Mu Yao

Long-term outcome of fetal cell transplantation on postinfarction ventricular remodeling and function

OBJECTIVES The purpose of this study was to determine the long-term outcome of fetal cell transplantation into myocardial infarction on left ventricular (LV) function and remodeling. BACKGROUND While neonatal cell transplantation improved function for acute myocardial infarction, long-term data on the effects of cell-transplant therapy using a more primitive cell on ventricular remodeling and function are needed.Methods. - Therefore, we injected 4 x 10(6) Fischer 344 fetal cardiac cells or medium into 1-week old infarcts in adult female Fischer rats to assess long-term outcome. RESULTS Ten months after transplantation histologic analysis showed that cell implants were readily visible within the infarct scar. Infarct wall thickness was greater in cell-treated at 0.69 +/- 0.05 mm (n = 11) vs. medium-treated hearts at 0.33 +/- 0.01 mm (n = 19; P = 0.0001). Postmortem LV volume was 0.41 +/- 0.04 ml in cell-treated vs. 0.51 +/- 0.03 ml in medium-treated hearts (P < 0.04). Ejection fraction assessed by LV angiography was 0.40 +/- 0.02 in cell-treated (n = 16) vs. 0.33 +/- 0.02 in medium-treated hearts (n = 24; P < 0.03) with trends towards smaller in vivo end-diastolic and end-systolic volumes in cell-treated vs. medium-treated hearts. Polymerase chain reaction analysis of the Sry gene of the Y chromosome was positive in four of five cell-treated and zero of five medium-treated hearts confirming viability of male cells in female donors. CONCLUSION Over the course of 10 months, fetal cardiac cell transplantation into infarcted hearts increased infarct wall thickness, reduced LV dilatation, and improved LV ejection fraction. Thus, fetal cell-transplant therapy mitigated the longer-term adverse effects of LV remodeling following a myocardial infarction.

Dp44mT targets the AKT, TGF- β and ERK pathways via the metastasis suppressor NDRG1 in normal prostate epithelial cells and prostate cancer cells

Background:Effective treatment of prostate cancer should be based on targeting interactions between tumour cell signalling pathways and key converging downstream effectors. Here, we determined how the tumourigenic phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), tumour-suppressive phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and transforming growth factor-β (TGF-β) pathways are integrated via the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1). Moreover, we assessed how the novel anti-tumour agent, Dp44mT, may target these integrated pathways by increasing NDRG1 expression.Methods:Protein expression in Dp44mT-treated normal human prostate epithelial cells and prostate cancer cells (PC-3, DU145) was assessed by western blotting. The role of NDRG1 was examined by transfection using an NDRG1 overexpression vector or shRNA.Results:Dp44mT increased levels of tumour-suppressive PTEN, and decreased phosphorylation of ERK1/2 and SMAD2L, which are regulated by oncogenic Ras/MAPK signalling. Importantly, the effects of Dp44mT on NDRG1 and p-SMAD2L expression were more marked in prostate cancer cells than normal prostate epithelial cells. This may partly explain the anti-tumour selectivity of these agents. Silencing NDRG1 expression increased phosphorylation of tumourigenic AKT, ERK1/2 and SMAD2L and decreased PTEN levels, whereas NDRG1 overexpression induced the opposite effect. Furthermore, NDRG1 silencing significantly reduced the ability of Dp44mT to suppress p-SMAD2L and p-ERK1/2 levels.Conclusion:NDRG1 has an important role in mediating the tumour-suppressive effects of Dp44mT in prostate cancer via selective targeting of the PI3K/AKT, TGF-β and ERK pathways.

Apoptosis of ventricular and atrial myocytes from pacing-induced canine heart failure

OBJECTIVE Rapid ventricular pacing in dogs results in a low output cardiomyopathic state similar to idiopathic dilated cardiomyopathy in man. Cell death by apoptosis may play an important role in the loss of cardiac function. This study investigates the molecular pathways involved in the regulation of apoptosis in dogs with pacing-induced heart failure. METHODS Apoptosis was identified by terminal transferase nick end-labelling (TUNEL) in the ventricles and atria of dog hearts affected by rapid-ventricular pacing. Western blots were used to determine expression of the components involved in the initiation (Fas, Fas-Ligand, FADD), regulation (Bcl-2, Bax) and execution (caspase-2 and caspase-3) of apoptosis. RESULTS Pacing-induced heart failure resulted in a significant increase in the number of ventricular and atrial myocyte nuclei undergoing apoptosis as measured by TUNEL. Compared to the samples from control hearts (n=6) the expression of Bcl-2, an inhibitor of apoptosis, was significantly reduced in ventricles from five dogs with pacing-induced heart failure. No change in the expression of the apoptotic inducer Bax was detected. Fas and FADD were significantly elevated in all paced ventricles, and Fas-L was only detected in the paced hearts. Both caspase-2 and caspase-3 were elevated following ventricular pacing. CONCLUSIONS We have identified components of the signalling pathways along which apoptosis proceeds following the induction of heart failure in dogs. Apoptosis was also detected in the atria raising the possibility that, like human dilated cardiomyopathy, the molecular changes are global.

