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外文文摘：0 o: F4 X& y6 t) N! H
Abstract Fatty acid synthase is a key synthetic enzyme that catalyzes the NADPH-dependent condensation of malonyl-CoA and acetyl-CoA to produce the 16-carbon saturated free fatty acid palmitate. FAS is downregulated in most normal human tissues, but is often highly expressed in human cancers including human hepatocellular carcinoma (HCC). The differential tissue distribution makes FAS an attractive target for cancer cells. C75, a stably synthetic, small-molecule inhibitor of FAS, has a good effect on antitumor and can induce cell cycle arrests and apoptosis in human tumor cells in vitro and in vivo. C75 is covalently attached to the active-site cystein residue and lies within a hydrophobic pocket at the FAS-dimer interface to result in FAS inhibition. Recent research showed that C75 not only targets the crucial condensation step catalyzed by the β -ketoacyl synthase, but also inactivates the enoyl reductase and thioesterase partial activities of FAS. Liver is a highly lipogenic tissue and exhibits high levels of FAS. Hepatocellular carcinoma (HCC), etiologically associated to hepatitis Β virus (HBV) and Hepatitis C virus (HCV), is one of the most common malignancies in the world. An implication of FAS-associated metabolic stresses in the growth and development of HCC has rarely yet been investigated, particularly much less is known about the correlation between FAS and growth arrest/apoptosis and its related signaling pathways. & o& q# {' Y( f% v& A# W& c# k
p53 gene, a tumor suppressor gene, has been most cared and well studied for a long time. p53 protein is known as cell cycle checkpoints with other cellular proteins.The cell cycle progress is halted at the checkpoints after stresses by restraining cell cycle transition. To clarify the role of p53 in growth arrests by C75, we use three HCC cell lines with different types of p53: HepG2 (wt-p53), SMMC7721 (mut-p53)and Hep3B (p53 null) cells. Additionally, p38 MAPK mediates tumor growth suppression in many stress responses, though it is unclear whether p38 MAPK is involved in the effect of C75. For these purposes, we made use of C75, preferentially targeting on three HCC cell lines to investigate the possible roles of FAS in hepatic cancer growth, and to understand the potential of anti-metabolic therapy in hepatic cancers. ( t r+ K8 n& ]4 N( b& f
First we detected the overexpression level and base activity of FAS in the three HCC cell lines, which are higher in HepG2 than other two HCC cells. FAS activity was rapidly inhibited by C75 at 15 min and more obviously at 30 min in three HCC cells. C75 produced the similar cytotoxic effect using MTT assay and the IC50 value was about 60 μM in HepG2 and SMMC7721 cells and 30 μM in Hep3B cells. Then we analyzed the cell growth suppression effects induced by CD75 using flow cytometry. The results showed that C75 induced G? phase growth arrest time and dose dependently in HepG2 and SMMC7721 cells, while G? phase arrest in Hep3B cells. The dose-effect analysis of C75 at 24 h indicated that the cell number of G?phase was elevated respectively from 12.8% to 24.6% in HepG2 cells and from 13.7 to 32.1% in SMMC7721 cells; the cell number of G? phase was from 37.7% up to 61.4%. However, the little apoptosis was detected within 24h below/at 60 μM C75 treatment. The apoptotic cells increased rapidly after 80 μM C75 treatment, whereas the ratio of growth arrests was declined or almost unchanged. Additionally, the time-effect analysis of 60 μM C75 treatment within 24 h indicated that the growth arrests occurred at the different time points in the HCC cells. The earliest time point is at 6 h in SMMC7721, 12 h in Hep3B and 24 h in HepG2 cells. The growth arrests became the strongest at 24 h. The cell number of G? phase was raised from 7.9% to 17.9% in HepG2 cells and from 18.7% to 43.4% in SMMC7721 cells. That of G?phase was from 45.0% up to 66.6% in Hep3B cells. The growth arrests induced by C75 were dose and time dependently in HCC cells. 6 f1 @" r' v# z; A' S1 T( ~+ @2 b
Despite different p53 types in HepG2 and SMMC7721 cells, C75 produced the similar cytotoxic effects and yielded the similar induction of G? phase arrest. It suggested that p53 might not be involved in the regulation of cell cycle arrests.Western blot assay showed that the expression of p53 was up-regulated in a time-dependent manner in two HCC cells treated by C75 within 24 h, but that of p-p53 was only elevated at 24 h in HepG2 cells and was not detected in SMMC7721 cells. To further observe the role of p53, we used siRNA to silence p53 gene in HepG2 cells. RNA interference markedly reduced p53 protein level, but did not change cell cycle delay by C75. It approved that p53 might not involve in the regulation of G? arrest aroused by C75. Besides, we investigated the expression levels of cell cycle proteins. Cyclin A and cyclin B1 were decreased time dependently corresponding to G? arrest in HepG2 and SMMC7721 cells. SiRNA targeting p53 did not have effect on the expression of these cell cycle proteins,suggesting that expression of cell cycle proteins varied with the level of growth arrest but not with the status of p53. C75 induced G? phase arrest in p53-deleted Hep3B cells, which further indicated a lack of the correlation of the G? phase growth arrest with p53 in these hepatic cancer cells. Cyclin D1 with close to G? phase progression was declined but cell cycle inhibitory protein p21〓 rose up time dependently.Presumably, these cycle proteins were regulated by other important signal moleculars but independent of p53. At present, it is still unclear whether p53 at a high level in the cellular can induce growth suppression or apoptosis. For this purpose, we thansfected p53 wild-type vectors in Hep3B cells and make p53 and p-p53 overexpression. But no growth arrest and apoptotic death were detected. Therefore,the growth suppression effects by C75 were exactly independent of p53.
