Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells.

Brain expressed X-linked (Bex) genes are newer group of pro-apoptotic genes. Role of any Bex gene in neuroblastoma and Bex4 and Bex6 in any cancer is completely unknown. Re-expression of all endogenous Bex genes by any nutraceutical is also unknown. Therefore, we investigated the induction of all endogenous Bex genes and associated mechanisms by curcumin using N2a, an aggressive neuroblastoma cell line. Curcumin induced all endogenous Bex genes prior to apoptosis in N2a cells in a dose- and time-dependent manner. Wortmannin (PI-3Kinases inhibitor), SP600125 (JNK inhibitor) and pifithrin-α (p53 inhibitor) abrogated curcumin-mediated induction of Bex genes. Inhibition of curcumin-mediated induction of Bex genes by pifithrin-α also inhibited N2a cells apoptosis suggesting, a direct role of Bex genes in N2a cells apoptosis and involvement of p53 in Bex genes induction. Curcumin treatment activated p53 through hyperphosphorylation at serine 15 before Bex genes induction indicating Bex genes are novel downstream targets of p53. Collectively, curcumin, a safe nutraceutical has the potential to induce all endogenous Bex genes to harness their anti-cancer properties in neuroblastoma cells. Re-expression of Bex genes by curcumin acts as tumor suppressors and may provide alternate strategy to treat neuroblastomas and other cancers with silenced Bex genes.

A Novel Anticancer Agent, 8-Methoxypyrimido[4',5':4,5]thieno(2,3-b) Quinoline-4(3H)-One Induces Neuro 2a Neuroblastoma Cell Death through p53-Dependent, Caspase-Dependent and -Independent Apoptotic Pathways.

Neuroblastoma is the most common cancer in infants and fourth most common cancer in children. Despite recent advances in cancer treatments, the prognosis of stage-IV neuroblastoma patients continues to be dismal which warrant new pharmacotherapy. A novel tetracyclic condensed quinoline compound, 8-methoxypyrimido [4',5':4,5]thieno(2,3-b) quinoline-4(3H)-one (MPTQ) is a structural analogue of an anticancer drug ellipticine and has been reported to posses anticancer property. Study on MPTQ on neuroblastoma cells is very limited and mechanisms related to its cytotoxicity on neuroblastoma cells are completely unknown. Here, we evaluated the anticancer property of MPTQ on mouse neuro 2a and human SH-SY5Y neuroblastoma cells and investigated the mechanisms underlying MPTQ-mediated neuro 2a cell death. MPTQ-mediated neuro 2a and SH-SY5Y cell deaths were found to be dose and time dependent. Moreover, MPTQ induced cell death reached approximately 99.8% and 90% in neuro 2a and SH-SY5Y cells respectively. Nuclear oligonucleosomal DNA fragmentation and Terminal dUTP Nick End Labelling assays indicated MPTQ-mediated neuro 2a cell death involved apoptosis. MPTQ-mediated apoptosis is associated with increased phosphorylation of p53 at Ser15 and Ser20 which correlates with the hyperphosphorylation of Ataxia-Telangiectasia mutated protein (ATM). Immunocytochemical analysis demonstrated the increased level of Bax protein in MPTQ treated neuro 2a cells. MPTQ-mediated apoptosis is also associated with increased activation of caspase-9, -3 and -7 but not caspase-2 and -8. Furthermore, increased level of caspase-3 and cleaved Poly (ADP Ribose) polymerase were observed in the nucleus of MPTQ treated neuro 2a cells, suggesting the involvement of caspase-dependent intrinsic but not extrinsic apoptotic pathway. Increased nuclear translocation of apoptosis inducing factor suggests additional involvement of caspase-independent apoptosis pathway in MPTQ treated neuro 2a cells. Collectively, MPTQ-induced neuro 2a cell death is mediated by ATM and p53 activation, and Bax-mediated activation of caspase-dependent and caspase-independent mitochondrial apoptosis pathways.

Mechanism of amyloid peptide induced CCR5 expression in monocytes and its inhibition by siRNA for Egr-1.

