Synthesis and Investigation of Anti-tumor Properties of Novel, Bicyclic Furopyrimidine, Pyrrolopyrimidine and Pyrimidopyridazine Nucleoside Analogues

A series of nine hitherto unknown bicylic pyrimidine nucleoside analogues (BCNAs) bearing bicyclic furo[2,3-d]pyrimidin-2(3H)-one, 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one and 5,6-dihydropyrimido[4,5-c]pyridazin-7(8H)-one bases were prepared in a straightforward approach. The synthesised compounds posses β-D-rybofuranose or β-D-2-deoxyrybofuranose or β-D-arabinofuranose moieties attached to each of the heterocylic ring systems. This is one of a few examples of synthesis of pyrrolo[2,3-d]pyrimidin-2(7H)-one and dihydropyrimido[4,5-c]pyridazin-7(8H)-one nucleosides, and the first example of such nucleosides possessing arabinose moiety. A key synthetic step was a Sonogashira coupling reaction. For coupling with 4-phenyl-1-butyne, we used deprotected 5-iodouridine, 2’-deoxy-5-iodouridine, and 5-iodoarabinouridine and this reaction was followed by cycloisomerization and subsequent conversion of the furane ring into a pyrole ring or a pyridiazine. This approach resulted in the creation of small library of compounds, which were evaluated for their antiproliferative properties against HL-60 and Jurkat E6.1 cell lines. Of all tested compounds, only 3-(β-D-rybofuranosyl)-6-(2-phenylethyl)furo[2,3-d]pyrimidin-2(3H)-one exhibited weak anti-proliferative activity, with IC50 values of 54 and 81 µM for HL-60 and Jurkat E6.1 cells, respectively

p53 and p73 display common and distinct requirements for sequence specific binding to DNA

Although p53 and p73 share considerable homology in their DNA-binding domains, there have been few studies examining their relative interactions with DNA as purified proteins. Comparing p53 and p73β proteins, our data show that zinc chelation by EDTA is significantly more detrimental to the ability of p73β than of p53 to bind DNA, most likely due to the greater effect that the loss of zinc has on the conformation of the DNA-binding domain of p73. Furthermore, prebinding to DNA strongly protects p73β but not p53 from chelation by EDTA suggesting that DNA renders the core domain of p73 less accessible to its environment. Further exploring these biochemical differences, a five-base sub-sequence was identified in the p53 consensus binding site that confers a greater DNA-binding stability on p73β than on full-length p53 in vitro. Surprisingly, p53 lacking its C-terminal non-specific DNA-binding domain (p53Δ30) demonstrates the same sequence discrimination as does p73β. In vivo, both p53 and p73β exhibit higher transactivation of a reporter with a binding site containing this sub-sequence, suggesting that lower in vitro dissociation translates to higher in vivo transactivation of sub-sequence-containing sites

Blood Magnesium, and the Interaction with Calcium, on the Risk of High-Grade Prostate Cancer

Ionized calcium (Ca) and magnesium (Mg) compete as essential messengers to regulate cell proliferation and inflammation. We hypothesized that inadequate Mg levels, perhaps relative to Ca levels (e.g. a high Ca/Mg ratio) are associated with greater prostate cancer risk.In this biomarker sub-study of the Nashville Men's Health Study (NMHS), we included 494 NMHS participants, consisting of 98 high-grade (Gleason≥7) and 100 low-grade cancer cases, 133 prostate intraepithelial neoplasia (PIN) cases, and 163 controls without cancer or PIN at biopsy. Linear and logistic regression were used to determine associations between blood Ca, Mg, and the Ca/Mg ratio across controls and case groups while adjusting for potential confounding factors.Serum Mg levels were significantly lower, while the Ca/Mg ratio was significantly higher, among high-grade cases vs. controls (p = 0.04, p = 0.01, respectively). Elevated Mg was significantly associated with a lower risk of high-grade prostate cancer (OR = 0.26 (0.09, 0.85)). An elevated Ca/Mg ratio was also associated with an increased risk of high-grade prostate cancer (OR = 2.81 (1.24, 6.36) adjusted for serum Ca and Mg). In contrast, blood Ca levels were not significantly associated with prostate cancer or PIN.Mg, Ca, or Ca/Mg levels were not associated with low-grade cancer, PIN, PSA levels, prostate volume, or BPH treatment.Low blood Mg levels and a high Ca/Mg ratio were significantly associated with high-grade prostate cancer. These findings suggest Mg affects prostate cancer risk perhaps through interacting with Ca

