<p>У овој дисертацији испитивана су антитуморска дејства антихипергликемијског лека метформина, вазодилататорног лека нитроглицерина, и комбинација ових лекова са дијагностичким средством 2-деокси-D-глукозом и/или радио и хемио сензибилизатором кофеином на хуманим културама аденокарцинома плућа (A549), колоректалног карцинома (HT29), аденокарцинома цервикса (HeLa), као и на контролној ћелијској култури нормалних фибробласта плућа (МRC 5). In vitro испитивање утицаја метформина, нитроглицерина, 2-деокси-D-глукозе и кофеина на проли- ферацију ћелија карцинома грлића материце (HeLa), ћелијској култури аденокарциномa плућа (A549) и ћелијској линији карцинома дебелог црева (HT29). Ћелије у експоненцијалној фази раста третиране су растућим концентрацијама метформина, нитроглицерина и 2-деокси-D-глукозе и утврдила се дозна зависност цитотоксичног ефекта. Метформин, кофеин и 2-деокси-D-глукоза су утицали на смањење процента преживљавања туморских ћелија, док је применом нитроглицерина овај ефекат изостао, иако у експериментима код истовремене примене нитроглицерина и кофеина постоји пад процента преживелих ћелија. Најпотентнији ефекат је постигнут код истовремене примене метформина и кофеина, док је разлог за одсуство снажног цитотоксичног ефекта метформина и 2-деокси-D-глукозе код комбиноване примене молекуларни механизам деловања појединачних супстанци. Снажан пролиферативни ефекат је евидентиран применом метформина и кофена на здравим фибробластима плућа.</p> / <p>U ovoj disertaciji ispitivana su antitumorska dejstva antihiperglikemijskog leka metformina, vazodilatatornog leka nitroglicerina, i kombinacija ovih lekova sa dijagnostičkim sredstvom 2-deoksi-D-glukozom i/ili radio i hemio senzibilizatorom kofeinom na humanim kulturama adenokarcinoma pluća (A549), kolorektalnog karcinoma (HT29), adenokarcinoma cerviksa (HeLa), kao i na kontrolnoj ćelijskoj kulturi normalnih fibroblasta pluća (MRC 5). In vitro ispitivanje uticaja metformina, nitroglicerina, 2-deoksi-D-glukoze i kofeina na proli- feraciju ćelija karcinoma grlića materice (HeLa), ćelijskoj kulturi adenokarcinoma pluća (A549) i ćelijskoj liniji karcinoma debelog creva (HT29). Ćelije u eksponencijalnoj fazi rasta tretirane su rastućim koncentracijama metformina, nitroglicerina i 2-deoksi-D-glukoze i utvrdila se dozna zavisnost citotoksičnog efekta. Metformin, kofein i 2-deoksi-D-glukoza su uticali na smanjenje procenta preživljavanja tumorskih ćelija, dok je primenom nitroglicerina ovaj efekat izostao, iako u eksperimentima kod istovremene primene nitroglicerina i kofeina postoji pad procenta preživelih ćelija. Najpotentniji efekat je postignut kod istovremene primene metformina i kofeina, dok je razlog za odsustvo snažnog citotoksičnog efekta metformina i 2-deoksi-D-glukoze kod kombinovane primene molekularni mehanizam delovanja pojedinačnih supstanci. Snažan proliferativni efekat je evidentiran primenom metformina i kofena na zdravim fibroblastima pluća.