+ X1 c. {# F) V8 x# o! ` p38 MAPK is one of three groups of mammalian MAPKs and induces growth arrests in many stress responses. Significantly, we found that C75 stimulated p38 MAPK activation in three HCC cell lines and p-p38 MAPK was elevated from 1.5 h and accumulated in a time-dependent manner. The cell cycle arrests were partly reversed by using SB203580, a special inhibitor of p38 MAPK. Although SB203580 did not affect HCC cell distribution alone, obviously inhibited p38 MAPK activity.This result showed that p38 MAPK partially mediated growth arrests induced by C75.The previous research reported that p38 MAPK may alterating the cell cycle progression by regulating directly or indirectly the transcription or expression of cell cycle proteins. So we observed the expression alteration of cell cycle proteins influenced by SB203580 in three HCC cells. The results revealed that SB203580 partly restored the protein levels of cyclii、cyclinB1、cyclinD1 and p21 comparing with only C75-treated group, indicating that p38 MAPK mediated partly growth arrests by regulating the expression of cell cycle proteins after C75 treatment.
0 P. K: I8 g9 d& o It has been reported that p53 and p38 MAPK interacts with each other. In our study, after p53 gene silence, the expression of p-p38 MAPK was still raised by C75 in HepG2 cells, which showed that p53 had no effect on the activity of p38 MAPK;The overexpression of p53 and p-p53 were not reversed by pretreatment of SB203580 in HepG2 and SMMC7721 cells. This result revealed that p53 and p38 MAPK might have no interaction with each other. $ H6 C8 G, h( c
The accumulation of malonyl-CoA, the committed substrate of FAS, not only inhibited carnitine palmitoyltransferase 1 (CPT-1) activity to decrease the oxidation of fatty acids, but also resulted in tumor cell apoptosis according to the previous research. To investigate the role of malonyl-CoA in cell cycle arrest, we used TOFA,a specific inhibitor of the acetyl-CoA carboxylase (ACC), to reduce the amount of malonyl-CoA. The result showed that TOFA pretreatment did not alter the cell cycle arrest by C75, indicating that high level of malonyl-CoA wasn't the reason of the growth arrests. ' N" j6 `0 i# o' S7 X, V3 E5 r- ^* C
One of the main reasons of cell cycle arrest is to initiate repair mechanism for DNA damage, which result in cell cycle delay or arrest. We next examined whether significant DNA damage occurred after C75 exposure using comet assay in HepG2 cells. We found that DNA damage was induced at 1.5 h and 3 h but not detected at 6 h when 60 μM C75 was added to the medium. Although growth arrest occurred at 24 h, it is difficult to determine that DNA damage was not related with it. Additionally,it is possible that the perturbation of normal lipid metabolism after FAS inhibited by C75 might be regarded as a metabolic stress to activate p38 MAPK and arouse growth arrests in HCC cells.
' w3 F* E. Y8 H+ W- d) K8 ] In the summary, C75 not only aroused G? phase arrests in HepG2 and SMMC7721 cells but also G? phase arrests in Hep3B cells. p53 might have little effect on the growth arrests induced by C75 in HCC cells while p38 MAPK played a predominant role via regulating the cell cycle proteins. In addition, we detected DNA damage in the early time after C75 treatment, which is a great difference from the previous study. Thus, the cell cycle arrest might be related with DNA damage or the metabolic disturbance after FAS inhibition, which activated p38 MAPK. The accumulation of malonyl-CoA was of little importance, indicating that effects of FAS inhibitor on cell cycle progression are distinct from those mediating apoptotic cell death. . ^9 H @: r6 O* r
Key words: C75; Fatty acid synthase (FAS); anti-tumor; Hepatocellular carcinoma (HCC); cell cycle arrest; p53; p38 MAPK; cell cycle regulatory protein;DNA damage |
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发表于 2012-9-26 18:49
发表于 2012-9-26 20:11
