Synthesis and characterization of 2-aminoethyl dihydrogen phosphate and analogues: evaluation of antitumour potential in triple-negative breast tumour cells

O 2-aminoetil dihidrogeno fosfato (2-AEH2F) é um monofosfoester de cadeia curta. Quimicamente o 2-AEH2F é zwitterion, eletricamente neutro, porém possui cargas opostas em diferentes átomos. Neste estudo, foi sintetizado utilizando o 2-aminoetanol e ácido fosfórico. O 2-AEH2F tem efeitos no turnover de fosfolipídios da membrana celular e apresenta efeitos antiproliferativos em uma variedade de linhagens de células tumorais. Desta forma, foram sintetizados 5 compostos análogos, o N-(2-hidroxietil)benzamida (N-2-HEB), o 2-bezamidoetil dihidrogeno fosfato (2-BEH2F), o 2-aminoetil dodecil hidrogeno fosfato (2- AE(C12)HF), o 2-aminoetil hexadecil hidrogeno fosfato (2-AE(C16)HF) e o 2- aminoetil octadecil hidrogeno fosfato (2-AE(C18)HF), sendo os últimos três diésteres de fósforo. Todos os compostos foram caracterizados por ressonância magnética nuclear de hidrogênio e carbono. As caracterizações foram capazes de determinar as estruturas devido aos acoplamentos heteronucleares do núcleo de fósforo com núcleos de hidrogênio e carbono. Para todos os compostos sintetizados, foram avaliados os efeitos de citotoxicidade e obtidas as concentrações inibitórias (IC50%). Modificações no ciclo celular, expressão de marcadores envolvidos na morte celular e o potencial elétrico da membrana mitocondrial foram avaliadas por citometria de fluxo. As alterações morfológicas proveniente do processo de morte celular programada, apoptose, foram avaliadas com o uso do marcador MitoRed para marcação das mitocôndrias e o DAPI para DNA e núcleo celular. Os compostos 2-AEH2F, N-2-HEB e 2- AE(C18)HF foram os que apresentaram efeitos citotóxicos específicos para a linhagem tumoral de mama triplo negativo humano (MDA-MB-231) e murino (4T1). O 2-AEH2F e o N-2-HEB modularam o ciclo celular da célula tumoral MDAMB-231 ocasionando a parada na fase G2/M, o 2-AE(C18)HF não mostrou mudanças significativas no ciclo celular. Para a linhagem 4T1 o 2-AEH2F promoveu a parada na fase G2/M, os demais compostos não causaram nenhuma alteração no ciclo desta linhagem. A expressão dos marcadores envolvidos na morte celular das células tumorais MDA-MB-231 foram modificadas pelos três compostos estudados. Estes promoveram o aumento na expressão dos marcadores de caspases 3 e 8 ativas, p53, liberação do citocromo c e Bad. O 2-AEH2F e N-2-HEB causaram uma redução na expressão de Bcl-2, contrário do 2-AE(C18)HF. O marcador Trail-DR4 teve sua expressão aumentada pelos compostos 2-AEH2F e N-2-HEB e não houve alteração para o 2-AE(C18)HF. Esses dados sugerem a ativação das vias intrínsecas e extrínsecas de morte celular, uma vez que aumentam a fosforilação de proteínas de morte via mitocondrial. A expressão dos marcadores VEGF-R1, VEGF-R2 e uPAR apresentaram modulações após o tratamento das células tumorais humanas MDA-MB-231 com os compostos 2-AEH2F e N-2-HEB o que pode sugerir o efeito na inibição da angiogênese e invasão tumoral. Com exceção do marcador VEGF-R2, envolvido principalmente na linfogênese, o composto 2-AEH2F não alterou sua expressão. O receptor ativador do plasminogênio do tipo uroquinase PAR teve uma diminuição quando expostos aos tratamentos com 2-AEH2F e N-2-HEB. As análises de microscopia confocal a laser das células tumorais MDA-MB-231, tratadas com os compostos 2-AEH2F, N-2-HEB e 2-AE(C18)HF promoveram modificações importantes no arranjo e na distribuição das mitocôndrias. Houve modificações morfológicas decorrente da despolarização e reorganização nuclear para região pericelular e redução potencial elétrico. Os tratamentos com os compostos 2-AEH2F, N-2-HEB e 2- AE(C18)HF se mostraram efetivos na modulação dos marcadores de morte celular e assim como na especificidade citotóxica para as linhagens