Estudos da resposta estringente de Bacillus subtilis e busca por pequenas moléculas moduladoras de RelA / Studies of the stringent response of Bacillus subtilis and search for small molecules capable of modulating RelA activity

Seja no meio ambiente, dentro de um hospedeiro ou em outro habitat, bactérias estarão frequentemente enfrentando condições adversas, como exposição a compostos antibacterianos ou carência nutricional. Em situações como essas, as bactérias são capazes de ativar a chamada resposta estringente, modulada pelo alarmônio (p)ppGpp. O acúmulo de (p)ppGpp promove a inibição da transcrição de rRNAs e tRNAs e a supressão do processo de tradução, e a ativação de operons de biossíntese de aminoácidos. Sabe-se também hoje que a resposta estringente está relacionada a outras importantes carências nutricionais em Escherichia coli, como a falta de ácidos graxos, porém não se sabe se o mesmo ocorre em Bacillus subtilis ou em outras Grampositivas. (p)ppGpp atua também direta e indiretamente em vários outros processos celulares, como motilidade, resistência a antibióticos, virulência e persistência, indicando que (p)ppGpp é um regulador central que integra informação metabólica e respostas adaptativas. O presente trabalho buscou estudar a correlação da resposta estringente de B. subtilis com a carência de ácidos graxos e a busca por pequenas moléculas capazes de modular RelA (a principal proteína envolvida na síntese de (p)ppGpp) e impedir o acúmulo de (p)ppGpp. Para a indução da carência de ácidos graxos, foram utilizadas duas estratégias; uso da droga Cerulenina (inibidor de FabF) e mutantes condicionais no gene FabF. Observou-se que mutantes incapazes de ativar a resposta estringente (cepa ppGpp(0) ou RelAD264G) apresentaram grande perda de viabilidade celular durante a carência de ácidos graxos, ao passo que a cepa selvagem manteve sua viabilidade celular. A causa da morte se deu majoritariamente devido ao colapso do potencial de membrana. Apesar de não termos observado aumento de (p)ppGpp nas células selvagens durante a carência de ácidos graxos, observou-se uma redução da razão GTP/ATP, ao passo que na cepa ppGpp(0), a razão GTP/ATP aumentou, devido ao acúmulo de GTP. O uso da droga decoinina, capaz de reduzir os níveis intracelulares de GTP, resgatou parcialmente a viabilidade da cepa e impediu a perda do potencial de membrana, indicando que os níveis de GTP são importantes durante a carência de ácidos graxos em B. subtilis. Para a triagem de pequenas moléculas inibidoras do acúmulo de (p)ppGpp, foi utilizada uma biblioteca de 2320 diferentes compostos químicos, e buscou-se drogas capazes de reverter o fenótipo de crescimento lento de cepas de B. subtilis que acumulam (p)ppGpp (via mutação pontual; mutante RelAH77A e via tratamento com o indutor hidroxamato de arginina) em meio rico. A primeira etapa selecionou 40 moléculas capazes de resgatar o crescimento de células tratadas com arginina-hidroxamato, porém apenas uma, salicilanilida, foi capaz de também resgatar o crescimento da cepa RelAH77A. Todavia, apesar de ser capaz de acelerar o crescimento de B. subtilis esse efeito é limitado. Diversos análogos de salicilanilida foram testados, porém não apresentaram efeito superior a salicilanilida para a reversão do fenótipo de crescimento lento de B. subtilis. Em adição, a droga não foi capaz de aumentar a sensibilidade dos organismos a diversos antibióticos testados, e aparentemente é incapaz de alterar os níveis internos de (p)ppGpp, porém é capaz de causar alterações nos níveis de ATP. Logo, acredita-se que o efeito observado para o crescimento das células seja devido a efeitos indiretos, possivelmente envolvendo alteração de outros nucleotídeos fosforilados

