Effects of cordycepin on RNA metabolism in …
It has also been reported that cordycepin lowers c-Myc mRNA levels in HeLa cells without affecting the levels of protein produced (). c-Myc mRNA contains an internal ribosome entry site, which could explain why it is resistant to the inhibition of cap-dependent translation initiation caused by 4EBP dephosphorylation ().
DNA, RNA, and the Flow of Genetic Information …
After treatment with cordycepin, the cells werecollected, washed with cold phosphate-buffered saline (PBS) andfixed in 75% ethanol at 4°C for 30 min. Prior to analysis, thecells were washed once again with PBS, suspended in a cold PIsolution containing 100 g/ml RNase A, 50 g/ml PI,0.1% (w/v) sodium citrate and 0.1% (v/v) NP-40, and furtherincubated on ice for 30 min in the dark. Flow cytometry analyseswere carried out using a flow cytometer (FACSCalibur;Becton-Dickinson). Cell-Quest software was used to determine therelative DNA content based on the presence of red fluorescence. Thesub-G1 population was calculated to estimate the apoptotic cellpopulation ().
To determine the effect of cordycepin on cellular translation, we measured radioactive amino acid incorporation for 10 min after treatment with cordycepin, adenosine, or actinomycin D for 2 h. As can be seen in A, cordycepin at 200 μ inhibited translation by up to 95%, whereas adenosine and actinomycin D had no effect. We then examined the effect of cordycepin on the association of mRNAs with ribosomes using high salt sucrose gradient centrifugation. In these gradients, ribosomes that are not engaged in translation dissociate into 40 and 60 S subunits (). B shows the polyribosome profile of untreated and treated cells. In treated cells there was a large increase in the free ribosomes (40 and 60 S) and a decrease in the polyribosome levels, indicating that there is repression of translation by cordycepin at the level of initiation. C shows the dose response of the effect of cordycepin on translation. From 50 μ onward there is a significant effect on translation, with smaller effects at 10 and 20 μ. This is confirmed in the time course in D, which also demonstrates that the maximal repression is reached after 1 h of cordycepin incubation. A similar dose response was seen in HeLa cells (E). These data demonstrate that cordycepin is a strong inhibitor of translation in mammalian cells.
Natural Products - Compound Libraries
The polyadenylation inhibitor cordycepin(3′-deoxyadenosine) is an active component of the caterpillarfungus . Due to the absence of oxygen inthe 3′ position of its ribose moiety, incorporation of cordycepinduring RNA synthesis results in termination of chain elongation(,). This activity has been well described with purified RNA polymerases and poly(A)polymerases from a number of organisms, including yeasts andmammals (,). Cordycepin has anticancer activitiesincluding induction of apoptosis, DNA double-strand break activity,and cell cycle arrest in cancer cells (–).This compound also inhibits metastasis and angiogenesis (–).Despite these observations, the molecular mechanisms underlying theanticancer effects of cordycepin on TRAIL-mediated apoptosis havenot been fully elucidated.
EGFR Mutation and Resistance of Non–Small-Cell Lung …
The fermentation broth of with a cordycepin concentration of ~0.2 mg/ml was centrifuged athigh speed (2,000 × g) for 15 min. The supernatant was concentratedunder vacuum to a concentration of 0.6 mg/ml. Then, 1,800 ml ofthis sample solution was passed through 300 ml macroporous resin(HPD-100) for the preliminary removal of impurities. The volumeflow rate of the loading sample was 2 BV/h, and the sample waseluted with ethanol at volume fraction of 25% at a volume flow of 3bed volumes/h. The eluted sample was concentrated and freeze-driedto obtain the crude extract samples of cordycepin. The cordycepincontents of the crude samples were determined by HPLC. A solutionwas prepared by dissolving 400 mg crude sample in 100 ml mobilephase, and ultrasonic vibration was used to ensure that the samplewas completely dissolved. This solution was subjected to furtherpurification.
Susumu Kobayashi, M.D., Ph.D., Titus J
Caspase activities were determined by colorimetricassays using caspase-3, -8 and -9 activation kits according to themanufacturer’s protocol. The kits utilize synthetic tetrapeptideslabeled with p-nitroaniline. Briefly, the cells were lysed in thesupplied lysis buffer. The supernatants were collected andincubated with the supplied reaction buffer containingdithiothreitol (DTT) and substrates at 37ºC. Caspase activity wasdetermined by measuring changes in absorbance at 405 nm using anELISA reader.