</p> / <p>In this dissertation, the anti-cancer effects of an antihyperglycaemic agent of metformin, a vasodilator drug nitroglycerin, and a combination of these drugs with a 2-deoxy-D-glucose diagnostic agent and / or radio and hemio sensitizer with caffeine on human cultures of adenocarcinoma of the lungs (A549), colorectal carcinoma (HT29), cervix adenocarcinoma (HeLa), as well as on the control cell culture of normal fibroblasts of the lungs (MRC 5). An in vitro study of the effects of metformin, nitroglycerin, 2-deoxy-D-glucose and caffeine on the proliferation of cervical cancer cells (HeLa), cell culture of the lung adenocarcinoma (A549), and colon cancer of the colon (HT29). The cells at the exponential growth stage were treated with rising concentrations of metformin, nitroglycerin and 2-deoxy-D-glucose, and the cytotoxic effect was determined. Metformin, caffeine, and 2-deoxy-D-glucose reduced the number of tumor cells, while nitroglycerin did not it could be concluded. Although there is a decrease in survival in experiments with the simultaneous administration of nitroglycerin and caffeine, the most effective effect is achieved in the simultaneous use of metformin and caffeine, while the reason for the absence of a potent cytotoxic effect of metformin and -deoxy-D-glucose is the molecular mechanism of the action of individual substances. The most significant effect was achieved with the simultaneous administration of metformin and caffeine to the cell culture of lung adenocarcinoma. A potent proliferative effect was recorded using metformin and 2-deoxy-Dglucose on healthy lung fibroblasts.</p>

AQ4N (banoxantrone) is a prodrug that, under hypoxic conditions, is enzymatically converted to a cytotoxic DNA-binding agent, AQ4. Incorporation of AQ4N into conventional chemoradiation protocols therefore targets both oxygenated and hypoxic regions of tumors, and potentially will increase the effectiveness of therapy. This current pharmacodynamic and efficacy study was designed to quantify tumor exposure to AQ4 following treatment with AQ4N, and to relate exposure to outcome of treatment. A single dose of 60 mg/kg AQ4N enhanced the response of RT112 (bladder) and Calu-6 (lung) xenografts to treatment with cisplatin and radiation therapy. AQ4N was also given to separate cohorts of tumor-bearing mice 24 hours before tumor excision for subsequent analysis of metabolite levels. AQ4 was detected by high performance liquid chromatography/mass spectrometry in all treated samples of RT112 and Calu-6 tumors at mean concentrations of 0.23 and 1.07 microg/g, respectively. These concentrations are comparable with those shown to be cytotoxic in vitro. AQ4-related nuclear fluorescence was observed in all treated tumors by confocal microscopy, which correlated with the high performance liquid chromatography/mass spectrometry data. The presence of the hypoxic marker Glut-1 was shown by immunohistochemistry in both Calu-6 tumors and RT112 tumors, and colocalization of AQ4 fluorescence and Glut-1 staining strongly suggested that AQ4N was activated in these putatively hypoxic areas. This is the first demonstration that AQ4N will increase the efficacy of chemoradiotherapy in preclinical models; the intratumoral levels of AQ4 found in this study are comparable with tumor AQ4 levels found in a recent phase I clinical study, which suggests that these levels could be potentially therapeutic.

