The role of proteomics in the discovery of new anti cancer drugs

S obzirom na to da je za potpuno razumijevanje bioloških sustava nužna i molekularna karakterizacija proteoma, proteomika je disciplina koja je nastala sa svrhom da komplementira istraživanje genoma. Iako je proteomika relati vno nova biološka znanstvena disciplina, ona ima velik potencijal u farmaceutskoj industriji. Različite tehnike proteomike stoga se koriste u svrhu razjašnjavanja molekularnih mehanizama koji upravljaju staničnim procesima, za karakterizaciju složenih proteinskih mreža u stanici, za otkrivanje proteina biomarkera te, naposljetku, za identifikaciju novih meta za razvoj novih lijekova. Upravo u razvoju protutumorskih lijekova proteomika može omogućiti identi fi kaciju proteina uključenih u patogenezu raka, a rekonstrukcija signalnih putova može olakšati otkrivanje novih meta na koje bi novi lijek djelovao, te omogućiti uvid u mehanizme djelovanja ti h lijekova, kao i njihovu toksičnost.The fi eld of proteomics has been thought as complementary to the genomic research since proteins are the executi ve molecules and players in biological systems. In particular, protein functionality is highly deregulated in cancer. Although proteomics is a relatively new research area it holds great potential in pharmaceutical industry due to its ability to quantitatively measure protein expression and identi fy proteins by mass spectrometry analyses as well as its ability to map the protein-protein interacti ons. As such, the proteomics technological platf orm has been widely applied to basic cancer research, cancer therapy and most importantly in the drug discovery process. Proteomics allows identi fi cati on of proteins involved in cancer pathogenesis and deregulated signalling pathways that facilitates the discovery of new drug targets, or it might be used as well to precisely elucidate the drug mechanisms of acti on and toxicology

Proteomics in drug development – optimizati on of sample preparation for the two-dimensional gel-electrophoresis

Uvod: Dvodimenzionalna gel-elektroforeza (2-DE) metoda je analize kompleksnih proteinskih smjesa izdvojenih iz stanica, tkiva, tjelesnih tekućina ili drugih bioloških uzoraka, koja objedinjuje dva različita elektroforetska postupka: izoelektrično fokusiranje i SDS-poliakrilamidnu gel-elektroforezu. Metode: U ovom radu ispitan je utjecaj četi riju različiti h pufera na proteomske profile dviju staničnih linija karcinoma kolona, SW 620 i HCT 116 uzgojenih klasičnim metodama uzgoja stanica in vitro uz pomoć metode 2-DE, u pH rasponu 3 – 10 NL, te rasponu molekulskih težina od 14 do 100 kDa. Rezultati : Dobiveni rezultati pokazuju kako se pojedini puferi međusobno razlikuju po prirodi proteina koji su specifi čno otopljeni, što je djelomično ovisilo i o staničnoj liniji. Diskusija: Topljivost proteina jedan je od najvećih problema pri korištenju 2-DE, što naročito dolazi do izražaja kod hidrofobnih membranskih proteina, proteina jezgre i proteina izrazito sklonih agregaciji. Jedan od načina poboljšanja topljivosti proteina je i opti mizacija pufera za izdvajanje proteina, pri čemu se u pufer dodaju zwitt er-ionski amfi fi lni surfaktanti poput CHAPS, SB 3-10 (kaprilil sulfobetain) i ASB-14, te uporabom ti ouree u kombinaciji s ureom. Gelovi dobiveni uz pomoć pufera koji sadrži CHAPS imali su najbolju rezoluciju i na njima je bilo moguće detekti rati najveći broj jedinstvenih proteinskih vrsta obiju ispitanih staničnih linija. Zaključak: Postupkom opti mizacije pripreme uzorka za analizu mehanizama učinka novosinteti ziranog protutumorskog spoja na razini proteoma uz pomoć 2-DE pokazano je kako je pufer sastava 7M urea + 2M ti ourea + 4% (w/v) CHAPS opti malan pufer za izolaciju i razdvajanje smjese proteina dobivene iz tumorskih staničnih linija kolona HCT-116 i SW 620.Introducti on: The two-dimensional gel-electrophoresis (2-DE) is a method of choice for complex protein mixture separati on from cells, ti ssue, body fl uids or other biological samples which comprises two diverse electrophoreti c principles: the isoelectric focusing and the SDS-polyacrilamide gel electrophoresis. Methods: In this paper we tested the effects of four diff erent 2-DE buff ers on the protein profi les obtained by 2-DE in the pH range 3-10 NL, and the molecular weight range 14-100 kDa. The proteins were isolated from two colon cancer cell lines SW 620 i HCT 116 grown in vitro by using standard cell propagation procedures. Results: The obtained results revealed diff erent 2-DE buff er properti es for each buff er tested as the protein profi les showed a diff erent patt ern both as the consequence of the buff er type and the type of cell line. Discussion: One of the major problems in 2-DE is protein solubility which is parti cularly emphasized when dealing with hydrophobic membrane proteins, the nuclear proteins as well as with proteins prone to form aggregates. One of the possible soluti ons to this problem is the opti mizati on of the 2-DE buff er by additi on of zwitt er-ionic amphyphilic surfactants like CHAPS, SB 3-10 (sulphobetain) and ASB-14, as well as by additi on of thiourea and urea. The gels obtained with the buff er containing CHAPS showed the best resoluti on properti es and had the best unique protein species score for both tested cell lines in comparison to all other gels. Conclusion: Through the 2-DE buffer optimization in the sample preparati on step for proteomic analysis of newly synthesized anti tumor drug mechanisms of acti on, it has been shown that the buff er containing 7M urea + 2M thiourea + 4% (w/v) CHAPS was the most eff ecti ve for isolati on and separati on of proteins from HCT-116 and SW 620 tumour cell lines

Unveiling the mechanisms of breast cancer pathogenesis by proteomics methods

Patogenezu složene i heterogene bolesti kao što je to rak dojke nije moguće u potpunosti razjasniti klasičnim metodološkim pristupima koji su se koristi li posljednjih desetljeća u molekularnoj medicini. Osobito je važno otkriti nove biljege koji bi pomogli u ranoj detekciji bolesti te služili za razvoj ciljane terapije. Dosadašnji rezultati iz područja istraživanja raka dojke u kombinaciji s globalnim metodama istraživanja statusa ekspresije gena i proteina pokazali su kako je u patogenezu raka dojke uključen ljudski hormon rasta (hGH) te mehanizmi autokrine regulacije preko proteina Pax-5. Također je identi fi ciran čitav niz novih potencijalnih biomarkera kao što su to HOXA1, CHOP SH3GLB1, kazein kinaze, p53, aneksin XI, CDC25C, eIF- 4E i MAP kinaze 7, 14-3-3e, galekti n-1, aneksin-5, aneksin-1, LDH-B, GST-pi, akti n, vimenti n, HSP70, CK18, moezin, SH3GLB1, SUB1, SND1 i TRIM28, osteoponti n i osteonekti nBreast cancer is a complex and heterogenic disease. Classical molecular medical approaches cannot be successfully used to completely understand its pathogenesis. In additi on, fi nding new biological markers that would help in early detecti on and creati on of guided and specifi c therapy is crucial for the future breast cancer management. The results of breast cancer research in combinati on with those obtained by large scale methods that examine the expression status of genes and proteins, have pointed to an important molecule, namely the human growth hormone (hGH), as a major player in the pathogenesis of breast cancer. It acts through the autocrine regulati on system where the acti vati on of Pax-5 gene has been recently confi rmed as well. A large number of new biomarkers, like HOXA1, CHOP SH3GLB1, casein kinase, p53, annexin XI, CDC25C, eIF-4E, MAP kinase 7, 14-3-3e, galecti n-1, annexin-5, annexin-1, LDH-B, GST-pi, acti n, vimenti n, HSP70, CK18, moesin, SH3GLB1, SUB1, SND1 i TRIM28, osteoponti n and osteonecti n, have been recently identi fi ed as well

