negative regulator of adipogenesis by repressing the transcription of CCAAT/enhancer binding protein (C/EBPα) gene. During adipogenesis, AP-2α is degraded, leading to transcriptional up-regulation of C/EBPα. However, the mechanism for AP-2α degradation is not clear. Here, using immunoprecipitation assay and mass spectrometry, we identified ring finger protein 20 (RNF20) as an AP-2α-interacting protein in 3T3-L1 preadipocytes. RNF20 has been proved to be an E3 ubiquitin ligase for both histone H2B and tumor suppressor ErbB3-binding protein 1 (Ebp1). In this study, we demonstrated that RNF20 co-localized and interacted with AP-2α, and promoted its polyubiquitination and proteasome-dependent degradation. Over-expression of RNF20 inhibited the activity of AP-2α and rescued the C/EBPα expression which was inhibited by AP-2α. These results suggested that RNF20 may play roles in adipocyte differentiation by stimulating ubiquitin-proteasome-dependent degradation of AP-2α.

The canonical Wnt signaling pathway controls normal embryonic development, cellular proliferation and growth, and its aberrant activity results in human carcinogenesis. The core component in regulation of this pathway is β-catenin, but molecular regulation mechanisms of β-catenin stability are not completely known. Here, our recent studies have shown that KCTD1 strongly inhibits TCF/LEF reporter activity. Moreover, KCTD1 interacted with β-catenin both in vivo by co-immunoprecipitation as well as in vitro through GST pull-down assays. We further mapped the interaction regions to the 1-9 armadillo repeats of β-catenin and the BTB domain of KCTD1, especially Position Ala-30 and His-33. Immunofluorescence analysis indicated that KCTD1 promotes the cytoplasmic accumulation of β-catenin. Furthermore, protein stability assays revealed that KCTD1 enhances the ubiquitination/degradation of β-catenin in a concentration-dependent manner in HeLa cells. And the degradation of β-catenin mediated by KCTD1 was alleviated by the proteasome inhibitor, MG132. In addition, KCTD1-mediated β-catenin degradation was dependent on casein kinase 1 (CK1)- and glycogen synthase kinase-3β (GSK-3β)-mediated phosphorylation and enhanced by the E3 ubiquitin ligase β-transducin repeat-containing protein (β-TrCP). Moreover, KCTD1 suppressed the expression of endogenous Wnt downstream genes and transcription factor AP-2α. Finally, we found that Wnt pathway member APC and tumor suppressor p53 influence KCTD1-mediated downregulation of β-catenin. These results suggest that KCTD1 functions as a novel inhibitor of Wnt signaling pathway.

Neuroglobin (Ngb) has been demonstrated to be a novel neuroprotective protein that protects against hypoxia/ischemia and oxidative stress-induced injury in the nervous system. However, the regulation mechanisms of Ngb gene expression under both normal resting and stress conditions have not been fully elucidated. The cyclic AMP response element binding protein (CREB) is a key transcription factor that regulates a variety of pro-survival genes, but its role in regulating the neuroprotective gene Ngb has not been studied. In this study we investigated the transcriptional regulation of mouse Ngb gene by CREB in mouse neuroblastoma cell line N2a. Our results showed that CREB knockdown decreased Ngb gene expression, and overexpression of the wild-type CREB, but not the mutant CREB, significantly increased Ngb gene expression in N2a cells. Moreover, a cAMP response element (CRE) site located at −854 in the promoter region of mouse Ngb gene was found to be responsible for both basal and CREB-induced Ngb promoter activity. Using chromatin immunopreciptation (ChIP) assays, we found that CREB could bind to the Ngb promoter region spanning from −1016 to −793 that harbors the CRE site. Taken together, our results suggested that transcription factor CREB participates in the transcriptional regulation of mouse Ngb gene.

