detection-small-gtpase-prenylation-gtp-binding-using-membrane

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

University of Manitoba

Isfahan University of Medical Sciences

Autophagy and apoptosis are basic physiologic processes contributing to the maintenance of cellular homeostasis. Autophagy encompasses pathways that target long-lived cytosolic proteins and damaged organelles. It involves a sequential set of events including double membrane formation, elongation, vesicle maturation and finally delivery of the targeted materials to the lysosome. Apoptotic cell death is best described through its morphology. It is characterized by cell rounding, membrane blebbing, cytoskeletal collapse, cytoplasmic condensation, and fragmentation, nuclear pyknosis, chromatin condensation/fragmentation, and formation of membrane-enveloped apoptotic bodies, that are rapidly phagocytosed by macrophages or neighboring cells. Neurodegenerative disorders are becoming increasingly prevalent, especially in the Western societies, with larger percentage of members living to an older age. They have to be seen not only as a health problem, but since they are care-intensive, they also carry a significant economic burden. Deregulation of autophagy plays a pivotal role in the etiology and/or progress of many of these diseases. Herein, we briefly review the latest findings that indicate the involvement of autophagy in neurodegenerative diseases. We provide a brief introduction to autophagy and apoptosis pathways focusing on the role of mitochondria and lysosomes. We then briefly highlight pathophysiology of common neurodegenerative disorders like Alzheimer's diseases, Parkinson's disease, Huntington's disease and Amyotrophic lateral sclerosis. Then, we describe functions of autophagy and apoptosis in brain homeostasis, especially in the context of the aforementioned disorders. Finally, we discuss different ways that autophagy and apoptosis modulation may be employed for therapeutic intervention during the maintenance of neurodegenerative disorders. 

Autophagy and apoptosis dysfunction in neurodegenerative disorders.

The cholesterol biosynthesis pathway, also known as the mevalonate (MVA) pathway, is an essential cellular pathway that is involved in diverse cell functions. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (HMGCR) is the rate-limiting step in cholesterol biosynthesis and catalyzes the conversion of HMG-CoA to MVA. Given its role in cholesterol and isoprenoid biosynthesis, the regulation of HMGCR has been intensely investigated. Because all cells require a steady supply of MVA, both the sterol (i.e. cholesterol) and non-sterol (i.e. isoprenoid) products of MVA metabolism exert coordinated feedback regulation on HMGCR through different mechanisms. The proper functioning of HMGCR as the proximal enzyme in the MVA pathway is essential under both normal physiologic conditions and in many diseases given its role in cell cycle pathways and cell proliferation, cholesterol biosynthesis and metabolism, cell cytoskeletal dynamics and stability, cell membrane structure and fluidity, mitochondrial function, proliferation, and cell fate. The blockbuster statin drugs ('statins') directly bind to and inhibit HMGCR, and their use for the past thirty years has revolutionized the treatment of hypercholesterolemia and cardiovascular diseases, in particular coronary heart disease. Initially thought to exert their effects through cholesterol reduction, recent evidence indicates that statins also have pleiotropic immunomodulatory properties independent of cholesterol lowering. In this review we will focus on the therapeutic applications and mechanisms involved in the MVA cascade including Rho GTPase and Rho kinase (ROCK) signaling, statin inhibition of HMGCR, geranylgeranyltransferase (GGTase) inhibition, and farnesyltransferase (FTase) inhibition in cardiovascular disease, pulmonary diseases (e.g. asthma and chronic obstructive pulmonary disease (COPD)), and cancer. 

Targeting the mevalonate cascade as a new therapeutic approach in heart disease, cancer and pulmonary disease.

Therapies that selectively target cancer cells for death have been the center of intense research recently. One potential therapy may involve apoptin proteins, which are able to induce apoptosis in cancer cells leaving normal cells unharmed. Apoptin was originally discovered in the Chicken anemia virus (CAV); however, human gyroviruses (HGyV) have recently been found that also harbor apoptin-like proteins. Although the cancer cell specific activity of these apoptins appears to be well conserved, the precise functions and mechanisms of action are yet to be fully elucidated. Strategies for both delivering apoptin to treat tumors and disseminating the protein inside the tumor body are now being developed, and have shown promise in preclinical animal studies. 

Apoptins: selective anticancer agents.

