Influence of fatty acid synthase inhibitor orlistat on the DNA repair enzyme O6-methylguanine-DNA methyltransferase in human normal or malignant cells in vitro
Giorgia CioccoloniLaura BonmassarElena PaganiSimona CaporaliMaria Pia FuggettaEnzo BonmassarStefania D’AtriAngelo Aquino
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Tetrahydrolipstatin (orlistat), an inhibitor of lipases and fatty acid synthase, is used orally for long-term treatment of obesity. Although the drug possesses striking antitumor activities in vitro against human cancer cells and in vitro and in vivo against animal tumors, it also induces precancerous lesions in rat colon. Therefore, we tested the in vitro effect of orlistat on the expression of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that plays an essential role in the control of mutagenesis and carcinogenesis. Western blot analysis demonstrated that 2-day continuous exposure to 40 µM orlistat did not affect MGMT levels in a human melanoma cell line, but downregulated the repair protein by 30-70% in human peripheral blood mononuclear cells, in two leukemia and two colon cancer cell lines. On the other hand, orlistat did not alter noticeably MGMT mRNA expression. Differently from lomeguatrib (a false substrate, strong inhibitor of MGMT) orlistat did not reduce substantially MGMT function after 2-h exposure of target cells to the agent, suggesting that this drug is not a competitive inhibitor of the repair protein. Combined treatment with orlistat and lomeguatrib showed additive reduction of MGMT levels. More importantly, orlistat-mediated downregulation of MGMT protein expression was markedly amplified when the drug was combined with a DNA methylating agent endowed with carcinogenic properties such as temozolomide. In conclusion, even if orlistat is scarcely absorbed by oral route, it is possible that this drug could reduce local MGMT-mediated protection against DNA damage provoked by DNA methylating compounds on gastrointestinal tract epithelial cells, thus favoring chemical carcinogenesis.Keywords:
Orlistat
Temozolomide
DNA methyltransferase
The EcaI GGTNACC‐specific DNA‐adenine modification methyltransferase has been purified to apparent homogeneity. The active form of the DNA methyltransferase is a single polypeptide. The enzyme has a pH optimum at pH 8.0 and a temperature optimum at 25°C. EcaI DNA methyltransferase transfers one methyl group to the adenine of the recognition site in a single binding event. The K m was 170 nM for DNA and 1.8 μM for the methyl donor S ‐adenosylmethionine. Methylated DNA is a competitive inhibitor with respect to DNA ( K i = 3.5 nM). The other product of the DNA‐methylation reaction, S ‐adenosylhomocysteine was found to be a competitive inhibitor with respect to S ‐adenosylmethionine ( K i = 2.7 μM). The S ‐adenosylmethionine analog sinefungin was shown to be a very strong inhibitor ( K i = 3.5 nM) of the DNA methyltransferase reaction.
DNA methyltransferase
DNMT1
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Abstract Fatty acid synthase (FASN), a seven domain enzyme, is the sole protein capable of de novo synthesis of free fatty acids, most commonly 16-carbon palmitate. The fatty acid synthesis cycle begins with the condensation of acetyl-CoA and malonyl-CoA, and continues with the elongation of the fatty acid chain, which is tethered to an acyl carrier protein domain (ACP), via a repeating cycle. At the end of elongation, the thioesterase (TE) domain of FASN, a member of the serine hydrolase family with an Asp-His-Ser catalytic triad, cleaves the bond between palmitate and ACP, and releases free palmitate. FASN has been found to be over-expressed in a wide variety of human cancers, and this over-expression is correlated to a higher metastatic potential and poorer prognosis in cancer patients. FASN over-expression is also associated with increased resistance toward cancer chemotherapeutics. Orlistat, an FDA approved drug for obesity treatment that prevents uptake of dietary fats by inhibiting pancreatic lipases in the gastrointestinal tract, is a compound found to act as a reversible inhibitor of FASN TE. A previous study using the co-crystal structure of the TE domain with orlistat found that orlistat was covalently bound to the active site Ser within the TE domain, indicating that inhibition of FASN by orlistat halts the fatty acid synthesis cycle by blocking the release of free palmitate from the ACP. A hydrolyzed form of orlistat was also observed in the active site of TE, demonstrating that orlistat is not a stable inhibitor of FASN. In this study, we examined the mechanism of orlistat hydrolysis within the TE domain of FASN using molecular dynamics simulations. We found that the hexanoyl tail of orlistat is capable of shifting while covalently bound to the active site Ser, and that this shift is accompanied by the destabilization of a hydrogen bond that exists between a hydroxyl moiety of orlistat and the active site His. Once this hydrogen bond is destabilized, a catalytic water molecule can enter the active site of TE with the proper orientation for catalysis of the covalent bond between orlistat and Ser. These findings suggest that the hexanoyl tail of orlistat plays an important role in its hydrolysis and may guide the future design of new inhibitors that target the TE domain of FASN with greater endurance for potential use in the treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3978. doi:1538-7445.AM2012-3978
Orlistat
Acyl carrier protein
Fatty acid synthesis
Serine hydrolase
Catalytic triad
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This work develops an electrochemical approach for rapid detection of the genomic DNA methylation level, assay of methyltransferase activity, and evaluation and screening of the inhibitors of methyltransferase. This method may be a help for the discovery of anticancer drugs.
