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.
Cerulenin ; pharmacology ; Hydroxybutyrates ; metabolism ; Polyesters ; metabolism ; Vibrio ; metabolism

OBJECTIVETo study the expression changes of apoptosis related genes induced by cerulenin in multiple myeloma cell line U266 and explore its molecular mechanism.

METHODSThe 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.

RESULTSAfter 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.

CONCLUSIONThe 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.

Apoptosis ; drug effects ; genetics ; Cell Line, Tumor ; Cerulenin ; pharmacology ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Multiple Myeloma ; genetics ; metabolism ; pathology ; Signal Transduction ; drug effects

OBJECTIVETo 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.

METHODSFAS 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).

RESULTSFAS 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.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cerulenin ; pharmacology ; Dose-Response Relationship, Drug ; Fatty Acid Synthases ; antagonists & inhibitors ; biosynthesis ; Humans ; Multiple Myeloma ; metabolism ; pathology ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Cells, Cultured

OBJECTIVETo determine fatty acid synthase (FAS) expression in human multiple myeloma and verify its potential as a therapeutic target in multiple myeloma.

METHODSFAS 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.

RESULTSBy 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.

CONCLUSIONIncreased 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.

Adult ; Aged ; Apoptosis ; drug effects ; Base Sequence ; Blotting, Western ; Case-Control Studies ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cerulenin ; pharmacology ; DNA Primers ; genetics ; Enzyme Inhibitors ; pharmacology ; Fatty Acid Synthase, Type I ; antagonists & inhibitors ; genetics ; metabolism ; Fatty Acid Synthesis Inhibitors ; pharmacology ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Multiple Myeloma ; drug therapy ; enzymology ; genetics ; pathology ; RNA, Messenger ; genetics ; metabolism ; RNA, Neoplasm ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction