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Molecular and Cellular Biochemistry: An International Journal for Chemical Biology in Health and Disease (v.342, #1-2)

Growth inhibition, morphology change, and cell cycle alterations in NFBD1-depleted human esophageal cancer cells by Zhengmei Yang; Youquan Bu; Changdong Wang; Geli Liu; Fangzhou Song (pp. 1-6).
NFBD1/MDC1 is a large nuclear protein mainly participating in DNA damage response, indicating its therapeutic potential as a radio-/chemosensitizer target in cancer field. Esophageal cancer ranks among one of the most frequent cause of cancer death in the world. In this study, we used three representative esophageal cancer cell lines to investigate the effects of NFBD1 silencing on cell proliferation, cell morphology, and cell cycle distribution. Synthetic small interfering RNA (siRNA) duplexes against NFBD1 were introduced into three esophageal cancer cell lines, which subsequently resulted in a significant inhibition in NFBD1 expression in the cells. Our results have shown that a targeted siRNA depletion of NFBD1 resulted in a significant growth inhibition, morphology change, and cell cycle alterations in esophageal cancer cells. Furthermore, NFBD1 depletion also sensitized all the three esophageal cancer cell lines to chemotherapeutic agents including adriamycin and cisplatin. Taken together, our study strongly suggested that NFBD1 may serve as a potential therapeutic target in human esophageal cancer.

Keywords: NFBD1; Esophageal cancer; RNA silencing; Cell cycle; Cell proliferation

Role of Rho kinase in sphingosine 1-phosphate-mediated endothelial and smooth muscle cell migration and differentiation by Kevin A. Harvey; Zachary Welch; Daniel Sliva; Rafat A. Siddiqui (pp. 7-19).
The role of sphingosine 1-phosphate (S1P)-induced Rho kinase (ROCK) activation in the angiogenic responses of pulmonary artery-derived endothelial cells (PAEC) and smooth muscle cells (PASMC) was examined. S1P, a biologically active phospholipid that regulates angiogenesis, promoted PAEC chemotaxis and capillary morphogenesis; furthermore, this activity was unaltered by pretreatment with the pharmacological inhibitor of ROCK, H1152. In contrast, S1P (500 nM) significantly inhibited spontaneous PASMC chemotaxis and differentiation; however, this inhibition was eradicated upon H1152 pretreatment. Similarly, PASMCs transfected with ROCK II siRNA diminished S1P-induced inhibition of the development of multi-cellular structures. Analysis by RT-PCR identified the presence of S1P1 and S1P3 receptors on both PAECs and PASMCs, while S1P2 receptor expression was confined to only PASMCs. Consistent with this observation, the S1P1 and S1P3 receptor antagonist, VPC23019, virtually abolished the S1P-initiated PAEC differentiation but did not impede the S1P-induced inhibition of PASMC differentiation. However, the S1P2 receptor antagonist, JTE013, had no effect on S1P-mediated differentiation of PAECs but abolished the S1P-induced inhibition of PASMC function. Co-cultured endothelial and smooth muscle cells differentiated into “neovascular-like” networks, which were significantly inhibited by S1P. The inhibition of co-culture differentiation in both PAECs and PASMCs was negated by H1152 pretreatment. However, when smooth muscle cells were added to S1P-initiated endothelial cell networks, additional S1P treatment did not inhibit the cellular networks generated by these cells. In conclusion, S1P-induced PAEC angiogenic responses are regulated by S1P1 and/or S1P3 receptors independent of Rho kinase activation, whereas S1P2 receptor-mediated curtailment of PASMC function by S1P.

Keywords: Sphingosine 1-phosphate; Rho kinase; Smooth muscle cells; Endothelial cells; Angiogenesis

Age-related deregulation of Aire and peripheral tissue antigen genes in the thymic stroma of non-obese diabetic (NOD) mice is associated with autoimmune type 1 diabetes mellitus (DM-1) by Thaís A. Fornari; Paula B. Donate; Claudia Macedo; Márcia M. C. Marques; Danielle A. Magalhães; Geraldo A. S. Passos (pp. 21-28).
Gene expression of peripheral tissue antigens (PTAs) in stromal medullary thymic epithelial cells (mTECs) is a key process to the negative selection of autoreactive thymocytes. This phenomenon was termed “promiscuous gene expression” (PGE), which is partially controlled by the Aire gene. Nevertheless, reasons for the correlation of Aire and PTAs with the emergence of autoimmune diseases are largely unknown, though it may be a result of a chronological effect. Although the effect of Aire mutations in pathogenic autoimmunity is well know, it could not be a unique cause for autoimmunity. Independently of mutations, temporal deregulation of Aire expression may imbalance Aire-dependent PTAs and/or wide PGE. This deregulation may be an early warning sign for autoimmune diseases as it guarantees autoantigen representation in the thymus. To assess this hypothesis, we studied the expression levels of Aire, Aire-dependent (Ins2) and Aire-independent (Gad67 and Col2a1) PTAs using real-time-PCR of the thymic stromal cells of NOD mice during the development of autoimmune type 1 diabetes mellitus (DM-1). Wide PGE was studied by microarrays in which the PTA genes were identified through parallel CD80+ mTEC 3.10 cell line expression profiling. The results show that Aire gene was down-regulated in young pre-autoimmune (pre-diabetic) NOD mice. PGE and specific PTA genes were down-regulated in adult autoimmune diabetic animals. These findings represent evidence indicating that chronological deregulation of genes important to negative selection may be associated with the development of an autoimmune disease (DM-1) in mice.