In the environment, inside a host or other habitat, bacteria will always face adverse conditions, as for example exposure to antimicrobials or starvation. In situations like those, bacteria activate the stringent response, modulated by the alarmone (p)ppGpp. (p)ppGpp accumulation promotes inhibition of rRNA and tRNA transcription and suppression of translational process, at the same time that it activates several amino acid biosynthesis operons. It is known also that the stringent response it is related to other starvation stress in Escherichia coli, like lack of fatty acids, but there is no knowledge if the same occurs for Bacillus subtilis or other gram-positive bacteria. ppGpp acts directly and indirectly affecting several other cellular process, as motility, resistance to antibiotics, virulence and persistence, indicating that (p)ppGpp is a central regulator that integrates metabolic information and adaptive responses. This work aimed to study the correlation between the stringent response in B. subtilis with fatty acid starvation, and search for small moleculas capable of modulating RelA (the main enzyme responsible for ppGpp synthesis) and stop (p)ppGpp production. For fatty acid starvation induction, two strategies were used; use of the drug Cerulenin (inhibitor of the FabF protein) and conditional mutants of the FabF gene. We observed that mutants incapable of activating the stringent response (strains ppGpp(0) ou RelAD264G) presented great loss of viability during fatty acid starvation, whereas the wild-type strain keeps its viability. The main cause of death is due membrane rupture in some cells, but mainly due to membrane potential collapse. Although we did not observed increase of (p)ppGpp in wild-type strains during fatty acid starvation, we observed reduction in GTP/ATP ratios, a hallmark of (p)ppGpp production in gram-positive bacteria. In the strain ppGpp(0) GTP/ATP ratio increased, mainly due to GTP increase. Using the drug decoyinine, capable of reducing GTP levels, partially recued viability and protects cells of losing its membrane potential, indicating that GTP levels plays an important role during fatty acid starvation in B. subtilis. For the screening of small molecules capable of inhibit (p)ppGpp production, a library of 2320 different chemical compounds were used, and we looked for drugs capable of reverting the slow growth phenotype of B. subtilis strains with (p)ppGpp accumulation (using a mutant RelAH77A; and using a stringent response inductor, arginine hidroxamate). The first step selected for 40 molecules capable of rescuing the growth of cells treated with arginine hidroxamate, but only one drug, salicilanilyde could also rescue the growth of the strain RelAH77A. Although capable of rescuing growth of B. subtilis that accumulates (p)ppGpp, this rescue is limited. Several analogues of salicilanilyde were tested, but none were stronger than salicilanilyde itself in rescuing growth of slow growing strains of B. subtilis. In addition, the drug was not capable of increasing antibiotic sensibility and it is incapable of changing intracellular (p)ppGpp levels, but it does shifts ATP levels. Therefore, we believe that the observed effects of salicilanilyde is due indirect action, probably involving other phosphorylated nucleotides, rather than modifying (p)ppGpp levels

Inhibition of fatty acid synthase supresses osteosarcoma cell invasion and migration.

Background : Fatty acid synthase (FASN) is overexpressed in a variety of human cancers, and may be involved in cancer metastasis. Hence, the strategies targeted on FASN may have therapeutic potential for treating cancer metastasis. Objectives : The aim of this study is to investigate the correlation of FASN expression with metastasis in human osteosarcoma. Materials and Methods : Human osteosarcoma cell lines U2-OS and osteosarcoma biopsy specimens were employed in this study. The expression of FASN protein in osteosarcoma specimens was detected by IHC (immunohistochemistry) and the relationship with metastasis was analyzed. We performed the cerulenin, an inhibitor of FASN, to inhibit FASN expression in U2-OS cells. Western blot and RT-PCR were performed to investigate the expression of FASN in U2-OS cells. Cells mobility was detected by wound healing and Transwell assays. Results : Results showed that the FASN expression level in the cases with pulmonary metastases was significantly higher than in those without metastasis. In vitro, the invasion and migration of U2-OS cells were suppressed by inhibiting FASN. Our findings suggested that FASN may be involved in osteosarcoma metastasis

Increased fatty acid synthase as a potential therapeutic target in multiple myeloma / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To determine fatty acid synthase (FAS) expression in human multiple myeloma and verify its potential as a therapeutic target in multiple myeloma.</p><p><b>METHODS</b>FAS expression was determined by immunohistochemistry, reverse-transcription polymerase chain reaction (RT-PCR) and immunoblot analysis in bone marrow samples obtained from 27 patients with multiple myeloma (MM patients) and peripheral blood mononuclear cells (PBMCs) obtained from 12 healthy donors. In parallel, additional analyses were performed on 2 human multiple myeloma cell lines, U266 and RPMI8226. U266 cells were treated with cerulenin at various concentrations (5 to 320 microg/ml) for 24 h, and metabolic activity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Apoptosis was evaluated by dual Annexin V/PI (propidium iodide) labeling and flow cytometry (FCM) in U266 cells treated with 20 (g/ml cerulenin for 12 h or 24 h.</p><p><b>RESULTS</b>By immunohistochemistry, we found that 19 of 27 bone marrow samples obtained from MM patients expressed significantly high levels of FAS. Similarly, by RT-PCR, 22 of 27 bone marrow samples obtained from MM patients, U266 and RPMI8226 showed FAS expression, whereas PBMC samples from 12 healthy donors did not express detectable level of FAS. FAS protein expression was confirmed by immunoblot analysis in 16 of 27 bone marrow samples obtained from MM patients, U266 and RPMI8226 cell lines, and no FAS protein expression was detected in PBMC samples from 12 healthy donors. U266 cells were highly sensitive to cerulenin treatment, with a dosage-related effect on metabolic activity, as a measure for cell proliferation. U266 cells treated with 20 microg/ml cerulenin for 12 and 24 h also showed early sign of apoptosis with 56.9% and 69.3% Annexin V(+)/PI(-) cells, and late apoptotic and necrotic cells with 3.2% and 17.6% Annexin V(+)/PI(+) cells.</p><p><b>CONCLUSION</b>Increased FAS expression existed in multiple myeloma samples and human myeloma cell lines. Cerulenin greatly inhibited metabolic activity/cell proliferation of U266 cells and induced apoptosis, suggesting that FAS is an effective target for pharmacological therapy in human multiple myeloma.</p>