<p>&Scaron;tavelj (Rumex crispus, Polygonaceae) je vi&scaron;egodi&scaron;nja zeljasta biljka, koja predstavlja bogat izvor fenolnih komponenti. Iako se smatra invazivnim korovom, mlado li&scaron;će &scaron;tavelja je jestivo i često se koristi kao salata. Dalje, upotreba plodova &scaron;tavelja opisana je u srpskoj i turskoj narodnoj medicini u lečenju gastrointestinalnih tegoba. Cilj ovog rada bio je procena in vitro i in vivo antioksidantne/prooksidantne i citotoksične aktivnosti, i određivanje eventualnog in vitro antiinflamatornog efekta vodenog ekstrakta ploda Rumex crispus. Ukupan sadržaj flavonoida određen je spektrofotometrijskom metodom. Kvalifikacija i kvantifikacija flavonoida potvrđena je visokoefikasnom tečnom hromatografijom (HPLC). Antioksidantna aktivnost vodenog ekstrakta ploda &scaron;tavelja procenjena je na osnovu in vitro testova: Ferric-reducing antioxidant power (FRAP), sposobnosti ekstrakta da neutrali&scaron;e slobodne radikale NO&bull;, OH&bull; i DPPH&bull; i uticaja na lipidnu peroksidaciju u lipozomima. Citotoksičnost ispitivanog ekstrakta je određena in vitro na tumorskim ćelijskim linijama: humani karcinom cerviksa (HeLa), adenokarcinom (HT-29) i adenokarcinom dojke (MCF7). Takođe, moguća in vivo hepatoprotektivna i antioksidantna svojstva ekstrakta određena su kod oksidativnog stresa izazvanog CCl4 kod eksperimentalnih životinja. Pored toga, proverena je hipoteza u kojoj testiran ekstrakt pokazuje in vivo antiproliferativnu aktivnost kod Ehrlich-ovih (EAC) i Hepatoma AS30D ćelija, merenjem zapremine ascitesa, procenta vijabilnih ćelija i nivoa nekoliko antioksidantnih enzima. Optimizovan in vitro test za određivanje potencijala inhibicije ciklooksigenaze-1 (COX-1) i 12-lipooksigenaze (12-LOX) preduzet je u svrhu procene antiinflamatornog efekta vodenog ekstrakta ploda R. crispus. HPLC analiza otkrila je da je mikvelianin najdominantniji flavonoidni konstituent ekstrakta. Testirani ekstrakt pokazao je potencijalnu antioksidantnu aktivnost rezultujući velikom moći u neutralizaciji slobodnih radikala, i sposobno&scaron;ću da smanji lipidnu peroksidaciju u lipozomima. Rezultati su ukazali na tkivno-selektivnu citotoksičnost ekstrakta ploda R. crispus in vitro. Najizraženija antitumorska aktivnost primećena je prema HeLa i MCF7 ćelijskim linijama. Podaci sugeri&scaron;u da bi se ispitivani ekstrakt mogao smatrati potencijalnim in vivo hepatoprotektivnim i antioksidantnim agensom, sprečavajući oksidativna o&scaron;tećenja jetre. S druge strane, pomenuti ekstrakt može pokazati in vivo prooksidantna svojstva, uzrokujući oksidativni stres u maligno transformisanim EAC i AS30D ćelijama i smanjujući zapreminu ascitesa i udeo vijabilnih ćelija, u poređenju sa kontrolnom grupom. Promene u aktivnosti antioksidantnih enzima su verovatno posledica indukovanog oksidativnog stresa u EAC i AS30D ćelijama, naročito kod pretretiranih životinja. Vodeni ekstrakt ploda &scaron;tavelja pokazao je COX-1, kao i 12-LOX inhibitornu aktivnost, navodeći da bi ispitivani ekstrakt mogao biti antiinflamatorni agens. Vodeni ekstrakt ploda R. crispus ima potencijalnu antioksidantnu, citotoksičnu i antiinflamatornu aktivnost. Ispoljavanje prooksidantnih svojstava predstavlja mogući mehanizam antiproliferativnog efekta ekstrakta.</p> / <p>Curly dock (Rumex crispus, Polygonaceae) is a wild perennial herbaceous plant, which products are described as a rich source of phenolic compounds. Apart from being considered a seriously invasive weed, young leaves of curly dock are edible and often used as salad. Furthermore, the use of its fruits has been described in Serbian and Turkish traditional medicine against stomach complaints. The objectives of this study were to evaluate in vitro and in vivo antioxidant/prooxidant and cytotoxic activities, and to determine an eventual in vitro anti-inflammatory effect of the aqueous extract of Rumex crispus fruits. Total flavonoid content was determined by spectrophotometric method. Qualification and quantification of flavonoids were confirmed using High performance liquid chromatography (HPLC). The aqueous extract of curly dock fruits was evaluated for its antioxidant activity by in vitro assays for Ferric-reducing antioxidant power (FRAP), NO&bull;, OH&bull; and DPPH&bull;-free radical scavenging activities and the influence on lipid peroxidation in liposomes. The cytotoxicity of tested extract was examined in vitro in human cervix carcinoma (HeLa), colon adenocarcinoma (HT-29) and breast adenocarcinoma (MCF7). Also, the potential in vivo hepatoprotective and antioxidant properties of investigated extract were determined on CCl4-induced oxidative stress in experimental animals. Furthermore, the hypothesis that the examined extract might show in vivo antiproliferative activity in Ehrlich carcinoma (EAC) and Hepatoma AS30D cells was tested by measuring volume of ascites, percentage of viable cells and level of several antioxidant enzymes. The optimized in vitro test for determination of cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) inhibition potency was undertaken in order to estimate an anti-inflammatory effect of aqueous extract of R. crispus fruits. HPLC analysis revealed miquelianin as the most abundant flavonoid constituent of the extract. The tested extract might have an antioxidant activity resulting in scavenging of free radicals and ability to decrease lipid peroxidation in liposomes. The results could indicate tissue-selective cytotoxicity of R. crispus fruit extract in vitro. The most prominent antitumor activity was observed towards HeLa and MCF7 cell lines. The data suggested that investigated extract may be considered as potential in vivo hepatoprotective and antioxidant agent due to prevention of the liver injuries induced by oxidative damage. On the other hand, mentioned extract could exhibit in vivo prooxidant property, causing the oxidative stress in malignant transformed EAC and AS30D cells and reducing volume of ascites and percentage of viable cells, in comparison with control group. Changes in activities of antioxidant enzymes might be the results of induced oxidative stress in EAC and AS30D cells, especially in the pretreated animals. The aqueous extract of curly dock fruits showed COX-1, as well as 12-LOX inhibitory activity, suggesting that tested extract might be an anti-inflammatory agent. It could be concluded that aqueous fruit extract of R. crispus might have antioxidant, cytotoxic and anti-inflammatory activities. The prooxidant properties of examined extract could be the mechanism of potential antiproliferative effect of extract.</p>

Thesis (Ph. D.)--Uniformed Services University of the Health Sciences, 2005. / Typescript (photocopy).

The current thesis addresses the various facets of the chemistry of photocytotoxic iron complexes including their syntheses, characterization, evaluation of the anti-proliferative activities in various cancer cell lines upon photo-exposure, mechanism of cell death, the cellular uptake, localization inside cells, the interaction with double stranded DNA and their ability to induce DNA photocleavage. Chapter I presents a general introduction to cancer and the anticancer agents. It covers various procedures available for cancer treatment and different aspects of chemotherapy are discussed in details. The mechanism of action of several chemotherapeutic agents, the DNA cleavage pathways and the anticancer activity of bleomycins are delineated. Photo-chemotherapy or photodynamic therapy which has emerged as an alternative treatment modality is described. It also contains a brief description of ideal photosensitizers and the ones that are currently approved. The potential of transition metal complexes as photo-chemotherapeutic agents is discussed based on the recent literature reports on the prospective photocytotoxic metal complexes, the photo-release of cytotoxic molecules from metal complexes, the DNA cleavage activities and their cytotoxicities. The biochemistry of iron and its medical utility which prompted the development of iron based cytotoxins has been presented. The objective of the present investigation is also defined in this chapter. Chapter II describes the syntheses, characterization, evaluation of visible light induced cytotoxicity and interaction with DNA of a series of iron(II) bis-terpyridine complexes. Some interesting redox behaviour observed for two of the complexes has been described in details and rationalized from theoretical calculations. The DNA binding affinities of the complexes and their ability to induce DNA photocleavage in green light are discussed. The importance of this work lies in the remarkable photocytotoxic behaviour of the iron(II) complexes with visible light which was not reported earlier. Chapter III addresses the syntheses of a series of iron(III) catecholate