1,721,009 research outputs found
Conformational Changes in Hemoglobin triggered by changing the Iron Charge, High and Low Affinity Hemoglobin
No full textK:D-Rib ON BIOLOGY OF HUMAN CANCER AND NOT CANCER CELL LINE
No full textThis work describes different effects of K:D-Rib solution treatment: from one side the slow down of cell proliferation and the reduction of chemoinvasive potential of human breast cancer cell line (HTB-126) and from the other the maintenance of normal proliferation and normal morphology in mammary human not cancer epithelial cell line (HTB-125). K:D-Rib is a water solution of D-ribose and KHCO
3
. The role of D-ribose on the energetic metabolism and its involvement into glycogen synthesis [1, 2], as well as the importance of K
+
into the cell physiology, are well known [3, 4]. It has been found that K
+
is essential to fold and to stabilize G-quadruplex [5] with a strong relevance for telomeric structures [2, 6] and for oncogenic promoter regions. Our results showed that K:D-Rib has a cytostatic effect on canine carcinoma cell line (A72), slows the colony formation ability of the HTB-126 cell line and has an antioxidant behaviour reducing MTT salt to formazan in absence of cells [1]. These results are confirmed by our most recent work, demonstrating that 5mM K:D-Rib increase the cell cycle time of HTB-126 cell line treated with K:D-Rib at the concentration of 5mM, from 44h to 59h. Here it will be show how K:D-Rib interferes both on HTB-126 cell line proliferation and cell morphology. Results on cell morphology using Atomic Force Microscopy (AFM) are presented. K:D-Rib is tested also on human mammary epithelial cell line (HTB-125). HTB-125 cells treated with 5 mM K:D-Rib do not display toxicity or notable cell proliferation decreasing rate compared to the control one. HTB-125 cell morphology is analyzed by AFM. As mentioned before a key point of cancer cells is the capability to invade tissues nearby or far from cancer formation site. Tumour motility is an important step in the intricate process leading to the formation of metastasis. It has been shown that metastatic cells are more motile than non-metastatic tumour cells and most motile of normal cells. Metastatic cells lose growth-inhibitory responses, undergo alterations in adhesiveness and demonstrate enhanced production of enzymes that can degrade extracellular matrix components. Since the development of metastatic disease in breast cancer is one of main responsible of cancer mortality, the stopping and the understanding of the mechanisms that facilitate metastatic tumour progression is of prime importance [7]. We have investigated if K:D-Rib solution within 9 days can modify the migration and the invasion ability. The experiments show HTB-126 cells are able to migrate across the coverslip toward the FBS
–
agar spot and to invade it within 48, but the relative cell number inside the AGAR-FBS decrease already after five days of treatment. After nine days of treatment with K:D-Rib the relative cell number, inside the AGAR-FBS spot is reduced to 25%, demonstrating that tumorigenic potential is highly decreased with K:D-Rib treatment. These results show that 5mM K:D-Rib causes the change of some aspects like migration, invasion and proliferation of HTB-126 cell line. Despite these evidences K:D-Rib does not interfere neither with the proliferation of HTB-125 cell line nor with cell morphology.
1. Croci S, Bruni L, Bussolati S, Castaldo M, Dondi M: Potassium bicarbonate and D-ribose effects on A72 canine and HTB-126 human cancer cell line proliferation in vitro.
Cancer Cell International
2011, 11. 2. Heiden MGV, Cantley LC, Thompson CB: Understanding the Warburg Effect: The Metabolic Requirements of Cell Proliferation.
Science
2009, 324(5930):1029-1033. 3. Dai JX CM, Yang DZ: Polymorphism of human telomeric quadruplex structures.
4.
Xianfeng Zhou FS, † Yanqing Tian
,* Cody Youngbull, Roger H. Johnson, and Deirdre R. Meldrum: A New Highly Selective Fluorescent K+ Sensor.
Journal of the American Chemical Society
2011(133, ):18530
–
18533. 5. Parkinson GN, Lee MPH, Neidle S: Crystal structure of parallel quadruplexes from human telomeric DNA.
Nature
2002, 417(6891):876-880. 6. Lipps HJ, Rhodes D: G-quadruplex structures: in vivo evidence and function.
