Current Signal Transduction Therapy - Volume 5, Issue 2, 2010

The limited efficacy of current therapeutic approaches for a number of socially relevant human diseases has required the exploration of alternative and more effective therapeutic strategies. In the last three decades, nucleic acid based drugs (NABDs) have emerged as an attractive and novel alternative with great therapeutic potential. NABDs which include antisense oligonucleotides, decoys, aptamers, triple helix forming oligonucleotides, DNAzymes, Ribozymes and small interfering RNAs, have been shown to be able to efficiently counteract pathological gene expression in many different experimental systems. Despite their broad potential applicability, NABD use in the clinic is limited by the lack of optimal delivery systems. Due to their hydrophilic nature, NABDs cannot efficiently cross cellular membrane for which appropriate carriers are needed. Moreover, their instability in serum requires a proper protection to prevent a fast degradation which would invariably lead to the abrogation of any significant therapeutic effect. In this work we will review the strategies currently proposed to circumvent NABD delivery problems. Although the NABD delivery issue can and should be optimized to bring NABD closer to the clinic, the encouraging results displayed so far in experimental models fully justify further efforts both in terms of investments and scientific work.

Until recently, heat shock protein 90 (Hsp90) has been mostly known as an abundant intracellular chaperone with more than 100 target proteins often involved in control of cell metabolism, survival, growth and differentiation. Hsp90 has been a target for anti-cancer drugs, since it was found either overexpressed or overactive in cancer cells. For more than a decade, geldanamycin (GM)-derived inhibitors of Hsp90's ATPase have entered various clinical trials around the world. Despite of high expectations, the efficacy of these inhibitors in humans has been less than what was hoped for. While newer generations of GM inhibitors are being developed and tested in several ongoing clinical trials, recent studies have discovered a surprising need for cancer cells to constitutively secrete Hsp90α for invasion and metastasis. A main function for secreted Hsp90 is to promote cell motility via the cell surface receptor LRP1 and/or secreted MMP2. Distinct from its intracellular chaperone function that requires the N-terminal ATPase and the C-terminal dimerization, the pro-motility activity of extracellular Hsp90α resides in a highly charged peptide between the linker region and the middle domain in Hsp90α. Selective inhibition of secreted Hsp90α blocks tumor cell invasion in vitro and in vivo. More importantly, since Hsp90α secretion does not occur in normal cells under physiological conditions, drugs that selectively target the secreted Hsp90α at its pro-motility region may prove to be more effective and less toxic for treatment of cancer patients.

In this review, we discuss the γ-secretase protease complexes, which are responsible for the generation of amyloid-β peptides that constitutes the amyloid plaques of Alzheimer's disease (AD). We begin with a brief review on the amyloid-β precursor protein (APP), its proteolytic cleavage and a brief discussion on the pathogenesis of AD. We then present the four major components of the γ-secretase complexes - presenilin, nicastrin, Aph-1, and Pen-2 and also introduce the importance of post-translational modification activity of the γ-secretase complexes. We then discuss the diversity of γ-secretase substrates and proposed biological functions. Next we review the current therapeutic strategies aimed at developing selective inhibitors of AD-associated γ-secretase activities. Finally, we present a discussion, which serves as a platform for further questions into the viability of γ-secretase as an AD therapeutic target.

Endometriosis, a disease affecting 3-10% of women of reproductive age, is characterized by the ectopic growth of endometrial tissue. Histologically, this disease is characterized by endometrial gland and stroma with surrounding dense fibrous tissue. During the development of endometriotic lesions, excess fibrosis may lead to scarring and to alteration of the tissue function. It has been suggested that type I collagen is a major contributor to endometriosis-associated fibrosis. One approach to understanding the pathogenesis of endometriosis is to investigate the mechanisms underlying fibrogenesis associated with this disease. Using three-dimensional collagen gel culture model, we have evaluated the extracellular matrix contractility and myofibroblastic differentiation of endometriotic stromal cells. Endometriotic stromal cells showed enhanced extracellular matrix contractility in comparison with normal endometrial stromal cells. Activation of the Ras homology (Rho)/Rho-associated coiled-coil-forming protein kinase (ROCK)-mediated signaling pathway with simultaneously enhanced myofibroblastic differentiation is involved in this mechanism. We have proposed the mevalonate-Rho/ROCK-mediated signaling pathways as possible therapeutic targets for the treatment of endometriosis-associated fibrosis. This paper is a review of the recent information concerning the mechanism of endometriosis-associated fibrosis, focusing on the contractility of endometriotic stromal cells. The modulators of mevalonate-Rho/ROCK signaling pathways were also evaluated as promising agents for the treatment and prevention of endometriosis-associated fibrosis.

