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Cordycepin inhibits protein synthesis and cell adhesion through effects on signal transduction.

Wong YY, Moon A, Duffin R, Barthet-Barateig A, Meijer HA, Clemens MJ, de Moor CH - J. Biol. Chem. (2009)

Bottom Line: In 4EBP knock-out cells, the effect of cordycepin on translation is strongly reduced, confirming the role of this modification.Inhibition of AMPK prevented translation repression by cordycepin and abolished 4EBP1 dephosphorylation, indicating that the effect of cordycepin on mTOR signaling and protein synthesis is mediated by AMPK activation.We conclude that many of the reported biological effects of cordycepin are likely to be due to its effects on mTOR and AMPK signaling.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
3'-Deoxyadenosine, also known as cordycepin, is a known polyadenylation inhibitor with a large spectrum of biological activities, including anti-proliferative, pro-apoptotic and anti-inflammatory effects. In this study we confirm that cordycepin reduces the length of poly(A) tails, with some mRNAs being much more sensitive than others. The low doses of cordycepin that cause poly(A) changes also reduce the proliferation of NIH3T3 fibroblasts. At higher doses of the drug we observed inhibition of cell attachment and a reduction of focal adhesions. Furthermore, we observed a strong inhibition of total protein synthesis that correlates with an inhibition of mammalian target of rapamycin (mTOR) signaling, as observed by reductions in Akt kinase and 4E-binding protein (4EBP) phosphorylation. In 4EBP knock-out cells, the effect of cordycepin on translation is strongly reduced, confirming the role of this modification. In addition, the AMP-activated kinase (AMPK) was shown to be activated. Inhibition of AMPK prevented translation repression by cordycepin and abolished 4EBP1 dephosphorylation, indicating that the effect of cordycepin on mTOR signaling and protein synthesis is mediated by AMPK activation. We conclude that many of the reported biological effects of cordycepin are likely to be due to its effects on mTOR and AMPK signaling.

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Cordycepin inhibits cell spreading. NIH3T3 cells were detached, suspended for 1 h in serum-free medium, and allowed to re-attach to coverslips with serum for 5 h in the presence or absence of cordycepin, cycloheximide, or actinomycin D at the concentrations indicated. After fixation, cells were stained with phalloidin to visualize the actin cytoskeleton. A, images of typical control and cordycepin-treated cells. B and C, quantitation of the percentage of unspread cells (largest diameter, 25 μm or less) in cells incubated with the indicated doses of drugs.
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Figure 3: Cordycepin inhibits cell spreading. NIH3T3 cells were detached, suspended for 1 h in serum-free medium, and allowed to re-attach to coverslips with serum for 5 h in the presence or absence of cordycepin, cycloheximide, or actinomycin D at the concentrations indicated. After fixation, cells were stained with phalloidin to visualize the actin cytoskeleton. A, images of typical control and cordycepin-treated cells. B and C, quantitation of the percentage of unspread cells (largest diameter, 25 μm or less) in cells incubated with the indicated doses of drugs.

Mentions: To study the effect of cordycepin on adhesion further, we examined the influence of cordycepin on cell adhesion and spreading after detachment, when focal adhesions are formed de novo. We detached NIH3T3 cells with trypsin, washed them, and kept them in suspension for 1 h to disassemble all pre-existing focal adhesions. Cells are completely rounded after this procedure. We then plated them on coverslips in the presence of cordycepin for 5 h, which is the time the cells normally need to spread fully, and stained the cells with fluorescent phalloidin (for filamentous actin). The morphology of untreated and cordycepin-treated cells was very different with the phalloidin stain, with a high level of disorganisation and a lack of spreading in the treated cells (Fig. 3A). We counted the number of incompletely spread cells as those that do not reach a diameter of 25 μm or more and expressed it as a percentage of the total number of cells. As can be seen in Fig. 3B, 10 μm cordycepin had no detectable effect on cell spreading, whereas 50 and especially 200 μm caused an increase in the number of incompletely spread cells. To investigate if this effect is due to cordycepin blocking transcription or translation of a crucial factor, we repeated this experiment with the transcription inhibitor actinomycin D and the translation inhibitor cycloheximide. As can be seen in Fig. 3C, actinomycin D had no effect on cell spreading, whereas cycloheximide had a moderate effect. This indicates that cordycepin has effects on cell spreading that are not mediated through inhibition of transcription.


Cordycepin inhibits protein synthesis and cell adhesion through effects on signal transduction.