Keywords: Aire gene; Chronological gene expression; Non-obese diabetic (NOD); Thymic stromal cells; Type 1 diabetes mellitus

Novel 1-alkyl-tryptophan derivatives downregulate IDO1 and IDO2 mRNA expression induced by interferon-gamma in dendritic cells by Ting Sun; Xiang-Hua Chen; Zheng-De Tang; Jiong Cai; Xiao-Yan Wang; Shih-Chen Wang; Zhao-Long Li (pp. 29-34).
Indoleamine 2,3-dioxygenase (IDO) is an enzyme that suppresses adaptive T-cell immunity by catabolizing tryptophan from the cellular microenvironment. Inhibition of IDO pathway might enhance the efficacy of immunotherapeutic strategies for cancer. We synthesized 1-alkyl-tryptophan targeted IDO inhibitors and compared their effects on IDO expression and activity in dendritic cells (DCs) with the common IDO inhibitor 1-methyl-dl-tryptophan (1-MT). The IDO gene expression was examined by RT-PCR and realtime PCR. The toxicity of these analogs on the proliferation of DCs was detected by MTT assay. All of these analogs inhibited IDO expression and activity induced by IFN-γ and showed no cytotoxicity to DCs at 100 μM. 1-MT intensively suppressed IDO1 expression and activity in DCs, and 1-propyl-tryptophan (1-PT) and 1-isopropyl-tryptophan (1-isoPT) moderately inhibited them. 1-Butyl-tryptophan (1-BT) and 1-ethyl-tryptophan (1-ET) mainly inhibited IDO2 expression. Our results suggest that those analogs differed in their inhibitory activity on IDO expression may give us a clue for developing active IDO inhibitors.

Keywords: 1-Alkyl-tryptophan; 1-Methyl-dl-tryptophan; Indoleamine 2,3-dioxygenase; Inhibitor

MK-801 effect on regional cerebral oxidative stress rate induced by different duration of global ischemia in gerbils by Vesna Selakovic; Branka Janac; Lidija Radenovic (pp. 35-50).
We investigated MK-801 effect on ischemia-induced oxidative stress—the most important factor that exacerbates brain damage by reperfusion. The common carotid arteries of gerbils were occluded for 5, 10, or 15 min. Immediately after the occlusion, MK-801 (3 mg/kg i.p.) or saline were given in normothermic conditions. The MK-801 effects were followed in vivo by monitoring the neurological status of animals and at the intracellular level by standard biochemical assays. We investigated nitric oxide levels, superoxide production, superoxide dismutase activity, index of lipid peroxidation (ILP), and reduced glutathione content in hippocampus, striatum, forebrain cortex, and cerebellum. The measurements took place at different times (1, 2, 4, 7, 14, and 28 days) after reperfusion. Increased duration of cerebral ischemia resulted in a progressive induction of oxidative stress. Our results revealed pattern of dynamic changes in each oxidative stress parameter level which corresponded with ischemia duration in all tested brain structures. Most sensitive oxidative stress parameters were ILP and superoxide production. Our study confirmed spatial distribution of ischemia-induced oxidative stress. Tested brain structures showed different sensitivity to each oxidative stress parameter. As judged by biochemical and neurological data, applied MK-801 showed neuroprotective efficiency by reduction of ischemia-induced oxidative stress in brain.

Keywords: Brain; Gerbils; Nitric oxide; NMDA; Reactive oxygen species; Stroke

In vitro effects of silver nanoparticles on the mitochondrial respiratory chain by Cláudio Sérgio Costa; João Vitor Vieira Ronconi; Juliana Felipe Daufenbach; Cinara Ludvig Gonçalves; Gislaine Tezza Rezin; Emilio Luiz Streck; Marcos Marques da Silva Paula (pp. 51-56).
Silver has been used for years in medicine; it has known antimicrobial properties. Additionally, silver has been used in water and air filtration to eliminate microorganisms, and, more recently, as a biocide to prevent infections in burns. In contact with the human body, nanoparticles can elicit a spectrum of tissue responses such as the generation of reactive oxygen species, decreased function of mitochondria and even cell death. Mitochondries are intracellular organelles that play a crucial role in ATP production. In the present work, we evaluate the in vitro effect of silver nanoparticles (AgN) on the activities of mitochondrial respiratory chain complexes from the brain, skeletal muscle, heart, and liver of rats. Our results demonstrated that AgN (10, 25, and 50 mg l−1) decreases the activity of mitochondrial respiratory chain complexes I, II, III, and IV from all tissues.

Keywords: Silver nanoparticles; Mitochondrial respiratory chain; Brain; Skeletal muscle; Heart

Targeted Sprouty1 overexpression in cardiac myocytes does not alter myocardial remodeling or function by Nathan J. Charles; Robert C. Huebert; Sangjin Lee; Neeta Adhikari; Sean Polster; James E. Rider; Elizabeth Braunlin; Ami Mariash; Maggie Robledo; David Schuweiler; Jennifer L. Hall (pp. 57-62).
The mitogen activated protein kinase (MAPK) signaling pathway regulates multiple events leading to heart failure including ventricular remodeling, contractility, hypertrophy, apoptosis, and fibrosis. The regulation of conserved intrinsic inhibitors of this pathway is poorly understood. We recently identified an up-regulation of Sprouty1 (Spry1) in a targeted approach for novel inhibitors of the MAPK signaling pathway in failing human hearts following reverse remodeling. The goal of this study was to test the hypothesis that up-regulated expression of Spry1 in cardiac myocytes would be sufficient to inhibit ERK1/2 activation and tissue remodeling. We established a murine model with up-regulated Spry1 expression in cardiac myocytes using the alpha-myosin heavy chain promoter (α-MHC). Heart weight and cardiac myocyte morphology were unchanged in adult male α-MHC–Spry1 mice compared to control mice. Ventricular function of α-MHC–Spry1 mice was unaltered at 8 weeks or 1 year of age. These findings were consistent with the lack of an effect of Spry1 on ERK1/2 activity. In summary, targeted up-regulation of Spry1 in cardiac myocytes is not sufficient to alter cell or tissue remodeling consistent with the lack of an effect on ERK1/2 activity.