Cytosolic Phospholipase A2-α: A Potential Therapeutic Target for Prostate Cancer

Purpose: Cytosolic phospholipase A2-α (cPLA2-α) provides intracellular arachidonic acid to supply both cyclooxygenase and lipoxygenase pathways. We aim to determine the expression and activation of cPLA2-α in prostate cancer cell lines and tissue and the effect of targeting cPLA2-α in vitro and in vivo. Experimental Design: The expression of cPLA2-α was determined in prostate cancer cells by reverse transcription-PCR, Western blot, and immunocytochemistry. Growth inhibition, apoptosis, and cPLA2-α activity were determined after inhibition with cPLA2-α small interfering RNA or inhibitor (Wyeth-1). Cytosolic PLA2-α inhibitor or vehicle was also administered to prostate cancer xenograft mouse models. Finally, the expression of phosphorylated cPLA2-α was determined by immunohistochemistry in human normal, androgen-sensitive and androgen-insensitive prostate cancer specimens. Results: cPLA2-α is present in all prostate cancer cells lines, but increased in androgen-insensitive cells. Inhibition with small interfering RNA or Wyeth-1 results in significant reductions in prostate cancer cell numbers, as a result of reduced proliferation as well as increased apoptosis, and this was also associated with a reduction in cPLA2-α activity. Expression of cyclin D1 and phosphorylation of Akt were also observed to decrease. Wyeth-1 inhibited PC3 xenograft growth by ∼33% and again, also reduced cyclin D1. Immunohistochemistry of human prostate tissue revealed that phosphorylated cPLA2-α is increased when hormone refractory is reached. Conclusions: Expression and activation of cPLA2-α are increased in the androgen-insensitive cancer cell line and tissue. Inhibition of cPLA2-α results in cells and xenograft tumor growth inhibition and serves as a potentially effective therapy for hormone refractory prostate cancer.

Cytosolic phospholipase A2-alpha: a potential therapeutic target for prostate cancer.

Purpose: Cytosolic phospholipase A2-α (cPLA2-α) provides intracellular arachidonic acid to supply both cyclooxygenase and lipoxygenase pathways. We aim to determine the expression and activation of cPLA2-α in prostate cancer cell lines and tissue and the effect of targeting cPLA2-α in vitro and in vivo. Experimental Design: The expression of cPLA2-α was determined in prostate cancer cells by reverse transcription-PCR, Western blot, and immunocytochemistry. Growth inhibition, apoptosis, and cPLA2-α activity were determined after inhibition with cPLA2-α small interfering RNA or inhibitor (Wyeth-1). Cytosolic PLA2-α inhibitor or vehicle was also administered to prostate cancer xenograft mouse models. Finally, the expression of phosphorylated cPLA2-α was determined by immunohistochemistry in human normal, androgen-sensitive and androgen-insensitive prostate cancer specimens. Results: cPLA2-α is present in all prostate cancer cells lines, but increased in androgen-insensitive cells. Inhibition with small interfering RNA or Wyeth-1 results in significant reductions in prostate cancer cell numbers, as a result of reduced proliferation as well as increased apoptosis, and this was also associated with a reduction in cPLA2-α activity. Expression of cyclin D1 and phosphorylation of Akt were also observed to decrease. Wyeth-1 inhibited PC3 xenograft growth by ∼33% and again, also reduced cyclin D1. Immunohistochemistry of human prostate tissue revealed that phosphorylated cPLA2-α is increased when hormone refractory is reached. Conclusions: Expression and activation of cPLA2-α are increased in the androgen-insensitive cancer cell line and tissue. Inhibition of cPLA2-α results in cells and xenograft tumor growth inhibition and serves as a potentially effective therapy for hormone refractory prostate cancer.

Heart failure and apoptosis: electrophoretic methods support data from micro- and macro-arrays. A critical review of genomics and proteomics.

The multiple causes and multiple consequences of mammalian heart failure make it an attractive proposition for analysis using gene array technology, especially where the failure is idiopathic in nature. However, gene arrays also hold potential artefacts, particularly when gene expression levels are low, and where changes in expression levels are modest. Also, at present, the number of genes available on arrays is not large enough to prevent potential sampling deficiencies. Thus, it may not be wise to place too much reliance on quantitative interpretations of gene array data. Also, recently doubts were raised about the qualitative reliability of array genes. Electrophoretic methods are slow, cumbersome and complex but they can provide confirmation that the trends and numbers arising from the new gene arrays are reliable. In this overview, we compare gene array data with data from protein activity assays such as zymograms, Western blots, two‐dimensional electrophoresis, and immunohistochemistry. Similar or complementary data from the same heart tissues analyzed by either microarrays or macroarrays can be reassuring to those interested in reliable molecular analyses of normal and failing hearts. Similar principles will apply to other tissues and cells.

p27(Kip1) signaling: Transcriptional and post-translational regulation.

