It is known that cordycepin affects mTOR signaling and components of the upstream PI3K pathway have been reported to be subject to regulation by the cytoplasmic polyadenylation element binding protein (Wong et al. 2010). We therefore decided to investigate the mRNAs involved in mTOR signalling and other candidates for regulation by CPEB like TWIST1 (Nairismagi et al. 2012) in an attempt to identify more target mRNAs for comparison of cordycepin with other treatments in MCF-7 cells. Cells were treated with cordycepin 50µM for 2 hours followed by total RNA isolation. RT-qPCR was performed normalized to GAPDH and untreated values were set at 1. The key players of mTOR pathway including cancer related and control mRNAs were investigated as shown in figure 5.10 as separated by a space. None of the tested mRNAs were sensitive to cordycepin except TWIST1 and to a lesser extent PIK3CB mRNA. Housekeeping mRNAs ACTB and RPL10A remain unchanged. Experiments were done in three independent biological replicates and none of the examined mRNAs were found to be significantly different from the untreated by paired T-test.
We also examined the cordycepin sensitivity of several mRNAs that were linked to the machinery of polyadenylation complexes. This was on the argument that there might be autoregulation, however we were unable to find any other sensitive mRNAs. Therefore in order to find cordycepin targeted mRNAs there is a need to create high throughput data in order to identify more target mRNAs of cordycepin, such as by microarray analysis.
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Figure 5.10: Cordycepin does not affect expression levels of mTOR and cancer mRNAs:
Total RNA was isolated from MCF-7 cells treated with cordycepin (50µM) for 2 hours. RT- qPCR was performed and the mRNA levels were expressed relative to GAPDH mRNA. Control values of untreated (no cordycepin) were set at 1. Error bars represent standard deviations of independent biological triplicates. Statistical significance compared to control (No cordycepin) was determined by paired T-test but none of the examined mRNAs were found to be significant.
5.11 Discussion
The half life of the cordycepin is very short due to its degradation in the blood by an enzyme called adenosine deaminase. To counteract this effect, cordycepin is usually administered in combination with the adenosine deaminases inhibitor deoxycoformycin or pentostatin, however there is some limitation in using these drugs due to their toxicity. To design more stable cordycepin analogues, we first need to firmly establish its mechanism of action. If cordycepin works by inhibiting polyadenylation, which PAPs is it targeting? Therefore other adenosine analogues were investigated which could be stable and could have different specificity for individual PAPs. Like cordycepin, other adenosine analogues also show different activities towards various cell lines. 8 amino adenosine, 8 azido adenosine and Se15 inhibit cellular rates of protein synthesis, perhaps by reducing the phosphorylation of 4EBP. In MCF-7 cells Se15 reduces the rate of protein synthesis by exerting its
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effects on P-4EBP. In MCF-7 cells cell proliferation was found to be sensitive to 8 aminoadenosine, 8 azidoadenosine and cladribine. Therefore, among all tested analogues 8 azidoadenosine may reduce cell proliferation by affecting the rate of protein synthesis and phosphorylation of 4EBP.
We also evaluated the effects on cell proliferation of poly(A) polymerase (PAP) alpha, gamma and Papd4 knockdowns and found all PAPs are necessary for cell proliferation. However no significant difference has been observed by PAP knock downs on total polyadenylation and no changes in levels of gene expression were observed. However we previously published that knockdown of PAPOLA reduces inflammatory response in ASM cells. We also investigated of the level at which cordycepin affects mRNA abundance and we observed post-transcriptional effects on all mRNAs we examined (CXCL1, IL8 and TWIST1). To elucidate the cordycepin mode of action in MCF7 cells in detail we screened several mRNAs in order to find cordycepin sensitive mRNAs but we only found 2 mRNAs among all examined. We therefore proceeded to conduct a microarray in order to find more cordycepin targetted mRNAs.
We therefore conclude that 8 aminoadenosine is the only compound that has similar cellular effects to cordycepin (protein synthesis, 4EBP level, proliferation and gene expression), as well as the only other compound that affects total poly(A) tail size. This indicates that the polyadenylation inhibition and the cellular effects are causally linked. Knockdown of 3 poly(A) polymerases in MCF-7 cell lines indicates that all are required for cell proliferation but that it does not have effects similar to cordycepin on gene expression. As both cordycepin and 8 aminoadenosine have been reported to act as chain terminators, it is possible that their effect is more due to a dominant negative effect mediated by an arrested complex. This would possibly be better mimicked by mutant PAPs, rather than by knockdown. Alternatively, there may be more redundancy in the PAPs in MCF7 cells, and multiple knockdowns should be tried. A genome wide study needs to be done to identify potential candidate mRNAs targeted by cordycepin.
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6 Identifying cordycepin sensitive mRNAs in MCF7
cells
In this chapter we attempt to elucidate the mechanism of action of cordycepin in more detail by performing microarray analysis, which allows the study of the expression of thousand of genes simultaneously in one sample. This experiment was done in collaboration with Professor Anne Willis (MRC Toxicology unit, Leicester) on MCF-7 cells treated with cordycepin (50µM) for 2 hours in a four independent biological replicates. Enrichment of genes in functional groups was also investigated using the DAVID (Database for Annotation, Visualisation and Integrated Discovery). The lists of significantly expressed genes were sorted according to fold change and the P-value for the significance of the change. Some significantly changed candidates were selected for validation by RT-qPCR. We selected four cordycepin targeted mRNAs and examined the effects of adenosine analogues and PAP knockdowns. To elucidate the mode of action of cordycepin on these target mRNAs in detail we also investigate the stage in mRNA processing that is affected by cordycepin.