Keywords: Spry1; Myocyte; Echocardiography; MAPkinase

17β-Estradiol inhibits prostaglandin E2-induced COX-2 expressions and cell migration by suppressing Akt and ERK1/2 signaling pathways in human LoVo colon cancer cells by Tung-Yuan Lai; Li-Mien Chen; Jing-Ying Lin; Bor-Show Tzang; James A. Lin; Chang-Hai Tsai; Yueh-Min Lin; Chih-Yang Huang; Chung-Jung Liu; Hsi-Hsien Hsu (pp. 63-70).
Epidemiological studies demonstrate that the incidence and mortality rates of colorectal cancer in women are lower than in men. However, it is unknown if 17β-estradiol treatment is sufficient to inhibit prostaglandin E2 (PGE2)-induced cellular motility in human colon cancer cells. Upregulation of cyclooxygenase-2 (COX-2) is reported to associate with the development of cancer cell mobility, metastasis, and subsequent malignant tumor. After administration of inhibitors including LY294002 (Akt activation inhibitor), U0126 (ERK1/2 inhibitor), SB203580 (p38 MAPK inhibitor), SP600125 (JNK1/2 inhibitor), or QNZ (NFκB inhibitor), we found that PGE2 treatment increases COX-2 via Akt and ERK1/2 pathways, thus promoting cellular motility in human LoVo cancer cells. We further observed that 17β-estradiol treatment inhibits PGE2-induced COX-2 expression and cellular motility via suppressing activation of Akt and ERK1/2 in human LoVo cancer cells. Collectively, these results suggest that 17β-estradiol treatment dramatically inhibits PGE2-induced progression of human LoVo colon cancer cells.

Keywords: Estrogen; Prostaglandin E2; Human colon cancer cell; COX-2; Cell motility

Differential gene expression of human keratinocyte HaCaT cells induced by fibroblast growth factor 10 treatment by Xia Chen; Jianzhong Li; Wei Hu; Shengli Yang; Yi Gong (pp. 71-85).
Fibroblast growth factor 10 (FGF10) has multiple biological activities involved in angiogenesis, mitogenesis, cellular differentiation, development, and tissue injury repair. Our previous studies revealed that treatment of FGF10 remarkably stimulated HaCaT cell proliferation and abbreviated cell apoptosis. However, the molecular mechanisms remain largely unknown. The aim of this study was to investigate FGF10-induced modifications in gene expression in the HaCaT cells by using the cDNA microarray technique. The microarray data showed that FGF10 modified the expression of 2117 genes, 861 being up-regulated and 1256 down-regulated, using a threshold of twofold. Eight of nine candidate genes, validated by real-time quantitative polymerase chain reaction (qPCR), were correlated well with the array data. The GenMAPP and MappFinder software packages were further used for pathway analysis of these significantly altered genes. In support of multiple biological functions for FGF10, several gene pathways were found to be involved in processes of cell cycle, DNA repair, apoptosis, development, and wound healing. These data also provide a basis to further investigation of FGF10 molecular mechanisms.

Keywords: FGF10; Microarray; Gene pathway; Expression profile; HaCaT

Nitric oxide contributes to the regulation of iron metabolism in skeletal muscle in vivo and in vitro by Haitao Wang; Xianglin Duan; Jianguo Liu; Huanbin Zhao; Yuqian Liu; Yanzhong Chang (pp. 87-94).
Nitric Oxide (NO) plays an important role in iron redistribution during exercise, while its molecular regulatory mechanism is still not clear. Our present studies were to investigate the effects of NO on iron metabolism and to elucidate the regulatory mechanism of iron transport in skeletal muscle both in vivo and in vitro. One group of male Wistar rats (300 ± 10 g) were subjected to an exercise of 30 min on a treadmill for 5 weeks (exercise group, EG, 6 rats) and the other one was placed on the treadmill without running (control group, CG, 6 rats). The cultured L6 rat skeletal muscle cells were treated with either 0.5 mM SNAP (NO donor) or not for 24 h, and their iron release and intake amount were examined by measuring radiolabelled 55Fe. The results showed: (1) The NO content (CG, 1.09 ± 0.18 μmol/g vs. EG, 1.49 ± 0.17 μmol/g) and non-heme iron in gastrocnemius (CG, 118.35 ± 11.41 μg/g vs. EG, 216.65 ± 11.10 μg/g) of EG were significantly increased compared with CG. (2) The expression of DMT1 (IRE) and TfR1 of EG was increased. (3) The iron intake was increased in L6 cells treated with SNAP (P < 0.01). (4) Western blot results showed the protein level of both TfR1 and DMT1 (IRE) in SNAP cells were up-regulated, while the expression of FPN1 was down-regulated (P < 0.05). The data suggested that the induced elevation of NO level by exercise lead to the up-regulation of both TfR1 and DMT1 (IRE), which in turn increasing the iron absorption in skeletal muscle.

Keywords: Nitric oxide; Iron transport proteins; Gastrocnemius; Exercise

l-Carnitine is essential to β-oxidation of quarried fatty acid from mitochondrial membrane by PLA2 by Hiromi Yano; Eri Oyanagi; Yasuko Kato; Yoshiyuki Samejima; Junzo Sasaki; Kozo Utsumi (pp. 95-100).
Mitochondrial β-oxidation is an important system involved in the energy production of various cells. In this system, the function of l-carnitine is essential for the uptake of fatty acids to mitochondria. However, it is unclear whether or not endogenous respiration, ADP-induced O2 consumption without substrates, is caused by l-carnitine treatment. In this study, we investigated whether l-carnitine is essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by phospholipase A2 (PLA2) using isolated mitochondria from the liver of rats. Intact mitochondria were incubated in a medium containing Pi, CoA and l-carnitine. The effect of l-carnitine treatment on ADP-induced mitochondrial respiration was observed without exogenous respiratory substrate. Increase in mitochondrial respiration was induced by treatment with l-carnitine in a concentration-dependent manner. Treatment with rotenone, a complex I blocker, completely inhibited ADP-induced oxygen consumption even in the presence of l-carnitine. Moreover, the l-carnitine dependent ADP-induced mitochondrial oxygen consumption did not increase when PLA2 inhibitors were treated before ADP treatment. The l-carnitine-dependent ADP-induced oxygen consumption did contribute to ATP productions but not heat generation via an uncoupling system. These results suggest that l-carnitine might be essential to the β-oxidation of quarried fatty acids from the mitochondrial membrane by PLA2.