The early growth response-1 (Egr-1) gene encodes a Cys2-His2-type zinc-finger transcription factor. A broad range of extra cellular stimuli are known to lead to its induction thus furthering growth, proliferation, differentiation or apoptosis. Accordingly, Egr-1 is involved in a variety of diseases such as atherosclerosis or cancer. Among other functional elements, five serum response elements (SRE) have been identified in the promoter region of Egr-1. The Rho/Rho-kinase pathway has been shown to regulate actin reorganization via LIM-kinase mediated cofilin phosphorylation. Recent studies revealed the actin binding striated muscle activator of Rho signaling (STARS) to promote translocation of myosin related transcription factors (MRTFs) into the nucleus leading to serum response factor (SRF) activation. The ternary complex factor (TCF) Elk-1 eventually bridges the gap between SRF mediated gene transcription and the Raf/MEK/ERK pathway. Furthermore, the Egr-1 promoter has two cAMP response elements (CREs), whose relevance for gene expression is still controversial. An Egr-1 binding site (EBS) is located on the Egr-1 promoter itself arguing for a negative feedback mechanism. The acquired knowledge on transcriptional regulation, however, is not entirely understood. In this review we highlight upstream and downstream signaling in vitro and in vivo associated with Egr-1.

In steroidogenic tissue the first and rate limiting step of steroid hormone synthesis is the transport of cholesterol from the outer mitochondrial membrane to the inner membrane where it is enzymatically converted by cytochrome P450 side chain cleavage (P450scc) to pregnenolone, which is the metabolic precursor for all steroid hormones. This step is accelerated by steroidogenic hormones but how the signal from the periphery is transmitted to the mitochondria is only partially established. The steroidogenic acute regulatory protein (StAR) is known to accelerate the export of cholesterol from the outer mitochondrial membrane. StAR may bind cholesterol in its hydrophobic pocket and then transport or propel cholesterol towards the inner membrane. This action is thought to be dependent on interaction with proteins in the contact sites between the inner and outer mitochondrial membranes, such as the translocator protein and the voltage-dependent anion channel. Mutations in StAR often rob the protein of its steroidogenic properties and result in lipoid congenital adrenal hyperplasia (LCAH), a condition that is characterized by a build up of cholesterol and cholesterol esters in steroidogenic cells, deficiencies in mineralocorticoids and glucocorticoids and XY genetic males with female phenotypic external genitalia. StAR expression in healthy individuals is known to be regulated by protein kinase A and the transcription factor Nurr77. A complete understanding the workings of the signal transduction machinery in steroidogenic cells may allow future plans to combat LCAH by using gene therapy to restore appropriate activity to StAR gene products.

Polycystic kidney diseases (PKDs) represent a group of progressive genetic renal disorders that are characterized by the development of renal cysts that lead to end-stage renal disease. The rapid advance in our understanding of the pathogenesis of PKDs is revealing possible targets to slow down the progression of PKD. Among them, the epidermal growth factor receptor (EGFR) family members (ErbBs) as well as their ligands, such as EGF, HB-EGF, TGF-α, have emerged as important mediators of cystic formation and growth in PKD. ErbB receptors are either overexpressed or mislocated to the apical surface of cystic tubular epithelium in PKD instead of the basolateral localization seen in the normal adult kidney. Preclinical studies have demonstrated that inhibition of ErbB tyrosine kinase activity and/or reduced ErbB ligand availability significantly reduced cystogenesis in several murine PKD models (pcy, jck and cpk) and in the Han: SPRD rat ADPKD model. In this review, we will discuss the expression patterns, possible roles and related signaling molecules of the ErbB axis in PKD and the pharmaceutical potentials for future therapeutic development.

The present study is aimed to determine the role of Ca2+ signaling evoked by hydrogen peroxide (H2O2) on caspase activation in human leukemia cell line HL-60. We have analysed cytosolic free Ca2+ concentration ([Ca2+]c) determination, mitochondrial membrane potential and caspase-3 and -9 activity by fluorimetric methods, using the fluorescent ratiometric Ca2+ indicator Fura-2, the dye JC-1, and specific fluorogenic substrate, respectively. Our results indicated that treatment of HL-60 cells with H2O2 induced a transient increase in [Ca2+]c due to Ca2+ release from internal stores. The stimulatory effect of H2O2 on Ca2+ signal was followed by a mitochondrial membrane depolarization. Our results also indicated that H2O2 was able to increase the caspase-3 and -9 activities. The effect of H2O2 on caspase activation was time dependent, reaching a maximal caspase activity after 120 min of stimulation. Loading of cells with dimethyl BAPTA, an intracellular Ca2+ chelator, significantly reduced H2O2-induced mitochondrial depolarization and caspase activation. Similar results were obtained when the cells were pretreated with Ru360, a specific blocker of Ca2+ uptake into mitochondria. The findings suggest that H2O2-induced caspase-3 and -9 activation and mitochondrial membrane depolarization is dependent on rises in [Ca2+]c in human myeloid HL-60 cells.