Current Pharmaceutical Biotechnology - Volume 12, Issue 11, 2011

An idea to prepare a Special Issue of Current Pharmaceutical Biotechnology containing results which are overwhelmingly of Polish origin may seem strange or even inappropriate in times of international consortia uniting individual and national efforts to challenge the most important scientific problems of the new millennium. Contemporary Poland has not been recognized for a particularly strong position in life sciences. Nevertheless, Polish biologists and biotechnologists feel well connected to deeply rooted tradition of high quality university teaching in home country, which produced such towering figures in biomedical field as Marceli Nencki1, Kazimierz Funk2, Rudolf Weigl3, Ludwik Fleck4, Ludwik Hirszfeld5, Waclaw Szybalski6 and Hilary Koprowski7. Amidst turmoil of the Poland's lost independence, the first and then the second world war some of the original thinkers and discoverers who laid foundations to modern biotechnology were fortunate enough to continue their research in the Western world. It is rewarding to observe that their pronounced interest in life sciences in general and medical and biochemical education in their home country in particular, has borne some fruit also in the 21st century, when world war wounds are finally healing and Europe is largely united. We are very grateful to Professor Zeno Földes-Papp for his initiative to edit the ‘Polish’ issue of the Current Pharmaceutical Biotechnology. Having in our disposal, as Guest Editors, a handful of original contributions chiefly from Polish laboratories, which were qualified as interesting and valuable by international reviewers we are proud to be able to present this collection to the faithful readers of CPB. We did our best to present an accurate representation of pharmaceutical biotechnology in Poland. We hope this unconventional move will help serve well to further enhance and inspire fruitful interactions between Polish and foreign scientists. Last but not least, we wish to thank our colleagues who so positively responded to the invitation to publish in the Issue.

The endoperoxide sesquiterpene lactone artemisinin which is isolated from the plant Artemisia annua, and its semi-synthetic derivatives, are potent, novel, antimalarial drugs. They are effective against multidrug-resistant Plasmodium strains and have become essential components of the so-called Artemisinin-based Combination Therapy, that is recommended by the World Health Organization as the treatment of choice for malaria tropica. Moreover, artemisinin and its derivatives show additional anti-parasite, antitumor, and anti-viral properties. The plants, however, are very poor resources for the drug, as the content of artemisinin is low (from 0,1 to 1,5 % of dried leaves) and dependent on seasonal and somatic variations as well as the infestation of bacteria, fungi and insects. A chemical synthesis of the compound is complex and uneconomic. Therefore, artemisinin is in short supply and remains unaffordable for most people in malariaendemic countries. Thus, many researchers have focused on enhancing the production of artemisinin, first, through traditional breeding and in in vitro plant tissue cultures and, then, by heterologous expression systems (a semi-synthetic approach) with the use of genetically-modified or transgenic microbes. In this review, we summarize the progress made in the production of artemisinin by the biotechnological approach.

The presented paper describes the drug delivery devices which can be considered as advanced or potentially “intelligent”. Due to the current development state and the legal problems of implantable drug releasing electronic devices the review is limited to the systems which delivers drugs through the skin or mucosa. The article shows the principle of operation and some construction details of such devices. It also discusses the possible methods of sampling body fluids across the drug delivery barriers to introduce a feedback loop which is necessary to react on the metabolic process in the human body and their malfunctioning. In the near future presented devices will evolve towards the highly sophisticated systems which will monitor our metabolism and deliver necessary drugs and hormones in the precisely calculated doses to regulate our body functions without absorbing our attention.

Multiple populations of stem cells have been indicated to potentially participate in regeneration of injured organs. Especially, embryonic stem cells (ESC) and recently inducible pluripotent stem cells (iPS) receive a marked attention from scientists and clinicians for regenerative medicine because of their high proliferative and differentiation capacities. Despite that ESC and iPS cells are expected to give rise into multiple regenerative applications when their side effects are overcame during appropriate preparation procedures, in fact their most recent application of human ESC may, however, reside in their use as a tool in drug development and disease modeling. This review focuses on the applications of stem cells in pharmaceutical biotechnology. We discuss possible relevance of pluripotent cell stem populations in developing physiological models for any human tissue cell type useful for pharmacological, metabolic and toxicity evaluation necessary in the earliest steps of drug development. The present models applied for preclinical drug testing consist of primary cells or immortalized cell lines that show limitations in terms of accessibility or relevance to their in vivo counterparts. The availability of renewable human cells with functional similarities to their in vivo counterparts is the first landmark for a new generation of cell-based assays. We discuss the approaches for using stem cells as valuable physiological targets of drug activity which may increase the strength of target validation and efficacy potentially resulting in introducing new safer remedies into clinical trials and the marketplace. Moreover, we discuss the possible applications of stem cells for elucidating mechanisms of disease pathogenesis. The knowledge about the mechanisms governing the development and progression of multitude disorders which would come from the cellular models established based on stem cells, may give rise to new therapeutical strategies for such diseases. All together, the applications of various cell types derived from patient specific pluripotent stem cells may lead to targeted drug and cellular therapies for certain individuals.

At present, about 3% of the human population are infected with hepatitis C virus (HCV). The first, acute stage of the disease is usually asymptomatic. However, only 15-25% of the infected eliminate the virus, while the remaining patients develop chronic hepatitis C (CHC). After 10-30 years of CHC, cirrhosis occurs in 20-30% of patients; 5-10% of this group eventually suffer from hepatocellular carcinoma. Unfortunately, up till now no effective methods protecting against HCV or allowing for efficient CHC treatment have been elaborated. This is primarily because not much is known about the mechanism of CHC emergence and the factors affecting anti-HCV therapy. There are several lines of evidence that some specific features of the virus, especially its high genetic variability might be responsible for the maintenance of HCV infection. Moreover, a few mechanisms which affect host-virus interactions and can additionally support CHC development have recently been identified. Hybridization between the host-encoded, liver-specific microRNA (miR-122) and the 5'-untranslated region of HCV genome was shown to be required for effective viral RNA replication. It was also postulated that HCV proteins mimic some of the human ones; that is why the virus is not eliminated. Another hypothesis assumes that interactions between HCV E2 glycoprotein and CD81 receptor modulate various cellular pathways, thus supporting viral propagation. There is no doubt that a better understanding of the mechanisms described above is of great importance for designing new therapeutic strategies and anti-HCV drugs.

The aminoacylation of bacterial polysaccharide antigens and its biological role are poorly understood, although it might be relevant in infection and immunity. Due to the lability of ester-linked substituents on glycoconjugate antigens, such groups usually escape detection during routine structural investigation. Of the few data available, those on the occurrence of glycine in the endotoxic lipopolysaccharides of Gram-negative bacteria are well documented. This article summarizes these data on glycine as an integral constituent of bacterial LPS and also some other amino-acid esters in the teichoic acids and phosphatidylglycerol of Gram-positive bacteria. The possible functions of such noncarbohydrate ester-linked substituents in bacterial antigens are discussed. Because glycine, an inherent component of bacterial lipopolysaccharides in the core region, is supposed to participate in epitope formation, such a structure may be considered for potential use in the construction of a vaccine with broad specificity.

Over the last two decades proteins have become increasingly important in human therapy and diagnosis. Engineering therapeutic proteins through improving their biological activity and stability has been a major interest in our group. In this mini-review we summarize our research on three proteins with pharmaceutical potential - serine protease inhibitor from squash seeds (CMTI), bovine pancreatic trypsin inhibitor (BPTI), and human fibroblast growth factor 1 (FGF1). To improve the functional properties of these proteins we used multiple techniques such as homology approach, rational design, total chemical synthesis, site-directed mutagenesis and phage display. The physicochemical properties of the obtained protein variants were evaluated using protein crystallography, spectroscopic techniques, enzymatic assays, stability measurements as well as numerous biological tests.