Keywords: Oxygen consumption; ADP; CoA; β-oxidation; Fatty acid; Rat liver

Demethylating agent 5-aza-2-deoxycytidine enhances susceptibility of breast cancer cells to anticancer agents by Sameer Mirza; Gayatri Sharma; Pranav Pandya; Ranju Ralhan (pp. 101-109).
DNA methylation plays an important role in regulation of gene expression and is increasingly being recognized as a determinant of chemosensitivity of human cancers. With the aim of improving the chemotherapeutic efficacy of breast carcinoma, the effect of DNA methyltransferase inhibitor, 5-Aza-2′-deoxycytidine (5-aza-CdR), on the chemosensitivity of anticancer drugs was investigated. The cytotoxicity of paclitaxel (PTX), adriamycin (ADR), and 5-fluorouracil (5-FU) was analyzed against human breast cancer cell lines, MDA MB 231 and MCF 7 cell lines using the MTT assay, and the synergy of 5-aza-CdR and these agents was determined by Drewinko’s fraction method. The effects of each single agent or the combined treatment on cell cycle arrest were analyzed by flow cytometric analysis. We also investigated the effect of each single agent or the combined treatment of anticancer drugs with 5-aza-CdR on the methylation status of the selected genes by methylation specific PCR. In MDA MB 231 cells, a synergistic antiproliferative effect was observed with a combination of 10 μM 5-aza-CdR and these three anticancer drugs, while in MCF 7 cells, a semiadditive effect was observed. Treatment with 5-aza-CdR and anticancer drug resulted in partial demethylation of a panel of genes including RARβ2, Slit2, GSTP1, and MGMT. Based on these findings, we propose that 5-aza-CdR enhances the chemosensitivity of anticancer drugs in breast cancer cells and may be a promising approach for increasing the chemotherapeutic potential of these anticancer agents for more effective management of breast carcinomas.

Keywords: Demethylating agent; 5-Aza-2′-deoxycytidine; Breast carcinoma; Anticancer agents; Chemosensitivity

Mitochondrial respiratory chain in the colonic mucosal of patients with ulcerative colitis by Karla G. Sifroni; Carlos R. Damiani; Cristhopher Stoffel; Mariane R. Cardoso; Gabriela K. Ferreira; Isabela C. Jeremias; Gislaine T. Rezin; Giselli Scaini; Patricia F. Schuck; Felipe Dal-Pizzol; Emilio L. Streck (pp. 111-115).
Ulcerative colitis (UC) is a chronic inflammatory disease of the large bowel. Its pathogenesis remains unclear, but it appears to result from a deregulated immune response, with infiltration of leukocytes into the mucosal interstitium. Several studies link oxidative stress and mitochondrial dysfunction to the pathogenesis of UC. Thus, the aim of this study was to evaluate the activities of mitochondrial respiratory chain complexes in the colonic mucosal of UC patients. Colonic biopsies were obtained from UC patients (n = 13). The control specimens were taken from patients without any history of inflammatory bowel disease (n = 8). Colon mucosal was removed by colonoscopy and homogenized. Mitochondrial respiratory chain complexes activities were then measured. Our results showed that the activity of complex I was not altered in UC patients, when compared to the control group. On the other hand, complexes II, III, and IV were decreased around 50–60% in the colonic mucosal of UC patients. Based on the present findings, we hypothesize that mitochondrial dysfunction may play a role in pathogenesis of UC.

Keywords: Ulcerative colitis; Mitochondria; Respiratory chain; Energy metabolism

Knockdown of transmembrane protein 132A by RNA interference facilitates serum starvation-induced cell death in Neuro2a cells by Kentaro Oh-hashi; Kazuhide Imai; Hisashi Koga; Yoko Hirata; Kazutoshi Kiuchi (pp. 117-123).
Transmembrane protein 132A (TMEM132A) is a novel GRP78 binding protein that we recently discovered. However, the biological functions of TMEM132A are merely characterized because it does not encode any known structural domains. In this study, we down regulated intrinsic TMEM132A by RNA interference and identified a variety of genes that fluctuated during TMEM132A gene silencing using microarray analysis. TMEM132A-knockdown in Neuro2a cells caused neurite-like projection without any stimuli and enhanced the expression of ATF6 mRNA, an ER stress transducer, and GADD153 mRNA, a stress inducible gene. Under serum-deprived condition, TMEM132A-knockdown cells gradually retarded neurite-like projection and decreased cell viability. Moreover, TMEM132A knockdown markedly induced GADD153 expression due to serum starvation without affecting the level of cleaved caspase-3. Our data suggest that TMEM132A is an important factor of cell survival in regulating certain ER stress-related gene expression in neuronal cells.

Keywords: Transmembrane protein 132A; RNA interference; Serum starvation; GADD153

Induction of apoptosis in hepatocellular carcinoma Smmc-7721 cells by vitamin K2 is associated with p53 and independent of the intrinsic apoptotic pathway by Lu Li; Zhiling Qi; Jin Qian; Fuyong Bi; Jun Lv; Lei Xu; Ling Zhang; Hongyu Chen; Renbing Jia (pp. 125-131).
Vitamin K2 (VK2) can exert cell growth inhibitory effects in various human cancer cells. In this study, we investigated the cell growth inhibitory effects of VK2 in hepatocellular carcinoma Smmc-7721 cells and the mechanisms involved. We found that VK2-inhibited cell proliferation in Smmc-7721 cells in a dose-dependent manner, and the IC50 of VK2 in Smmc-7721 cells was 9.73 μM at 24 h. The data from flow cytometric analyses, DNA fragmentation assays, and caspase 3 activity assays revealed that apoptosis was the determining factor in VK2 activity. Furthermore, a significant increase in p53 phosphorylation and protein level was exhibited in apoptotic cells treated with VK2, although there were no changes in p53 mRNA expression. Bax expression was unaffected by VK2 in Smmc-7721 cells. In addition, our study showed that caspase 3 was activated by caspase 8, not caspase 9, in Smmc-7721 cells treated with VK2. In summary, these data suggested that VK2 can inhibit the growth of Smmc-7721 cells by induction of apoptosis involving caspase 8 activation and p53. This apoptotic process was not mediated by the intrinsic apoptotic pathway.

