Silencing survivin gene expression promotes apoptosis of human breast cancer cells through a caspase-independent pathway

Survivin is recognized as an attractive target in cancer therapy because of its selective overexpression in the majority of tumors. Upregulated expression of this protein correlates with increased tumor grade, recurrence risk and decreased cancer patients survival. In this study, we assessed the efficacy of two survivin-specific small interfering RNA (siRNA) constructs to inhibit T47D human breast cancer cell growth. After siRNA transfection, T47D cells showed a significant reduction in proliferation and survival exhibiting clear signs of apoptosis. pSil_1 that targeted exon 1 exhibited a stronger inhibitory effect on cell growth, and increased cell apoptosis compared to pSil_30 that targeted exon 4. Cell apoptosis was found to be mediated by translocation of the mitochondrial apoptosis inducing factor (AIF), while no changes were observed in caspase-3 activation and Bid cleavage. Thus, silencing survivin expression using siRNA strategies represents a suitable therapeutic approach to selectively modulate the survival and growth of human breast cancer cells.     

Caveolin-1 promotes Rfng expression via Erk-Jnk-p38 signaling pathway in mouse hepatocarcinoma cells

Journal of Physiology and Biochemistry - Caveolin-1 (Cav-1) is a critical structural protein of caveolae and plays an oncogene-like role by participating in abnormal protein glycosylation in...     

Polycystin-1 promotes PKCalpha-mediated NF-kappaB activation in kidney cells

Polycystin-1 (PC1), the PKD1 gene product, is a membrane receptor which regulates many cell functions, including cell proliferation and apoptosis, both typically increased in cyst lining cells in autosomal dominant polycystic kidney disease. Here we show that PC1 upregulates the NF-kappaB signalling pathway in kidney cells to prevent cell death. Human embryonic kidney cell lines (HEK293(CTT)), stably expressing a PC1 cytoplasmic terminal tail (CTT), presented increased NF-kappaB nuclear levels and NF-kappaB-mediated luciferase promoter activity. This, consistently, was reduced in HEK293 cells in which the endogenous PC1 was depleted by RNA interference. CTT-dependent NF-kappaB promoter activation was mediated by PKCalpha because it was blocked by its specific inhibitor Ro-320432. Furthermore, it was observed that apoptosis, which was increased in PC1-depleted cells, was reduced in HEK293(CTT) cells and in porcine kidney LtTA cells expressing a doxycycline-regulated CTT. Staurosporine, a PKC inhibitor, and parthenolide, a NF-kappaB inhibitor, significantly reduced the CTT-dependent antiapoptotic effect. These data reveal, therefore, a novel pathway by which polycystin-1 activates a PKCalpha-mediated NF-kappaB signalling and cell survival.     

Immunohistochemical examination of osteopontin and sirtuin-1 expression in cattle tuberculosis

We evaluated osteopontin (OPN) and sirtuin-1 (SIRT-1) expression in bovine tuberculosis lesions. The organs of cattle with tuberculosis (TB) were examined for morphology, histopathology and immunohistochemistry of OPN and SIRT-1 expression. Macroscopic lesions commonly were localized in the lungs and mediastinal lymph nodes as well as in livers and hearts. Mycobacterial agents were detected in lesions using the Ziehl-Neelsen method. No OPN or SIRT-1 expression was detected by immunohistochemistry in normal tissues, whereas a marked increase in their expressions was observed in tuberculous lesions. The most intense immunopositive cells were Langerhans giant cells and inflammatory cells. Our findings indicate that OPN and SIRT-1 participate in the pathogenesis of bovine TB.     