Trends in Cell Biology
2009, 19(8):414-422. 7. Fernandis AZ, Prasad A, Band H, Klosel R, Ganju RK: Regulation of CXCR4-mediated chemotaxis and chemoinvasion of breast cancer cells.
Oncogene
2004, 23(1):157-167
K:D-Rib un inibitore della proliferazione delle cellule tumorali ed un promotore del folding di DNAzima.
No full textLe cellule tumorali mostrano un aumento della glicolisi anche in presenza d’ossigeno disponibile. Questo processo chiamato “Effetto Warburg” è uno dei fondamentali switch metabolici che una cellula cancerosa manifesta se paragonata ad una cellula non cancerosa. Tra i trasportatori del glucosio c’è la famiglia SGLT (cotrasportatore Na+/glucosio). L’elevata concentrazione totale di sodio in lesioni mammarie tumorali misurata con 23Na-MRI sembra essere un indicatore del livello cellulare di malignità [1].
Il ruolo del D-ribosio e dello ione potassio (K+) sono ampiamente noti: il D-ribosio è un aldopentoso, assiste il metabolismo energetico della cellula oltre ad esser un precursore d’alcuni aminoacidi; lo ione K+ è coinvolto in molti processi tra cui apoptosi, genesi e mantenimento del potenziale di membrana e stabilizza il folding dei G-quadruplex. C'è un interesse crescente nell’utilizzo di G-quadruplex anche come sensori per lo ione K+ [2].
Per saggiare l’effetto del K:D-Rib sulla proliferazione delle linee cellulari non si sono potuti utilizzare i comuni saggi metabolici (MTT assay, WST-1 assay ecc) in quanto K:D-Rib interagisce con il bromuro di tetrazolio riducendolo a formazan [3].
Ci siamo perciò avvalsi di un metodo d’uso comune: le cellule (4000 cell/ml trattate con K:D-Rib 5mM e incubate per 12 gg) sono state contate utilizzando il programma ImageJ ed in seguito si è calcolato il tempo di duplicazione. Il controllo ha mostrato un tempo di duplicazione di 44 ore, mentre il trattato di 59 ore. L’effetto rilevato è un progressivo rallentamento della proliferazione cellulare del trattato rispetto al controllo.
Il saggio delle colonie, nel quale si contano gruppi cellulari di almeno 50 unità, ha confermato i risultati delle curve di crescita, mostrando ancora una volta un effetto antiproliferativo del K:D-Rib sia sulla linea HTB-126 [3] che HTB-30 (metastasi pleurica di carcinoma mammario). Quest’ultima linea
66 |
cellulare ha mostrato 15 colonie per il controllo e nessuna colonia per i trattati. Questi dati sperimentali unitamente ad una ricca bibliografia sui canali K+ [2] e sul ruolo stesso del K+ ci hanno portato a ipotizzare che una fine regolazione della concentrazione di K+ stia alla base del “corretto funzionamento” delle cellule. Da qui l’esigenza di valutare se K+ entri o no nelle cellule in seguito al trattamento con K:D-Rib. La concentrazione di K+ è stata misurata sfruttando la formazione di una macromolecola chiamata DNAzima [4]. Dalla concentrazione di DNAzima si è in grado, attraverso spettroscopia UV-VIS, di misurare se lo ione K+ è presente nella soluzione. Abbiamo quindi verificato la formazione di questa molecola in presenza di K:D-Rib 5mM. Il DNAzima così formato può essere utilizzato come biosensore per la misura della concentrazione del K+ nel mezzo cellulare, prima e dopo il trattamento. Risultati preliminari sembrano indicare che il surnatante delle HTB-126 trattate con K:D-Rib 5mM per 48h mostrano una concentrazione di DNAzima inferiore rispetto al DMEM (terreno di coltura delle cellule) con K:D-Rib 5mM, ma superiore al solo DMEM ed al surnatante del controllo (HTB-126 non trattate). Possiamo dire che una parte di K+ entri all’interno della cellula e che il DNAzima possa essere utilizzato come biosensore di K+.
