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 ...

Medical University of Silesia

Centre national de la recherche scientifique (CNRS) CS80054

LinkoCare Life Sciences AB

Apoptin, a small protein from chicken anemia virus, has attracted great attention, because it specifically kills tumor cells while leaving normal cells unharmed. The subcellular localization of apoptin appears to be crucial for this tumor-selective activity. In normal cells, apoptin resides in the cytoplasm, whereas in cancerous cells it translocates into the nucleus. The nuclear translocation of apoptin is largely controlled by its phosphorylation. In tumor cells, apoptin causes the nuclear accumulation of survival kinases including Akt and is phosphorylated by CDK2. Thereby, apoptin redirects survival signals into cell death responses. Apoptin also binds as a multimeric complex to DNA and interacts with several nuclear targets, such as the anaphase-promoting complex, resulting in a G2/M phase arrest. The proapoptotic signal of apoptin is then transduced from the nucleus to cytoplasm by Nur77, which triggers a p53-independent mitochondrial death pathway. In this review, we summarize recent discoveries of apoptin's mechanism of action that might provide intriguing insights for the development of novel tumor-selective anticancer drugs.

Apoptin, a tumor-selective killer.

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.

Methotrexate (MTX) is an antimetabolite broadly used in treatment of cancer and autoimmune diseases. MTX-induced hepatotoxicity limits its application. We investigated hepatoprotective effects of turmeric in MTX-induced liver toxicity.

Pre-administration of turmeric prevents methotrexate-induced liver toxicity and oxidative stress.

One of the most distinct features of middle ear cholesteatoma is bone destruction. Aetiology of cholesteatoma is thought to be multifactorial. Endotoxins produced by bacteria are thought to initiate the inflammation process in the middle ear leading to cholesteatoma. There are physiological differences in bone metabolism between men and women. The aim of our study was the immunohistochemical evaluation of the contents of two key components of the OPG/RANK/RANKL triad-RANKL and OPG in cholesteatoma, to analyse if there are any differences between the sexes and to evaluate the bacteria species isolated from cholesteatoma just before surgical treatment and to evaluate their plausible influence on the expression of OPG and RANKL in cholesteatoma. Twenty-one adult patients with acquired cholesteatoma who underwent surgery were analysed. There were no statistically significant differences in the expression of both regulators of osteoclastogenesis between the sexes. In 38.1 % patients cholesteatoma was not infected, whereas in 61.9 % patients various bacterial infections or mycosis were found. The most frequently isolated species was Pseudomonas aeruginosa (14.29 % infections) followed by Staphylococcus aureus (9.52 % infections). There were no statistically significant differences in expression of both OPG and RANKL between uninfected and infected cholesteatomas. 

Bacterial Infections and Osteoclastogenesis Regulators in Men and Women with Cholesteatoma.

Micro-RNAs (miRs) represent an innovative class of genes that act as regulators of gene expression. Recently, the aberrant expression of several miRs has been associated with different types of cancers. In this study, we show that miR301 inhibition influences PI3K-Akt pathway activity. Akt overexpression in MCF7 and MDAMB468 cells caused downregulation of miR301 expression. This effect was confirmed by co-transfection of miR301-modulators in the presence of Akt. Cells overexpressing miR301-inhibitor and Akt, exhibited increased migration and proliferation. Experimental results also confirmed PI3K, PTEN and FoxF2 as regulatory targets for miR301. Furthermore, Akt expression in conjunction with miR301-inhibitor increased nuclear accumulation of PTEN, thus preventing it from downregulating the PI3K-signalling. In summary, our data emphasize the importance of miR301 inhibition on PI3K-Akt pathway-mediated cellular functions. Hence, it opens new avenues for the development of new anti-cancer agents preferentially targeting PI3K-Akt pathway. 

Inhibition of miR301 enhances Akt-mediated cell proliferation by accumulation of PTEN in nucleus and its effects on cell-cycle regulatory proteins.

The cancer stem cell (CSC) hypothesis considers CSCs as the main culprits of tumor initiation, propagation, metastasis and therapy failure. CSCs represent a minority subpopulation of cells within a tumor. Their detection, characterization and monitoring are crucial steps toward a better understanding of the biological roles of these special cells in the development and propagation of tumors which, in turn, improves clinical reasoning and treatment options. Nowadays, in vitro and in vivo assays are available that address the self-renewal and differentiation potential of CSCs, and advanced in vivo molecular imaging technology facilitates the detection and provides an unprecedented in vivo observation platform to study the behavior of CSCs in their natural environment. Here, we provide a brief overview of CSCs and describe modern cellular models and labeling techniques to study and trace CSCs.