complexes which upon irradiation with red light can initiate photoreactions to generate cytotoxic species and induce death in HeLa, HaCaT, MCF-7 and A549 cells. The mechanisms of cell death, effect of the complexes on the cell cycle under various conditions, the uptake inside cells and the cellular localization of the complexes are studied. The DNA binding affinities of the five complexes and their ability to induce DNA photocleavage in red light are also presented here. These are the first iron based complexes to show red light induced photocytotoxicity. Chapter IV addresses the drawbacks associated with the aforementioned iron(III) catecholates and their modification with a mitochondria targeting triphenylphosphonium unit. The synthesis, characterization, photocytotoxicities in HeLa, HaCaT, MCF-7 and A549, cell death mechanisms and cellular uptake and localization of four iron(III) complexes are discussed. Chapter V describes the syntheses, characterization and the biological activities of carbohydrate appended iron(III) complexes and their non-glucose analogues. The selective and faster internalization of the glyco-conjugated complexes in HeLa cells has been studied using various spectroscopic and microscopic techniques. The red light induced cytotoxicities of the complexes, their effect on the progression of the cell cycle with and without irradiation and the mechanisms of cell death are explored. DNA binding abilities and photocleavage of DNA are also discussed. Chapter VI presents the syntheses, characterization of a series of iron(III) complexes of a pyridoxal derivative and their salicyldehyde analogues for exploring their differential photocytotoxicity and cellular uptake in cancer cells compared to normal cells. The visible light induced cytotoxicities of the complexes in HeLa, HaCaT, MCF-7 A549 cells and HPL1D cells, their effect on the progression of the cell cycle in dark and light, the mechanisms of cell death and the localization of the complexes inside the cells are explored. The references have been compiled at the end of each chapter and given as superscripts in the text. The complexes presented in this thesis are indicated by bold-faced numbers. Crystallography data of the complexes that are structurally characterized by single crystal X-ray crystallography are given in CIF format in the enclosed CD (Appendix-I). Due acknowledgements have been made wherever the work described is based on the findings of other investigators. Any unintentional omission that might have happened due to oversight is regretted. INDEX WORDS: Iron complexes • Crystal structure • Red light induced cytotoxicity • Cellular imaging • DNA binding • DNA photocleavage.

No / PURPOSE: Cytochrome P450 2W1 (CYP2W1) is a monooxygenase detected in 30% of colon cancers, whereas its expression in nontransformed adult tissues is absent, rendering it a tumor-specific drug target for development of novel colon cancer chemotherapy. Previously, we have identified duocarmycin synthetic derivatives as CYP2W1 substrates. In this study, we investigated whether two of these compounds, ICT2705 and ICT2706, could be activated by CYP2W1 into potent antitumor agents. EXPERIMENTAL DESIGN: The cytotoxic activity of ICT2705 and ICT2706 in vitro was tested in colon cancer cell lines expressing CYP2W1, and in vivo studies with ICT2706 were conducted on severe combined immunodeficient mice bearing CYP2W1-positive colon cancer xenografts. RESULTS: Cells expressing CYP2W1 suffer rapid loss of viability following treatment with ICT2705 and ICT2706, whereas the CYP2W1-positive human colon cancer xenografts display arrested growth in the mice treated with ICT2706. The specific cytotoxic metabolite generated by CYP2W1 metabolism of ICT2706 was identified in vitro. The cytotoxic events were accompanied by an accumulation of phosphorylated H2A.X histone, indicating DNA damage as a mechanism for cancer cell toxicity. This cytotoxic effect is most likely propagated by a bystander killing mechanism shown in colon cancer cells. Pharmacokinetic analysis of ICT2706 in mice identified higher concentration of the compound in tumor than in plasma, indicating preferential accumulation of drug in the target tissue. CONCLUSION: Our findings suggest a novel approach for treatment of colon cancer that uses a locoregional activation of systemically inactive prodrug by the tumor-specific activator enzyme CYP2W1.