Well-established evidence links extracellular nucleotides to numerous vascular pathologies, including restenosis associated with angioplasty, atherosclerosis and transplant arteriosclerosis. Through activation of purinergic P2 receptors, extracellular nucleotides contribute to the pathogenesis of occlusive vascular diseases by mediating thrombosis, and vascular smooth muscle proliferation and migration. Therefore, there is a growing interest in the enzymes that hydrolyze nucleotides for their capability to modulate nucleotide-triggered pathologies. In this review, we present the current data addressing the therapeutic potential of nucleoside triphosphate diphosphohydrolases (NTPDases) to prevent intimal hyperplasia and treat vascular intimal disease. In addition, we discuss the mechanisms by which NTPDases exert protective effects in vascular function.

Malignant gliomas are the deadliest brain tumors, which are characterized by highly invasive growth, a rampant genetic instability and intense resistance to apoptosis. Such an aggressive behavior of malignant gliomas is reflected in the resistance to chemo- and radiotherapy and weak prognosis in spite of cytoreduction through surgery. Brain tumors preferentially express a number of specific protein and RNA markers, that may be exploited as potential therapeutic targets in design of the new treatment modalities based on nucleic acids. For almost three decades, a possibility to apply DNA and RNA molecules as anticancer therapeutics have been studied. A variety of antisense oligonucleotides, ribozymes, DNAzymes, and aptamers can be designed to trigger the sequence-specific inhibition of particular mRNA of interest. RNA interference (RNAi) is the latest and the most promising technique in the long line of nucleic acid-based therapeutic technologies. Recently, we designed and implemented the experimental therapy of patients suffering from malignant brain tumors based on application of double-stranded RNA (dsRNA) specific for tenascin-C (TN-C) mRNA. That therapeutic agent, called ATN-RNA, induces RNAi pathway to inhibit the synthesis of TN-C, the extracellular matrix protein which is highly overexpressed in brain tumor tissue. In the chapter specific problems of application of nucleic acid-based technologies in glioma tumors treatment will be discussed.

Progress in many therapies, in particular in the therapies based on peptides, proteins and nucleic acids used as bioactive compounds, strongly depends on development of appropriate carriers which would be suitable for controlled delivery of the intact abovementioned compounds to required tissues, cells and intracellular compartments. This review presents last ten years' achievements and problems in development and application of synthetic polymer nanoparticulate carriers for oral, pulmonary and nasal delivery routes of oligopeptides and proteins. Whereas some traditional synthetic polymer carriers are only briefly recalled the main attention is concentrated on nanoparticles produced from functional copolymers mostly with hydroxyl, carboxyl and amino groups, suitable for immobilization of targeting moieties and for assuring prolonged circulation of nanoparticles in blood. Formulations of various nanoparticulate systems are described, including solid particles, polymer micelles, nanovesicles and nanogels, especially systems allowing drug release induced by external stimuli. Discussed are properties of these species, in particular stability in buffers and models of body fluids, loading with drugs and with drug models, drug release processes and results of biological studies. There are also discussed systems for gene delivery with special attention devoted to polymers suitable for compacting nucleic acids into nanoparticles as well as the relations between chemical structure of polymer carriers and ability of the latter for crossing cell membranes and for endosomal escape.

A major advantage of recombinant DNA technology is its flexibility allowing for “on demand” production of specific proteins with theurapeutic value in heterologous expression systems. Gene expression vectors based on baculovirus, insect virus attacking mostly lepidopteran species, are frequently used for relatively inexpensive and fast production of such proteins. This expression system is recognized as one of the most powerful technologies for commercial synthesis of glycoproteins originating from vertebrate themselves or from vertebrate viruses. Glycosylation pathways utilized by insects are not identical, though they are similar to vertebrate glycosylation pathways. In the review special attention is given to the development of new virus-like particles (VLPs) potential vaccines which represent a novel class of subunit vaccines that are able to stimulate efficiently cellular and humoral immune responses against viral agents. Apart from production of vertebrate proteins or VLPs “on demand ” in insect cells, a new exciting field of using baculovirus as gene delivery system to vertebrate cells was recently open which has a great potential for future uses of baculovirus as effective gene therapy vector.

