2013 Fiscal Year Research-status Report
Limonoids, a novel type of inhibitors of sphingomyelin biosynthesis to study the membrane dynamics of sphingolipid domains
| Project/Area Number |
25460081
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
FHULL IN・MATSUDA 独立行政法人理化学研究所, 小林脂質生物学研究室, 客員研究員 (30469928)
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| Project Period (FY) |
2013-04-01 – 2016-03-31
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| Keywords | sphingolipids / sphingomyelin / ceramide / cancer / infection / lipid domains / transbilayer movement / lipid dynamics |
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| Research Abstract |
In order to precise the therapeutic effects of Limonoids, a novel type of inhibitors of sphingomyelin biosynthesis that we recently characterized, we analyzed their effects on various sphingolipid-dependent events. (1) Limonoid (Gedunin) pretreatment increased the toxicity of Doxorubicin on HeLa cells, but did not improve the efficiency of Paclitaxel treatment on other cancer cells such as A 549, Neuro 2A, or the toxicity of UV irradiation on HeLa cells, suggesting that modulation of sphingolipid metabolism can improve the efficiency of some types of anticancer treatment. (2) Limonoid (Gedunin) treatment of erythrocytes and HeLa cells inhibited, respectively, the growth of Plasmodium and Toxoplasma, 2 types of parasites whose growth is dependent of the sphingolipid content of the host cells. We previously reported that Limonoids favored the formation of ceramide-rich domains in the membrane. (3) We studied more in detail the formation and dynamics of these domains and especially the effect of Limonoids on the ceramide transbilayer movement using sulfhydryl analogues of ceramides synthetized in the laboratory. We succeeded to set up the experimental conditions in order to study the ceramide flip-flop in large and small unilamellar vesicles with different lipid composition mimicking that of different organelles (endoplasmic reticulum, Golgi, late endosomes, mitochondria ) implicated in sphingolipid metabolism and will try now the effect of Limonoids.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The first phase of the research project concerning the therapeutical effects of Limonoid treatment is progressing as expected since efficient modulation of sphingomyelin synthesis by some limonoids compounds could improve the treatment with anticancer drugs depending of the cellular models. Furthermore, the limonoid-induced decrease of sphingomyelin in the host cells seemed to limit the growth of parasites such as Plasmodium and Toxoplasma. In parallel, we started in vitro models to analyze the effect of Limonoids on ceramide dynamics in the membrane.
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| Strategy for Future Research Activity |
In the second phase of the project, we will study more in detail the secondary effects of the limonoid treatment on the cell growth of non-cancer cells (mimicking the surrounding tissue of a tumor) as well as on the host cells in the case of parasite infection. We will analyze the effect of Limonoids on the flip-flop or transbilayer movement of ceramides in liposome mimicking the lipid composition of different organelles (endoplasmic reticulum, Golgi, late endosomes, mitochondria ) since it is know that ceramide affects differently the membrane organization according to the surrounding lipids.
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| Expenditure Plans for the Next FY Research Funding |
Finally the amount of radioactive compounds to study the effects of Limonoid compounds on sphingolipid synthesis was less than I expected.
Fluorescent lipid reagents and fluorescent-tagged secondary antibodies are needed to study the effect of the drugs on ceramide trafficking and membrane fluidity by fluorescence confocal microscopy, fluorimetry or fluorescence polarization. In addition I will need to purchase lipid standards and new GC column for lipid analysis.
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[Journal Article] Sphingomyelin regulates the transbilayer movement of diacylglycerol in the plasma membrane of Madin-Darby canine kidney cells.2013
Author(s)
Ueda, Y., Makino, A., Murase-Tamada, K., Sakai, S., Inaba, T., Hullin-Matsuda, F., and Kobayashi, T.
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Journal Title
FASEB J
Volume: 27
Pages: 3284-3297
DOI
Peer Reviewed
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