Cancer stem cells, cancer-initiating cells and methods for their detection.

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?

The human gyrovirus-Apoptin (HGyv-Apoptin) is a protein that gained attention because it is selectively cytotoxic toward cancer cells. In this study, we have investigated the effect of HGyv-Apoptin on cell cycle progression of cancer cells. We also compared HGyv-Apoptin's action to its homologue chicken anemia virus Apoptin (CAV-Apoptin). We show that HGyv-Apoptin induces G2/M arrest in cancer cells. This is at least in part due to the fact that HGyv-Apoptin induces an abnormal spindle formation in mitotic cells that do not progress properly throughout the cell cycle. HGyv-Apoptin most likely inhibits APC function leading to a sustained cyclin-B1-expression. These results indicate that HGyv-Apoptin has a similar mechanism of action as its homolog CAV-Apoptin and further supports its cancer therapeutic potential.

Human Gyrovirus-Apoptin Interferes with the Cell Cycle and Induces G2/M Arrest Prior to Apoptosis.

Reprogramming, or generation of induced pluripotent stem (iPS) cells (functionally similar to embryonic stem cells or ES cells) by the use of transcription factors (typically: Oct3/4, Sox2, c-Myc, Klf4) called "Yamanaka factors" (OSKM), has revolutionized regenerative medicine. However, factors used to induce stemness are also overexpressed in cancer. Both, ES cells and iPS cells cause teratoma formation when injected to tissues. This raises a safety concern for therapies based on iPS derivates. Transdifferentiation (lineage reprogramming, or -conversion), is a process in which one mature, specialized cell type changes into another without entering a pluripotent state. This process involves an ectopic expression of transcription factors and/or other stimuli. Unlike in the case of reprogramming, tissues obtained by this method do not carry the risk of subsequent teratomagenesis.

Transdifferentiation and reprogramming: Overview of the processes, their similarities and differences.

Adipose tissue is a promising source of mesenchymal stem cells. Their potential to differentiate and regenerate other types of tissues may be affected by several factors. This may be due to in vitro cell-culture conditions, especially the supplementation with antibiotics. The aim of our study was to evaluate the effects of a penicillin-streptomycin mixture (PS), amphotericin B (AmB), a complex of AmB with copper (II) ions (AmB-Cu) and various combinations of these antibiotics on the proliferation and differentiation of adipose-derived stem cells in vitro. Normal human adipose-derived stem cells (ADSC, Lonza) were routinely maintained in a Dulbecco's Modified Eagle Medium (DMEM) that was either supplemented with selected antibiotics or without antibiotics. The ADSC that were used for the experiment were at the second passage. The effect of antibiotics on proliferation was analyzed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and sulforhodamine-B (SRB) tests. Differentiation was evaluated based on Alizarin Red staining, Oil Red O staining and determination of the expression of ADSC, osteoblast and adipocyte markers by real-time RT-qPCR. The obtained results indicate that the influence of antibiotics on adipose-derived stem cells depends on the duration of exposure and on the combination of applied compounds. We show that antibiotics alter the proliferation of cells and also promote natural osteogenesis, and adipogenesis, and that this effect is also noticeable in stimulated osteogenesis.

Impact of Antibiotics on the Proliferation and Differentiation of Human Adipose-Derived Mesenchymal Stem Cells.

With the rapid advancement of regenerative medicine technologies, there is an urgent need for the development of new, cell-friendly techniques for obtaining nanofibers-the raw material for an artificial extracellular matrix production. We investigated the structure and properties of PCL nanofibers, PCL/PCL core-shell type nanofibers, as well as PCL/PCL nanofibres prepared by electrospinning. For the production of the fiber variants, a 5-10% solution of polycaprolactone (PCL) (M = 70,000-90,000), dissolved in a mixture of formic acid and acetic acid at a ratio of 70:30 m/m was used. In order to obtain fibers containing PCL 1% of silver nanoparticles was added. The electrospin was conducted using the above-described solutions at the electrostatic field. The subsequent bio-analysis shows that synthesis of core-shell nanofibers PCL/PCL, and the silver-doped variant nanofiber core shell PCL/PCL, by using organic acids as solvents, is a robust technique. Furthermore, the incorporation of silver nanoparticles into PCL/PCL makes such nanofibers toxic to model microbes without compromising its biocompatibility. Nanofibers obtained such way may then be used in regenerative medicine, for the preparation of extracellular scaffolds: (i) for controlled bone regeneration due to the long decay time of the PCL, (ii) as bioscaffolds for generation of other types of artificial tissues, (iii) and as carriers of nanocapsules for local drug delivery. Furthermore, the used solvents are significantly less toxic than the solvents for polycaprolactone currently commonly used in electrospin, like for example chloroform (CHCl), methanol (CHOH), dimethylformamide (CHNO) or tetrahydrofuran (CHO), hence the presented here electrospin technique may allow for the production of multilayer nanofibres more suitable for the use in medical field.

