Studies on Matrix Metalloproteinase (MMP-2, -9 and -14) and β2 Integrin Targeting as Potential Anticancer Therapeutics

Proteolytic enzymes, such as matrix metalloproteinases (MMP), are associated to the progression of several cancers. They degrade extracellular components, which helps tumors to expand and cancer cells to escape from the primary site. Of all MMPs, gelatinases (MMP-2 and -9) and membrane type-1 matrix metalloproteinase (MT1-MMP, MMP-14), in particular, are often associated to more aggressive types of head and neck carcinomas as well as to a poorer outcome in patient survival. Although therapies during the last decades have advanced, the mortality of the disease is still rather high and adjuvant therapies are searched for continuously. MMP-9 and MT1-MMP are also involved in neo-angiogenesis, which is necessary for tumor expansion. For this reason, we have identified synthetic peptides-targeting gelatinases and MT1-MMP, and have also evaluated their anticancer effects in vitro and in vivo. Antigelatinolytic peptides effectively inhibited tongue-carcinoma cell invasion and reduced the growth of xenografted tumors. In tumor samples of mice that were treated with antigelatinolytic peptides, the micro-vessel density was significantly reduced. We also identified a novel MT1-MMP targeting peptide and demonstrated that it exerted anticancer effects against several malignant cell lines in vitro. The effects of MT1-MMP inhibition on tongue-squamous cell carcinomas were evaluated by using xenograft tumors, which it effectively inhibited. Tranexamic acid was also demonstrated to inhibit tongue-squamous cell carcinoma invasion, most probably due to its ability to prevent the plasmin-mediated activation of proMMP-9. Leukocyte β2 integrins are another interesting option when evaluating targets for the therapeutic intervention of inflammatory conditions or malignancies of hematopoietic origin, since β2 integrins are expressed mainly by leukocytes. We identified a novel technique for screening small-molecule libraries against β2 integrins, and by using this technique we identified a novel αMβ2 integrin-binding chemical (IMB-10). IMB-10 significantly enhances leukocyte adhesion and inhibits their motility. We also demonstrated that IMB-10 can be used to inhibit inflammation and lymphoma growth in vivo. Interestingly, IMB-10 also reduced leukocyte tumor infiltration and inhibited tumor invasion.

Stress responses of Gram-positive bacteria to cationic antimicrobial peptides

As the resistance of bacteria to conventional antibiotics has become an increasing problem, new antimicrobial drugs are urgently needed. One possible source of new antibacterial agents is a group of cationic antimicrobial peptides (CAMPs) produced by practically all living organisms. These peptides are typically small, amphipathic and positively charged and contain well defined a-helical or b-sheet secondary structures. The main antibacterial action mechanism of CAMPs is considered to be disruption of the cell membrane, but other targets of CAMPs also exist. Some bacterial species have evolved defence mechanisms against the harmful effects of CAMPs. One of the most effective defence mechanisms is reduction of the net negative charge of bacterial cell surfaces. Global analysis of gene expression of two Gram-positive bacteria, Bacillus subtilis and Staphylococcus aureus, was used to further study the stress responses induced by different types of CAMPs. B. subtilis cells were treated with sublethal concentrations of a-helical peptide LL-37, b-sheet peptide protegrin 1 or synthetic analogue poly-L-lysine, and the changes in gene expression were studied using DNA macroarrays. In the case of S. aureus, three different a-helical peptides were selected for the transcriptome analyses: temporin L, ovispirin-1 and dermaseptin K4-S4(1-16). Transcriptional changes caused by peptide stress were examined using oligo DNA microarrays. The transcriptome analysis revealed two main cell signalling mechanisms mediating CAMP stress responses in Gram-positive bacteria: extracytoplasmic function (ECF)sigma factors and two-component systems (TCSs). In B. subtilis, ECF sigma factors sigW and sigM as well as TCS LiaRS responded to the cell membrane disruption caused by CAMPs. In S. aureus, CAMPs caused a similar stress response to antibiotics interfering in cell wall synthesis, and TCS VraSR was strongly activated. All of these transcriptional regulators are known to respond to several compounds other than CAMPs interfering with cell envelope integrity, suggesting that they sense cell envelope stress in general. Among the most strongly induced genes were yxdLM (in B. subtilis) and vraDE (in S. aureus) encoding homologous ABC transporters. Transcription of yxdLM and vraDE operons is controlled by TCSs YxdJK and ApsRS, respectively. These TCSs seemed to be responsible for the direct recognition of CAMPs. The yxdLM operon was specifically induced by LL-37, but its role in CAMP resistance remained unclear. VraDE was proven to be a bacitracin transporter. We also showed that the net positive charge of the cell wall affects the signalrecognition of different TCSs responding to cell envelope stress. Inactivation of the Dlt system responsible for the D-alanylation of teichoic acids had a strong and differential effect on the activity of the studied TCSs, depending on their functional role in cells and the stimuli they sense.