Hypoxia-induced Bcl-2 expression in endothelial cells via p38 MAPK pathway

Angiogenesis and apoptosis are reciprocal processes in endothelial cells. Bcl-2, an anti-apoptotic protein, has been found to have angiogenic activities. The purpose of this study was to determine the role of Bcl-2 in hypoxia-induced angiogenesis in endothelial cells and to investigate the underlying mechanisms. Human aortic endothelial cells (HAECs) were exposed to hypoxia followed by reoxygenation. Myocardial ischemia and reperfusion mouse model was used and Bcl-2 expression was assessed. Bcl-2 expression increased in a time-dependent manner in response to hypoxia from 2 to 72 h. Peak expression occurred at 12 h (3- to 4-fold, p < 0.05). p38 inhibitor (SB203580) blocked hypoxia-induced Bcl-2 expression, whereas PKC, ERK1/2 and PI3K inhibitors did not. Knockdown of Bcl-2 resulted in decreased HAECs’ proliferation and migration. Over-expression of Bcl-2 increased HAECs’ tubule formation, whereas knockdown of Bcl-2 inhibited this process. In this model of myocardial ischemia and reperfusion, Bcl-2 expression was increased and was associated with increased p38 MAPK activation.Our results showed that hypoxia induces Bcl-2 expression in HAECs via p38 MAPK pathway.     

Delta-like 1 expression promotes goblet cell differentiation in Notch-inactivated human colonic epithelial cells

Notch signaling has previously been implicated in the regulation of the cell fate of intestinal epithelial cells. However, the expression and function of Notch ligands in the human intestine remain largely unknown. In the present study, we showed that Notch ligands Delta-like 1 (Dll1) and Delta-like 4 (Dll4) are expressed in a goblet cell-specific manner in human colonic tissue. Additionally, we found that Dll1 and Dll4 expression was regulated in-parallel with Atoh1 and MUC2, which are both under the control of the Notch-Hes1 signaling pathway. Because knockdown of Dll1 expression completely abrogated the acquisition of the goblet cell phenotype in Notch-inactivated colonic epithelial cells, we postulate that Dll1 might function as a cis-acting regulatory element that induces undifferentiated cells to become goblet cells. Our results suggest a link between Dll1 expression and human goblet cell differentiation that might be mediated by a function that is distinct from its role as a Notch receptor ligand.     

Ectopic expression of angiopoietin-1 promotes neuronal differentiation in neural progenitor cells through the Akt pathway

In regions of adult neurogenesis, neural progenitor cells (NPCs) are found in close proximity to blood vessels within a so-called ‘vascular niche’. Neurogenesis is linked to angiogenesis via certain growth factors. We propose that angiopoietin-1 (Ang1), which is similar to VEGF, has a unique role in neurogenesis independent of its role in angiogenesis. In this study, primary cultures of NPCs were transduced with recombinant adenoviruses expressing Ang1 and induced to differentiate with dibutyryl cyclic AMP (dbcAMP). Neuronal differentiation was evaluated by quantitative PCR, immunofluorescence microscopy and Western blot analysis. The results show that ectopic expression of Ang1 promotes neuronal differentiation and neurite outgrowth in NPCs, while this effect was blocked by the presence of anti-Tie2 receptor antibody or the PI3-K inhibitor, LY294002. Our results suggest that Ang1, identified originally as an angiogenic factor, can also stimulate in vitro neurogenesis in NPCs through the Akt pathway.     

Conditional gene silencing in mammalian cells mediated by a stress-inducible promoter

MicroRNAs (miRNAs) are endogenous non-coding small RNAs, which negatively regulate gene expression in a sequence-specific manner through the RNA interference (RNAi) pathway. Here we describe a new miRNA-based conditional RNAi expression system that relies on cellular stress-response mechanisms in mammalian cells. In our constructs, expression of miRNA mimics is tightly controlled by a heat shock-inducible promoter. This system is highly effective in silencing permanently or conditionally expressed luciferase. The stress inducible vectors also effectively deplete co-expressed pro-apoptotic protein CHOP with heat shock. Furthermore, we demonstrate cloning of a protein-coding sequence between the stress-inducible promoter and the miRNA expression cassette allows simultaneous silencing of a target gene and activation of synthesis of a protein of choice in response to stress stimulation. This new conditional gene silencing approach could be an invaluable tool for various areas of basic and applied research and for therapeutic intervention.     