tumorais estudadas, quando comparadas a linhagens de células não tumoraisThe 2-aminoethyl dihydrogen phosphate (2-AEH2F) is a short-chain monophosphoester. Chemically, 2-AEH2F is zwitterion, electrically neutral, but it has charges placed on different atoms. In this study, it was synthesized using 2-aminoethanol and phosphoric acid. The 2-AEH2F has effects on the phospholipidic cell membrane turnover and exhibits antiproliferative effects on a variety of tumour cell lines. In such manner, 5 analogous compounds were synthesised, N-(2-hydroxyethyl)benzamide (N-2-HEB), 2-bezamidoethyl dihydrogen phosphate (2-BEH2F), 2-aminoethyl dodecyl hydrogen phosphate (2-AE(C12) HF), 2-aminoethyl hexadecyl hydrogen phosphate (2-AE(C16)HF) and 2-aminoethyl octadecyl hydrogen phosphate (2-AE(C18)HF), the last three being phosphorus diesters. All compounds were characterized by hydrogen and carbon nuclear magnetic resonance. The characterizations were able to determine the structures of the molecules due to the heteronuclear couplings of the phosphorus nucleus with hydrogen and carbon nuclei. For all synthesised compounds, the cytotoxicity effects were evaluated and the inhibitory concentrations (IC50%) were acquired. Changes in the cell cycle, expression of markers involved in cell death and the electrical potential of the mitochondrial membrane were evaluated by flow cytometry. Morphological alterations resulting from the process of programmed cell death, apoptosis, were evaluated using the MitoRed marker for the mitochondria and DAPI for DNA and cell nucleus. The compounds 2-AEH2F, N-2-HEB and 2-AE(C18)HF were the ones that showed specific cytotoxic effects for human triple-negative breast cancer (MDA-MB-231) and murine (4T1). Therefore, further experiments were conducted with the compounds 2-AEH2F, N-2-HEB and 2-AE(C18)HF. 2-AEH2F and N-2-HEB modulated the cell cycle of the MDA-MB-231 tumour cell line, causing the arrest in the G2/M phase however 2-AE(C18)HF did not show significant changes in the cell cycle. For the 4T1 cell line, 2-AEH2F promoted the arrest in the G2/M phase, the other compounds did not cause any change in the cycle of this cell line. The expression of markers involved in cell death of MDA-MB-231 tumour cells were modified by the three studied compounds. These compounds promoted an increase in the expression of active caspase 3 and 8 markers, p53, cytochrome c release and Bad. 2-AEH2F and N-2-HEB caused a reduction in Bcl-2 expression, contrary to 2-AE(C18)HF. The Trail-DR4 marker had its expression increased by the compounds 2-AEH2F and N-2-HEB and there was no alteration for 2-AE(C18)HF. These data suggest the activation of intrinsic and extrinsic pathways of cell death, as they increase the phosphorylation of proteins involved in mitochondrial cell death. The expression of VEGF-R1, VEGF-R2 and uPAR markers showed modulations after the treatment of human tumour cells MDA-MB-231 with the compounds 2-AEH2F and N-2-HEB, which may suggest the effect on the inhibition of angiogenesis and tumour invasion. With the exception of the VEGF-R2 marker, mainly involved in lymphangiogenesis, the compound 2-AEH2F did not alter its expression. The urokinase-type plasminogen PAR had a reduction in its expression when exposed to treatments with 2-AEH2F and N-2-HEB. The confocal laser microscopy analyses of MDA-MB-231 tumour cells, treated with the compounds 2-AEH2F, N-2-HEB and 2-AE(C18)HF promoted important changes in the arrangement and distribution of mitochondria. There were morphological changes due to depolarization and nuclear reorganization to the pericellular region and reduced electrical potential. The treatments with the compounds 2-AEH2F, N-2-HEB and 2-AE(C18)HF were effective, modulating cell death markers and as well as in the cytotoxic specificity for the studied tumour cell lines, when compared to non-tumour cell line