It was only in December 2008 that the European Union regulated the approval procedure for tissue engineered products (TEPs). Due to this regulation, TEP is classified as an advanced therapy medicinal product and as such may be recognized as a tool in pharmaceutical biotechnology. This paper gives a short review of the concept, the experimental evaluation and the clinical potency of tissue engineering (TE), with a particular focus on bone tissue engineered products. After a period of great enthusiasm about TE at the end of the 20th century a slight disappointment followed in the early 2000s. The review refers also to the continuously growing scientific interest, accompanied by the still modest representation of TEPs on the medical market. Some remarks are given on a bench-to-clinic road, including criticism concerning data originating from animal experiments. An attempt is made to foresee the still promising future of bone tissue engineered products (BTEPs) in practical use.

Mucopolysaccharidoses (MPS) are inherited metabolic disorders, caused by mutations leading to dysfunction of one of enzymes involved in degradation of glycosaminoglycans (GAGs) in lysosomes. Due to their impaired degradation, GAGs accumulate in cells of patients, which results in dysfunction of tissues and organs, including the heart, respiratory system, bones, joints and central nervous system. Depending on the kind of deficient enzyme, 11 types and subtypes of MPS are currently recognized. Although enzyme replacement therapy has been developed for 3 types of MPS (types I, II and VI), this treatment was found to be effective only in management of somatic symptoms. Since all MPS types except IVA, IVB and VI are characterized by various problems with functioning of the central nervous system (CNS), a search for effective treatment of this system is highly desirable. Recent discoveries suggested that substrate reduction therapy may be an efficient method for treatment of MPS patients, including their CNS. In this review, different variants of this therapy will be discussed in the light of recently published reports.

Despite more than a century of research in metastasis, the majority of cancer patients succumb due to complications of metastatic disease. It seems surprising that there remain so many questions related to the biology of metastasis. It is expected that a better understanding of the metastatic process will allow the development of specific therapies, improving the quality of life and extending the survival of cancer patients. In this special issue of Current Pharmaceutical Biotechnology we provide to a broad spectrum of readers with several reviews that bring together the most recent findings about metastasis. Gueron and co-workers focus their review on the key events responsible for the metastasis of tumors. They examine novel gene signatures identified in metastases, central inflammatory factors, mechanisms of cell plasticity and the role of microRNAs. They also point to new tools developed for cancer diagnostics and treatment, such as nanoparticle imaging and bioinformatic analysis of metastasis-related proteins. Gomes et al. as well as Krasnapolski and colleagues discuss a very hot topic in cancer research: Epithelial-Mesenchymal Transition (EMT). We largely accept that extensive tumor-stromal interactions and cross-talk are required to coordinate the multiple events of the metastatic cascade. Carlini and co-workers brought together recent findings that highlight the relevance of the cross-talk between the malignant cells and the microenvironment in the metastatic target organ. The ability to leave the primary tumor and then grow another tumor indicates that stem cell properties are likely to be important, as discussed Leiros & Balana. The authors provide an extensive revision of the literature describing the cancer stem cell (CSC) theory, and its possible role in the metastatic process. The present knowledge about the interactions between immune cells and tumors, particularly in the context of dissemination and metastasis development is revised by Croci & Salatino. They pay special attention to the latest information concerning several mechanisms involved in tumor immune evasion and the induction of distinct suppressive cells. A role of metalloproteinases (MMPs) in metastatic dissemination is widely accepted. Raffo and colleagues review all aspects of MMPs biology from the basic mechanisms of activation and actions to clinical applications. They discuss the results of clinical trials using MMPs inhibitors and therapeutic possibilities for the future. Given that primary tumor formation and metastasis are distinct processes, underlying molecular processes would be different and distinguishable. My group reviews a new class of molecules that reduces the metastatic propensity: metastasis suppressors. Finally, two reviews focused on therapeutic aspects of metastasis are included. In the first, Coluccio Leskow and collaborators describes the mechanistic theory behind the therapeutic potential of PKC inhibitors. In the second, Alonso and colleagues clearly review the benefits of a cancer peri-operative treatment with desmopressin (DDAVP), a vasopressin analog widely used in clinical settings. From the data presented in these reviews, we realize that considerable research to date has led to the identification of several aspects of the metastatic dissemination. However, we should keep in mind that although substantial progress has been made, great efforts are still needed to advance to the clinical setting. Therefore, I hope that this special issue of Current Pharmaceutical Biotechnology will be useful to those readers already working on metastasis, stimulating discussion and further studies, but mainly serving to attract new researchers from other fields interested in exploring new therapeutic applications to target metastatic disease.