p27(Kip1) is an inhibitor of a broad spectrum of cyclin-dependent kinases (CDKs), and the loss of a single p27(Kip1) allele is thereby sufficient to increase tumor incidence via CDK-mediated cell cycle entry. As such, down-regulation of p27(Kip1) protein levels, in particular nuclear expressed p27(Kip1), is implicated in both disease progression and poor prognosis in a variety of cancers. p27(Kip1) expression is positively regulated by the transcription factor MENIN, and inhibited by oncogenic transcription factors MYC and PIM. However, regulation of p27(Kip1) protein expression and function is predominantly through post-translational modifications that alter both the cellular localization and the extent of E3 ubiquitin ligase-mediated degradation. Phosphorylation of p27(Kip1) at Thr(187) and Ser(10) is a prerequisite for its degradation via the E3 ubiquitin ligases SKP2 (nuclear) and KPC (cytoplasmic), respectively. Additionally, Ser(10) phosphorylated p27(Kip1) is predominantly localized in the cytoplasm due to the nuclear export protein CRM1. Another E3 ubiquitin ligase, PIRH2, degrades p27(Kip1) in both the cytoplasm and nucleus independent of phosphorylation state. As such, inhibition of cell cycle entry and progression in a variety of cancers may be achieved with therapies designed to correct p27(Kip1) localization and/or block its degradation.

Ubiquitin-conjugating enzyme E2C: A potential cancer biomarker

The ubiquitin-conjugating enzymes 2C (UBE2C) is an integral component of the ubiquitin proteasome system. UBE2C consists of a conserved core domain containing the catalytic Cys residue and an N-terminal extension. The core domain is required for ubiquitin adduct formation by interacting with the ubiquitin-fold domain in the E1 enzyme, and contributes to the E3 enzyme binding. UBE2C N-terminal extension regulates E3 enzyme activity as a part of an intrinsic inhibitory mechanism. UBE2C is required for the destruction of mitotic cyclins and securin, which are essential for spindle assembly checkpoint and mitotic exit. The UBE2C mRNA and/or protein levels are aberrantly increased in many cancer types with poor clinical outcomes. Accumulation of UBE2C stimulates cell proliferation and anchorage-independent growth. UBE2C transgenic mice are prone to develop spontaneous tumors and carcinogen-induced tumor with evidence of chromosome aneuploidy.

ER stress‐induced autophagy in melanoma

The activation of RAF‐MEK–extracellular signal‐regulated kinase (ERK) mitogen‐activated protein kinase cascade by v‐raf murine sarcoma viral oncogene homolog B1 (BRAF)V600E mutation is a key alteration in melanoma. Although BRAF inhibitor (BRAFi) has achieved remarkable clinical success, the positive response to BRAFi is not sustainable, and the initial clinical benefit is eventually barred by the development of resistance to BRAFi. There is growing evidence to suggest that endoplasmic reticulum (ER) stress‐induced autophagy could be a potential pro‐survival mechanism that contributes to genesis of melanoma and to the resistance to BRAFi. ER stress‐induced autophagy is an evolutionarily conserved membrane process. By degrading and recycling proteins and organelles via the formation of autophagous vesicles and their fusion with lysosomes, the autophagy plays a key role in homeostasis as well as pathological processes. In this review, we examine the autophagy phenomenon in melanocytic nevus, primary and metastatic melanoma, and its significance in BRAFi‐resistant melanoma.

Decrease in expression or activity of cytosolic phospholipase A2α increases cyclooxygenase-1 action : a cross-talk between key enzymes in arachidonic acid pathway in prostate cancer cells

The eicosanoid pathway is activated in many types of cancers including prostate. Eicosanoids are synthesized from intracellular arachidonic acid (AA), which is released from membrane glycerophospholipids mainly by the action of cytosolic phospholipase A(2)alpha (cPLA(2)alpha). Thus, targeting cPLA(2)alpha has been proposed as a treatment option. The aim of this study was to determine the effect of cPLA(2)alpha inhibition on cyclooxygenase (COX) expression and PGE(2) production. Inhibition of cPLA(2)alpha expression by siRNA or activity by Efipladib in prostate cancer cell lines (PC3 and LNCaP) led to an increase in COX-1 protein and PGE(2) levels in a dose-dependent manner from 24 to 72 h. The COX-2 response was less evident. Efipladib treatment increased COX-1 promoter transcriptional activity without changing the rate of COX-1 protein degradation. Treatment with Efipladib also led to a decrease in most LOX products (HETEs) as measured by LC/MS/MS. Replenishing 5- and 12-HETEs abolished Efipladib-induced COX-1 and PGE(2) levels. Decreasing 5- and 12-HETE production, as a result of treating cells with inhibitors MK886 and Baicalein, respectively, mimicked the effect of Efipladib on COX-1 and PGE(2) levels. Hence, the mechanism underlying the cPLA(2)alpha inhibition-induced COX-1 is likely due to a decrease in LOX products, which may exert a negative feedback on COX-1 gene expression in prostate cancer cells. Considering that PGE(2) is a potent promoter of cancer cell proliferation and survival, understanding the mechanism coupling cPLA(2)alpha with COX-1 is of potential clinical significance.