Keywords: Apoptosis; Vitamin K2 ; Smmc-7721 cells; p53; Intrinsic apoptotic pathway

4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH) targets mRNA of the c-FLIP variants and induces apoptosis in MCF-7 human breast cancer cells by Khadijeh Bijangi-Vishehsaraei; Mohammad Reza Saadatzadeh; Su Huang; Michael P. Murphy; Ahmad R. Safa (pp. 133-142).
Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP) is a major resistance factor for the tumor necrosis factor-related apoptosis-inducing ligand TRAIL and in drug resistance in human malignancies. c-FLIP is an antagonist of caspases-8 and -10, which inhibits apoptosis and is expressed as long (c-FLIPL) and short (c-FLIPS) splice forms. c-FLIP is often overexpressed in various human cancers, including breast cancer. Several studies have shown that silencing c-FLIP by specific siRNAs sensitizes cancer cells to TRAIL and anticancer agents. However, systemic use of siRNA as a therapeutic agent is not practical at present. In order to reduce or inhibit c-FLIP expression, small molecules are needed to allow targeting c-FLIP without inhibiting caspases-8 and -10. We used a small molecule inhibitor of c-FLIP, 4-(4-chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH), and show that CMH, but not its inactive analog, downregulated c-FLIPL and c-FLIPS mRNA and protein levels, caused poly(ADP-ribose) polymerase (PARP) degradation, reduced cell survival, and induced apoptosis in MCF-7 breast cancer cells. These results revealed that c-FLIP is a critical apoptosis regulator that can serve as a target for small molecule inhibitors that downregulate its expression and serve as effective targeted therapeutics against breast cancer cells.

Keywords: c-FLIP; Breast cancer; Apoptosis; 4-(4-Chloro-2-methylphenoxy)-N-hydroxybutanamide (CMH); Caspases; Death receptors

Functionality of unliganded VDR in breast cancer cells: repressive action on CYP24 basal transcription by Fatouma Alimirah; Avani Vaishnav; Michael McCormick; Ibtissam Echchgadda; Bandana Chatterjee; Rajendra G. Mehta; Xinjian Peng (pp. 143-150).
It is well-established that CYP24, an immediate target gene of VDR is upregulated by VDR ligands. This study is focused on the functional role of unliganded VDR by investigating the correlation between the expression of VDR protein and basal mRNA levels of CYP24 in breast cancer cell lines. Analyses of multiple breast cancer cell lines demonstrated an inverse correlation between VDR protein expression and CYP24 mRNA expression levels; while in the presence of ligand, VDR protein level was positively correlated with CYP24 expression. In MCF-7 cells, VDR was mainly distributed in the nuclei in the absence of ligand. VDR overexpression in MCF-7 cells and MDA-MB231 cells decreased CYP24 mRNA expression levels and CYP24 promoter activity. Conversely, knock-down of VDR using siRNA techniques in MCF-7 and T47D cells significantly increased CYP24 mRNA expression. We also found that overexpression of VDR with a polymorphic site (FokI-FF) at its AF-1 domain, which makes VDR shorter by three amino acids, failed to repress CYP24 promoter activity. This report provides conclusive evidence for the repressive action of unliganded VDR on the expression of its target gene CYP24 and the importance of an intact VDR AF-1 domain for its repressive action.

Keywords: Vitamin D receptor; CYP24; Breast cancer cells; Unliganded VDR

Epigenetics in anoxia tolerance: a role for histone deacetylases by Anastasia Krivoruchko; Kenneth B. Storey (pp. 151-161).
The importance of epigenetics has been established in many key biological processes but the relevance of this regulatory mechanism to animal survival of low oxygen conditions has never been examined. To establish whether epigenetic mechanisms could be involved in natural anoxia tolerance, we have examined the anoxia-responsive expression of the transcriptional silencers, histone deacetylases (HDACs), in tissues of a unique model for anoxia tolerance, the freshwater turtle Trachemys scripta elegans. Transcript and protein levels of all five HDACs rose by 1.3–4.6 and 1.7–3.5-fold, respectively, in skeletal muscle in response to 20 h of anoxia exposure. In addition, HDAC activity in the muscle increased by 1.5-fold in response to 20 h of anoxia and levels of acetylated histone H3 (Lys 9 or Lys 23) decreased to 40–60% of control values. The liver displayed a milder response with HDAC1, -4, and -5 protein levels increasing by 1.6-2.1-fold after 5 h anoxia exposure; acetylated histone H3 levels also decreased to 50–75% of control values. Only HDAC5 responded to anoxia exposure in the heart; Hdac5 transcript levels increased 2.1–2.3-fold and HDAC5 protein rose by 3.3-fold. Overall, our results show a tissue-specific pattern of HDAC upregulation in response to anoxia exposure in T.s. elegans, suggesting that these enzymes play a key role in anoxia tolerance, probably by contributing to the transcriptional silencing necessary in this hypometabolic state.

Keywords: Trachemys scripta elegans ; Anoxia tolerance; Metabolic rate depression; Epigenetics; Histone deacetylase

Cramoll 1,4 lectin increases ROS production, calcium levels, and cytokine expression in treated spleen cells of rats by Cristiane Moutinho Lagos de Melo; Bruno Alves Paim; Karina Gotardelo Zecchin; Joseani Morari; Marcos R. Chiaratti; Maria Tereza Santos Correia; Luana Cassandra B. Barroso Coelho; Patrícia Maria Guedes Paiva (pp. 163-169).
This study reports the in vivo stimulatory effects of Cramoll 1,4 on rat spleen lymphocytes as evidenced by an increase in intracellular reactive oxygen species (ROS) production, Ca2+ levels, and interleukin (IL)-1β expression. Cramoll 1,4 extracted from seeds of the Leguminosae Cratylia mollis Mart., is a lectin with antitumor and lymphocyte mitogenic activities. Animals (Nine-week-old male albino Wistar rats, Rattus norvegicus) were treated with intraperitoneal injection of Cramoll 1,4 (235 μg ml−1 single dose) and, 7 days later, spleen lymphocytes were isolated and analyzed for intracellular ROS, cytosolic Ca2+, and IL-6, IL-10, and IL-1 mRNAs. Cell viability was investigated by annexin V-FITC and 7-amino-actinomycin D staining. The data showed that in lymphocytes activated by Cramoll 1,4 the increase in cytosolic and mitochondrial ROS was related to higher cytosolic Ca2+ levels. Apoptosis and necrosis were not detected in statistically significant values and thus the lectin effector activities did not induce lymphocyte death. In vivo Cramoll 1,4 treatment led to a significant increase in IL-1β but IL-6 and -10 levels did not change. Cramoll 1,4 had modulator activities on spleen lymphocytes and stimulated the Th2 response.