The metastatic cascade and colonization remains a major challenge in clinical therapeutics. The formation of metastasis has many rate limiting steps. The expression of metastases initiation genes in primary tumors is driven by the need for cell motility, invasiveness, handling the shear stress in the vasculature and lymphatic circulation, and the survival and persistent growth in the distant organ. However, the expression of the progression genes in the primary tumors has a more complex basis. These metastasis-prone genes support primary tumor growth through one particular effect, whereas they enhance distant metastasis through another effect. The boundaries between metastasis initiation and metastasis progression genes are not rigid. In this review, we examine novel gene signatures identified in metastases, address key inflammatory factors mastering homing selection, gain further mechanistic insights into cell plasticity and evaluate the role of microRNAs. Moreover, we also describe the recent progress in developing nanoparticle imaging substantiating a promising theranostic platform for future cancer diagnostics and treatment, and assess the relevance of the bioinformatic analysis of metastasis-related proteins with an eye toward the metastatic niche. All these tools will provide valuable biological information of the progression of the disease, helping find potential therapeutic targets and improving surgical procedures. In a near future the understanding of the molecular mechanisms in tumor dissemination will be pivotal for the translation of these methods to the clinic and will help to overcome the barriers in clinical therapy of metastases.

During the past few years, Epithelial-Mesenchymal Transition (EMT) has emerged as one of the most hot spots in clinical research. Its existence in human tumors can form the basis for explaining characteristics of cancer progression and metastasis, as well as certain cases of drug resistance and relapses after treatment. These cellular responses are tightly regulated by intracellular signaling pathways evoked by humoral factors that include growth factors, chemokines and cytokines. Indeed, several gene regulatory programs known to promote EMT during development have recently been discovered to play key roles in cancer progression. A deeper understanding of the cellular and molecular basis of these different programs should aid in both the development of better diagnosis methods, as well as of specific treatments for invasive cancer. In this review we set out to summarize recent novel insights into the molecular players underlying EMT and its relation with cancer progression and metastasis.

Epithelial-to-mesenchymal transition (EMT) is a transdifferentiation process by which a fully differentiated epithelial cell acquires mesenchymal traits, and therefore, mesenchymal abilities such as motility and invasiveness. It is a pivotal physiological process involved in embryogenesis (Type 1 EMT) and in wound healing and tissue remodeling (Type 2 EMT), which, some authors claim, but there are still some controversies, has also been co-opted by tumor cells to increase their malignant potential (Type 3 EMT). Many biomarkers of Type 3 EMT have been characterized and classified into functional categories (i.e., extracellular proteins, cell surface molecules, cytoskeletal markers, transcriptional factors, and, recently, micro RNAs). The extra and intracellular signals that lead to EMT are only starting to be understood, but there is a consensus that Ras and TGF-beta signaling must converge with NF-κB in order to achieve a full EMT. The most classical experimental model is the induction of EMT by TGF-beta in cultures of epithelial cells. Other pathways involving GSK3b, and Wnt/beta-catenin, are also implicated. Ultimately, every EMT-inducing pathway will activate any of the E-cadherin transcriptional repressors (ZEB1, ZEB2, Twist, Snail or Slug). Although in the pre-clinical setting, EMT has also been related to an accelerated tumor progression and to an increased resistance to conventional chemotherapy. In this sense, several groups are beginning to use EMT as a predictive marker of response to treatment. Finally, two chemicals targeting TGF-beta are in clinical trials and many laboratories have initiated studies to use other EMT-related molecules as a therapeutic target for the cancer patient with some modest, but encouraging results.