We aimed to compare the effects of oral ethanol (Eth) alone or combined with the phytoestrogen resveratrol (Rsv) on the expression of various brain-derived neurotrophic factor (BDNF) transcripts and the encoded protein pro-BDNF in the hippocampus of pregnant and embryonic rats. A low (0.25 g/kg body weight (BW)/day) dose of Eth produced an increase in the expression of BDNF exons I, III and IV and a decrease in that of the exon IX in embryos, but failed to affect BDNF transcript and pro-BDNF protein expression in adults. However, co-administration of Eth 0.25 g/kg·BW/day and Rsv led to increased expression of BDNF exons I, III and IV and to a small but significant increase in the level of pro-BDNF protein in maternal rats. A high (2.5 g/kg·BW/day) dose of Eth increased the expression of BDNF exons III and IV in embryos, but it decreased the expression of exon IX containing BDNF mRNAs in the maternal rats. While the high dose of Eth alone reduced the level of pro-BDNF in adults, it failed to change the levels of pro-BDNF in embryos. Eth differentially affects the expression pattern of BDNF transcripts and levels of pro-BDNF in the hippocampus of both adult and embryonic rats.

Effects of Ethanol on the Expression Level of Various BDNF mRNA Isoforms and Their Encoded Protein in the Hippocampus of Adult and Embryonic Rats.

The mevalonate cascade is a key metabolic pathway that regulates a variety of cellular functions and is thereby implicated in the pathophysiology of most brain diseases, including neurodevelopmental and neurodegenerative disorders. Emerging lines of evidence suggest that statins and Rho GTPase inhibitors are efficacious and have advantageous properties in treatment of different pathologic conditions that are relevant to the central nervous system. Beyond the original role of statins in lowering cholesterol synthesis, they have anti-inflammatory, antioxidant and modulatory effects on signaling pathways. Additionally, Rho GTPase inhibitors and statins share the mevalonate pathway as a common target of their therapeutic actions. In this review, we discuss potential mechanisms through which these drugs, via their role in the mevalonate pathway, exert their neuroprotective effects in neurodegenerative and neurodevelopmental disorders.

Mevalonate Cascade and Neurodevelopmental and Neurodegenerative Diseases: Future Targets for Therapeutic Application.

Natural products have a long history of use in traditional medicines and their activities against different diseases have been the focus of many basic and clinical researches in past few decades. The essential oils, volatile liquid containing aroma compound from plants, are known as active ingredients in the herbal medicine. Perillyl alcohol (POH) is usually available through dietary sources and is being explored for its cancer chemoprevention, tumor growth suppression, and regression. Citrus peels are the waste product of juice manufacturing industries and have been considered as a critical problem for environmental green ecology policies for years. One of the most well-known approaches to overcome this problem is transformation of these monoterpene by the use of specific strains of bacteria or yeasts. Limonene (1-methyl-4-isopropyl-cyclohexene) is a monoterpene, as other monoterpenes consists of two isoprene units, that comprises more than 90% of citrus essential oil and it exists in many fruits and vegetables. Although, the anticancer activity of d-limonene has identified nearly two decades ago, it has recently attracted much more attention in translational medicine. In this chapter, we will overview the anticancer effects of POH and d-limonene. Later, we will address the pharmacokinetics of these compounds, highlight the signaling pathways which are targeted by these proteins, review the clinical trials which have been done for these compounds in different cancer models, and finally discuss the future directions of the research in this field that might be more applicable in future cancer therapy strategies. 

Perillyl Alcohol (Monoterpene Alcohol), Limonene.