CNS injury, γ1 laminin, and its KDI peptide

Nisäkkäillä keskushermoston uudistuminen on rajallista. Keskushermostovamman jälkeen aktivoituu monien paranemista edistävien tekijöiden lisäksi myös estäviä tekijöitä. Monella molekyylillä, kuten laminiinilla, on keskushermoston paranemista tehostava vaikutus. Laminiinit ovat myös kehon tyvikalvojen oleellisia rakennuskomponentteja. Keskushermoston laminiinit ovat tärkeitä sikiökehityksen aikana, esimerkiksi hermosäikeiden ohjauksessa. Myöhemmin ne osallistuvat veriaivoesteen ylläpitoon sekä vammojen jälkeiseen kudosreaktioon. Väitöskirjatutkimuksessani olen selvittänyt lamiiniinien, erityisesti γ1 laminiinin ja sen KDI peptidin, ekspressiota keskushermoston vammatilanteissa. Kokeellisessa soluviljelmäasetelmassa, joka simuloi vammautunutta keskushermostoympäristöä, osoitimme että KDI peptidi voimistaa sekä hermosolujen selviytymistä että hermosäikeiden kasvua. Kainihappo on glutamaattianalogi, ja glutamaattitoksisuudella uskotaan olevan tärkeä merkitys keskushermoston eri vamma- ja sairaustilanteissa tapahtuvassa hermosolukuolemassa. Toisessa väitöskirjani osatyössä osoitimme eläinmallissa KDI peptidin suojaavan rotan aivojen hippokampuksen hermosoluja kainihapon aiheuttamalta solutuholta. Elektrofysiologisilla mittauksilla osoitimme kolmannessa osatyössäni, että KDI peptidi estää glutamaattireseptorivirtoja ja suojaa siten glutamaattitoksisuudelta. Aivoveritulpan aiheuttama aivovaurio on yleinen syy aivohalvaukseen. Viimeisessä osatyössäni tutkimme eläinmallissa laminiinien ekspressiota iskemian vaurioittamassa aivokudoksessa. Laminiiniekspression todettiin voimistuvan vaurion jälkeen sekä tyvikalvo- että soluväliainerakenteissa. Vaurion ympärillä havaittiin astrosyyttejä, jotka jo melko aikaisessa vaiheessa vamman jälkeen ekspressoivat γ1 laminiinia ja KDI peptidiä. Tästä voidaan päätellä laminiinien osallistuvan aivoiskeemisen vaurion patofysiologiaan. Yleisesti väitöskirjatyöni kartoitti laminiinien ekspressiota sekä terveessä että vammautuneessa keskushermostossa. Väitöskirjatyöni tukee hypoteesia, jonka mukaan KDI peptidi suojaa keskushermostoa vaurioilta.

Role of γ1 Laminin and Its KDI Peptide in the Central Nervous System

Since the 1980 s, laminin-1 has been linked to regeneration of the central nervous system (CNS) and promotion of neuronal migration and axon guidance during CNS development. In this thesis, we clarify the role of γ1 laminin and its KDI tripeptide in development of human embryonic spinal cord, in regeneration of adult rat spinal cord injury (SCI), in kainic acid-induced neuronal death, and in the spinal cord tissue of amyotrophic lateral sclerosis (ALS). We demonstrated that γ1 laminin together with α1, β1, and β3 laminins localize at the floor plate region in human embryonic spinal cord. This localization of γ1 laminin is in spatial and temporal correlation with development of the spinal cord and indicates that γ1 laminin may participate in commissural axon guidance during the embryonic development of the human CNS. With in vitro studies using the Matrigel culture system, we demonstrated that the KDI tripeptide of γ1 laminin provides a chemotrophic guidance cue for neurites of the human embryonic dorsal spinal cord, verifying the functional ability of γ1 laminin to guide commissural axons. Results from our experimental SCI model demonstrate that the KDI tripeptide enhanced functional recovery and promoted neurite outgrowth across the mechanically injured area in the adult rat spinal cord. Furthermore, our findings indicate that the KDI tripeptide as a non-competitive inhibitor of the ionotropic glutamate receptors can provide when administered in adequate concentrations an effective method to protect neurons against glutamate-induced excitotoxic cell death. Human postmortem samples were used to study motor neuron disease, ALS (IV), and the study revealed that in human ALS spinal cord, γ1 laminin was selectively over-expressed by reactive astrocytes, and that this over-expression may correlate with disease severity. The multiple ways by which γ1 laminin and its KDI tripeptide provide neurotrophic protection and enhance neuronal viability suggest that the over-expression of γ1 laminin may be a glial attempt to provide protection for neurons against ALS pathology. The KDI tripeptide is effective therapeutically thus far in animal models only. However, because KDI containing γ1 laminin exists naturally in the human CNS, KDI therapies are unlikely to be toxic or allergenic. Results from our animal models are encouraging, with no toxic side-effects detected even at high concentrations, but the ultimate confirmation can be achieved only after clinical trials. More research is still needed until the KDI tripeptide is refined into a clinically applicable method to treat various neurological disorders.