Keywords: Cratylia mollis ; Calcium; Interleukin; Lectin; Reactive oxygen species

oxHDL decreases the expression of CD36 on human macrophages through PPARγ and p38 MAP kinase dependent mechanisms by Jingyi Ren; Wenying Jin; Hong Chen (pp. 171-181).
CD36, belongs to class B scavenger receptor family, is a macrophage receptor for oxidized low-density lipoprotein (oxLDL) and has been proven to play a critical role in atherosclerotic foam cell formation. In addition, CD36 expression is regulated by many factors including oxLDL and HDL. A recent study suggests that CD36 can also bind with oxidized high-density lipoprotein (oxHDL). However, the direct role of oxHDL in atherosclerosis is still not clear and it is not known whether oxHDL has any influence on the expression of CD36 in macrophages. Here, we performed experiments to investigate the effect of oxHDL on the expression of CD36 on human peripheral blood monocytes–macrophages and the possible mechanisms. Our results suggest that the uptake of oxHDL by CD36 on macrophages accelerates foam cell formation. In addition, oxHDL can down-regulate both the mRNA and surface protein expression of CD36 on human peripheral macrophages in vitro. oxHDL increased the mRNA expression and protein phosphorylation of peroxisome proliferators-activated receptor-γ (PPARγ). Using different mitogen-activated protein kinase (MAPK) inhibitors, we demonstrated that oxHDL regulated CD36 and PPARγ expression in a p38-MAP kinase dependent mechanism.

Keywords: oxHDL; CD36; PPARγ; MAP kinase; Macrophages; Atherosclerosis

Altered expression of pro- and anti-inflammatory cytokines is associated with reduced cardiac function in rats following coronary microembolization by Lang Li; Xianming Zhao; Yongguang Lu; Weiqiang Huang; Weiming Wen (pp. 183-190).
Coronary microembolization (CME) is a spontaneous event in patients with ischemic heart disease and a potential iatrogenic complication in patients undergoing coronary interventions. CME induces an obvious inflammatory reaction, characterized by cellular infiltration, particularly of eosinophils, and multifocal microinfarcts. However, little is known on the correlation between pro- and anti-inflammatory cytokines and cardiac function following CME. In this study, microspheres with a diameter of 42 μm were intracoronarily injected into the apex of the left ventricle to induce CME (CME group). Rats injected with normal saline served as controls (sham operated control group). Expression of pro-inflammatory cytokines, TNF-α and IL-1β, and an anti-inflammatory cytokine, IL-10, was measured at 3, 6, 12, 24 h, and 4 weeks post-injection by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. At the same time points, cardiac function and histological changes were evaluated by echocardiographic imagining and H&E staining, respectively. It was observed that the mRNA and protein expressions of TNF-α, IL-1β, and IL-10 all started to increase at 3 h, reached to the peak levels at 12 h, and returned to the normal levels at 4 weeks post-injection. The left ventricular ejection fraction (LVEF) was significantly lower in the CME group than in the control group at 3, 6, 12, 24 h, and 4 weeks post-injection. Obvious myocardial microinfarcts with surrounding myocardial edema and degeneration, caryolysis, and infiltration of neutrophils and monocytes were observed in the CME group at 3, 6, 12, 24 h, and 4 weeks post-injection. Moreover, mRNA expression of TNF-α and IL-1β was negatively correlated with LVEF, although not with myocardial microinfarcts, in the CME group. It is concluded that both protein and mRNA expression of TNF-α IL-1β and IL-10 are dynamically changed following CME, which is associated with reduced cardiac function. Therefore, these cytokines may be potential therapeutic targets for clinical treatment of CME.

Keywords: Coronary microembolization; TNF-α; IL-1β; IL-10; Cardiac function; Myocardial microinfarcts

Osteogenic role of endosomal chloride channels in MC3T3-E1 cells by Huan Wang; Na Huo; Feifei Li; Shanmin Fu; Yang Xue; Ting Yang; Xuan Wen; Yin Ding; Xiaohong Duan (pp. 191-199).
ClC-3, ClC-4, and ClC-5 belong to the voltage gated chloride channels (ClCs) and facilitate the endosomal acidification. The mutations of these endosomal chloride channel genes cause different genetic diseases with various bone disorders. We hypothesized that these endosomal ClCs might be involved in the bone development or osteoblast differentiation. Here we used MC3T3-E1 osteoprogenitor cell line and primarily cultured mouse osteoblasts and detected the expression of Clcn3, Clcn4, and Clcn5 in these cells. We analyzed the relationships between three endosomal ClCs and the osteogenic phenotype using osteoinductive treatment, overexpressing of ClCs and RNAi of ClCs. We found the increased mRNA levels of osteogenic markers [alkaline phosphatase (Alp), osteocalcin (Oc), bone sialoprotein (Bsp), and runt-related transcription factor 2 (Runx2)] were in parallel to that of Clcn3, Clcn4 and Clcn5 with osteoinductive treatment and overexpressed ClCs. Overexpressed ClCs were localized in intracellular periphery and also promoted the mineralization of cells in vitro. While RNAi mediated gene silencing of ClC-3, ClC-4, and ClC-5 down regulated the expression of the four osteogenic markers. The positive relationship between endosomal ClCs and the osteogenic markers suggested a new function of endosomal ClCs in osteogenic differentiation.