Understanding the connection between metabolic pathways and cancer is very important for the development of new therapeutic approaches based on regulatory enzymes in pathways associated with tumorigenesis. The mevalonate cascade and its rate-liming enzyme HMG CoA-reductase has recently drawn the attention of cancer researchers because strong evidences arising mostly from epidemiologic studies, show that it could promote transformation. Hence, these studies pinpoint HMG CoA-reductase as a candidate proto-oncogene. Several recent epidemiological studies, in different populations, have proven that statins are beneficial for the treatment-outcome of various cancers, and may improve common cancer therapy strategies involving alkylating agents, and antimetabolites. Cancer stem cells/cancer initiating cells (CSC) are key to cancer progression and metastasis. Therefore, in the current review we address the different effects of statins on cancer stem cells. The mevalonate cascade is among the most pleiotropic, and highly interconnected signaling pathways. Through G-protein-coupled receptors (GRCP), it integrates extra-, and intracellular signals. The mevalonate pathway is implicated in cell stemness, cell proliferation, and organ size regulation through the Hippo pathway (e.g. Yap/Taz signaling axis). This pathway is a prime preventive target through the administration of statins for the prophylaxis of obesity-related cardiovascular diseases. Its prominent role in regulation of cell growth and stemness also invokes its role in cancer development and progression. The mevalonate pathway affects cancer metastasis in several ways by: (i) affecting epithelial-to-mesenchymal transition (EMT), (ii) affecting remodeling of the cytoskeleton as well as cell motility, (iii) affecting cell polarity (non-canonical Wnt/planar pathway), and (iv) modulation of mesenchymal-to-epithelial transition (MET). Herein we provide an overview of the mevalonate signaling network. We then briefly highlight diverse functions of various elements of this mevalonate pathway. We further discuss in detail the role of elements of the mevalonate cascade in stemness, carcinogenesis, cancer progression, metastasis and maintenance of cancer stem cells.

Could drugs inhibiting the mevalonate pathway also target cancer stem cells?

Idiopathic pulmonary fibrosis (IPF) is a lethal fibrotic lung disease in adults with limited treatment options. Autophagy and the unfolded protein response (UPR), fundamental processes induced by cell stress, are dysregulated in lung fibroblasts and epithelial cells from humans with IPF. Human primary cultured lung parenchymal and airway fibroblasts from non-IPF and IPF donors were stimulated with transforming growth factor-β (TGF-β) with or without inhibitors of autophagy or UPR (IRE1 inhibitor). Using immunoblotting, we monitored temporal changes in abundance of protein markers of autophagy (LC3βII and Atg5-12), UPR (BIP, IRE1α, and cleaved XBP1), and fibrosis (collagen 1α2 and fibronectin). Using fluorescent immunohistochemistry, we profiled autophagy (LC3βII) and UPR (BIP and XBP1) markers in human non-IPF and IPF lung tissue. TGF-β-induced collagen 1α2 and fibronectin protein production was significantly higher in IPF lung fibroblasts compared with lung and airway fibroblasts from non-IPF donors. TGF-β induced the accumulation of LC3βII in parallel with collagen 1α2 and fibronectin, but autophagy marker content was significantly lower in lung fibroblasts from IPF subjects. TGF-β-induced collagen and fibronectin biosynthesis was significantly reduced by inhibiting autophagy flux in fibroblasts from the lungs of non-IPF and IPF donors. Conversely, only in lung fibroblasts from IPF donors did TGF-β induce UPR markers. Treatment with an IRE1 inhibitor decreased TGF-β1-induced collagen 1α2 and fibronectin biosynthesis in IPF lung fibroblasts but not those from non-IPF donors. The IRE1 arm of the UPR response is uniquely induced by TGF-β in lung fibroblasts from human IPF donors and is required for excessive biosynthesis of collagen and fibronectin in these cells.

Autophagy and the unfolded protein response promote profibrotic effects of TGF-β in human lung fibroblasts.

Despite advances in neurosurgical techniques and radio-/chemotherapy, the treatment of brain tumors remains a challenge. This is particularly true for the most frequent and fatal adult brain tumor, glioblastoma (GB). Upon diagnosis, the average survival time of GB patients remains only approximately 15months. The alkylating drug temozolomide (TMZ) is routinely used in brain tumor patients and induces apoptosis, autophagy and unfolded protein response (UPR). Here, we review these cellular mechanisms and their contributions to TMZ chemoresistance in brain tumors, with a particular emphasis on TMZ chemoresistance in glioma stem cells and GB.

Glioblastoma and chemoresistance to alkylating agents: Involvement of apoptosis, autophagy, and unfolded protein response.

Autophagy is increasingly identified as a central player in many cellular activities from cell proliferation to cell division, migration, and differentiation. However, it is also considered as a double-edged sword in cancer biology which either promotes oncogenesis/invasion or sensitizes the tumor cells to chemotherapy induced apoptosis. Recent investigations have provided direct evidence for regulation of cellular phenotype via autophagy pathway. One of the most important types of phenotype conversion is Epithelial-Mesenchymal-Transition (EMT), resulting in alteration of epithelial cell properties to a more mesenchymal form. In the current chapter, we provide a method which is established and being used in our laboratory for detection of autophagy and EMT in lung epithelial cells and show the involvement of autophagy in modulation of cellular phenotype.