Cerulenin changes apoptosis related genes expression in multiple myeloma cell line U266 / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the expression changes of apoptosis related genes induced by cerulenin in multiple myeloma cell line U266 and explore its molecular mechanism.</p><p><b>METHODS</b>The expression changes of 96 apoptosis related genes were analyzed by superArray cDNA in U266 cells treated with cerulenin (20 microg/ml) for 12 h. Semi-quantitative RT-PCR was used to confirm the representative expression changes genes, Rip2, caspase 9 and TRAF2.</p><p><b>RESULTS</b>After treated with cerulenin for 12 h, 44 apoptosis related genes expression in the U266 cells were changed, among which 41 were over 2 fold increase and 3 over 2 fold decrease. The expression of caspase 9 was increased markedly, indicating that mitochondria pathway played a key role in cerulenin inducing apoptosis and TRAF2 expression change suggested that nuclear factor (NF) participates in cerulenin inducing apoptosis.</p><p><b>CONCLUSION</b>The death acceptor signaling pathway and the death acceptor non-dependence signaling pathway co-regulate cerulenin inducing apoptosis in U266 cells. Mitochondria pathway played the key role and nuclear factor (NF) participates in the apoptosis process.</p>

Study on proliferation inhibiting and apoptosis inducing effects of cerulenin on multiple myeloma cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To determine whether fatty acid synthase (FAS) is expressed in human multiple myeloma( MM) cells and investigate the proliferation inhibition effect of fatty acid synthase inhibitor cerulenin on multiple myeloma cell line U266 and its mechanism.</p><p><b>METHODS</b>FAS mRNA expression in human MM cell line U266, RPMI8226 cell was assayed by RT-PCR. The proliferation inhibition rate of U266 cells was assayed by MTr analysis. Cell apoptosis and cycle distribution were evaluated by flow cytometry (FCM).</p><p><b>RESULTS</b>FAS mRNA was highly expressed in human multiple myeloma cell lines as compared with healthy donor PBMNCs. After U266 cells were treated with cerulenin (the concentrations from 5 microg/ml to 640 microg/ ml) for 24 h, the cell proliferation was markedly inhibited with a dose related manner, while the inhibition rate of human skin fibroblast cells were all lower than 30%. When U266 cells were treated with 20 pjg/ml cerulenin for 12 h and 24 h, the early apoptosis rate revealed by Annexin V/PI were 56. 9% and 69. 3% respectively, being higher than that of the blank controls (4. 3% and 1.8%, P < 0. 01). Cell cycle analysis showed it was blocked in S phase. Conclusion FAS is highly expressed in human MM. Cerulenin could induce apoptosis and inhibit proliferation of U266 cells. FAS might be a new potential target for multiple myeloma treatment.</p>

Biosynthesis and accumulation of poly(3-hydroxybutyrate) in Vibrio natriegens / 生物工程学报

Accumulation of poly(3-hydroxybutyrate) [poly(3HB)] by V. natriegens was studied. Results indicated that V. natriegens used glucose, gluconate, fructose and molasses as carbon sources for poly(3HB) synthesis. When molasses was used, up to 28.4% of poly(3HB) to cellular dry weight was accumulated. The accumulation of poly(3HB) followed, was not simultaneously to, the cell growth. Analysis of the PHA polymerase, beta-ketothiolase, and acetoacetyl-CoA reductase showed that the poly(3HB) accumulation was correlated to the increase of their activities in cells. Poly(3HB) accumulation was also related to the de novo fatty acid synthesis, as revealed by the results that cerulenin, a specific inhibitor to the de novo fatty acid synthesis, significantly reduced accumulation of poly(3HB). Based on the results from this study, the synthetic pathway of poly(3HB) was proposed.

Effect of supplementation of ergosterol on miconazole action in vivo and in vitro in Candida albicans.

Ergosterol was observed to alter the lipid composition of C. albicans 3153 selectively, resulting in steep rise in ergosterol content with marginal changes in other lipids content. Supplementation of ergosterol in presence or absence of cerulenin made cells more protective towards the miconazole drug. However, when the same experiments were carried out with liposomes prepared from the lipid extracts of control and supplemented cells, the pattern obtained show a little deviation from the in vivo experiments thus indicating that in addition to lipids, other cell components like proteins also affect interaction of miconazole with C. albicans.

Influence of lipid composition on the sensitivity of Candida albicans to antifungal agents.

Cerulenin, a specific inhibitor of fatty acid and sterol biosyntheses, inhibited growth and lipid synthesis in C. albicans, which on supplementing the growth medium with optimum concentrations of fatty acids was reversed. Significant changes in the levels of phospholipids and sterols were observed in fatty acid-supplemented cells. Altered phospholipids and their fatty acid profile rendered cells more resistant to miconazole and thereby more permeable to [3H]proline. Thus it appears that fatty acid composition plays an important role in determining the permeability susceptibility of C. albicans to drugs.