Immunogenic epitope scanning in bacteriolytic enzymes Pal and Cpl-1 and engineering Pal to escape antibody responses

Bacteriolytic enzymes are promising antibacterial agents, but they can cause a typical immune response in vivo. In this study, we used a targeted modification method for two antibacterial endolysins, Pal and Cpl-1. We identified the key immunogenic amino acids, and designed and tested new, bacteriolytic variants with altered immunogenicity. One new variant of Pal (257-259 MKS → TFG) demonstrated decreased immunogenicity while a similar mutant (257-259 MKS → TFK) demonstrated increased immunogenicity. A third variant (280-282 DKP → GGA) demonstrated significantly increased antibacterial activity and it was not cross-neutralized by antibodies induced by the wild-type enzyme. We propose this variant as a new engineered endolysin with increased antibacterial activity that is capable of escaping cross-neutralization by antibodies induced by wild-type Pal. We show that efficient antibacterial enzymes that avoid cross-neutralization by IgG can be developed by epitope scanning, in silico design, and substitutions of identified key amino acids with a high rate of success. Importantly, this universal approach can be applied to many proteins beyond endolysins and has the potential for design of numerous biological drugs

Rapid copper acquisition by developing murine mesothelioma: Decreasing bioavailable copper slows tumor growth, normalizes vessels and promotes T cell infiltration

Copper, an essential trace element acquired through nutrition, is an important co-factor for pro-angiogenic factors including vascular endothelial growth factor (VEGF). Decreasing bioavailable copper has been used as an antiangiogenic and anti-cancer strategy with promising results. However, the role of copper and its potential as a therapy in mesothelioma is not yet well understood. Therefore, we monitored copper levels in progressing murine mesothelioma tumors and analyzed the effects of lowering bioavailable copper. Copper levels in tumors and organs were assayed using atomic absorption spectrophotometry. Mesothelioma tumors rapidly sequestered copper at early stages of development, the copper was then dispersed throughout growing tumor tissues. These data imply that copper uptake may play an important role in early tumor development. Lowering bioavailable copper using the copper chelators, penicillamine, trientine or tetrathiomolybdate, slowed in vivo mesothelioma growth but did not provide any cures similar to using cisplatin chemotherapy or anti-VEGF receptor antibody therapy. The impact of copper lowering on tumor blood vessels and tumor infiltrating T cells was measured using flow cytometry and confocal microscopy. Copper lowering was associated with reduced tumor vessel diameter, reduced endothelial cell proliferation (reduced Ki67 expression) and lower surface ICAM/CD54 expression implying reduced endothelial cell activation, in a process similar to endothelial normalization. Copper lowering was also associated with a CD4+ T cell infiltrate. In conclusion, these data suggest copper lowering is a potentially useful anti-mesothelioma treatment strategy that slows tumor growth to provide a window of opportunity for inclusion of other treatment modalities to improve patient outcomes

1-methylnicotinamide and its structural analog 1,4-dimethylpyridine for the prevention of cancer metastasis