Antiproliferative and proapoptotic effects of DODAC/synthetic phosphoethanolamine on hepatocellular carcinoma cells

Abstract Background Current studies have demonstrated that DODAC/PHO-S (Dioctadecyldimethylammonium Chloride/Synthetic phosphoethanolamine) liposomes induces cytotoxicity in Hepa1c1c7 and B16F10 murine tumor cells, with a higher proportion than PHO-S. Therefore, our aim was to evaluate the potential of DODAC/PHO-S to elucidate the mechanism of cell death whereby the liposomes induces cytotoxicity in hepatocellular carcinoma Hepa1c1c7, compared to the PHO-S alone. Methods Liposomes (DODAC/PHO-S) were prepared by ultrasonication. The cell cycle phases, protein expression and types of cell’s death on Hepa1c1c7 were analyzed by flow cytometry. The internalisation of liposomes, mitochondrial electrical potential and lysosomal stability were also evaluated by confocal laser scanning microscopy. Results After treatment with liposomes (DODAC/PHO-S), we observed a significant increase in the population of Hepa1c1c7 cells experiencing cell cycle arrest in the S and G2/M phases, and this treatment was significantly more effective to promote cell death by apoptosis. There also was a decrease in the mitochondrial electrical potential; changes in the lysosomes; nuclear fragmentation and catastrophic changes in Hepa1c1c7 cells. The liposomes additionally promoted increases in the expression of DR4 receptor, caspases 3 and 8, cytochrome c, p53, p21, p27 and Bax. There was also a decrease in the expression of Bcl-2, cyclin D1, CD90 and CD44 proteins. Conclusion The overall results showed that DODAC/PHO-S liposomes were more effective than PHO-S alone, in promoting cytotoxicity Hepa1c1c7 tumor cells, activating the intrinsic and extrinsic pathways of programmed cell death

Modulation of pro-apoptotic effects and mitochondrial potential on B16F10 cells by DODAC/PHO-S liposomes

Abstract Objective We aimed to evaluate the potential of DODAC/PHO-S liposomes on the modulation of the expression of pro-apoptotic proteins, loss of lysosomal integrity and the mitochondrial electrical potential, compared with phosphoethanolamine. Results The results of this study demonstrate that DODAC/PHO-S liposomes have exhibited broad cytotoxic potential in B16F10 murine melanoma cells, with significantly greater proportions than treatment with PHO-S. The treatment with the DODAC/PHO-S 2.0 mM liposomal formulation was more efficient in decreasing mitochondrial electrical potential at the same concentrations and treatment time than PHO-S The liposomal formulation DODAC/PHO-S (2.0 mM) was more efficient to promote morphological changes in the cells, without presenting intact lysosomes, at the same time of treatment and concentration as PHO-S Our results demonstrated that the liposomal formulation increased DR4 receptor expression and activated caspases 8 and 3, resulting in the release of cytochrome c in B16F10 tumour cells, when compared to treatment with PHO-S The data obtained prove that the use of DODAC as carrier can maximize the cytotoxic effects of PHO-S This was demonstrated by the translocation of cytochrome c to the cytoplasm and activation of caspase-3 and 8, decreasing the mitochondrial electrical potential and generating morphological changes, in B16F10 cells

Observation of Cationic Transition Metal–Alkane Complexes with Moderate Stability in Hydrofluorocarbon Solution

In seeking to create more-stable transition metal–alkane complexes, we generated cationic alkane complexes of the type [(HEB)­Re­(CO)₂­(alkane)]­[Al­(OR^f)₄] (HEB = η⁶-hexa­ethyl­benzene; alkane = cyclo­pentane (<b>16</b>) or pentane (<b>17</b>–<b>19</b>); OR^f = perfluoro-<i>tert</i>-butoxy) via photolysis of the precursor complex [(HEB)­Re­(CO)₃]­[Al­(OR^f)₄] (<b>15</b>) in the presence of the added alkane. The alkane complexes were generated in a hydro­fluoro­carbon (HFC) solvent, most often CF₃CH₂CF₃, which is capable of simultaneously dissolving the ionic complex <b>15</b> and a small amount of alkane at low temperature (183 K). Use of the HFC solvent in tandem with the highly fluorinated, solubilizing, weakly coordinating [Al­(OR^f)₄]⁻ anion overcomes the technical difficulty of combining ionic species with alkanes in solution without the solvent molecules rapidly displacing the bound alkane ligand, as the alkanes bind in preference to the HFCs to the organo­metallic fragments employed in this study. The [(HEB)­Re­(CO)₂­(alkane)]⁺ complexes are more long-lived than the corresponding neutral alkane complexes [(HEB)­W­(CO)₂­(alkane)] and [CpRe­(CO)₂­(alkane)] (Cp = η⁵-cyclo­penta­dienyl), with samples of [CpRe­(CO)₂­(cyclo­pentane)] decaying significantly more rapidly than [(HEB)­Re­(CO)₂­(alkane)]⁺ when present in the same solution. Intra­molecular exchange of the methylene group bound to the metal within the cyclo­pentane ligand in <b>16</b> was observed at 212 K, with the 1,2 shifts appearing to be faster than 1,3 shifts