Structure and properties of slow-resorbing nanofibers obtained by (co-axial) electrospinning as tissue scaffolds in regenerative medicine.

This study was designed to evaluate the relationship between Programmed cell death protein 6 (PDCD6) polymorphisms and cancer susceptibility. The online databases were searched for relevant case-control studies published up to November 2017. Review Manage (RevMan) 5.3 was used to conduct the statistical analysis. The pooled odds ratio (OR) with its 95% confidence interval (CI) was employed to calculate the strength of association. Overall, our results indicate that PDCD6 rs3756712 T>G polymorphism was significantly associated with decreased risk of cancer under codominant (OR = 0.82, 95%CI = 0.70-0.96,  = 0.01, TG vs TT; OR = 0.53, 95%CI = 0.39-0.72,  < 0.0001, GG vs TT), dominant (OR = 0.76, 95%CI = 0.66-0.89,  = 0.0004, TG+GG vs TT), recessive (OR = 0.57, 95%CI = 0.43-0.78,  = 0.0003, GG vs TT+TG), and allele (OR = 0.76, 95%CI = 0.67-0.86,  < 0.00001, G vs T) genetic model. The finding did not support an association between rs4957014 T>G polymorphism of PDCD6, and different cancers risk.

Association of PDCD6 polymorphisms with the risk of cancer: Evidence from a meta-analysis.

LAPTM4B gene polymorphism augments the risk of cancer: Evidence from an updated meta-analysis.

Psoriasis, an autoimmune diseases of the skin, characterized by patches of abnormal/inflammed skin, although not usually life-threatening, it causes severe discomfort, esthetic impairments, and may lead to impaired social functions and social withdrawal. Besides UV-phototherapy, various anti-inflammatory treatments are applied, depending on the severity of symptoms. In 2008, adalimumab (fully humanized human anti-TNF antibody) was launched for the treatment of psoriasis. In the quest to better understand the pathomechanism of adalimumab's therapeutic effects, and the acquired resistance to the drug, we have investigated how its administration affect the regulation of the expression of selected caspases, including those activated by inflammosome.

Article	Year
Apoptin, a tumor-selective killer. Biochimica et biophysica acta\| PubMed ID: 19374922	2009
Autophagy and apoptosis dysfunction in neurodegenerative disorders. Progress in neurobiology\| PubMed ID: 24211851	2014
Pre-administration of turmeric prevents methotrexate-induced liver toxicity and oxidative stress. BMC complementary and alternative medicine\| PubMed ID: 26199067	2015
Bacterial Infections and Osteoclastogenesis Regulators in Men and Women with Cholesteatoma. Archivum immunologiae et therapiae experimentalis\| PubMed ID: 26584851	2016
Inhibition of miR301 enhances Akt-mediated cell proliferation by accumulation of PTEN in nucleus and its effects on cell-cycle regulatory proteins. Oncotarget\| PubMed ID: 26967567	2016
Cancer stem cells, cancer-initiating cells and methods for their detection. Drug discovery today\| PubMed ID: 26976692	2016
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
Human Gyrovirus-Apoptin Interferes with the Cell Cycle and Induces G2/M Arrest Prior to Apoptosis. Archivum immunologiae et therapiae experimentalis\| PubMed ID: 28386695	2017
Transdifferentiation and reprogramming: Overview of the processes, their similarities and differences. Biochimica et biophysica acta. Molecular cell research\| PubMed ID: 28460880	2017
Impact of Antibiotics on the Proliferation and Differentiation of Human Adipose-Derived Mesenchymal Stem Cells. International journal of molecular sciences\| PubMed ID: 29186789	2017
Structure and properties of slow-resorbing nanofibers obtained by (co-axial) electrospinning as tissue scaffolds in regenerative medicine. PeerJ\| PubMed ID: 29302386	2017
Association of PDCD6 polymorphisms with the risk of cancer: Evidence from a meta-analysis. Oncotarget\| PubMed ID: 29872511	2018
LAPTM4B gene polymorphism augments the risk of cancer: Evidence from an updated meta-analysis. Journal of cellular and molecular medicine\| PubMed ID: 30255662	2018
Psoriasis Treatment Changes the Expression Profile of Selected Caspases and their Regulatory MicroRNAs. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology\| PubMed ID: 30308514	2018

Marek J. Los

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