This review presents recent information about the cross-talk between the tumor cells and the microenvironment in the target organ of metastasis at the premetastatic and metastatic stage. The development of metastatic foci is driven not only by the tumor cells intrinsic properties, but also by the interplay with resident and foreign cells located at particular niches in the target organ. The primary tumor modulates the metastatic target through the production of soluble factors that mobilize cells from distant organs like the bone marrow, which in turn localize in the metastatic niche. There is also strong evidence indicating that some primary tumors induce a fertile ground for the tumor cell at the target organ even before the arrival of the disseminated tumor cell (premetastatic niche). The relationship between the players of the metastatic setting is dynamic and shows a high degree of plasticity. Tumor cells change through the acquisition of genetic and/or epigenetic alterations that provide adaptive advantages and the metastatic niche is remodeled by incoming cell types or newly secreted soluble mediators, as a result a reciprocal dialogue is established that invokes new levels of molecular and cellular complexity. Unraveling the mechanisms that sustain the metastatic niche will allow a better understanding of the biology of the disseminated tumor cell, the design of new therapeutic approaches and, hopefully, the improvement of cancer patients' survival.

The cancer stem cell (CSC) hypothesis, predicts that a small subpopulation of cancer cells that possess “stemlike” characteristics, are responsible for initiating and maintaining cancer growth. According to the CSC model the many cell populations found in a tumour might represent diverse stages of differentiation. From the cellular point of view metastasis is considered a highly inefficient process and only a subset of tumour cells is capable of successfully traversing the entire metastatic cascade and eventually re-initiates tumour growth at distant sites. Some similar features of both normal and malignant stem cells suggest that CSCs are not only responsible for tumorigenesis, but also for metastases. The CSC theory proposes that the ability of a tumour to metastasize is an inherent property of a subset of CSCs. The similar biological characteristics shared by normal stem cells (NSCs) and CSCs mainly implicate self-renewal and differentiation potential, survival ability, niche-specific microenvironment requirements and specific homing to metastatic sites and may have important implications in terms of new approaches to cancer therapy in the metastatic setting. There are several agents targeting many of these CSC features that have shown to be effective both in vitro and in vivo. Although clinical trials results are still preliminary and continue under investigation, these new therapies are very promising. The identification of new therapeutic targets and drugs based on CSC model constitutes a great challenge.

Immune cells actively influence, among other factors, each step of tumor development determining the chance of a cancer cell to survive in a threaten microenvironment. Antitumor immune-mediated mechanisms are activated as soon as the first cancer cell is detected and operate both during primary tumor formation and during metastasis. However, when both innate and adaptive immunity becomes impaired, tumor development occurs. In this sense, compelling evidences indicate that tumor cells employ mechanisms that circumvent or thwart the immune response to enhance their own growth. These mechanisms include the secretion of immunosuppressive factors and the induction of distinct regulatory lymphoid or myeloid cells and, as occur with the immune response, they operate both during primary tumor formation and metastasis. Interestingly, cellular and molecular mechanisms of the immune response are important components of the tumor microenvironment and have the ability to promote or suppress tumor progression depending of the context of each cell interaction. In that sense, researchers are focusing their attention in the study of the influence of the tumor microenvironment in tumor growth and metastasis to better understand cancer biology and to formulate novel therapeutic approach. This review will focus on the present knowledge about interaction between immune cells and tumors in the context of metastasis, discussing the participation of different components of innate and adaptive immune response in the process of metastasis formation and dissemination.