Assessment of In-House Natural Product and Synthetic Compound Libraries Based on In vitro Inhibition of Cholinesterases

The first line medication for mild to moderate Alzheimer s disease (AD) is based on cholinesterase inhibitors which prolong the effect of the neurotransmitter acetylcholine in cholinergic nerve synapses which relieves the symptoms of the disease. Implications of cholinesterases involvement in disease modifying processes has increased interest in this research area. The drug discovery and development process is a long and expensive process that takes on average 13.5 years and costs approximately 0.9 billion US dollars. Drug attritions in the clinical phases are common due to several reasons, e.g., poor bioavailability of compounds leading to low efficacy or toxic effects. Thus, improvements in the early drug discovery process are needed to create highly potent non-toxic compounds with predicted drug-like properties. Nature has been a good source for the discovery of new medicines accounting for around half of the new drugs approved to market during the last three decades. These compounds are direct isolates from the nature, their synthetic derivatives or natural mimics. Synthetic chemistry is an alternative way to produce compounds for drug discovery purposes. Both sources have pros and cons. The screening of new bioactive compounds in vitro is based on assaying compound libraries against targets. Assay set-up has to be adapted and validated for each screen to produce high quality data. Depending on the size of the library, miniaturization and automation are often requirements to reduce solvent and compound amounts and fasten the process. In this contribution, natural extract, natural pure compound and synthetic compound libraries were assessed as sources for new bioactive compounds. The libraries were screened primarily for acetylcholinesterase inhibitory effect and secondarily for butyrylcholinesterase inhibitory effect. To be able to screen the libraries, two assays were evaluated as screening tools and adapted to be compatible with special features of each library. The assays were validated to create high quality data. Cholinesterase inhibitors with various potencies and selectivity were found in natural product and synthetic compound libraries which indicates that the two sources complement each other. It is acknowledged that natural compounds differ structurally from compounds in synthetic compound libraries which further support the view of complementation especially if a high diversity of structures is the criterion for selection of compounds in a library.

Synthetic Modifications of Estrogens and Androgens

Estrogens the female sex hormones have numerous biological actions. Estradiol is the most abundant estrogen in women before menopause. It influences the development, maturation and function of the female reproductive tract. It also plays a role in mammary cancer. Accordingly determinations of estradiol level in body fluids assist in the evaluation of ovarian function and diagnosis for malignancies. Estriol is the primary estrogen in pregnant women and secreted from the fetoplacental unit. Measurement of estriol in maternal body fluids is the basis of fetoplacental monitoring test. Concentration of estrogens in body fluids is determined by immunoassay. Accuracy of this measurement depends on the availability of a specific antibody. As estrogens are not antigenic, their derivatives (haptens) are coupled with a carrier and this hapten-protein conjugate is used to generate antibodies. Specificity of the generated antibody largely depends on the structure of hapten. Therefore the synthesis of a hapten with a right structure is crucial for the accurate measurement of a steroid. We have synthesised new haptens for estradiol and estriol by adding an alkyl or alkoxy side chain at the C-7 of estrane skeleton. The side chains carry a terminal amino group, which can be used for conjugation with a carrier molecule. Estrogens and their biosynthetic precursor androgens both exist as fatty acid esters. They are known to act as hormone storage but their physiological role is not completely known yet. Our collaborator is studying their effect in cardiovascular diseases. We synthesised fatty acid ester derivatives of several steroids in high yield by a very rapid procedure (in 1 min) under microwave irradiation in an ionic liquid (IL). An expedient regioselective hydrolysis at C-3 of estradiol diesters is also reported. 8-Isoestrogens are compounds of pharmaceutical interests, their synthesis, structure, conformation and biological activity studies are ongoing. 7-Hydroxy-8-isoestradiol and 7-alkyl ether of it were synthesised as well. During this study we have developed a selective O-debenzylation method. A mild route for selective removal of benzylic protection on phenol in presence of benzyl protected alcohol was explored.