Background: 1-methylnicotinamide (1-MNA), an endogenous metabolite of nicotinamide, has recently gained interest due to its anti-inflammatory and anti-thrombotic activities linked to the COX-2/PGI2 pathway. Given the previously reported anti-metastatic activity of prostacyclin (PGI2), we aimed to assess the effects of 1-MNA and its structurally related analog, 1,4-dimethylpyridine (1,4-DMP), in the prevention of cancer metastasis. Methods: All the studies on the anti-tumor and anti-metastatic activity of 1-MNA and 1,4-DMP were conducted using the model of murine mammary gland cancer (4T1) transplanted either orthotopically or intravenously into female BALB/c mouse. Additionally, the effect of the investigated molecules on cancer cell-induced angiogenesis was estimated using the matrigel plug assay utilizing 4T1 cells as a source of pro-angiogenic factors. Results: Neither 1-MNA nor 1,4-DMP, when given in a monotherapy of metastatic cancer, influenced the growth of 4T1 primary tumors transplanted orthotopically; however, both compounds tended to inhibit 4T1 metastases formation in lungs of mice that were orthotopically or intravenously inoculated with 4T1 or 4T1-luc2-tdTomato cells, respectively. Additionally, while 1-MNA enhanced tumor vasculature formation and markedly increased PGI2 generation, 1,4-DMP did not have such an effect. The anti-metastatic activity of 1-MNA and 1,4-DMP was further confirmed when both agents were applied with a cytostatic drug in a combined treatment of 4T1 murine mammary gland cancer what resulted in up to 80 % diminution of lung metastases formation. Conclusions: The results of the studies presented below indicate that 1-MNA and its structural analog 1,4-DMP prevent metastasis and might be beneficially implemented into the treatment of metastatic breast cancer to ensure a comprehensive strategy of metastasis control

Redox cycling metals: Pedaling their roles in metabolism and their use in the development of novel therapeutics

Essential metals, such as iron and copper, play a critical role in a plethora of cellular processes including cell growth and proliferation. However, concomitantly, excess of these metal ions in the body can have deleterious effects due to their ability to generate cytotoxic reactive oxygen species (ROS). Thus, the human body has evolved a very well-orchestrated metabolic system that keeps tight control on the levels of these metal ions. Considering their very high proliferation rate, cancer cells require a high abundance of these metals compared to their normal counterparts. Interestingly, new anti-cancer agents that take advantage of the sensitivity of cancer cells to metal sequestration and their susceptibility to ROS have been developed. These ligands can avidly bind metal ions to form redox active metal complexes, which lead to generation of cytotoxic ROS. Furthermore, these agents also act as potent metastasis suppressors due to their ability to up-regulate the metastasis suppressor gene, N-myc downstream regulated gene 1. This review discusses the importance of iron and copper in the metabolism and progression of cancer, how they can be exploited to target tumors and the clinical translation of novel anti-cancer chemotherapeutics

Étude des relations entre le statut en magnésium et les cancers expérimentaux chez la souris