Keywords: Chloride channel; Endosome; Osteoblast; Differentiation; Osteogenic gene

The effects of ox-LDL in human atherosclerosis may be mediated in part via the toll-like receptor 4 pathway by Honglian Geng; Aihua Wang; Guanghua Rong; Bei Zhu; Yan Deng; Jun Chen; Renqian Zhong (pp. 201-206).
Toll-like receptor 4 (TLR4) may provide a potential pathophysiological link between lipids and infection/inflammation and atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) makes it more atherogenic than its native form. The purpose of the study was to investigate the relationship between the effects of ox-LDL in human atherosclerosis and the expression of TLR4. We studied the relationship between TLR4 and ox-LDL, pro and con, using both real-time quantitative RT-PCR and RNA interference technology through in vitro cell culture. Nuclear factor kappa B (NF-κ B) activity and the concentrations of monocyte chemoattractant protein-1(MCP-1) and interleukin-8 (IL-8) were detected by ELISA. The results showed that the expression of TLR4 increased in response to ox-LDL. Simultaneously, NF-κ B relative activity and the concentrations of MCP-1 and IL-8 in cell supernatant were upregulated by ox-LDL in a dose-dependent manner. TLR4 expression was inhibited by small interference RNA(siRNA) plasmid expression vectors; NF-κ B activity and the secretions of MCP-1 and IL-8 in response to ox-LDL were significantly lower in the group whereinTLR4 expression has been inhibited than that in the group wherein TLR4 expression has not been inhibited. We suggest that the atherogenic effects of ox-LDL could be mediated in part via the TLR4 pathway. Furthermore, inhibition of TLR4 expression may downregulate the NF-κ B activity and secretions of MCP-1 and IL-8 in monocytes due to oxidized LDL, resulting in the alleviation of the progress of atherosclerosis.

Keywords: ox-LDL; Toll-like receptors; Atherosclerosis; NF-kappa B; siRNA

Contribution of rpoS and bolA genes in biofilm formation in Escherichia coli K-12 MG1655 by Mohd Adnan; Glyn Morton; Jaipaul Singh; Sibte Hadi (pp. 207-213).
Flexibility of gene expression in bacteria permits its survival in varied environments. The genetic adaptation of bacteria through systematized gene expression is not only important, but also clinically relevant in their ability to grow biofilms in stress environments. Stress responses enable their survival under more severe conditions, enhanced resistance and/or virulence. In Escherichia coli (E. coli), two of the possible important genes for biofilm growth are rpoS and bolA gene. RpoS is also called as a master regulator of general stress response. Even though many studies have revealed the importance of rpoS in planktonic cells, little is known about the functions of rpoS in biofilms. In contrast, bolA which is a morphogene in E. coli is overexpressed under stressed environments resulting in round morphology. The hypothesis is that bolA could be implicated in biofilm development. This study reviewed the literature with the aim of understanding the stress tolerance response of E. coli in relation with rpoS and bolA genes in different environmental conditions including heat shock, cold shock, and stress in response to oxidation, acidic condition and in presence of cadmium. Knowledge of the genetic regulation of biofilm formation may lead to the understanding of the factors that drive the bacteria to switch to the biofilm mode of growth.

Keywords: E. coli ; Biofilm; Stress environment; rpoS ; bolA

The envelope glycoprotein domain III of dengue virus type 2 induced the expression of anticoagulant molecules in endothelial cells by Lien-Cheng Chen; Trai-Ming Yeh; Yi-Ying Lin; Yi-Fen Wang; Shu-Jem Su; Chang-Yu Chen; Kuan-Hua Lin; Miao-Chen Chou; Huey-Wen Shyu (pp. 215-221).
Dengue virus (DV) causes a non-specific febrile illness known as Dengue fever (DF), and a severe life-threatening illness, Dengue hemorrhagic fever/Dengue shock syndrome (DHF/DSS). Hemostatic changes induced by this virus involve three main factors: thrombocytopenia, endothelial cell damage, and significant abnormalities of the coagulation and fibrinolysis systems. The pathogenesis of bleeding in DV infections remains unknown. In this article, we focused on the DV activating endothelial cells and altering the parameters of hemostasis system. The expression of hemostasis-related factors, Thrombomodulin, TF, TFPI, t-PA, and PAI-1, in DV-infected cells were determined by RT-PCR. Flow cytometry analysis and immunofluorescence staining confirmed that the expression levels of TM in the DV-infected HMEC-1 and THP-1 cells were increased. In addition, the purified recombinant domain III of the envelope glycoprotein of DV (EIII) could induce the expression of TM in the HMEC-1 cells and THP-1 cells. The TM expression induced by DV or EIII in the endothelial cells and monocytic cells suggests that the EIII of DV plays an important role in the pathogenesis of DHF/DSS.

Keywords: DV-2; Human microvascular endothelial cell line (HMEC-1); Thrombomodulin

Over-expression of transglutaminase in the Drosophila eye imaginal disc induces a rough eye phenotype by Masaru Umehara; Akira Ichikawa; Harunobu Sakamoto; Ayako Yamada; Yasuhide Yoshioka; Masamitsu Yamaguchi; Koji Ikura (pp. 223-232).
Transglutaminases (TGs) catalyze the cross-linking of proteins and are involved in various biological processes in mammals. In invertebrates, except for the involvement in the hemolymph clotting, the functions of TG have not been revealed. Drosophila has a single TG gene (CG7356), from which two kinds of mRNAs (dTG-RA and dTG-RB) are formed. RT-PCR analyses indicated that both dTGs-RA and -RB are synthesized in all the developmental stages tested. To reveal the roles of dTG during the development, we examined a phenotype induced through the ectopic expression of dTG by using a GAL4-UAS targeted expression system. Over-expression of dTG-A in the eye imaginal disc of larva induced a rough eye phenotype in adult compound eyes. Co-expression of P35, an inhibitor of apoptosis, suppressed the rough eye phenotype, suggesting that the rough eye phenotype induced by the over-expression of dTG-A in the eye imaginal disc is due to the occurrence of apoptosis. The rough eye phenotype induced by the over-expression of dTG-A was suppressed by the crossing with mutant fly lines lacking Drosophila JNK gene basket (bsk) or Drosophila JNKK gene hemipterous. FLP-out experiments using an enhancer trap line showed that the over-expression of dTG-A in the eye imaginal disc increased the puckered enhancer activity, a reporter of Bsk activity. These results suggested that the rough eye phenotype induced by the over-expression of dTG-A is related to an enhancement of JNK signaling pathway.