Eps8 was initially identified as a substrate of the epidermal growth factor receptor. Overexpression of Eps8 leads to increased mitogenic signaling and malignant transformation. However, little is known concerning the importance of Eps8 in human gliomas. In this study, we found that Eps8 was overexpressed in 56.6% of human gliomas (WHO grades III and IV) compared with adjacent normal brain tissues by immunohistochemical analysis. The U251 human glioma cell line stably expressing Eps8 was established by G418 screening, and the ectopic expression of Eps8 enhanced U251 glioma cell growth and survival by cell survival, MTT and liquid colony formation assays. By contrast, the lentiviral expression of Eps8 siRNA in SHG-44 cells resulted in a significant reduction in cellular growth and proliferation. Furthermore, Eps8 modulated the levels of phosphorylated extracellular signal-regulated protein kinase (ERK), phosphorylated serine-threonine protein kinase Akt and β-catenin expression in glioma cell lines and tissues. These results suggest that Eps8 is overexpressed in human gliomas, and affects glioma cell growth possibly by regulating ERK and Akt/β-catenin signaling. Therefore, Eps8 may represent a novel potential target in human glioma therapy.

Recent studies indicate that cancer stem cells (CSCs) exist in most hematological and solid tumors. CSCs are characterized by their ability to self-renew and their capacity to differentiate into the multitude of cells that comprise the tumor mass. Moreover, these cells have been shown to be intrinsically resistant to conventional anticancer therapies. Despite their fundamental role in cancer pathogenesis, the cellular origin of CSCs remains highly controversial. The aim of this study was to examine whether heterogeneous cancer cells can acquire stem cell-like properties in response to chemotherapy. We demonstrate that carboplatin can induce the self-renewal (spherogenesis) and pluripotency (Sox2 and Oct3/4 expression) of hepatocellular carcinoma (HCC) cells grown under stem cell culture conditions. Moreover, we show that non-CSC cells, obtained by side population flow cytometric sorting using Hoechst 33342, can acquire stem-like properties after exposure to carboplatin. Finally, we show that knockdown of Sox2 and Oct3/4 gene expression in HCC cells can reduce carboplatin-mediated increases in sphere formation and increase cellular sensitivity to chemotherapy. Taken together, our data indicate that bulk cancer cells may be an important source of CSCs during tumor development, and that targeting Sox2 and/or Oct3/4 may be a promising approach for targeting CSCs in clinical cancer treatment.

The broad-complex, tramtrack, and bric-a-brac/poxvirus and zinc finger domain-containing protein tumor necrosis factor, alpha-induced protein 1 (TNFAIP1) was first identified as a gene whose expression can be induced by the tumor necrosis factor alpha. Some studies showed that TNFAIP1 may function in DNA replication, apoptosis and human diseases. However, the definite functions and the mechanisms of TNFAIP1 are poorly known. In this study, we performed a yeast two-hybrid assay and used TNFAIP1 as the bait to screen human brain cDNA library. Potassium channel tetramerisation domain containing 10 (KCTD10) was identified as TNFAIP1-interacting partner. The KCTD10–TNFAIP1 interaction was then confirmed by the in vitro GST pull-down assays and the in vivo co-immunoprecipitation and colocalization assays. In addition, protein degradation and ubiquitin assays revealed TNFAIP1 overexpression resulted in ubiquitin-mediated degradation of KCTD10 proteins, which was significantly alleviated with the proteasome inhibitor MG132 treatment. Furthermore, transient transfection assays with two reporters showed that TNFAIP1 and KCTD10 inhibited the transcriptional activities of nuclear factor kappa B (NF-κB) and activating protein-1 reporters. Taken together, our results indicated the novel interaction and function between KCTD10 and TNFAIP1 in human PDIP1 family.