Novosti u mozaiku metastaziranja zloćudnih tumora

Despite the abundance of attention that cancer has attracted, it continues to constitute one of the deadliest scourges of the modern era. Tumour heterogeneity greatly contributes to the ineffectiveness of current therapies and hampers the study and treatment of cancer. There are two models accounting for tumour heterogeneity and propagation, namely clonal evolution model and cancer stem cell model. In particular, cancer stem theory has attracted much attention lately, as these cells with self-renewal and differentiation abilities are responsible for the initiation of tumour development, growth, and its ability to metastasize and reoccur, and provide a reasonable explanation for poor prognosis for patients in advanced stages of solid tumours. Advances in technologies such as proteomics open new avenues in metastasis research by specifically revealing complex protein networks involved in tumour progression, which should facilitate early diagnosis and provide the basis for designing more effective treatment strategies.Novosti u mozaiku metastaziranja zloćudnih tumora Unatoč napretku u istraživanju i proučavanju zloćudnih tumora, ta je bolest i dalje velik izazov modernoj medicini. Biološka raznolikost, ali i klasično poimanje mehanizama metastaziranja tumora, razlog su neučinkovitosti postojećih načina liječenja. Dva su modela kojima se objašnjava ta raznolikost – model klonske evolucije te model matičnih stanica novotvorina. Ovaj drugi u posljednje je vrijeme privukao pozornost jer su matične stanice novotvorina, zbog svojih sposobnosti samostalnog obnavljanja i diferenciranja, odgovorne za nastanak i razvoj tumora te njihovu sposobnost metastaziranja i pojave recidiva. Metode globalnih analiza poput proteomike otvaraju nove mogućnosti u istraživanju procesa metastaziranja, jer omogućuju identifikaciju složenih mreža proteina uključenih u progresiju novotvorina, što može pridonijeti ranoj dijagnostici i omogućiti razvoj učinkovitijih lijekova protiv metastaza

Novel Bis- and Mono-Pyrrolo[2,3-d]pyrimidine and Purine Derivatives: Synthesis, Computational Analysis and Antiproliferative Evaluation.

Novel symmetrical bis-pyrrolo[2,3-d]pyrimidines and bis-purines and their monomers were synthesized and evaluated for their antiproliferative activity in human lung adenocarcinoma (A549), cervical carcinoma (HeLa), ductal pancreatic adenocarcinoma (CFPAC-1) and metastatic colorectal adenocarcinoma (SW620) cells. The use of ultrasound irradiation as alternative energy input in Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) shortened the reaction time, increased the reaction efficiency and led to the formation of exclusively symmetric bis-heterocycles. DFT calculations showed that triazole formation is exceedingly exergonic and confirmed that the presence of Cu(I) ions is required to overcome high kinetic requirements and allow the reaction to proceed. The influence of various linkers and 6-substituted purine and regioisomeric 7-deazapurine on their cytostatic activity was revealed. Among all the evaluated compounds, the 4-chloropyrrolo[2,3-d]pyrimidine monomer 5f with 4,4'-bis(oxymethylene)biphenyl had the most pronounced, although not selective, growth-inhibitory effect on pancreatic adenocarcinoma (CFPAC-1) cells (IC50 = 0.79 µM). Annexin V assay results revealed that its strong growth inhibitory activity against CFPAC-1 cells could be associated with induction of apoptosis and primary necrosis. Further structural optimization of bis-chloropyrrolo[2,3-d]pyrimidine with aromatic linker is required to develop novel efficient and non-toxic agent against pancreatic cancer

Critical Review on Zeolite Clinoptilolite Safety and Medical Applications in vivo

Unique and outstanding physical and chemical properties of zeolite materials make them extremely useful in a variety of applications including agronomy, ecology, manufacturing, and industrial processes. Recently, a more specific application of one naturally occurring zeolite material, clinoptilolite, has been widely studied in veterinary and human medicine. Due to a number of positive effects on health, including detoxification properties, the usage of clinoptilolite-based products in vivo has increased enormously. However, concerns have been raised in the public about the safety of clinoptilolite materials for in vivo applications. Here, we review the scientific literature on the health effects and safety in medical applications of different clinoptilolite-based materials and propose some comprehensive, scientifically-based hypotheses on possible biological mechanisms underlying the observed effects on the health and body homeostasis. We focus on the safety of the clinoptilolite material and the positive medical effects related to detoxification, immune response, and the general health status

Critical Review on Zeolite Clinoptilolite Safety and Medical Applications in vivo

Unique and outstanding physical and chemical properties of zeolite materials make them extremely useful in a variety of applications including agronomy, ecology, manufacturing, and industrial processes. Recently, a more specific application of one naturally occurring zeolite material, clinoptilolite, has been widely studied in veterinary and human medicine. Due to a number of positive effects on health, including detoxification properties, the usage of clinoptilolite-based products in vivo has increased enormously. However, concerns have been raised in the public about the safety of clinoptilolite materials for in vivo applications. Here, we review the scientific literature on the health effects and safety in medical applications of different clinoptilolite-based materials and propose some comprehensive, scientifically-based hypotheses on possible biological mechanisms underlying the observed effects on the health and body homeostasis. We focus on the safety of the clinoptilolite material and the positive medical effects related to detoxification, immune response, and the general health status