Silencing of Bcl-XL expression in human MGC-803 gastric cancer cells by siRNA

To investigate the inhibitory effect of the Bcl-XL small interfering RNA （siRNA） on Bcl-XL gene expression in the human gastric cancer cell line MGC-803, green fluorescent protein （GFP） siRNA was constructed and transfected into MGC-803 cells, together with GFP expression vector pTrace SV40.GFP expression levels were observed using fluorescence microscopy. Bcl-XL siRNA and negative siRNA were then constructed and stably transfected into MGC-803 cells. RT-PCR and immunofluorescence were used to detect the expression of Bcl-XL. Spontaneous apoptosis was detected by acridine orange （AO） and flow cytometry. Results were as follows： （1） 48 h after GFP expression vector and GFP siRNA co-transfection,the expression level of GFP in the GFP siRNA group was much lower than the negative siRNA group,according to fluorescence microscopy results. The mRNA and protein levels of Bcl-XL in Bcl-XL siRNA stable transfectants were reduced to almost background level compared with negative siRNA transfectants or untreated cells. （2） Changes in nucleus morphology was observed by AO staining nucleic and flow cytometry analysis, which showed that stable Bcl-XL siRNA transfectants have an increased spontaneous apoptosis （21.17%± 1.26% vs. 1.19%±0.18% and 1.56%±0.15% respectively, P〈0.05 vs. negative siRNA or untreated control）, siRNA targeting GFP or Bcl-XL genes can specifically suppress GFP or Bcl-XL expression in MGC-803 cells, and Bcl-XL siRNA can increase spontaneous apoptosis. Bcl-XL siRNA may be a beneficial agent against human gastric adenocarcinoma.     

LFA-1 expression on target cells promotes human immunodeficiency virus type 1 infection and transmission

While CD4 and the chemokine receptors are the principal receptors for human immunodeficiency virus (HIV), other cellular proteins, such as LFA-1, are also involved in HIV infection. LFA-1 and its ligands, ICAM-1, ICAM-2, and ICAM-3, can be expressed on the cells infected by HIV, as well as on the HIV virions themselves. To examine the role of LFA-1 expressed on target cells in HIV infection, Jurkat-derived Jbeta2.7 T-cell lines that express either wild-type LFA-1, a constitutively active mutant LFA-1, or no LFA-1 were used. The presence of wild-type LFA-1 enhanced the initial processes of HIV infection, as well as the subsequent replication and transmission from cell to cell. In contrast, the constitutively active LFA-1 mutant failed to promote virus replication and spread, even though this mutant could help HIV enter cells and establish the initial infection. This study clearly demonstrates the contribution of LFA-1 in the different stages of HIV infection. Moreover, not only is LFA-1 expression important for initial HIV-cell interaction, subsequent replication, and transmission, but its activity must also be properly regulated.     

Two human MARs effectively increase transgene expression in transfected CHO cells

Matrix attachment regions (MARs) can enhance the expression level of transgene in Chinese hamster ovaries (CHO) cell expression system. However, improvements in function and analyses of the mechanism remains unclear. In this study, we screened two new and more functional MAR elements from the human genome DNA. The human MAR‐3 and MAR‐7 element were cloned and inserted downstream of the polyA site in a eukaryotic vector. The constructs were transfected into CHO cells, and screened under G418 to produce the stably transfected cell pools. The expression levels and stability of enhanced green fluorescent protein (eGFP) were detected by flow cytometry. The transgene copy number and transgene expression at mRNA level were detected by quantitative real‐time PCR. The results showed that the expression level of eGFP of cells transfected with MAR‐containing vectors were all higher than those of the vectors without MARs under transient and stably transfection. The enhancing effect of MAR‐7 was higher than that of MAR‐3. Additionally, we found that MAR significantly increased eGFP copy numbers and eGFP gene mRNA expression level as compared with the vector without. In conclusion, MAR‐3 and MAR‐7 gene can promote the expression of transgene in transfected CHO cells, and its effect may be related to the increase of the number of copies.     