Yachun Liu1,2,*, Tao Wang1,*, Fangli He1,*, Qian Liu1,*, Dexi Zhang2, Shuanglin Xiang1, Shengpei Su2, Jian Zhang11Key Laboratory of Protein Chemistry and Developmental Biology, Ministry of Education of China, College of Life Sciences; 2Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research and Key Laboratory of Sustainable Resources Processing and Advanced Materials of Hunan Province, Ministry of Education of China, College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, Hunan, China*These authors contributed equally to this workBackground: Smaller nanoparticles facilitate the delivery of DNA into cells through endocytosis and improve transfection efficiency. The aim of this study was to determine whether protamine sulfate-coated calcium phosphate (PS-CaP) could stabilize particle size and enhance transfection efficiency.Methods: pEGFP-C1 green fluorescence protein was employed as an indicator of transfection efficiency. Atomic force microscopy was used to evaluate the morphology and the size of the particles, and an MTT assay was introduced to detect cell viability and inhibition. The classical calcium phosphate method was used as the control.Results: Atomic force microscopy images showed that the PS-CaP were much smaller than classical calcium phosphate particles. In 293 FT, HEK 293, and NIH 3T3 cells, the transfection efficiency of PS-CaP was higher than for the classical calcium phosphate particles. The difference in efficiencies implies that the smaller nanoparticles may promote the delivery of DNA into cells through endocytosis and could improve transfection efficiency. In addition, PS-CaP could be used to transfect HEK 293 cells after one week of storage at 4°C with a lesser extent of efficiency loss compared with classical calcium phosphate, indicating that protamine sulfate may increase the stability of calcium phosphate nanoparticles. The cell viability inhibition assay indicated that both nanoparticles show similar low cell toxicity.Conclusion: PS-CaP can be used as a better nonviral transfection vector compared with classical calcium phosphate.Keywords: transfection efficiency, particle size, protamine sulfate, stability

RNA interference (RNAi) is a potent and specific mechanism for eliminating the mRNA of specific genes. This gene silencing mechanism occurs naturally and is highly conserved from plants to human cells, holding promise for functional genomics and for revolutionizing medicine due to its unlimited potential to treat genetic, epigenetic, and infectious disease. However, efforts to unleash the enormous potential of RNAi have met with significant challenges. Delivery is problematic because short interfering RNAs (siRNA) are negatively charged polymers that inefficiently enter cells and undergo rapid enzymatic degradation in vivo. In addition, the synthesis of siRNAs is expensive for long-term research and therapeutic applications. Recently, we have shown that nonpathogenic bacteria can be engineered to activate RNAi in mammalian cells (TransKingdom RNA interference; tkRNAi). This new approach offers several advantages and has significant implications. First, this method allows the establishment of a long-term stable gene silencing system in the laboratory against genes of interests in vitro and in vivo, and enables high-throughput functional genomics screening in mammalian systems. RNAi libraries can be constructed, stored, reproduced, amplified, and used with the help of E. coli as currently done with gene cloning. Second, this technology provides a clinically compatible way to achieve RNAi for therapeutic applications due to the proven clinical safety ofnonpathogenic bacteria as a gene carrier, tkRNAi also eliminates the siRNA manufacture issue, and may circumvent or mitigate host interferon-like responses since siRNA is produced intracellularly.

Tumor necrosis factor, alpha-induced protein 1 (TNFAIP1) is an immediate-early response gene of endothelium induced by TNF alpha. However, little is really known concerning the TNFAIP1 expression regulation. To better understand how TNFAIP1 expression is regulated, we functionally characterized the promoter region of human TNFAIP1 gene. Deletion mutation analysis, gel electrophoretic mobility shift, and site-directed mutagenesis assays allowed the identification of one functional Sp1-binding site within the human TNFAIP1 core promoter region. Moreover, chromatin immunoprecipitation analysis indicated that Sp1 was associated in vivo with the TNFAIP1 promoter. Further, Sp1 overexpression enhanced TNFAIP1 promoter activity. These findings suggest that Sp1 is implicated in the control of basal TNFAIP1 gene expression. Accordingly, Sp1 is supposed to be involved in the elevation of TNFAIP1 in response to TNF alpha induction, and thus participate in inflammation-associated angiogenesis.

Potassium channel tetramerization domain-containing 10 gene (KCTD10) belongs to the polymerase delta-interacting protein 1 (PDIP1) gene family. Mouse KCTD10 was thought to interact with proliferating cell nuclear antigen and the small subunit of polymerase delta, and to have roles in DNA repair, DNA replication and cell-cycle control. To better understand the regulatory mechanism of KCTD10 expression, we characterized the promoter of human KCTD10 containing a 639 bp fragment of the 5'-flanking region (-609/+30). A primer extension assay identified the transcription start site as an A, 63 bp upstream of the start codon. The promoter region contains neither a TATA box nor a CCAAT box, but a CpG island was found near to the transcription start site. Deletion mutagenesis showed that the region from -108 to +30 was indispensable to the promoter activity, and site-directed mutation analysis in this proximal promoter region of KCTD10 revealed two important transcription regulatory elements of specificity protein 1 (SP1) and activating protein-2 (AP-2). An in vivo chromatin immunoprecipitation assay further demonstrated that SP1 and AP-2alpha could bind to this proximal promoter region. Moreover, using a luciferase reporter assay, quantitative real-time RT-PCR and western blot analysis, both overexpression and RNA interference of SP1 and AP-2alpha indicated that the binding of SP1 to the proximal promoter region stimulated the promoter activity and endogenous KCTD10 expression, whereas binding of AP-2alpha to this region showed opposite effects.