Bibliografia:
[1] Ronald Ouwerkerk, Michael A. Jacobs , Katarzyna J. Macura, Antonio C. Wolff, Vered Stearn, Sarah D. Mezban, Nagi F. Khouri, David A. Bluemke, Paul A. Bottomley Elevated tissue sodium concentration in malignant breast lesions detected with non-invasive 23Na MRI. Breast Cancer Res Treat (2007) 106:151–160.
[2] Zhiguo Wang Roles of K+ channels in regulating tumour cell proliferation and apoptosis. Eur J Physiol (2004) 448: 274–28:
[3] S. Croci, L. Bruni, S. Bussolati, M. Castaldo, M. Dondi Potassium bicarbonate and D-ribose effects on A72 canine and HTB-126 human cancer cell line proliferation in vitro. Cancer Cell International (2011) vol. 11 11-30.
[4] Travascio P, Li Y, Sen D.. DNA-enhanced peroxidase activity of a DNA-aptamer-hemin complex. Chem Biol (1998) 5:5 05–517
D-ribose acts as trojan horse equilibrating K+ into cancer cells.
No full textCancer cells have a glycolysis enhancement even in the presence of available oxygen. The “aerobic glycolysis” is known as the Warburg effect and it considered a fundamental metabolic alteration during malignant transformation. The up-regulation of glycolysis in tumour cells is well known and it is the rationale of F18-FDG PET diagnostic technique. This propriety is strictly link to the increase of sugar transporters GLUT in most tumours. Also SGLT family transporters, which include the sodium-glucose symporters, are deregulated in many tumours. The influx of Na+ into tumour cells leads to a change into ions equilibrium, with particular reference to K+ intracellular concentration. We demonstrate that the synergic action of potassium bicarbonate and D-ribose (K:D-Rib) has a cytostatic effect on human cancer cell line (HTB-126), reducing by 30% the doubling population time of treated cancer cells respect to the control, after fourteen days of treatment. The clonogenic assay shows that the colonies formed during the incubation with 5 mM K:D-Rib, are significantly less respect to control but not if fructose is used instead of D-ribose.
Cell membranes are permeable to D-ribose that is a carbon source but in contrast to glucose without sodium symport. In addition a significant increase in K+ channel expression, K+ current and /or K+ efflux, can be correlated with tumorigenesis and proliferation. We show that the re-equilibrium of potassium intracellular concentration could be one of the key point to control cancer cell proliferation. Results also report the correlation between morphological cell changes and K+ ion intracellular concentration
PS2.M: Looking for a potassium biosensor
No full textDNA sequences with guanine repeats are able to fold as G-quadruplex (G4) structures. This is an alternative DNA conformation in which four guanines are arranged as a tetrad, the structural unit of G4; two or more stacked tetrads form a G4 structure. The hydrogen bonds characterizing G4 are called Hoogsten bonds but more interactions are involved in the G4 structure stabilization. For example, cations work as G4 stabilizers and their role is not restricted to the structural folding. They coordinate the guanines’ carbonilic oxygens located towards the hydrophobic channel of the G4 inner structure. This feature suggests that the G4 could work as a cation biosensor. Biological media are characterized by the simultaneous presence of K+and Na+exhibiting different affinities and thus promoting different topological arrangements in the folding solution. In this article we explore the possibility of using PS2.M, an 18 base long synthetic oligonucleotide, as a detector of K+at concentrations in the range 0mM to 10mM. Our intent is therefore to study a biosensor that is made of the G4 sequence only, without intervention of other coupled molecules. As with most guanine-rich oligonucleotides, also PS2.M shows different structures depending on the folding conditions. In agreement with the literature data, our results outline the expected behavior and the key role of K+and Na+in promoting the folding, with Na+promoting the antiparallel structure formation of PS2.M, even at high concentrations. On the other hand, if PS2.M is folded in 10mM to 100mM KCl solutions, the parallel conformation sets in becoming more and more relevant as concentration grows. In a complex solution of G4 in the presence of both K+and Na+ions, spectra display the coexistence of parallel, antiparallel and mixed type conformations. Results show that the CD spectra values at this wavelength can be diagrammed as a function of the K+concentration to construct a biosensor calibration curve. In conclusion, given a Na+concentration in the range 50mM to 80mM and K+concentration in the range 0mM to 10mM, the measured CD signal at 263.6nm permits a K+concentration measurement with a resolution of ∼ 1 mM
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