Activation of the cAMP pathway by variant human MC1R alleles expressed in HEK and in melanoma cells

Alpha-melanocyte-stimulating hormone (alpha-MSH) activates the melanocortin-1 receptor (MC1R) on melanocytes to promote a switch from red/yellow pheomelanin synthesis to darker eumelanins via positive coupling to adenylate cyclase. The human MC1R locus is highly polymorphic with the specific variants associated with red hair and fair skin (RHC phenotype) postulated to be loss-of-function receptors. We have examined the ability of MC1R variants to activate the cAMP pathway in stably transfected HEK293 cells. The RHC associated variants, Arg151Cys, Arg160Trp and Asp294His, demonstrated agonist-mediated increases in cAMP and phosphorylation of cAMP-responsive element-binding protein (CREB). Whereas the Asp294His variant showed severely impaired functional responses, the Arg151Cys and Arg160Trp variants retained considerable signaling capacity. Melanoma cells homozygous for either the Arg151Cys variant or consensus sequence both elicited CREB phosphorylation in response to alpha-MSH in the presence of IBMX. The common RHC alleles, Arg151Cys, Arg160Trp and Asp294His, are neither complete loss-of-function receptors nor are they functionally equivalent.     

Heat-induced phosphorylation of NBS1 in human skin fibroblast cells

NBS1 is known to be involved in DNA damage-induced cellular responses after exposure to ionizing radiation (IR). Phosphorylation of NBS1 contributes to cell-cycle checkpoints. The aim of this study was to determine whether heat exposure induces or stimulates cellular responses mediated by the phosphorylation of NBS1 in human skin fibroblast cell lines. The results of immunofluorescent staining and Western blot analysis showed that NBS1 proteins are phosphorylated after exposure to heat in the nucleus of a normal skin fibroblast cell line (82-6 cells). This suggests that the NBS1-mediated signal transduction could be induced by heat. We further examined whether a deficiency in the NBS1 protein modifies heat sensitivity in human skin fibroblast cell lines. A skin fibroblast cell line (Gmtert), derived from a Nijmegen breakage syndrome (NBS) patient containing mutant NBS1, showed higher sensitivity to heat than the same cell line transfected with the wild-type copy of the NBS1 gene. We also showed that transfection of a DNA cassette expressing small interference RNA (siRNA) targeted to NBS1 into 82-6 cells enhanced cell sensitivity to heat. These results suggest that NBS1 is involved in cellular responses to DNA damage which is induced by heat exposure as well as by radiation exposure in human skin fibroblast cells.     

Quercetin inhibits inducible ICAM-1 expression in human endothelial cells through the JNK pathway

The cell adhesion molecule intercellular adhesion molecule-1(ICAM-1) plays a pivotal role in inflammatory responses. Quercetin (3,3',4',5,7-pentahydroxyflavone), a naturally occurringdietary flavonol, has potent anti-inflammatory properties. The effect of quercetin on ICAM-1 expression induced by agonists phorbol 12-myristate 13-acetate (PMA) and tumor necrosis factor- (TNF-) in human endothelial cell line ECV304 (ECV) wasinvestigated. Quercetin treatment downregulated both PMA-and TNF--induced surface expression, as well as the ICAM-1 mRNAlevels, in ECV cells in a dose-dependent (10-50 µM) manner.Quercetin had no effect on PMA- or TNF--induced nuclear factor-B(NF-B) activation. However, under similar conditions a remarkabledose-dependent downregulation of activator protein-1 (AP-1) activationwas observed. This decrease in AP-1 activation was observed to beassociated with the inhibitory effects of quercetin on the c-JunNH₂-terminal kinase (JNK) pathway.These results suggest that quercetin downregulates both PMA- andTNF--induced ICAM-1 expression via inhibiting both AP-1 activationand the JNK pathway.