Wong YY, Moon A, Duffin R, Barthet-Barateig A, Meijer HA, Clemens MJ, de Moor CH - J. Biol. Chem. (2009)

Cordycepin inhibits cell spreading. NIH3T3 cells were detached, suspended for 1 h in serum-free medium, and allowed to re-attach to coverslips with serum for 5 h in the presence or absence of cordycepin, cycloheximide, or actinomycin D at the concentrations indicated. After fixation, cells were stained with phalloidin to visualize the actin cytoskeleton. A, images of typical control and cordycepin-treated cells. B and C, quantitation of the percentage of unspread cells (largest diameter, 25 μm or less) in cells incubated with the indicated doses of drugs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2807318&req=5

Figure 3: Cordycepin inhibits cell spreading. NIH3T3 cells were detached, suspended for 1 h in serum-free medium, and allowed to re-attach to coverslips with serum for 5 h in the presence or absence of cordycepin, cycloheximide, or actinomycin D at the concentrations indicated. After fixation, cells were stained with phalloidin to visualize the actin cytoskeleton. A, images of typical control and cordycepin-treated cells. B and C, quantitation of the percentage of unspread cells (largest diameter, 25 μm or less) in cells incubated with the indicated doses of drugs.
Mentions: To study the effect of cordycepin on adhesion further, we examined the influence of cordycepin on cell adhesion and spreading after detachment, when focal adhesions are formed de novo. We detached NIH3T3 cells with trypsin, washed them, and kept them in suspension for 1 h to disassemble all pre-existing focal adhesions. Cells are completely rounded after this procedure. We then plated them on coverslips in the presence of cordycepin for 5 h, which is the time the cells normally need to spread fully, and stained the cells with fluorescent phalloidin (for filamentous actin). The morphology of untreated and cordycepin-treated cells was very different with the phalloidin stain, with a high level of disorganisation and a lack of spreading in the treated cells (Fig. 3A). We counted the number of incompletely spread cells as those that do not reach a diameter of 25 μm or more and expressed it as a percentage of the total number of cells. As can be seen in Fig. 3B, 10 μm cordycepin had no detectable effect on cell spreading, whereas 50 and especially 200 μm caused an increase in the number of incompletely spread cells. To investigate if this effect is due to cordycepin blocking transcription or translation of a crucial factor, we repeated this experiment with the transcription inhibitor actinomycin D and the translation inhibitor cycloheximide. As can be seen in Fig. 3C, actinomycin D had no effect on cell spreading, whereas cycloheximide had a moderate effect. This indicates that cordycepin has effects on cell spreading that are not mediated through inhibition of transcription.

Bottom Line: In 4EBP knock-out cells, the effect of cordycepin on translation is strongly reduced, confirming the role of this modification.Inhibition of AMPK prevented translation repression by cordycepin and abolished 4EBP1 dephosphorylation, indicating that the effect of cordycepin on mTOR signaling and protein synthesis is mediated by AMPK activation.We conclude that many of the reported biological effects of cordycepin are likely to be due to its effects on mTOR and AMPK signaling.

View Article: PubMed Central - PubMed

Affiliation: School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK.

ABSTRACT
3'-Deoxyadenosine, also known as cordycepin, is a known polyadenylation inhibitor with a large spectrum of biological activities, including anti-proliferative, pro-apoptotic and anti-inflammatory effects. In this study we confirm that cordycepin reduces the length of poly(A) tails, with some mRNAs being much more sensitive than others. The low doses of cordycepin that cause poly(A) changes also reduce the proliferation of NIH3T3 fibroblasts. At higher doses of the drug we observed inhibition of cell attachment and a reduction of focal adhesions. Furthermore, we observed a strong inhibition of total protein synthesis that correlates with an inhibition of mammalian target of rapamycin (mTOR) signaling, as observed by reductions in Akt kinase and 4E-binding protein (4EBP) phosphorylation. In 4EBP knock-out cells, the effect of cordycepin on translation is strongly reduced, confirming the role of this modification. In addition, the AMP-activated kinase (AMPK) was shown to be activated. Inhibition of AMPK prevented translation repression by cordycepin and abolished 4EBP1 dephosphorylation, indicating that the effect of cordycepin on mTOR signaling and protein synthesis is mediated by AMPK activation. We conclude that many of the reported biological effects of cordycepin are likely to be due to its effects on mTOR and AMPK signaling.

Show MeSH
Related in: MedlinePlus