Les relations entre le statut en magnésium et le développement des cancers sont complexes et non complètement élucidées. D'une part la progression des cancers entraîne des modifications dans le métabolisme du magnésium et d'autre aprt des modifications du statut en magnésium pourraient affecter l'apparition et la progression des cancers. Les travaux présentés dans cette thèse concernent donc ces 2 aspects. Nos travaux ont montré que la croissance de cancer du poumon LLC chez la souris C57B1/6 entraîne des modifications du métabolisme du magnésium. Une de ces manifestations est l'augmentation de la teneur en magnésium dans les érythrocytes liée à des modifications des flux de cet élément. Les souris portant cette tumeur développent une réaction inflammatoire avec une leucocytose résultant de l'augmentation du nombre de granulocytes et de monocytes. Elles présentent également une anémie hypochrome. Par la suite nous avons montré que la carence en magnésium peut être induite facilement par un régime carencé en cet élément. Cette carence conduit à une réaction inflammatoire se caractérisant par l'augmentation du nombre de granulocytes et de monocytes. Chez les animaux carencés nous avons observé des modifications histologiques dans les poumons témoignant d'un état inflammatoire de ce tissu. Ces modifications s'accompagnent de modifications dans l'expression des gènes impliqués dans la régulation du cycle cellulaire, le stress oxydant et les phénomènes d'adhésion. Nous avons montré que la carence en magnésium ralentit la croissance de plusieurs tumeurs solides : LLC, C38, B16 et 16/C. par contre elle n'affecte pas le développement de la leucémie P338. L'étude réalisée sur le modèle LLC montre que la supplémentation en magnésium chez ces animaux conduit à un développement explosif de cette tumeur. Nous observons également que même si la carence (3 semaines) entraîne une hypomagnésemie sévère, la diminution de la concentration en magnésium dans la tumeur est très limitée. Nos résultats suggèrent que l'inhibition de la croissance tumorale par un statut faible en magnésium est en relation avec l'inhibition de la progression du cycle cellulaire et un processus d'angiogenèse affecté. Nous montrons également que même si la carence en magnésium inhibe la croissance tumorale elle favorise le processus de métastase. Cette augmentation de potentiel de métastase pourrait être liée à l'inflammation induite par cette carence. En effet, l'inflammation est responsable de l'activation de l'endothélium vasculaire ce qui facilite l'adhésion de cellules cancéreuses à l'endothélium. Dans ce contexte nous avons montré que la carence expérimentale in vitro conduit à l'augmentation de l'expression de molécule d'adhésion VCAM dans les cellules de l'endothélium microvasculaire de souris 1G11. L'activation de l'endothélium et les conséquences de l'inflammation au niveau pulmonaire peuvent donc en partie expliquer pourquoi la carence en magnésium favorise les métastases. En conclusion, nous montrons que le développement d'une tumeur conduit à des perturbations du métabolisme du magnésium. Le statut en magnésium affecte la croissance des tumeurs solides mais favorise le processus de métastases. Nous avons apporté des premiers éléments afin d'expliquer les mécanismes responsables de ces phénomènes, cependant d'autres études au niveau moléculaire sont nécessaires afin d'approfondir les relations entre magnésium et cancer.CLERMONT FD-BCIU-Santé (631132104) / SudocPARIS-BIUP (751062107) / SudocSudocFrancePolandFRP

Bicyclic cytarabine analogues: synthesis and investigation of antitumor properties of novel, 6-aryl- and 6-alkyl-3H-pyrrolo[2,3-d] pyrimidin-2(7H)-one arabinosides.

A series of sixteen hitherto unknown Cytarabine analogues bearing a bicyclic 3H-pyrrolo[2,3-d]pyrimidin-2(7H)-one base modified with aryl, pyridyl, benzyl and alkyl substituents was prepared in a straightforward approach. This is the one of the few examples of the synthesis of pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleosides and the first example of pyrrolo[2,3-d]pyrimidin-2(7H)-one nucleosides possessing arabinose moiety. For the first time, the conversion of the furopyrimidine arabinoside products to a series of novel pyrrolopyrimidines by ammonolysis reaction was thoroughly investigated using aqueous and methanolic reaction conditions under classical and micro-wave heating. This approach resulted in a small library of compounds, which were evaluated for their antiproliferative properties against HL-60 and Jurkat E6.1 cell lines. All synthesised compounds exhibited a weaker cytotoxic effect in comparision to the mother compound. Of all the tested compounds, the derivative bearing a 4-n-pentylphenyl substituent exhibited the highest antiproliferative activity

Magnesium and microvascular endothelial cells: A role in inflammation and angiogenesis

Microvascular endothelial cells are protagonists in inflammation and angiogenesis. Since magnesium (Mg) deficiency promotes inflammation and impairs angiogenesis in vivo, we evaluated the effect of different concentrations of the cation on microvascular 1G11 cells. We found that low Mg inhibits endothelial growth and migration, while it increases some inflammatory markers. In particular we show that low Mg stimulates the synthesis of interleukin 1a and 6, of nitric oxide, a mediator of inflammatory responses, and of VCAM, which mediates monocyte/endothelial interactions. On the contrary, high Mg stimulates proliferation and migration and sensitizes microvascular cells to migratory signals, thus inducing crucial events in angiogenesis. Our results demonstrate a direct role of Mg in modulating microvascular functions and provide a molecular explanation to the link among Mg, angiogenesis and inflammation observed in in vivo models