DNA methyltransferase
genomic DNA
DNMT1
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Abnormal DNA methylation plays an extremely important role in tumors.DNA methylation is completed through DNA methyltransferases(DNA methyhransferases,DMNTs).The miR-29 had a certain relationship with the expression of methylation transferase.In this study,an adenovirus vector carrying miR-29b is constructed and the virus is used to infect the liver cancer cell PLC.Then qRT-PCR is exploited to detect the relative quantity of miR-29b and methyltransferase in the infected PLC cells.The results show that the adenovirus carrying miR-29b can effectively elevate the cellular miR-29b and inhibit the expression of methyltransferases.
DNA methyltransferase
O-6-methylguanine-DNA methyltransferase
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Temozolomide
O-6-methylguanine-DNA methyltransferase
DNA methyltransferase
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Abstract Methyltransferase inhibitors : Short double‐stranded oligonucleotides that have a hemimethylated target sequence and 5‐fluoro‐2′‐deoxycytidine as a suicide inhibitor as well as their phosphorothioated analogues were tested for their ability to inhibit the bacterial methyltransferase M.HhaI and the human Dnmt1 in vitro. magnified image The cytidine analogue 5‐fluoro‐2′‐deoxycytidine (dC F ) is a mechanism‐based inhibitor of DNA methyltransferases. We report the synthesis of short 18‐mer dsDNA oligomers containing a triple‐hemimethylated CpG motive as a recognition sequence for the human methyltransferase Dnmt1. The DNA strands carry within these CpG islands dC F building blocks that function as mechanism‐based inhibitors of the analyzed methyltransferases. In addition, we replaced the phosphodiester backbones at defined positions by phosphorothioates. These hypermodified DNA strands were investigated as inhibitors of the DNA methyltransferases M.HhaI and Dnmt1 in vitro. We could show that both methylases behave substantially differently in respect to the amount of DNA backbone modification.
DNMT1
DNA methyltransferase
CpG site
Phosphodiester bond
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DNA methyltransferase
O-6-methylguanine-DNA methyltransferase
Human placenta
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The DNA repair protein O(6)-methylguanine-DNA methyltransferase is a drug-resistant protein, which protects the tumors from chemotherapeutic alkylating agents, such as temozolomide. The methylation status of O(6)-methylguanine-DNA methyltransferase promoter has been shown to be a major predictive factor for clinical outcome in glioma patients when treated by alkylating agents. Thereby, there were many reports on O(6)-methylguanine-DNA methyltransferase promoter methylation and mRNA expression in primary glioma, in contrast, there were only a few studies in recurrent glioma.We evaluated the O(6)-methylguanine-DNA methyltransferase mRNA expression and promoter methylation status in glioma patients before and after recurrence by quantitative real-time PCR and methylation-specific PCR assay. Thirteen paired primary and recurrent glioma patients were analyzed, including four patients in whom malignant transformation occurred from Grade II to Grade III.Methylation-specific PCR assay demonstrated that the status of O(6)-methylguanine-DNA methyltransferase promoter changed from methylated to unmethylated in 10 of 13 samples when the tumor relapsed. Moreover, intra-individual O(6)-methylguanine-DNA methyltransferase mRNA level increased in recurrent gliomas than in primary ones (P = 0.016). O(6)-methylguanine-DNA methyltransferase mRNA level was correlated with the methylation status (P = 0.012).Our results give the evidence that the increase of O(6)-methylguanine-DNA methyltransferase mRNA expression caused by methylation changes in recurrence may be associated with chemoresistance in the recurrent glioma.
DNA methyltransferase
Temozolomide
O-6-methylguanine-DNA methyltransferase
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The levels of DNA methyltransferase in nuclei from 9 tumorigenic and 9 nontumorigenic cell lines were examined. In all but 2 cases, the extractable methyltransferase activity was 4-3000-fold higher in tumorigenic than in nontumorigenic cells. Tumorigenic and nontumorigenic cells from four species were grown in the presence of various concentrations (10(-8)-10(-6) M) of an inhibitor of the methylase enzyme, 5-aza-2'-deoxycytidine (5-aza-dCyd). The reduction of 5-methylcytosine content in newly replicated DNA in the presence of 5-aza-dCyd was used to determine the relative methylase activity in each cell line. In all 4 cases, tumorigenic cells required larger doses of drug to inhibit DNA methylation to the same extent as their nontumorigenic counterparts. The relative rates of incorporation of [3H]5-aza-dCyd were determined for each cell line, and tumorigenic cells were shown to incorporate equal or greater amounts of 5-aza-dCyd into DNA compared to nontumorigenic cells. These results showed that the differences in the inhibition of DNA methylation in response to 5-aza-dCyd were not due to differences in the ability of these cells to incorporate the drug. Thus, it was demonstrated by two independent methods that tumorigenic cells contained higher levels of methylating capacity than nontumorigenic cells. This overabundance of methyltransferase may alter DNA methylation patterns and affect phenotypic stability.
DNA methyltransferase
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Orlistat
Cerulenin
Viability assay
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