In Alzheimer's disease (AD), one finds increased presence of monocytes/macrophages and activated microglial cells in the brain. Immunohistochemical studies show increased expression of chemokine receptor 5 (CCR5) on reactive microglia associated with amyloid deposits in AD, suggesting that CCR5 may play a role in the regulation of the immune response in AD. In this study, we used peripheral blood monocytes and human monocytic THP-1 cell line as a model of microglia to delineate the cellular signaling mechanism of Abeta-induced CCR5 expression and the latter's role in the chemotaxis of monocytes. We observed that Abeta peptides at pathophysiological concentrations (125 nM) increased CCR5 mRNA and cell surface protein expression. The cellular signaling involved activation of c-Raf, ERK-1/ERK-2, and c-Jun NH(2)-terminal kinase. Analysis of some transcription factors associated with CCR5 promoter revealed that Abeta increased DNA binding activity of Egr-1 and AP-1. In addition, we show that CCR5 promoter contains an Egr-1 like consensus sequence GCGGGGGTG as demonstrated by 1) electrophoretic mobility shift assay, 2) transfection studies with truncated CCR5 gene promoter construct, and 3) chromatin immunoprecipitation analysis. Moreover, transfection of Egr-1 siRNA, but not of scrambled Egr-1 siRNA, in THP-1 cells resulted in >75% reduction in both Abeta-mediated CCR5 expression and concomitant chemotaxis to its ligands. We suggest that inhibition of Egr-1 by either Egr-1 siRNA or pharmacological agents may reduce activation of monocytes/microglia and possibly ameliorate the inflammation and progression of AD.

Metal-catalyzed oxidation of extracellular matrix components perturbs hepatocyte survival with activation of intracellular signaling pathways.

To investigate whether oxidative manipulation of extracellular matrix components could affect cell survival, we studied primary rat hepatocytes cultured on dishes coated with collagen type 1, which was oxidized with a metal-based system. Culture of hepatocytes on oxidized collagen led to decreased cellular catalase activity along with impaired cell survival. The fraction of polyploid hepatocytes decreased early followed by greater reaccumulation of polyploid cells. Cells cultured on oxidized collagen showed greater susceptibility to additional oxidant stress induced by tert.-butyl-hydroperoxide. The capacity of hepatocytes for growth factor-induced DNA synthesis was unaffected by culture on oxidized collagen. In response to culture on oxidized matrix, AP-1, Egr-1, CREB, and NF-kappaB transcription factor activity was rapidly increased. This change in transcription factor activity was ameliorated by treatment of collagen with a free radical spin trap, N-tert.-butyl-alpha-phenylnitrone, prior to oxidation. Moreover, culture of hepatocytes with aminoguanidine, an antioxidant drug, decreased cell injury. These findings established that exposure of primary hepatocytes to oxidized extracellular matrix components rapidly activates cell signaling events with loss of hepatocyte subpopulations. Such cell-extracellular matrix interactions may play roles in organ homeostasis and oncogenetic progression.

Amyloid peptide-induced cytokine and chemokine expression in THP-1 monocytes is blocked by small inhibitory RNA duplexes for early growth response-1 messenger RNA.

In Alzheimer's disease (AD) one finds increased deposition of A beta and also an increased presence of monocytes/macrophages in the vessel wall and activated microglial cells in the brain. AD patients show increased levels of proinflammatory cytokines by activated microglia. Here we used a human monocytic THP-1 cell line as a model for microglia to delineate the cellular signaling mechanism involved in amyloid peptides (A beta(1-40) and A beta(1-42))-induced expression of inflammatory cytokines and chemokines. We observed that A beta peptides at physiological concentrations (125 nM) increased mRNA expression of cytokines (TNF-alpha, and IL-1 beta) and chemokines (monocyte chemoattractant protein-1 (MCP-1), IL-8, and macrophage inflammatory protein-1 beta (MIP-1 beta)). The cellular signaling involved activation of c-Raf, extracellular signal-regulated kinase-1 (ERK-1)/ERK-2, and c-Jun N-terminal kinase, but not p38 mitogen-activated protein kinase. This is further supported by the data showing that A beta causes phosphorylation of ERK-1/ERK-2, which, in turn, activates Elk-1. Furthermore, A beta mediated a time-dependent increase in DNA binding activity of early growth response-1 (Egr-1) and AP-1, but not of NF-kappa B and CREB. Moreover, A beta-induced Egr-1 DNA binding activity was reduced >60% in THP-1 cells transfected with small interfering RNA duplexes for Egr-1 mRNA. We show that A beta-induced expression of TNF-alpha, IL-1 beta, MCP-1, IL-8, and MIP-1 beta was abrogated in Egr-1 small inhibitory RNA-transfected cells. Our results indicate that A beta-induced expression of cytokines (TNF-alpha and IL-1 beta) and chemokines (MCP-1, IL-8, and MIP-1 beta) in THP-1 monocytes involves activation of ERK-1/ERK-2 and downstream activation of Egr-1. The inhibition of Egr-1 by Egr-1 small inhibitory RNA may represent a potential therapeutic target to ameliorate the inflammation and progression of AD.