Keywords: Transglutaminase; GAL4-UAS targeted expression system; Apoptosis; JNK signaling pathway

The functional expression of calcium-sensing receptor in the differentiated THP-1 cells by Yu-hui Xi; Hong-zhu Li; Wei-hua Zhang; Li-na Wang; Li Zhang; Yan Lin; Shu-zhi Bai; Hong-xia Li; Ling-yun Wu; Rui Wang; Chang-qing Xu (pp. 233-240).
The expression and function of calcium-sensing receptor (CaSR) in differentiated THP-1 (human acute monocytic leukemia cell line) cells are unknown currently. This study investigated above-mentioned issues using TRAP staining, immunofluorescence staining, Western blotting, ELISA, and Laser Confocal Scanning Microscopy techniques. We found that CaSR protein was expressed, and mainly located in the membrane and cytoplasm in differentiated THP-1 cells. Elevated extracellular calcium or GdCl3 (an agonist of CaSR) raised intracellular calcium concentration. And this increase was inhibited or abolished by NPS2390 (an inhibitor of CaSR), U73122 (a specific inhibitor of phospholipase C, PLC) or thapsigargin (a Ca2+-ATPase inhibitor). The extracellular GdCl3 elevation stimulated both of IL-1β and TNFα release, and this effect of GdCl3 was inhibited by NPS2390. In conclusion, CaSR is functionally expressed in differentiated THP-1 cells, and the activated CaSR contributes to intracellular calcium increment through Gq-PLC- inositol triphosphate (IP3) pathway and commits to cytokine secretion. These results suggest that CaSR might be involved in a variety of pathological processes mediated by activated monocyte-macrophages.

Keywords: Calcium-sensing receptor; Monocyte/macrophage; Intracellular calcium; Cytokine; Human acute monocytic leukemia cell line (THP-1)

Differentiation linked regulation of telomerase activity by Makorin-1 by Jose Salvatico; Joo Hee Kim; In Kwon Chung; Mark T. Muller (pp. 241-250).
To understand telomere homeostasis, a significant aspect of cancer and growth control, it is important to examine telomerase induction as well as mechanisms of regulated elimination. Makorin-1 (MKRN1) was previously shown to be an E3 ubiquitin ligase that targets the telomerase catalytic subunit (hTERT) for proteasome processing (Kim et al., Genes Dev 19:776–781, 2005). In this study we examined expression and regulation of endogenous MKRN1 during the cell cycle and terminal differentiation. When WI-38 cells transition from active growth into a resting G1 state, basal levels of MKRN1 were found to increase by sixfold. In contrast, cancer cells typically contained low or in some cases undetectable levels of MKRN1 protein. HL-60 cells growing exponentially in culture contain no detectable MKRN1; however, following terminal differentiation, MKRN1 mRNA and protein levels are strongly up-regulated while hTERT mRNA, hTERC, and telomerase are shut down. The initial decrease in telomerase activity is due to a gradual reduction in transcription of the hTERT gene that occurs during the first 12 h of terminal differentiation. MKRN1 protein appears between 12 and 24 h and is attended by a more rapid loss of telomerase activity. As more MKRN1 protein accumulates, significantly less telomerase activity is seen. Addition of the proteasome inhibitor, MG132, reverses the loss of telomerase activity; therefore, reductions in telomerase activity are dynamic, ongoing, and correlated with robust up-regulation of MKRN1 as the cells terminally differentiate. The data are consistent with the idea that MKRN1 represents a telomerase elimination pathway to rapidly draw down the activity during differentiation or cell cycle arrest when telomerase action at chromosome ends is no longer necessary.

Keywords: Makorin-1; hTERT; Ubiquitin; Ubiquitin ligase; Telomerase; Proteasome

Hearts of surviving MLP-KO mice show transient changes of intracellular calcium handling by Péter Kemecsei; Zsuzsanna Miklós; Tamás Bíró; Rita Marincsák; Balázs I. Tóth; Edina Komlódi-Pásztor; Enikő Barnucz; Éva Mirk; Ger J. Van der Vusse; László Ligeti; Tamás Ivanics (pp. 251-260).
The muscle Lim protein knock-out (MLP-KO) mouse model is extensively used for studying the pathophysiology of dilated cardiomyopathy. However, explanation is lacking for the observed long survival of the diseased mice which develop until adulthood despite the gene defect, which theoretically predestines them to early death due to heart failure. We hypothesized that adaptive changes of cardiac intracellular calcium (Ca i 2+ ) handling might explain the phenomenon. In order to study the progression of changes in cardiac function and Ca i 2+ cycling, myocardial Ca i 2+ -transients recorded by Indo-1 surface fluorometry were assessed with concomitant measurement of hemodynamic performance in isolated Langendorff-perfused hearts of 3- and 9-month old MLP-KO animals. Hearts were challenged with β-agonist isoproterenol and the sarcoplasmic reticular Ca2+-ATPase (SERCA2a) inhibitor cyclopiazonic acid (CPA). Cardiac mRNA content and levels of key Ca2+ handling proteins were also measured. A decline in lusitropic function was observed in 3-month old, but not in 9-month old MLP-KO mice under unchallenged conditions. β-adrenergic responses to isoproterenol were similar in all the studied groups. The CPA induced an increase in end-diastolic Ca i 2+ -level and a decrease in Ca2+-sequestration capacity in 3-month old MLP-KO mice compared to age-matched controls. This unfavorable condition was absent at 9 months of age. SERCA2a expression was lower in 3-month old MLP-KO than in the corresponding controls and in 9-month old MLP-KO hearts. Our results show time-related recovery of hemodynamic function and an age-dependent compensatory upregulation of Ca i 2+ handling in hearts of MLP-KO mice, which most likely involve the normalization of the expression of SERCA2a in the affected hearts.

Keywords: Ca i 2+ handling; Isolated hearts; MLP-KO

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