RNA interference (RNAi) is a potent and specific mechanism for eliminating the mRNA of specific genes. This gene silencing mechanism occurs naturally and is highly conserved from plants to human cells, holding promise for functional genomics and for revolutionizing medicine due to its unlimited potential to treat genetic, epigenetic, and infectious disease. However, efforts to unleash the enormous potential of RNAi have met with significant challenges. Delivery is problematic because short interfering RNAs (siRNA) are negatively charged polymers that inefficiently enter cells and undergo rapid enzymatic degradation in vivo. In addition, the synthesis of siRNAs is expensive for long-term research and therapeutic applications. Recently, we have shown that nonpathogenic bacteria can be engineered to activate RNAi in mammalian cells (TransKingdom RNA interference; tkRNAi). This new approach offers several advantages and has significant implications. First, this method allows the establishment of a long-term stable gene silencing system in the laboratory against genes of interests in vitro and in vivo, and enables high-throughput functional genomics screening in mammalian systems. RNAi libraries can be constructed, stored, reproduced, amplified, and used with the help of E. coli as currently done with gene cloning. Second, this technology provides a clinically compatible way to achieve RNAi for therapeutic applications due to the proven clinical safety ofnonpathogenic bacteria as a gene carrier, tkRNAi also eliminates the siRNA manufacture issue, and may circumvent or mitigate host interferon-like responses since siRNA is produced intracellularly.

Signal regulatory protein α (SIRPα) is a critical immune inhibitory receptor on macrophages, and its interaction with CD47, a ligand for SIRPα, prevents autologous phagocytosis. We hypothesized that interspecies incompatibility of CD47 may contribute to the rejection of xenogeneic cells by macrophages. Here, we show that pig CD47 does not interact with mouse SIPRα. Similar to CD47−/− mouse cells, porcine red blood cells (RBCs) failed to induce SIRPα tyrosine phosphorylation in mouse macrophages. Blocking SIRPα with antimouse SIRPα mAb (P84) significantly enhanced the phagocytosis of CD47+/+ mouse cells, but did not affect the engulfment of porcine or CD47−/− mouse cells by mouse macrophages. CD47-deficient mice, whose macrophages do not phagocytose CD47−/− mouse cells, showed markedly delayed clearance of porcine RBCs compared with wild-type mouse recipients. Furthermore, mouse CD47 expression on porcine cells markedly reduced their phagocytosis by mouse macrophages both in vitro and in vivo. These results indicate that interspecies incompatibility of CD47 contributes significantly to phagocytosis of xenogeneic cells by macrophages and suggest that genetic manipulation of donor CD47 to improve its interaction with the recipient SIRPα may provide a novel approach to prevent phagocyte-mediated xenograft rejection.

RNA-interference (RNAi) is a potent mechanism, conserved from plants to humans for specific silencing of genes, which holds promise for functional genomics and gene-targeted therapies. Here we show that bacteria engineered to produce a short hairpin RNA (shRNA) targeting a mammalian gene induce trans-kingdom RNAi in vitro and in vivo. Nonpathogenic Escherichia coli were engineered to transcribe shRNAs from a plasmid containing the invasin gene Inv and the listeriolysin O gene HlyA, which encode two bacterial factors needed for successful transfer of the shRNAs into mammalian cells. Upon oral or intravenous administration, E. coli encoding shRNA against CTNNB1 (catenin beta-1) induce significant gene silencing in the intestinal epithelium and in human colon cancer xenografts in mice. These results provide an example of trans-kingdom RNAi in higher organisms and suggest the potential of bacteria-mediated RNAi for functional genomics, therapeutic target validation and development of clinically compatible RNAi-based therapies.