Reversal of hyperglycemia in mice by using human expandable insulin-producing cells differentiated from fetal liver progenitor cells.

Beta-cell replacement is considered to be the most promising approach for treatment of type 1 diabetes. Its application on a large scale is hindered by a shortage of cells for transplantation. Activation of insulin expression, storage, and regulated secretion in stem/progenitor cells offers novel ways to overcome this shortage. We explored whether fetal human progenitor liver cells (FH) could be induced to differentiate into insulin-producing cells after expression of the pancreatic duodenal homeobox 1 (Pdx1) gene, which is a key regulator of pancreatic development and insulin expression in beta cells. FH cells possess a considerable replication capacity, and this was further extended by introduction of the gene for the catalytic subunit of human telomerase. Immortalized FH cells expressing Pdx1 activated multiple beta-cell genes, produced and stored considerable amounts of insulin, and released insulin in a regulated manner in response to glucose. When transplanted into hyperglycemic immunodeficient mice, the cells restored and maintained euglycemia for prolonged periods. Quantitation of human C-peptide in the mouse serum confirmed that the glycemia was normalized by the transplanted human cells. This approach offers the potential of a novel source of cells for transplantation into patients with type 1 diabetes.

Mechanism of monocyte activation and expression of proinflammatory cytochemokines by placenta growth factor.

Monocytes from patients with sickle cell disease (SCD) are in an activated state. However, the mechanism of activation of monocytes in SCD is not known. Our studies showed that placenta growth factor (PlGF) activated monocytes and increased mRNA levels of cytokines (tumor necrosis factor-alpha [TNF-alpha] and interleukin-1beta [IL-1beta]) and chemokines (monocyte chemotactic protein-1 [MCP-1], IL-8, and macrophage inflammatory protein-1beta [MIP-1beta]) in both normal monocytes and in the THP-1 monocytic cell line. This increase in mRNA expression of cytochemokines was also reflected in monocytes derived from subjects with SCD. We studied the PlGF-mediated downstream cellular signaling events that caused increased transcription of inflammatory cytochemokines and chemotaxis of THP-1 monocytes. PlGF-mediated cytochemokine mRNA and protein expression was inhibited by PD98059 and wortmannin, inhibitors of mitogen-activated protein kinase kinase (MAPK/MEK) kinase and phosphatidylinositol-3 (PI3) kinase, respectively, but not by SB203580, a p38 kinase inhibitor. PlGF caused a time-dependent transient increase in phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2), which was completely inhibited by wortmannin, indicating that activation of PI3 kinase preceded MEK activation. PlGF also induced transient phosphorylation of AKT. MEK and PI3 kinase inhibitors and antibody to Flt-1 abrogated PlGF-induced chemotaxis of THP-1 monocytes. Overexpression of a dominant-negative AKT or a dominant-negative PI3 kinase p85 subunit in THP-1 monocytes attenuated the PlGF-mediated phosphorylation of ERK-1/2, cytochemokine secretion, and chemotaxis. Taken together, these data show that activation of monocytes by PlGF occurs via activation of Flt-1, which results in activation of PI3 kinase/AKT and ERK-1/2 pathways. Therefore, we propose that increased levels of PlGF in circulation play an important role in the inflammation observed in SCD via its effects on monocytes.