Simultaneous Detection of Autophagy and Epithelial to Mesenchymal Transition in the Non-small Cell Lung Cancer Cells.

Lung cancer is considered one of the most frequent causes of cancer-related death worldwide and Non-Small Cell Lung Cancer (NSCLC) accounts for 80% of all lung cancer cases. Autophagy is a cellular process responsible for the recycling of damaged organelles and protein aggregates. Transforming growth factor beta-1 (TGFβ) is involved in Epithelial to Mesenchymal Transition (EMT) and autophagy induction in different cancer models and plays an important role in the pathogenesis of NSCLC. It is not clear how autophagy can regulate EMT in NSCLC cells. In the present study, we have investigated the regulatory role of autophagy in EMT induction in NSCLC and show that TGFβ can simultaneously induce both autophagy and EMT in the NSCL lines A549 and H1975. Upon chemical inhibition of autophagy using Bafilomycin-A1, the expression of the mesenchymal marker vimentin and N-cadherin was reduced. Immunoblotting and immunocytochemistry (ICC) showed that the mesenchymal marker vimentin was significantly downregulated upon TGFβ treatment in ATG7 knockdown cells when compared to corresponding cells treated with scramble shRNA (negative control), while E-cadherin was unchanged. Furthermore, autophagy inhibition (Bafilomycin A1 and ATG7 knockdown) decreased two important mesenchymal functions, migration and contraction, of NSCLC cells upon TGFβ treatment. This study identified a crucial role of autophagy as a potential positive regulator of TGFβ-induced EMT in NSCLC cells and identifies inhibitors of autophagy as promising new drugs in antagonizing the role of EMT inducers, like TGFβ, in the clinical progression of NSCLC.

Autophagy modulates transforming growth factor beta 1 induced epithelial to mesenchymal transition in non-small cell lung cancer cells.

Patients with glioblastoma multiforme (GBM) have an average life expectancy of approximately 15 months. Recently, statins have emerged as a potential adjuvant cancer therapy due to their ability to inhibit cell proliferation and induce apoptosis in many types of cancer. The exact mechanisms that mediate the inhibitory actions of statins in cancer cells are largely unknown. The purpose of this proceeding paper is to discuss some of the known anticancer effects of statins, while focusing on GBM therapy that includes adjunct therapy of statins with chemotherapeutic agents.

Article	Year
Autophagy and apoptosis dysfunction in neurodegenerative disorders. Progress in neurobiology\| PubMed ID: 24211851	2014
Targeting the mevalonate cascade as a new therapeutic approach in heart disease, cancer and pulmonary disease. Pharmacology & therapeutics\| PubMed ID: 24582968	2014
Apoptins: selective anticancer agents. Trends in molecular medicine\| PubMed ID: 25164066	2014
Effects of Ethanol on the Expression Level of Various BDNF mRNA Isoforms and Their Encoded Protein in the Hippocampus of Adult and Embryonic Rats. International journal of molecular sciences\| PubMed ID: 26703578	2015
Mevalonate Cascade and Neurodevelopmental and Neurodegenerative Diseases: Future Targets for Therapeutic Application. Current molecular pharmacology\| PubMed ID: 26758948	2017
Perillyl Alcohol (Monoterpene Alcohol), Limonene. The Enzymes\| PubMed ID: 27102697	2014
Could drugs inhibiting the mevalonate pathway also target cancer stem cells? Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy\| PubMed ID: 27155373	2016
Autophagy and the unfolded protein response promote profibrotic effects of TGF-β in human lung fibroblasts. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 29074489	2018
Glioblastoma and chemoresistance to alkylating agents: Involvement of apoptosis, autophagy, and unfolded protein response. Pharmacology & therapeutics\| PubMed ID: 29080702	2018
Simultaneous Detection of Autophagy and Epithelial to Mesenchymal Transition in the Non-small Cell Lung Cancer Cells. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29101677	2019
Autophagy modulates transforming growth factor beta 1 induced epithelial to mesenchymal transition in non-small cell lung cancer cells. Biochimica et biophysica acta. Molecular cell research\| PubMed ID: 29481833	2018
Statins: a new approach to combat temozolomide chemoresistance in glioblastoma. Journal of investigative medicine : the official publication of the American Federation for Clinical Research\| PubMed ID: 30368483	2018

Shahla Shojaei

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